Efficient Succinate Production From Pentose In Engineered Corynebacterium Glutamicum

Lignocellulosic biomass from agricultural waste is a widely abundant andpotentially attractive source of renewable feedstocks for production of biofuel andcommodity chemicals. The non-medical corynebacteria are Gram-positive bacteria thatbelong to the Actinomycetes subdivision of the eubacteria. Corynebacteriumglutamicum has been widely used for the industrial production of various amino acidsand organic acids. Focusing on efficient succinate production from pentose, SAZ3which could efficiently produce succinate under anaerobic conditions was engineeredas the starting strain, aiming at enhancing the xylose consumption, succinate yield andproductivity with C.glutamicum ATCC13032by metabolic engineering.To improve xylose utilization, different xylose isomerase genes and xylulokinasegenes were test in C.glutamicum. The genes orginating from Xanthomonas campestriswere shown to have the highest effect, resulting in growth rates of0.23h-1, followed bygenes from Escherichia coli, Paenibacillus polymyxa SC2and Streptomyces coelicolor.The newly engineered strain I-xcb was shown to grow significantly faster(0.229±0.009h-1)on minimal medium containing xylose as sole carbon source compared with thepreviously described strain CRX2(0.2h-1) expressing xylAB from E. coli.Succinic acid, an important four carbon dicarboxylic acid, is a valuable platformchemical for multiple applications,such as pharmaceutical, agricultural and foodindustry. Firstly, the improved plasmid pX-xcbAB was transformed into the modelsuccinate producer CGL01. The reconstruction strain CGL11can consumpte30g L-1in20h, and accumulated up to28g L-1succinate under anaerobic fermentation, and theyield of succinate was0.93g succinate（g Dxylose）-1, reaching83%of the theoreticalyield. Further overexpression of the non-pentose phosphate pathway in C.glutamicumincreased the yield of succinate expectedly. This modification of tal and tktoverexpression also increased xylose consumption rate by22%(2.06g L-1h-1versus1.69g L-1h-1and succinate productivity by12%(1.72g L-1h-1versus1.54g L-1h-1). Thirdly, to improve simultaneous utilization of mixed sugars, the B.subtilis L-arabinose transporter gene, araE, was independently integrated into the IISKL. Theresulted strain CGL31maintained a high yield of succinic acid and utilization ofxylose.Besides, this modification increased the succinate productivity by20% compared with CGL21(1.72g L-1h-1versus2.06g L-1h-1).To expand the substrate utilization, the araBAD operon from Escherichia coli andxylAB operon from Xanthomonas campestris were introduced into an succinate-producing C. glutamicum, which enabled anaerobic production of succinate usingarabinose,xylose and glucose,the yield of succinate was0.79g succinate（g total sugar）-1, making it a promising platform for the anaerobic production of succinate from thehydrolysis of lignocellulosic.