CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris

The important methylotrophic yeast <i>Pichia pastoris</i> 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 <i>P. pastoris</i>, 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 % (<i>Δaox1Δgut1</i>), 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 <i>P. pastoris</i> as an attractive cell factory for multiplexed compound biosynthesis and will serve as a valuable tool for enhancing one-carbon (C1) bio-utilization.