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Cell and Tissue Reactor Engineering
© 2003 University of Minnesota

Metabolic Engineering Review Article


By Anurag Khetan and Wei-Shou Hu

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METABOLIC ENGINEERING OF ANTIBIOTIC BIOSYNTHESIS FOR
PROCESS IMPROVEMENT

Anurag Khetan and Wei-Shou Hu


Department of Chemical Engineering and Materials Science
University of Minnesota
Contents

1. Introduction
2. A brief overview of conventional strain improvement
3. Facilitators for metabolic engineering of secondary metabolite producesrs
3.1 Knowledge of biochemical and genetic nature of biosynthesis
3.2 Knowledge of regulation
3.3 Knowledge of flux along the pathway
3.4 Mathematical modeling and simulation
4. Examples of metabolic engineering of antiobiotic biosynthesis
4.1 Increasing the metabolic flux
4.1.1 Enhancing enzymatic activity
4.1.2 Manipulating regulatory genes
4.1.3 Enhanced antibiotic resistance
4.2 Increasing product selectivity
4.3 Biosynthesis of a product previously made semi synthetically
4.4 Synthesizing antibiotics in heterologous strains
4.5 Solving scaleup challenges
5. General principles derived from examples of metabolic engineering
6. Opportunities
6.1 Impact of new tools
6.1 Integration of metabolic engineering and process engineering
7. Conclusions
8. References
1. INTRODUCTION
Since the discovery of penicillin, thousands of molecules with antibiotic
properties have been identified. Although a wide variety of microorganisms
synthesize antibiotics, the majority of clinically useful antibiotics are produced by
the eubacteria Actinomycetes, in particular Streptomyces, and the filamentous
fungi. Among the most important antibiotics are β-lactams, polyketides and the
aminoglycosides (Figure 1). Most antibiotics are secondary metabolites which are
synthesized from primary metabolite precursors. The disruption of antibiotic
biosynthetic pathways does not affect the viability of the producer organism.
H
N S CH 3
CH 3
O
N
O COOH
O

(1) CH 3
CH 3

H3C
HO
O N(CH3) 2
H2 HO CH 3
H3C C H3C O O
O
O
OCH 3 CH 3
HO O OH
H3C CH 3
H3CO O
O OH

(2)
CH 3 CH 3
H H 3C N
OH H H
OH

CONH 2 HO OH
OH
OH HO O OH
OH O O O
(3) HO HO O NH 2
OH O
NH 2
H 2N

H 2N

(4)

Figure 1: Representative antibiotics (1) Penicillin G - a β-lactam antibiotic (2) Tylosin - a macrolide
polyketide (3) Tetracycline – an aromatic polyketide (4) Kanamycin A – an aminoglycoside

With recombinant DNA technology it is possible to genetically alter


antibiotic producing organisms in a directed manner. Metabolic engineering of
secondary metabolism could possibly lead to the synthesis of new or known

1
antibiotic(s) in an organism predisposed to producing another antibiotic or in one
not producing any at all. By metabolic engineering, an organism can also be
altered to synthesize an intermediate which previously had to be supplied
exogenously. One may apply metabolic engineering to eliminate the biosynthesis
of undesirable byproducts. It is also possible to change flux distributions of
compounds involved in antibiotic biosynthesis to increase the yields or
productivity.
The application of recombinant DNA technology to microorganisms
producing antibiotics has been lagging behind those involving other industrial
biochemicals. This has been largely due to the complicated biosynthetic pathways
leading to the final product and the relatively slow development of genetic tools.
Except in a few cases, most of the biochemical pathways involved in antibiotic
biosynthesis are not completely characterized, the enzymes involved are not
isolated, and the loci of genes encoding these enzymes are not known.
Furthermore, most secondary metabolites are produced by microorganisms which
undergo differentiation in their growth cycle. The regulation of gene expression
for the biosynthesis of secondary metabolites and its relation to differentiation is
little known. The interface between the primary and secondary metabolism is also
poorly understood. Despite these difficulties in studying secondary metabolism,
research over the past decade has made substantial advancement. Examples of
genetic manipulations for the synthesis of new antibiotics or for process
improvements are appearing with increasing frequency in the published literature.
New molecules with antibiotic properties have been constantly sought since
the discovery of penicillin. This has typically been accomplished by screening the
natural diversity of microorganisms and their products. More recently, it has
become possible to genetically alter microorganisms to produce new variants of
the original molecule, which would have enhanced potency, a more favorable
antimicrobial spectrum of activity or reduced toxicity. Mutasynthesis1 is a strategy
by which analogs of metabolites are produced by eliminating endogenous
synthesis of one of the precursors of the metabolite by mutation, and replacing the
missing building block with exogenously added analogs. By disrupting the genes
in the precursor pathways, mutants have been produced, which can synthesize
analogs of the antibiotic. Methods to make new antibiotics with different carbon
chain lengths have been specially researched for polyketide and non ribosomal
peptide antibiotics. Reviews dealing with this aspect have appeared recently.2, 3
This review concentrates on knowledge-based metabolic engineering for
improving antibiotic biosynthesis processes. Because of the breadth of the subject
matter, only representative examples are covered to illustrate some principal
applications of metabolic engineering in antibiotic biosynthesis. In particular

2
many examples are drawn from studies of β-lactam antibiotics, including
penicillins and cephalosporins, which have been amongst the most studied
antibiotics. Readers are referred to other recent reviews on secondary
metabolism.4-13

2. A BRIEF OVERVIEW OF CONVENTIONAL STRAIN


IMPROVEMENT
For most antibiotics from the time of their discovery to the time they reach
clinical or field application, the titer of the producing strain has increased by
orders of magnitude. While medium development and process engineering have
had major roles, strain improvement has been the key to increase the final titer of
antibiotics in fermentation. It is also through strain improvement that the
producing strains are adapted to economically desirable substrates or to eliminate
undesirable byproducts. A typical strain improvement program involves first
generating genotype variants in the population using physically or chemically
induced mutations or by recombination among strains. This is followed by
selection or screening of those with improved phenotype properties.
Many possible mechanisms can contribute to an increased antibiotic
production in a producing microorganism. An increased flux of a precursor
primary metabolite may lead to an increased productivity. Some antibiotics are
toxic even to the producing microorganisms. An increased resistance of the
producer to the antibiotics can lead to an enhanced productivity. Another possible
mechanism for increased production is to enhance gene expression and the
resulting concentrations of enzymes involved in antibiotic biosynthesis. Besides
random screening for mutants with the desired phenotype properties, selection
strategies can be designed to facilitate the isolation of mutants having one of the
above mechanisms. For example, selection of mutants resistant to analogs
involved in the precursor synthesis, may enhance the probability of isolating a
mutant which is deregulated in the biosynthesis of the precursor and overproduces
it. Another method used is the selection of auxotrophs of the precursor followed
by isolating a revertant of the prototrophic microorganism. In a strain used in
commercial production, often both the precursor fluxes and the antibiotic
biosynthetic machinery have been greatly enhanced. A number of reviews have
focused on the applications of such conventional methodology.14-16 The strain
improvement program carried out on penicillin production using Penicillium
strains has also been described in detail.17

3
3. FACILITATORS FOR METABOLIC ENGINEERING OF
SECONDARY METABOLITE PRODUCESRS
All the mechanisms contributing to the increase of antibiotic production as
described above can be exploited by metabolic engineering. However, since
metabolic engineering aims to manipulate the genes involved in the antibiotic
biosynthesis, detailed knowledge of the biochemical pathways, the genes encoding
for the enzymes, and the regulation of the expression of these genes is beneficial.
In addition to such biochemical knowledge and the availability of genetic tools,
the employment of various analytical tools for determining the bottlenecks of the
biosynthesis is also important.

3.1 KNOWLEDGE OF BIOCHEMICAL AND GENETIC NATURE OF BIOSYNTHESIS


In the past, elucidation of the biochemical pathways relied on understanding
the incorporation of labeled precursors and on the use of blocked mutants which
accumulate an intermediate(s) in the culture medium. With a collection of such
mutants, the identity of the intermediates accumulated in those mutants, and the
knowledge on chemical conversions of these compounds, it is possible to put the
puzzle pieces together to elucidate the biochemical pathway. In the last two
decades, the availability of the various genetic tools has greatly facilitated the
elucidation of the biochemical pathway of secondary metabolite synthesis. The
fact that many genes involved in the biosynthesis of an antibiotic, or a group of
antibiotics, were often clustered in the genome of microorganism has eased the
elucidation of pathways significantly. Furthermore, the resistance genes which
shield the producing microorganism from the harmful effects of it’s own antibiotic
are often clustered with the biosynthetic genes.
Techniques used in identification of the genes involved include screening of
cosmids via degenerate synthetic oligonucleotide probes (multiple nucleotide
sequences each of which codes for the same peptide sequence) based on partial
amino acid sequence of biosynthetic enzymes. This approach is sometimes termed
as “reverse genetics." It is possible to employ heterologous probes which include
conserved sequence of genes in similar pathways in another organism. This
allows one to locate the genes locus of pathways which are hypothesized to
include evolutionarily related enzymes. Complementation of blocked mutants
with a gene library from the original wild type can also be used to identify the
responsible genes. Since most antibiotic biosynthetic genes have been found
clustered together with resistance encoding genes, another successful technique
has relied on cloning of the resistance gene fragment by phenotype selection in a
heterologous host. This is then followed by screening of library clones to identify
biosynthetic genes. Screening for heterologous expression of the entire antibiotic

4
pathway via cosmids in another strain with a null background has also been used.
Mutational cloning is another technique which has been used with success.18 In
this approach, random insertional disruption is followed by screening of the clones
for disruption of antibiotic biosynthesis. Another approach using differential
screening of the transcripts produced during the exponential versus the stationary
phase has been shown to work in isolating eucaryotic penicillin biosynthetic
genes.19 Using all of these techniques the number of pathways elucidated have
increased.8, 12 Concurrently it is becoming easier to use the available information
and techniques to decipher unknown pathways rapidly.

3.2 KNOWLEDGE OF REGULATION


Most of the microorganisms producing secondary metabolites undergo
differentiation during their life cycle. The onset of secondary metabolism is
frequently associated with the differentiation events. It is not surprising that the
regulation of the secondary metabolism is rather complicated. Regulation occurs
at both genetic and biochemical levels. In Streptomyces, accumulated evidence
has been reviewed6, 7, 20 and points to a hierarchy in the regulation of antibiotic
biosynthesis and differentiation. At the global level, the production of secondary
metabolite may be coregulated with other differentiation phenomena such as
sporulation; a global regulatory gene which turns on sporulation possibly also
turns on the production of secondary metabolite.6 In Streptomyces which produce
multiple secondary metabolites, pleiotropic regulators control some or all of those
biosynthetic pathways. Lowermost in the hierarchy of regulation are the pathway
specific activators, which control the production of only a single antibiotic, or a
number of related antibiotics which share segments of the same biosynthetic
pathway. In addition to pathway specific activators, antibiotic biosynthesis in
some cases is also regulated by pathway specific repressors.
Although most regulatory molecules which control production are
intracellular, there are a few extracellular effector molecules with hormone like
properties. Notable examples are A-factor in S. griseus21 and Virginia butanolides
in Streptomyces virginiae.22 A-factor and Virginia butanolides are autoregulators
whose synthesis turns on their own production and triggers both sporulation and
antibiotic production. They are excreted extracellularly to induce antibiotic
production and sporulation in neighboring cells.6
On the genetic level, regulation involves the induction or repression of the
expression of the enzymes in the biosynthetic pathways. Available evidence
suggests that the activity of the enzymes is coordinately regulated by the pathway
specific regulators.7 The regulatory circuit controls not only the level of enzyme
synthesized but also the temporal profiles of these enzymes, i.e., when they are

5
synthesized and when they are turned over to shut down biosynthesis. Although
evidence is scarce, it is conceivable that regulation occurs also at the spatial level,
i.e., only certain portions of the organisms are actively producing secondary
metabolite. The temporal regulation of secondary metabolism is well known;
indeed it is such a profile where metabolite synthesis lags behind the initial rapid
expansion of biomass that gives the term “secondary metabolism.” In a detailed
study with the β-lactam cephamycin C producer Streptomyces clavuligerus,
activities of the biosynthetic pathway enzymes were shown to increase after the
initial rapid cell growth, reaching a peak at the end of growth phase before their
decline.23 Similarly, the temporal profiles of mRNA concentrations also reached a
peak about the same time.24 However, the global level factors regulating the
temporal nature of secondary metabolism are not understood very well.
The regulation at the biochemical level involves the control of enzyme
activity including feedback inhibition, activation and inactivation.
Microorganisms also control the flux of precursors and cofactors into secondary
metabolism by a variety of means to ensure that these precursors and cofactors are
not diverted when they are needed for cell growth. This type of control differs
from conventional feedback inhibition, repression and induction in that it is
affected by the balance of fluxes in a metabolic network rather than involving only
a single biosynthetic pathway. One example of such regulation is the β-lactam
antibiotic biosynthesis. The three precursors from primary metabolism for β-
lactam antibiotics are L-Lysine, L-Cysteine and L-Valine. These amino acids are
also needed for cellular protein synthesis. The competing reactions for these
amino acids are therefore the binding of these amino acids to tRNA through
aminoacyl-tRNA synthetases. The Km for the binding of the amino acid to the
tRNA is usually in the range of micromolar, whereas the Km’s for these amino
acids for β-lactam biosynthetic enzymes (L-Lysine aminotransferase (LAT) and
ACV synthetase) are all in millimolars. Since the Km for these amino acids for
secondary metabolism are at least an order of magnitude higher than those for
protein synthesis, diversion of precursors preferentially towards secondary
metabolism at the expense of primary metabolism is not favored. A similar
phenomenon is observed in the case of α-ketoglutarate. α-Ketoglutarate is the
cosubstrate for the first enzyme (LAT) involved in the β-lactam biosynthesis. It is
also a cofactor for expandase and hydroxylase, involved in cepholosporin
biosynthesis. In primary metabolism, it is a component in the citric acid cycle and
is involved in amino transfer reactions. It is interesting to note that the K m of α-
ketoglutarate for LAT is as high as 8 mM (Khetan et al, submitted) whereas those
for the other enzymes involved in the primary metabolism and secondary
metabolism are all in the range of 0.01–1.0 mM. Again, it is an example of flux

6
distribution regulation: α-ketoglutarate will be diverted significantly to the
formation of secondary metabolite and be utilized by LAT only if the demands of
primary metabolism are low.
From the regulatory point of view, there are many approaches of metabolic
engineering which may lead to increased productivity. It is possible to increase
the copy number of the positive regulatory gene to enhance antibiotic production.
It is also conceivable to convert the regulatory gene to an inducible one so that
time profile of antibiotic production can be manipulated. However our current
level of understanding on the regulatory mechanism of secondary metabolism and
the interactions between the metabolic networks of the primary metabolism and
secondary does not provide us with a way of predicting the consequences of
altering the regulatory structure. Only in a few cases, the potential effect of
altering the expression of a regulatory gene can be qualitatively assessed. In most
cases, the consequences have to be observed experimentally and no a priori
quantitative analysis is currently feasible.

3.3 KNOWLEDGE OF FLUX ALONG THE PATHWAY


As discussed above, a thorough understanding of the biochemical pathway,
the genetic makeup, and the regulation of the biosynthesis is extremely valuable
for metabolic engineering of secondary metabolites. However, except for a few
very well studied antibiotics, such knowledge is usually incomplete. In many
cases, even the pathway is not completely known. The identity of precursors and
possible intermediates leading to the final product can often be inferred from their
molecular structure and radioisotope labeling studies. When such information is
available, it is possible to measure the fluxes of the precursors and the
intermediates involved. Analytical tools such as HPLC and NMR can be applied
to determine the intracellular fluxes. With these metabolic fluxes known, it is
possible to identify the rate controlling steps by pertubation experiments. Such
studies may shed light on the controlling steps during various stages of cultivation.
The flux information is particularly useful when assessing the interactions
between the various controlling steps.

3.4 MATHEMATICAL MODELING AND SIMULATION


Antibiotic biosynthetic pathways typically consist of a large number of
enzyme mediated reactions. Identification of the rate limiting steps is critical to
the enhancement of flux by metabolic engineering. A kinetic model for the
biosynthetic system can help identify the rate- limiting steps. Since perturbation
of a number of reaction steps may have a positive effect on the metabolic flux, it
may not be easy to identify the one with the maximum effect on the flux.

7
Sensitivity Analysis, or Metabolic Control Analysis (MCA)25-27, can be carried
out to identify steps which are most likely to exert the strongest influence on the
overall rate of biosynthesis.
A kinetic analysis was carried out by Malmberg and Hu for cephalosporin
biosynthesis, for both Streptomyces clavuligerus28 and the fungus,
Cephalosporium acremonium.29 Mass balance equations were set up for the
intermediates in the reaction series making up the biosynthetic pathway. The
intracellular concentration of each intermediate was a balance of its synthesis, its
consumption by subsequent reactions, and the dilution due to biomass expansion.
A set of simultaneous differential equations was used to describe the change of
concentrations of the intermediates as a function of time. The kinetic
characteristics of each of the enzymes were assumed to be Michaelis-Menten type,
and the kinetic parameters determined in vitro were used for calculations.
Literature values of precursor concentrations and time profiles of enzyme
activities were used. All the co-factors were assumed to be at saturation
concentrations. The time profile of the specific cephamycin production rate
obtained by model simulation was very similar to that of experimental
observation, thus giving credence to the validity of the model. Subsequently
metabolic control analysis was performed to assess the possible rate controlling
steps by calculating the control coefficient of every enzyme and precursor. The
control coefficient is basically the fractional change of the cephamycin formation
rate caused by a fractional change of the concentration of a particular enzyme or
precursor. A large value of control coefficient implies that the particular element
has a larger influence on the flux than those with a lower value. This allowed
them to identify the condensation reaction forming ACV tripeptide as the rate
limiting step for both organisms. More recently such an analysis has also been
attempted for the penicillin biosynthetic pathway.30, 31
Metabolic flux analysis as described in a preceding chapter can be carried
out to analyze flux distribution in the antibiotic producer. This approach is useful
to find out the maximum theoretical yields on a substrate as well as to identify the
segments of pathway that need to be changed in order to enhance the final
antibiotic flux. There are two basic methodologies: One treats cells as black box
models and the other uses all the major biochemical flux information in the cell.32
Both approaches have been developed for Penicillium chrysogenum.33, 34
Experimentally measured substrate uptake rates and product excretion rates are
introduced as measured constraints. Using a stoichiometric model which accounts
for the major reactions, the fluxes through various pathways can be solved for a
given pseudo steady state. This can then give information on the expected

8
theoretical yields, which can be used as a benchmark for directing the metabolic
engineering efforts.

4. EXAMPLES OF METABOLIC ENGINEERING OF ANTIOBIOTIC


BIOSYNTHESIS
4.1 INCREASING THE METABOLIC FLUX
Many attempts have been made using metabolic engineering to enhance
secondary metabolite production. As discussed above, there are many possible
ways by which biosynthesis of secondary metabolites may be enhanced. These
possibilities include increasing the supply of precursors through primary
metabolism, redirecting the flux of the needed cofactors to antibiotic biosynthesis,
the amplification of one or more of the enzymes involved in the biochemical
pathways, and improving the kinetic characteristics of the enzyme. Following is a
brief review on some efforts in these areas.

4.1.1 Enhancing enzymatic activity


β-lactam antibiotics, being one of the most important industrial antibiotics
and most studied secondary metabolite, have also drawn most attempts to apply
metabolic engineering to enhance its production. The biosynthetic pathway of the
β-lactams penicillin G, cephalosporin C and cephamycin C is shown on Figure 2.
Because cephalosporin products are of higher commercial value, more studies on
metabolic engineering have been directed toward cephalosporin rather than
penicillin. The synthesis of penicillin and cephaolsporin share part of the
pathway. The sequential actions of three enzymes, ACV synthetase (ACVS),
Isopenicillin N synthase (IPNS) and Acyltransferase(AAT) convert three amino
acid precursors to penicillin. Cephalosporin C is synthesized by six enzymes in
series, using the same amino acid precursors.

9
Lysine
Lysine ε-aminotransferase (lat)

1-piperideine-6-carboxylate
Dehydrogenase

Cysteine + Valine + α-aminoadipic acid


ACV synthetase (pcbAB)

ACV tripeptide
Isopenicillin N synthase (pcbC)

Isopenicillin N
Acyltransferase
Isopenicillin N epimerase (cefD)
(pen DE)

H
Penicillin N
N S CH 3
CH3 DAOC synthase (cefE)
O
N
O COOH
Deacetoxycephalosporin C
Penicillin G DAOC hydroxylase (cefF)

DAC acetyl
Deacetylcephalosporin C
transferase O-carbamoyltransferase
(cefG) (cmcH)
O-carbamoyl-DAC
Cephalosporin-7-α -
H
N S
H2 N

COOH O
hydroxylase (cmcI)
O
N
CH 2 OCOCH 3 7-α-hydroxy-OCDAC
COOH
Methyltransferase
Cephalosporin C (cmcJ)

H OCH 3
N S
H2 N

COOH O
N
O CH 2OCONH 2

COOH

Cephamycin C

Figure 2: The β-lactam antibiotics penicillin G, cephalosporin C and cephamycin C share parts of a
common biosynthetic pathway. The segment from Lysine to α-aminoadipic acid is present in the
eubacteria cephamycin C producers. In fungi, α-aminoadipic acid is an intermediate of the Lysine
biosynthetic pathway. Names in parentheses are the genes coding for corresponding enzymes.

10
In a divergent branched pathway, one branch usually leads to the final
product, while the other results in wasted resources. The reaction intermediates
can be channeled to the final product either by blocking the undesirable branch, or
by increasing the reaction rate toward the final product. In cephalosporin C
production with a commercial strain of Cephalosporium acremonium an
intermediate in the cephalosporin biosynthesis, penicillin N, accumulated in the
fermentation broth significantly. The average molar ratio of extracellular
penicillin N to cephalosporin C was found to be 0.3.13 From this observation it
was reasoned that the conversion of penicillin N to the six membered ring
containing deacetoxycephalosporin was slower as compared to the upstream
pathway. As a result, penicillin N accumulated intracellularly, resulting in high-
level secretion to the culture broth. To reduce the diversion of intracellular
penicillin N to excretion, enzyme DAOC synthase (DAOCS) was overexpressed
by introducing plasmids with the gene cefEF. In a transformant which had the
plasmid integrated on the chromosome, the production of the antibiotic increased
by 15% in pilot plant scale fermentations. The recombinant strain had an
enhanced level of DAOCS and did not accumulate intracellular penicillin N.
This is an elegant example of amplifying the enzyme to redirect metabolic
flux to a desired product. It is interesting to note that the branched pathway
involved in this case is atypical in the sense that the side reaction is excretion.
This strategy of redirecting metabolic flux was successful, partly due to the
absence of strong feed back regulation along the pathway. If the branching point
had been a “rigid node,”35 merely increasing the enzyme level of downstream
reaction may not have resulted in a favorable redistribution of fluxes. More recent
work indicates that the excretion of β-lactam antibiotics is mediated by an active-
transport system.36 This suggests that the intracellular penicillin N level in the
parent strain was at a level below the saturation concentration for the transport
system. In other words, the magnitude of the control strength for excretion of
Penicillin N is probably lower than or about the same as that for DAOCS.
Otherwise, the excretion rate might not have been affected unless the intracellular
Penicillin N concentration was reduced drastically by a large increase in DAOCS
level. Despite these other possible causes which could have complicated this
metabolic engineering attempt, the results were clearly successful. This work
clearly demonstrates the importance of intuitive or heuristic approach in
metabolically engineering secondary metabolism.
In the above example the researchers had substantial physiological insight.
They used empirical evidence to speculate on the potential rate limiting steps and
successfully applied metabolic engineering approach to increase antibiotic
production. In another case detailed kinetic information was available to allow for

11
calculation of the control strengths of various factors involved in antibiotic
biosynthesis. An approach based on kinetic modeling of the pathway dynamics
was demonstrated for rationally enhancing flux of the cephamycin C biosynthetic
pathway in wild type Streptomyces clavuligerus. Malmberg and Hu28 modeled the
in vivo kinetics of the cephamycin biosynthesis and subsequently carried out
sensitivity analysis, identifying the concentration of an intermediate, α-
aminoadipic acid (α-AAA) as rate limiting. To enhance the concentration of α-
AAA, an additional copy of the lat gene was introduced into the S. clavuligerus
chromosome by homologous integration37, lat codes for lysine ε-aminotransferase
(LAT), an enzyme that mediates the first step in the conversion from lysine to α-
aminoadipic acid. The recombinant strain showed 2–5-fold enhanced
productivity38 and an enhanced intracellular level of the α-AAA. An interesting
finding in this study was that the intracellular concentrations of the three amino
acid precursors (lysine, valine and cysteine), all remained constant even though
the antibiotic flux was enhanced. As the antibiotic titer increased by two-fold, the
precursor flux should have increased by the same level. Nevertheless, the
intracellular precursor concentrations remained relatively constant. The increased
fluxes of the precursors in the recombinant strain did not perturb their intracellular
pool. This suggests that either their fluxes into the secondary metabolism were
relatively small compared to the total consumption, or that increased flux was
compensated by increased biosynthetic rate of these amino acids.
In developing the kinetic model and performing the theoretical analysis,
these researchers made several significant assumptions.28, 39 They assumed that the
cofactors including ATP, NADP and α-ketoglutarate were at saturation
concentrations. Furthermore, it was assumed that the kinetic parameters,
including the half saturation constants (Km) and their Vmax’s measured in vitro
were applicable to in vivo. With these limitations their theoretical analysis and
prediction of the rate limiting steps can only be considered as a first
approximation. Nevertheless, this approach provided a rational basis for selecting
the target enzyme for metabolic engineering. The subsequent success in
genetically modifying the organism for enhanced cephamycin C production lends
credence to the value of theoretical analysis in metabolic engineering.
For cephalosporin biosynthesis in wild type Cephalosporium acremonium,
the last step mediated by acetyltransferase has been speculated to be rate
controlling.40 The cefG gene coding for DAC acetyltransferase was cloned from
C. acremonium and reintroduced into the wild type strain on a plasmid. A plasmid
copy number dependent increase in the cephalosporin C titer was observed. The
titer increased from 0.625 g/l in the wild type strain to 1.9 g/l in a transformant
with 5-6 copies of the plasmid. While no apriori logic was used to identify this

12
step as rate limiting, the results suggest that acetyltransferase significantly
influences the flux of cephalosporin. Such cloning and retransformation into
producer strains demonstrate that empirical cloning and reintroducing genes may
identify and even relax the rate controlling steps.
Enhancement of penicillin titer by increasing the copy number of
biosynthetic genes has also been demonstrated in Penicillium chrysogenum.41
Amplification of the pcbC-penDE gene cluster of P. chrysogenum in one of the
early production strains led to as much as 40% increase in product titer. However,
the individual clones showed variability, which was speculated to be caused by
differences in the integrated plasmid copy number or the site of integration.
Metabolic engineering on β-lactam antibiotic biosynthesis has also been
applied to penicillin producing Aspergillus nidulans. ACVS was overexpressed
by replacing its native promoter with an inducible ethanol dehydrogenase
promoter, alcAp.42 A. nidulans grown with 10 mM cyclopentanone inducer was
estimated to have a 100-fold enhanced ACVS level compared to the control that
was grown without the inducer. Penicillin final titers were increased 30-fold as
compared to wild type when fermentations were carried out under induction
conditions. When the same approach was used to overexpress two downstream
enzymes IPNS and AAT, no significant enhancement in penicillin titers was
observed even though the transcript level and the enzyme activity were
considerably increased.43
Another approach of metabolic engineering is to amplify the entire pathway,
or a large portion of the pathway in a gene cluster, instead of amplifying only the
gene encoding for the rate-controlling enzyme. As an example, the entire gene
cluster coding for all the enzymes in the cephamycin pathway from Streptomyces
cattleya was cloned as a 29.3 kb DNA segment.44 This was accomplished by
screening for cephamycin producing transformants derived from a cephamycin
non-producing S. lividans, after “shot gun” cloning. Subsequently, an extra copy
of this whole biosynthetic gene cluster was introduced to Streptomyces.
lactamgens, a native producer of cephamycin C, and led to a 2–3-fold increase in
yields. However, the cause of the increase was not characterized; it could be due
to extra copies of either the structural genes or regulatory genes.
Studies on enhancing productivity by supplementing with additional
structural genes have not been limited to the β-lactams. In the production of the
macrolide tylosin by Streptomyces fradiae, accumulation of the intermediate
macrosin was observed in the culture broth. A terminal O-methylation in the
tylosin pathway converts macrosin to tylosin. A pronounced correlation was
observed between increased tylosin production and increased macrosin O-
methyltransferase specific activity among mutants isolated by conventional

13
approaches, suggesting this enzyme was rate limiting.45 Solenberg et al46 used
transposon Tn5099 to identify and clone a neutral genomic site in the producer
strain for the insertion of a second copy of the tylF gene, which codes for the
required methyltransferase. The introduction of second copy of the tylF gene was
carried out by homologous recombination with the transposon at the neutral site.
S. fradiae recombinants containing two copies of the tylF gene carried out the
normally rate limiting conversion of macrocin to tylosin very efficiently,
substantially increasing the tylosin yield (~30%) while reducing that of the
macrosin.
In another study involving tetracenomycin, a polyketide synthesized by a
type II polyketide multienzyme complex in Streptomyces glaucescens,
enhancement of intermediates and the final product was observed on
complementation with extra copies of the biosynthetic genes.47 While a variety of
antibiotics are synthesized by type II polyketide-like systems, the biosynthesis is
complex and a clear role for individual enzymatic components leading to the final
product has not been completely elucidated. Polyketide synthase (β-ketoacyl:acyl
carrier protein(ACP) synthase) and the acyl carrier protein have been postulated to
play a role in the early steps. By enhancing the gene dosage of these enzymes on
multicopy plasmids with their expression under the control of a strong promoter,
the accumulation of an intermediate TcmD3 increased by almost 30-fold and
tetracenomycin C production increased by 30%. Although this level of increase in
titer was modest, it was still substantial compared to the control transformed with
the plasmid vector alone. The control strain, however, had a titer lower than that
of the wild type strain due to a postulated plasmid effect. A similar plasmid effect
on antibiotic production has been observed in other antibiotic producers.48

4.1.2 Manipulating regulatory genes


Antibiotic regulator genes have been identified in many polyketide
antibiotic producing Streptomyces.20 Two DNA segments dnrR1 and dnrR2 were
isolated from Streptomyces peucetius based on their ability to enhance the
production of daunorubicin and the intermediate ε-rhodomycinone when
introduced back into their parent strain. Sequencing of dnrRI revealed a putative
pathway specific regulator gene dnrI and another gene.49 Introduction of dnrI on a
high copy plasmid caused a more than 100-fold increase in the production of a
pathway intermediate ε-rhodomycinone as compared to that in the wild type strain.
The enhancment effect was an order of magnitude lower with the same gene
segment on a low copy plasmid. Insertional inactivation of dnrI in the wild type
strain abolished ε-rhodomycinone and daunorubicin production.50 This insertional
inactivation of the regulator gene also eliminated the transcripts of the known

14
daunorubicin biosynthetic genes. Gel shift assays using purified DnrI protein
showed that it binds to upstream region of biosynthetic genes of the daunorubicin
gene cluster.51
A regulatory gene srmR has been identified to have a role in the
biosynthesis of spiramycin, a macrolide produced by Streptomyces ambofaciens.
A mutant of S. ambofaciens derived by disrupting srmR was blocked in
spiramycin production and was unable to complement any of the other mutants
which accumulated different biosynthetic pathway intermediates in the medium.
The product of srmR was identified as a transcriptional activator required for the
initiation of transcription of the biosynthetic genes.52 Introduction of the gene on a
multicopy vector increased the final spiramycin titer from 100 to 500 µg/ml. A
similar overproduction of the antibiotic has been previously observed in
Streptomyces coelicolor where multiple copies of redD caused multifold increase
in undecylprodigiosin,53 whereas that of actII-ORF4 led to overproduction of the
blue pigment actinorhodin.54 A pathway specific regulator ccaR, has also been
identified recently in the Streptomyces clavuligerus cephamycin C gene
cluster.55,56 Additional copies of this regulatory gene on a multicopy plasmid led
to almost two-fold enhanced production of both cephamycin and clavulanic acid.56
The disruption of the ccaR gene led to the complete elimination of the
biosynthesis of both compounds.
Besides the pathway specific regulators, global and pleiotropic regulators in
Streptomyces are also potential targets of genetic manipulation for enhancing
secondary metabolite production. The absA gene in S. coelicolor 57 appears to
code for one of the components of an eubacterial two-component sensor kinase-
response regulator involved in the negative regulation of antibiotic production.
Disruption of the absA gene led to the overproduction of the antibiotics
actinorhodin and undecylprodigiosin. A similar two component signal tranduction
system cutRS has been found in Streptomyces lividans.58 Insertion disruption of
either of the two genes resulted in about three-fold increase in actinorhodin
synthesis. In S. lividans another such pleiotropic regulator, afsR2 has been
identified that encodes a 63 amino-acid protein. When expressed on a high copy
number plasmid it stimulates both actinorhodin and undecylprodigiosin
production.59 For actinorhodin, the increase was mediated by an increase in the
pathway specific regulator actII-ORF4 transcription. In another such example a
Streptomyces griseus mutant deficient in the A-factor binding protein produced
almost ten times streptomycin as compared to the wild type.6, 60 The proposed
explanation is that the binding protein acts as a repressor in the signal transduction
cascade responsible for activating the streptomycin biosynthesis.

15
4.1.3 Enhanced antibiotic resistance
Many Antibiotic producers have resistance mechanisms to protect
themselves from the adverse effects of the antibiotics they produce. Mechanisms
of cellular self-protection include drug inactivation by chemical modification,
target site modification, drug binding and reduction of the intracellular
concentration using an efflux pump system and/or a low permeability to the
antibiotic.61 Gene coding for antibiotic biosynthesis and resistance are often
clustered and their expression is interdependent physiologically. A molecular
genetic basis for this interdependence has been demonstrated for the streptomycin
producer Streptomyces griseus. A regulatory protein SmA controls the
transcription of the shared promoter of the resistance gene and a pathway specific
activator, the latter in turn activates the biosynthetic genes.62
Enhanced antibiotic resistance have been shown to be correlated to
enhanced production for a number of antibiotic producing species.63 In
Streptomyces kanamyceticus and Streptomyces fradiae, producers of kanamycin
and neomycin respectively, aminoglycoside 6’-N-acetyltransferase provides the
host with resistance to these antibiotics by N-acetylation of the compound.
Crameri and Davies64 subcloned the gene for aminoglycoside 6’-N-
acetyltranferase and introduced it into the producers on a high copy vector pIJ702.
The transformed S. kanamyceticus showed 3–4-fold enhanced final titers of
kanamycin. Transformed S. fradiae had upto 7-fold enhanced titers of neomycin.
The transformed strains showed an increased level of resistance against the
antibiotics, too. The obvious explanation for the enhanced titers is that higher
levels of resistance allowed for higher antibiotic flux and intracellular
accumulation. However, the possibility exists that these antibiotics are actually
synthesized and transported out in an inactive form, and are activated only
subsequently in the medium. The acetyltranferase would then be an enzyme in the
biosynthesis of these antibiotics. In such a case, the enhanced production might
simply be a consequence of a relaxed rate controlling step in the biosynthesis of
these antibiotics.
In many cases, cellular resistance to the produced antibiotic is inducible.
Constitutive expression of the resistance gene in such producers may lead to
enhanced productivity. Constitutively resistant strains of S. fradiae and S.
lincolnensis had higher yields of tylosin and lincomycin respectively.65

4.2 INCREASING PRODUCT SELECTIVITY


Many antibiotic producers produce a number of antibiotics. Some of them
may be the intermediates in the biosynthetic pathway, some byproducts in
branched pathways, others entirely different molecules. In many cases only one or

16
a small number are desired. The presence of the undesired byproducts burdens
downstream separation and constrains the overall yield. It is desirable to increase
selectivity of production. S. pristinaespiralis produces antibiotic pristinamycin,
which is a mixture of two macrocyclic lactone peptolide, pristinamycins I (PI) and
pristinamycins II (PII). PII is an 80:20 mixture of PIIA and PIIB, PIIA being
synthesized from PIIB. Pristinamycin is poorly soluble in water and cannot be
administered orally. A soluble semisynthetic derivative has been developed by
chemical modification of PIIA. For synthesizing the semisynthetic antibiotic, a
strain that produces only PIIA was desirable. The enzyme PIIA synthase mediating
the conversion of PIIB to PIIA, and the genes snaA,B encoding the enzyme had
previously been identified. By overexpressing the genes snaA,B under the control
of a strong and constitutive promoter ermE*, the accumulation of PIIB was
completely eliminated.66 This was accomplished by using a site specific
integration plasmid vector to introduce a single additional copy of the gene coding
for PIIA synthase.
Another successful effort of enhancing product selectivity involved the
production of antiparasitic avermectins. S. avermitilis produces eight different but
structurally very similar avermectins, A1a, A1b, A2a, A2b, B1a, B2b, B2a and
B2b. Of these only B1a and B1b possess useful antiparasitic activity. In addition
B2a is useful as the substrate for the manufacture of semisynthetic avermectin,
Invermectin B1a, which is the most potent anthelmintic compound among the
products. The process challenge is to separate the useful components from the rest
and to remove an additional antibiotic, oligomycin, which is also produced by the
strain. Since the major components of the biosynthetic pathway have been
elucidated and the gene cluster coding for the enzymes has been located, it is
possible to devise a rational metabolic engineering strategy. Ikeda, Omura and
their colleagues67 used random mutagenesis to isolate a strain K2021 producing
only A1a, A2a, B1a and B2a, and another strain K2034 producing only B1a, B1b,
B2a and B2b. The strain of K2034 appears to have a mutation in a structural gene
aveD for biosynthetic enzyme, while K2021 is speculated to be disrupted in the
incorporation of a branched-chain fatty acid precursor derived from L-valine.
Recombinants that had both the mutations were derived by protoplast fusion from
these strains. They were shown to produce only two components, B1a and B2a.
Subsequently, the gene aveC responsible for a dehydration step converting B2a to
B1a was disrupted. The resulting clone K2099 produced only a single avermectin
B2a. However this clone still produced the toxic polyketide oligomycin. To
disrupt oligomycin production, a transposon Tn4560 was used to produce
disruptant clones in the wild type S. avermitilis. Chromosomal DNA fragments
were subsequently subcloned from these disruptants into a temperature sensitive

17
plasmid and used to disrupt the oligomycin biosynthesis in K2099, yielding a
strain producing only a single desired avermectin.

4.3 BIOSYNTHESIS OF A PRODUCT PREVIOUSLY MADE SEMI SYNTHETICALLY


Biotransformation and chemical conversion of secondary metabolites are
frequently practiced to give antibiotics more desirable properties. 7-amino
deacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-
ACA) are the two main starting materials for semi-synthetic cephalosporins.
These starting materials are prepared from cephalosporins by removing D-α-
aminoadipyl side chain chemically or enzymatically. To devise an alternative
biosynthesis route, Crawford et al68 introduced additional genes coding for DAOC
synthase from Streptomyces clavuligerus, and DAOC synthase-hydroxylase and
DAC acetyltransferase from Cephalosporium acremonium into a penicillin
producing Penicillium chrysogenum. By feeding adipate to the developed strain,
cephalosporins were produced instead of penicillin G. This strategy tapped into
the high productivity of penicillin in P. chrysogenum to produce cephalosporins
with an adipyl side chain which can be removed enzymatically to produce 7-ACA
and 7-ADCA.
Another biosynthetic process for the production of ACA has been
developed previously. Isogai et al69 introduced two heterologous bacterial genes
coding for a D-amino oxidase and a cephalosporin acylase into a producer strain
of C. acremonium to produce 7-ACA from cephalosporin C. To ensure expression
of the genes in the fungal strain, the genes were modified by the addition of
expression signals from the cloned C. acremonium alkaline protease gene. The
developed strain synthesized and secreted 7ACA, demonstrating the potential for
using metabolic engineering to introduce additional antibiotic biosynthetic
pathway segments.

4.4 SYNTHESIZING ANTIBIOTICS IN HETEROLOGOUS STRAINS


Heterologous expression of entire gene clusters for a complete pathway or
segment of a pathway has been used for the production of non-native antibiotics in
a number of streptomyces and fungi.70, 71 Many reasons make it desirable to
produce antibiotics in heterologous strains. Among these are higher precursor
flux, better resistance to the end product, and fewer by products in the recipient
organism. Also it might be attractive to carry out all metabolic engineering in a
heterologous host for which better genetic tools are available or the physiological
characteristics are better known.
Deacetoxycephalosporin C (DAOC) was produced in an industrial strain of
Penicillium chrysogenum by transforming it with two hybrid genes, cefDh and

18
cefEh, coding respectively for an isopenicillin N epimerase and DAOC synthase.72
A new branch was introduced in the pathway to siphon off the intermediate
isopenicillin N resulting in the production of DAOC. However, the resulting
strain retained the capability of producing penicillin V because the pathway
leading to Penicillin V could not be disrupted. The disruption was rendered
difficult because the strain had multiple copies of the genes coding for
acyltransferase, responsible for converting the isopenicillin N to penicillin V. The
authors suggested the use of antisense transcription to block acyltransferase and
eliminate penicillin V production.
In a reverse example, benzyl penicillin was produced in Cephalosporium
acremonium by expressing the penDE gene from P. chrysogenum.73 The total
amount of benzylpenicillin and cephalosporin produced in the transformant was
approximately equal to that from the parent strain. Again in this case, the
disruption of the original pathway by inactivating isopenicillin N epimerase or
expandase would probably lead to enhanced production of the new product.

4.5 SOLVING SCALEUP CHALLENGES


In antibiotic production by filamentous microorganisms, oxygen transfer is
a major technical challenge in large scale operations. Since the dissolved oxygen
level is often rather low in large reactors, it is desirable to enhance the ability of
microorganisms to utilize oxygen under such conditions. By introducing a
bacterial haemoglobin into Acremonium chrysogenum, cephalosporin production
was improved.74 In shaker flask cultures, A. chrysogenum transformed with an
integration vector containing the Vitreoscilla hemoglobin gene produced higher
levels of cephalosporin C compared to the controls. A similar positive effect on
actinorhodin production was seen in Streptomyces coelicolor75 under low aeration
conditions. While the mechanism for the beneficial effects has yet to be
elucidated, it illustrated the concept of applying metabolic engineering to
overcome process engineering obstacles.

5. GENERAL PRINCIPLES DERIVED FROM EXAMPLES OF


METABOLIC ENGINEERING
The examples presented above cover a variety of antibiotics. The
approaches taken by different researchers are many. The outcome of those
metabolic engineering efforts also varied widely. Nevertheless, some general
lessons can be derived from those examples, and some guiding principles can be
enunciated from those efforts. It is clear that when the kinetic and biochemical
information on the pathway leading to the product is available, a quantitative
analysis to identify the rate limiting factors involved can drastically increase the

19
probability of success of metabolic engineering. A notable example is the
metabolic engineering work by Malmberg, Sherman and Hu.28, 37 Although the
example was carried out on a wild type strain of Streptomyces, a similar approach
can be readily be applied to an industrial producing strain provided that such
kinetic information is also available. However, more than often detailed
biochemical pathway is not known, or the kinetic parameters for the enzymes
involved are not available. Under those conditions, one has to resort to using
empirical evidence to probe the rate controlling steps. As has been shown by the
Lilly example on cephalosporin biosynthesis,76 the wasteful accumulation of an
intermediate in the fermentation broth can be decreased by increasing the flux
towards downstream biosynthesis of the final product.
Many industrial microorganisms produce antibiotic at levels exceeding tens
of grams per liter at a similar level of biomass. The antibiotic produced is almost
in the same order of magnitude as the biomass. It is obvious that significant
fractions of the precursors, metabolic energy (ATP) and cofactors have been
diverted from primary metabolism to secondary metabolism. Even without a
detailed kinetic analysis it is apparent that this can happen only if the precursor
flux towards the secondary metabolism has been enhanced. It is likely that the
regulation of these primary precursor biosynthesis have been altered to allow for
such diversion of a large fraction of the precursors. Finally, from the examples
presented above it is also evident that the amplification of a positive
transcriptional regulator or the deletion of the negative transcriptional regulator
can have a profound effect on the antibiotic biosynthesis. Most studies presented
above involve only one of these aspects of metabolic engineering. It is likely in
the future that the metabolic engineering of the secondary metabolism will involve
not only the relaxation of the rate controlling steps in the structural genes, but also
the deregulation and increased flux of the precursor biosynthesis. As more
regulatory genes governing the biosynthesis of secondary metabolites are being
unearthed, more emphasis on the deregulation or restructuring of the regulatory
network is likely.

6. OPPORTUNITIES
6.1 IMPACT OF NEW TOOLS
In the last few years there has been significant advances in the genetic tools
for metabolic engineering. A major difficulty in metabolically engineering
microorganisms for secondary metabolite production is that the biosynthetic
pathway is often unknown or sketchy at best. The lack of knowledge on
biosynthetic pathway does not deter the conventional strain improvement program.
For most industrially important antibiotics, a large number of strains have been

20
isolated in the course of strain improvement. “Reverse engineering” can possibly
be combined with traditional strain improvement program to lead to a more
“rational” approach of metabolic engineering. Comparative genome sequence
analysis of wild type and various high-producing strains will be instructive in
understanding the mechanistic causes for the differences in the phenotype. High
producing strains of Penicillium chrysogenum have been shown to have multiple
copies of biosynthetic genes and higher transcript levels.77-79 Recent advances in
high throughput hybridization methods80, 81 should also lead to a cost effective
comparative sequence analysis. Comparative transcriptional analysis of different
mutants should also be facilitated by this technology.82 Genomic analysis and
sequencing will facilitate the elucidation of the pathways and their regulations.
Recently the whole genome of S. coelicolor has been organized into ordered
cosmids and efforts are underway to sequence the entire genome.83 Another tool
that will expand the horizon of metabolic engineering of secondary metabolites is
protein engineering. One can speculate on many potential applications; altering
the substrate specificity to eliminate the synthesis of undesirable side products, or
manipulating the half saturation constant of the enzyme (Km) to channel the
precursors to the biosynthesis of the product

6.1 INTEGRATION OF METABOLIC ENGINEERING AND PROCESS ENGINEERING


Heterologous expression of proteins for secondary metabolite production is
closer to reality. It may enable microorganisms to produce an antibiotic that is not
normally produced by itself. To date the application of this strategy is largely
restricted to the introduction of single enzymes into the host microorganism. The
possibility of expressing the entire pathway or a major portion of the pathway to a
new host organism that exhibits more desirable characteristics has just begun to be
exploited. The original producer may not have the desired properties for large-
scale cultivation in industrial reactors, or may not have the sufficient fluxes of the
precursors. Heterologous expression enables a better host to be used for the
production. It may also allow one segment each from two different pathways to be
combined. Each of the particular segments of the pathway may offer better
reaction characteristics. The two segments may arise from the same or different
species.
In future metabolic engineering efforts, we will also see better integration of
cellular physiology and process engineering. The biosynthetic machinery of
secondary metabolites is temporally regulated. Recent evidence (Khetan, Sherman
and Hu, unpublished) suggests that the biosynthetic gene expression may also be
spatially distributed, implying that only a portion of the cells are actively
producing antibiotics at a given time. Such temporal and spatially distributed

21
profile may vary in the laboratory and in production reactor due to changes in
physical and chemical environment related to scale change. An integrated
metabolic engineering approach takes all these factors into consideration. For
example, the case of an enzyme which requires oxygen as a cosubstrate being the
rate limiting enzyme. Amplification of this enzyme may lead to an enhanced
productivity in a laboratory investigation but not in a production reactor. A more
integrated approach would consider both the amplification of the enzyme as well
as altering the Km for oxygen. One may also consider altering mycelial
morphology from the dispersed mycelia to a pellet form to improve the
characteristics of oxygen transfer culture broth. To date such alteration has been
done largely through the manipulation of environmental factors. It is conceivable
that through genetic means, cells can be induced to form loose pellets to enhance
mass transfer in the bulk culture broth and facilitate diffusion. This would make
the introduction of oxygen requiring enzymes via metabolic engineering more
meaningful.

7. CONCLUSIONS
The integration of metabolic engineering along with other traditional
approaches for process improvement of antibiotic biosynthesis is poised at an
exciting stage. Contributing to this is the increasing understanding of secondary
metabolism pathways and their physiological regulation, the relative maturity in
the available tools to manipulate them and the accumulating experience in the
effects of directed changes. While advances in molecular biology techniques have
made it feasible to create defined changes at the genetic level, the challenge is to
predict quantitatively the changes that need to be made. In this regard, the cellular
basis for the increase in productivity of one of the best-known antibiotics
penicillin by more than 30,000-fold from the original 0.0012g/l to about 50g/l is
not yet fully understood. Progress in quantitative characterization of basic and
altered physiology of producer strains is critical for major successes to be achieved
by metabolic engineering.

22
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