Improving product yields on <scp>D</scp>‐glucose in Escherichia coli via knockout of pgi and zwf and feeding of supplemental carbon sources

Shiue, Eric; Brockman, Irene M.; Prather, Kristala L. J.

doi:10.1002/bit.25470

Cited by 36 publications

(25 citation statements)

References 23 publications

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“…The plasmid pRSFDCarb-IN-Udh, encoding enzymes MIPS and Udh, was modified from pRSFDuet-IN-Udh (29), where the kanamycin resistance gene was swapped for carbenicillin resistance using CPEC. The bla gene encoding carbenicillin resistance was PCR amplified from pET-Duet with primers Carb-C-RSFD and Carb-N-RSFD.…”

Section: Methodsmentioning

confidence: 99%

Layered dynamic regulation for improving metabolic pathway productivity in Escherichia coli

Doong

Gupta

Prather

2018

Proc. Natl. Acad. Sci. U.S.A.

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176

148

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Microbial production of value-added chemicals from biomass is a sustainable alternative to chemical synthesis. To improve product titer, yield, and selectivity, the pathways engineered into microbes must be optimized. One strategy for optimization is dynamic pathway regulation, which modulates expression of pathway-relevant enzymes over the course of fermentation. Metabolic engineers have used dynamic regulation to redirect endogenous flux toward product formation, balance the production and consumption rates of key intermediates, and suppress production of toxic intermediates until later in the fermentation. Most cases, however, have utilized a single strategy for dynamically regulating pathway fluxes. Here we layer two orthogonal, autonomous, and tunable dynamic regulation strategies to independently modulate expression of two different enzymes to improve production of D-glucaric acid from a heterologous pathway. The first strategy uses a previously described pathway-independent quorum sensing system to dynamically knock down glycolytic flux and redirect carbon into production of glucaric acid, thereby switching cells from "growth" to "production" mode. The second strategy, developed in this work, uses a biosensor for -inositol (MI), an intermediate in the glucaric acid production pathway, to induce expression of a downstream enzyme upon sufficient buildup of MI. The latter, pathway-dependent strategy leads to a 2.5-fold increase in titer when used in isolation and a fourfold increase when added to a strain employing the former, pathway-independent regulatory system. The dual-regulation strain produces nearly 2 g/L glucaric acid, representing the highest glucaric acid titer reported to date in K-12 strains.

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Section: Methodsmentioning

confidence: 99%

Layered dynamic regulation for improving metabolic pathway productivity in Escherichia coli

Doong

Gupta

Prather

2018

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

176

148

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“…More consistent success with chromosomal modifications in the K strains led to the initial construction of the Pfk-I control system in that background, but additional improvements in glucaric acid titer, both with and without Pfk knockdown, can likely be achieved by transferring the genetic modifications of IB1486 to an E. coli B strain. In previous work, wild-type BL21 has outperformed MG1655 containing the same pathway genes (Moon et al, 2009; Raman et al, 2014; Shiue et al, 2015). …”

Section: Discussionmentioning

confidence: 94%

“…By eliminating the pathways for glucose catabolism in the production strain, and feeding alternative carbon sources, higher yields of glucaric acid from glucose could be achieved (Shiue et al, 2015). However, the rate of glucose uptake in this K-12 host strain was quite slow, especially in minimal medium, and its use was limited to mixed sugar substrates.…”

Section: Introductionmentioning

confidence: 99%

“…While gene knockouts provide a static solution for redirecting fluxes in the cell (Kogure et al, 2007; Shiue et al, 2015), under many conditions, it may be advantageous to develop cells where dynamic changes in enzyme levels can be used to switch between substrate consumption for biomass formation and substrate conversion into product. Dynamic control of key enzymes can be used to facilitate more rapid initial accumulation of biomass, overcoming potential reductions in growth rate, and can eliminate the need for supplementation of the medium or addition of secondary carbon sources required with some gene knockouts (Anesiadis et al, 2008; Gadkar et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

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Improvement of glucaric acid production in E. coli via dynamic control of metabolic fluxes

Reizman

Stenger

Reisch

et al. 2015

Metabolic Engineering Communications

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D-glucaric acid can be used as a building block for biopolymers as well as in the formulation of detergents and corrosion inhibitors. A biosynthetic route for production in E. coli has been developed (Moon et al., 2009), but previous work with the glucaric acid pathway has indicated that competition with endogenous metabolism may limit carbon flux into the pathway. Our group has recently developed an E. coli strain where phosphofructokinase (Pfk) activity can be dynamically controlled and demonstrated its use for improving yields and titers of the glucaric acid precursor myo-inositol on glucose minimal medium. In this work, we have explored the further applicability of this strain for glucaric acid production in a supplemented medium more relevant for scale-up studies, both under batch conditions and with glucose feeding via in situ enzymatic starch hydrolysis. It was found that glucaric acid titers could be improved by up to 42% with appropriately timed knockdown of Pfk activity during glucose feeding. The glucose feeding protocol could also be used for reduction of acetate production in the wild type and modified E. coli strains.

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“…[17] In addition, Udh is able to convert d-glucuronic acid to d-glucaric acid. [18] Recently,t hree additional Udhs that could serve as alternative enzymes in our whole-cellb iocatalyst werec haracterized. [19] We chose the Udh enzyme from A. tumefaciens because of its high activity towards d-galacturonic acid (the Michaelis-Menten constants for d-galacturonica cida re K m = 0.5 mM, V max = 124 Umg À1 ).…”

Section: Development Of Recombinant E Coli-basedb Iocatalystmentioning

confidence: 99%

Production of Adipic Acid from Sugar Beet Residue by Combined Biological and Chemical Catalysis

Zhang¹,

Su³

et al. 2016

ChemCatChem

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Adipic acid is one of the most important industrial dicarboxylic acids and is used mainly as a precursor to nylon‐6,6. Currently, commercial adipic acid is produced primarily from benzene by a chemical route that is associated with environmental, health, and safety concerns. Herein, we report a new process to produce adipic acid from an inexpensive renewable feedstock, sugar beet residue by combining an engineered Escherichia coli strain and Re‐based chemical catalysts. The engineered E. coli converted d‐galacturonic acid to mucic acid, which was precipitated easily with acid, and the mucic acid was further converted to adipic acid by a deoxydehydration reaction catalyzed by an oxorhenium complex followed by a Pt/C‐catalyzed hydrogenation reaction under mild conditions. A high selectivity to the free acid products was achieved by tuning the acidity of the Re‐based catalysts. Finally, adipic acid was produced directly from sugar beet residue that was hydrolyzed enzymatically with engineered E. coli and two chemical catalysts in a yield of 8.4 %, which signifies a new route for the production of adipic acid.

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Improving product yields on D‐glucose in Escherichia coli via knockout of pgi and zwf and feeding of supplemental carbon sources

Cited by 36 publications

References 23 publications

Layered dynamic regulation for improving metabolic pathway productivity in Escherichia coli

Layered dynamic regulation for improving metabolic pathway productivity in Escherichia coli

Improvement of glucaric acid production in E. coli via dynamic control of metabolic fluxes

Production of Adipic Acid from Sugar Beet Residue by Combined Biological and Chemical Catalysis

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