Membrane remodelling for increased methanol tolerance in Komagataella phaffii

Methanol is a promising C1 feedstock for bioproduction but its cytotoxicity limits applications. Here we enhanced Komagataella phaffii GS115 methanol tolerance to 10–13% (v/v) by overexpressing four genes (phosphatidylethanolamine methyltransferase ( PET2 ), phosphatidylserine synthase ( PSA ), PAS_chr1-4_017 8 ( 0178 ) and PAS_chr2-2_0267 ( 0267 )) (S33 strain). Our results show that, under high methanol stress, S33 undergoes membrane lipid remodelling-driven metabolic reprogramming, with central carbon flux being redistributed from the pentose phosphate pathway and citric acid cycle towards glycolysis. Within this framework, 0178 encodes a vacuolar transporter of sulfur-containing amino acids, which we propose supports phospholipid methylation and redox balance. Meanwhile, 0267 encodes a peroxisomal thiolase-degrading long‑chain fatty acyl‑CoAs, limiting toxic long-chain sphingolipid accumulation, maintaining membrane stability. These genes enable S33 to achieve lipid titre of approximately 40 g l −1 under non-sterile fed‑batch fermentation conditions. This work positions S33 as a methanol-tolerant K. phaffii chassis for further strain and process development.