L-高丝氨酸是一种非必需手性氨基酸,在农业、化工、生物医药等领域具有重要的用途。近年来,利用微生物发酵法生产L-高丝氨酸越来越受到人们的关注。氨基酸转运蛋白不仅可以降低反馈抑制和细胞毒性,还能提高氨基酸产量,已成为代谢工程改造的重要靶点。目前,谷氨酸棒杆菌中L-高丝氨酸转运蛋白仍然未知。该研究通过生物信息学分析,从谷氨酸棒杆菌中筛选出10个L-高丝氨酸转运蛋白候选基因,经过体外和体内L-高丝氨酸耐受性生长实验,结果显示在大肠杆菌三重高丝氨酸转运缺陷突变体中,异源过表达brnFE、azlCD和lysE基因,能显著恢复大肠杆菌突变体的生长缺陷,而在谷氨酸棒杆菌体内,只有lysE敲除菌较对照生物量下降16%,推测在体内LysE蛋白具有更重要的高丝氨酸外排能力。进一步对LysE的外排活性进行分析,发现lysE敲除菌的外排能力降低了18%。最后,在高丝氨酸底盘菌中,评估了敲除或过表达lysE基因对L-高丝氨酸产量的影响,结果发现lysE基因缺失导致L-高丝氨酸的产量降低了65%,而过表达lysE使L-高丝氨酸产量提高了22%。综上所述,谷氨酸棒杆菌LysE蛋白具有转运L-高丝氨酸的能力,相关结果为开发高效的L-高丝氨酸细胞工厂提供了新靶点。
L-homoserine is a non-essential chiral amino acid that plays a very important role in agriculture, the chemical industry, biomedicine, and other fields.In recent years, the production of L-homoserine using microbial fermentation has attracted more and more attention.Amino acid transporters can not only reduce feedback inhibition and cytotoxicity but also increase amino acid production, which have become important targets for metabolic engineering.So far, the L-homoserine transporter in Corynebacterium glutamicum remains unknown.In this study, ten candidate genes involved in L-homoserine transport were selected from Corynebacterium glutamicum through bioinformatics analysis.By L-homoserine tolerance growth experiments in vitro and in vivo, this study found that heterologous overexpression of brnFE, azlCD, and lysE genes could significantly restore the growth defects of the triple homoserine transporter mutant of Escherichia coli strain, however, only deletion of lysE in Corynebacterium glutamicum reduced the biomass by 16% compared with the control, suggesting that the LysE protein showed more important homoserine efflux ability in vivo.Further analysis of the efflux activity of LysE revealed that the efflux ability of the lysE deletion strain was reduced by 18%.Finally, the effect of deletion or overexpression of lysE on L-homoserine production was evaluated in an L-homoserine-producing chassis strain.It was found that deletion of lysE reduced the production of L-homoserine by 65%, while overexpression of lysE increased the production of L-homoserine by 22%.In summary, the LysE protein in Corynebacterium glutamicum exhibited the ability to transport L-homoserine, and the relevant results provide a new target for constructing efficient L-homoserine cell factories.
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