Dynamically Regulating\nHomologous Recombination Enables\nPrecise Genome Editing in Ogataea polymorpha

Methylotrophic yeast Ogataea polymorpha has become a promising cell factory due to its efficient utilization\nof methanol to produce high value-added chemicals. However, the low\nhomologous recombination (HR) efficiency in O. polymorpha greatly hinders extensive metabolic engineering for industrial applications.\nOverexpression of HR-related genes successfully improved HR efficiency,\nwhich however brought cellular stress and reduced chemical production\ndue to constitutive expression of the HR-related gene. Here, we engineered\nan HR repair pathway using the dynamically regulated gene <i>ScRAD51</i> under the control of the l-rhamnose-induced\npromoter P<sub><i>LRA3</i></sub> based on the previously\nconstructed CRISPR-Cas9 system in O. polymorpha. Under the optimal inducible conditions, the appropriate expression\nlevel of <i>ScRAD51</i> achieved up to 60% of HR rates without\nany detectable influence on cell growth in methanol, which was 10-fold\nhigher than that of the wild-type strain. While adopting as the chassis\nstrain for bioproductions, the dynamically regulated recombination\nsystem had 50% higher titers of fatty alcohols than that static regulation\nsystem. Therefore, this study provided a feasible platform in O. polymorpha for convenient genetic manipulation\nwithout perturbing cellular fitness.