Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms

There is a growing interest in establishing the methylotrophic yeast Pichia pastoris as microbial cell factories for producing fuels, chemicals, and natural products, particularly with methanol as the feedstock. Although CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technology has been established for the integration of multigene biosynthetic pathways, long (500–1000 bp) homology arms are generally required, probably due to low homologous recombination (HR) efficiency in P. pastoris. To achieve efficient genome integration of heterologous genes with short homology arms, we aimed to enhance HR efficiency by introducing the recombination machinery from Saccharomyces cerevisiae. First, we overexpressed HR related genes, including RAD52, RAD59, MRE11, and SAE2, and evaluated their effects on genome integration efficiency. Then, we constructed HR efficiency enhanced P. pastoris, which enabled single-, two-, and three-loci integration of heterologous gene expression cassettes with ∼40 bp homology arms with efficiencies as high as 100%, ∼98%, and ∼81%, respectively. Finally, we demonstrated the construction of β-carotene producing strain and the optimization of betaxanthin producing strain in a single step. The HR efficiency enhanced P. pastoris strains can be used for the construction of robust cell factories, and our machinery engineering strategy can be employed for the modification of other nonconventional yeasts.