文献类型：期刊文献

英文题名：CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii

作者：Zhou, Wei[1];Li, Yuanyi[1];Liu, Guosong[1];Qin, Weichuang[1];Wei, Dongzhi[1];Wang, Fengqing[1];Gao, Bei[1]

机构：[1]East China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, 130 Meilong Rd,POB 311, Shanghai 200237, Peoples R China

年份：2024

卷号：108

期号：1

外文期刊名：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

收录：;EI(收录号：20240715561144);WOS:【SCI-EXPANDED(收录号:WOS:001158514300005)】；

基金：This work was supported by the National Key Research and Development Program of China (No. 2023YFA0914100), and the Natural Science Foundation of Shanghai (No. 21ZR1416500)

语种：英文

外文关键词：Komagataella phaffii; CRISPR/Cas9; Visual marker recycling; Multi-fragment integration; Large DNA fragment knockout; Yeast assembled libraries

摘要：Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of Ku70 and DNA ligase IV, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in K. phaffii by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in K. phaffii using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73 mg/L in shake flask fermentation.