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multiple tools to the pathway to get the best results. The following examples show how
different tools were applied.
Production of Glucaric Acid
A culmination of some of these pathway construction and optimization methods is
exemplified in the production of glucaric acid. This compound has been identified as a
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by the US Department of Energy, 81 and is used
in cholesterol-reduction studies 82 and cancer therapies. 83,84 Currently, its primary use is as a
starting material for hydroxylated nylons. 40 Moon et al. used the ReBiT program to identify
three enzymes from different sources that can form a novel metabolic pathway to produce
glucaric acid in E. coli . 16,40,80 The pathway design process was based on an organic chemist
Top Value-Added Chemical from Biomass
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s
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methodology-retrosynthesis, which considers the product first and then designs a synthetic
route backwards towards the substrate. It was hypothesized that glucaric acid can be
produced from D-glucuronic acid by uronate dehydrogenase (UDH). The D-glucuronic acid
can be produced from myo -inositol by the myo- inositol oxygenase (MIOX), whereas the
myo -inositol can be produced from glucose via a combination of endogenous enzymes and
a heterologous enzyme: myo- inositol-1-phosphate synthase (INO1) ( Fig. 3.2A ).
The MIOX gene was identified from a mouse genome and was synthesized with codon
optimization. The INO1 gene was cloned from the cDNA of S. cerevisiae , while the UDH
gene was cloned from the cDNA of Pseudomonas syringae . Traditional cloning techniques
incorporated these enzymes into both high- and low-copy plasmids, with a T7 promoter
for each enzyme. The resulting E. coli strains produced 0.73 g/L of glucaric acid. Initial
optimization by varying the isopropyl-
-D-thiogalactopyranoside (IPTG) concentrations
increased the production to 1.13 g/L. 40 Experiments determined the bottleneck to be the
MIOX-catalyzed step. 80 The high MIOX activity in E. coli was found to be strongly
influenced by exposure to high concentrations of its substrate myo- inositol. Thus it was
hypothesized that by reducing diffusion distance and transit time, the recruitment of the
pathway enzymes to a protein synthetic scaffold could increase the concentration of
myo- inositol concentrated around the active site of the bottleneck enzyme. The scaffolds
were constructed by assembling three protein
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protein interaction domains linked together
by glycine-serine linkers. The strains were constructed with varying numbers of proteins
linked and ratios of INO1 and MIOX in the scaffold; UDH was determined to be the most
active and thus did not need to be overexpressed in the scaffold. It was found that a 1:1 of
INO1 to MIOX ratio was the best, with four of each protein linked together. The glucaric
acid production increased five-fold compared to the nonscaffold system. The results
suggested that the improved production was not only due to increased stability of the MIOX
enzyme, but also because the substrate of the enzyme was in close proximity to the enzyme
(shown by high titers with increasing the number of INO1 linked to the scaffold). 80,85
Production of 1,4-Butanediol
Another example of a microbe producing a nonnatural compound is 1,4-butanediol (BDO).
BDO is a major commodity chemical used to annually make over 2.5 million tons of
valuable polymers such as polyesters, plastics, and spandex fibers. 86 It is currently produced
from petroleum-based substrates such as acetylene, butane, propylene, and butadiene.
Due to the instability of petroleum-based substrates, it is desirable to identify a metabolic
pathway for BDO production from biomass. The design of this novel metabolic pathway
was carried out by Genomatica using an in-house pathway prediction software called
SimPheny Biopathway Predictor, which elucidated all the potential pathways from the
E. coli central metabolism that could possibly produce BDO. The Biopathway Predictor
algorithm is based on transformations of functional groups by known chemistry, termed
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The algorithm identified over 10 000 pathways within four to six steps
for the synthesis of BDO from substrates acetyl-CoA,
reaction operators.
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α
-ketoglutarate, succinyl-CoA, and
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