CRISPR‐Cas9 mediated genome editing of Kluyveromyces marxianus for iterative, multiplexed gene disruption and pathway integration

Abstract Kluyveromyces marxianus , a thermotolerant, fast‐growing, Crabtree‐negative yeast, is a promising chassis for the manufacture of various bioproducts. Although several genome editing tools are available for this yeast, these tools still require refinement to enable more convenient and efficient genetic modification. In this study, we engineered the K. marxianus NBRC 104275 strain by impairing the nonhomologous end joining and enhancing the homologous recombination machinery, which resulted in improved homology‐directed repair effective on homology arms of up to 40 bp in length. Additionally, we simplified the CRISPR‐Cas9 editing system by constructing a strain for integrative expression of Cas9 nuclease and plasmids bearing different selection markers for gRNA expression, thereby facilitating iterative genome editing without the need for plasmid curing. We demonstrated that tRNA was more effective than the hammerhead ribozyme for processing gRNA primary transcripts, and readily assembled tRNA‐gRNA arrays were used for multiplexed editing of at least four targets. This editing tool was further employed for simultaneous scarless in vivo assembly of a 12‐kb cassette from three fragments and marker‐free integration for expressing a fusion variant of fatty acid synthase, as well as the integration of genes for starch hydrolysis. Together, the genome editing tool developed in this study makes K. marxianus more amenable to genetic modification and will facilitate more extensive engineering of this nonconventional yeast for chemical production.