BIOTEKNOLOGI DALAM PEMBUATAN WINE RENDAH ALKOHOL

Eklesia Vida Kurnia (200802101)

Fakultas Teknobiologi, Universitas Atma Jaya Yogyakarta.

PENDAHULUAN
Wine yang dijual di pasaran telah mengalami peningkatan kadar alkohol rata-rata
sekitar 1% (v/v) setiap 10 tahun, dan hingga saat ini telah terjadi peningkatan kadar total
3-4%. Hal ini dapat terjadi akibat pemanasan global yang mempengaruhi proses
pematangan buah anggur, dimana akumulasi gula yang lebih besar. Kandungan gula yang
tinggi dalam buah anggur matang, menyebabkan kadar alkohol tinggi karena semua
kandungan gula akan difermentasi oleh yeast.
Kadar alkohol yang terlalu tinggi dapat menjadi masalah bagi industri wine. Dari
segi sensori, tingginya kadar alkhol akan meningkatkan rasa pahit yang berlebihan, serta
meningkatkan rasa manis, astringency, dan panas, sehingga menutupi rasa buah anggur itu
sendiri. Dari segi kesehatan, tingginya kadar alkohol pada wine meningkatkan risiko
kesehatan konsumen dan mengancam keselamatan dalam berkemudi. Selain itu, dari segi
ekonomi, kadar alkohol yang makin tinggi akan meningkatkan pajak di beberapa negara
yang berorientasi pada kadar alkohol produk. Oleh karena itu, perlu dilakukan tindakan
untuk mencegah kadar alkhol yang terlalu tinggi pada akhir proses fermentasi wine.

PENDEKATAN BIOTEKNOLOGI
Dalam beberapa tahun terakhir, telah dilakukan ada upaya pendekatan bioteknolgi untuk
membatasi kandungan akohol dalam wine. Pendekatan yang cukup popular yaitu dari sisi
mikrobiologi, seperti rekayasa genetika yeast dan pencarian spesies dan galur yeast yang
tepat dalam proses fermentasi.

1. Penghapusan PDC2 pada Saccharomyces cerevisiae

Salah satu proses dalam fermentasi yaitu piruvat menjadi asetaldehida, yang
dikatalisis oleh piruvat dekarboksilase (PDC). PDC2 merupakan pengkode faktor
transkripsi yang terlibat dalam gen PDC. Penghapusan PDC2, pengkodean faktor
transkripsi yang terlibat dalam ekspresi gen PDC, menghasilkan aktivitas PDC 19%,
dengan pengurangan yang jelas dalam kandungan etanol, dikombinasikan dengan
peningkatan pelepasan gliserol dan piruvat. Gliserol berkontribusi positif dalam
viskositas dan rasa manis wine. Efek ini ditingkatkan dengan peningkatan aktivitas
 gliserol-3-fosfat dehidrogenase, melalui overekspresi GPD1, tanpa produksi berlebih
dari asam asetat dan asetaldehida.

2. Mutasi Faktor Transkripsi Spt15p

Global transcriptional machinery engineering (gTME) digunakan untuk
mengembangkan strain S. cerevisiae dengan kemampuan produksi etanol pada proses
fermentasi berkurang. Sasaran teknologi ini adalah memutasi faktor transkripsi umum
Spt15p (TATA binding protein) yang berperan dalam kerja RNA polimerase dan
sebagai salah satu protein pengikat DNA utama yang mengatur spesifisitas promotor
dalam ragi. Strain yang digunakan yaitu S. cerevisiae YS59 (MATα; ura3-52, leu2-3,
dan 5-519).
Gen SPT15 ditransfer ke plasmid pada situs restriksi antara BamHI dan EcoRI
menggunakan vektor pY16, yang diapit oleh promotor TEF1 dan terminator CYC1
(pY16-SPT15). Dilakukan mutagenesis acak (error-prone PCR) dari gen SPT15
menggunakan kit DiversifyTM PCR Random Mutagenesis (Clontech) dengan pY16-
SPT15 sebagai templat. Plasmid yang diperoleh ditransfer ke Escherichia coli JM109,
kemudian koloni dipilih secara acak untuk identifikasi mutasi.
Plasmid yang mengandung SPT15 termutasi, kembali ditransfer ke S. cerevisiae
YS59 dengan metode lithium asetat, kemudian diinkubasi pada 25◦C pada medium SD
padat untuk menghasilkan yeast library for SPT15 mutant. Setelah masa inkubasi,
diambil 20 mutan dengan biomassa tertinggi. Dipilih biomassa tertinggi karena strain
dengan biomassa tingi umumnya memiliki kapasitas produksi etanol yang lebih rendah
akibat persaingan untuk sumber karbon antara produksi biomassa dan etanol.
Dilakukan uji kapasitas produksi etanol dengan memfermentasi masing-masing dalam
10 mL media Triple M, kemudian strain dengan hasil etanol terendah diidentifikasi
dan diapatkan strain YS59-409. Strain ini dibandingkan dengan strain kontrol,
menunjukkan produksi etanol rendah yaitu berkurang secara signifikan sebesar 34,9%.

3. Mutasi Faktor Transkripsi Pdc2p

Sasaran teknologi ini merupakan protein transkripsi Pdc2p. Faktor transkripsi
Pdc2p yang diubah secara struktural dapat menunjukkan penurunan aktivitas regulasi
positif PDC1 dan PDC5. Penurunan aktivitas regulasi ini mengarah pada pengurangan
moderat aktivitas enzimatik PDC dan akibatnya pengurangan produksi etanol.
 Protein Pdc2p mutasi, pdc2Δ519, diintegrasikan ke dalam genom yeast melalui
rekombinasi homolog. Yeast yang digunakan adalah Saccharomyces cerevisiae
BY4743. Strain mutan BY4743pdc2Δ519 menunjukkan penurunan sekitar 1-2% (v/v)
kandungan etanol wine, tanpa ada pengaruh signifikan terhadap konsentrasi sisa gula
dan asam asetat. Selanjutnya, dilakukan pemeriksaan terhadap fenotipe untuk dapat
direproduksi pada jenis yeast lain yang sering digunakan pada fermentasi wine pada
skala industri. Mutasi pdc2Δ519 diintegrasikan dengan transformasi dan rekombinasi
homolog ke dalam genom EC1118 komersial dan ragi anggur asli Mab2C.
Setelah penyisipan satu salinan mutasi pdc2Δ519, diamati kurva pertumbuhan
dalam kondisi aerobik dan vinifikasi skala lab untuk membandingkan EC1118Δ519
dan Mab2CΔ519 yang dimodifikasi secara genetik dengan galur kontrol berupa wild
type. Hasil menunjukkan bahwa mutan EC1118Δ519 dan Mab2CΔ519 tumbuh normal
di media YPD, serta tidak menunjukkan perbedaan dengan kontrol. Analisis parameter
fermentasi menunjukkan bahwa EC1118Δ519 dapat menghasilkan pengurangan total
etanol sebesar 15%, sedangkan Mab2CΔ519 dapat menghasilkan pengurangan total
etanol sebesar 10%, dibandingkan dengan kontrol. Konsentrasi asam asetat
EC1118Δ519 dan Mab2CΔ519 juga tidak terpengaruh oleh mutasi.

4. Delesi Gen ADH

Langkah enzimatik terakhir dalam pembentukan etanol yaitu kerja dari enzim alkohol
dehidrogenase (ADH). Penghapusan gen pengkode ADH menyebabkan pengalihan
seluruh karbon untuk digunakan dalam pembentukan gliserol dan asetal-dehida.
Namun, teknologi ini memiliki kekurangan karena memiliki sensori yang kurang baik
akibat tingginya kadar asetal dehida.

5. Penggunaan Spesies Saccharomyces Non-Konvensional

Salah satu spesies Saccharomyces yang dapat digunakan yaitu Saccharomyces
uvarum. S. uvarum telah terbukti menghasilkan wine dengan konsentrasi alkohol
(<0,7% v/v) dan asiditas volatil (asam asetat) yang lebih rendah dibandingkan wine
yang difermentasi dengan S. cerevisiae. Wine yang diproduksi dengan S. uvarum
memiliki atribut sensori “off-flavor” yang unik dan tidak dimiliki wine yang diproduksi
dengan S.cerevisiae. Namun, atribut ini dapat menjadi suatu hal negatif dalam industri
karena rasa yang berbeda dari wine konvensional.
6. Penggunaan Jenis Yeast Lain
Jenis yeast lain yang digunakan untuk menghasilkan wine rendah alkohol yaitu
Metschnikowia pulcherrima (FLAVIA MP346). Metschnikowia pulcherrima
diinokulasikan terlebih dahulu, kemudian setelah 48 jam, diinokulasikan
Saccharomyces bayanus, kemudian setelah 96 jam diinokulasikan Saccharomyces
cerevisiae. Hasil fermentasi dengan 3 yeast ini menghasilkan produksi anggur dengan
kandungan etanol yang rendah (penurunan 0,9% v/v dibandingkan wine kontrol).
Namun, teknologi ini memiliki kekurangan karena terdapat perbedaan kandungan
senyawa aromatik tertentu, serta rasa dan aroma yang sering dianggap negatif oleh
industri.

SIMPULAN
Berdasarkan studi pustaka yang telah dilakukan, diketahui bahwa penerapan bioteknologi,
khususnya pendekatan mikrobiologi yang paling efektif untuk produksi wine rendah
alkohol adalah dengan melakukan mutasi pada faktor transkripsi Pdc2p. Hal ini
dikarenakan teknologi mutasi Pdc2p berhasil menurunkan kadar alkohol pada produk
akhir wine pada berbagai strain, baik skala lab maupun skala industri. Teknologi ini
berhasil menurunkan kadar alkohol dalam wine sebesar 1-15% dibandingkan wine
komersil umumnya.

DAFTAR PUSTAKA
Cuello, R. A., Montero, K. J. F., Mercado, L. A., Combina, M. dan Ciklic, I. F. 2017.
Construction of low-ethanol–wine yeasts through partial deletion of the
Saccharomyces cerevisiae PDC2 gene. AMB Express 7 (67): 1-11.

Du, Q., Liu, Y., Song, Y. dan Qin, Y. 2020. Creation of a low-alcohol-production yeast by
a mutated SPT15 transcription regulator triggers transcriptional and metabolic
changes during wine fermentation. Frontiers in Microbiology 11 (597828): 1-10.

Gonzalez, R., Guindal, A. M., Tronchoni, J. dan Morales, P. 2021. Biotechnological

approaches to lowering the ethanol yield during wine fermentation. Biomolecules
11 (1569): 1-16.
Puškaš, V. S., Miljić, U. D., Djuran, J. J. dan Vučurović, V. M. 2019. The aptitude of
commercial yeast strains for lowering the ethanol content of wine. Food Science
& Nutrition 2020 (8):1489–1498.

Varela, J. dan Varela, C. 2019. Microbiological strategies to produce beer and wine with
reduced ethanol concentration. Current Opinion in Biotechnology 2019 (56):88–
96.
Cuello et al. AMB Expr (2017) 7:67
DOI 10.1186/s13568-017-0369-2

ORIGINAL ARTICLE Open Access

Construction of low‑ethanol–wine yeasts

through partial deletion of the Saccharomyces
cerevisiae PDC2 gene
Raúl Andrés Cuello1,2, Karina Johana Flores Montero2, Laura Analía Mercado2, Mariana Combina1,2
and Iván Francisco Ciklic2* 

Abstract 
We propose an alternative GMO based strategy to obtain Saccharomyces cerevisiae mutant strains with a slight reduc-
tion in their ability to produce ethanol, but with a moderate impact on the yeast metabolism. Through homologous
recombination, two truncated Pdc2p proteins Pdc2pΔ344 and Pdc2pΔ519 were obtained and transformed into
haploid and diploid lab yeast strains. In the pdc2Δ344 mutants the DNA-binding and transactivation site of the protein
remain intact, whereas in pdc2Δ519 only the DNA-binding site is conserved. Compared to the control, the diploid
BY4743pdc2Δ519 mutant strain reduced up to 7.4% the total ethanol content in lab scale-vinifications. The residual
sugar and volatile acidity was not significantly affected by this ethanol reduction. Remarkably, we got a much higher
ethanol reduction of 10 and 15% when the pdc2Δ519 mutation was tested in a native and a commercial wine yeast
strain against their respective controls. Our results demonstrate that the insertion of the pdc2Δ519 mutation in wine
yeast strains can reduce the ethanol concentration up to 1.89% (v/v) without affecting the fermentation performance.
In contrast to non-GMO based strategies, our approach permits the insertion of the pdc2Δ519 mutation in any locally
selected wine strain, making possible to produce quality wines with regional characteristics and lower alcohol con-
tent. Thus, we consider our work a valuable contribution to the problem of high ethanol concentration in wine.
Keywords:  Yeast, Ethanol, Metabolism, Genetic engineering, Wine

Introduction global warming, this effect is particularly exacerbated in

Nowadays there is a growing demand for softer wines
with reduced ethanol content. However, during the last
twenty years there has been an increment in the alcohol
concentration of wine of about 2% (v/v) (Kutyna et  al.
2010; Tilloy et  al. 2014). Current viticultural practices
favor the harvest of very mature grapes to obtain wines
with sweet tannins, as demanded by the consumers.
The biosynthesis of polyphenols is usually delayed with
respect to sugar production, leading to a harvest of grapes
with high sugar amounts. This sugar excess in turn, pro-
duces wines with high ethanol. As a consequence of
*Correspondence: ciklic.ivan@inta.gob.ar
2
Laboratorio de Biotecnología, Estación Experimental Agropecuaria
Mendoza, Instituto Nacional de Tecnología Agropecuaria (INTA), San
Martín 3853, Luján de Cuyo, Mendoza, Argentina
Full list of author information is available at the end of the article
regions with hot summers (Mira de Orduña 2010). A high
concentration of alcohol in wine can have many negative
consequences. The quality of the product may be com-
promised, e.g. the perception of viscosity and hotness
could be enhanced, in detriment of acidity, aroma, inten-
sity of flavors, sweetness and other organoleptic proper-
ties (Gawel et al. 2007a, b). Production costs may rise in
countries where taxes are applied according to the etha-
nol content. Sluggish or stuck fermentations might also
happen as a result of yeast inhibition provoked by high
ethanol levels (Buescher et  al. 2001). In addition, con-
sumption of wine with high ethanol content can also have
a negative impact on health. This combination of quality,
economic and health problems associated with high alco-
hol in wine, has promoted a significant interest in devel-
oping technologies to reduce ethanol, but conserving all

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.
Cuello et al. AMB Expr (2017) 7:67
desirable sensory characteristics of wine (Kutyna et  al.
2010). Indeed, this problem represents a major chal-
lenge for the wine industry in its attempt to counteract
some of the negative effects of global warming. Differ-
ent approaches have been tried, such as the application
of alternative viticultural practices (Kontoudakis et  al.
2011), or the implementation of physical methods like
dealcoholization (Bogianchini et  al. 2011). Neverthe-
less, the microbiological approach is probably the most
attractive because of its easy implementation and low
costs (Kutyna et al. 2010). The microbiological approach
includes the use of non-GMO (genetically modified
organisms) strategies like sequential inoculations and
co-inoculations of S. cerevisiae with non-Saccharomyces
yeasts, as well as GMO-based strategies with the use of
genetically modified yeasts. Considering the first case,
sequential inoculations and co-inoculations have become
quite popular in recent years (Comitini et al. 2011; Mag-
yar and Tóth 2011; Di Maio et  al. 2012; Sadoudi et  al.
2012 and reviewed in Ciani et  al. 2016). Unfortunately,
low yields of ethanol are usually the result of wines with
high residual sugar concentrations (Ciani and Ferraro
1996; Ciani et al. 2006). In some cases, reduction in the
ethanol concentration varied only between 0.2 and 0.7%
(v/v) (Benito et al. 2011; Gobbi et al. 2013). However, in
recent works, ethanol reduction between 1.5 and 2.2%
(v/v) has been achieved through different strategies like
sequential fermentation with immobilized non-Saccharo-
myces yeasts (Canonico et  al. 2016) and the use of non-
Saccharomyces yeasts combined with S. cerevisiae under
controlled aeration conditions (Contreras et  al. 2015;
Morales et al. 2015). With regards to GMO-based strate-
gies, most of the studies have concentrated in redirecting
part of the carbon flux from the ethanol metabolic path-
way to other secondary products like glycerol. The over-
expression of GPD1 and/or GDP2 genes, which encode
the glycerol-3-phosphate dehydrogenase isozymes,
reduce the ethanol and enhance the glycerol production
(Remize et al. 1999; Lopes et al. 2000). However, the con-
centrations of some undesirable by-products, particularly
acetic acid, are also increased. There were some efforts to
inhibit the formation of acetic acid by deleting the ALD6
gene, but this increased the synthesis of other oxidized
compounds like acetoin, which has negative organoleptic
properties (Cambon, et al. 2006; Eglinton, et al. 2002). In
a previous study, a large number of genetic modifications
were generated and evaluated in order to reduce ethanol
concentration in wine (Varela et al. 2012). Using the same
genetic background, 41 different alterations in different
combinations were tested. Of all the strategies tried, the
most successful to reduce ethanol were those designed to
increase glycerol formation. Still, like in previous works,
the increase of glycerol formation was accompanied with
Page 2 of 11
high levels of oxidized by-products like acetic acid, ace-
toin and acetaldehyde, and this effect could not be com-
pletely neutralized with additional genetic modifications.
In the present work, we propose an alternative GMO
based strategy to obtain S. cerevisiae mutant strains with
a slight reduction in their ability to produce ethanol, but
with a moderate impact on the yeast metabolism. For this
purpose, we designed two functional strategic mutations
at the C-terminus of the PDC2 gene in order to alter the
structure of the encoded protein. PDC2 encodes a tran-
scription factor (Hohmann 1993) that regulates the avail-
ability of the pyruvate decarboxylase (PDC) isozymes
Pdc1p and Pdc5p, which catalyze the reaction of pyruvate
to acetaldehyde in the ethanol biosynthetic pathway [the
main active form during glucose catabolism is PDC1 and
PDC5 is a secondary form which is only expressed under
thiamine starvation (Mojzita and Hohmann 2006)]. The
full Δpdc2 deletion has already been tested to study the
genetic factors affecting the glycerol formation and its
overproduction (Nevoigt and Stahl 1996). Although the
glycerol formation is enhanced without affecting the ace-
tic acid concentration, the Δpdc2 deletion exhibits a phe-
notype incompatible for yeasts with oenological purposes
(drastic reduction of PDC specific activity and ethanol
concentration, as well as strong inhibition of growth in
aerobic conditions). A structurally altered version of the
Pdc2p transcription factor may display a reduced posi-
tive regulatory activity of PDC1 and PDC5, leading to a
moderate reduction of the PDC enzymatic activity and
consequently a reduction in ethanol production. Through
homologous recombination, two truncated Pdc2p pro-
teins lacking 344 (pdc2Δ344) or 519 (pdc2Δ519) amino
acids at the C-terminus were obtained and transformed
into lab yeast strains. In the case of pdc2Δ344 the DNA-
binding and transactivation site are both intact, whereas
in pdc2Δ519 only the DNA-binding site is conserved.
Subsequently, these mutants were tested in lab-scale vini-
fications to select low-ethanol yeasts. The selected muta-
tion was then tested in both native and commercial wine
yeast strains.
Materials and methods
Strains, media and growth conditions
Saccharomyces cerevisiae laboratory strains BY4741
(haploid, MATa; his3Δ 1; leu2Δ 0; met15Δ 0; ura3Δ 0)
and BY4743 (diploid, MATa/MATα his3Δ 1/his3Δ 1;
leu2Δ0/leu2Δ 0; met15Δ 0/MET15; LYS2/lys2Δ 0; ura3Δ
0/ura3Δ 0) were used to construct the mutants and as
control strains during the vinification experiments. The
commercial wine yeast EC1118 (Lallemand, Denmark)
and the native Mab2C strain previously selected in our
laboratory, were used to test the pdc2Δ519 mutation in
native genetic backgrounds. All yeast strains were grown
Cuello et al. AMB Expr (2017) 7:67
at 30  °C and 150  rpm in YPD medium (2% glucose, 2%
peptone and 1% yeast extract). YPD supplemented with
200  mg/L of G-418 was used for selection and mainte-
nance of transformants.
Construction and genomic integration of the pdc2Δ344
and pdc2Δ519 mutations
The pdc2Δ344 and pdc2Δ519 mutations were integrated
into the yeast genome through homologous recombi-
nation following the method described by Güldener
et  al. (1996). The disrupting fragment was obtained by
PCR amplification of the KanMx resistance cassette
present in the pUG6 plasmid (Güldener et  al. 1996).
The primers used were, forward 5′-ACAGAATACT-
GTTGATAATAGTACCAAAA CAGGTAACCCTT-
GAAGCTGAAGCTCGTACGC-3 for pdc2Δ344 or
5′-TTGGGAT GATATACCCGTTGATGCTATCAAA-
GCAAATTGGTGAAGCTGAAGCTTCGTACGC-3 for
pdc2Δ519 with the reverse primer 5′-CTAAAAAAA-
GCCTGTGT TACCAGGTAAGTGTAAGTTATTAG-
CATAGGCCACTAGTGGATCTG-3′. A specific PDC2
homologous sequence was inserted at the 5′-end of
each primer to generate the recombination event at the
expected C-terminal region of the PDC2 gene. Besides,
two stop codons were placed downstream to the homolo-
gous region of each forward primer to generate the spe-
cific truncated proteins at the C-terminus. After the PCR
reaction, the disrupting fragments were transformed by
a slightly modified version of the lithium acetate method
(Gietz and Woods 2002) selecting the G-418 resistant
transformants. The correct insertion of the fragment was
confirmed by PCR using the forward primer 5′-GCGTG-
GTCGACTCAAAACCAATAGCTGCTTAAAAA-3′
which binds upstream of the PDC2 gene and the reverse
5′-GGATGTATGGGCTAAATG-3′ which binds inside
the KanMx resistance cassette.
Growth curves in YPD rich medium
Yeast cultures were inoculated into 10  mL YPD and
grown overnight. These cultures were then used to
inoculate 50 mL of YPD in 100 mL Erlenmeyer flasks at
an initial OD600 of 0.2 (Spectrophotometer UV–visible
T60U PG Instruments, Leicestershire, UK). The cultures
were grown with 150 rpm at 30 °C and aliquots of 100 µL
were taken at different intervals for the measurement of
the OD600.
Laboratory scale vinifications
Following the recommendation of Vazquez et al. (2001),
Lab-scale vinifications were carried out using concen-
trated white must as substrate, diluted to a final sugar
concentration of 20°Bx and supplemented with 1  g/L of
yeast extract. The vinifications were performed in 500 mL
Page 3 of 11
flasks plugged with glass fermentation traps so that only
CO2 could evolve from the system, and they were kept
at 28  °C without agitation (Vaughan-Martini and Mar-
tini 1998). All vinification experiments were performed
under the described conditions but comparing different
strains (V1, V2, V3 and V4). Fermentation kinetics was
monitored by measuring the daily CO2 weight loss. Alco-
hol concentration, acetic acid and residual sugar were
measured according to standard methods (OIV 2015),
whereas glycerol was measured with spectrophotometry
(WinescanTM Foss, Hillerød, Denmark). Several derived
fermentative parameters such as carbon balance, glucose
and ethanol yield were calculated (Vazquez et  al. 2001).
Carbon balance was calculated as the ratio between
carbon moles of fermentation by-products and carbon
moles of glucose. Meanwhile, glucose yield results from
the amount of glucose required (g) to produce 1% (v/v) of
ethanol, and ethanol yield, from the ratio between grams
of produced ethanol and grams of consumed glucose. All
assays were performed at least in triplicates (three inde-
pendent cultures).
Statistical analysis of data
An ANOVA and a LSD Fisher test with a p value <0.05
was performed for the analysis and media comparison
of the growth, fermentative and kinetics parameters.
Growth parameters as well as kinetic parameters were
estimated using the reparameterized Gompertz equation
as proposed by Zwietering et al. (1990):
y = D∗ exp −exp[((µmax ∗ e)/D) ∗ ( − t) + 1]
where y = ln (ODt/OD0) OD0 is the initial OD and ODt is
the OD at time t; D = ln (ODmax/DO0) is the curve maxi-
mum asymptotic, μmax is the maximum specific growth
rate (1/h), and λ is the lag phase period (h). Growth and
kinetics data were fitted by nonlinear regression proce-
dure, minimizing the sum of squares of the difference
between the experimental data and the fitted model.
Results
Growth of haploid and diploid pdc2Δ519 and pdc2Δ344
mutants in aerobic conditions
The Δpdc2 complete deletion causes a drastic reduction
of the PDC specific activity, an accumulation of pyruvate
and a strong inhibition of growth in aerobic conditions
(Hohmann 1993; Nevoigt and Stahl 1996). The ability to
grow in aerobic conditions is essential for a yeast strain
to be selected to develop wine yeast starters. Although
the conditions of wine fermentation are predominantly
anaerobic, the biomass production is performed in
aerobic conditions. Hence, before quantifying the fer-
mentative parameters of each strain we wanted to test
the growth capability of the pdc2Δ519 and pdc2Δ344
Cuello et al. AMB Expr (2017) 7:67
mutants under aerobic conditions with glucose as carbon
source. Figure 1 shows the growth curves in YPD medium
for the haploid mutant strains BY4741pdc2Δ344 and
BY4741pdc2Δ519 as well as the diploid BY4743pdc2Δ344
and BY4743pdc2Δ519 with their respective BY4741 and
BY4743 controls. Remarkably, all mutants displayed
growth pattern similar to the controls, and consequently
no statistical difference was detected among the kinetic
parameters analyzed (λ, and µmax) (data not shown). This
result demonstrates that all mutant strains grow as good
as the controls in aerobic conditions, and therefore they
are suitable for further experimentation.
Quantification of fermentative parameters from lab‑scale
vinifications and selection of low ethanol pdc2 mutants
In order to select low ethanol pdc2 mutant strains, we
performed a series of microvinifications with concen-
trated must diluted to 20°Bx and supplemented with
Fig. 1  Growth of parental and mutant laboratory strains in YPD at 30 °C and 150 rpm shaking. Each point represents the average value of two
independent cultures
Page 4 of 11
0.1% (w/v) of yeast extract (Vazquez et al. 2001). The con-
centration of ethanol, acetic acid, glycerol and residual
sugar were determined, and other derived fermentative
parameters like carbon balance and glucose yield were
calculated. We searched for a mutant strain with a slight
inefficiency to produce ethanol in order to reduce the
alcohol content of wine between 1 and 2% (v/v). During
the first vinification experiment (V1) the haploid and dip-
loid pdc2Δ519 mutants were assayed against their respec-
tive BY4741 and BY4743 controls (Table 1). Considering
the ethanol production, BY4741pdc2Δ519 showed no
statistical difference when compared to the control,
whereas BY4743pdc2Δ519 displayed a statistically sig-
nificant ethanol reduction (p  <  0.05) of around 7%. The
moderate reduction observed in BY4743pdc2Δ519 was
within the expected, however, it is surprising we didn’t
observe any phenotype for the haploid BY4741pdc2Δ519,
which carries only the mutant copy of the PDC2 gene.
Table 1  Fermentative parameters of lab-scale vinification assays in 20°Bx white must by parental and mutant laboratory yeast strains
Parameter BY4741 BY4741 BY4743 BY4743 BY4741 BY4741 BY4743 BY4743 BY4743
pdc2Δ519 pdc2Δ519 pdc2Δ344 pdc2Δ344 pdc2Δ519
Cuello et al. AMB Expr (2017) 7:67

Main compounds V1 V1 V1 V1 V2 V2 V2 V2 V2
(g/Liter)
 Consumed 153.95 ± 0.87A 146.49 ± 0.63B 147.22 ± 0.00B 143.84 ± 1.66C 172.04 ± 4.64B,C 154.90 ± 4.22D 179.56 ± 3.08A 170.98 ± 1.98C 176.05 ± 4.71A,B
sugar
 CO2 71.11 ± 1.92A 73.33 ± 5.77A 66.67 ± 0.00A,B 62.22 ± 3.85B 81.88 ± 3.89A,B 72.48 ± 2.52C 83.45 ± 1.60A 78.68 ± 2.84B 83.75 ± 2.20A
A A A B A C A A,B
 Ethanol 75.61 ± 0.91 75.35 ± 2.23 74.56 ± 1.58 69.04 ± 0.79 87.58 ± 5.69 76.007 ± 2.28 87.74 ± 1.41 84.27 ± 2.58 81.27 ± 1.48B
A A,B A,B B A B,C C B
 Acetate 1.11 ± 0.16 0.91 ± 0.06 0.88 ± 0.24 0.78 ± 0.07 1.11 ± 0.11 0.63 ± 0.06 0.58 ± 0.12 0.83 ± 0.22 0.60 ± 0.09C
b B B,C B A
 Glycerol ND ND ND ND 2.67 ± 0.20 2.44 ± 0.074 2.58 ± 0.14 3.16 ± 0.11 2.24 ± 0.18C
a
Balance (%)
 Carbon 97.18 ± 1.27B 101.43 ± 3.18A 97.64 ± 0.72A,B 95.26 ± 1.13B 99.32 ± 2.50A 97.33 ± 1.27A,B 96.40 ± 0.96A,B 96.67 ± 2.66A,B 95.18 ± 2.37B
Yield
 Ethanol produc- 9.58 ± 0.12A 9.55 ± 0.20A 9.45 ± 0.20A 8.80 ± 0.10B 11.1 ± 0.72A 9.63 ± 0.29C 11.12 ± 0.18A 10.68 ± 0.33A,B 10.30 ± 0.19B
tion (%[v/v])
 EtOH (g/g 0.49 ± 0.008A 0.51 ± 0.011A 0.51 ± 0.009A 0.48 ± 0.009A 0.51 ± 0.02A 0.49 ± 0.02A,B 0.49 ± 0.008B 0.49 ± 0.019A,B 0.46 ± 0.012C
glucose
consumed)
 Glucose (g) 16.07 ± 0.28A,B 15.34 ± 0.27C 15.58 ± 0.33B,C 16.35 ± 0.32A 15.52 ± 0.62B 16.09 ± 0.59B 16.15 ± 0.25B 16.02 ± 0.62B 17.09 ± 0.42A
required
for 1% (v/v)
ethanol
production
 Glucose (g) ND ND ND ND 65.64 ± 4.21B 69.49 ± 2.30A,B 64.56 ± 3.83B 54.57 ± 1.99C 73.64 ± 3.38A
required for 62.65 ± 0.87C 70.11 ± 0.63B 69.38 ± 0.00B 72.76 ± 1.66A 44.56 ± 4.64B,C 61.7 ± 4.22A 37.04 ± 3.08D 45.62 ± 1.98B 40.55 ± 4.71C,D
1 g/L glycerol
Residual sugar
(g/Liter)
ND not determined
Distinct letters correspond to statistical significant difference for a Fischer test with p < 0.05
a
  Carbon balance represents the ratio between carbon moles of fermentation by-products and carbon moles of glucose
b
  Glycerol was measured in an independent experiment
Page 5 of 11
Cuello et al. AMB Expr (2017) 7:67
In correspondence with the observed ethanol reduc-
tion, BY4743pdc2Δ519 also presented the lowest value
of carbon balance and it was the least efficient to pro-
duce ethanol. It is important to note that this ethanol
reduction didn’t cause an increase of the volatile acid-
ity. Taken together, this was a promising result and as a
consequence BY4743pdc2Δ519 was selected for further
experimentation. Continuing with the mutant’s char-
acterization we performed a second vinification experi-
ment (V2), with the selected BY4743pdc2Δ519, the still
uncharacterized BY4741pdc2Δ344, BY4743pdc2Δ344d
strains, and both haploid BY4741 and BY4743 diploid
controls (Table  1). The same trend was observed for
the mutant BY4743pdc2Δ519, which again is the least
efficient to produce ethanol, showing statistically sig-
nificant lower values of glucose yield compared with the
control and the rest of the strains. With regards to the
ethanol production of the pdc2Δ344 mutants, the hap-
loid produced significantly less ethanol than the BY4741
control, whereas the mutant diploid showed no statisti-
cal difference comparing with the diploid BY4743 con-
trol. Nonetheless, the ethanol reduction observed for
BY4741pdc2Δ344 was not reflected in the efficiency of
the mutant strains, since its glucose yield was not sig-
nificantly different from the control. This compensation
in the glucose yield production is explained by the fact
that BY4741pdc2Δ344 displayed considerably higher val-
ues of residual sugar compared with BY4741. Up to this
point, the BY4743pdc2Δ519 mutant showed a consistent
phenotype of ethanol reduction being the least efficient
in both vinifications. According to the value obtained for
the glucose yield, BY4743pdc2Δ519 would reduce the
ethanol almost 1 degree (0.85) for a wine with a predic-
tion of 15.5% (v/v). The BY4743pdc2Δ344 mutant showed
an intermediate phenotype producing 4% less ethanol
than the control, but there was no statistically significant
difference in the glucose yield. In summary, the vinifica-
tion experiments showed that BY4743pdc2Δ519 is the
most promising mutant strain displaying a small but con-
sistent reduction of the ethanol concentration, as a result
of a slight inefficiency for its production. Importantly,
this ethanol reduction did not cause an increment of the
concentration of acetic acid, which is perhaps the most
undesirable by-product of the wine fermentation.
Most of the efforts to deviate the carbon flux away from
ethanol have concentrated in the production of glycerol,
a desirable secondary metabolite of fermentation. There-
fore, the glycerol produced in lab-scale vinifications was
also measured (Table  1). The quantification of glycerol
could give as a clue of what is happening to the carbon
flux of the pdc2Δ mutants, particularly BY4743pdc2Δ519
which showed a consistent ethanol reduction. Surpris-
ingly, the glycerol production of BY4743pdc2Δ519 was
Page 6 of 11
not increased but reduced, showing statistically sig-
nificant differences with the control in the final concen-
tration and the glucose yield for glycerol. In contrast,
BY4743pdc2Δ344 which previously showed no ethanol
reduction produced more glycerol than the control with
statistically significant higher values. At least for the
Δpdc2 diploid mutants, there seems to be no clear cor-
relation between ethanol and glycerol production and the
ethanol reduction observed in BY4743pdc2Δ519 is not a
consequence of an increment of glycerol concentration.
Kinetics analysis of the vinifications by CO2 weight loss
The progression of the vinifications was daily moni-
tored by measuring the CO2 weight loss. Figure 2 shows
the curves of accumulated CO2 weight loss obtained for
each vinification experiment. As seen in the graphs (pan-
els a, b), all fermentations were very slow, lasting around
three weeks (an industrial fermentation performed with
wine yeasts usually last 7–10  days). Nevertheless, this
result is not surprising considering we used lab strains,
which are not specialized for wine fermentation. We cal-
culated for each curve the mean value of the three main
kinetic parameters (Zwietering et  al. 1990) lag phase
(λ), maximum CO2 weight loss speed (μmax) and total
accumulated CO2 weight loss (A, for asymptote). After
performing an ANOVA, the mean values of the kinetic
parameters were compared (Table  2). With few excep-
tions, the kinetics of the fermentations was very similar
between the mutants and their controls. In the first vini-
fication (V1) there was no statistical difference between
the haploid strains BY4741pdc2Δ519h and BY4741, but
they lost more CO2 than the diploid strains. The diploid
mutant BY4743pdc2Δ519 lost less CO2 than its control
but the difference was not statistically significant. Con-
sidering that this strain also produces less ethanol, we
expected a higher CO2 reduction for BY4743pdc2Δ519,
which was not the case. In the second vinification,
BY4743pdc2Δ519 showed again similar values of CO2
and μmax when compared to the control. It is interest-
ing to note that despite the ethanol reduction observed
for BY4743pdc2Δ519, there was little (vinification 1) or
no difference (vinification 2) in the total CO2 weight loss
comparing with the BY4743 control. Apparently, another
decarboxylation reaction is compensating the CO2
formed during the ethanol biosynthesis, and this is a clue
which could help us to reveal how the carbon flux has
been modified in the BY4743pdc2Δ519 mutant strain.
Insertion of the pdc2Δ519 mutation in the commercial
EC1118 and native Mab2C wine yeast strains
The BY4743pdc2Δ519 mutant strain showed a pheno-
type according to our aim of reducing around 1–2%
(v/v) the ethanol content of wine, without significantly
Cuello et al. AMB Expr (2017) 7:67 Page 7 of 11

Fig. 2  Accumulated CO2 weight loss curves along the experiments of vinifications 1 (a), 2 (b), 3 (c) and 4 (d) with parental and mutant strains. The
evolution of each vinification was daily monitored by measuring the CO2 weight loss. Each point represents the average value of three independent
cultures

Table 2  Quantification and statistical analyses of fermentation parameters

Parameter BY4741 BY4741 BY4743 BY4743
pdc2Δ519 pdc2Δ519

Vinification 1
 A 21.96 ± 0.59A,B 22.83 ± 1.57A 21.31 ± 0.52A,B 19.79 ± 1.74B
 µmax 1.8 ± 0.05A 1.74 ± 0.13A 1.44 ± 0.05B 1.39 ± 0.01B
 λ 2.66 ± 0.26A 2.38 ± 0.01A 2.47 ± 0.39A 2.86 ± 0.40A
BY4741 BY4741 BY4743 BY4743 BY4743
pdc2Δ344 pdc2Δ344 pdc2Δ519

Vinification 2
 A 25.24 ± 1.10A,B 22.05 ± 0.68C 26.11 ± 0.49A 24.38 ± 0.88B 26.07 ± 0.74A
B,C C A,B B
 µmax 1.78 ± 013 1.64 ± 0.09 1.91 ± 0.06 1.80 ± 0.09 1.98 ± 0.06A
A A,B B A,B
 λ 2.97 ± 1.11 2.30 ± 0.17 1.80 ± 0.05 1.97 ± 0.05 2.04 ± 0.23B
A = Asymptote, µmax = maximum specific growth rate and (λ) = lag time, for the three vinifications
Distinct letters correspond to statistical significant difference for a Fischer test with p < 0.05
Cuello et al. AMB Expr (2017) 7:67 Page 8 of 11

Table 3 Determination of  fermentative parameters for  mutant and  wild type wine yeast strains EC1118 and  Mab2C
in two independent lab-scale vinifications
Parameter EC1118 EC1118Δ519 Mab2C Mab2CΔ519
V3 V3 V4 V4

Main compounds (g/L)

 Consumed sugar 210.38 ± 0.64A 203.95 ± 2.51B 212.03 ± 0.44A 209.01 ± 0.63B
A A A
 CO2 94.84 ± 2.05 91.56 ± 3.01 95.60 ± 3.50 93.51 ± 1.40A
A B A
 Ethanol 102.31 ± 2.99 87.05 ± 2.77 95.73 ± 4.05 86.00 ± 1.58B
A A A
 Acetate 1.13 ± 0.16 1.12 ± 0.19 0.83 ± 0.17 0.86 ± 0.19A
Balance (%)
 Carbon 94.42 ± 1.81A 88.81 ± 1.48B 90.82 ± 3.19A 86.78 ± 0.47A
Yield
 Ethanol production (% [v/v]) 12.97 ± 0.38A 11.03 ± 0.35B 12.13 ± 0.51A 10.90 ± 0.20B
A B A
 EtOH (g/g glucose consumed) 0.49 ± 0.013 0.43 ± 0.08 0.47 ± 0.021 0.42 ± 0.008B
A B B
 Glucose (g) required for 1% (v/v) ethanol production 16.23 ± 0.42 18.49 ± 0.36 17.50 ± 0.71 19.18 ± 0.32A
A B B
 Residual sugar (g/L) 6.22 ± 0.64 12.65 ± 2.51 4.57 ± 0.44 7.59 ± 0.63A
Distinct letters correspond to statistical significant difference for a Fischer test with p < 0.05

affecting the concentration of residual sugar and ace-
tic acid. This is already a positive result, but we should
bear in mind that all the previous experiments were per-
formed with laboratory yeast strains which are geneti-
cally quite different from wine yeasts. Therefore, our
next challenge was to check whether this phenotype
could be reproduced or even improved in a wine yeast
genetic background. This way, we would obtain wine
yeasts suitable for industrial fermentation and capable
of reducing the ethanol content of wine. To test this, the
pdc2Δ519 mutation was first integrated by transforma-
tion and homologous recombination into the genome
of the commercial EC1118 and the native Mab2C wine
yeasts. Considering the ploidy of these strains, EC1118
has been reported to be a diploid (Novo et al. 2009) and
according to a PCR test performed in our lab, Mab2C
would be at least a diploid since both mating types were
present. After the insertion of one pdc2Δ519 mutation
copy, we performed a growth curve in aerobic condi-
tions and lab-scale vinifications to compare the geneti-
cally modified EC1118Δ519 and Mab2CΔ519 with their
respective wild type control strains. The experiments
were performed with the same conditions used for the
lab strains. With respect to the growth in aerobic condi-
tions, both EC1118Δ519 and Mab2CΔ519 mutants grew
normally in YPD medium showing no difference with
their respective wild type controls (data not shown). As
for the vinification experiments V3 and V4, Fig. 2 shows
the curve for the accumulated CO2 weight loss (panels
c, d) and Table 3 summarizes the value obtained for the
main fermentative parameters. In contrast with the lab
strains, the vinifications were in this case much faster and
lasted around eight days, which is the expected time for
well adapted wine yeasts. Interestingly, the mutants were
not affected by the mutation and their kinetics was very
similar to the wild type strains. The analysis of the fer-
mentative parameters revealed a remarkable 15% total
ethanol reduction for the mutant EC1118Δ519 and 10%
for Mab2CΔ519 comparing with the wild type controls.
Both EC1118Δ519 and Mab2CΔ519 mutants were also
less efficient to produce ethanol showing statistically
significant lower values of glucose yield. According to
the values obtained for the glucose yield, EC1118Δ519
would reduce the ethanol almost 2 degrees (1.89) and
Mab2CΔ519 almost 1.5 degrees (1.36) for a wine with a
prediction of 15.5% (v/v). As it happened with the labo-
ratory BY4743pdc2Δ519 mutant strain, the acetic acid
concentrations of EC1118Δ519 and Mab2CΔ519 were
also unaffected by the mutation. The ethanol reduction
obtained with EC1118Δ519 was about two-fold higher
than that of the laboratory BY4743pdc2Δ519 mutant
strain. The Mab2CΔ519 native strain also displayed a
higher ethanol reduction (about 1.5-fold) when com-
pared with the laboratory BY4743pdc2Δ519 mutant
strain. Thus, we were not only able to reproduce the phe-
notype observed in the lab strain, but also we improved
it. In agreement with the ethanol reduction displayed,
the carbon balance of the mutants are considerable lower
than the wild type controls indicating that part of the car-
bon flux is being redirected away from the ethanol bio-
synthesis pathway.
Discussion
Before quantifying the fermentative parameters of each
laboratory mutant strain we tested the growth capability
of the pdc2Δ519 and pdc2Δ344 mutants under aerobic
Cuello et al. AMB Expr (2017) 7:67
conditions with glucose as carbon source. All mutants
were able to grow normally showing no difference with
their respective controls. This was already a positive
result considering the inability of the full Δpdc2 deletion
to grow under such conditions (Hohmann 1993; Nevoigt
and Stahl 1996). Nevertheless, it is quite surprising that
none of the mutants showed a growth defect, especially
BY4741pdc2Δ519 that carries only the mutant version of
Pdc2p with a deletion of 519 amino acids which accounts
for 56% of the wild type protein. Apparently, the activity
of the binding site alone is sufficient to sustain a normal
growth of the yeast.
With respect to the mutant’s fermentative param-
eters, they were determined by lab-scale vinifications.
The phenotypic analysis showed that BY4743pdc2Δ519
is the most interesting mutant strain, displaying a con-
sistent reduction of the ethanol concentration of up to
7.4%, as a result of a slight inefficiency for its production.
It is important to remark that this ethanol reduction did
not provoke an increment of the concentration of acetic
acid, in contrast to previous GMO based strategies where
the carbon flux was diverted to glycerol (Remize et  al.
1999; Lopes et  al. 2000; Varela et  al. 2012). The moder-
ate reduction observed in BY4743pdc2Δ519 was within
the expectable, and it could be the result of a competence
phenomenon between the wild type and the mutant
Pdc2p protein for the DNA binding site and some acti-
vating protein. Although it is just a speculation, there is
some indirect evidence which supports this proposition.
On one hand, it has been shown that the Pdc2p DNA-
binding site alone retains some DNA binding activity,
and on the other hand, an activating protein has been
proposed at least for the regulation by PDC2 of the THI
genes (Nosaka et  al. 2012). In any case, the molecular
mechanism underlying the regulation of PDC1 by PDC2
is still unknown (Brion et  al. 2014) and more investiga-
tion would be required to clarify this matter.
In view of the good results obtained with the
BY4743pdc2Δ519 strain our next goal was to test this
mutation in wine yeast strains. For this purpose the
pdc2Δ519 mutation was transformed into the com-
mercial EC1118 and native Mab2C wine yeast strains.
EC1118 has been widely used in the wine industry and
it is known for its reliability and excellent fermenta-
tion performance (Aceituno et  al. 2012). Meanwhile,
Mab2C is a native strain previously selected in our
laboratory for its excellent oenological properties in
the elaboration of Malbec wine. Both EC1118Δ519 and
Mab2CΔ519 mutants grew normally in aerobic condi-
tions with glucose as a carbon source and displayed typ-
ical fermentation kinetics for a wine yeast strain. As it
happened with the laboratory BY4743pdc2Δ519 mutant
Page 9 of 11
strain, the acetic acid concentrations of EC1118Δ519
and Mab2CΔ519 were also unaffected by the mutation.
The ethanol reduction obtained with EC1118Δ519 and
Mab2CΔ519 was about two-fold higher than that of the
laboratory BY4743pdc2Δ519 mutant strain. Thus, we
were not only able to reproduce the phenotype observed
in the lab strain, but also we improved it. These results
demonstrate that the wine yeasts mutants EC1118Δ519
and Mab2CΔ519 are good candidates to develop a yeast
starter for the elaboration of wines with reduced etha-
nol content. Still, it will be necessary to determine how
the carbon flux is being redirected. In an exploratory
experiment, we determined the glycerol concentra-
tion for the laboratory strain BY4743pdc2Δ519 and we
found no significant increment compared to the con-
trol. Perhaps, a clue could come from the analysis of
the CO2 weight loss data. Despite the ethanol reduc-
tion observed for BY4743pdc2Δ519, EC1118Δ519 and
Mab2CΔ519 mutants, there was little or no difference in
the total CO2 weight loss comparing with the wild type
controls. It seems that another decarboxylation reac-
tion is compensating the CO2 formed during the etha-
nol biosynthesis. Following this reasoning, two good
candidates could be acetoin and 2,3-butanediol. Both
compounds are derived from the secondary metabolism
of yeast, and are produced from pyruvate in a series
of chemical reactions where at least one decarboxyla-
tion reaction is involved (Romano and Suzzi 1996). In
a recent work, an ethanol reduction of 1.3% (v/v) was
achieved by combining adaptive laboratory evolution
strategies with hybridization (Tilloy et  al. 2014). Inter-
estingly, the enhancement of glycerol production in
the selected yeast was accompanied by an increment in
2,3-butanediol, which is consider a neutral organoleptic
compound.
In this study we present an alternative microbiological
strategy to reduce the ethanol content in wine, through
a genetic modification of the S. cerevisiae Pdc2p tran-
scription factor. Our results demonstrate that the inser-
tion of the pdc2Δ519d mutation in a wine yeast strain can
reduce the ethanol concentration up to 1.89% v/v with-
out affecting the fermentation performance. In contrast
to non-GMO based strategies, our approach permits the
insertion of the selected mutation in any locally selected
wine strain, making possible to produce quality wines
with regional characteristics and lower alcohol content.
This makes our work a valuable contribution to the prob-
lem of high ethanol concentration in wine. Nevertheless,
pilot-scale trials complemented with sensorial analysis
of the produced wines are required for a full evaluation
of our strain’s potential for its application in the wine
industry.
Cuello et al. AMB Expr (2017) 7:67
Abbreviations
ANOVA: analysis of variance; GMO: genetically modified organism; LSD: least
significant difference; PDC: pyruvate decarboxylase.
Authors’ contributions
RAC Participated in the design and coordination of the study, performed
the experiments, interpreted the data, and drafted the manuscript. KJFM
performed the experiments and the data analysis. LAM performed the experi-
ments and the data analysis. MC participated in its design and coordination,
interpreted the data, and helped to draft the manuscript. IFC conceived of the
study, participated in its design and coordination, interpreted the data, and
drafted the manuscript. All authors read and approved the final manuscript.
Author details
1
 Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET),
Buenos Aires, Argentina. 2 Laboratorio de Biotecnología, Estación Experimental
Agropecuaria Mendoza, Instituto Nacional de Tecnología Agropecuaria (INTA),
San Martín 3853, Luján de Cuyo, Mendoza, Argentina.
Competing interests
The authors declare that they have no competing interests.
Funding
This worked was supported by a PICT 2008-206 project of the Fondo para la
investigación Científica y Tecnológica (FonCyT) Argentina. R.A.C. is a fellow of
CONICET.
Received: 7 March 2017 Accepted: 13 March 2017
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Edited by:
Hui Wu,
East China University of Science
and Technology, China
Reviewed by:
Xinqing Zhao,
Shanghai Jiao Tong University, China
Estéfani García Ríos,
Consejo Superior de Investigaciones
Científicas (CSIC), Spain
Soo Rin Kim,
Kyungpook National University,
South Korea
*Correspondence:
Yi Qin
qinyi@nwsuaf.edu.cn
Specialty section:
This article was submitted to
Microbial Physiology and Metabolism,
a section of the journal
Frontiers in Microbiology
Received: 22 August 2020
Accepted: 23 November 2020
Published: 14 December 2020
Citation:
Du Q, Liu Y, Song Y and Qin Y
(2020) Creation of a
Low-Alcohol-Production Yeast by
a Mutated SPT15 Transcription
Regulator Triggers Transcriptional
and Metabolic Changes During Wine
Fermentation.
Front. Microbiol. 11:597828.
doi: 10.3389/fmicb.2020.597828
Frontiers in Microbiology | www.frontiersin.org
ORIGINAL RESEARCH
published: 14 December 2020
doi: 10.3389/fmicb.2020.597828
Creation of a
Low-Alcohol-Production Yeast by a
Mutated SPT15 Transcription
Regulator Triggers Transcriptional
and Metabolic Changes During Wine
Fermentation
Qing Du 1,2,3 , Yanlin Liu 1,2,3 , Yuyang Song 1,2,3 and Yi Qin 1,2,3*
1
College of Enology, Northwest A&F University, Yangling, China, 2 Shaanxi Engineering Research Center for Viti-Viniculture,
Yangling, China, 3 National Forestry and Grassland Administration Engineering Research Center for Viti-Viniculture, Yangling,
China
There is significant interest in the wine industry to develop methods to reduce the ethanol
content of wine. Here the global transcription machinery engineering (gTME) technology
was used to engineer a yeast strain with decreased ethanol yield, based on the mutation
of the SPT15 gene. We created a strain of Saccharomyces cerevisiae (YS59-409), which
possessed ethanol yield reduced by 34.9%; this was accompanied by the increase
in CO2 , biomass, and glycerol formation. Five mutation sites were identified in the
mutated SPT15 gene of YS59-409. RNA-Seq and metabolome analysis of YS59-409
were conducted compared with control strain, suggesting that ribosome biogenesis,
nucleotide metabolism, glycolysis flux, Crabtree effect, NAD+ /NADH homeostasis and
energy metabolism might be regulated by the mutagenesis of SPT15 gene. Furthermore,
two genes related to energy metabolism, RGI1 and RGI2, were found to be associated
with the weakened ethanol production capacity, although the precise mechanisms
involved need to be further elucidated. This study highlighted the importance of applying
gTME technology when attempting to reduce ethanol production by yeast, possibly
reprogramming yeast’s metabolism at the global level.
Keywords: low-alcohol, SPT15, RNA-seq, metabolome, wine, Saccharomyces cerevisiae
INTRODUCTION
Over recent decades, the alcohol concentration of wines produced by many warm regions around
the world has increased by approximately 2% (v/v) (Goold et al., 2017). This is mainly due to
the increasing preference of consumers for well-structured, full-bodied, and ripe-fruit wines and
those wines are generally made from more mature grapes. Also, this has been exacerbated by
global warming, which leads to higher sugar content in the grape varieties used to make wine
1 December 2020 | Volume 11 | Article 597828
Du et al.
(Varela et al., 2015; Rolle et al., 2017). Prompted by various
reasons regarding wine quality, economic variables, and
health concerns caused by high levels of alcohol, strategies
aimed at reducing ethanol concentrations without impairing
wine organoleptic quality have been performed in different
ways (Varela et al., 2015; Dequin et al., 2017). Generally,
microbiological strategies relating to the isolation and/or
generation of the yeast strains used to make wine have proved
to be the simplest and most economical methods, including
Saccharomyces cerevisiae and non-conventional yeast species
(Tilloy et al., 2015; Varela and Varela, 2018).
Engineering yeast strains with the capacity of redirecting
carbon away from ethanol production to other endpoints
is an effective approach and thus far, enhancing glycerol
production has testified to be the most effective method
(Otterstedt et al., 2004; Varela et al., 2012; Tan et al., 2016).
Classical gene modification (GM) technologies have achieved
increasing glycerol formation to reduce ethanol production by
the manipulation of single or several genes. However, high
concentrations of by-products, such as acetate, acetaldehyde, and
acetoin, were generated in these previous experiments (Cambon
et al., 2006; Varela et al., 2012). Since these by-products could
have a significant negative effect on the flavor of wine, concerted
efforts have been made to reduce the formation of these by-
products (Cambon et al., 2006; Ehsani et al., 2009). Additionally,
a combination of adaptive evolution and breeding strategies
is applied to develop a low-alcohol yeast for wine making
with higher levels of glycerol production; this strain reduced
ethanol production by 1.3% (v/v) without the formation of
undesirable by-products (Tilloy et al., 2014). Other researchers
have focused on non-Saccharomyces strains in an attempt to
reduce the ethanol content of wine; this is because such strains
are known to exhibit different respiro-fermentative regulatory
mechanisms when compared to S. cerevisiae (Quiros et al., 2014).
Nevertheless, those non-Saccharomyces strains generally possess
a weak capacity to complete wine fermentation on their own
and must be accompanied by S. cerevisiae (Hranilovic et al.,
2020). Consequently, there is significant interest in developing
more effective strategies to balance low-ethanol wine production
efficiency with good organoleptic qualities.
In this study, an alternative approach, global transcriptional
machinery engineering (gTME), was used to develop strains
of S. cerevisiae with reduced ethanol-production ability. The
gTME technology was carried out by mutating the general
transcription factor Spt15p, the TATA-binding protein (Alper
et al., 2006); this protein plays a key role in the action of
RNA polymerase and is one of the main DNA binding proteins
that regulate promoter specificity in yeast (Eisenmann et al.,
1989). The gTME technology is first used to improve the
glucose/ethanol tolerance of S. cerevisiae, which shows the
ability to re-program global gene transcription and change the
complex phenotype of yeast strains (Alper et al., 2006; Alper
and Stephanopoulos, 2007). Since then, several research studies
have used the gTME approach to optimize the ethanol tolerance
and ethanol production capacity of S. cerevisiae, and successfully
demonstrated that gTME is advantageous when attempting to
regulate the ethanol metabolism of yeast strains (Yang et al., 2011;
Frontiers in Microbiology | www.frontiersin.org
Low-Alcohol-Production Yeast
Seong et al., 2017). In the present study, we used gTME
technology to weaken the capacity of yeast to produce ethanol
and ultimately created a strain of S. cerevisiae (YS59-409) with
a low yield of ethanol production. RNA-Seq and metabolomic
analysis were also conducted in an attempt to understand the
metabolic mechanisms underlying the modified phenotype of
YS59-409. This study highlighted the critical role of the SPT15
regulator in reducing ethanol production in yeast and provided
comprehensive insights to understand the molecular mechanisms
of a new low-ethanol yeast.
MATERIALS AND METHODS
Plasmids, Strains, SPT15 Mutant Library
Construction and Culture Conditions
We used S. cerevisiae YS59 (MATα; ura3-52, leu2-3, and his 5-
519) (Liu et al., 2007) as the host strain and then amplify the
open reading frame of SPT15 gene from genomic DNA of YS59.
The SPT15 gene was inserted into the restriction sites between
BamHI and EcoRI using the pY16 vector, which was flanked with
TEF1 promoter and CYC1 terminator (pY16-SPT15); the plasmid
has a URA3 selective marker, an ampicillin resistant marker and
a CEN/ARS element (low copy).
The yeast mutant library was created by random mutagenesis
(error-prone PCR) of the SPT15 gene. Firstly, the SPT15 mutant
library was generated using the DiversifyTM PCR Random
Mutagenesis kit (Clontech) with pY16-SPT15 as template.
Plasmids obtained were transformed into Escherichia coli JM109
to produce a primary library for SPT15 mutants. From the
sequencing of 20 randomly selected colonies, mutations were
found at 2–10 sites without preference. Then library plasmids
were transformed into S. cerevisiae YS59 and incubated at 25◦ C
on solid SD to generate a yeast library for SPT15 mutant.
The plasmid was transformed into yeast cells by the
lithium acetate method (Gietz and Woods, 2001). The strains
and plasmids used in this procedure are summarized in
Supplementary Table 1. S. cerevisiae strains were pre-cultured
overnight in YPD medium (1% yeast extract, 2% peptone, and
2% glucose) at 30◦ C for non-selective propagation. The selective
culture of engineered strains was conducted in SD medium
(0.67% YNB, 0.077% Ura DO Supplement, and 2% glucose)
at 30◦ C.
Alcoholic Fermentation
Alcoholic fermentation was carried out using an inoculum of
5 × 105 cells/mL in a shaking incubator at 25◦ C at 150 rpm;
all cultures were carried out in triplicate. The medium was
similar to Triple M medium as reported previously (Spiropoulos
et al., 2000), and consisted of 75 g/L glucose, 75 g/L fructose,
6 g/L tartaric acid, 3 g/L malic acid, 0.5 g/L citric acid, 1.7 g/L
yeast nitrogen base without amino acids, 1 g/L ammonium
phosphate, 2 g/L casamino acids, 0.8 g/L L-arginine,1 g/L L-
proline, 0.1 g/L Tryptophan, and 4 mL ergo stock (composed
of 250 mL/L Tween80, 750 mL/L 95% ethanol and 2.5 g/L
ergosterol), with pH 3.25.
2 December 2020 | Volume 11 | Article 597828
Du et al.
Site-Directed Mutagenesis of the SPT15
Gene and Fermentation Assays for
Recombinant Strains
Mutations in SPT15 in strain YS59-409 were identified by DNA
sequencing. For site-directed mutagenesis, plasmid pY16-SPT15
(containing the non-mutated SPT15 gene) was used as a template
with primers designed to the target nucleotide substitutions
(Supplementary Table 2); these reactions were carried out with
a Mut Express II Fast Mutagenesis Kit (Vazyme, China) in
accordance with the manufacturer’s directions. The reconstructed
plasmids were then transformed into S. cerevisiae YS59 in order
to obtain recombinant strains (Supplementary Table 1). The
effects of mutation on the ethanol production capacity of strains
were performed in Triple M media.
RNA-Seq Analysis
RNA-Seq analysis was used to investigate the transcriptional
differences between the low-ethanol-production strain YS59-
409 and the control strain YS59-pY16. Three independent
samples were collected from the mid-log phase of fermentation
for RNA extraction. Total RNA was extracted using the
procedures described previously (Li et al., 2019). Agarose
gel (1%) electrophoresis and a spectrophotometer (NanoDrop
ND1000, United States) were then used to detect the purity
and concentration of samples. Bioanalyzer (Agilent 2100,
United States) was used to detect the RNA integrity numbers
(>8.0) of these samples to satisfy the particular requirements of
RNA-seq. The cDNA library construction and RNA sequencing
were carried out by Beijing Genomics Institute (Shenzhen,
China) using standard protocols. The RNA-Seq data generated in
this study were submitted to NCBI Sequence Read Archive (SRA)
under the accession number PRJNA548495.
To compare the transcriptomes of the mutant and control
yeast strains, we used Bowtie2 to map clean reads to the
reference gene and then used HISAT to reference the genome
of S. cerevisiae S288c. Gene expression levels were quantified
using the FPKM method (Trapnell et al., 2012). Screening
of differentially expressed genes (DEGs) between the two
strains was conducted using the NOISeq method (Tarazona
et al., 2011) based on a foldchange (log2 Ratio) ≥1.5 and a
divergent probability ≥0.8. Functional enrichment analysis of
Gene Ontology (GO) and KEGG pathway enrichment analysis
of DEGs were conducted by comparison with the entire genome
background (Bonferroni-corrected P-value < 0.05).
Metabolomics Analysis
The method used to prepare samples (six independent replicates)
from the low-ethanol-production strain for metabolomic analysis
was consistent with that used for RNA-seq analysis. Metabolites
were extracted by vortex blending approximately 1 g of cells
(fresh weight) in 1 mL of cold methyl alcohol for 30 s. Cells
were then lysed by ultrasonication for 15 min and centrifuged
for 15 min at 12,000 × g at 4◦ C; an aliquot of 200 µL of
the supernatant was used for further analysis. Samples were
analyzed by Shanghai Biocluster Biotech Co., Ltd. (China) using
the Ultimate 3000 LC system coupled with an Orbitrap Elite
Frontiers in Microbiology | www.frontiersin.org
Low-Alcohol-Production Yeast
mass spectrometer (Thermo, United States). The separation was
performed on a Hypergod C18 column (100 mm × 4.6 mm
3 µm) at 40◦ C. A flow rate of 0.3 mL/min, and an injection
volume of 4 µL, were used in all analyses; this was followed by
auto-sampling at 4◦ C. Mass spectrometry detection used negative
polarity with the following parameters: heater temperature,
300◦ C; sheath gas flow rate, 45 arb; auxiliary gas flow rate, 15
arb; sweep gas flow rate, 1 arb; spray voltage, 3.2 kV; capillary
temperature, 350◦ C, and an S-Lens RF Level of 60%.
With regards to multivariate statistical analysis, principal
component analysis (PCA) and orthogonal partial least squares
discriminant analysis (OPLS-DA) were performed using SIMCA-
P version 13.0 software (Umetrics AB, Sweden) to separate the
two groups of data. We then searched for differential metabolites
using the variable importance in the projection (VIP) value of the
OPLS-DA model (VIP > 1) in combination with the p-value of
the t-test (p < 0.05). Qualitative metabolites were characterized
by searching an online database1 . Metabolic pathway analysis was
carried out using MetaboAnalyst 3.02 .
Analytical Methods
We measured the weight loss in CO2 from each sample
by weighing the fermenters on a daily basis. Cell growth
was recorded by a microplate reader at OD600 nm (BioTek,
ELx800, United States). Ethanol yields were analyzed using
an SBA-40C biological sensor analyzer (Biology Institute of
Shandong Academy of Sciences, China). The content of reducing
sugar in each sample was measured by the DNS method
(Hu et al., 2008) with glucose as a standard using an
ultra-violet spectrophotometer. Concentrations of glycerol and
acetic acid were determined with an Enology Analyzer Y15
(BioSystems, Spain).
Data Analysis
Statistically significant differences between the wild-type (YS59-
pY16) and mutant yeast strains (YS59-409) were determined
using the Student’s t-test. The effect of site-directed mutagenesis
and gene knockout on the fermentation characteristics of the two
strains were further determined by one-way analysis of variance
(ANOVA) and Duncan’s test. The confidence level for both tests
was 95% and all analyses were carried out using SPSS version 19.0
software (SPSS Inc., United States).
RESULTS AND DISCUSSION
Selection and the Characteristics of the
Low-Ethanol-Yield Strain
Previously, we produced a yeast mutant library (>1,000 clones)
that was based on S. cerevisiae YS59 and created by random
mutagenesis (error-prone PCR) of the SPT15 gene with the
pY16 plasmid backbone using gTME technology. Preliminary
screening using SD media in 24-well plates was performed to
identify the 20 mutants with the highest biomass; these were
1
http://metlin.scripps.edu
2
http://www.metaboanalyst.ca
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Du et al.
the strains that were supposed to have lower ethanol-production
capacity due to competition for carbon sources between
biomass and ethanol production (Supplementary Figure 1). To
determine the ethanol production capacity of these 20 strains, we
fermented each in 10-mL of Triple M media (approximately one-
fifth of the tube’s total volume; this allowed the strains to grow
fully) in the tubes. Finally, the strain with the lowest ethanol yield
was identified (Supplementary Table 3). Compared with the
control strain, the ethanol yield of the low-ethanol-production
strain (YS59-409) was significantly reduced by 34.9% (Table 1,
p < 0.05). Interestingly, the strain featuring SPT15 gene mutation
exhibited a greater CO2 weight loss than the control group
(Figure 1). In the wine industry, CO2 weight loss is generally
used as an indicator of the fermentation capacity of yeast. It is
worth noting that the YS59-409 strain produced a larger amount
of CO2 than the control but had a lower ethanol production. This
suggests that other decarboxylation pathways might contribute
to the CO2 loss in the mutant strain. In addition, the glycerol
content of the YS59-409 strain was 43% higher than the control
strain, although there were no significant differences with regards
to acetic acid production (Table 1). These findings were not
consistent with the classical theory that higher glycerol synthesis
is associated with increased acetic acid production (Nevoigt and
Stahl, 1996). Commonly, the main by-product, glycerol, confers
positive sensory effects, body, and sweetness, to wines when
present in appropriate amounts and has been used as a target to
redirect carbon sources away from ethanol production by many
researchers in the wine industry (Tilloy et al., 2015; Goold et al.,
2017). Our mutant strain (YS59-409) not only produced more
glycerol than the control strain, but also did not accumulate
overmuch acetic acid, an undesirable compound; these findings
were also in agreement with those from previous studies (Ehsani
et al., 2009; Tilloy et al., 2014). During wine fermentation, most of
the sugars are used for the production of ethanol and CO2 , with
a small part for biomass and glycerol forming, including minute
amounts of other byproducts. Therefore, we can speculate that
strain YS59-409 uses more sugar for CO2 , biomass, and glycerol
synthesis, thus shunting the carbon source away from ethanol
synthesis and resulting in low ethanol yield.
Sequence Analysis of the SPT15 Gene in
the YS59-409 Strain
The SPT15 gene in YS59-409 was amplified from the pY16-409
plasmid and then was sequenced. There were 5 mutational sites
in the structural domain of the mutated SPT15 gene as shown
in Figure 2, in which methionine is substituted for isoleucine
(Ile46 Met), and similarly, Asp56 Gly, Ser118 Pro, Tyr195 His, and
Leu205 Ser (I46M, D56G, S118P, Y195H, and L205S, respectively).
Three point mutations were located in the highly conserved
domain (amino acids 61–240) and two mutations were located
in the non-conserved domain (amino acids 1–60). To explore
which of these sites conferred the most desirable phenotype to the
low-ethanol-yielding strain, we constructed five strains featuring
single point mutations (Supplementary Table 1). However, none
of these mutants led to a reduction in ethanol yield; instead,
I46M, D56G, and L205S, led to enhanced ethanol yield compared
Frontiers in Microbiology | www.frontiersin.org
Low-Alcohol-Production Yeast
with the control under fermentation conditions (Table 2). Of all
these mutational sites, Y195H has been reported to work together
with F177S and K218R to improve glucose/ethanol tolerance and
the efficiency of glucose conversion to ethanol of yeast (Alper
et al., 2006); and Leu-205 site has shown to be important for
DNA binding specificity of Spt15p and mutating this site to other
different amino acids can cause various degrees of changes in
yeast growth (Arndt et al., 1992). Although our results showed
that single mutation of Y195H or L205S did not produce the low-
alcohol phenotype, we hypothesized that these two sites might
play key roles in the mutant strain and that the desired phenotype
might be obtained through double or multiple mutations based
on previous reports (Arndt et al., 1992; Alper et al., 2006). In
addition, the same mutation on SPT15 may lead to different
phenotypes in strains with different genetic backgrounds. In a
word, these results illustrate that the combined action of the
five separate mutations, or at least in part, conferred the desired
phenotype to the mutant strain.
Transcriptional and Metabolic Analysis of
the Mutated Strain
As described in previous research, gTME can trigger overall
disturbances at the transcriptional level and can be used to
unravel complex phenotypes (Alper and Stephanopoulos, 2007).
In order to investigate the mechanisms underlying the observed
phenotype, we used RNA-Seq and metabolomic analysis to
analyze the differences in transcription and metabolism between
mutant strain (YS59-409) and the control (YS59-pY16). Under
fermentation conditions, we observed significant changes in
the transcription and metabolism of the mutant; these factors
are likely to have made an important contribution to the
target phenotype of the mutant. Subsequently, we would discuss
the overall transcriptional and metabolic regulation and the
specific pathways and function change associated with ethanol
metabolism answering to the mutation of SPT15 gene.
The Overall Analysis of Transcription and Metabolism
in Strain YS59-409
A total of 964 genes showed significantly different expression
levels when compared between the mutant and control strains;
636 genes were up-regulated and 328 genes were down-regulated
in the mutant YS59-409 strain (Supplementary Table 4). These
differentially expressed genes (DEGs) demonstrated that the
mutation of the SPT15 gene, a transcription factor, led to the
global reprogramming of transcription at genes and may have
contributed to the regulation of ethanol metabolism in this
mutant strain. GO analysis and KEGG pathway enrichment
analysis were used to further analyze the functional enrichment of
DEGs. KEGG pathway enrichment analysis revealed that most of
the DEGs participated in those key metabolic pathways, including
translation, transcription, carbohydrate metabolism, nucleotide
metabolism, and amino acid metabolism (Table 3).
There were 41 differential metabolites identified (16
upregulated and 25 downregulated), as shown in Supplementary
Table 6. OPLS-DA and PCA analysis were conducted to
identify differences in the metabolome that were caused by
random mutagenesis of the SPT15 gene. We found there
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Du et al.
TABLE 1 | Fermentation characteristics of the mutant strain S. cerevisiae YS59-409.
Strains OD600 Residual sugar (g/L) Ethanol (g/L)
YS59-pY16 4.52 ± 0.34 1.13 ± 0.35 54.67 ± 0.94
YS59-409 5.45 ± 0.40* 1.90 ± 0.30 35.33 ± 3.09*
a Ethanolyield represents the ratio of ethanol production (g) to the sugar consumption (g).
*Student’s t-test showed significant difference at the level of 0.05.
FIGURE 1 | CO2 weight loss (g). Black square represents S. cerevisiae
YS59-409 and black round represents S. cerevisiae YS59-pY16 (control
strain).
were distinct discrepancies between the mutant and control
strains in terms of metabolic profile (Supplementary Figure 2).
Indeed, we found that several metabolites related to amino
acid metabolism (L-glutamine, L-glutamate, L-tryptophan,
L -histidine, L -lysine, kynurenine, glutathione, succinic acid
semialdehyde, and adenylosuccinate) (Supplementary Table 6)
showed significant differences when compared between the two
strains, corresponding to transcriptional alterations in amino
acid metabolism.
Transcription, Translation, and Nucleotide
Metabolism, Were Activated in Strain YS59-409
In total, 14 and 135 genes were functionally annotated
into transcription and translation, respectively (Table 3 and
Supplementary Tables 4, 5). Genes related to RNA polymerase
were upregulated, thus indicating the transcriptional activation
of the mutant strain. This could be explained by the upregulation
FIGURE 2 | Mutation sites in the SPT15 gene of mutant strain (arrows). The schematic of structural domain is referred to the previous study (Alper et al., 2006).
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Low-Alcohol-Production Yeast
Glycerol (g/L) Acetate (g/L) Ethanol yielda (g/g Sugar) Change in ethanol
yield
2.13 ± 0.08 0.493 ± 0.109 0.367 ± 0.007 0
3.05 ± 0.07* 0.487 ± 0.066 0.239 ± 0.021* −34.9%
of SPT15 in the mutant YS59-409 strain (Supplementary
Table 4) for its regulatory effect on RNA polymerase and
gene transcription levels (Eisenmann et al., 1989). In addition,
RPL and RPS genes encoding ribosome biogenesis proteins
were significantly over-expressed in the low ethanol-production
strain (Supplementary Tables 4, 5), which reflected the
stimulation of relative translational activity at certain time
points (Backhus et al., 2001). Coincidently, the upregulation
of RNA polymerase metabolism and ribosome biogenesis were
reported in a low-ethanol-production yeast by a previous study
(Varela et al., 2018). Furthermore, most of the genes associated
with nucleotide metabolism were expressed at high levels
(Table 3 and Supplementary Tables 4, 5), of which purines
and pyrimidines are the basic components (Ljungdahl and
Daignan-Fornier, 2012), exhibiting the activation of purine and
pyrimidine metabolism. Collectively, these results above indicate
that transcription, translation, and nucleotide metabolism,
were activated in the mutant YS59-409 strain. This probably
demonstrates that the mutation of the SPT15 gene leads to the
reprogramming of global gene expression and metabolism.
The Fermentative Pathways Leading to Ethanol
Production Was Reduced in Strain YS59-409
Glycolytic flux relies on glucose uptake rate, which is regulated
by a family of hexose transporters encoded by HXT genes. In
the present study, we found that several hexose transporter genes
were significantly downregulated in the YS59-409 strain (HXT2,
HXT4, HXT5, HXT6, HXT7, HXT9, HXT11, and HXT13; Figure 3
and Supplementary Table 4); of these, HXT4, HXT6, and HXT7,
are known to be vital for the uptake of glucose (Özcan and
Johnston, 1999). Previous research has reported that a deficiency
of hexose transporters in S. cerevisiae could reduce the levels of
sugar uptake and thus regulate the glycolysis flux, particularly
with regards to HXT7, which eventually resulted in a reduction
in ethanol production (Otterstedt et al., 2004). Consequently, the
significant downregulation of several HXT genes in YS59-409
is likely to result in a decrease of glycolysis flux. Besides, the
expression of HXK1 was downregulated by 4.7-fold (Figure 3
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Du et al. Low-Alcohol-Production Yeast

TABLE 2 | Fermentation characteristics of point-mutated strains.

Strains Residual sugar (g/L) Ethanol (g/L) Ethanol yield (g/g sugar) Changes in ethanol yield

409-46 0.56 ± 0.91a 53.09 ± 0.36b 0.348 ± 0.005ac 8.10%

409-56 1.10 ± 1.67a 52.23 ± 1.09b 0.357 ± 0.018a 11.00%
409-118 1.04 ± 0.01a 51.42 ± 1.76ab 0.325 ± 0.005b 1.10%
409-195 1.03 ± 0.02a 50.19 ± 0.49ab 0.332 ± 0.003bc 3.10%
409-205 1.03 ± 0.02a 53.34 ± 3.19b 0.355 ± 0.013a 10.40%
YS59-pY16 1.02 ± 0.02a 48.43 ± 0.24a 0.322 ± 0.003b 0
a,b,c Different letters indicate significant differences according to the Duncan test (p < 0.05). Data are mean ± SD of independent triplicate.

and Supplementary Table 4); this gene encodes hexokinase consumption under conditions of excess glucose, even in the
1, which is responsible for catalyzing the phosphorylation of presence of oxygen (also referred to as ‘overflow metabolism’)
glucose in the first irreversible step of glycolysis (Rodríguez et al., (de Deken, 1966). In the present study, we observed indications
2001). The downregulation of HXK1 also might contribute to the that the Crabtree effect of the YS59-409 strain might be
reduced glycolysis metabolism in the mutant; this observation is disturbed. On the one hand, the attenuated glycolysis flux
supported by a previous study that the repression of hexokinase in the YS59-409 strain, in combination with the higher CO2
activity resulted in reduced glycolysis flux (Tan et al., 2016). production and the lower ethanol formation rate, appeared
So, the downregulation of HXTs and HXK1 in strain YS59- to suggest that the TCA cycle was enhanced. This deduction
409 probably lead to a reduction in glycolysis flux compared could be supported by an earlier study showing that when
to the control strain, thus well explaining the reduced yield of glucose uptake rates were reduced, then the CO2 /ethanol ratio
ethanol. Varela et al. (2018) have reported that the low ethanol increased more than 50% and the net flux through the TCA
strain produced in their study exhibited reduced glycolysis cycle increased significantly (Heyland et al., 2009). That is to
activity, which agrees well with the present study. What’s more, say, the overflow metabolism of the YS59-409 strain might
a reduction in pyruvate content (Figure 3 and Supplementary shift toward respiratory metabolism. Our specific experimental
Table 6) provides further evidence of the weaker glycolysis flux conditions (the headspace of the 24-well plates and test tubes)
(Otterstedt et al., 2004). We hypothesize that the glycolysis flux could provide a micro-oxygen environment that can support
of the YS59-409 strain is reduced by mutation of the SPT15 gene, the respiration of yeast to some extent, as also noted by a
thus resulting in a weakened ethanol fermentation pathway of the previous study (Heyland et al., 2009). Respiratory metabolism
YS59-409 strain. plays an important role in terms of producing energy in the
form of ATP in aerobic growing cells. Intriguingly, we found
The Low-Ethanol-Producing Strain YS59-409 that the energetic metabolism of YS59-409 may be enhanced
Exhibited a Disturbance in the Crabtree Effect compared to the control strain. As is well-known, the histidine
Saccharomyces cerevisiae is a Crabtree-positive yeast, which and nucleotide biosynthetic pathways are connected (Ljungdahl
generally utilizes the ethanol fermentation pathway for glucose and Daignan-Fornier, 2012). The upregulation of genes related
to histidine metabolism (such as HIS1, HIS2, HIS5, and HIS7),
accompanied by the increased histidine content in YS59-409
TABLE 3 | KEGG pathway enrichment analysis (p-value < 0.05). (Figure 3), provided strong evidence for an enhancement in the
synthesis of histidine. And KEGG pathway enrichment analysis
Pathway P-value Number of Gene match indicated that purine synthesis metabolism was reinforced in
genes (genome match)a
the mutant strain (Table 3 and Supplementary Tables 4, 5).
Translation So, with the viewpoint that the de novo purine pathway feeds
Ribosome 1.00E-71 135 19.77 (4.33) into the histidine pathway and branches to allow ATP synthesis
Transcription (Ljungdahl and Daignan-Fornier, 2012), we can speculate that
RNA polymerase 5.87E-05 14 2.05 (0.69) ATP production in the YS59-409 strain is boosted. In addition,
Carbohydrate metabolism the upregulation of the ADE4 gene in the first step of the
Starch and sucrose metabolism 6.27E-10 22 3.22 (0.84) ATP formation pathway (Ljungdahl and Daignan-Fornier, 2012)
Galactose metabolism 6.11E-04 13 1.90 (0.74) provided supplementary evidence to the enhanced ATP synthesis.
Nucleotide metabolism What’s more, high ATP yields may result in excess biomass
Pyrimidine metabolism 1.28E-04 24 3.51 (1.65) formation at the expense of product yield (de Kok et al., 2012),
Purine metabolism 3.23E-02 24 3.51 (2.41) which further supports the fact that YS59-409 showed higher
Amino acid metabolism biomasses and lower ethanol production. Therefore, we can
Histidine metabolism 4.08E-03 6 0.88 (0.26) infer that the characteristic of Crabtree effect of the mutant
Arginine and proline metabolism 4.58E-02 6 0.88 (0.41) strain have been changed. On the other hand, some Crabtree-
a Thepercentage of DEGs involved in individual pathway account for all DEGs with negative yeast, such as Hanseniaspora uvarum and Metschnikowia
pathway annotation (683) and all genes with pathway annotation (4184). pulcherrima, possess different respiro-fermentative regulatory

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Du et al. Low-Alcohol-Production Yeast

FIGURE 3 | Changes in genes and metabolites of mutant strain YS59-409. Blue and green represent up- and down-regulation, respectively. The values in brackets
represent the fold change of expression level of mutant strain compared to control strain.

mechanisms than S. cerevisiae (Quiros et al., 2014). Researchers

have used these non-conventional species to reduce ethanol
production by partial controlling the aeration of grape juice
(Quiros et al., 2014). However, non-conventional yeasts cannot
generally complete alcoholic fermentation. We created a new
mutant strain (YS59-409), which might not only have similar
ethanol-producing properties as non-conventional yeasts, but
also is capable of finishing fermentation alone. Thus, these results
provide a new concept for the creation of new low-ethanol-
production strains.

NAD+ /NADH Homeostasis Was Disturbed in Strain

YS59-409
Sugar fermentation in S. cerevisiae is a redox neutral process
that is influenced by NAD+ /NADH balance, in which glycerol
plays important roles (Goold et al., 2017). We found those key
genes in the synthesis pathway of glycerol, GPD1, encoding
glycerol-3-phosphate dehydrogenase, along with GPP1 and
FIGURE 4 | CO2 weight loss (g). Black asterisk and black triangle represent
GPP2, encoding glycerol-3-phosphate phosphatase (Nevoigt knockout strains (YS59-1rgi1 and YS59-1rgi2, respectively) and black
and Stahl, 1997), were overexpressed in the low-ethanol- square represents control strain (YS59).
producing strain (Figure 3 and Supplementary Table 4).
Simultaneously, GUT1 and GUT2, genes that encode for glycerol
kinase for glycerol catabolism (Nevoigt and Stahl, 1997) were
both downregulated (Figure 3 and Supplementary Table 4). YS59-409 strain (Table 1). Higher production of glycerol is
Changes in the expression of those genes related to glycerol likely due to the need to balance cytosolic NADH produced
metabolism demonstrated the high production of glycerol; this and consumed. What is noticeable is that the de novo
was confirmed by the increased glycerol concentration in the pathway of NAD+ (namely, the kynurenine pathway) might

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Du et al.
TABLE 4 | Fermentation characteristics of knockout strains.
Strains Residual sugar (g/L)
YS59 1.96 ± 0.68a
YS59-1rgi1 0.99 ± 0.19a
YS59-1rgi2 1.62 ± 0.40a
a,b,c Values
followed by different letters within the same column are significantly different using Duncan’s multiple range test at the level of 0.05. Data are mean ± SD of
independent triplicate.
have been disturbed. In yeast, NAD+ can be synthesized de
novo from tryptophan (Panozzoa et al., 2002). We found
that tryptophan content was increased in the mutant strain
(Figure 3 and Supplementary Table 6); this was consistent
with the fact that genes involved in the synthesis of tryptophan
were also increased (Figure 3 and Supplementary Table 4).
Moreover, as the key participator in the de novo synthesis of
NAD+ from L-tryptophan (Panozzoa et al., 2002), kynurenine
was found to be the increased metabolite with the highest
fold-change in this study (Figure 3 and Supplementary
Table 6). Previous studies have shown that the de novo
pathway plays only a minor role if a functional salvage
pathway is present (Sporty et al., 2009); and one of the
key requirements in the formation of kynurenine is that the
kynurenine pathway needs oxygen (Panozzoa et al., 2002).
Therefore, the increased production of kynurenine and L-
tryptophan in the mutant strain probably shows the activation
of the de novo pathway for the synthesis of NAD+ under our
experimental conditions. The strengthening trend of NAD+
level might further explain the enhanced synthesis of ATP
synthesis for the reason that ATP synthesis and redox potential
are directly proportional to the intracellular concentration of
NAD+ (Gonzalez Esquivel et al., 2017). Collectively, those data
indicate that NAD+ /NADH equilibrium might be disturbed
in the mutant strain. Certainly, the NAD+ /NADH balance of
the YS59-409 strain depends on a range of factors, including
biomass formation, respiration, ATP production, and the
generation of some metabolites, such as ethanol, glycerol,
and amino acids. Additionally, researchers have deployed
methods to intentionally perturb the NAD+ /NADH balance
to reduce ethanol production (Goold et al., 2017), which
also emphasizes the importance of NAD+ /NADH balance for
ethanol metabolism.
The Effects of Deleting the RGI1/2 Gene
on the Ethanol-Production Capacity of
Yeast
To further identify key genes in the regulatory network of our
new mutant strain, we constructed a protein-protein interaction
(PPI) network using the STRING database V113 and Cytoscape
tools; the core gene module was then excavated using the
MCODE plugin in Cytoscape. Nine genes were identified
(Supplementary Table 7); of these, AQY3 (Yfl054c) and RGI2
(Yil057c) were shown to be associated with glycerol and energy
metabolism, which was corresponded with the changes in
3
https://string-db.org/
Frontiers in Microbiology | www.frontiersin.org
Low-Alcohol-Production Yeast
Ethanol (g/L) Ethanol yield (g/g Sugar) Change in ethanol yield
52.04 ± 1.43c 0.351 ± 0.008c 0
40.59 ± 0.96a 0.272 ± 0.006a −22.0%
43.43 ± 0.77b 0.292 ± 0.004b −16.5%
glycerol and energy metabolism observed in YS59-409. Then, we
found that only RGI2 exerted influence over ethanol production.
The RGI2 gene possesses very little data concerning protein
function or biological processes involved for itself, which was
found to be significantly down-regulated by 41.9-fold in this
study (Supplementary Table 4). Noteworthily, the homologous
gene of RGI2, RGI1 (Yer067w), was significantly down-regulated
by 4.3-fold (Supplementary Table 4), which shared 70% identity
with the sequence of RGI2 (Domitrovic et al., 2010). RGI1 gene
is reported to be regulated transcriptionally by SPT15 under
conditions involving ethanol stress (Yang et al., 2011). Previous
studies show that Rgi1p and Rgi2p proteins most likely belong
to the same complex and/or operate in the same pathway, and
that these proteins are involved in the control of energetic
metabolism, particularly under respiratory growth conditions
(Domitrovic et al., 2010). Therefore, the effects of those two
genes on ethanol metabolism were conducted in the present
study. We performed single gene knock-outs for RGI1 and
RGI2 genes in strain YS59 using Cre/loxP-mediated technology
in order to evaluate their effects on ethanol production in
S. cerevisiae, generating two new strains: YS59-1rgi1 and YS59-
1rgi2 (Supplementary Table 1). As follow, we compared the
performance of strain YS59-1rgi1 and strain YS59-1rgi2 in
Triple M media (250-mL flasks containing 150-mL of media).
Compared with the control strain YS59, the weight loss of
CO2 was both reduced in the two knockout strains (Figure 4),
thus illustrating their reduced fermentation capacity; this was
further supported by the fact that these strains showed a
22.0 and 16.5% reduction in ethanol productivities, respectively
(Table 4). The results implied that perturbation of the RGI1
or RGI2 gene could elicit alterations in ethanol metabolism,
which showed the positive effects of RGI1/2 deletion with regards
to weakening the ethanol-yielding ability of yeast. However,
these two knockout strains may have a different low-yield
ethanol mechanism than the YS59-409 strain, because they
had different CO2 production models; this possibility requires
further investigation.
CONCLUSION
In this study, we applied gTME technology to engineer a low-
ethanol-production strain of S. cerevisiae achieved by mutating
the transcription factor SPT15 to change gene expression in
a global level. We provide a novel insight into the use of
gTME technology to modulate ethanol metabolism, which could
not only facilitate the construction of a low-ethanol-production
strain for the wine industry, but also, enhance our understanding
8 December 2020 | Volume 11 | Article 597828
Du et al.
of the mechanisms underlying reduced ethanol production by
yeast. We created a new strain of S. cerevisiae, YS59-409, with
weakened ethanol production capacity. The ethanol-production
capacity of this strain was reduced by 34.9% compared
to the control strain, which was caused by comprehensive
changes associated with the regulation of transcription and
metabolism. Sequence analysis was performed on the mutated
SPT15 gene, demonstrating that the five mutation sites may
work collectively, or at least partly, to create the specific
characteristics of YS59-409, including a higher CO2 release,
biomass, and glycerol formation. The integration of RNA-Seq
and metabolomics analysis showed that the specific phenotype
of the new mutant strain featured changes in ribosome
biogenesis, nucleotide metabolism, glycolysis flux, the Crabtree
effect, NAD+ /NADH homeostasis, and energy metabolism.
Remarkably, we also found that RGI1 and RGI2 genes,
which play key roles in energy metabolism, were significantly
down-regulated; it is possible that this was linked with low
ethanol metabolism, although this needs to be investigated
further in future research. Although public attitudes toward
the use of GMOs (Genetically Modified Organisms) in wines
are often less than positive, this study demonstrated that it
is possible to reduce ethanol yields in yeast using gTME
technology, while also reprogramming the metabolism of this
new mutant strain. Currently, we can get some knowledge
from this study, which could be used to direct strategies
for generating wine yeast with weakened ethanol production
capacity using other approaches, such as adaptive evolution.
In summary, this study highlights the potential to use gTME
technology to reduce the ethanol content of yeast for the wine-
making industry.
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DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in
online repositories. The names of the repository/repositories
and accession number(s) can be found in the article/
Supplementary Material.
AUTHOR CONTRIBUTIONS
QD: experimental operation and writing – original draft. YL:
supervision. YS: data analysis. YQ: experimental design and
supervision. All authors edited and approved the final version
of the manuscript.
FUNDING
This project was supported by National Key Research and
Development Project (2019YFD1002500), National Natural
Science Foundation of China (31301541 and 31960470), the
Fundamental Research Funds for the Central Universities
(2452020177), and China Agriculture Research System (grant
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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10 December 2020 | Volume 11 | Article 597828
 biomolecules
Review
Biotechnological Approaches to Lowering the Ethanol Yield
during Wine Fermentation
Ramon Gonzalez 1 , Andrea M. Guindal 1 , Jordi Tronchoni 2






Citation: Gonzalez, R.; Guindal,
A.M.; Tronchoni, J.; Morales, P.
Biotechnological Approaches to
Lowering the Ethanol Yield during
Wine Fermentation. Biomolecules 2021,
11, 1569. https://doi.org/10.3390/
biom11111569
Academic Editors: Encarna
Gómez-Plaza and Rocio Gil-Muñoz
Received: 26 September 2021
Accepted: 20 October 2021
Published: 22 October 2021
Publisher’s Note: MDPI stays neutral
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Biomolecules 2021, 11, 1569. https://doi.org/10.3390/biom11111569
and Pilar Morales 1, *
1 Instituto de Ciencias de la Vid y del Vino (CSIC, Gobierno de la Rioja, Universidad de La Rioja),
26007 La Rioja, Spain; rgonzalez@icvv.es (R.G.); andrea.martin@icvv.es (A.M.G.)
2 Faculty of Health Sciences, Valencian International University (VIU), 46002 Valencia, Spain;
jtronchoni@universidadviu.com
* Correspondence: pilar.morales@icvv.es
Abstract: One of the most prominent consequences of global climate warming for the wine industry
is a clear increase of the sugar content in grapes, and thus the alcohol level in wines. Among the
several approaches to address this important issue, this review focuses on biotechnological solutions,
mostly relying on the selection and improvement of wine yeast strains for reduced ethanol yields.
Other possibilities are also presented. Researchers are resorting to both S. cerevisiae and alternative
wine yeast species for the lowering of alcohol yields. In addition to the use of selected strains under
more or less standard fermentation conditions, aerobic fermentation is increasingly being explored for
this purpose. Genetic improvement is also playing a role in the development of biotechnological tools
to counter the increase in the wine alcohol levels. The use of recombinant wine yeasts is restricted to
research, but its contribution to the advancement of the field is still relevant. Furthermore, genetic
improvement by non-GMO approaches is providing some interesting results, and will probably
result in the development of commercial yeast strains with a lower alcohol yield in the near future.
The optimization of fermentation processes using natural isolates is, anyway, the most probable
source of advancement in the short term for the production of wines with lower alcohol contents.
Keywords: wine; low-alcohol; fermentation; global warming
1. Introduction
Commercial wines have been experiencing a steady increase in alcohol levels since the
1980s. On average, the gain in alcohol has been about 1% (v/v) every ten years, currently
reaching a total increase of 3–4% (v/v). There are two main reasons behind this trend.
From one side, global climate warming affects, in different ways, the different physiological
processes involved in grape berry ripening. Both sugar accumulation and phenolic and
aromatic maturity develop faster, but the impact is greater for sugars. This imbalance
prevents early harvesting from being an adequate solution to this problem, as it would lead
to organoleptic defects associated with unripe berries. On the other hand, the consumer
demand for tasty, structured, and full-bodied wines has pushed the market towards
winemaking styles that require the harvest of very ripe grapes. As a result, quality grapes
today tend to be richer in sugar content, leading to the observed increase in wine alcohol
levels when all this sugar is fermented by yeasts [1].
Excess alcohol in wines is becoming a problem for the industry from several points
of view. Wine balance is a delicate equilibrium between sweetness, acidity, bitterness,
and aroma compounds, with alcohol as a vehicle for the perception of the latter. Ethanol
surplus can lead to a sensory imbalance by promoting bitterness and masking fruit per-
ceptions, and increasing the perception of sweetness, astringency and hotness. Besides
this, sticking to moderate alcohol consumption becomes more difficult, and can therefore
lead to increased risks for consumer health and road safety. Taxation based on alcohol
levels in several importing countries is an additional incentive to look for wines with lower
https://www.mdpi.com/journal/biomolecules
Biomolecules 2021, 11, 1569 2 of 16

ethanol contents. Taken together, all of these factors might contribute to discourage wine
consumption. Furthermore, from a bioprocess point of view, high levels of ethanol could
lead to sluggish or stuck fermentation processes, with significant economic consequences.
In recent years, there has been a collective effort by researchers, engineers, and wine-
makers to develop approaches to limit the ethanol content of wines [2]. This community
is targeting almost every stage of the production cycle, including, among other examples,
grapevine clonal selection, vineyard management, winemaking practices adapted to unripe
grapes, the use of yeast strains with a lower ethanol yield (often recombinant) or metabolic
inhibitors, and partial dealcoholisation by physical means. Unfortunately, straightforward
solutions, such as early harvesting or post-fermentation treatments, often have a negative
impact on the wine quality (e.g., a green character and altered aromatic profile). Apart from
sweet wines, most wines on the market are dry wines, which should contain very low
levels of residual sugar. Solutions that result in less alcohol but an increase in residual
sugars (i.e., stopping fermentation before completion) will drastically change the wine
style, and will not be considered in this review. Here, we will present biotechnological
solutions related to the fermentation process, mainly involving the selection and improve-
ment of wine yeast strains for reduced ethanol yield, but also the use of some enzymes or
metabolic inhibitors.
In this context, a pivotal concept is alcohol yield, which is the amount of ethanol
produced per unit of sugar consumed. Most microbiological approaches to the reduction
of the alcohol level in wines will target the ethanol yield of S. cerevisiae or alternative
wine yeast species. Associated with the lowering of the alcohol yield is the concept of
alternative carbon sinks (carbon-containing metabolic end products other than ethanol).
Under standard fermentation conditions, about 60% of the hexose carbon consumed by
S. cerevisiae ends up in the form of ethanol, 30% goes to carbon dioxide, and the remainder
goes to biomass and less abundant metabolites. This led to the rule of thumb which states
that for every 17 g/L of sugar in the grape must, an additional 1% v/v of alcohol wine is to
be expected in the wine. Reducing the alcohol yield requires the diversion of the metabolic
flux from ethanol to alternative carbon sinks.
However, alcoholic fermentation is the metabolic hallmark of Saccharomyces cerevisiae,
the yeast species dominating most wine fermentation processes in the industry, either spon-
taneously or because it is used as a starter culture. This species always shows a high ethanol
yield during fermentation, with very little biological diversity [3,4]. As an example, a sur-
vey of 72 S. cerevisiae strains from various geographical origins and ecological niches found
great phenotypic diversity for other traits, but almost no differences in ethanol yield [4].
Below, we describe the different efforts made to overcome this biotechnological hurdle.

2. Genetic Improvement of S. cerevisiae

2.1. Genetic Engineering
Since the 1990s, it has been a constant in wine biotechnology that product improvement
and problem solving tend to be addressed, in the first instance, by the genetic engineering
of wine yeasts. However, the wine industry is not the ideal breeding ground for genetically
modified organisms due to restrictions to GMOs on food production in most countries,
and their negative perception by wine consumers [5]. The only two recombinant wine
yeasts that have been commercialised so far do not seem to have become bestsellers in the
markets they were introduced in [5,6]. Therefore, none of the recombinant strains described
in this section were intended for direct commercialization. Nevertheless, their study
provided useful information for the improvement of yeast strains by alternative approaches.
Indeed, in several instances, the reduction in alcohol levels appeared as a side-effect of
genetic manipulations which were not intended for that purpose. As mentioned above,
when it comes to reducing the alcohol level, a key issue to guide genetic engineering is
the choice of carbon sinks. Figure 1 shows a summary of the main enzymatic reactions
involved in alcoholic fermentation, including those referred to on the following lines.
Biomolecules 2021, 11, 1569 3 of 16

Alcohol dehydrogenase (ADH) catalyses the final step in the production of ethanol
during alcoholic fermentation. It is therefore a rational deletion target for limiting ethanol
production. However, cell metabolism is complex and depends on a set of biochemical
reactions that must be balanced in several ways, especially regarding the redox balance.
For this reason, simple logics often lead to unwanted results. For example, alcohol dehy-
drogenase deletion leads to a reduction in ethanol production and an increase in that of
glycerol, but these recombinant strains are unable to grow under anaerobic conditions [7].
This genetic modification also results in higher levels of acetic acid and acetaldehyde,
far beyond levels suitable for commercial wines.
In addition to the modulation of the expression of endogenous genes by shutting
down or upregulating their expression, genetic engineering allows for the heterologous
expression of genes from other biological species. In 1994, Dequin and Barre [8] expressed
lactate dehydrogenase from Lactobacillus casei in S. cerevisiae to divert some of the pyruvate
formed in glycolysis to lactate formation. The engineered yeast strain simultaneously
performed alcoholic and lactic fermentations. The redox balance was not affected, as the
formation of lactic acid is compensated by the disappearance of ethanol in equimolecular
amounts, for the same consumption of NADH. Thus, a reduction of 1% ethanol leads to an
increase in 15 g/L lactic acid. The increased lactate content of these wines contributed to a
higher total acidity. This feature could be useful to compensate for low acidity, which is a
problem affecting some grape varieties and growing regions, and is also related to global
warming. However, this also sets a limit to this approach, as excessive acidity would make
the wines unpalatable.
Glycerol contributes positively to the viscosity, body, and sweetness of wine. It has
been a preferred carbon sink for the genetic engineering of wine yeasts. The overexpression
of GPD1, coding for glycerol 3-P dehydrogenase, results in a significant increase in glycerol
(plus 28 g/L) and a reduction in ethanol (minus 15 g/L), and also in the appearance of
excess acetate and acetoin to restore the intracellular redox balance during fermentation [9].
Researchers identified Ald6 as the aldehyde dehydrogenase isoenzyme responsible for most
of the acetate production of S. cerevisiae [10]. However, the knockout of ALD6 resulted in an
additional increase in acetoin production which, at high concentrations, negatively affects
wine aroma, providing buttery notes [11]. Following this line of research, the problem of
acetoin overproduction was tackled by overexpressing BDH1, which codes for butanediol
dehydrogenase [12]. This enzyme catalyses the conversion of acetoin to 2,3-butanediol,
a compound with no apparent sensory impact. Using this recombinant strain, wines show
a significant reduction in ethanol, and above 26 g/L of glycerol content. The number of
different genetic modifications required to achieve this objective is in itself an indication of
the difficulty of using this as a general approach to developing wine yeast strains with a
low ethanol yield.
The different adjustments that were required to develop the above-described recom-
binant yeasts respond to metabolic constraints concerning the redox balance and the
availability of an adequate pool of cofactors. Therefore, some authors have tried to act
directly on the reactions of the redox system. By cloning an NADH oxidase for the regen-
eration of the reducing power, and with controlled oxygenation, not only is a decrease in
ethanol obtained but also a significant increase in the levels of acetaldehyde, acetic acid,
and acetoin [13].
There are two enzymatic steps from pyruvate to ethanol during alcoholic fermentation
(Figure 1). The final one catalysed by alcohol dehydrogenase has been discussed above.
The first, from pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase (PDC),
was also taken as an improvement target by some researchers. In the absence of PDC
activity, the Crabtree effect is abolished, but this activity is required for acetyl-CoA synthesis
in the cytoplasm and NAD+ regeneration. It is therefore essential for S. cerevisiae growing
on glucose [14]. The deletion of PDC1 resulted in a fourfold reduction of the PDC activity,
with an increase in pyruvate levels, but no reduction of ethanol levels [10]. The deletion of
PDC2, coding for a transcription factor involved in PDC gene expression, results in 19%
Biomolecules 2021, 11, 1569 4 of 16

PDC activity, with a clear reduction in the ethanol content, combined with an increase
in glycerol and pyruvate release [15]. This effect is enhanced by increased glycerol-3-
phosphate dehydrogenase activity, through the overexpression of GPD1, without the
over-production of acetic acid and acetaldehyde associated with GPD1 over-expression
in the original strain. Cuello et al. [16] replaced one of the native PDC2 alleles in different
diploid wine yeast strains by a truncated PDC2 allele. These engineered strains showed
a 2% ABV reduction without any increase in acetate production. An alternative way to
produce cytosolic acetyl CoA in pdc- strains was developed by substituting Acs1 and Acs2
(acetyl coA synthetase) with a heterologous pyruvate carboxylase and a transacetylase [17].
This strain produces glycerol, succinate, pyruvate and acetate from glucose, but not ethanol.
The additional deletion of gpp1 and gpp2 (glycerol 3p phosphatase) avoids the production
of these metabolites, including glycerol, and increases the growth rate.
Crabtree-negative strains of S. cerevisiae were also constructed by substituting all of the
active hexose transport genes with a single chimeric one [18]. This strain does not produce
ethanol as long as oxygen is available for respiration. However, its growth kinetics are
extremely slow. Further possibilities of using the respiratory metabolism of yeast to reduce
the ethanol yield during wine fermentation will be examined below in a specific section.
Triose-phosphate isomerase, encoded by TPI1, is the enzyme catalysing the inter-
conversion between the two products resulting from the breakdown of fructose 1,6-
bisphosphate: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (Figure 1).
The topology of the pathway suggests that TPI1 deletion would result in equimolar levels
of glycerol and ethanol precursors. However, in practice, such deletion strains are unable
to grow on glucose due to a redox imbalance [19]. In order to improve industrial glyc-
erol yields, additional genetic modifications have been introduced in strains deleted for
TPI1 [20]. These authors deleted ADH1 (coding for alcohol dehydrogenase) and TPI1 in a
yeast strain while overexpressing GPD1 (coding for glycerol-3-phosphate dehydrogenase),
FPS1 (coding for a glycerol transporter) and ALD3 (coding for an aldehyde dehydrogenase).
These strains produce very low levels of ethanol (about 25% of the parent strain), and their
glycerol levels are multiplied by 30. However, their growth rates were strongly reduced.
Another alternative carbon sink is yeast biomass. Despite the portion of carbon going
to biomass being quantitatively small during wine fermentation, some authors have tar-
geted reserve sugars to divert the carbon flux from ethanol. A moderate overexpression of
TPS1, the gene coding for trehalose-6-phosphate synthase, involved in trehalose biosynthe-
sis, resulted in a clear reduction in the ethanol yield, without an apparent impact on the
redox balance, according to the profile of the byproducts analysed [21].
Global transcription machinery engineering (gTME) technology has recently been
employed to construct a low-alcohol yeast strain. One strain with a reduced ethanol yield
was selected from a library of mutants in SPT15 (coding for a transcription factor) generated
by random directed mutagenesis [22]. The pleiotropic effect of this mutation downregulates
hexose transport and ethanol production, and upregulates glycerol production. Acetic acid
is not increased, but the mutant shows slower growth kinetics than the parent strain.
The treatment of must with glucose oxidase is an alternative method to decrease the
alcohol content of wine that will be discussed in the corresponding section. Based on
this method, Malherbe et al. [23] cloned a glucose oxidase gene from Aspergillus niger in a
laboratory strain of S. cerevisiae. Wines produced with the recombinant strain contained 2%
less ethanol. The recombinant strain showed antimicrobial activity against acetic and lactic
acid bacteria, probably due to the H2 O2 released by the oxidation reaction.
The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/
CRISPR associated protein 9) genetic modification system has already been implemented
in S. cerevisiae, including wine yeasts [24,25]. The basic CRISPR/Cas9 system consists of a
guide RNA molecule (gRNA) and an endonuclease (Cas9). The endonuclease mediates a
double strand break guided by the hybridization of gRNA with genomic DNA. This triggers
the DNA repair mechanisms of the host cell. The system can be designed to target the
nuclease to the gene of interest, and to perform more or less large insertions and deletions.
Biomolecules 2021, 11, 1569 5 of 16

An advantage of this system is that, unlike other genetic engineering techniques, it can
easily be tuned to leave no trace of the ancillary genetic material. In addition, it is less
dependent on traditional transformation markers, and makes easier the manipulation
of diploid loci. Nonetheless, microorganisms obtained with this system still fall under
European Union GMO regulations. The genes GPD1 and ATF1 have been cloned in a
haploid wine strain using this technique [26], with no differences in ethanol production
between the strains, contrary to the results obtained in previous works [9,15]. Other works
applying this technique for the reduction of wine alcohol content will certainly appear soon.

2.2. Random Mutagenesis

Genetic improvement by random mutagenesis involves the artificial increase in
mutation rates by treatment with physical or chemical agents (UV radiation, X-rays,
ethyl methane sulfonate, nitrosoguanidine) coupled with a suitable selection strategy [27].
To be effective, the intensity of the treatments must be lethal for most of the cells in the
population. However, given the random nature of the process, the number of cells that
must remain viable in order to capture the expected mutations is often still too high for
strain-by-strain analysis. It is then critical to establish appropriate selection criteria that
link selectable or detectable phenotypes to technologically relevant features of the mutant
strains. While this seems like an easy task for some improvement outcomes—for example,
those related to the robustness of yeast strains—improvements in wine quality, including
the reduction of alcohol levels, require indirect and non-obvious selection criteria.
An improved strain of S. bayanus was developed by random mutagenesis, and was
marketed as a low ethanol producer (Oenoferm® LA-HOG from Erbslöh, Geisenheim,
Germany). According to commercial information, the genetic improvement process in-
volved two stages of chemical mutagenesis. The first one, with selection under hypersaline
conditions, was intended to recover strains with an improved HOG (High Osmolarity
Glycerol) response. In the second, the selection pressure was established with pyrazole,
an inhibitor of alcohol dehydrogenase. The commercial strain was selected among the
40 strains recovered from this last step. During the fermentation of the Pinot noir grape
juice, this strain produced 12.9 g/L glycerol and 94.6 g/L ethanol, compared to the 6.4 g/L
and 102.4 g/L, respectively, produced by the parent strain.

2.3. Adaptive Laboratory Evolution

Adaptive laboratory evolution (also known as experimental evolution) consists of
recreating in the laboratory, in an accelerated way, the natural selection process, promot-
ing the selection of the mutations that are most useful to our purpose. This technique
shares several features with random mutagenesis [27], including some limitations, such as
that it cannot be easily targeted to specific genes, that dominant mutations are favoured,
and the difficulties of connecting readily selectable phenotypes to technological traits. How-
ever, it also offers advantages, as the strains obtained in this way do not fall under GMO
regulations. In this case, mutations appear spontaneously in the population during each
round of duplication (although it can eventually be combined with random mutagenesis).
Yeasts are grown in a selective environment in which the desired metabolic changes would
favour their growth. Once such a mutation appears, and after several generations under
the same selection conditions, many individuals in the population will harbour the desired
mutation. In addition, new variants can arise from the original one, and in this way the
favourable mutations for the intended technological objective can be accumulated in the
evolving lineage. The random nature of spontaneous mutations, and the fact that many
of them can be added over time during experimental evolution, gives some advantages
to this approach over genetic engineering. As indicated above, the challenge of this tech-
nology is to anticipate the metabolic pathways to be altered in order to reach the expected
technological output, and the growth conditions that will give advantage to the yeast cells
improved in that way.
Biomolecules 2021, 11, 1569 6 of 16

In the context of alcohol level reduction, adaptive laboratory evolution has been
used to channel carbon flux towards the pentose phosphate pathway (PPP; Figure 1).
The selective pressure was established using gluconate as the only carbon source [28].
Gluconate is a substrate poorly assimilated by S. cerevisiae and metabolized by the PPP.
The rationale for targeting PPP was that, compared to glycolysis, an additional CO2
molecule is released for each glucose molecule entering the pathway (meaning that this
carbon will not contribute to ethanol production). The selected strain showed a 1.5-fold
increase in flux through the PPP compared to the ancestral strain, corresponding to a
reduction of 2 g/L in ethanol levels. A fivefold increase in flux through the PPP would
have been necessary to reduce the ethanol level of wine by approximately 1% (vol/vol).
However, selected evolved strains did not show a reduced ethanol yield, but an increased
production of esters [29], which can be useful, but not for the original purpose.
Other authors have used experimental evolution to push carbon flux towards glyc-
erol production. The process was based on a long-standing strategy for the industrial
production of glycerol based on sulphite addition [30]. The reaction between sulphite
and acetaldehyde decreases the availability of acetaldehyde for ethanol production and
NADH oxidation, and cells must compensate the redox balance by increasing glycerol
production. Kutyna et al. [31] used this principle to drive the evolution of a laboratory
strain, with sodium sulphite as a selective agent, at an alkaline pH. Strains evolved in this
way did show a relative increase in glycerol yields, but there was a very limited impact on
ethanol yield. In addition, genetic analysis showed that the sulphite tolerance and glycerol
yield were not linked in the evolved strains.
Glycerol is the major osmolyte in yeasts [32]. Tilloy et al. [33] targeted glycerol
production to reduce the ethanol yield from a wine yeast strain. In this case, the selective
pressure was osmotic stress and the HOG intracellular signal transduction pathway. Strains
evolved in a hypersaline medium were selected and further improved by sporulation and
back-crossing. The resulting strains showed reduced ethanol yield, increased glycerol
yield, and did not produce acetic acid. However, the metabolic analysis revealed that,
contrary to expectations, the overproduction of glycerol was not due to the activation
of the HOG pathway. Compared to recombinant strategies which were also based on
glycerol overproduction, the authors found the evolved strains did not show the drawbacks
of GPD1-overexpressing strains. At the pilot scale, these strains produced wines with
0.4% (v/v) to 1.3% (v/v) less alcohol than the control (for Syrah and Grenache grapes,
respectively). Grenache wines were tasted, and no organoleptic defects were detected.
This strain has been commercialized.
 amounts, for the same consumption of NADH. Thus, a reduction of 1% ethanol leads to
an increase in 15 g/L lactic acid. The increased lactate content of these wines contributed
to a higher total acidity. This feature could be useful to compensate for low acidity, which
is a problem affecting some grape varieties and growing regions, and is also related to
global warming. However, this also sets a limit to this approach, as excessive acidity
Biomolecules 2021, 11, 1569 7 of 16
would make the wines unpalatable.

Figure
Figure 1.
1. Diagram
Diagram of
of the
the alcoholic
alcoholic fermentation
fermentation pathway
pathway with
with indications
indications of
of the
the steps
steps affected
affected by
by
the
thedifferent
differentgenetic
geneticmodifications
modificationsassayed
assayedtoto
lower
lowerethanol yields.
ethanol References
yields. References in black: genetic
in black: en-
genetic
gineering.
engineering.References in yellow:
References adaptive
in yellow: evolution.
adaptive Metabolites
evolution. outside
Metabolites of the
outside of central ethanol
the central fer-
ethanol
mentation
fermentationpathway areare
pathway indicated in purple.
indicated in purple.

3. Alternative Wine Yeast Species

Microbial ecology studies of grape juice and wine fermentation clearly indicate that,
most often, S. cerevisiae is a minor species at the beginning of the fermentation process.
In contrast, it is almost invariably the dominant one at the end of spontaneous fermenta-
tions. Therefore, this species is considered particularly well adapted to the process, and has
been selected for use in inoculated fermentations. The other yeast species present during
the initial stages of fermentation were traditionally considered to be spoilage microorgan-
isms, and therefore undesirable. Over time, after inoculation with S. cerevisiae became
common practice, non-Saccharomyces wine yeast species have been found to harbour in-
teresting characteristics, such as their contribution to the aroma and complexity of the
wine, and their ability as microbiological control agents [34]. Several commercial strains
of non-Saccharomyces yeast are currently available for use in wine fermentation, including
Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima, among the
most common yeast species [35]. In addition to their features related to the sensorial quality
of wines, one potentially interesting feature of some non-Saccharomyces species is their
lower ethanol yield compared to S. cerevisiae. However, only during the last decade has the
possibility of using alternative yeast species to reduce alcohol content been considered [36].
A summary of the results described below, using different non-Saccharomyces yeast species
to obtain lower alcohol wines, is shown in Table 1.
Biomolecules 2021, 11, 1569 8 of 16

Table 1. Fermentation conditions and the results obtained for alcohol level reduction using strains of different non-
Saccharomyces wine yeast species on natural grape must.

Time for S. Ethanol

Sugar Content Sensory
Single Species Conditions cerevisiae Reduction Reference
(g/L) Analysis c
Addition (% ABV)
Saccharomyces uvarum 171 a standard Negative No. Pure culture 0.5 [37]
240 b standard Negativec No. Pure culture 1.7 [38]
210–240 a standard No c at 50% sugars 0.8–0.9 [39]
Metschnikowia pulcherrima 230–240 a standard No c at 50% sugars 0.9–1.6 [40]
210–240 a standard No c at 50% sugars 1.0–1.1 [39]
240 b standard No different c 0 h (1/10) 1.0 [38]
230 a standard No c 3 days 0.8–1.25 [41]
264 standard No different c 3 days 1.0 [42]
20 VVH discont
260 a No 0h 3.7 [43]
48 h
212 a DO = 20% 3 days Negative c 72 h 3.6 [44]
220 a 3 VVH 72 h No c 72 h 1.5 [45]
Starmerella bacillaris 244 standard Different 24 h 0.5–0.6 [46]
(Candida zemplinina) 250 standard No 48 h 0.5 [47]
DO = 20% 2–3
212 a Negative c 69–52 h 0.6 [44]
days
Starmerella bombicola 218 1.2 VVH O2 72 h No c 72 h 1.5 [48]
Hanseniaspora uvarum 230–236 standard Different c 7 days 0.8–1.1 [49]
238 standard Positive c 48 h 0.9 [50]
Hanseniaspora opuntiae 230–236 standard Different c 7 days 0.6–1.3 [49]
Hanseniaspora vineae 252 a 20 VVH 24 h No c 24 h 2.5 [51]
Torulaspora delbrueckii 223 standard Positive c 4 days 0.5 [52]
195 a standard No different c No. Pure culture 0.5 [53]
220 a 1.5–3 VVH 72 h No c 72 h 0.9–1.0 [45]
Candida oleophila 206 a DO = 20% 5 days Negative c 120 h 1.4 [44]
Pichia kudriavzevii 230–236 standard Different c 7 days 0.4–0.6 [49]
Pichia guilliermondii 206 a DO = 20% 5 days Negative c 120 h 3.6 [44]
212 a DO = 20% 3 days Negative c 72 h 1.8 [44]
Pichia kluyveri 212 a DO = 20% 3 days Negative c 72 h 2.7 [44]
Lachancea thermotolerans 222 standard Higher acidity c 48 h 0.7 [54]
220 a standard Negative c 6 days 1.2 [55]
Meyerozyma guilliermondii 230 a standard No c 3 days 1.2 [41]
Zygosaccharomyces bailii 220 a 3 VVH 72 h No c 72 h 1.2 [45]
Two Species
M. pulcherrima/S. uvarum 210–240 a standard No c at 50% sugars 1.8 [39]
M. pulcherrima/S. bayanus 195 a standard Positive c 96 h 0.9 [53]
Immobilized
Hanseniaspora osmophila 202 standard No c 72 h 1.0 [56]
Hanseniaspora uvarum 202 standard No c 72 h 1.2 [56]
Starmerella bombicola 202 standard No c 72 h 1.6 [56]
Metschnikowia pulcherrima 202 standard No c 72 h 1.5 [56]
204 a 1.2 VVH 72 h No c 72 h 1.4 [57]
Schizosaccharomyces
240 a standard No c 0 h/48 h 1.7/2.4 [58]
japonicus
a Heat treated or filter sterilized; b DMDC treated; c Volatile Compound Analysis performed; DO: dissolved oxygen; VVH: volume per

volume per hour.

The cryophilic species Saccharomyces uvarum has been described as a low ethanol,
low acetate, and high glycerol producer yeast [37,59,60]. In experimental fermentations,
the ethanol contents of wines produced with two strains of S. uvarum were 0.5 and 0.3%
ABV lower than S. cerevisiae wine, and their hybrids showed ethanol contents which were
intermediate between the parent species [37]. An important reduction (1.7% ABV) was
obtained with a strain of S uvarum in Merlot wine, but it showed predominantly negative
attributes in a sensory analysis, with aromas of meat and barnyard [38]. Another cryophilic
species, Saccharomyces kudriavzevii, has been described as a high glycerol producer [61].
Although S. kudriavzevii is not found in winemaking, S. cerevisiae × S. kudriavzevii hybrids
are often isolated from cold-climate wine environments. However, the ethanol yield of
these hybrids has been found to be higher than that of S. cerevisiae [62].
Biomolecules 2021, 11, 1569 9 of 16

In 2011, Magyar and Toth [63] studied the ethanol yield of several strains of S. cerevisiae,
S. uvarum/bayanus, Candida zemplinina (currently Starmerella bacillaris) and Candida stellata.
All of the other species showed a lower ethanol yield than S. cerevisiae, in particular
C. zemplinina isolates. In addition, this species is fructophilic and shows a high glycerol yield.
A great number of strains of this species have been explored for ethanol production [64,65].
Wines made with the selected strains contained, at best, 0.6% ABV less than the control
wine [46,47].
Gobbi et al. (2014) [66] assessed the ethanol yield of 32 strains belonging to 8 dif-
ferent species, compared to a control strain of S. cerevisiae, and found that the species
Hanseniaspora uvarum showed the lowest yield, followed by Zygosaccharomyces sapae and
Zygosaccharomyces bisporus. The authors highlight the variability among strains of the
same species, and the correlation found between ethanol yield and secondary metabolite
production. Mestre et al. (2017) [67] also selected a strain of H. uvarum among 117 strains
belonging to 32 different species. Malbec wine produced with the selected strain [50] had a
0.9% drop in ethanol in compared to that made from S. cerevisiae, and a good qualification
in a sensory analysis.
Contreras et al. (2014) [40] studied the ethanol yield and sugar consumption of
50 yeast strains, with the aim of selecting suitable strains to lower the alcohol content of
wine. The authors highlighted the potential of strains of the species C. stellata, Schizosaccha-
romyces malidevorans and, mainly, M. pulcherrima. A selected strain of M. pulcherrima was
used for sequential fermentations with S. cerevisiae, and allowed for an ethanol reduction
of 1.6% (v/v) for Shiraz must and 0.9% (v/v) for Chardonnay must. An increase in ethyl
acetate and 2-methyl propanol was observed, which can negatively impact the wine quality.
More recently, they fermented Merlot must with M. pulcherrima in a sequential inocu-
lation with S. cerevisiae [38]. Compared to wine inoculated with S. cerevisiae, wine from
spontaneous fermentation contained 0.7% (v/v) less ethanol, and wine fermented by M. pul-
cherrima contained 1% (v/v) less. In the sensory analysis, spontaneously fermented and
M. pulcherrima wines were indistinguishable, and very similar to the control. Coinoculation
with M. pulcherrima and S. uvarum, in sequential inoculation with S. cerevisiae, allowed a
stronger reduction (1.7% v/v) than using these strains separately (0.8–1.0% v/v) [39].
Rossouw and Bauer (2016) [49] studied the ethanol yield of 91 yeast strains, including
some isolates of S. cerevisiae and two commercial strains as control strains. The authors
highlight the variability in the rate and extent of sugar utilisation between strains of the
same species, except for S. cerevisiae species, and the differences in aroma compounds
produced by strains affecting the wine aroma. Wines made with selected strains of Hanse-
niaspora opuntiae, Hanseniaspora uvarum, and Pichia kudriavzevii, in sequential inoculation
with S. cerevisiae, contained between 0.4 and 1.5% ABV less than the control wines.
There are currently several strains of T. delbrueckii for winemaking in the market which
have been selected for their contribution to the aromatic complexity of wine. Regarding
the ability to lower the alcohol content, there was a 0.5% v/v reduction in the ethanol
content compared to the S. cerevisiae control wine [52]. Puškaš et al. (2019) [53] studied
the effectiveness of one commercial strain of T. delbrueckii and one strain of M. pulcherrima,
in co-inoculation with S. cerevisiae and/or S. bayanus. In pasteurised must, a reduction
of 0.9% (v/v) in ethanol concentration was achieved with Metschnikowia and 0.5% with
Torulaspora, but this effect was lost in natural must, probably due to the low dominance of
the selected strains.
L. thermotolerans has also been introduced in the market because its positive effects
on wine aroma, total acidity, and volatile acidity [68]. Wines obtained with the sequential
inoculation of L. thermotolerans and S. cerevisiae contained 1.2% less ethanol than the control
wine, but suffered a decrement in aroma quality [55].
Some authors are also exploring yeast cell immobilization to improve effectiveness
and process control (Table 1). Canonico et al. (2016) [56] studied the use of immobilised
alternative yeasts, together with free S. cerevisiae in sequential inoculation. Using Starmerella
bombicola, H. uvarum, Hanseniaspora osmophila and M. pulcherrima, ethanol reduction values
Biomolecules 2021, 11, 1569 10 of 16

from 1.0 to 1.6% (v/v) were achieved. Changes in the volatile compounds were also
observed. It was predicted that these could potentially affect the sensory properties of
the wines, either positively or negatively, depending on the case, but the study did not
include a sensory evaluation. Finally, the authors indicated that this technology makes
the process more expensive. Domizio et al. (2018) [58] obtained a 1.7–2.4% reduction of
ethanol levels with immobilized cells of Schizosaccharomyces japonicus in co-inoculation and
sequential inoculation with free S. cerevisiae. The levels of acetoin, ethyl acetate, and acetate
esters were higher in both wines, and acetic acid and glycerol were higher in wine made by
sequential inoculation.
Several of the studies described above show some promise for non-Saccharomyces
yeast used in sequential or co-inoculation with S. cerevisiae for the reduction of ethanol
yields during wine fermentation, and hence the alcohol content of wines. However,
even under laboratory controlled experimental conditions, the reduction values were
generally moderate (Table 1), and difficulties associated to scale-up were often reported.
Fairly different reduction values are published by different laboratories, which is not
surprising because the authors did not follow standardized conditions (the optimization
of fermentation conditions is usually a secondary objective of their work), and different
strains were used in each case. According to the available information, when developing
protocols aiming at alcohol level reduction, attention should also be paid to the impact of
strains and fermentation conditions on the sensory properties of the wines. In addition,
the use of non-Saccharomyces species for alcohol reduction faces technological challenges
common to other oenological applications of these strains. These include difficulties for
the industrial production of most of them as active dry yeast, a generally low tolerance to
sulphiting agents, low imposition, or considerations about compatibility with S. cerevisiae
starters (which will almost always be necessary, in sequential or simultaneous inoculation
with non-Saccharomyces, to ensure that fermentation is completed).

4. Aerobic Fermentation
4.1. Non-Saccharomyces Species
Glycerol has been the target in many attempts to reduce ethanol yields by genetic
improvement (see above). However, reducing the alcohol content by 1% (v/v), by diverting
all excess carbon to glycerol, can result in more than 15 g/L extra glycerol in the wine.
This sets a technological limit to this strategy, as a further increase in the glycerol yield
would lead to values well above the commercially acceptable levels. However, almost
every alternative carbon sink can have a detrimental sensory impact if produced in excess,
even for compounds with positive sensory attributes.
In this context, some authors, including our research group [69,70], proposed CO2
generated by the aerobic respiration of yeast as a neutral carbon sink. By respiration,
all of the carbon present in sugar is converted to CO2 , in a process that requires molecular
oxygen. However, S. cerevisiae is a Crabtree-positive yeast. This implies that, except
under very small concentrations of glucose, it will mostly ferment sugars and produce
ethanol, even in the presence of oxygen. Therefore, the proposal was made to perform a
multi-starter fermentation with S. cerevisiae and alternative yeasts with higher respiration
capacities [70]. The appropriate yeast would be inoculated into the must, and during the
first hours, oxygen would be provided to the fermentation tank. After some time, the forced
aeration would be stopped, and the traditional process would follow. S. cerevisiae could be
inoculated from the beginning (co-inoculation), or when the aeration is stopped (sequential
inoculation). The more sugar is respired during the aeration process, the lower the ethanol
yield of the overall process will be.
The availability of suitable yeast strains was a prerequisite to reach this goal. To be
useful, these strains must be able to respire in the stressful conditions of grape must,
with no negative impact on the wine sensory properties. In a screening of 65 strains from
28 yeast species, Quirós et al. (2014) [71] found an interesting result for some S. cerevisiae
strains. Despite the Crabtree effect, the residual respiration activity of some of them was
Biomolecules 2021, 11, 1569 11 of 16

enough to allow for a substantial reduction in the ethanol yield according to the measured
respiration quotient (RQ) values. However, S. cerevisiae strains showed a boost in their
acetic acid production when provided with air. In contrast, several non-Saccharomyces
strains showed a combination of metabolic features suggesting that they might be suited
for use in the reduction of alcohol levels. Namely, some strains showed a moderate-to-high
sugar consumption rate, very low acetic acid production, and a low RQ (which means
a low expected ethanol yield). Indeed, one strain of M. pulcherrima was used in a co-
inoculation with S. cerevisiae in laboratory-scale bioreactors for the fermentation of natural
grape must (260 g/L sugar content). Several inoculation and aeration rates were assayed.
The alcohol level reduction reached 3.6% (v/v) with a somehow too-high volatile acidity,
or 2.2% (v/v) with acceptable volatile acidity levels [43]. Some environmental factors that
can be controlled during wine fermentation—such as nutrients, temperature, and dissolved
oxygen levels—will certainly affect both the ethanol and acetic acid yield by different
yeast strains. Interestingly, strains of M. pulcherrima and Candida sake show low acetic acid
production almost independently of the fermentation conditions [72].
Fermentation conditions leading to lower ethanol yields based on the use of non-
Saccharomyces strains under aerobic conditions have been studied by these and other authors,
using T. delbrueckii, M. pulcherrima, Zygosaccharomyces bailii, C. zemplinina, M. pulcherrima,
Pichia guilliermondii, Pichia kluyveri, Candida oleophila, Starmerella bombicola, or Hanseniaspora
vineae [44,45,48,51,57,73,74]. The authors have worked with different aeration times and air
flows, free or immobilized cells, and different varieties of white grapes (Table 1). As a trend,
increased aeration leads to better results in terms of reduced alcohol levels, but also to a
greater impact on the volatile compounds (including volatile acidity) and a lower rating
from sensory panels. It can be concluded that the results are promising, but the process
needs to be further developed, including the criteria for the selection of non-Saccharomyces
strains for this application, in order to develop useful protocols for the industry.

4.2. S. cerevisiae Strains Improved for Aerobic Fermentation

As mentioned above, one major drawback for the use of S. cerevisiae under the aerobic
growth conditions required for respiration was that strains of this species produced un-
acceptable acetic acid levels under these conditions. In order to overcome this problem,
the researchers turned to other wine yeast species. Although this strategy is yielding some
promising results (see the previous section), and though the use of non-Saccharomyces
species could bring some other benefits to the wine, it also hinders the fermentation man-
agement in the winery. The use of a single yeast starter that will at the same time be able
to reduce alcohol levels, and drive fermentation to the end, while not increasing volatile
acidity, would be an optimal alternative.
A wine strain of S. cerevisiae improved for lower acetic acid production was expected
to be able to meet all of these requirements. Indeed, recombinant wine yeast strains partly
defective in carbon catabolite repression (CCR) due to REG1 gene knockout were shown to
produce reduced levels of acetic acid under anaerobic conditions [75]. Interestingly, reg1
mutant strains can grow on non-preferred carbon sources (glycerol or galactose, for ex-
ample) in the presence of the non-metabolizable glucose analogue 2-deoxyglucose (2DG).
In contrast, strains with an intact CCR will be inhibited under these growth conditions.
Although this link between CCR and acetic acid production under aerobic conditions is
not yet clarified, it is possible to take a practical advantage of it to design experimental
evolution strategies to develop S. cerevisiae wine yeast strains which are better suited for
alcohol level reduction. This was recently performed by Guindal et al. [76] using galactose
as a carbon source in the presence of growing amounts of 2DG for around 100 genera-
tions. Several evolved strains from different wine yeast genetic backgrounds were indeed
derepressed for glucose, and had been indirectly selected for lower acetic acid produc-
tion under aerobic conditions. As a trend, the glycerol yields were also increased for the
selected evolved strains. However, some strains had to be discarded due to impaired
fermentation kinetics. Indeed, there is always a risk of unwanted change during adaptive
Biomolecules 2021, 11, 1569 12 of 16

laboratory evolution. This must be managed by the careful design of experimental condi-
tions and a thorough characterization of the derived strains before they can by promoted
to industrial applications.
On the other side, in the framework of the European project CoolWine (ERA Co-
BioTech), researchers are working on the development of rational strategies for adaptive
laboratory evolution aiming at alcohol level reduction. For S. cerevisiae, the aim is to develop
new strategies to reduce the above-mentioned problem of excess acetic acid production in
the presence of oxygen, while for non-Saccharomyces strains, the aim is to improve their
survival and competitiveness in the context of winemaking.
Although the general trend for S. cerevisiae strains is the production of high volatile
acidity under aerated wine fermentation conditions, we recently found rare isolates showing
acceptable acetic acid yields under aerobic conditions [77]. These strains show promise
to be used for alcohol level reduction by sugar respiration, without resourcing to non-
Saccharomyces yeast or the requirement for genetic improvement. However, genetic improve-
ment will still be necessary to ensure enough genetic diversity for industrial applications
(a single strain cannot fulfil all industry requirements), and to take advantage of the features
of commercial strains that have previously shown optimal features for winemaking.

5. Enzymatic Treatment of Grape Must

One conceptually simple approach for the reduction of the ethanol content of wines
is to remove excess sugar from the grape juice before fermentation. Biotechnologically,
this goal can be achieved using enzymes, and the enzymatic activity proposed for this
application is glucose oxidase. This enzyme catalyses the oxidation of glucose to gluconic
acid. The reaction involves molecular oxygen as a co-substrate, and releases hydrogen
peroxide as a by-product.
The use of glucose oxidase during the pre-fermentation stages to produce low-alcohol
wines was first proposed by Villetaz (1986) [78], and was optimised by this and other
authors (as reviewed in [79]). However, the use of glucose oxidase is not among the
procedures included in the International Code of Oenological Practices [80].
The procedure proposed by Pickering et al. [81] involved an initial grape must deacid-
ification to accommodate the pH to that which is optimal for the enzyme used, as well as
aeration during the enzymatic treatment. Catalase was added with glucose oxidase to re-
move the hydrogen peroxide formed in the reaction. The removal of the hydrogen peroxide
improves the yeast survival and the activity of glucose oxidase enzyme itself. By treating
Riesling grape juice for 10 h under these conditions, these authors produced wines with an
alcohol content reduced by 3.7% (v/v) [82]. However, gluconic acid negatively affected the
taste of the wine, fruity aromas were perceived with less intensity, and their persistence
was also reduced [83]. A second deacidification or a sweetening were proposed to alleviate
the effect of gluconic acid [81].
Röcker et al. [84] assayed several methods of deacidification following glucose oxidase
treatment. However, the wines were more acidic than the control, and lost part of their
fruitiness. Other authors have used encapsulated enzymes in order to improve glucose
oxidase activity without prior pH correction [85,86]. Wines obtained from juices treated
for two days under these conditions showed a 0.68% (v/v) reduction in their ethanol
content [85].

6. Metabolic Inhibitors
Depending on their mode of action, sublethal concentrations of some metabolic
inhibitors might be expected to alter the ethanol yield of S. cerevisiae. This is the case of
furfural, an aromatic aldehyde resulting from sugar dehydration. It can be found in wines
at concentrations below the perception limit (20 mg/L). Furfural is toxic for S. cerevisiae,
and the detoxification mechanism involves its transformation to furfuryl alcohol by alcohol
dehydrogenase, the same enzyme catalysing the final step of alcoholic fermentation from
acetaldehyde to ethanol. The use of furfural has been proposed to reduce the ethanol yield
Biomolecules 2021, 11, 1569 13 of 16

during wine fermentation [87]. However, the suggested dose, 50 mg/L, is clearly above
the perception limit, for a modest reduction in ethanol content of 0.6% (v/v). The same
research group suggested other metabolic inhibitors to be used for the same purpose [88],
but no experimental results have been published yet.

7. Conclusions
The metabolic versatility of oenological yeasts, considering both non-Saccharomyces
and Saccharomyces species, and our growing knowledge about it, can be exploited to
address the problem of the rising alcohol levels observed over the last few decades in
wine. Research groups have approached the problem from various perspectives: the
exploration of natural genetic diversity, focusing mainly on non-Saccharomyces yeasts;
the genetic improvement of S. cerevisiae by genetic engineering, random mutagenesis,
or experimental evolution; or the modification of the environmental conditions (metabolic
inhibitors, aerobiosis). A combination of aerobic conditions with yeast genetic improvement
(of both Saccharomyces and non-Saccharomyces yeast strains) is currently one of the most
promising options. However, considering the metabolic diversity of wine yeast species and
the impact of oxygen on yeast metabolism, any technological improvement developed in
the laboratories must be validated by the sensory analysis of the wines produced at the
pilot or industrial scale.

Author Contributions: P.M. and R.G. conceived the idea, wrote the first draft, and supervised the
writing. A.M.G. and J.T. contributed to the writing of several sections. All authors have read and
agreed to the published version of the manuscript.
Funding: The work of the Microwine group is currently funded by the Spanish Government through
grants PID2019-105159RB-I00 and PCI2018-092949 (co-funded by ERA-CoBioTech). JT is funded by
FGCSIC by the COMFUTURO program. AMG’s predoctoral contract is funded by Consejería de
Desarrollo Económico e Innovación de la Comunidad Autónoma de La Rioja.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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 | |
Received: 17 July 2019    Revised: 18 December 2019    Accepted: 27 December 2019

DOI: 10.1002/fsn3.1433

ORIGINAL RESEARCH

The aptitude of commercial yeast strains for lowering the

ethanol content of wine

Vladimir S. Puškaš | Uroš D. Miljić  | Jovana J. Djuran | Vesna M. Vučurović

Faculty of Technology Novi Sad, University

of Novi Sad, Novi Sad, Serbia
Correspondence
Uroš D. Miljić, Faculty of Technology Novi
Sad, University of Novi Sad, Blvd. cara
Lazara 1, Novi Sad 21000, Serbia.
Email: urosmiljic@yahoo.com
Funding information
Ministry of Education, Science and
Technological Development of the Republic
of Serbia, Grant/Award Number: Project
TR-31002
Abstract
The high alcohol content in wine usually has a negative impact on its sensory prop-
erties, but can also affect the general health of the consumers. The possibility to
reduce ethanol production in wines during fermentation involves the use of different
yeast strains characterized by the increased production of fermentation by-products
(glycerol, 2,3-butanediol, etc.) from the available sugar. The activity of these strains
should not impair the sensory properties of the wine. In general, the use of geneti-
cally and evolutionarily (non-GM) engineered Saccharomyces cerevisiae strains is still
not close enough to commercial application, and therefore, it is unavailable for wine
producers. Thus, the aim of this study was to examine the possibility of reducing
the production of ethanol in wines using different selected yeast strains (S. cerevi-
siae, Saccharomyces bayanus, Torulaspora delbrueckii, and Metschnikowia pulcherrima)
available at the market. The application of individual yeast and sequential inoculation
for wine alcoholic fermentation was examined. The achieved effects were evaluated
by determining the content of ethanol, as well as fermentation by-products (glyc-
erol and volatile acids) and aromatic components in wine samples. Depending on the
strain/s used, a decrease in ethanol content of up to 0.9% v/v was recorded in com-
parison with fermentation by S. cerevisiae alone. The sensory analysis of produced
wine showed significant differences in taste and flavor. The results of the experiment
conducted at the laboratory level and with the use of sterile must were compared to
the ones from the scale-up experiment in real vinification conditions. The observed
differences in the alcohol content of produced wines were significantly lower.

KEYWORDS

commercial yeast strains, global warming, lower-alcohol wines, sequential inoculation

1 |  I NTRO D U C TI O N
Climate change, new market trends, and technologies' progress now-
adays strongly affect the wine industry. Over recent decades, the av-
erage alcohol content of table wines has increased by about 2% (v/v),
due to the high sugar content of the grapes currently used in the most
wine-growing regions (Goold et al., 2017). A wide range of factors sig-
nificantly affects sugar accumulation in the grape such as warm climate
conditions combined with the lengthy maturation periods used to sat-
isfy the consumer demand for rich and ripe fruit flavor in the wine.
The high alcohol content in wine has several negative consequences.
One of the major issues of higher alcohol content in wines is its effect

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2020 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc.

Food Sci Nutr. 2020;8:1489–1498.  |

www.foodscience-nutrition.com     1489
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1490       PUŠKAŠ et al.
on the sensory properties, which increases the perception of the heat
and alters the perception of wine aroma complexity (Goldner, Zamora,
Di Leo Lira, Gianninoto, & Bandoni, 2009). Also, excessive alcohol
intake through the consumption of wines with higher ethanol levels
is often associated with undesirable implications on human health.
Furthermore, higher alcohol in wine may increase costs in countries
where taxes are levied according to alcohol concentration. Thus, the
combination of quality, health, and economic issues associated with
high-alcohol wines has created significant interest in the development
of technologies for the production of reduced ethanol wines.
Different approaches to reduce alcohol levels in wines have been
proposed at all stages of the winemaking process. These mainly fit
into four basic groups as viticultural, prefermentation, fermentation,
and postfermentation strategies (Varela et al., 2015). Viticulture
strategies, as promising but long-term techniques, are based on the
selection of new grape varieties with low sugar accumulation, viticul-
tural practices adapted to unripe grapes, and different agronomical
methods (Olego et al., 2016). On the other hand, postfermentation
strategies such as reverse osmosis, nanofiltration, and distillation
represent a short-term perspective dependent on the current EU
and OIV regulations. Moreover, these procedures may increase pro-
duction costs and also can compromise the wine organoleptic quality
due to the elimination of volatile compounds (Schmidtke, Blackman,
& Agboola, 2012). Considering possible approaches, the application
of yeast strains characterized by lower sugar-to-ethanol transforma-
tion rates has been imposed as an attractive way to deal with the
problem of high-alcohol wines (Kutyna, Varela, Henschke, Chambers,
& Stanley, 2010). Lower ethanol-producing yeast strains could be
isolated and characterized from spontaneous wine alcoholic fermen-
tations or obtained through the application of adaptive evolution
(development of the low-alcohol variants of existing Saccharomyces
cerevisiae strains) and genetic modification techniques (GMO ap-
proaches) (Ozturk & Anli, 2014; Varela et al., 2015). Due to poor
consumer acceptance of GMO foods and beverages, there is a need
to investigate and develop the non-GMO approaches for the gener-
ation of wine yeasts that produce less ethanol (Kutyna et al., 2010).
Nonconventional yeasts such as Kloeckera, Pichia, Candida,
Metschnikowia, Schizosaccharomyces, and Torulaspora species are
among the main representatives of grape natural microbiota. In gen-
eral, their pronounced sensitivity to antimicrobial agents (e.g., SO2)
and higher alcohol contents prevent the complete transformation of
grape sugars into ethanol during alcoholic fermentation. Therefore,
their application in co-inoculation or sequential inoculation with
S. cerevisiae is increasingly getting popular especially regarding their
potential positive effects on wine flavor (Ciani et al., 2016). On the
other hand, species, such as Saccharomyces bayanus, are associ-
ated with spontaneous fermentation of must and have been shown
to be of oenological interest (González, Barrio, Gafner, & Querol,
2006). The use of mixed cultures of selected Saccharomyces and
non-Saccharomyces yeasts for wine fermentation can result in the
formation of higher amounts of undesired compounds (e.g., volatile
phenols and ethyl acetate) which can affect both structure and the
aromatic profile of the wines. Therefore, ensuring the expression of
appropriate metabolic characteristics of different yeast strains indi-
vidually or in mixed cultures might serve as an efficient mechanism
for reducing ethanol production in wines (Ciani & Comitini, 2015).
The application of non-Saccharomyces species for decreased alcohol
production is possible through both aerobic (respiration) and anaer-
obic (fermentation) metabolism (Ciani et al., 2016).
The aim of this research was to examine the possibility of reduc-
ing ethanol production in wines using different selected yeast strains
(Saccharomyces cerevisiae, Saccharomyces bayanus, Torulaspora del-
brueckii, and Metschnikowia pulcherrima) which are currently available
at the market. The experiments implied a series of wine fermentations
carried out both by the activity of chosen strains individually and in the
form of mixed cultures applied through sequential inoculation.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Inoculation strategies for the fermentation of
experimental wines
In order to investigate the possibility of reducing the production of
ethanol in wine using different yeasts as producing microorganisms,
the following commercial strains were used:
1. Saccharomyces cerevisiae Oenoferm Bouquet (Erbslöh, Germany),
shortened CER.
2. Saccharomyces bayanus LittoLevure CHA (Erbslöh, Germany),
shortened BAY.
3. Torulaspora delbrueckii Oenoferm Wild and Pure (Erbslöh,
Germany), shortened TOR.
4. Metschnikowia pulcherrima FLAVIA MP346 (Lallemand, France),
shortened MET.
The yeast inoculation was carried out according to the plan shown
in Table 1, for both laboratory and scale-up experiments. Production
TA B L E 1   Inoculation plan the fermentation of experimental
wines
Experimental
sample Plan of inoculation with selected strains
CER Saccharomyces cerevisiae
BAY Saccharomyces bayanus
MET+CER Metschnikowia pulcherrima and then 48 hr after
the first inoculation of S. cerevisiae
MET+BAY Metschnikowia pulcherrima and then 48 hr after
the first inoculation of S. bayanus
TOR Torulaspora delbrueckii
TOR+BAY Torulaspora delbrueckii and then 48 hr after the
first inoculation of S. bayanus
MET+BAY+CER Metschnikowia pulcherrima, then 48 hr after the
first inoculation of S. bayanus, and then 96 hr
after the first inoculation of S. cerevisiae
PUŠKAŠ et al.
of experimental wines was carried out under identical conditions for
all samples (laboratory and scale-up level) and was done in triplicate.
2.2 | Laboratory-scale experiment
Wines were produced from the Serbian white grape variety Sila (Vitis
vinifera L.), from grapes originating from the experimental vineyards of
the Faculty of Agriculture, University of Novi Sad, located in Sremski
Karlovci. Grapes were harvested at the stage of technological maturity
(optimal ratio between sugar and acids, phenolic and aromatic maturity
also ensured), at the end of September 2016. The processing of grapes
included crushing and destemming (Zambelli Gamma 30), followed by
pressing in classical vertical basket press (capacity 100 kg). The sugar
content in the must was 19.5%, total acidity 5.1 g/L (as tartaric acid),
and assimilable nitrogen 225 mg/L. The must was sulfited by the addi-
tion of potassium metabisulfite (0.1 g/L) in order to prevent oxidation.
The clarification of must was carried out by classic precipitation with
the addition of the Trenolin FastFlow pectolytic enzyme (Erbslöh) in
the amount of 5 ml/hl. The must was pasteurized (Grant instruments,
75°C during 15 min) to neutralize the influence of autochthonous
yeasts on the experimental fermentations. Fermentation was carried
out in 10-liter glass vessels. Must samples were inoculated with se-
lected yeasts and rehydrated according to the manufacturer's instruc-
tions, and in the amounts suggested. The yeast nutrient VitaFerm Ultra
(Erbslöh) was added after one-third of sugar fermented (sugar content
120 g/L), in the amount of 0.2 g/L. The fermentation temperature was
maintained at 16–18°C. During alcoholic fermentation, several param-
eters were evaluated in duplicate: sugars, glycerol, volatile acidity, and
ethanol. After the end of fermentation, the wines were racked, sulfited
(0.08 g/L potassium metabisulfite), and stored in 2-L glass bottles. The
analysis of the major volatile compounds and the sensory analysis of
produced wines were carried out after 1 month.
2.3 | Scale-up experiment
The experiments conducted at laboratory level and with the use of
pasteurized must were followed by the scale-up trials. The goal was to
evaluate the previously obtained results in real winemaking conditions
which do not imply the pasteurization of the must. For this purpose, ex-
periments were carried out in 200-liter stainless still tanks. Grape pro-
cessing and wine fermentation were conducted in an identical way as
in the case of the laboratory experiment. The only difference was the
use of fresh unpasteurized grape must. After the end of fermentation,
the content of ethanol, glycerol, and volatile acids was determined.
2.4 | Analyses
Total sugars, total acidity (expressed as tartaric acid), volatile acidity (ex-
pressed as acetic acid), and ethanol content (hydrostatic balance Densi
Alcomat; Gibertini) of the must and produced wines were determined
|
      1491
using official OIV methods (OIV, 2016). The content of yeast assimila-
ble nitrogen in the must was determined by formol titration method
(Zoecklein, Fugelsang, Gump, & Nury, 1999). The content of glycerol
was determined by a commercial enzyme test (Megazyme, CO).
Concentration of methanol, ethanol, and higher alcohols was de-
termined by gas chromatographic analysis using gas chromatograph
Agilent 7890A equipped with flame ionization detector (FID) and a
split/splitless injector, while a capillary column HP-INNOWax (poly-
ethylene glycol; 30  m  ×  250  µm i.d. with 0.25  µm film thickness)
was used. The GC-FID conditions were previously described (Miljić,
Puškaš, Vučurović, & Muzalevski, 2017).
2.5 | Wine sensory evaluation
Buxbaum model of positive ranking, described in the paper of Amerine
and Roessler (1983), was applied for the evaluation of sensory prop-
erties of experimental wines. Sensory evaluation was performed by a
panel of five qualified testers (officially certified tasters authorized for
wine sensory analysis by Serbian Ministry of Agriculture). Four sen-
sorial experiences rated up making a maximum of 20 points (up to 2
points for color, up to 2 points for clearness, up to 4 points for aroma,
and up to 12 points for overall flavor). Overall flavor implies both taste
and aroma components evaluated by retronasal olfaction. The minor
unit of the scale is 0.1. The better the parameter is rated, the higher
the mark is given. The bottom part of the evaluation sheet contained
the space for tasters' comments since they were asked to highlight the
most dominant aroma descriptors of the assessed wines.
Wines were presented to panelists in ISO standard wine glasses,
in isolated booths, and under daylight-type lighting. All wine sam-
ples were evaluated by the panel (seven trial sets, Table 1) with ran-
domized presentation order. Three sessions were employed in three
consecutive days where the panelist evaluated all seven individual
wines each day in two sets. Each replicate presented on three con-
secutive days of tasting was poured from a separate bottle.
2.6 | Statistical analysis
Statistical analysis in the present study was performed using Statistica
12.0 (StatSoft). The statistical difference between mean values of pa-
rameters was estimated by analyses of variance (ANOVA), at the 95%
confidence level. Values detected as significantly different by the use of
Duncan multiple range test were marked with different letters (a, b, c …).
3 | R E S U LT S A N D D I S CU S S I O N
3.1 | The influence of different yeast strains’
metabolic activity on ethanol production
Different selected yeast strains were used in wine fermentation tri-
als, and the effect of their metabolic activity on the content of most
 |
1492       PUŠKAŠ et al.
important fermentation products was assessed. At the same time,
the activity of autochthonous microbiota was suppressed by pas-
teurization of the must.
Sugar consumption profiles during the fermentation of must inoc-
ulated according to the defined inoculation plan are shown in Figure 1.
A different rate of sugar consumption between individual and sequen-
tial inoculation regime was observed. The use of individual yeasts
S. cerevisiae and S. bayanus resulted in the shortest time of fermen-
tation, that is, the highest rate of sugar consumption. Also, the sug-
ars were almost consumed completely in 6 days of fermentation by
these individual yeast strains. The activity of M. pulcherrima in the first
48–72 hr of fermentation, before the inoculation with S. cerevisiae and
S. bayanus, resulted in a slight decrease in sugar content. After sequen-
tial inoculation, M. pulcherrima ferments (MET+CER, MET+BAY, and
MET+CER+BAY) completed fermentation in 8–9  days. T. delbrueckii
showed similar fermentation activity as M. pulcherrima with the slower
sugar consumption in the first 3 days of fermentation. These results
may be the consequence of a higher sensitivity to SO2 of M. pulcher-
rima and T. delbrueckii. Higher sensitivity to SO2 increases the time
necessary for yeasts to adapt to the environmental conditions and to
start the alcoholic fermentation, which is associated with the reduced
consumption of sugars. In the end, it is important to emphasize that all
treatments produced wines that had 0.5–1.0 g/L of reducing sugars.
Figures 2‒4 show changes in the content of the observed com-
ponents (concentration of ethanol and glycerol and volatile acidity
value) during the alcoholic fermentation of experimental wines.
Through the comparison of the final values of observed parameters,
the efficiency of applied yeast strains in the production of wines
with reduced ethanol concentration was assessed.
The highest content of ethanol (11.92% v/v) was determined in
the experimental wine produced only using a commercial strain of
F I G U R E 1   Sugar consumption profiles during the alcoholic
fermentation of pasteurized must inoculated with different yeast
strains in laboratory conditions. Marks describing each trial set
are given in the figure legend: Saccharomyces cerevisiae—CER;
Saccharomyces bayanus—BAY; Torulaspora delbrueckii—TOR;
Metschnikowia pulcherrima—MET; a detailed inoculation plan is
given in Table 1
S. cerevisiae, while sequential inoculation of grape must with M. pul-
cherrima, S. bayanus, and S. cerevisiae resulted in the production of
the lowest amount of this compound in the test wines (11.01% v/v).
A significant reduction in the production of ethanol is also registered
in the experimental wines produced by the activity of M. pulcherrima
and S. bayanus (MET+BAY) and T. delbrueckii with the final ethanol
concentration of 11.30% v/v and 11.40% v/v, respectively (Figure 2).
The highest rate of ethanol production (fermentative power) was
observed in wines produced only with S. cerevisiae, where approx-
imately 70% of the ethanol content formed within the first 3 days
of fermentation. Fermentation of grape must inoculated only with
S. bayanus showed the lower fermentative power (about 50% of the
total ethanol) in the first 3 days compared with fermentation using a
pure culture of S. cerevisiae.
In the grape must fermented with M. pulcherrima, the ethanol con-
centration in the first 3 days of fermentation was only 1.30%–1.70%
v/v, while it is assumed that the rest of the formed alcohol is produced
mainly by the activity of S. bayanus and S. cerevisiae after sequential
inoculation. The activity of T. delbrueckii resulted in an ethanol concen-
tration of 0.8%–0.9% v/v in the early days of fermentation. The high-
est ethanol production rate with a pure culture of T. delbrueckii was
achieved between the third and sixth days of fermentation.
Previous studies (Canonico, Comitini, Oro, & Ciani, 2016;
Contreras, Curtin, & Varela, 2015; Contreras et al., 2014; Gobbi et
al., 2013; Varela, Barker, Tran, Borneman, & Curtin, 2017) have re-
ported a significant reduction in ethanol yield (0.3%–1.7% v/v) when
using non-Saccharomyces and S. cerevisiae strains in co-inoculated
or sequential cultures. The use of M. pulcherrima co-inoculated with
S. cerevisiae in the pilot-scale production of Merlot wines resulted
in 1.0% v/v lower ethanol content compared to wines fermented by
S. cerevisiae alone (Varela et al., 2017).
Contreras et al. (2014) have reported that sequential inoculation of
the selected strain of M. pulcherrima AWRI1149 and S. cerevisiae, after
3  days, resulted in the production of wines with a lower concentra-
tion of ethanol in relation to wine produced only by the application
of S. cerevisiae (in Chardonnay, the decrease was 0.9% v/v, while in
Shiraz, this reduction was 1.60% v/v). Canonico et al. (2016) investi-
gated the use of immobilized selected strains of non-Saccharomyces
yeasts (Starmerella bombicola, M. pulcherrima, Hanseniaspora osmophila,
and Hanseniaspora uvarum) to start fermentation, followed by inocula-
tion of free S. cerevisiae cells. The sequential inoculations of S. bombi-
cola- and M. pulcherrima-immobilized cells and S. cerevisiae-free cells
showed the best reductions in the final ethanol content (1.6% and
1.4% v/v, respectively). Also, lower ethanol production was recorded
in ferments obtained by joint activity of different Saccharomyces spe-
cies. Sequential inoculation of Saccharomyces uvarum (AWRI 2846) and
S. cerevisiae resulted in ethanol reduction of 0.8% v/v and an increase in
glycerol content for 6.4 g/L (Contreras, Curtin, et al., 2015). Moreover,
wines fermented with S. uvarum in the study of Varela et al. (2017)
had a 1.7% v/v lower ethanol concentration than S. cerevisiae ferment.
Previous works also showed that the application of non-Saccharomy-
ces yeast in fermentations with controlled aeration can be an efficient
approach for the production of wines with decreased alcohol content
PUŠKAŠ et al. |
      1493

F I G U R E 2   Changes in ethanol content during alcoholic fermentations carried out by different commercial yeast strains used in
laboratory conditions. Marks describing each trial set are given in the figure legend: Saccharomyces cerevisiae—CER; Saccharomyces bayanus—
BAY; Torulaspora delbrueckii—TOR; Metschnikowia pulcherrima—MET; a detailed inoculation plan is given in Table 1.a,b,c different letters for the
results in every specific day of alcoholic fermentation indicate significant differences between values (p < .05)

F I G U R E 3   Changes in glycerol content during alcoholic fermentations carried out by different commercial yeast strains used in
laboratory conditions. Marks describing each trial set are given in the figure legend: Saccharomyces cerevisiae—CER; Saccharomyces bayanus—
BAY; Torulaspora delbrueckii—TOR; Metschnikowia pulcherrima—MET; a detailed inoculation plan is given in Table 1. a,b,c different letters for
the results in every specific day of alcoholic fermentation indicate significant differences between values (p < .05)

(Canonico, Comitini, & Ciani, 2019; Canonico, Solomon, Comitini, Ciani,
& Varela, 2019; Contreras, Hidalgo, et al., 2015). The following exper-
iments generally implied controlled aeration (1–10 ml L-1 min-1) during
the first 24–72 hr of fermentation. Under these conditions, M. pulcher-
rima, T. delbrueckii, and Z. bailii strains produced wines with 0.9%–2.0%
v/v lower ethanol content compared to S. cerevisiae wines.
Glycerol is nonvolatile three-hydroxy alcohol which indirectly
contributes to the sensory character of a wine. At high concentra-
tion, it contributes significantly to the sweetness, body, and fullness
of wines. For these reasons, glycerol production is one of the desir-
able features during grape must fermentation. Furthermore, of all
the microbiological strategies to reduce ethanol yield, glycerol over-
production was proven to be the most effective (Varela et al., 2012).
Among experimental wines produced in this study, the highest glyc-
erol content (6.99 g/L) was obtained during sequential fermentation
with T. delbrueckii and S. bayanus (Figure 3). Sequential inoculation
of strain S. bayanus, M. pulcherrima, and S. cerevisiae also resulted in
relatively high value (6.7 g/L) of glycerol. The lowest production of
this compound (5.7 g/L) was established in the wine produced only
with S. cerevisiae. Similar results for glycerol levels were reported by
the study of Canonico et al. (2016). From the obtained results, it can
be noticed that S. bayanus, among the tested yeasts, was the most
efficient glycerol producer. The individual inoculation with both
S. cerevisiae and S. bayanus led to an intensive glycerol production in
the first 3 days of fermentation which resulted in formation of more
than 70% of total glycerol amount in this period. Available literature
(Ribéreau-Gayon, Dubourdieu, Donèche, & Lonvaud, 2006) points
out that the production of glycerol is associated with fermentation
of the first 50 g/L of sugar from must, and these data relate primarily
on S. cerevisiae yeast activity. From the obtained results, it can be no-
ticed that the production of glycerol in the ferments with T. delbruec-
kii during the first 3 days was minimal. T. delbrueckii was shown to be
a lower producer of secondary by-products of fermentation (Ciani et
al., 2016). Glycerol production by M. pulcherrima in the first 3 days of
 |
1494       PUŠKAŠ et al.

F I G U R E 4   Changes in volatile acidity during alcoholic fermentations carried out by different commercial yeast strains used in laboratory
conditions. Marks describing each trial set are given in the figure legend: Saccharomyces cerevisiae—CER; Saccharomyces bayanus—BAY;
Torulaspora delbrueckii—TOR; Metschnikowia pulcherrima—MET; A detailed inoculation plan is given in Table 1. a,b,c different letters for the
results in every specific day of alcoholic fermentation indicate significant differences between values (p < .05)

fermentation was also low (1.3–1.75 g/L), compared to the individual
fermentations by S. cerevisiae and S. bayanus. Positive linear correla-
tion between the production of ethanol and glycerol was recorded in
fermentations started by S. cerevisiae, S. bayanus, and M. pulcherrima
(0.991, 0.918, and 0.933, respectively). On the other hand, signifi-
cantly lower degree of linear correlation (.516) was determined be-
tween ethanol and glycerol content in T. delbrueckii ferments.
Volatile acidity (VA) is a parameter which significantly deter-
mines the quality of wine, and, in general, it represents the content
of acetic acid as a by-product of alcoholic fermentation (more than
80% of VA originates from acetic acid). If adequate vinification
practices had been employed, the resulting values of this param-
eter are below the ones that can negatively impact the organo-
leptic properties of the wine. According to Zoecklein et al. (1999),
the sensory detection threshold of volatile acidity in the wine is in
the range of 0.7–1.1 g/L. Analysis of produced experimental wines
(Figure 4) showed the highest volatile acidity in wines obtained
by the activity of T. delbrueckii (0.44 g/L), but similar values were
determined also for the other ferments (ranging 0.30–0.40  g/L).
It should be emphasized that higher glycerol production in some
ferments (TOR+BAY and MET+BAY+CER) was not followed by the
increase in volatile acidity. This is very important, especially from
the organoleptic point of view. The changes of the volatile acidity
recorded during fermentations with individual or mixed yeast cul-
tures clearly indicate that the most intensive production of these
compounds is in the first 3 days in all fermentations.
3.2 | The impact of used yeast strains on the
composition of volatile compounds in wines
The aroma is one of the main characteristics that determine the qual-
ity and value of a wine. The aroma of wine is a unique mixture of
volatile compounds originating from grape and secondary products
formed during the wine fermentation and aging. A large number of
volatile compounds are formed by yeast during alcoholic fermenta-
tion, and they significantly impact the aroma and overall quality of
wines. The most important volatile compounds synthesized by wine
yeast include higher alcohols, acetate esters, ethyl esters, and alde-
hydes among others. The influence of commercial selected yeast
strains used individual or sequential inoculation, on the composition
of the aromatic compounds in experimental wines, is shown in Table 2.
Metabolic activity of commercial selected yeast strains during
fermentation caused significant differences in the content of aro-
matic compounds in produced wines. As shown, ten higher alcohols
and two aldehydes were detected in the analyzed wine samples;
meanwhile, the compounds such as 2-propanol, 1-butanol, 2-buta-
nol, 1-pentanol, and 1-hexanol were not detected. The produced
wines were characterized by relatively uniform content of ethyl ac-
etate, 2-methyl-1-propanol, that is, isobutanol, octanol, and deca-
nol. The use of commercial non-Saccharomyces strains did not lead
to the increase in the ethyl acetate levels, and the amounts deter-
mined (about 50 mg/L) were far below the threshold associated with
a negative impact on wine sensory characteristics (above 150 mg/L).
The content of acetaldehyde was the highest in the MET+CER and
MET+BAY+CER samples (15–20  mg/L); however, even the highest
levels determined were significantly lower than the ones associ-
ated with oxidative faults (“over-ripe bruised apples,” “sherry,” and
“nut-like” characters). At lower levels (below 50 mg/L), acetaldehyde
can contribute pleasant fruity aromas to a wine. As for comparison,
Varela et al. (2017) and Canonico et al. (2016) reported that wines
produced with M. pulcherrima showed higher concentrations of ethyl
acetate, total esters, total higher alcohols, and total sulfur compounds
compared to S. cerevisiae ferments. The wine obtained by S. bayanus
activity had a significantly higher concentration of 1-propanol (sam-
ples BAY and TOR+BAY) and 1-heptanol (sample BAY) compared
to the rest of the experimental wines. Also, the wines BAY and
TOR+BAY had significantly higher amounts of benzaldehyde (76.4
and 87.3 mg/L, respectively), in comparison with other experimen-
tal wines. The highest contents of 3-methyl-1-butanol (24–28 mg/L)
PUŠKAŠ et al. |
      1495

TA B L E 2   Concentration of the major volatile compounds in wines produced in laboratory-scale experiment

Volatile compound
(mg/L) CER BAY MET+CER MET+BAY TOR TOR+BAY MET+BAY+CER
a bc c a b a
Acetaldehyde 6.5 ± 0.3 13 ± 1.2 18 ± 0.9 7.4 ± 0.7 10.7 ± 0.8 7 ± 0 15 ± 0.5c
Ethyl acetate 49 ± 1.5a 49 ± 0.7a 48.2 ± 2.3a 49.4 ± 1.9a 46.1 ± 1.1a 49.8 ± 1.5a 47.5 ± 1.1a
2-Butanol nd nd nd nd nd nd nd
2-Propanol nd nd nd nd nd nd nd
1-Propanol 0.7 ± 0.1a 4.5 ± 0.4b 1.0 ± 0.2a 1.1 ± 0a 0.4 ± 0.1a 3.7 ± 0.3b 0.9 ± 0a
a a a a a a
2-Methyl−1-propanol 11.7 ± 0.8 11.6 ± 0.7 12.2 ± 1.2 11.6 ± 1.4 10.9 ± 0.3 11.5 ± 0.7 11.4 ± 0.8a
1-Butanol nd nd nd nd nd nd nd
b b c b ab a
3-Methyl−1-butanol 26.4 ± 2.4 23.9 ± 1.3 28.1 ± 0.7 22.8 ± 1 20.6 ± 1.5 18.9 ± 0.6 24 ± 1.5b
1-Pentanol nd nd nd nd nd nd nd
1-Hexanol nd nd nd nd nd nd nd
1-Heptanol 10.7 ± 0.1a 23 ± 1.9d 19.5 ± 1.0 c 14.5 ± 0.5b 19.7 ± 1c 9 ± 0.8a 16.4 ± 0.4bc
a a a a a a
Furfural 0.6 ± 0.2 0.7 ± 0.2 0.8 ± 0.3 0.6 ± 0.2 1.1 ± 0.3 0.9 ± 0.3 0.4 ± 0.1a
Benzaldehyde 48.5 ± 2.1a 76.4 ± 1d 49.5 ± 0.7a 56.8 ± 1.4b 61.2 ± 1.5c 87.3 ± 1.9e 51.4 ± 0.9a
a a a a a a
1-Octanol 22 ± 1.2 23.2 ± 1 21.7 ± 1.5 21.5 ± 0.8 21.2 ± 0.3 23.5 ± 1.2 20.5 ± 0.8a
1-Nonanol 5.2 ± 0.4a nd nd 5.4 ± 0.7a 5.1 ± 0.4a nd nd
a a a a a a
1-Decanol 6.2 ± 0.2 5.9 ± 0.6 5.7 ± 0 6.3 ± 0.8 6.7 ± 0.2 5.9 ± 0.7 5.4 ± 0.2a
Benzyl alcohol 10.5 ± 0.6c 8 ± 0.4b 11.2 ± 0.8c 9.1 ± 2b 8 ± 0.7b 5.8 ± 0.3a 9.7 ± 0.8bc

Note: Saccharomyces cerevisiae—CER; Saccharomyces bayanus—BAY; Torulaspora delbrueckii—TOR; Metschnikowia pulcherrima—MET. Different letters
in the same row indicate significant differences between values (p < .05). nd, not detected.

TA B L E 3   Sensory evaluation of wines produced in laboratory-scale experiment

Clearness (max 2 Overall flavor (max 12 Total (max

  Color (max 2 points) points) Aroma (max 4 points) points) 20 points)

CER 2.0 ± 0.0a 2.0 ± 0.0a 3.2 ± 0.2a 11.3 ± 0.2ab 18.5 ± 0.2a

BAY 2.0 ± 0.0a 2.0 ± 0.0a 3.6 ± 0.1b 11.4 ± 0.0 b 19.0 ± 0.1bc
a a a a
MET+CER 2.0 ± 0.0 2.0 ± 0.0 3.2 ± 0.1 11.2 ± 0.1 18.4 ± 0.1a
MET+BAY 2.0 ± 0.0a 2.0 ± 0.0a 3.7 ± 0.0 b 11.5 ± 0.1bc 19.2 ± 0.1c
a a a b
TOR 2.0 ± 0.0 2.0 ± 0.0 3.4 ± 0.1 11.4 ± 0.2 18.8 ± 0.2b
TOR+BAY 2.0 ± 0.0a 2.0 ± 0.0a 3.7 ± 0.1b 11.6 ± 0.0 c 19.3 ± 0.1c
a a a a
MET+BAY+CER 2.0 ± 0.0 2.0 ± 0.0 3.4 ± 0.2 11.0 ± 0.2 18.4 ± 0.2a

Note: The data represent the average values of the scores given by five members of the panel. Different letters in the same column indicate
significant differences between values (p < .05).

and benzyl alcohol (9.7–11.2 mg/L) were detected in wines produced
by the activity of S. cerevisiae, in both individual and sequential in-
oculations (samples CER, MET+CER, and MET+BAY+CER). The pres-
ence of furfural was detected in small amounts in all wine samples.
One of the main sources of furfural in wines is toasted oak wood.
Considering the fact that wines in this study were not in contact with
wood, the possible explanation for furfural occurrence is the pas-
teurization of must before inoculation of yeast strains.
3.3 | Wine sensory analysis
The results of the sensory analysis (Table 3) indicate that a dif-
ference in the aroma, as well as taste and flavor, was recorded in
experimental wines produced using different commercially available
yeast strains in laboratory conditions. On the other hand, the color
differences could be considered insignificant. In terms of aroma,
the best-evaluated wines were produced by the activity of yeasts
MET+BAY and TOR+BAY. The lowest marks were given to MET+CER
and MET+BAY+CER wines, which can be due to the higher content
of acetaldehyde compared to other wines. Wine MET+BAY was
characterized by citrus flavors, while in MET+CER, melon and ba-
nana flavor dominated. Wines MET+BAY and TOR+BAY had fuller,
more complex, and rounded flavors compared to other experimental
wines. These properties could partly be impacted by the higher con-
tents of 1-propanal, 1-heptanol (flower tones), and benzaldehyde (al-
mond flavor) compared to other wines. The use of M. pulcherrima and
T. delbrueckii in co-inoculation with S. bayanus commercial strains
 |
1496       PUŠKAŠ et al.

gave the complexity to the sensory profiles of produced wines which

was confirmed by the highest taste and overall flavor marks, as well
as the highest overall marks (19.2–19.3). Varela et al. (2017) used a
consensus-based descriptive methodology and demonstrated that
apart from being able to produce wine with reduced alcohol concen-
tration, M. pulcherrima in co-inoculation with S. cerevisiae gave wines
with similar sensory profiles (not differing significantly in any attrib-
ute and with high scores given for desirable sensory descriptors) as
uninoculated and control S. cerevisiae ferments.

3.4 | Scale-up experiment

Evaluation of the results obtained in the laboratory experiments

was done by simulating the same vinification conditions in the
scale-up experiment. The aim was to assess the activity of ap-
plied yeast strains in a real must sample and in the presence of
autochthonous microbiota. The final concentration of ethanol and
glycerol, and value of volatile acidity in these wines are shown
in Figure 5. The aptitude of commercial yeast strains for lower-
ing the ethanol content of wine was lower compared to ferments
based on sterile must use. The competitiveness between inocu-
lated non-Saccharomyces yeasts and yeasts from autochthonous
microbiota resulted in a slower start of fermentation than in the
laboratory experiment (results not shown). Moreover, the content
of ethanol was in a pretty narrow range 11.4%–11.8% v/v, while
the content of glycerol was 3.78–4.89  g/L which is significantly
lower than that obtained in wines from laboratory-scale experi-
ment (5.7–6.99 g/L). The value of volatile acidity was very simi-
lar in both experimental sets (0.3–0.45  g/L). Similar differences
among the results of experiments with model and real must sam-
ples were reported (Ciani & Ferraro, 1998; Ferraro, Fatichenti, &
Ciani, 2000). The use of Starmerella bombicola (formerly C. stellata)
and S. cerevisiae resulted in a high production of glycerol, succinic
acid, and different by-products (interactions involving acetalde- F I G U R E 5   Final concentrations of glycerol, volatile acids, and
ethanol in wines produced by different commercial yeast strains
hyde and acetoin) with a consequent reduction of final ethanol
used in scale-up conditions. Marks describing each trial in the figure
amount. The reduction in ethanol content in these experiments
legend: Saccharomyces cerevisiae—CER; Saccharomyces bayanus—
varied from 0.64% v/v at pilot scale in natural grape juice to 1.60% BAY; Torulaspora delbrueckii—TOR; Metschnikowia pulcherrima—MET;
v/v at laboratory scale using synthetic grape juice. Furthermore, a detailed inoculation plan is given in Table 1. a,b,c different letters
different factors affecting the metabolism of M. pulcherrima indicate significant differences between values (p < .05)
AWRI1149 during fermentation of nonsterile Shiraz must were
evaluated (Contreras, Curtin, et al., 2015). Among different in-
oculation regimes which were applied, only initial inoculation with 4 | CO N C LU S I O N
1 × 10 6 cells/mL of M. pulcherrima AWRI1149, followed by S. cer-
evisiae after 50% sugar consumption, leads to a significant ethanol In summary, this work evaluated the potential of commercial
concentration reduction. Canonico et al. (2016) showed that appli- selected yeast strain application for purpose of wines with de-
cation of immobilized selected strains of M. pulcherrima, followed creased alcohol content production. Sequential inoculation of the
by inoculation of free S. cerevisiae cells, resulted in a decrease in must with M. pulcherrima, S. bayanus, and S. cerevisiae resulted in
ethanol content for 1.3% v/v when synthetic grape juice was used, the production of wines with the lowest ethanol content among
while in the case of natural grape juice the reduction was 1% v/v experimental samples (decrease of 0.9% v/v compared to the con-
(in both trials, the beads of immobilized yeast were removed after trol wine). Significant differences in the content of certain aro-
72 hr from inoculation). matic compounds, as well as in taste and flavor, were also found
PUŠKAŠ et al.
in produced wines. The experiment in real conditions showed that
used commercially available Saccharomyces and non-Saccharomy-
ces were not effective enough in lowering ethanol concentration
in wines due to interactions and competitiveness with yeasts from
autochthonous microbiota. Already evident problems of wines
with very high alcohol content will force wine producers in near
future to find more efficient ways of directing alcoholic fermenta-
tion to the production of wines with lower alcohol content and not
worsened sensory properties. The application of Saccharomyces
and non-Saccharomyces strains obtained by adaptive evolution
principles (non-GM) for this purpose will need further investiga-
tion and commercialization.
AC K N OW L E D G M E N T
Financial support from the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Project TR-
31002) is greatly appreciated.
C O N FL I C T O F I N T E R E S T
The authors declare that they have no conflicts of interest.
E T H I C A L A P P R OVA L
This study does not involve any human or animal testing.
ORCID
Uroš D. Miljić  https://orcid.org/0000-0002-1947-8122
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 Available online at www.sciencedirect.com
ScienceDirect
Microbiological strategies to produce beer and wine
with reduced ethanol concentration
Javier Varela1 and Cristian Varela2
Changes in consumer preferences, government policies and
environmental conditions have driven research efforts towards
producing alcoholic beverages with reduced alcohol content,
namely wine and beer. While the strategies available to
accomplish this goal vary for wine and beer, a common
approach relies on the use of yeast strains which are less
efficient at producing ethanol. Here we discuss current
research on the isolation and/or generation of yeast strains able
to produce beer or wine with reduced ethanol concentration.
Particular consideration is given to the impact of ‘low-ethanol’
yeasts on volatile composition and sensory profile of beer and
wine.
Addresses
1
School of Microbiology/Centre for Synthetic Biology and
Biotechnology/Environmental Research Institute/APC Microbiome
Institute, University College Cork, Cork T12 YN60, Ireland
2
The Australian Wine Research Institute, P.O. Box 197, Glen Osmond,
Adelaide, SA 5064, Australia
Corresponding author: Varela, Cristian (cristian.varela@awri.com.au)
Current Opinion in Biotechnology 2019, 56:88–96
This review comes from a themed issue on Food biotechnology
Edited by Rute Neves and Herwig Bachmann
https://doi.org/10.1016/j.copbio.2018.10.003
0958-1669/ã 2018 Elsevier Ltd. All rights reserved.
Introduction
Health concerns related to alcohol consumption, no-alco-
hol policies advocated for some consumers (e.g. drivers,
pregnant women) and growing market demand, have
focused research attention on reducing alcohol concentra-
tion in beer and wine [1,2]. Additional reasons for this
focus, particularly in wine, include the effect of high
alcohol levels on flavor perception, sometimes impacting
negatively on wine sensory properties [3,4], and greater
taxes for wines with high alcohol concentration.
Reduced-alcohol beers are often classified according to
their alcohol content, with low-alcohol beers and alco-
hol-free beers containing <1.2% v/v and <0.5% v/v of
alcohol, respectively. These beers can be produced
through several strategies which, are broadly categorized
Current Opinion in Biotechnology 2019, 56:88–96
as either biological or physical processes [reviewed by
Branyik et al. 5]. Biological processes are based on
limiting alcohol formation, while physical methods
involve ethanol removal from regular beer. Biological
processes cover arrested or limited fermentation, the use
of non-conventional yeast species and continuous fer-
mentation, whereas physical methods include: vacuum
rectification and evaporation, spinning cone column
distillation, osmotic distillation, dialysis and reverse
osmosis [1,5].
Strategies aimed at reducing wine alcohol concentration
include: viticultural practices, such as decreasing the leaf
area to fruit weight ratio in order to lower sugar concen-
tration in the berry; pre-fermentation and winemaking
practices, including dilution and sugar removal from the
grape must; microbiological strategies such as the use of
yeasts that are less efficient at ethanol production; and
post-fermentation practices and processing technologies,
including the physical removal of alcohol after fermenta-
tion [reviewed by Varela et al. 6].
Yeast diversity during brewing and
winemaking
The first beers and wines were the products of spontane-
ous fermentations; microorganisms present on the surface
of grains and fruits, or unintentionally introduced by
human action, were likely responsible for their produc-
tion [7]. Spontaneous fermentation involves the sequen-
tial action of several yeast species, which not only gener-
ate ethanol but also produce numerous secondary
metabolites that shape the aroma and flavor of beer
and wine [7,8].
Two Saccharomyces species are associated with the pro-
duction of beer. Saccharomyces carlbergensis is employed in
ale beer production, while Saccharomyces pastorianus (syn.
S. carlbergensis), which is an interspecies hybrid between
S. cerevisiae and Saccharomyces eubayanus [9], is used for the
production of lager beers. Inoculation is performed with
pure starter cultures which ensures a dominant yeast
population from the beginning of fermentation. Although
most beers are produced by inoculation, certain beer
styles (e.g. Belgium lambic beer and American Coolship
Ales) rely on spontaneous fermentation. Several yeast
species have been found during these latter fermenta-
tions including Meyerozyma guilliermondii, Debaryomyces
spp., Pichia spp., Wickerhamomyces anomalus, Brettanomyces
anomalus, Brettanomyces custersii, Brettanomyces bruxellensis,
Candida krusei, Cryptococcus keutzingii, Saccharomyces
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 Microbiological strategies for reducing alcohol concentration Varela and Varela 89
pastorianus, Naumovia castellii, Kazachstania servazzii and
Rhodotorula mucilaginosa [10,11].
Until the 1980s wine was largely produced by sponta-
neous fermentation. This process is characterized by a
rich microbial diversity, with more than 40 of the
1500 known yeast species isolated from fermenting
grape must [8]. Fermentation usually commences by
the action of different non-Saccharomyces yeasts until
indigenous S. cerevisiae yeasts dominate. The micro-
bial communities responsible for this originate in the
vineyard and the winery [12]. While several species
live on the surfaces of vines, grapes and vineyard soil
[13,14], others reside in the winery environment with
different yeast species isolated from equipment, soil
and air [15–19].
Until recently, non-Saccharomyces yeasts were regarded as
responsible for microbial-related problems during wine
production, which resulted in the almost global use of
pure S. cerevisiae yeast starter cultures. However, over the
past 5–10 years thanks to numerous reports describing the
potential of these so-called non-conventional yeasts to
introduce desirable aromas and flavors to wine during
fermentation, their role in the production of fermented
beverages has been revised [8,20–22]. This has generated
a growing interest in isolating and characterizing non-
conventional yeasts for development of starter cultures
that increase flavor diversity in beer and wine. Given the
enormous diversity of non-conventional yeasts, these
species are also being studied to determine their ability
to produce beer and wine with reduced ethanol
concentration.
Generating S. cerevisiae yeasts with reduced
ethanol production
S. cerevisiae is the main yeast used for wine fermentation.
Depending on the environmental conditions S. cerevisiae
can either respire or ferment sugar. If oxygen is available
and sugar concentration is low, respiration takes place,
whereas under anaerobic conditions fermentation is
favored (Crabtree effect). Aeration is a common practice
in commercial wine production, performed through
‘pump-overs’ or macro-oxygenation techniques.
Although air addition can lower ethanol production by
S. cerevisiae, the amount of air required to achieve this also
increases the concentration of acetic acid, which can
negatively affect wine aroma [23,24]. There are numerous
strains of this species commercially available, however
they all generate similar ethanol yields, which translates
to comparable wine ethanol concentrations [25,26].
Although there are no published studies describing the
ethanol productivity of S. cerevisiae brewing strains, it is
safe to assume that ethanol yields are comparable. Thus,
initial efforts to produce ‘low-ethanol’ yeasts focused
primarily on generating new S. cerevisiae strains. It is
key to highlight that microbial strategies described here
www.sciencedirect.com
target complete wine fermentation, therefore minimising
residual sugar and any potential impact on wine
sweetness.
In wine, gene modification technologies (GM
approaches) have been used to partially divert carbon
metabolism away from ethanol formation, by redirect-
ing carbon to other end points, while attempting to
maintain yeast fermentation performance and wine
quality [reviewed by Varela et al. [6] and Kutyna
et al. [27]]. Therefore, researchers not only have to
find suitable end-point metabolites, they also have to
determine whether or not the introduced gene mod-
ifications affect the formation of other metabolites
which will in turn affect wine quality. The most rele-
vant GM strategies to decrease ethanol production in S.
cerevisiae are summarized in Figure 1, while gene targets
and their location in the yeast metabolic network are
detailed in [6]. Successful strategies proven to
decrease ethanol production include: increasing glyc-
erol production, diverting carbon to the tricarboxylic
acids (TCA) cycle, relieving glucose repression,
increasing trehalose production, increasing lactic acid
production and expressing genes from other species.
Although not yet tested, increasing the production of
complex carbohydrates, such as glycogen and cellulose,
could also provide an alternative to decrease ethanol
production in yeast.
GM approaches have also been used to generate S.
cerevisiae strains able to produce beer with reduced alco-
hol concentration (Figure 1). Strains lacking the genes
FUM1, and KGD1/KGD2, encoding fumarase and alpha-
ketoglutarate dehydrogenase, respectively, produced
beer with an alcohol concentration below 0.5% v/v [28
]. In these strains, carbon was directed towards the
formation of lactic acid, a compound that effectively
masks wort-like off-flavors. However, ‘side effects’ were
observed, including increased concentration of the off-
flavor diacetyl and depleted production of ethyl acetate
and isoamyl acetate, two key beer flavor molecules.
Deletion of genes encoding alcohol dehydrogenase
(ADH), the last enzymatic step in ethanol formation,
has also been applied to successfully reduce beer alcohol
concentration (US Patent No. 4814188). Strains lacking
ADH genes divert carbon towards the formation of glyc-
erol, and unfortunately also to the production of acetal-
dehyde, an off-flavor associated with ‘bruised apple’
sensory descriptors.
GM approaches have proven very effective at reducing
wine ethanol concentration with some engineered strains
producing 1.5–2.5 % v/v less ethanol than their parent
strains. Although most GM strategies have reported an
effect on the formation of volatile compounds in addition
to reduced alcohol concentration, none have described
the sensory profile of the resulting wines. Thus, it is not
Current Opinion in Biotechnology 2019, 56:88–96
90 Food biotechnology

Figure 1

Increasing glycerol production

GPD overexpression
Sugar Ethanol PDC deletion
ADH deletion
TPI1 deletion
FPS1 modification
Diverting carbon to TCA cycle

Sugar Ethanol MDH2 overexpression

FRD1 overexpression
Relieving glucose repression
HXT1/7 chimera
Other carbon HXK2 deletion
metabolites MIG1 deletion
Increasing trehalose production
TPS1 overexpression
Increasing lactic acid production
FUM1 deletion
KGD1/KGD2 deletion
LDH overexpression
Expression of genes from other species
GOX expression
NoxE expression
Increasing complex carbohydrates
Glycogen
Cellulose
Formate production through
formate lyase expression
Current Opinion in Biotechnology

GM strategies to decrease ethanol production in S. cerevisiae. The different genetic modifications aiming to divert carbon away from ethanol
production are shown. Several of these strategies have been tested and proven successful in reducing ethanol in wine and beer (ticked boxes)
while others have not been evaluated yet (empty boxes). Gene targets and their location in the yeast metabolic network are detailed in Ref. [6].

possible to completely assess the success of these engi-
neered strains.
Considering the negative public perception toward geneti-
cally modified organisms (GMOs) in food and beverage
production it is not surprising that only one article [29] has
been published in this area in recent years. Perhaps the
possibility of using CRISPR-Cas9 strategies to generate
yeast strains, which could be potentially regarded as non-
GM [30,31] could revitalize research on this topic. Yeast
strains which, in addition to lower ethanol concentration,
generate products with added value, may be attractive for
the fermented beverage industries. For example, expres-
sing the enzyme pyruvate-formate lyase would divert yeast
carbon metabolism towards the formation of formate [32],
which then could be cleaved by formate hydrogenlyase
resulting in the production of carbon dioxide and hydrogen
[33]. Potentially, hydrogen can be then stored and sold or
used to provide energy in-house.
Recently, GM approaches have been used to generate a
yeast strain able to produce the molecules that impart hop
flavor in beer [34]. Hops are an expensive ingredient in
beer and the increasing preference for beers with high
hoppy flavors (IPA and Pale Ale varieties) has put
pressure on producing enough hops to cope with the
market demand. This GM strain represents an interesting
alternative for brewing in a more controlled and sustain-
able manner.
Adaptive evolution offers another potential strategy to
generate yeast strains that produce reduced amounts of
ethanol. This approach relies on applying a selection
pressure that favors a metabolic diversion away from
ethanol production. Several reagents can be used to shift
yeast carbon metabolism and potentially generate ‘low
alcohol’ strains [reviewed by Kutyna et al. 27]. Although
different agents/conditions have been tested [35,36], only
one adaptively evolved strain has been shown to decrease
ethanol levels significantly and become a commercial
product [37]. After 200 generations under hyperosmotic
stress caused by potassium chloride, an evolved strain
showing increased glycerol production and a reduced
ethanol yield was isolated. This strain was then subjected
to two rounds of spore mating generating a hybrid strain
able to produce Shiraz wines with 1.3% v/v less ethanol,
and increased acidity and glycerol concentration than the
parental wine strain [37]. These findings suggest that the
generation of ‘low-ethanol’ strains by adaptive evolution
is not an easy task, and that promising strain isolates may

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 Microbiological strategies for reducing alcohol concentration Varela and Varela 91
need further rounds of improvement to create a commer-
cial yeast product.
Use of non-conventional Saccharomyces
yeasts to reduce ethanol concentration
Members of the Saccharomyces clade, which includes
Saccharomyces kudriavzevii, Saccharomyces cariocanus, Sac-
charomyces mikatae, Saccharomyces eubayanus, Saccharomyces
uvarum and Saccharomyces paradoxus, can provide an addi-
tional source of novel wine yeast strains with reduced
ethanol production.
S. uvarum has been shown to produced Malvasia delle
Lipari wines with lower alcohol concentration (0.7% v/v
less) and volatile acidity (acetic acid) than wines fermented
with S. cerevisiae [38]. S. uvarum wines showed increased
positive sensory attributes and decreased negative attri-
butes compared to S. cerevisiae wines [38]. A study by Varela
et al. [39] also showed the ability of S. uvarum to produce
wines with reduced ethanol concentration; Merlot wines
fermented with this yeast had 1.7% v/v less ethanol than S.
cerevisiae wines and increased glycerol concentration [39].
However, S. uvarum wines were characterized by a sensory
profile mostly dominated by unusual and negative sensory
attributes that would conventionally be considered off-
flavors. Nonetheless, these reports demonstrate the poten-
tial of S. uvarum to produce wines with decreased alcohol
concentration, but they highlight the importance of sensory
analysis of such wines.
Although not found during wine fermentation S. kudriav-
zevii presents several traits relevant for winemaking [40],
including the potential to reduce ethanol concentration in
wine [41]. S. kudriavzevii is not able to compete well with S.
cerevisiae during fermentation and it usually requires par-
ticular environmental conditions, such as low temperature
and aeration, to grow competitively and improve its per-
formance [40,42]. Mixed fermentations inoculated with S.
kudriavzevii and S. cerevisiae and supplied with limited
aeration, produced wines with 1% v/v lower ethanol con-
centration than wines fermented with S. cerevisiae alone and
increased glycerol concentration [42]. Although S. kudriav-
zevii has shown potential to reduce wine ethanol concen-
tration at lab scale, it has not been yet tested in grape juice
or at pilot-scale.
A strategy to eliminate unwanted traits in Saccharomyces
yeasts, such as production of off-flavor compounds or
poor performance, is the generation of interspecific
yeast hybrids. These can be generated from crosses
between S. cerevisiae and other members of the Saccha-
romyces clade [21]. This approach has been used to
generate robust hybrids with novel sensory attributes
[43,44], to increase cryotolerance and reduce volatile
acidity [45], and to produce hybrids with enhanced
flavor diversity and reduced hydrogen sulfide produc-
tion [46]. Although the generation of ‘low-ethanol’
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Saccharomyces hybrids has not been shown, a natural
S. cerevisiae x S. kudriavzevii hybrid has been described
which, relative to S. cerevisiae, produced more glycerol
and potentially less ethanol during wine fermentation
at low temperature (14  C) [41].
Similarly, hybridization has been used to improve the
fermentation performance, stress tolerance and flavor
profile of brewing strains [reviewed by Krogerus et al.
47]. However, a set of S. cerevisiae x S. eubayanus hybrids
generated to increase aroma in lager beers showed vari-
able ethanol production, with some strains producing up
to 6% v/v ethanol and others only 3% v/v [48]. This
suggest that generating hybrids for low-ethanol beer
production is a viable option.
Non-Saccharomyces species exhibiting
decreased ethanol yield
Despite an initial bad reputation, non-Saccharomyces
yeasts are now considered to contribute positively during
the production of alcoholic beverages [7,8,49]. These
species can be used for controlling spoilage microorgan-
isms, to conduct malolactic fermentation, which is the
transformation of L-malic acid to L-lactic acid intended
mainly to deacidify wines, to improve wine clarification
and filtration, to shape beer and wine sensory profile, and
to reduce the ethanol concentration in fermented bev-
erages [7,50].
Table 1 lists recent studies reporting the use of non-
Saccharomyces yeast species to produce wine with reduced
ethanol concentration and indicates their impact on wine
composition and/or wine sensory properties. While non-
Saccharomyces yeasts are generally able to ferment wort to
produce beer, they are not capable of completing wine
fermentation on their own. Instead they have to be
accompanied by a S. cerevisiae wine strain, which is added
sequentially (inoculating first with the non-Saccharomyces
yeast followed by S. cerevisiae) or simultaneously [7,50].
Thus, different inoculation regimes have been trialled to
reduced ethanol concentration and shape flavor profile
[51]. Additionally, several reports have explored the oxi-
dative metabolism observed in some non-Saccharomyces
species, which can respire sugar at high sugar concentra-
tions and therefore, ‘burn off’ carbon that would other-
wise go into ethanol formation [23,52–54]. While aeration
can enhance the growth and the persistence of non-
Saccharomyces yeasts during wine fermentation [55], it
can also negatively impact wine sensory profile increasing
the concentration of some off-flavors [53,54]. The pro-
duction of some of these off-flavors is related to amino
acid metabolism and it has been suggested that supple-
mentation with carefully formulated nitrogen sources
could be used to reduce their formation [54]. In fact,
nitrogen sources can affect the fermentation performance
of mixed yeast cultures, stimulate nitrogen consumption
Current Opinion in Biotechnology 2019, 56:88–96
92 Food biotechnology

Table 1

Summary of recent studies evaluating the use of non-Saccharomyces yeasts to lower wine alcohol concentration and their impact on
wine composition and/or wine sensory properties

Non-Saccharomyces Scale and Grape variety Ethanol Impact on chemical composition References
yeast a conditions of reduction b or sensory attributes c
fermentation
Candida sake Lab-scale Tempranillo 2.4% v/v Increased sorbitol, low production of ethyl [62]
esters. No sensory analysis reported
Candida stellata Medium-scale Chardonnay 0.7% v/v Increased glycerol concentration, decreased [63]
‘floral’ and ‘fruity’, increased ‘ethyl acetate’
sensory descriptors
Candida stellata Pilot-scale Trebbiano 0.6% v/v Increased glycerol and succinic acid content, [64]
immobilized cells decreased esters and higher alcohols. No
sensory reported
Candida zemplinina Pilot-scale Riesling 0.8% v/v Decreased ‘purity’ and increased ‘oxidation’ [53]
aeration and ‘solvent’ sensory attributes
Hanseniaspora opuntiae Medium-scale Pinotage 0.6% v/v Increased ‘hazelnut’, ‘coffee’, ‘caramel’, [65]
‘cherry’ and ‘acetone’ sensory descriptors
Hanseniaspora uvarum Medium-scale Pinotage 0.8% v/v Increased ‘hazelnut’, ‘coffee’, ‘caramel’, [65]
‘cherry’ and ‘acetone’ sensory descriptors
Lachancea Lab-scale Tempranillo 1.2% v/v Reduced ‘aromatic intensity’ and ‘aromatic [51]
thermotolerans quality’, increased ‘herbaceous’ character
Lachancea Industrial-scale Sangiovese 0.7% v/v Increased ‘spicy’ and ‘acidity’ sensory [66]
thermotolerans attributes
Metschnikowia Lab-scale Chardonnay 0.9% v/v Increased concentration of esters and higher [67]
pulcherrima alcohols, decreased volatile acids content. No
sensory reported
Metschnikowia Lab-scale Shiraz 1.6% v/v Increased concentration of higher alcohols, [67]
pulcherrima decreased volatile acids content. No sensory
reported
Metschnikowia Lab-scale Verdicchio 1.4% v/v Increased geraniol and acetaldehyde content. [68]
pulcherrima immobilized cells No sensory reported
Metschnikowia Lab-scale Malvasia/Viura 2.2% v/v No volatile composition or sensory analysis [52]
pulcherrima aeration blend reported
Metschnikowia Pilot-scale Merlot 1.0% v/v Decreased ‘brown tint’ and increased ‘red fruit [39]
pulcherrima aroma’ sensory attributes
Metschnikowia Pilot-scale Riesling 3.8% v/v Decreased ‘purity’ and ‘fruity’, and increased [53]
pulcherrima aeration ‘vinegar’, ‘oxidation’ and ‘solvent’ sensory
attributes
Metschnikowia Pilot-scale Viura-Malvası́a 0.8% v/v Increased glycerol content, decreased ‘tropical [54]
pulcherrima Aeration fruit’ and ‘white fruits’, and increased
‘oxidation’ sensory attributes
Pichia guilliermodii Pilot-scale Riesling 2.0% v/v Decreased ‘purity’ and ‘fruity’, and increased [53]
aeration ‘vinegar’ and ‘reductive’ sensory attributes
Pichia kluyveri Pilot-scale Riesling 3.0% v/v Decreased ‘purity’ and ‘fruity’, and increased [53]
aeration ‘vinegar’, ‘oxidation’ and ‘solvent’ sensory
attributes
Schizosaccharomyces Medium-scale Airen 0.7% v/v Decreased malic acid concentration, increased [69]
pombe ‘bitterness’ and decreased ‘acidity’ sensory
attributes
Starmerella bacillaris Lab-scale Barbera 0.7% v/v Increased glycerol content. No volatile [70]
composition or sensory analysis reported
Starmerella bacillaris Pilot- scale Barbera 0.5% v/v Increased glycerol content. No volatile [70]
composition or sensory analysis reported
Starmerella bacillaris Pilot- scale Barbera 0.3% v/v Reduced concentration of higher alcohols. No [71]
sensory analysis reported
Starmerella bombicola Lab-scale Verdicchio 1.6% v/v Increased ethyl acetate and isoamyl acetate [68]
immobilized cells concentration. No sensory reported
Torulaspora delbrueckii Pilot-scale Airen 0.3% v/v Increased ‘aromatic intensity’ and ‘fruity’ [72]
sensory attributes
Torulaspora delbrueckii Pilot-scale Viura-Malvası́a 0.5% v/v Increased glycerol content, decreased ‘tropical [54]
Aeration fruit’ and ‘white fruits’, and increased
‘reduction’ and ‘dried fruits’ sensory attributes
a
Depending on the study non-Saccharomyces yeasts were used in single, sequential or simultaneous inoculation.
b
Ethanol concentration compared to control wines fermented with S. cerevisiae.
c
Chemical composition or sensory attributes compared to control wines fermented with S. cerevisiae.

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 Microbiological strategies for reducing alcohol concentration Varela and Varela 93

Table 2

Summary of recent studies evaluating the use of non-Saccharomyces yeasts to decrease ethanol concentration in beer and the impact on
beer sensory properties

Non-Saccharomyces yeast Maltose Ethanol concentration Impact on chemical References

Candida tropicalis
Candida shehatae
Pichia kluyveri
Saccharomycodes ludwigii
Torulaspora delbruenckii
Torulaspora delbruenckii
Williopsis saturnus
Zygosaccharomyces rouxii
a
Variable maltose utilization is indicated as ‘v’ and refers to the use of maltose positive and negative strains in the same study.
utilizationa composition or sensory attributes
– 0.2 % v/v Low content of aroma compounds [73]
– <0.5 % v/v ‘Sweet’, no ‘wort-like flavors’ were Patent CN102220198B
identified
– 0.1 % v/v ‘Fruity’. High concentrations of esters Patent US9580675B2
– 0.5–1.4 % v/v High ester concentration, low diacetyl [1]
– 2.66 % v/v High levels of phenethyl acetate, ethyl [20]
hexanoate and
ethyl octaonate. ‘Fruity’, ‘Hoppy’
v 0.9–4.0 % v/v Slight ‘wort-like’ flavor. [74]
‘Honey’ and ‘pear-like’ flavors
– 1.7% v/v High levels of terpenes and terpernoids [75]
– 0.9–3.3 % v/v High diacetyl levels [1]

Figure 2

Engineered and adaptively Non-conventional Novel Sacchromyces

evolved novel S. cerevisiae yeast species hybrids

Screening for low-ethenol

producing strains
Ethanol concentration (% v/v)

16

12

0
Sc

Y1

Y2

Y3

Y4

Y5

Y6

Y7
LA

LA

LA

LA

LA

LA

LA

Assessing sensory
properties

Fruity Fruity

Worty Sweet Vegetal Floral

Beer sensory Wine sensory

analysis analysis
Current Opinion in Biotechnology

The process of selecting low-alcohol yeasts. Novel Saccharomyces cerevisiae strains generated by adaptive evolution or gene modification; non-
conventional Saccharomyces and non-Saccharomyces yeast species; and new Saccharomyces hybrids are screened for ethanol production in
wort or grape must. Strains showing reduced ethanol formation are then evaluated for beer or wine sensory profile.

www.sciencedirect.com Current Opinion in Biotechnology 2019, 56:88–96

94 Food biotechnology
in S. cerevisiae and alter the competitiveness of non-
Saccharomyces yeasts [56,57].
Several non-Saccharomyces species have been tested for the
production of low-alcohol and alcohol-free beers (Table 2)
[58]. The rationale was to use yeasts species that are
unable to utilize maltose and/or maltotriose, the main
sugars present in wort. Thus, ethanol production is limited
by the amount of other sugars, including glucose, fructose
and sucrose, which accounts for less than 20% of the
carbohydrates in wort. Beers produced this way contain
high amounts of maltose, but this does not translate into a
higher calorie content compared to standard beers. In fact,
since the calorific value of ethanol is higher than carbo-
hydrates, low-alcohol beers tend to have a lower calorie
content [59]. Regardless, high amounts of maltose often
result in higher susceptibility to microbial contamination
and in unpleasant ‘wort-like’ flavors. The latter can be
overcome by using yeasts that produce high amounts of
esters that mask ‘wort-like’ flavors. An example of this is
the use of Saccharomycodes ludwigii to produce alcohol-free
beer [1], which is now commercially available. Beer
produced with this yeast contains very low alcohol con-
centration (<0.1% v/v) and shows high amounts of esters
that contribute to a ‘fruity’ character. Similarly, beer
produced with Pichia kluyveri contain reduced alcohol
concentration and increased concentration of esters (US
Patent No. 9580675B2). Although all of the examples
listed in Table 2 have used single inoculation with
non-Saccharomyces species, sequential fermentations with
S. cerevisiae have also been investigated. Beers produced
using this strategy contained a more complex flavor profile
compared to single fermentations with S. cerevisiae and, in
some cases, lower alcohol content [60].
Concluding remarks
Several yeast-based strategies have been shown to be effec-
tive in producing beer and/or wine with reduced ethanol
concentration. Regardless of the approach, ‘low-ethanol’
yeast strains should always be evaluated for sensory impacts
they have, ideally, at pilot-scale to determine their real
contribution to the flavor characteristics of the final product
(Figure 2). Reduction in alcohol content has been widely
studied in wine but production of low-alcohol and alcohol-
free beers has received less attention. This difference can be
explained by the different priorities of the wine and beer
industries. While the wine industry sees production of
reduced-alcohol wine as a necessity, the beer industry seems
to focus mainly in producing beers with new sensory attri-
butes (e.g. new flavors). Nevertheless, the expansion of the
beer industry into new markets (e.g. beer production in
countries where alcohol consumption is banned) could moti-
vate researchers to look for new strategies to reduce alcohol
content in beer.
Recent advances in next-generation sequencing techniques
have enabled to investigate at great depth microbial
Current Opinion in Biotechnology 2019, 56:88–96
communities during fermentation, enhancing our under-
standing of microbial ecology and population dynamics
[10,61]. Applying these metagenomics approaches not only
to samples taken during alcoholic fermentation, but also to
environmental samples promises to increase our knowledge
on microbial diversity. In turn, systematic characterization of
this diversity could lead to the isolation or development of
novel ‘low-ethanol’ yeasts for the production of beer and
wine with reduced ethanol concentration.
Conflict of interest statement
Nothing declared.
Acknowledgements
The authors would like to thank Dr Paul Cambers for reviewing the
manuscript. The AWRI, a member of the Wine Innovation Cluster in
Adelaide, is supported by Australia’s grape growers and winemakers through
their investment body Wine Australia with matching funds from the
Australian Government. J.V works on the CHASSY project, which has
received funding from the European Union’s Horizon 2020 Framework
Programme for Research and Innovation – Grant Agreement No. 720824.
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