L-丝氨酸是化学和材料领域中最重要的30个骨架化合物之一。为探索谷氨酸棒杆菌高产L-丝氨酸的机理,通过对2株不同表型的产L-丝氨酸谷氨酸棒杆菌进行全基因组比较分析,采用回复突变、基因敲除及增强表达探索基因差异与表型差异之间的关系,明确影响L-丝氨酸产量和菌株生长的关键因素。在对出发菌株ΔSSAAI与突变株A36进行全基因组比较分析基础上,采用pK18mobsacB质粒在出发菌株ΔSSAAI上对与转运或代谢相关的4个差异基因faS、folC、rhtA和tyrP进行回复突变,得到4个回复突变菌株Kf、Ko、Kr和Kt。结果表明,4个突变菌株的生物量与出发菌株ΔSSAAI相比变化不显著,Kf和Ko突变株的L-丝氨酸产量也没有显著变化,而突变株Kr和Kt的L-丝氨酸产量分别为31.1和31.04 g/L,分别提高18.5%和18.3%。继而在ΔSSAAI中敲除rhtA与tyrP构建敲除菌株ΔrhtA与ΔtyrP。结果表明,2个敲除菌株中L-丝氨酸产量均无明显变化。而在ΔSSAAI中加强表达rhtA或tyrP基因时,L-丝氨酸产量有提高,进一步在高产突变株A36中分别加强表达rhtA与tyrP基因,重组菌株L-丝氨酸产量分别为32.55和33.7 g/L,比菌株A36分别提高6.5%和10.3%。然而与菌株A36相比,加强表达rhtA与tyrP基因导致菌株生物量分别下降13.88%和24.5%,表明加强rhtA与tyrP有利于谷氨酸棒杆菌产L-丝氨酸,而不利于菌株生长。
L-serine is one of the 30 most important skeleton compounds in chemical and materials industries. In order to explore the mechanism of L-serine production by Corynebacterium glutamicum, the whole-genome sequencing and comparative genomics analysis were performed, and the relationship between genotype and phenotype was studied through reverse mutation, gene deletion and overexpression. The pK18mobsacB plasmid was used for reverse mutation of four genes (faS, folC, rhtA, and tyrP) related to transport or metabolism in the starting strain ΔSSAAI, and corresponding mutants Kf, Ko, Kr, and Kt were obtained. The fermentation of the mutants showed that, compared with the starting strain ΔSSAAI, the biomass of Kf, Ko, Kr, and Kt did not change significantly, and the L-serine titer of Kf and Ko did not change significantly, while that of Kr and Kt were 31.1 g/L and 31.04 g/L, increased by 18.5% and 18.3%, respectively. The deletion strains ΔrhtA and ΔtyrP were further constructed, and the fermentation results showed that there was no significant change in the L-serine titer. Then rhtA and tyrP were overexpressed in ΔSSAAI, L-serine titer improved slightly. rhtA and tyrP were overexpressed in themutantstrainA36,andtherecombinantstrainspD-RandpD-Twere obtained, resulting in L-serine titer of 32.55 g/L and 33.7 g/L, which was 6.5% and 10.3% higher than that of A36. At the same time, rhtA and tyrP overexpression resulted in a decrease of the biomass by 13.88% and 24.5%, respectively. The results indicated that rhtA and tyrP were related to the cell growth and L-serine production.
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