CRISPR/Cas9介导的高产谷胱甘肽原养型酵母工程菌的构建  

Construction of prototrophic glutathione-high-producing yeast strain mediated by CRISPR/Cas9

作　　者：周文龙 唐亮 成凯 刘忞之 杨燕 王伟 Wenlong Zhou;Liang Tang;Kai Cheng;Minzhi Liu;Yan Yang;Wei Wang(State Key Laboratory of Bioaetive Substance and Function of Natural Medieines ＆ Key Laboratory, of Biosynthesis of Natural Products o/National Health and Family Planning Commission, Institute of Materia Medica, Chinese Academv of Medical Science and Peking Union Medical College, Beoing 100050, China)

机构地区：[1]中国医学科学院北京协和医学院药物研究所天然药物活性物质与功能国家重点实验室/国家卫生和计划生育委员会天然药物生物合成重点实验室,北京100050

出　　处：《生物工程学报》2017年第12期1999-2008,共10页Chinese Journal of Biotechnology

基　　金：中国医学科学院医学与健康科技创新工程项目(No.2016-I2M-3-012);北京协和医学院"协和青年基金"项目(No.3332016057)资助~~

摘　　要：谷胱甘肽(Glutathione,GSH)是具有多种生理功能的非蛋白质类巯基化合物,已广泛应用于药品、食品等行业,且市场需求量逐年增加。遗传工程育种是提高细胞内GSH含量的重要策略,但在遗传操作过程中使用到的营养缺陷型遗传标记可能会影响菌株的正常生长,且不利于高密度发酵的进行。为回复工程菌株的营养缺陷型,利用g RNA转录表达框和靶基因同源DNA片段直接共转化酵母细胞,由细胞内表达的Ⅱ型CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9)介导的基因组编辑技术将营养缺陷型GSH工程菌株W303-1b/FGP回复为原养型菌株。结果显示,与营养缺陷型菌株相比,原养型菌株生长周期缩短,且可以利用简单的合成培养基进行培养,方便菌株的大规模培养。Glutathione（GSH）, a non-protein thiol product with various biological activities, has been widely used in pharmaceutical and food industries. Recently, genetic engineering becomes an important strategy for obtaining GSH-high-producing strains. However, auxotrophic selection markers used may result in reduced cell growth or GSH production. In the present study, clustered regularly interspaced short palindromic repeats（CRISPR） and Cas9-associated-system（CRISPR-Cas）, in which g RNA expression constructs and homologous DNA fragments of target genes were co-transformed into Saccharomyces cerevisiae cells, was used for the construction of the prototrophic strain derived from the engineered auxotrophic strain W303-1 b/FGP. As a result, the prototrophic strain W303-1 b/FGP^（PT） showed a significantly shorter culture cycle compared with the auxotrophic strain. Furthermore, chemically defined medium could be used to culture strain W303-1 b/FGP^（PT） that might have great interests in industrial fermentation.

关 键 词：CRISPR/Cas9 谷胱甘肽 酿酒酵母 营养缺陷型 

分 类 号：Q78[生物学—分子生物学]