• Over 100 g·L −1 succinic acid were produced from methanol in Komagataella phaffii. • Engineering a cytosolic reductive TCA pathway enabled high-titer production. • Dicarboxylate transporters were key for efficient acid export. • Fumarase and fumarate reductase drove flux to succinate. Succinic acid is a versatile platform chemical produced either by petrochemistry or via microbial fermentation of sugars. In light of increasing anthropogenic CO 2 emissions, sustainable alternatives using non-food, carbon–neutral substrates are urgently needed. Here, we report the efficient biosynthesis of succinic acid from methanol—a promising C 1 feedstock derivable from CO 2 —using the native methylotrophic yeast Komagataella phaffii. By integrating dicarboxylic acid transporters from Aspergillus niger and Schizosaccharomyces pombe, alongside a heterologous cytosolic reductive tricarboxylic acid pathway we achieved titers exceeding 100 g·L −1 . Further, by employing a top down approach, we could show that the expression of a transporter is crucial for succinic acid export in K. phaffii , combined with the expression of a fumarase and fumarate reductase, while expression of a malate dehydrogenase contributed minimally to pathway flux. Our work establishes K. phaffii as a robust host for C 1 -based bioproduction of succinic acid and highlights the potential of methanol in building a more sustainable future of bioprocesses.
Bachleitner et al. (Wed,) studied this question.