AUTHOR=Li Ya-Wen , Yang Cai-Ling , Shen Qi , Peng Qian-Qian , Guo Qi , Nie Zhi-Kui , Sun Xiao-Man , Shi Tian-Qiong , Ji Xiao-Jun , Huang He TITLE=YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica JOURNAL=Frontiers in Bioengineering and Biotechnology VOLUME=9 YEAR=2022 URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.816980 DOI=10.3389/fbioe.2021.816980 ISSN=2296-4185 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.