Reprint from

D a i l y B i o t e c h U p d a t e s . . . w w w. g e n e n g n e w s . c o m
Volume 24, Number 2
January 15, 2004

BIOPROCESSING
Pathway Engineering through Rational Design
Tutorial: Designing Figure 1.
1,3-propanediol production from glucose
and Building Cell
Factories for Biobased
Production
Karl Sanford, Ph.D., Fernando
Valle, and Roopa Ghirnikar

he multidisciplinary field of path-

T way engineering is exploiting life in

unprecedented ways, essentially
forcing microbial cells to bypass processes
resulting from billions of years of evolu-
tion and allowing them to be exploited for
human use. The concept is simple: using a
set of enabling technologies, researchers
manipulate a microorganism to use any
carbon source to produce a range of
desired biomolecules.
The single substrate glucose, for exam-
ple, can be converted into a wide range of
products including enzymes, therapeutic
proteins, peptides, antibiotics, vitamins,
amino acids like lysine and phenylala-
nine, and organic acids, like citric, glu-
conic, ascorbic, succinic, and lactic. Fur-
thermore, the manufacture of biofuels,
such as ethanol and hydrogen, is also
possible, making this discipline a new
paradigm for more efficient production
of desired bioproducts.
Rational Design
In technical terms, pathway engineering
encompasses directed modification of cel-
lular physiology through the introduction,
deletion, and/or modification of metabol-
ic pathways or regulatory functions of a
cell. The rational design is based on an
understanding of the cellular physiology
under conditions identical or close to
those that will be used in the commercial
manufacturing process. This requires col-
laborative input from scientists from a
variety of disciplines, such as biochemistry,
molecular biology, chemical engineering,
 Figure 2.
1,3-Propanediol production in engineered E. coli
and computational sciences.
Researches use numerous techniques
to study and modify cells. For example,
global analyses of the transcriptome and
proteome provide information of the
cells’ behavior during production.
Improved strains are being made by site-
directed modifications, directed evolu-
tion, and protein engineering, while
mathematical models predict and/or
explain cellular responses. Knowledge
then gained about the physiology of the
microorganism enables scientists to
make improvements to a process, obtain
new products, or solve unexpected prob-
lems that may arise during process scale-
up or transfer.
Biocatalysts and bioprocesses are
used for manufacturing several con-
sumer and industrial products. Their
ability to deliver sustainable systems, by
diminishing dependence on fossil fuels,
lowering greenhouse-gas emissions, and
decreasing hazardous waste streams, is
well recognized.
Microbes, however, have evolved over
the years to sustain their propagation
within the confines of their environ-
ment—a process never intended to
include overproduction of industrially
useful bioproducts. Thus the challenge is
to convert microorganisms into “pro-
duction hosts,” that is, organisms with a
single purpose: to convert substrate to
product with maximum efficiency, in a
manufacturing process that is economi-
cally feasible.
Pathway-Engineering Project
The first step is establishing the best
pathway from a substrate to a given
product. With that accomplished, the sci-
ence then becomes more complex.
The initial process involves cloning the
genes of interest into the production
host, to eliminate or diminish those that
are detrimental. The amount or rate of
production of the molecule is optimized
by fine-tuning the expression of a gene or
genes, or eliminating transcriptional
and/or allosteric regulatory mechanisms.
Understanding the regulation of genes
(in particular, the interaction of promot-
ers and regulatory proteins) is also essen-
tial. Furthermore, enhancing committed
steps from central metabolism or branch
points, identifying and relieving rate-
limiting steps, and preventing carbon
loss to competing pathways, are just
some of the routine tricks used to
enhance product yield.
While molecular biologists and bio-
chemists work on the organism, chemical
engineers simultaneously work to optimize
the production process. The goals are
always the same: to maximize the conver-
sion of the substrate into the desired prod-
uct, in a process capable of fulfilling all
requirements for commercialization. These
requirements will depend on the product
being made, scale, regulatory and environ-
mental regulations, application, etc.
Case 1: Polyester Monomer from
Glucose
Recently a significant pathway-engi-
neering project was accomplished—the
engineering of an E. coli strain to pro-
duce 1, 3-propanediol (PDO) from glu-
cose—that highlighted the coordination
of scientific disciplines and technical
efforts. PDO is a monomer utilized for
the production of polyester fibers such
as Sorona™1 by DuPont (Wilmington,
DE).
Although scientists at Genencor Inter-
national (Palo Alto, CA) and DuPont
knew of certain chemical and biological
approaches to make PDO, these
approaches were not well suited for
industrial-scale production because they
were energy-intensive and required
expensive starting materials. Thus, there
was a need to develop a new process that
would use one microorganism with the
ability to convert the basic carbon source
into the desired PDO end product.
The choice of a wild-type strain of E.
coli to build a pathway from glucose to
PDO came after an initial phase where
several production microorganisms were
compared. The fact that E. coli is a highly
studied organism with a rich set of genet-
ic tools also strengthened the decision.
Since E. coli normally does not pro-
duce 1,3-propanediol, not Figure 3.
2-keto-L-gulonic
only did the the metabolism
acid production
of E. coli have to be modified, in Pantoea citrea

but relevant genes from other

organisms had to be import-
ed. The combination in a sin-
gle host of a natural biological
pathway present in yeast for
the production of glycerol
from dihydroxyacetone (a
n o r m a l
E. coli metabolite), and a path-
way present in the bacteria
Klebsiella pneumoniae to con-
vert glycerol into PDO, creat-
ed a novel and unique process
(Figure 1).
Assembly of Genes and Pathways
The assembly process required state-
of-the-art technology as well as the
development of several new molecular
biology tools that enabled the fine-tun-
ing of critical pathways, elimination of
undesired enzymes, and careful deregu-
lation of ancillary systems of the E. coli-
based cell factory.
The genetic modifications necessary
in E. coli were initially defined using
mathematical models that provided a
road map for the process. Enzymology
techniques helped in establishing the
pathway components for the conversion
of glycerol to PDO. Microarray-based
transcriptome analyses aided in the
characterization of some of the regulato-
ry pathways, as well as the identification
of stress responses that occurred during
the cultivation of microbes.
Proteomics, which studies the charac-
teristics, function, localization, and inter-
actions of proteins was used to identify
isozymes. Information gained from these
studies resulted in the introduction of
eight new genes into a plasmid to convert
the intermediate, dihydroxyacetone
phosphate, into PDO.
With the PDO-production pathway in
place, scientists turned to the fine-tuning
of the host to maximize product forma-
tion. Modifications were made to more
than 70 E. coli genes. Intensive analysis of
various combinations of the modified
genes resulted in a production organism
that had direct modifications to 18 genes.
Some of these modifications, however,
were made to regulatory systems that
indirectly affected the function of hun-
dreds of other genes. All this led to the
development of a strain of E. coli that
produced 135 g/L of PDO at 3.5 g/L/h
and 51% yield (Figure 2), which repre-
sents a significant bioprocessing yield.
The assimilation of these diverse
improvements resulted in a biobased
process that has enabled the manufacture
of PDO.
Case 2: Vitamin C Intermediate
from Glucose
An equally aggressive approach was
taken to develop a commercial process to
produce 2-keto-L-gulonic acid, an inter-
mediate in the production of vitamin C. In
this example, a new production platform
was developed by using Pantoea citrea, a
gram-negative bacteria capable of produc-
ing keto sugars.
Using multiple approaches that includ-
ed the sequencing of the P. citrea genome
and the elimination of competing reac-
tions, strains capable of converting 97%
of the glucose into product were devel-
oped (Figure 3). Importantly, along the
way, strains capable of overproducing
other commercially relevant products,
like gluconic acid, 2-keto-gluconate, and
2,5 di-keto-gluconate, were also obtained.
Integrated Biotech Approach
Although successful pathway-engineer-
ing applications have been around for the
last two decades, previous strategies relied
mainly on experimentation and a trial-
and-error approach. With advances in
gene-sequencing technologies and the
ability to characterize gene expression and
protein interactions on a whole-cell basis,
it is now possible to use a directed and
rational approach to modify the physiolo-
gy of a microorganism.
Technological advances in the areas of
metabolic reconstruction, metabolic-flux
modeling, predictive modeling, and path-
way design are assisting scientists in nar-
rowing the possible pathways to be con-
sidered for modification and allow them
to identify precisely key genes that need to
be modified in order to achieve the
desired change. This has tremendously
shortened the time required to do these
manipulations and develop the optimal
strain. The result is an approach that inte-
grates strain development with product
yield and process optimization and allows
for simple and efficient downstream
recovery of the product.
The amalgamation of biology, chem-
istry, engineering, metabolomics, phys-
iomics, and bioinformatics to produce
biomolecules efficiently is the key com-
ponent that allows this versatile
approach to compete on a global scale.
The fundamental nature of engineered
life is to grow, reproduce, and produce
metabolites.
By engineering a microbe, however,
scientists limit its growth, streamline the
carbon flow, and focus energy into the
production of a single product. In short,
they build nanocell factories that can
replicate under defined conditions, with
an “operating system” and “software”
defined by the metabolic engineer. The
result is a new paradigm for the manu-
facture of chemicals and materials con-
sistent with the demands for improved
sustainability. GEN
Karl Sanford, Ph.D., is vp of technology development, Fer-
nando Valle is staff scientist for process science, and
Roopa Ghirnikar is scientific writer, at Genencor Interna-
tional (Palo Alto, CA). Phone: (650) 846-7500. Fax: (650)
845-6500. Website: www.genencor.com.
NOTES
1. Sorona™ is a trademark of EI DuPont de Nemours &
Co., a DuPont company.