Development of a CRISPR/Cas12a genome editing toolbox in <i>Kluyveromyces marxianus</i> and its application in succinic acid biosynthesis.

ABSTRACT: Kluyveromyces marxianus is a promising thermotolerant yeast for industrial biotechnology, but lacks efficient genome engineering tools. A CRISPR/Cas12a genome editing toolbox for K. marxianus was developed for the first time in this study. A plasmid-free transient system achieved single-gene knockout efficiencies of about 50 %-100 % in Δku70 strain. Even with homology arms as short as 35 bp, the knockout efficiency remained 66.67 %. Chromosomal integration of Cas12a enabled single-to-triple fragment knock-ins efficiency of 82.93-85.70 % and 94.50 % for large fragment (>5 kb) integrations. Applying this system, the roles of succinate dehydrogenase (SDH) genes SDH1-SDH5 were elucidated. Combinatorial SDH genes knockouts redirected carbon flux

SUBMITTER: Zha H 

PROVIDER: S-EPMC12510058 | biostudies-literature | 2026 Mar

REPOSITORIES: biostudies-literature