Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity

The CreBC (carbon source-responsive) two-component regulation system of Escherichia coli affects a number of functions, including intermediary carbon catabolism. The impacts of different creC mutations (a ⌬creC mutant and a mutant carrying the constitutive creC510 allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carrying creC510 and shown to be affected by oxygen availability. The ⌬creC strain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P) ؉ ratios showed that the creC510 strain had a more reduced intracellular redox state, while the opposite was observed for the ⌬creC mutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase from E. coli and a NADH-forming formate dehydrogenase from Candida boidinii in the ⌬creC mutant further increased the yield of succinate on glucose. Interestingly, the elimination of ackA and adhE did not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network of E. coli make it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate.T he survival of an organism depends, at least in part, on its ability to sense and respond to changes in the environment. In bacteria, global regulators control the transcription of genes in response to specific external stimuli and metabolic signals, finely tuning different aspects of their physiology to overcome environmental challenges. In Escherichia coli, seven global regulators (ArcA, Crp, Fis, Fnr, Ihf, Lrp, and NarL) directly modulate the expression of about one-half of all genes (1). This facultative aerobe is able to adapt its metabolism to different oxygen availability conditions through the concerted actions of a network of regulators, including the global regulators ArcAB and Fnr (2-4). These regulators affect many metabolic pathways, allowing the cells to reach an adequate redox balance under any given conditions. There is a very close association between carbon and electron flows, and even small differences in oxygen availability have been observed to elicit profound effects on the distribution of carbon fluxes (5).CreBC (for carbon source responsive) is a global sensing and regulation system affecting genes involved in a variety of functions, including enzymes of intermediary catabolism (6). Previous studies have shown that the creABCD operon is activated (i) during growth in minimal mediu...

Section: Discussionmentioning

confidence: 99%

Section: Ec and Fdh1mentioning

confidence: 99%

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

Godoy

Nikel

Gomez

et al. 2016

“…One of the most beneficial approaches was based on the design of a metabolic pathway which is functionally similar to that of natural producers from the Pasteurellaceae family, such as A. succinogenes (64). Another approach used a combination of production pathways, such as the reductive tricarboxylic acid (TCA) cycle with the glyoxylate shunt (45) and the introduction of a heterologous carboxylation reaction (53). A further interesting approach for the optimization of succinate production with E. coli combined rational metabolic engineering with directed evolution techniques (23).…”

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confidence: 99%

Toward Homosuccinate Fermentation: Metabolic Engineering of Corynebacterium glutamicum for Anaerobic Production of Succinate from Glucose and Formate

Litsanov

Brocker

Bott

2012

189

131

ABSTRACTPrevious studies have demonstrated the capability ofCorynebacterium glutamicumfor anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (ΔcatΔpqoΔpta-ackAΔldhA), was constructed. Chromosomal integration of the pyruvate carboxylase genepycP458Sinto BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, thefdhgene fromMycobacterium vaccae, coding for an NAD+-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase genegapA. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-gapaccumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose).

“…Lin et al (17) showed that succinic acid could be aerobically produced by utilizing the glyoxylate cycle in E. coli. Also, Sanchez et al (23) reported impressive results for anaerobic production of succinic acid with a cumulative yield of 160 mM of succinic acid from 100 mM glucose in 24 h by repeated feeding fermentation of recombinant E. coli.…”

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confidence: 99%

Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation

Lee

Kim³

et al. 2005

277

136

Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.Succinic acid is one of the fermentation products of anaerobic metabolism as well as an intermediate of the tricarboxylic acid cycle. It has been used as a precursor for various chemicals, a food additive, an ion chelator, and a supplement to pharmaceuticals (34). Succinic acid has mostly been produced chemically from maleic anhydride. Recently, fermentative production of succinic acid has been receiving much research attention, as several bacteria can produce succinic acid as a major fermentation product. The naturally isolated obligate anaerobe Anaerobiospirillum succiniciproducens (6) and facultative anaerobes belonging to the family Pasteurellaceae, such as Actinobacillus succinogenes (10) and Mannheimia succiniciproducens (16), have been shown to be able to produce succinic acid efficiently.Escherichia coli also produces succinic acid but as a minor fermentation product. E. coli prefers to produce much more acetic acid, formic acid, lactic acid, and ethanol rather than succinic acid during anaerobic fermentation. Thus, it is necessary to redirect metabolic fluxes for increasing succinic acid production as well as reducing formation of other metabolites. Toward this goal, an ldhA and pfl double mutant E. coli strain NZN111 was developed to block the formation of lactic, acetic, and formic acids (2). However, cell growth was relatively slow, possibly due to the inactivation of enzymes involved in pyruvate dissimilation (3). Chatterjee and coworkers (4) reported that an additional ptsG gene mutation recovered the growth of NZN111 to some extent. However, acetic acid, formic acid, and ethanol were still...