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 optimised 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 optimisation 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.
Mauri et al. (Sun,) studied this question.