Bioconversion of the C1 substrate methanol to succinic acid with engineered Komagataella phaffii.

Succinic acid is a versatile platform chemical produced either by petrochemistry or via microbial fermentation of sugars. In light of increasing anthropogenic CO2 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 C1 feedstock derivable from CO2-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 C1-based bioproduction of succinic acid and highlights the potential of methanol in building a more sustainable future of bioprocesses.