Efficient CRISPR-Cas9 mediated multiplex genome editing in yeasts----中国科学院微生物研究所

科研成果

论文题目:	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 , and Wen Tingyi*
联系作者:
刊物名称:	Biotechnology for Biofuels
期:	11
卷:
页:
年份:	2018
影响因子:	6.661
论文下载:
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.