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

backgroundMethanol 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.

resultsThrough 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 stress. Notably, the fastest-growing isolate, designated PM7a, was capable of growing in a minimal medium containing 5% methanol as the sole carbon source, whereas the wild-type strain was unable to do so. Furthermore, the introduction of the β-carotene biosynthesis pathway into both the wild-type and PM7a strains, cultured in a minimal medium containing methanol as the sole carbon source, resulted in a 4.91-fold higher titer in PM7a.

conclusionsTaken together, our findings demonstrate that ALE facilitates the development of strains with enhanced methanol tolerance, growth, and biosynthetic performance. These findings highlight the potential of the evolved P. pastoris strain as a robust chassis for methanol-based biomanufacturing.