A CRISPR-Cas12a system for multi-gene editing (CCMGE) and metabolic pathway assembly in Starmerella bombicola  

作　　者：Min Zhang Yibo Shi Lihua Zhang Shiying Zhu Haiquan Yang Wei Shen Yuanyuan Xia Xianzhong Chen 

机构地区：[1]Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,No.1800 Lihu Avenue,Wuxi 214122,Jiangsu,China [2]School of Biotechnology,Jiangnan University,Wuxi 214122,China

出　　处：《Systems Microbiology and Biomanufacturing》2022年第4期665-675,共11页系统微生物学与生物制造（英文）

基　　金：The work was supported by the 111 Project(No.111-2-06);National Natural Science Foundation of China(32001064);Key Research and Development Program of China(2021YFC2100102-03);China Postdoctoral Science Foundation(2020M671331);Postgraduate Research and Practice Innovation Program of Jiangsu Province(No.KYCX20-1807).

摘　　要：The traditional homologous recombination(HR)gene-editing method faces problems such as low editing efficiency and absence of marker genes.CRISPR-Cas9-editing efficiency is high and has been widely used in bacteria and yeast.In com-parison with CRISPR-Cas9,CRISPR-Cas12a has many outstanding advantages.Here,we report an Acidaminococcus sp.BV3L6(As)Cas12a-based genome-editing method used for Starmerella bombicola.To demonstrate the high efficiency of the CCMGE system,we verified a counter-selectable marker in S.bombicola,orotidine 5’-phosphate decarboxylase(100%for URA3).We also tested the common gene UDP-glucosyltransferase(100%for UGTA)using a 300 bp donor containing hygromycin expression cassette.This toolkit was further extended to simultaneously edit two genes(18%for UGTA and leu)and three genes(13.8%for UGTA,leu and URA3).The system greatly reduces the screening time for such multi-site editing.Based on the CCMGE system,the PHA(polyhydroxyalkanoate)-producing strain was constructed by increasing the copy number of the PHA synthase(PHAC).The PHA content and DCW reached 11.8%and 30.1 g/L,respectively.The yield of PHA was about three times higher than that of the single-copy strain using the same fermentation method.

关 键 词：Starmerella bombicola CRISPR-Cas12a Genome editing PHA 

分 类 号：Q78[生物学—分子生物学]