Metabolic Engineering Of Corynebacterium Glutamicum For High-level Production Of Succinate

Starting with the wild-type Corynebacterium glutamicum ATCC13032, a seriesof metabolically engineered C. glutamicum mutants were constructed that couldefficiently produce succinate under anaerobic conditions and aerobic conditions. Withthe comparative analysis of central carbon metabolism and single gene disruptionanalysis, the pathway for anaerobic biosynthesis of succinate was identified. Severalgene targets were predicted by the combination of elementary mode analysis andexperimental metabolic flux analysis, and all of them were genetically engineered stepby step. Subsequently, the designed mutants were experimentally tested via anaerobicfermentation and metabolic flux analysis to confirm the accuracy of the prediction.With the aim at increasing succinate titer, high-cell-density fermentation was carriedout. On the other hand, a pathway from Bacillus subtilis for acetate assimilation wasintroduced into succinate-producing C. glutamicum to recycle wasted carbon underaerobic conditions. The citrate synthase was further overexpressed to reduce pyruvateaccumulation and increase TCA cycle flux. Finally, the feasibility of aerobicproduction of succinate at large scale was evaluated by fed-batch culture. The mainresults were shown as follows:Through the comparison of central metabolism of C. glutamicum and naturallyisolated succinate producers and rational analysis, potential targets that involved insuccinate production were recognized. After testing these single gene disruptionstrains, the reductive TCA cycle was identified as the major pathway for anaerobicsuccinate production. Inactivation of the enzymes of pyruvate: quinoneoxidoreductase, acetyltransferase, acetyl-CoA: CoA transferase and L-lactatedehydrogenase, acetate production by strain C. glutamicum SAZ1was significantlyreduced and succinate yield of0.99mol (mol glucose)-1was achieved.A refined metabolic network was constructed based on the genome-scalenetwork. Three targets, carboxylation pathway, glyoxylate pathway and citratesynthase were identified by elementary mode analysis and metabolic flux analysis.Based on prediction, metabolic engineering and fermentation were performed. Furtheroverexpression of succinate exporter leading to strain C. glutamicum SAZ2(pEpycgltAsucE，pXaceAB), which produced succinate with a yield of1.43mol (molglucose)-1and had more identical flux distribution to the optimal EMs. The reductionof intracellular succinate alleviated its restriction to glyoxylate pathway. Usingtwo-stage high-cell-density fermentation, succinate titer up to930mM was achieved with a yield of1.32mol (mol glucose)-1and a productivity of9.48mM h-1.By inactivating the succinate dehydrogenase complex, the pathways for acetateand lactate formation, replacing the native promoters of pyruvate carboxylase andPEP carboxylase with strong promoter sod, introducing the acetate assimilationpathway from B. subtilis and overexpressing the native citrate synthase, the succinateyield of strain C. glutamicum ZX1(pEacsAgltA) increased to0.61mol (molglucose)-1. In fed-batch culture,241mM succinate with a yied of0.63mol (molglucose)-1was produced. The araBAD operon from Escherichia coli was introducedinto succinate-producing C. glutamicum, which enabled aerobic production ofsuccinate using arabinose as sole carbon source.