Studies On Construction Of Escherichia Coli To Produce Succinate With Glycerol As Substrate And Metabolic Regulation In Fermentation

Succinate is a C4 dicarboxylic acid and an intermediate of the tricarboxylic acid cycle,and it plays very important roles in metabolism.It is one of the key platform chemicals and widely used in production of foods,pharmaceuticals,and biodegradable plastics.Traditionally,succinate is synthesized from petroleum-derived maleic anhydride,which is unrenewable.With the depletion of oil resources,researchers pay more attentions to the production of succinate by microbial fermentation of renewable resources.With the rapid development of biodiesel industry,glycerol has been produced as a major by-product of biodiesel,and the price of glycerol has declined rapidly.Glycerol can be converted to varieties of important products by the metabolism of some microbes under anaerobic conditions.E.coli is able to dissimilate glycerol under aerobic conditions,however,it grows extremely slowly on glycerol under anaerobic conditions.Because glycerol is a high reductive carbon source and in the absence of exogenous electron acceptors,NADH can not be effectively utilized and regenerated in E.coli,and the accumulation of NADH inhibited the metabolism of glycerol.Hence,it is necessary to make E.coli obtain the ability to rapidly consume glycerol and to produce the target products under anaerobic conditions.In this study,the metabolic pathway of wild-type MG1655 was reconstructed.The genes of ldhA encoding lactate dehydrogenase and pflB encoding pyruvate-formate lyase were knocked out,and the double mutation strain was named MLB.We used two-stage fermentation strategy to improve the glycerol consumption and succinate production.In order to further improve glycerol consumption and succinate production,the genes of key enzymes involved in succinate production were overexpressed in MLB,and it was found that overexpression of phosphoenolpyruvate carboxykinase(PCK)greatly improved glycerol consumption and succinate production.Enzyme activity assay showed that enzyme activity of isocitrate lyase(ICL),PCK,malic enzyme(ME)and malate dehydrogenase(MDH)were 5.9,4.0,1.5 and 1.5-fold higher with overexpression of PCK.In addition,overexpression of pyruvate carboxylate(PYC)also could improve succinate production,but its effect was a little less than that of PCK.Glycerol dissimilation pathways of E.coli include respiratory pathways(anaerobic pathway:GlpK-GlpABC and aerobic pathway:GIpK-GlpD)and fermentative pathway(GldA-DhaKLM).E.coli could not grow using glycerol as carbon source with the deletion of glpK.The deletion of dhaM and ppc were also failed to increase glycerol consumption when PCK was overexpressed,indicating the essential role of GldA-DhaKLM and PPC in glycerol metabolism of the two stage fermentation.In the aerobic phase of the two stage fermentation,the mixture of acetate and glycerol were used as carbon sources for culture.The experimental results showed that the mixed carbon sources could effectively improve glycerol consumption and succinate production in the anaerobic stage of two-stage fermentation.Oxygen is very important to regulation of the glycerol dissimilation pathways.In the induction phase of PCK overexpression,restriction of oxygen supply by adopting a suitable aeration rate could effectively increase glycerol consumption and succinate production in the subsequent anaerobic stage.When MLB/pTrc99a-pck was grown in an acetate-glycerol-based medium together with restriction of oxygen supply in the PCK induction phase,926.7 mmol·L-1(109.4 g·L-1)succinate was produced in the anaerobic stage with a yield of 0.90 mol·mol-1,significantly increased 9.1-fold compared with that ever reported succinate titer under anaerobic condition.The experimental results of qRT-PCR showed that the respiratory and fermentative pathways were all involved in glycerol dissimilation,and the GldA-DhaKLM pathway played the primary role in glycerol dissimilation in the anaerobic stage of two-stage fermentation.Crude glycerol was also used to produce succinate.However,crude glycerol could not effectively produce succinate because of the existence of toxic materials.After adsorption with activated carbon,the treated glycerol was used for two-stage fermentation,and the titer and yield of succinate achieved 566 mmol·L-1(66.8 g·L-1)and 0.97 mol·mol-1,respectively,suggesting the feasibility of constructed strains and methods of fermentation control.