Abstract
Corynebacterium glutamicum lacking the succinate dehydrogenase complex can produce succinate aerobically with acetate representing the major byproduct. Efforts to increase succinate production involved deletion of acetate formation pathways and overexpression of anaplerotic pathways, but acetate formation could not be completely eliminated. To address this issue, we constructed a pathway for recycling wasted carbon in succinate-producing C. glutamicum. The acetyl-CoA synthetase from Bacillus subtilis was heterologously introduced into C. glutamicum for the first time. The engineered strain ZX1 (pEacsA) did not secrete acetate and produced succinate with a yield of 0.50 mol (mol glucose)−1. Moreover, in order to drive more carbon towards succinate biosynthesis, the native citrate synthase encoded by gltA was overexpressed, leading to strain ZX1 (pEacsAgltA), which showed a 22% increase in succinate yield and a 62% decrease in pyruvate yield compared to strain ZX1 (pEacsA). In fed-batch cultivations, strain ZX1 (pEacsAgltA) produced 241 mM succinate with an average volumetric productivity of 3.55 mM h−1 and an average yield of 0.63 mol (mol glucose) −1, making it a promising platform for the aerobic production of succinate at large scale.
Highlights
Succinic acid is considered as one of the most important platform chemicals because of its extensive applications in many industrial fields
Plasmid DNA transferring into C. glutamicum ATCC 13032 was carried out by electroporation, and the recombinant strains were selected on brain heart infusionsorbitol (BHIS) agar plates containing 25 mg mL21 kanamycin
ATCC 13032 under aerobic conditions have been made by disrupting the succinate dehydrogenase complex and pathways leading to the synthesis of acetate, as well as co-overexpressing pyruvate carboxylase and PEP carboxylase [13]
Summary
Succinic acid is considered as one of the most important platform chemicals because of its extensive applications in many industrial fields. Succinic acid production is accomplished by chemical synthesis using petroleum-derived maleic anhydride. This process requires high temperature, high pressure and heavy metal catalysts, which makes the conversion costly and ecologically questionable [1,3]. The remaining acetate formation pathways were still unclear. Several genes of C. glutamicum ATCC 13032 were annotated as putative acetyltransferases, hydrolases or dehydrogenases and could be involved in the conversion of acetyl-CoA to acetate [13,21,22]. Inactivation of one or more of these genes might reduce acetate formation, whereas the influence was uncertain [13,21]. Introduction of an acetate assimilation pathway could address this conundrum. Even though C. glutamicum ATCC 13032 utilized the native acetate kinase-phosphotransacetylase (PTA-ACK) pathway to uptake acetate under aerobic conditions [18], this reversible pathway contributed to acetate formation in the sdhCAB-deficient mutant
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