The Research Of High-yield Succinate Production By Systematically Regulating Multiple Metabolic Pathways In Escherichia Coli

Succinate and its derivatives are widely used as specialty chemicals in the field of food,pharmaceutical,and cosmetic industries.Recent studies on metabolic engineering of Escherichia coli for succinate production have been carried out due to the ease of genetic manipulation coupled to its rapid growth rate,standardized cultivation techniques,and inexpensive media.While the maximum theoretical succinate yield under anaerobic conditions through the glycolysis and reductive TCA cycle is limited to 1 mol/mol glucose due to NADH limitation.In this study,we firstly report that the stoichiometric maximum succinate yield of 1.714 mol/mol glucose can be obtained if the carbon flux ratio between PP pathway and glycolysis is 6:1.Based on this theoretical guidance,we strategically regulating multiple metabolic pathways for succinate production.Systems metabolic engineering including overexpressing key genes in the oxidative part of the PP pathway and carboxylation pathway,improving anaerobic glucose transport velocity and activating anaerobic C4-dicarboxylates efflux transportation were followed to increase 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.The deregulated genes zwf243-gnd361 from Corynebacterium glutamicum were firstly introduced into E.coli for succinate production.We compared the effects of overexpression of native zwf-gnd and deregulated zwf243-gnd361 on succinate production and NADH/NAD+ ratio.It was found that overexpression of deregulated zwf243-gnd361 was more efficient to achieve a high succinate yield with significantly increased intracellular NADH availability.Three critical genes,pgl,tkt A and tal B were then overexpressed to redirect more carbon flux towards PP pathway and further improved succinate yield to 1.21 mol/mol glucose.Introducing Actinobacillus succinogenes pepck,overexpressing sth A and inactivating acetate formation genes ack A-pta,the succinate yield of resultant strain WSA150 increased to 1.36 mol/mol glucose.The specific glucose uptake rate increased by 12% after galp overexpression.Heterogenously expressing pyc from C.glutamicum led to improved succinate yield to 1.4 mol/mol glucose(amounting to 82% of the maximum stoichiometric yield)with reducing byproduct pyruvate by half.We additionally targeted to activate anaerobic C4-dicarboxylates efflux transportation to maximize succinate yield.Succinate molar yield reached the value of 1.53 mol/mol glucose after dcu B overexpression,which is 89% of stoichiometric maximum.Overexpressing dcu C increased succinate yield to 1.46 mol/mol glucose,amounting to 85% of stoichiometric maximum yield.Synchronously overexpressing dcu B and dcu C 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.Finally,deleting the repressor protein binding site dramatically leads to removed IPTG induction,the specific growth rate and specific glucose uptake rate increased by 63% and 13%,while the succinate yield decreased slightly and maintained at 1.50 mol/mol glucose,which represents 88% of the maximum stoichiometric yield.The systems metabolic engineering strategy described in this work may also be useful for the synthesis of other related NADHdemanding products in E.coli...