Engineering <i>Escherichia coli</i> for propionic acid production through the Wood–Werkman cycle

Gonzalez-Garcia, R. Axayacatl; McCubbin, Tim; Turner, Mark S.; Nielsen, Lars K.; Marcellin, Esteban

doi:10.1002/bit.27182

Cited by 20 publications

(16 citation statements)

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“…This advantage comes with the limitation to operate in anaerobic conditions considering one of the enzymes within the cycle (mutase) is inhibited by oxygen, therefore limiting the availability of ATP required to support the synthesis of polyketides. However, the Wood-Werkman cycle provides an alternative for the generation of energy by allowing the operation of the electron transport chain by using fumarate as terminal electron acceptor [17]. Here, we tested whether that energetic surplus could make it possible to sustain polyketide production during a single-stage anaerobic process.…”

Section: Discussionmentioning

confidence: 99%

“…We evaluated the feasibility of using a synthetic Wood-Werkman cycle as a source of propionyl-CoA and S-methylmalonyl-CoA, the intermediaries required for the synthesis of 6-dEB. Previously, we demonstrated that under anaerobic conditions, E. coli can produce propionate via the Wood-Werkman cycle using a plasmid system which encodes a single operon (10 kb) under the control of the P BAD promoter [17]. We decided to use the same expression system in 6-dEB producer strain, E. coli 207-3.…”

Section: Assembling a Three-plasmid System For The Production Of 6-deb In E Colimentioning

confidence: 99%

“…We have recently shown that E. coli can produce propionate via the Wood-Werkman cycle [17]. Through the cycle, pyruvate is carboxylated to oxaloacetate via a methylmalonyl-CoA carboxyltransferase (MTCA, MTCB, MTCC, MTCD) and then reduced to succinate through the reductive tricarboxylic acid cycle.…”

Section: Introductionmentioning

confidence: 99%

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Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

2020

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Polyketides are a remarkable class of natural products with diverse functional and structural diversity. The class includes many medicinally important molecules with antiviral, antimicrobial, antifungal and anticancer properties. Native bacterial, fungal and plant hosts are often difficult to cultivate and coax into producing the desired product. As a result, Escherichia coli has been used for the heterologous production of polyketides, with the production of 6-deoxyerythronolide B (6-dEB) being the first example. Current strategies for production in E. coli require feeding of exogenous propionate as a source for the precursors propionyl-CoA and S-methylmalonyl-CoA. Here, we show that heterologous polyketide production is possible from glucose as the sole carbon source. The heterologous expression of eight genes from the Wood-Werkman cycle found in Propionibacteria, in combination with expression of the 6-dEB synthases DEBS1, DEBS2 and DEBS3 resulted in 6-dEB formation from glucose as the sole carbon source. Our results show that the Wood-Werkman cycle provides the required propionyl-CoA and the extender unit S-methylmalonyl-CoA to produce up to 0.81 mg/L of 6-dEB in a chemically defined media.

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Section: Discussionmentioning

confidence: 99%

Section: Assembling a Three-plasmid System For The Production Of 6-deb In E Colimentioning

confidence: 99%

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Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

2020

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“…Their combination with glucose and glycerol as carbon sources results in increased acid concentration and higher productivity ( 70 ). Productivity can also be improved by the application of metabolic engineering, for which Escherichia coli is the most widely used host ( 71 ). Phosphoenolpyruvate carboxylase enzyme from Escherichia coli has been cloned into Propionibacterium freudenreichii .…”

Section: Process Parameters Influencing Microbial Production and Prodmentioning

confidence: 99%

Propionic Acid

Ranaei

Pilevar

Khaneghah

et al. 2020

Food technol. biotechnol. (Online)

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During the past years, there has been a growing interest in the bioproduction of propionic acid by Propionibacterium. One of the major limitations of the existing models lies in their low productivity yield. Hence, many strategies have been proposed in order to circumvent this obstacle. This article provides a comprehensive synthesis and review of important biotechnological aspects of propionic acid production as a common ingredient in food and biotechnology industries. We first discuss some of the most important production processes, mainly focusing on biological production. Then, we provide a summary of important propionic acid producers, including Propionibacterium freudenreichii and Propionibacterium acidipropionici, as well as a wide range of reported growth/production media. Furthermore, we describe bioprocess variables that can have impact on the production yield. Finally, we propose methods for the extraction and analysis of propionic acid and put forward strategies for overcoming the limitations of competitive microbial production from the economical point of view. Several factors influence the propionic acid concentration and productivity such as culture conditions, type and bioreactor scale; however, the pH value and temperature are the most important ones. Given that there are many reports about propionic acid production from glucose, whey permeate, glycerol, lactic acid, hemicelluloses, hydrolyzed corn meal, lactose, sugarcane molasses and enzymatically hydrolyzed whole wheat flour, only few review articles evaluate biotechnological aspects, i.e. bioprocess variables.

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“…Currently, pathway design and optimization are essential to obtain microbial cell factories with excellent performance for industrial production of commercial chemicals ( 15 , 16 ). A series of attempts have been made to construct novel producers for biobased chemicals as the technologies in support of synthesizing new materials and manipulating genes have matured ( 17 – 19 ). Indeed, some strategies have been applied to optimize the biosynthetic pathways, which consist of large numbers of genes, or to reprogram gene expression to manipulate complex phenotypes.…”

Section: Introductionmentioning

confidence: 99%

Multiplex Design of the Metabolic Network for Production of l -Homoserine in Escherichia coli

Liu

Zhang

Yao

et al. 2020

Appl Environ Microbiol

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L-homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. In this study, systematic metabolic engineering was applied to target E. coli W3110 for production of L-homoserine. Initially, the basic L-homoserine producer was engineered through the strategies overexpressing thrA (encoding homoserine dehydrogenase), removing the degradative and competitive pathways by knocking out metA (encoding homoserine O-succinyltransferase) and thrB (encoding homoserine kinase), reinforcing the transport system and redirecting the carbon flux by deleting iclR (encoding isocitrate lyase regulator). The resulted strain constructed by these strategies yielded 3.21 g/L of L-homoserine in batch cultures. Moreover, based on CRISPR-Cas9/dCas9 mediated gene repression for 50 genes, the iterative genetic modifications of biosynthesis pathways stepwise improved the L-homoserine yield. The rational integration of glucose uptake and recovery of L-glutamate increased L-homoserine production to 7.25 g/L in shake-flask cultivation. Furthermore, the intracellular metabolic analysis further provided target for strain modification by introducing the anaplerotic route afforded by pyruvate carboxylase to oxaloacetate formation, which resulted in accumulating 8.54 g/L L-homoserine (0.33 g/g glucose, 62.4% of the maximum theoretical yield) in shake-flask cultivation. Finally, rationally designed strain gave 37.57 g/L L-homoserine under fed-batch fermentation, with a yield of 0.31 g/g glucose. Importance In this study, the bottlenecks that sequentially limited the L-homoserine biosynthesis were identified and resolved, based on the rational and efficient metabolic engineering strategies coupled with CRISPRi-based systematic analysis. The metabolomics data largely expanded the understanding of metabolic effects and revealed relevant targets for further modification to achieve a preferable performance. The systematic analysis strategy as well as metabolomics analysis can be used to rationally design cell factories for the production of highly valuable chemicals.

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Engineering Escherichia coli for propionic acid production through the Wood–Werkman cycle

Cited by 20 publications

References 50 publications

Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

Propionic Acid

Multiplex Design of the Metabolic Network for Production of l -Homoserine in Escherichia coli

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