Engineering of CO recycling and formate metabolism for succinic acid production in Yarrowia lipolytica.

Succinic acid (SA), a key platform chemical in biorefineries, faces industrial production challenges stemming from inefficient CO fixation and redox imbalances in microbial cell factories. Here, we report a novel strategy to enhance SA biosynthesis in unconventional yeast Yarrowia lipolytica by cooperatively integrating carbonic anhydrase (CA)-mediated CO recycling with formate metabolism. Initially, the expression of CA improved the carboxylation efficiency of reductive TCA (rTCA) pathway, resulting in the strain Hi-SA-YlCA, which produced 89.75 g/L of SA with a yield of 1.01 g/g glucose under 5 % CO conditions-the highest yield reported for yeast to date. Subsequently, formate was utilized as an auxiliary carbon source, generating both CO and NADH via formate dehydrogenase (FDH) catalysis. Through systematic strain engineering and fed-batch optimization, the engineered strain consumed 6 g formic acid in a 5-L bioreactor, yielding an SA titer of 97.54 g/L with yield of 0.64 g/g mixed carbon source (Glucose and formic acid). This corresponds to a 10.5 % increase in titer and a 4.6 % enhancement in yield relative to the control. Notably, this study pioneers the integration of CA-enhanced carbon fixation with formate-driven NADH regeneration, simultaneously improving CO availability and alleviating redox balance limitations. Our findings provide a scalable framework for sustainable SA production and advance the application of one-carbon compounds in industrial biomanufacturing.