A Study On How GPI-anchored Protein Gcw13p Effects The Methanol Utilization And Growth Of Pichia Pastoris

The methylotrophic yeast Pichia pastoris is one of the few yeast species that can use methanol as the sole source of energy and carbon.It is widely used as a heterologous expression platform for industrial production of a range of valuable proteins.Pichia pastoris auxotrophic mutant GS115 is a common strain for studying the regulation of methanol metabolism,peroxisome proliferation/autophagy.The growth of GS115 in methanol medium is slow.Previous studies in our laboratory showed that knocking out of Gcw13 p resulted in an increasing of growth rate of GS115 when grown on methanol,which could be reversed by complementing Gcw13 p to the knock-out strain.Based on this finding,we study on on how GPI-anchored Protein Gcw13 p effects the growth of Pichia pastoris on methanol by analyzing the of the structure and properties of Gcw13 p,the differential expression profiles of Gcw13 p knockout strains,the effect of Gcw13 p defeciency on methanol metabolism,amino acid metabolism and autophagy.The main results and conclusions of this study are:(1)Through the replenishment of GCW13 in the GCW13? strain,the relationship between Gcw13 p and methanol growth phenotypic change was confirmed.By analyzing the structure and properties of Gcw13 p,it was found that Gcw13 p was mainly distributed in cell membrane and vacuole,and it was probably cell membrane protein.Bl AST analysis showed that Gcw13 p has no homology to known proteins.(2)The gene expression difference between gcw13? strain and GS115 on methanol as a carbon source was analyzed by RNA-seq.It was found that the deletion of Gcw13 p had no significant effect on the gene expression of the methanol utilization-related pathway,but significantly down-regulated the expression of genes involved in the amino acid anabolic pathway.It is indicated that the methanol growth phenotypic changes caused by Gcw13 p deletion are not related to methanol metabolism,but may be related to amino acid metabolism and utilization.(3)By studying the effect of Gcw13 p defeciency on methanol metabolism,amino acid absorption and autophagy,it was found that the growth of GS115 was inhibited when using methanol as a carbon source,which may be the amino acid starvation due to histidine synthesis defeciency and decrease of amino acid uptake,which causes a decrease in TORC1 activity,which in turn causes autophagy.The deletion of Gcw13 p up-regulated the expression of amino acid permease Gap1 p and reduced its endocytosis,which increased the uptake of Gap1-dependent amino acids,thereby increasing intracellular amino acid concentration and promoting the growth in methanol medium.At the same time,the viability of Gcw13 p knockout strain in heat stress and nitrogen starvation decreased,indicating that Gcw13 p may be related to stress stress response.