Recently, we developed novel genetic tools for precise genome editing in Pichia pastoris (syn.Komagataella phaffii) by enhancing homologous recombination (HR) and engineering the multiple intrusion-induced rearrangement (MIR) processes. With this advanced gene editing systems, we can theoretically realize the stable loading of more than 100 exogenous genes and the precise regulation of gene expression in P. pastoris.

P. pastoris, a model methylotrophic yeast, can easily achieve high density fermentation, and thus is considered as a promising chassis cell for efficient methanol biotransformation. However, inefficient gene editing and lack of synthetic biology tools hinder its metabolic engineering toward industrial application.

This study identified the key gene RAD52, which plays crucial role in HR repair in P. pastoris, and its overexpression can improve the efficiency of single gene editing to 90%. Further knockout of MPH1 increased the efficiency of multi-fragment recombination at one site by 13.5 times. In addition, 46 neutral sites and 18 promoters were characterized for genome integration and gene expression. Finally, a two-factorial regulation system was developed for regulating fatty alcohol biosynthesis in P. pastoris from different carbon sources. (Text and image by Peng Cai)