摘要：SummaryMethanol biotransformation can expand biorefinery substrate spectrum other than biomass by using methylotrophic microbes.Ogataea(Hansenula)polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but the 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 inO. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ) increased 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 system has been applied for homologous integration of large fragments andin vivoassembly of multiple fragments, which enables the production of fatty alcohols inO. polymorpha. These findings will simplify genetic engineering in non-conventional yeasts and facilitate the adoption ofO. polymorphaas an attractive cell factory for industrial application.Graphical abstractDisplay OmittedHighlights•Establishing an efficient and convenient CRISPR-Cas9 system inOgataea polymorpha•Enhancing homologous recombination for precise genome editing inO. polymorpha•Realizing seamless deletion and assembly of multiple fragments inO. polymorphaBiological sciences; Molecular Microbiology; Bioengineering; Metabolic Engineering; Biotechnology; Microbial Biotechnology
