Optimization and scale up of L-malic acid production from methanol by the methylotrophic yeast Ogataea polymorpha

Industrial production of malic acid remains dependent on fossil resources or, when performed microbiologically, on sugar-based feedstocks. Both routes come with caveats, generating emissions and competing with food supply. The use of CO₂-derived one-carbon substrates offers a promising alternative to circumvent these constraints. In this study, the malic acid production process from methanol in metabolically engineered Ogataea polymorpha NYCY495 LEU-ΔSTE12 Pyc Mdh MAE1 strain was optimized and scaled up. A two-phase cultivation strategy, using glycerol for biomass formation and methanol for product synthesis, was established in shake flasks and subsequently transferred to a 1 L bioreactor. Process optimization through automated feeding strategies was evaluated. DO-based feeding was the most effective approach, using a combination of methanol and glycerol, achieving a final molar yield of 0.1 molMA molMeOH ⁻¹ and a maximum productivity of 0.5 g l⁻¹ h⁻¹. This successful fermentation strategy was validated using green methanol, showcasing the feasibility of "closing the loop" as envisioned in the bioeconomy. Finally, a comparative study of the effect of glycerol, methanol, and their mixture on O. polymorpha NYCY495 LEU-ΔSTE12 Pyc Mdh MAE1 methanol metabolism, peroxisome biogenesis, and cellular redox balance is presented, supporting the positive cumulative effect of both on gene transcription.