L-丝氨酸是化学和材料领域中最为重要的30个骨架化合物之一,但至今尚未实现微生物发酵法工业化生产L-丝氨酸。氨基脱氧分支酸合成酶(aminodeoxychorismate synthase,ADC synthase)是L-丝氨酸分解代谢相关的关键酶,研究前期发现,与出发菌株SSAAI相比,通过ARTP诱变获得的高产L-丝氨酸突变株A36中氨基脱氧分支酸合成酶编码基因pabAB 426位发生点突变(T426I),而氨基脱氧分支酸合成酶基因突变对酶活力、菌株生长及产L-丝氨酸的影响尚未见报道。该文采用pK18 mobsacB质粒在出发菌株SSAAI上对pabAB进行426位定点突变(T426I)研究,结果发现,pabAB定点突变株的生物量比出发菌株SSAAI下降29.3%,L-丝氨酸产量提高15.9%。同时对氨基脱氧分支酸合成酶酶活力进行测定,发现氨基脱氧分支酸合成酶编码基因pabAB 426位的T426I突变导致酶的比活力下降。进一步采用不同强度的启动子调控高产菌株A36中氨基脱氧分支酸合成酶的活力,构建重组菌株A36-Pkan和A36-Dap-e;与出发菌株A36相比,重组菌氨基脱氧分支酸合成酶的比酶活力均有提高,发酵120 h时,生物量分别提高8.3%和16%,而L-丝氨酸产量分别下降18.2%和22.6%,说明提高氨基脱氧分支酸合成酶酶活力有利于谷氨酸棒杆菌生长却不利于产L-丝氨酸。
L-serine is one of the 30 most important skeletal compounds in chemistry and materials, but the industrial production of L-serine by microbial fermentation has not yet been realized. Aminodeoxychorismate(ADC) synthase is a key enzyme related to the catabolism of L-serine. Previous study revealed that the gene pabAB encoding ADC synthase was mutated in the high-yielding L-serine mutant strain A36, but the effect of mutation of ADC synthase gene on enzyme activity, strain growth, and L-serine production has not been investigated. In this paper, the pK18mobsacB plasmid was used for the side mutation of pabAB (T426I) on the parent strain SSAAI, and the results showed that L-serine production increased by 15.9% while the biomass of the strain with pabAB 426 side mutation, decreased by 29.3% compared with the parent strain SSAAI. The enzyme activity of ADC synthase was also measured, and it was found that the T426I mutation at position 426 of ADC synthase resulted in a decrease in specific enzyme activity. The recombinant strains A36-Pkan and A36-Dap-e were constructed by using promoters of different strengths to regulate the activity of ADC synthase. The specific enzyme activities of the recombinant ADC synthase increased compared with the parent strain, and the biomass increased by 8.3% and 16%, respectively, while the L-serine production decreased by 18.6% and 22.6% compared with the parent strain. This indicates that increasing the enzymatic activity of ADC synthase is beneficial to the growth of Corynebacterium glutamicum but not to the production of L-serine.
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