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

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 toward succinic acid (SA), but increased glycerol/acetate byproducts. Subsequent GPD1 / ACH1/ADH2A co-knockout in a Δsdh1,3,5,4A,2 strain with NDE1 overexpression (YZH43) yielded a chassis producing 32.38 g/L SA from glucose at 37°C, which is the highest reported titer in K. marxianus , while reducing ethanol, acetate, and glycerol by 60.79%, 89.24%, and 67.5%, respectively. At 46°C, YZH43 produced 20.51 g/L SA through simultaneous saccharification and fermentation (SSF) using cellulose as substrate. This work provides a high-efficiency CRISPR/Cas12a platform for K. marxianus , enabling rapid metabolic engineering for value-added chemical production, and demonstrates its utility in developing thermotolerant SA-overproducing strains.