基于常压室温等离子体诱变和高通量筛选选育L-色氨酸高产菌株  

Breeding of L-tryptophan high-yield strain by ARTP mutagenesis and high-throughput screening

作　　者：叶慧敏 杜军 唐玉景 袁鑫 张铮 李璐[3] 冀颐之[1] YE Huimin;DU Jun;TANG Yujing;YUAN Xin;ZHANG Zheng;LI Lu;JI Yizhi(Beijing Key Laboratory of Biomass Waste Resource Utilization,College of Biochemical Engineering,Beijing Union University,Beijing 100023,China;Research Institute,Beijing Tsingke Biotech Co.,Ltd.,Beijing 100000,China;Key Laboratory for Industrial Biocatalysis,Ministry of Education,Department of Chemical Engineering,Center for Synthetic&Systems Biology,Institute of Biochemical Engineering,Tsinghua University,Beijing 100084,China)

机构地区：[1]北京联合大学生物化学工程学院、生物质废弃物资源化利用北京市重点实验室,北京100023 [2]北京擎科生物科技股份有限公司研究院,北京100000 [3]清华大学化工系生物化工研究所、清华大学合成与系统生物学研究中心、工业生物催化教育部重点实验室,北京100084

出　　处：《生物工程学报》2024年第9期3201-3215,共15页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2021YFC2102101,2021YFC2102102)。

摘　　要：L-色氨酸是人体中不可或缺的必需氨基酸,由于其广泛的应用和国内外巨大的需求,L-色氨酸已成为备受关注的研究和产业发展方向。尽管非理性诱变育种策略是一种开发工业菌株的有效手段,但是如何筛选具有理想表型的菌株依然是一个重大挑战。为了提高筛选L-色氨酸高产菌株的效率与准确性,本研究通过常压室温等离子体诱变构建随机突变文库,并结合深孔板高通量筛选,基于能够特异性响应L-色氨酸的拟荧光蛋白传感器从随机突变文库中成功筛选出一株L-色氨酸高产菌株,其产量在摇瓶中达到1.99 g/L,较出发菌株提高了41.77%。还通过基因组与转录组的比较组学分析,进一步对菌株的高产机理进行了解析。本研究基于常压室温等离子体诱变和高通量筛选策略,成功筛选出L-色氨酸高产菌株,为后续进一步选育和开发相关优质的L-色氨酸生产菌株资源提供了坚实的研究基础。L-tryptophan is an indispensable essential amino acid with a wide range of applications,which leads to a high demand.Accordingly,the production of L-tryptophan becomes a much-anticipated direction in research and industrial development.While irrational mutagenesis is an effective means to breed industrial strains,how to screen the strains with desirable phenotypes is still a major challenge.In order to improve the efficiency and accuracy of screening L-tryptophan high-yield strains,we used atmospheric and room temperature plasma mutagenesis to construct a random mutant library and then combined it with high-throughput screening in deep-well plates.Using a pseudo-fluorescent protein sensor capable of responding specifically to L-tryptophan,we successfully screened out a strain producing L-tryptophan at a high yield from a random mutagenesis library.The fermentation with the strain in shake flasks produced L-tryptophan at a yield of 1.99 g/L,which was 41.77%higher than that of the starting strain.Finally,the mechanism of high yield of the strain was deciphered by comparative genomics and transcriptomics.The above strategies provide a solid research foundation for further selection and development of high quality L-tryptophan producing strains.

关 键 词：拟荧光蛋白传感器 常压室温等离子体诱变 高通量筛选 L-色氨酸 

分 类 号：Q939.9[生物学—微生物学]