Manipulating pyruvate to acetyl-CoA conversion in Escherichia coli for anaerobic succinate biosynthesis from glucose with the yield close to the stoichiometric maximum
“…Succinate synthesis through the reductive branch of the TCA cycle with the CO 2 fixation is an attractive option under anaerobic conditions [ 4 , 24 ]. However, the maximum theoretical yield of succinate through the glycolysis and reductive TCA cycle is 1 mol/mol glucose resulting from the NADH limitation [ 14 , 24 , 38 ]. It is well known that the excess NADH would upset the flux distribution among the end products: succinate, acetate and ethanol [ 9 ].…”
Section: Discussionmentioning
confidence: 99%
“…In addition, the glyoxylate pathway can also form succinate, which is essentially active under aerobic condition, and conserves NADH consumption relative to reductive TCA branch in succinate formation [ 14 , 15 ]. It has been reported that succinate yield can reach its maximum level 1.714 mol/mol glucose by redirecting 71.4 % of carbon flows to the reductive TCA branch and 28.6 % of the carbon flows to the glyoxylate pathway [ 10 ], while employment of the glyoxylate bypass for succinate synthesis is accompanied by the CO 2 loss [ 24 ]. Fig.…”
BackgroundSuccinate has been identified by the U.S. Department of Energy as one of the top 12 building block chemicals, which can be used as a specialty chemical in the agricultural, food, and pharmaceutical industries. Escherichia coli are now one of the most important succinate producing candidates. However, the stoichiometric maximum succinate yield under anaerobic conditions through the reductive branch of the TCA cycle is restricted by NADH supply in E. coli.ResultsIn the present work, we report a rational approach to increase succinate yield by regulating NADH supply via pentose phosphate (PP) pathway and enhancing flux towards succinate. The deregulated genes zwf243 (encoding glucose-6-phosphate dehydrogenase) and gnd361 (encoding 6-phosphogluconate dehydrogenase) involved in NADPH generation from Corynebacterium glutamicum were firstly introduced into E. coli for succinate production. Co-expression of beneficial mutated dehydrogenases, which removed feedback inhibition in the oxidative part of the PP pathway, increased succinate yield from 1.01 to 1.16 mol/mol glucose. Three critical genes, pgl (encoding 6-phosphogluconolactonase), tktA (encoding transketolase) and talB (encoding transaldolase) were then overexpressed to redirect more carbon flux towards PP pathway and further improved succinate yield to 1.21 mol/mol glucose. Furthermore, introducing Actinobacillus succinogenes pepck (encoding phosphoenolpyruvate carboxykinase) together with overexpressing sthA (encoding soluble transhydrogenase), further increased succinate yield to 1.31 mol/mol glucose. In addition, removing byproduct formation through inactivating acetate formation genes ackA-pta and heterogenously expressing pyc (encoding pyruvate carboxylase) from C. glutamicum led to improved succinate yield to 1.4 mol/mol glucose. Finally, synchronously overexpressing dcuB and dcuC encoding succinate exporters enhanced succinate yield to 1.54 mol/mol glucose, representing 52 % increase relative to the parent strain and amounting to 90 % of the strain-specific stoichiometric maximum (1.714 mol/mol glucose).ConclusionsIt’s the first time to rationally regulate pentose phosphate pathway to improve NADH supply for succinate synthesis in E. coli. 90 % of stoichiometric maximum succinate yield was achieved by combining further flux increase towards succinate and engineering its export. Regulation of NADH supply via PP pathway is therefore recommended for the production of products that are NADH-demanding in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0536-1) contains supplementary material, which is available to authorized users.
“…Succinate synthesis through the reductive branch of the TCA cycle with the CO 2 fixation is an attractive option under anaerobic conditions [ 4 , 24 ]. However, the maximum theoretical yield of succinate through the glycolysis and reductive TCA cycle is 1 mol/mol glucose resulting from the NADH limitation [ 14 , 24 , 38 ]. It is well known that the excess NADH would upset the flux distribution among the end products: succinate, acetate and ethanol [ 9 ].…”
Section: Discussionmentioning
confidence: 99%
“…In addition, the glyoxylate pathway can also form succinate, which is essentially active under aerobic condition, and conserves NADH consumption relative to reductive TCA branch in succinate formation [ 14 , 15 ]. It has been reported that succinate yield can reach its maximum level 1.714 mol/mol glucose by redirecting 71.4 % of carbon flows to the reductive TCA branch and 28.6 % of the carbon flows to the glyoxylate pathway [ 10 ], while employment of the glyoxylate bypass for succinate synthesis is accompanied by the CO 2 loss [ 24 ]. Fig.…”
BackgroundSuccinate has been identified by the U.S. Department of Energy as one of the top 12 building block chemicals, which can be used as a specialty chemical in the agricultural, food, and pharmaceutical industries. Escherichia coli are now one of the most important succinate producing candidates. However, the stoichiometric maximum succinate yield under anaerobic conditions through the reductive branch of the TCA cycle is restricted by NADH supply in E. coli.ResultsIn the present work, we report a rational approach to increase succinate yield by regulating NADH supply via pentose phosphate (PP) pathway and enhancing flux towards succinate. The deregulated genes zwf243 (encoding glucose-6-phosphate dehydrogenase) and gnd361 (encoding 6-phosphogluconate dehydrogenase) involved in NADPH generation from Corynebacterium glutamicum were firstly introduced into E. coli for succinate production. Co-expression of beneficial mutated dehydrogenases, which removed feedback inhibition in the oxidative part of the PP pathway, increased succinate yield from 1.01 to 1.16 mol/mol glucose. Three critical genes, pgl (encoding 6-phosphogluconolactonase), tktA (encoding transketolase) and talB (encoding transaldolase) were then overexpressed to redirect more carbon flux towards PP pathway and further improved succinate yield to 1.21 mol/mol glucose. Furthermore, introducing Actinobacillus succinogenes pepck (encoding phosphoenolpyruvate carboxykinase) together with overexpressing sthA (encoding soluble transhydrogenase), further increased succinate yield to 1.31 mol/mol glucose. In addition, removing byproduct formation through inactivating acetate formation genes ackA-pta and heterogenously expressing pyc (encoding pyruvate carboxylase) from C. glutamicum led to improved succinate yield to 1.4 mol/mol glucose. Finally, synchronously overexpressing dcuB and dcuC encoding succinate exporters enhanced succinate yield to 1.54 mol/mol glucose, representing 52 % increase relative to the parent strain and amounting to 90 % of the strain-specific stoichiometric maximum (1.714 mol/mol glucose).ConclusionsIt’s the first time to rationally regulate pentose phosphate pathway to improve NADH supply for succinate synthesis in E. coli. 90 % of stoichiometric maximum succinate yield was achieved by combining further flux increase towards succinate and engineering its export. Regulation of NADH supply via PP pathway is therefore recommended for the production of products that are NADH-demanding in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0536-1) contains supplementary material, which is available to authorized users.
“…Under anaerobic conditions, the dissimilated carbon from glycolysis enters the TCA cycle in the form of phosphoenolpyruvate (PEP), subjecting to carboxylation to form oxaloacetate, and then proceeds with reactions in the reductive TCA branch to generate succinate as a final product (Cheng, Wang, Zeng, & Zhang, 2013). Although the reductive TCA branch can potentially yield high‐level succinate, this pathway is generally unfavorable as it is limited by the availability of reducing equivalents (i.e., NADH; Skorokhodova, Morzhakova, Gulevich, & Debabov, 2015). However, under aerobic conditions, acetyl‐CoA derived from glycolysis enters the oxidative TCA cycle to generate succinate as a cycle intermediate (Thakker, Martínez, San, & Bennett, 2012).…”
A propanologenic (i.e., 1‐propanol‐producing) bacterium Escherichia coli strain was previously derived by activating the genomic sleeping beauty mutase (Sbm) operon. The activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl‐CoA node to form propionyl‐CoA and its derived metabolites of 1‐propanol and propionate. In this study, we targeted several TCA cycle genes encoding enzymes near the succinyl‐CoA node for genetic manipulation to identify the individual contribution of the carbon flux into the Sbm pathway from the three TCA metabolic routes, that is, oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. For the control strain CPC‐Sbm, in which propionate biosynthesis occurred under relatively anaerobic conditions, the carbon flux into the Sbm pathway was primarily derived from the reductive TCA branch, and both succinate availability and the SucCD‐mediated interconversion of succinate/succinyl‐CoA were critical for such carbon flux redirection. Although the oxidative TCA cycle normally had a minimal contribution to the carbon flux redirection, the glyoxylate shunt could be an alternative and effective carbon flux contributor under aerobic conditions. With mechanistic understanding of such carbon flux redirection, metabolic strategies based on blocking the oxidative TCA cycle (via ∆sdhA mutation) and deregulating the glyoxylate shunt (via ∆iclR mutation) were developed to enhance the carbon flux redirection and therefore propionate biosynthesis, achieving a high propionate titer of 30.9 g/L with an overall propionate yield of 49.7% upon fed‐batch cultivation of the double mutant strain CPC‐Sbm∆sdhA∆iclR under aerobic conditions. The results also suggest that the Sbm pathway could be metabolically active under both aerobic and anaerobic conditions.
“…Apart from anaerobic strategies of succinate production also aerobic and microaerobic strategies have been applied. In these strategies the production of acetate is prohibited, accompanied by a block in the tricarboxylic acid cycle and an enhancement of the glyoxylate shunt [ 10 – 13 ]. Aerobic strategies were motivated to overcome the severe growth problems of succinate production strains under anaerobic conditions.…”
BackgroundGlucose is the main carbon source of E. coli and a typical substrate in production processes. The main glucose uptake system is the glucose specific phosphotransferase system (Glc-PTS). The PTS couples glucose uptake with its phosphorylation. This is achieved by the concomitant conversion of phosphoenolpyruvate (PEP) to pyruvate. The Glc-PTS is hence unfavorable for the production of succinate as this product is derived from PEP.ResultsWe studied, in a systematic manner, the effect of knocking out the Glc-PTS and of replacing it with the glucose facilitator (Glf) of Zymomonas mobilis on succinate yield and productivity. For this study a set of strains derived from MG1655, carrying deletions of ackA-pta, adhE and ldhA that prevent the synthesis of competing fermentation products, were constructed and tested in two-stage cultivations. The data show that inactivation of the Glc-PTS achieved a considerable increase in succinate yield and productivity. On the other hand, aerobic growth of this strain on glucose was strongly decreased. Expression of the alternative glucose transporter, Glf, in this strain enhanced aerobic growth but productivity and yield under anaerobic conditions were slightly decreased. This decrease in succinate yield was accompanied by pyruvate production. Yield could be increased in both Glc-PTS mutants by overexpressing phosphoenolpyruvate carboxykinase (Pck). Productivity on the other hand, was decreased in the strain without alternative glucose transporter but strongly increased in the strain expressing Glf. The experiments were complemented by flux balance analysis in order to check the observed yields against the maximal theoretical yields. Furthermore, the phosphorylation state of EIIAGlc was determined. The data indicate that the ratio of PEP to pyruvate is correlating with pyruvate excretion. This ratio is affected by the PTS reaction as well as by further reactions at the PEP/pyruvate node.ConclusionsThe results show that for optimization of succinate yield and productivity it is not sufficient to knock out or introduce single reactions. Rather, balancing of the fluxes of central metabolism most important at the PEP/pyruvate node is important.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0980-1) contains supplementary material, which is available to authorized users.
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