Engineering Corynebacterium Glutamicum For Efficient Aerobic Succinate Production

Succinic acid is an important four-carbon platform compound which has widelyapplication in the pharmaceutical, agricultural and food industry. Biological processesfor succinic acid production show a good prospect for its social, environmental andeconomic benefits. As a traditional amino acid production bacterium,Corynebacterium glutamicum can synthesize various chemicals and it is thusbecoming a desired platform strain for industrial production of value-added chemicals.Although most reported efforts to enhance bio-production of succinate have beenperformed under anaerobic conditions, they have often been hampered by thelimitations of NADH availability, poor cell growth, and slow production. In contrast,aerobic cell culture conditions have many advantages over anaerobic conditions thatfavor implementation on an industrial scale due to higher biomass generation, fastercarbon throughput and product formation.Corynebacterium glutamicum lacking the succinate dehydrogenase complex canproduce succinate aerobically with acetate representing the major byproduct. Effortsto increase succinate production involved deletion of acetate formation pathways andoverexpression of anaplerotic pathways, but acetate formation could not becompletely eliminated. In this study, a new approach to reduce acetate accumulationby engineering an acetate assimilation pathway in C. glutamicum was presented. Twoenzymes of the acetyl-CoA synthase from Escherichia coli (acs) and Bacillus subtilis(acsA) were introduced into the strain ZX1(that is sdhABCD ldhA pta cat pqoPsod-ppcPsod-pyc) background, resulting strain ZX1(pEacs) and ZX1(pEacsA). Bothmodifications resulted in the elimination of acetate formation and overexpression ofacsA had a higher succinate yield with0.50mol (mol glucose)-1. Then, in order todrive more carbon towards succinate biosynthesis, the native citrate synthase encodedby gltA was overexpressed, leading to strain ZX1(pEacsAgltA), which showed a22%increase in succinate yield and a62%decrease in pyruvate yield compared to strainZX1(pEacsA). In fed-batch cultivations, strain ZX1(pEacsAgltA) produced241mMsuccinate with an average volumetric productivity of3.55mM h-1and an averageyield of0.63mol (mol glucose)-1, making it a promising platform for the aerobicproduction of succinate at large scale.Arabinose is considered as an ideal feedstock for the microbial production ofvalue-added chemicals due to its aboundance in agricultural wastes. In this study, the araBAD operon from Escherichia coli was introduced into an succinate-producing C.glutamicum, which enabled aerobic production of succinate using arabinose as solecarbon source. Also, the native citrate synthase and the acetyl-CoA synthetase fromBacillus subtilis were co-overexpressed to divert more carbon towards succinatebiosynthesis. After culture condition optimization, the engineered strain ZX1(pXaraBAD, pEacsAgltA) produced82.3mM succinate with a yield of0.64mol (molL-arabinose)-1(76.8%of the maximum yield) and accumulated tiny byproducts.Moreover, this strain produced110.23mM succinate using glucose and arabinose ascosubstrates in shake-flask batch fermentation. To date, this is the highest succinateproduction under aerobic conditions in minimal medium.