Transcriptome analysis of Pichia pastoris delta-FLD adaptation strain with high methanol utilization and tolerance

Pichia pastoris (komagataella phaffii) is a very potential methylotrophic cell factories for methanol-based biomanufacturing, but its assimilation pathway is much lower than dissimilation pathway. To further improve methanol assimilation, we mutated formaldehyde dehydrogenase FLD to block methanol dissimilation pathway and forced the mutant to utilize methanol only by methanol assimilation via adaptive laboratory evolution strategy. Our results showed that we successfully blocked the methanol dissimilation by FLD mutant and obtained the evolved strain delta-FLDV6 with high methanol utilization and tolerance. delta-FLDV6 can grow on 4% methanol medium with 2 folds OD600 more than GS115. For better understanding of the transcriptional regulation, we focused on the transcriptome analysis of delta-FLD and delta-FLDV6 on high concentration of methanol. The expression profile of delta-FLD showed that genes were globally downregulated across all the time periods. Adaptation allowed delta-FLDV6 to obtain energy from amino acid metabolism to restore most of gene expression via some of the key enzymes of TCA pathway, such as KGD1, KGD2, LSC1, LSC2 and PDA1. At the same time, peroxisome, alcohol oxidase AOX1 and AOX2, PPP pathway, amino acids biosynthesis were strongly up-regulated compared to GS115. The ability of resistance to ROS was also improved. The glycolysis was furtherly downregulated in delta-FLDV6 compared with GS115. We assumed that all those were regulated by several key transcriptional factors with MET32, MET4 and CBF1 playing the core role in the regulation of complex metabolic networks. This study successfully improved the methanol assimilation by blocking methanol dissimilation pathway and changed the energy obtainments from amino acids metabolism. We made a preliminary attempt to elucidate metabolic networks of the transcriptional regulation on the energy obtainments and protection of cells avoid formaldehyde toxify and ROS damage in Pichia pastoris on high methanol condition. This study provided new understanding of the genetic regulation on Pichia pastoris and genetic targets for metabolic engineering of native and synthetic methylotrophs to enhance the methanol utilization and tolerance.