CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris
Kaidi Chen, Gulikezi Maimaitirexiati, Qiannan Zhang, Yi Li, Xiangjian Liu, Hongting Tang, Xiang Gao, Bo Wang, Yu Tao, Shuyuan Guo
Abstract
The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate. However, the lack of convenient and efficient genome engineering tools has hindered further applications of P. pastoris , especially in complex and multistep metabolic engineering scenarios. Hence, we developed a rapid and convenient multi-gene editing system based on CRISPR/Cas9 by optimizing the guide RNA processing strategy, which can achieve dual-gene knockout or multi-gene integration in single step. Firstly, we found that the HgH (HH-sgRNA-HDV) structure achieved the highest single-gene knockout efficiency (95.8 %) among the three sgRNA processing cassettes, including a tRNA-sgRNA-tRNA (tgt) array, HgH structure and tRNA-sgRNA-HDV (tgH) structure. Furthermore, the dHgH structure (double HgH) enabled one-step dual-gene disruption and multi-gene integration. The efficiency of dual-site knockout ranged from 60 % to 100 %, with functional genes knockout achieving approximately 60 % ( Δaox1Δgut1 ), while dual neutral sites knockout reached 100 %. Finally, we applied the system for one-step production of fatty acids and 5-hydroxytryptophan. The yield of FFAs reached 23 mg/L/μg protein/OD, while the yield of 5-hydroxytryptophan was 13.3 mg/L. The system will contribute to the application of P. pastoris as an attractive cell factory for multiplexed compound biosynthesis and will serve as a valuable tool for enhancing one-carbon (C1) bio-utilization.