Analyses Of Comparative Transcriptome And Metabolome On The Methanol Dissimilation Pathway Of Pichia Pastoris

The methylotrophic yeast Pichia pastoris can use methanol as the sole source of carbon and energy for growth and metabolism.Methanol is oxidized to formaldehyde in the peroxisome,which is fixed intracellularly via the assimilation pathway,or completely oxidised in the cytoplasm to CO₂ for release extracellularly via dissimilation pathway.In this project,the CRISPR Cas9 system was constructed in P.pastoris,and the genes related to the dissimilation pathway were knocked out one by one.The phenotypic,transcriptomic and metabolomic differences between knockouts and GS115 under glucose and methanol incubation were compared using GO analysis and KEGG pathway analysis to mine and resolve the relevant genes and pathways affecting methanol utilization.The main findings are as follows.（1）Using the CRISPR Cas9 system,the formaldehyde dehydrogenase knockout strain（Δfld）,s-hydroxymethylglutathione hydrolase knockout strain（Δfgh）and formic acid dehydrogenase knockout strain（Δfdh）were successfully constructed in this project.When methanol was used as the main carbon source,the OD₆₀₀ growth rates ofΔfld,Δfgh andΔfdh were lower than those of GS115 at 60.98%,23.66%and 5.69%respectively.GS115 growth levels were significantly better thanΔfld andΔfgh.（2）The comparative transcriptome results are consistent with the phenotype.FLD was involved not only in methanol metabolism but also in organic nitrogen metabolism and amide biosynthesis;FGH regenerated GSH,assisted FLD in supplying energy and contributed to formaldehyde and hydrogen peroxide metabolism;FDH mainly affected intracellular acid metabolism,such as aldehydes,carboxylic acids and various amino acids.Comparative transcriptome results showed that FLD knockout caused differences in ABC transporters,amino acid biosynthesis,and protein digestion and absorption.（3）Multi-omics analysis revealed that the blockage of the dissimilation pathway resulted in transcriptional down-regulation of the assimilation pathway,down-regulation of the key metabolites D-glucose-6-phosphate and glyceraldehyde,and consequent down-regulation of central carbon metabolism,resulting in inefficient fixation of methanol to biomass.At the same time oxidative phosphorylation is significantly down-regulated,mitochondrial enzyme subunit activity is reduced,ATP supply is inadequate and the genetic replication process is compromised.Both of these aspects may be the main reasons for the low biomass of the knockout strains.（4）Formaldehyde not only complexes with glutathione,but also reacts with DNA and protein.Knockdown of FLD promotes amino acid metabolism and protein digestion and absorption.Glutathione and amino acids are up-regulated under formaldehyde exposure,contributing to formaldehyde complexation.Transcriptional up-regulation of proteasomes and autophagy is used to resolve formaldehyde-induced DNA-protein cross-linking.In this study,we investigated the differences in multigene transcript levels and small molecule metabolism during methanol metabolism in dissimilation pathway-deficient strains in a multidimensional manner,and resolved the relationship between the dissimilation pathway and the assimilation pathway,providing a theoretical basis for understanding the effects of dissimilation pathway blockade on intracellular metabolic networks.