Dynamically Regulating Homologous Recombination Enables Precise Genome Editing in .

Methylotrophic yeast has become a promising cell factory due to its efficient utilization of methanol to produce high value-added chemicals. However, the low homologous recombination (HR) efficiency in greatly hinders extensive metabolic engineering for industrial applications. Overexpression of HR-related genes successfully improved HR efficiency, which however brought cellular stress and reduced chemical production due to constitutive expression of the HR-related gene. Here, we engineered an HR repair pathway using the dynamically regulated gene under the control of the l-rhamnose-induced promoter P based on the previously constructed CRISPR-Cas9 system in . Under the optimal inducible conditions, the appropriate expression level of achieved up to 60% of HR rates without any detectable influence on cell growth in methanol, which was 10-fold higher than that of the wild-type strain. While adopting as the chassis strain for bioproductions, the dynamically regulated recombination system had 50% higher titers of fatty alcohols than that static regulation system. Therefore, this study provided a feasible platform in for convenient genetic manipulation without perturbing cellular fitness.