CRISPR-Cas9系统介导的工业酵母营养缺陷型菌株构建  

Construction of an Auxotrophic Mutant from an Industrial Saccharomyces cerevisiae Strain by CRISPR-Cas9 System

作　　者：陆海燕 李佳蔓 孙思凡 章小毛 丁娟娟 邹少兰 LU Hai-yan;LI Jia-man;SUN Si-fan;ZHANG Xiao-mao;DING Juan-juan;ZOU Shao-lan(Key Laboratory of Systems Bioengineering,Ministry of Education,School of Chemical Engineering and Technology,Tianjin University,Tianjin 300072,China)

机构地区：[1]天津大学化工学院系统生物工程教育部重点实验室

出　　处：《中国生物工程杂志》2019年第10期67-74,共8页China Biotechnology

基　　金：天津市科技计划项目(18YFZCNC01240);国家自然科学基金(31470208)资助项目

摘　　要：以组氨酸营养缺陷型菌株构建为例,在前期分离、诱变和筛选得到的安琪酵母工业菌株衍生菌株K-a中试用CRISPR-Cas9系统进行基因修饰。针对菌株K-a为单倍体、ura3和对潮霉素B敏感的特点,构建了以URA3为选择标记的Cas9表达载体YCplac33-Cas9、以hphNT1为选择标记的gRNA表达载体pRS42H-gHIS1,使用PCR方法合成donor DNA片段。使用醋酸锂法制备感受态细胞K-a(YCplac33-Cas9)、将pRS42H-gHIS1和donor DNA共转化,涂布(CMG-URA+300μg/ml潮霉素B)平板,经表型筛选和PCR产物测序证明筛选平板生长菌落为目的转化子K-a(his1)的比例为74.4%,初步建立了适于利用CRISPR/Cas 9系统进行基因修饰的工业菌株宿主平台和相应的简便、快速进行基因修饰的操作技术流程。CRISPR-Cas9 system was first attempted to use in Angel industrial yeast-derived strain K-a,and HIS1 gene knockout was carried out.Because strain K-a is haploid,ura3 and sensitive to hygromycin B,the three key factors were designed as followings:Cas9 expressing vector YCplac33-Cas9 with URA3 marker,gRNA expressing vector pRS42H-gHIS1 with hph NT1 marker,and donor DNA fragment by PCR.By using lithium acetate method,pRS42H-gHIS1 and donor DNA was co-transformed into K-a(YCplac33-Cas9)competent cells and(CMG-URA+300μg/ml hygromycin B)plate was used for screening.The target transformants were proved to obtain and the ratio was 74.4%by phenotype screening and PCR product sequencing.So the industrial host platform utilizing CRISPR-Cas9 system was constructed and the simple and rapid gene manipulation protocol was preliminarily formed.

关 键 词：酿酒酵母 CRISPR-Cas9 his1 基因敲除 

分 类 号：Q812[生物学—生物工程]