Manipulating redox and ATP balancing for improved production of succinate in E. coli

Singh, Amarjeet; Soh, Keng Cher; Hatzimanikatis, Vassily; Gill, Ryan T.

doi:10.1016/j.ymben.2010.10.006

Cited by 114 publications

(88 citation statements)

References 30 publications

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“…Anaerobic processes can be operated with lower energy input but show formation of unwanted by-products [2]. Microorganisms form by-products to balance their redox metabolism [3] which reduces yields and can cause product inhibition [4][5][6]. An alternative may be offered by electro-fermentation, where an electrode provides an electron source or sink to overcome bottlenecks or drive fermentations towards particular desired products [7,8].…”

Section: Introductionmentioning

confidence: 99%

Anodic respiration of Pseudomonas putida KT2440 in a stirred-tank bioreactor

Hintermayer

Krömer

et al. 2016

Biochemical Engineering Journal

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Section: Introductionmentioning

confidence: 99%

Anodic respiration of Pseudomonas putida KT2440 in a stirred-tank bioreactor

Hintermayer

Krömer

et al. 2016

Biochemical Engineering Journal

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“…However, as the available ATP in anaerobic fermentation can be generated only from substrate-level phosphorylation, the biomass concentration in anaerobic fermentation is usually much lower than that in aerobic fermentation (2,7). Although a lower energy charge in anaerobic fermentation is beneficial for increasing the glycolysis rate (8)(9)(10), it has been shown that increasing the ATP concentration can improve protein synthesis and increase biomass flux during anaerobic fermentation (11,12). This suggests that factors affecting the intracellular ATP level may well be the targets for engineering to increase the efficiency of anaerobic fermentation processes.…”

mentioning

confidence: 99%

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

Zhao

Dong

et al. 2017

Appl Environ Microbiol

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Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli. PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA::Tn5 mutant in a screen for increased yield of an end product derived from pyruvate (n-butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n-butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products.KEYWORDS ATP, anaerobic condition, Escherichia coli, physiological role, PykA I ndustrial fermentation aims to produce valuable products from cheap feedstocks by utilizing the diverse functions of microbes. Products such as antibiotics, amino acids, and vitamins are mostly produced through aerobic fermentations, while products such as alcohols (ethanol, butanol, butanediol) are mainly produced through anaerobic fermentation (1-3). Organic acids such as lactic acid and succinic acid are also produced through anaerobic fermentation because higher yields could be obtained (4,5).During anaerobic fermentation, the reducing power is directed mostly to producing synthesis rather than to oxidization, resulting in a higher yield of target products. In addition, the low energy level due to the absence of oxidative phosphorylation often leads to a higher glycolysis rate, resulting in higher productivity (6). However, as the available ATP in anaerobic fermentation can be generated only from substrate-level phosphorylation, the biomass concentration in anaerobic fermentation is usually much

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“…To improve the production of succi- nate under these conditions, genetic modifications based on an energy-conserving strategy may be effective. Previous studies indicated that replacing glucose uptake from PTS with glucose permease to increase the PEP supply resulted in an increase net yield of ATP because of the dominant PEP carboxylation by PCK with ATP generation in E. coli (41)(42)(43)(44). In the near future, we will construct a further engineered strain based on the energy-conserving strategy and evaluate the impact of increase net ATP yield on succinate production under weakly acidic (pH Ͻ6.0) and anaerobic conditions.…”

Section: Discussionmentioning

confidence: 99%

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

Tajima

Yamamoto

Fukui

et al. 2015

Appl Environ Microbiol

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cLowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ⌬adhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ⌬adhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK؉PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production. There is an increasing interest in bio-based chemicals from renewable carbon sources because of the increasing price of petroleum and the negative impact of petrochemical production on the environment (1, 2). Succinate, a C 4 -dicarboxylic acid, which is an intermediate metabolite in the tricarboxylic acid cycle, is potentially useful as a chemical precursor for many commodity chemicals, such as ␥-butyrolactone, tetrahydrofuran, and 1,4-butanediol. These chemicals can, in turn, be converted into a wide variety of products, such as green solvents, pharmaceuticals, and biodegradable plastics (3, 4). Lowering the pH of microbial cultures has been considered a feasible approach to reducing the total costs of succinate production by limiting the use of alkali and acids in the fermentation and recovery processes (5, 6). Although anaerobic succinate production by Escherichia coli, Corynebacterium glutamicum, Actinobacillus succinogenes, and Anaerobiospirillum succiniciproducens has been studied with pHs ranging from 6.0 to 7.0 (7-9), few studies have focused on the effect of weakly acidic pH (pH Ͻ6.0) on succinate production by bacteria. This is because these bacteria are sensitive to acidic stress and are unable to grow and assimilate carbon sources effectively under weakly acidic conditions (10, 11). One potential solution to this limitation is to develop a new p...

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Manipulating redox and ATP balancing for improved production of succinate in E. coli

Cited by 114 publications

References 30 publications

Anodic respiration of Pseudomonas putida KT2440 in a stirred-tank bioreactor

Anodic respiration of Pseudomonas putida KT2440 in a stirred-tank bioreactor

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

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