L-高丝氨酸是一种非蛋白质手性氨基酸,在食品、饲料、医药和农业等领域中具有广阔的应用潜力。但目前改造食品安全微生物谷氨酸棒杆菌发酵生产L-高丝氨酸的实例仍然较少。该研究以野生型谷氨酸棒杆菌为出发菌,通过敲除thrB、alaT和ddh弱化L-高丝氨酸竞争途径,并过表达lysC、hom和ppc强化L-高丝氨酸合成代谢流,实现了L-高丝氨酸的有效积累。随后,通过增强胞内NADPH供应以及促进L-高丝氨酸外排效率,进一步提升L-高丝氨酸生产。最后,通过改造和重塑葡萄糖摄取和利用系统,在谷氨酸棒杆菌中实现了L-高丝氨酸的高效合成。最优基因工程菌在摇瓶条件下可积累9.29 g/L L-高丝氨酸,在5 L发酵罐条件下可积累77.53 g/L L-高丝氨酸,生产强度达到1.29 g/(L·h),是已报道谷氨酸棒杆菌生产L-高丝氨酸的最高水平。该研究将为构建L-高丝氨酸高产菌株提供一定参考依据。
L-homoserine is a non-protein chiral amino acid with broad application potential in the fields of food, feed, medicine, and agriculture.However, there are still limited examples of exploring the GRAS (generally regarded as safe) Corynebacterium glutamicum for fermentative production of L-homoserine.This study employed the wild-type C.glutamicum ATCC 13032 as an initial strain for designing and constructing L-homoserine producer.Firstly, this study attenuated the L-homoserine competitive pathway by knocking out thrB, alaT, and ddh, and overexpressed lysC, hom, and ppc to enhance L-homoserine biosynthetic flux, thus achieving effective L-homoserine accumulation.Subsequently, this study further enhanced L-homoserine production by improving intracellular NADPH supply and enhancing L-homoserine efflux capacity.Finally, through engineering and reshaping the glucose uptake and utilization system, an efficient biosynthesis of L-homoserine was achieved in C.glutamicum.The optimal engineered strain could accumulate 9.29 g/L L-homoserine in shake flasks and 77.53 g/L L-homoserine in a 5-L fermenter with a production rate of 1.29 g/L/h, representing the highest reported level of L-homoserine production by C.glutamicum.This study will provide a valuable reference for the construction of high-producing L-homoserine strains.
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