In Vivo Multi-Enzymatic Catalysis for Bioproduction of L-Lactic Acid from Sole Methanol

Microbial biotransformation of methanol for lactate production presents a promising carbon-neutral strategy to mitigate "white pollution". However, this approach has been hindered by poor cell growth on methanol, largely due to the methanol toxicity and possible competition of the precursor pyruvate between the tricarboxylic acid cycle and lactate synthesis. To address this, we developed an in vivo multi-enzymatic catalysis system in the methylotrophic yeast Ogataea polymorpha for direct L-lactate production from methanol. We determined that the optimal process involves cell culture and enzyme induction in a mixed glucose and methanol, followed by a dedicated cell catalysis phase. Finally, by integrating CO₂ hydrogenation with this multi-enzymatic pathway, we achieved an L-lactate titer of 19 g/L from methanol in a 5 L bioreactor within 46 h, with a yield of 0.30 g/g and a productivity of 407 mg/L/h. This established in vivo catalysis platform demonstrates significant potential for expanding methanol biotransformation into a wider array of valuable chemicals.