Recombination Machinery Engineering for Precise Genome Editing in Methylotrophic Yeast <i>Ogataea Polymorpha</i>

Abstract

Methanol biotransformation can expand biorefinary substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR/Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ), increase HR rates from 20-30% to 60-70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated systems has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the first production of fatty alcohols in O. polymorpha. These findings will simplify the genetic engineering in non-conventional yeasts, and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.