Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production

Wong, Matthew S.; Wu, Steven C.; Causey, Thomas B.; Bennett, George N.; San, Ka‐Yiu

doi:10.1016/j.ymben.2007.10.003

Cited by 58 publications

(38 citation statements)

References 29 publications

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“…For example, by deletion of the pstHI operon in E. coli GJT 001, Wong and coworkers increased protein production yield by 25-fold in a batch bioreactor. This high productivity was attributed to low acetate accumulation, although cell growth was compromised [124]. The E. coli B strain was Wrst designed and engineered by Studier and MoVatt, and has been a model strain for studying phage sensitivity, restriction-modiWcation systems, bacterial evolution and recombinant protein expression in laboratories as well as in the biotech industry [100].…”

Section: Expression Considerations For Recombinant Therapeutics In Ementioning

confidence: 99%

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

Huang

Lin

Yang

2012

Journal of Industrial Microbiology and Biotechnology

363

248

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Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics.

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Section: Expression Considerations For Recombinant Therapeutics In Ementioning

confidence: 99%

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

Huang

Lin

Yang

2012

Journal of Industrial Microbiology and Biotechnology

363

248

View full text Add to dashboard Cite

show abstract

“…Consequently, a bottleneck occurs at the pyruvate node, and pyruvate is converted to acetate instead of being processed to acetyl-CoA. By deleting one of the phosphotransferase system (PTS) genes, e.g., ptsG, ptsH, or ptsI, the uptake through the major glucose transporter is severely impeded, resulting in a reduced glycolytic flux and a reduced acetate production [29, 148,178]. However, one of the major drawbacks of this approach is the strong reduction in growth rate.…”

Section: Reduction Of Acetate Formation By Genetic Engineeringmentioning

confidence: 99%

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Waegeman¹,

Soetaert²

2011

J Ind Microbiol Biotechnol

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Different hosts have been used for recombinant protein production, ranging from simple bacteria, such as Escherichia coli and Bacillus subtilis, to more advanced eukaryotes as Saccharomyces cerevisiae and Pichia pastoris, to very complex insect and animal cells. All have their advantages and drawbacks and not one seems to be the perfect host for all purposes. In this review we compare the characteristics of all hosts used in commercial applications of recombinant protein production, both in the area of biopharmaceuticals and industrial enzymes. Although the bacterium E. coli remains a very often used organism, several drawbacks limit its possibility to be the first-choice host. Furthermore, we show what E. coli strains are typically used in high cell density cultivations and compare their genetic and physiological differences. In addition, we summarize the research efforts that have been done to improve yields of heterologous protein in E. coli, to reduce acetate formation, to secrete the recombinant protein into the periplasm or extracellular milieu, and to perform post-translational modifications. We conclude that great progress has been made in the incorporation of eukaryotic features into E. coli, which might allow the bacterium to regain its first-choice status, on the condition that these research efforts continue to gain momentum.

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“…Alternatively, the fermentative pathway is functional particularly under anaerobic conditions, and it includes glycerol dehydrogenase (encoded by gldA) and a PTS-like phosphorelay system (with various enzymes encoded by dhaKLM, ptsI, and hpr) for phosphorylation of dihydroxyacetone (DHA) using phosphoenolpyruvate (PEP) as a phosphate donor [12,17]. It is generally perceived that uptake and dissimilation of the major carbon source during E. coli cultivation could critically affect the metabolite profile and even culture performance, such as titer/yield of recombinant proteins and target metabolites [4,7,25,33,42]. Herein, we investigated these genetic effects on Sbm-mediated production of propionate by knocking out or overexpressing various genes involved in glycerol dissimilation.…”

Section: Introductionmentioning

confidence: 99%

Engineering Escherichia coli for high-level production of propionate

Akawi

Srirangan

Liu

et al. 2015

Journal of Industrial Microbiology and Biotechnology

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Mounting environmental concerns associated with the use of petroleum-based chemical manufacturing practices has generated significant interest in the development of biological alternatives for the production of propionate. However, biological platforms for propionate production have been limited to strict anaerobes, such as Propionibacteria and select Clostridia. In this work, we demonstrated high-level heterologous production of propionate under microaerobic conditions in engineered Escherichia coli. Activation of the native Sleeping beauty mutase (Sbm) operon not only transformed E. coli to be propionogenic (i.e., propionate-producing) but also introduced an intracellular "flux competition" between the traditional C2-fermentative pathway and the novel C3-fermentative pathway. Dissimilation of the major carbon source of glycerol was identified to critically affect such "flux competition" and, therefore, propionate synthesis. As a result, the propionogenic E. coli was further engineered by inactivation or overexpression of various genes involved in the glycerol dissimilation pathways and their individual genetic effects on propionate production were investigated. Generally, knocking out genes involved in glycerol dissimilation (except glpA) can minimize levels of solventogenesis and shift more dissimilated carbon flux toward the C3-fermentative pathway. For optimal propionate production with high C3:C2-fermentative product ratios, glycerol dissimilation should be channeled through the respiratory pathway and, upon suppressed solventogenesis with minimal production of highly reduced alcohols, the alternative NADH-consuming route associated with propionate synthesis can be critical for more flexible redox balancing. With the implementation of various biochemical and genetic strategies, high propionate titers of more than 11 g/L with high yields up to 0.4 g-propionate/g-glycerol (accounting for ~50 % of dissimilated glycerol) were achieved, demonstrating the potential for industrial application. To our knowledge, this represents the most effective engineered microbial system for propionate production with titers and yields comparable to those achieved by anaerobic batch cultivation of various native propionate-producing strains of Propionibacteria.

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Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production

Cited by 58 publications

References 29 publications

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Engineering Escherichia coli for high-level production of propionate

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