| Komagataella phaffii is one of the most potent systems for protein expression.In particular,it is of great importance and widely used in food,fermentation and pharmaceutical industries.However,little is known about the mechanism of highly efficient heterologous protein in K.phaffii.In this study,the K.phaffii with high yield phospholipase was selected as the research object,and obtained the transcriptional expression level of all the genes using RNA-Seq technology.We next studied the effects of different carbon source conditions(glycerol phase and methanol phase)and different phospholipase A2 expression levels(different phospholipase gene copy number)on gene differential expression to comprehensively explore the mechanism of efficient expression of phospholipase in K.phaffii.The main research results are as follows:(1)There were 857 significant differential genes in the comparison of methanol induction and glycerol culture.The results showed that,through the KEGG cluster analysis of differential genes,ribosomal,methanol metabolism,pentose phosphate pathway and endoplasmic reticulum protein processing were significantly up-regulated,while the glycerol metabolic pathway and glyoxylic acid circulating metabolic pathway were significantly downregulated,but the TCA cycle has no change.When methanol was used as the sole carbon source,methanol was not only used for cell growth,but also induced expression of AOX promoter.At the same time,the expression level of protein synthesis pathway was increased and the expression of phospholipase A2 was promoted.(2)A total of 164 genes were significantly changed compared with the strains with different phospholipase A2 gene copy number.Through the KEGG cluster analysis of differential genes,the results showed that metabolic pathways mainly focused on DNA replication,endoplasmic reticulum protein processing and ribosome metabolism.The phospholipase A2 gene copy number affected the growth status of the strain.First,DNA replication and base repair related gene were downregulation.Secondly,ribosomal protein synthesis and ribosome component gene expression decreased and protein translation decreased.Thirdly,the expression of heat shock protein and cell stress related protein was significantly up-regulated by overexpressing heterologous protein.Finally,under the stress state of the cells,endoplasmic reticulum protein processing was up-regulated,increased the ability of protein folding,promoted the expression of protein phospholipase A2 gene.At the same time,triggering the ERAD effect,improved the ability to degrade the wrong folding protein.(3)Up-regulated genes were enriched in heat shock protein-related genes in high copy phospholipase A2 strains.Therefore,the effects of the expression of heterologous protein phospholipase A2 and prolyl endopeptidase on K.phaffii were studied by co-expression of heat shock protein-related genes.The effect of co-expression of heat shock protein on phospholipase A2 expression was from high to low: STI1,FES1,SSA1 and CPR6.Phospholipase A2 protein expression increased by 35%,enzyme activity increased by 50% with co-expression of RPL10.Co-expression of FES resulted in the protein concentration of extracellular total protein was increased from 0.20 g?L-1 to 0.26 g?L-1,The activity of the prolyl endopeptidase was 14.28% higher than that of the control strain,reaching 200 U·L-1.(4)According to the results of different protein expression sequencing,it was found that the down-regulated genes were concentrated in the ribosomal protein-related genes.The crude phospholipase A2 and prolyl endopeptidase were expressed as K.phaffii,and the highest concentration of proline endopeptidase protein was K.phaffii GS115/pPIC9K-MLMH/pPICZRPL10,the total extracellular protein concentration was 0.25 g?L-1,which was 8.6% higher than that of the control strain,reaching 190 U?L-1. |
