The methylotrophic yeast Pichia pastoris (also known as Komagataella pastoris) is an ideal host for producing proteins and natural products. Enhancing homologous recombination (HR) is helpful for improving the precision of genome editing, but results in stress to cellular fitness and is harmful for industrial applications. To overcome these challenges, we developed a tetracycline repressor protein (TetR)/tetO2 inducible system to dynamically regulate the HR-related gene RAD52 in P. pastoris. This approach significantly improved the positivity rate of single gene deletion to 81%. Furthermore, inducible overexpression of endogenous MUS81-MMS4 resulted in high-efficiency (81%) genome assembly of multiple genes. This inducible system had no adverse effect on cell growth in different media and resulted in greater fatty alcohol production from methanol compared with a strain constitutively overexpressing HR-related genes. We anticipate that this inducible regulation is applicable for enhancing HR for precise genome editing in P. pastoris and other non-conventional microbes without compromising cellular fitness.
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