Enhanced methanol tolerance and utilization in Pichia pastoris revealed by adaptive laboratory evolution

BACKGROUND: Methanol is a promising renewable C1 feedstock for biomanufacturing. Native methylotrophs such as Pichia pastoris are considered potential chassis strains for methanol utilization. However, the cytotoxicity of methanol limits its usable concentration, posing a major bottleneck in bioproduction. In this study, we aimed to develop a P. pastoris strain with enhanced methanol tolerance and utilization by adaptive laboratory evolution. RESULTS: Through serial passaging under increasing methanol concentrations, we obtained an evolved strain that exhibited a specific growth rate of 0.101 h− 1 in a 7% (v/v) methanol, a condition under which the wild-type strain failed to grow. Genome resequencing identified the mutations, and the introduction of individual mutations into the wild-type background demonstrated that mutations in PSR1 and BFA1 significantly improved growth under methanol…