YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica

Ya-Wen Li; Cai-Ling Yang; Qi Shen; Qian-Qian Peng; Qi Guo; Zhi-Kui Nie; Xiao-Man Sun; Tian-Qiong Shi; Xiao-Jun Ji; He Huang

doi:10.3389/fbioe.2021.816980

YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica

Front Bioeng Biotechnol. 2022 Mar 2:9:816980. doi: 10.3389/fbioe.2021.816980. eCollection 2021.

Authors

Ya-Wen Li¹, Cai-Ling Yang¹, Qi Shen¹, Qian-Qian Peng¹, Qi Guo², Zhi-Kui Nie³, Xiao-Man Sun¹, Tian-Qiong Shi¹, Xiao-Jun Ji², He Huang^{1

4}

Affiliations

¹ School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China.
² College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China.
³ Jiangxi New Reyphon Biochemical Co., Ltd., Salt and Chemical Industry, Xingan, China.
⁴ College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China.

Abstract

Non-homologous end-joining (NHEJ)-mediated random integration in Yarrowia lipolytica has been demonstrated to be an effective strategy for screening hyperproducer strains. However, there was no multigene assembly method applied for NHEJ integration, which made it challenging to construct and integrate metabolic pathways. In this study, a Golden Gate modular cloning system (YALIcloneNHEJ) was established to develop a robust DNA assembly platform in Y. lipolytica. By optimizing key factors, including the amounts of ligase and the reaction cycles, the assembly efficiency of 4, 7, and 10 fragments reached up to 90, 75, and 50%, respectively. This YALIcloneNHEJ system was subsequently applied for the overproduction of the sesquiterpene (-)-α-bisabolol by constructing a biosynthesis route and enhancing the flux in the mevalonate pathway. The resulting strain produced 4.4 g/L (-)-α-bisabolol, the highest titer reported in yeast to date. Our study expands the toolbox of metabolic engineering and is expected to enable a highly efficient production of various terpenoids.

Keywords: (-)-α-bisabolol; Golden Gate cloning; Y. lipolytica; non-homologous end-joining; sesquiterpene.