Sorbitol uptake and oxygen transfer shape AOX1 promoter induction under methanol-free conditions in Komagataella phaffii lacking formate dehydrogenase

For decades, expression systems based on the methanol-regulated AOX1 promoter (p AOX1 ) from the alcohol oxidase 1 gene have served as a benchmark for recombinant protein (rProt) production in Komagataella phaffii . However, methanol-free processes are increasingly being developed to overcome the drawbacks of methanol utilization, particularly its toxicity and flammability. The use of formate as a p AOX1 inducer in combination with sorbitol, a non-repressive carbon source, has emerged as a promising alternative to methanol-based expression systems. Recently, we demonstrated that formate derived from the tetrahydrofolate-mediated one-carbon (THF-C1) metabolism accumulates in K. phaffii cells deficient in formate dehydrogenase (FdhKO) when grown in sorbitol-based methanol-free medium. Using the lipase CalB from Candida antarctica as a model protein, we observed that rProt productivity in an FdhKO strain grown on sorbitol was comparable to that of an Fdh-proficient strain grown on methanol. However, sorbitol is inefficiently metabolized in K. phaffii , leading to a low growth rate and potentially limiting rProt productivity due to insufficient energy and carbon supply.

Here, we increased sorbitol uptake rate, and thus improved sorbitol metabolism, by overexpressing the gene encoding sorbitol dehydrogenase ( SOR1 ) in an FdhKO strain. Our results demonstrate that while increased sorbitol metabolism promotes biomass formation, it reduces p AOX1 induction, as evidenced by lower formate accumulation and decreased rProt productivity, both for intracellular eGFP and secreted proteins namely CalB lipase and glucose oxidase GOx from Aspergillus niger in SOR1 -overexpressing strains. Additionally, oxygen availability for cells influences these dynamics, with lower oxygen transfer favoring higher p AOX1 induction due to increased formate accumulation in an FdhKO strain. Our data also suggests that at low oxygen transfer and low sorbitol uptake rate, the proportion of cells in an induced state increased significantly. This work provides valuable insights into the interplay between sorbitol metabolism and oxygen transfer conditions, contributing to the development of improved recombinant protein production strategies in K. phaffii .