Abstract
Kluyveromyces marxianus, a non-conventional thermotolerant yeast, is potentially useful for production of ethanol and other products. This species has a strong tendency to randomly integrate transforming DNA fragments, making necessary the development of more precise methods for gene targeting. In this study, we first demonstrated that K. marxianus NBRC1777 is cold-tolerant, and then established a highly efficient and precise technique for gene editing by introducing genes encoding deaminase-mediated targeted point mutagenesis (Target-AID) and clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas9). We used Target-AID to introduce targeted point mutations that disrupted Nej1 or Dnl4, genes that are involved in non-homologous end-joining (NHEJ). Both of the resulting mutant strains showed enhanced proportions of homology-mediated integration compared to the wild-type parent. In combination with target cleavage by CRISPR-Cas9, markerless integration was performed using short (~50 bp) flanking homologous sequences. Together, these tools render this species fully tractable for gene manipulation, permitting targeted genetic changes in the cold- and thermo-tolerant yeast K. marxianus.