Efficient CRISPR-Cas9 mediated multiplex genome editing in yeasts

作     者：Wang, Laiyou Deng, Aihua Zhang, Yun Liu, Shuwen Liang, Yong Bai, Hua Cui, Di Qiu, Qidi Shang, Xiuling Yang, Zhao He, Xiuping Wen, Tingyi 

作者机构：Chinese Acad Sci Inst Microbiol CAS Key Lab Pathogen Microbiol & Immunol Beijing 100101 Peoples R China Univ Chinese Acad Sci Beijing 100049 Peoples R China Chinese Acad Sci Inst Microbiol CAS Key Lab Microbial Physiol & Metab Engn Beijing 100101 Peoples R China Univ Chinese Acad Sci Savaid Med Sch Beijing 100049 Peoples R China 

出 版 物：《BIOTECHNOLOGY FOR BIOFUELS》 (生物燃料)

年 卷 期：2018年第11卷第1期

页      面：277-277页

核心收录：

学科分类：0820[工学-石油与天然气工程] 080703[工学-动力机械及工程] 08[工学] 0807[工学-动力工程及工程热物理] 0836[工学-生物工程] 

基　　金：Chinese Academy of Sciences [XDA17010503] National Natural Science Foundation of China National Hi-Tech Research and Development Program of China [2014AA021203] Science and Technology Service Network Initiative [KFJ-STS-QYZD-047] 

主　　题：CRISPR-Cas9-assisted multiplex genome editing Markerless multi-locus integration Markerless multi-copy integration Ogataea polymorpha Saccharomyces cerevisiae 

摘      要：Background: The thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications. It is generally recognized as an efficient and safe cell factory in fermentative productions of chemicals, biofuels and other bio-products. However, it is difficult to genetically engineer for the deficiency of an efficient and versatile genome editing technology. Results: In this study, we developed a CRISPR-Cas9-assisted multiplex genome editing (CMGE) approach including multiplex genes knock-outs, multi-locus (ML) and multi-copy (MC) integration methods in yeasts. Based on CMGE, various genome modifications, including gene deletion, integration, and precise point mutation, were performed in O. polymorpha. Using the CMGE-ML integration method, three genes TAL from Herpetosiphon aurantiacus, 4CL from Arabidopsis thaliana and STS from Vitis vinifera of resveratrol biosynthetic pathway were simultaneously integrated at three different loci, firstly achieving the biosynthesis of resveratrol in O. polymorpha. Using the CMGE-MC method,similar to 10 copies of the fusion expression cassette P-ScTEF1-TAL-P-ScTPI1-4CL-P-ScTEF2-STS were integrated into the genome. Resveratrol production was increased similar to 20 fold compared to the one copy integrant and reached 97.23 +/- 4.84 mg/L. Moreover, the biosynthesis of human serum albumin and cadaverine were achieved in O. polymorpha using CMGE-MC to integrate genes HSA and cadA, respectively. In addition, the CMGE-MC method was successfully developed in Saccharomyces cerevisiae. Conclusions: An efficient and versatile multiplex genome editing method was developed in yeasts. The method would provide an efficient toolkit for genetic engineering and synthetic biology researches of O. polymorpha and other yeast species.