A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in .

The methylotrophic budding yeast has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter (pAOX1). However, it is difficult to use as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination (HR) efficiency and the limitation of handy selective markers, especially in the condition of multistep biosynthetic pathways. Hence, we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker (HiEE-ReSM) to facilitate cell factory in . Firstly, we improved the HR rates of through knocking out the non-homologous-end-joining gene () and overexpressing HR-related proteins ( and ), resulting in higher positive rate compared to the basal strain, achieved 97%. Then, we used the uracil biosynthetic genes as the reverse screening marker, which can improve the recycling efficiency of marker. Meanwhile, the HR rate is still 100% in uracil auxotrophic yeast. Specially, we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter () to increase the uptake of exogenous uracil from medium. Meanwhile, we explored the optimal concentration of uracil (90 mg/L) for strain growth. In the end, the HiEE-ReSM system has been applied for the inositol production (250 mg/L) derived from methanol in . The systems will contribute to as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon (C1) bio-utilization.