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

Overview

Journal Front Bioeng Biotechnol

Date 2022 Mar 21

PMID 35308823

Authors

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

Affiliations

Soon will be listed here.

Abstract

Non-homologous end-joining (NHEJ)-mediated random integration in 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 . 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.

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