Engineering Escherichia Coli For Improving Succinate Productivity And Yield

Succinate is a four-carbon dicarboxylate that has been reported as one of12most valuable bulk chemicals by the U.S. Department of Energy. Succinate has many applications in food, agricultural, pharmaceutical, and biodegradable plastic industries. Currently, succinate is mainly produced from petroleum derived maleic anhydride, resulting in high energy consumption and environmental pollution. Microbial production of succinate has attracted lots of interests, especially engineering Escherichia coli for succinate production. In this work, pyruvate dehydrogenase and C4-dicarboxylates transporters of E. coli were engineered to improve succinate yield and productivity.Reducing equivalent is an essential cofactor for chemical synthesis. Increasing supply of reducing equivalent could contribute to improving succinate yield. PDH is the key enzyme of pyruvate decarboxylation reaction, which can convert NAD+to NADH. However, the activity of PDH is very low in anaerobic growth, and sensitive to high NADH concentration. Through analysis of various combinations of LpdA mutations, we obtained a mutant with high PDH activity under anaerobic condition and decreased sensitivity to NADH. The PDH activity of this mutant increased1.9times compared to the wild type.The PDH enzyme was then engineered at the transcriptional level based on an ethonal-producing strain. The PDH activity increased to9.9μmol/mg/min, which was then used for improving succinate production. The PDH activity increased579-fold, leading to23%and25%increase of succinate titer and yield, respectively.E. coli contains four Dcu carries (encoded by dcuA, dcuB, dcuC and dcuD genes) for C4-dicarboxylates (succinate, fumarate and malate) transportation. The uptake, antiport and efflux of C4-dicarboxylates are mediated by the Dcu transporters. These four Dcu transporters were investigated for their succinate efflux capabilities through gene knock-out experiments. Deleting dcuA and dcuD genes had nearly no influence, while deleting dcuB and dcuC genes led to15%and11%decrease of succinate titer, respectively. Deleting both dcuB and dcuC genes resulted in90%decrease of succinate titer, suggesting that DcuB and DcuC were the main transporters for succinate efflux.Furthermore, RBS library was used to modulate dcuB and dcuC genes for improving succinate production. Activating DcuB individually led to8%and11%increase of succinate titer and yield, respectively. Activating DcuC individually led to18%and19%increase of succinate titer and yield, respectively. Modulating these two genes in combination led to34%and26%increase of succinate titer and yield, respectively.