Metabolic engineering of the non-conventional yeast Kluyveromyces marxianus for enhancing the biosynthesis of succinic acid

Succinic acid is a crucial four-carbon dicarboxylic acid with widespread applications in the detergent, food, and pharmaceutical industries. However, its microbial production on a large scale is limited by the utilization of neutralizing agents and high cooling costs. In this study, we conducted metabolic engineering of the thermotolerant and acid-tolerant yeast Kluyveromyces marxianus to facilitate the efficient biosynthesis of succinic acid at high temperature and low pH. A robust genetic manipulation platform for K. marxianus was established by developing gene editing tools, characterizing neutral integration sites, and identifying endogenous promoters. Leveraging this efficient platform, we systematically constructed and optimized the biosynthetic pathway of succinic acid through many metabolic engineering strategies, such as the knockout of byproduct pathways, the redistribution of carbon flux, and the enhancement of the succinic acid transport system. Finally, the engineered strain K. marxianus KmSA12 was able to produce 50.6 g/L succinic acid with a yield of 0.31 g/g and productivity of 0.42 g/L/h in a 5-L bioreactor. These results demonstrate the potential of K. marxianus as a promising platform for the large-scale production of various organic acids.