Studies On Thermophily Of Chaetomium Thermophilum Based On Proteome And N-glycoproteome

Extreme microorganisms are a group of microorganisms that grow normally in extreme environments.Its special genes,unique physiological and biochemical mechanisms,and the biological macromolecules to adapt to special environments are of special significance in the study of biological origin and evolution.According to their living environment,they are mainly divided into the following categories: thermophilic microorganisms,psychrotrophic microorganisms,acidophilic microorganisms,alkaliphilic microorganisms,halophilic microorganisms and piezotrophic microorganisms.Thermophilic microorganisms usually refer to strains whose optimum growth temperature is above 50 ?.Chaetomium thermophilum is a thermophilic fungus of the phylum Ascomycota that grows at temperatures up to 55?.C.thermophilum was the first thermophilic fungus to obtain a genome sequence,and the availability of complete genome sequences of several mesophilic fungi closely related to C.thermophilum makes it a preferred choice for high temperature adaptation studies.C.thermophilum is suitable for large-scale production and has a wide range of applications due to its unique fermentation characteristics.In this study,the proteome and N-glycoproteome of C.thermophilum cultured at the lowest(30?),optimal(50?)and highest growth temperature(55?)were determined.4302 proteins were identified in C.thermophilum proteome,of which 3878 proteins were quantified by TMT labeling.Taking 1.5 times as the change threshold and t-test p-value<0.05 as the standard,among the differentially expressed proteins,there are 660,680 and 443up-regulated proteins in T50/T30,T55/T30 and T55/T50,and 614,688 and 630down-regulated proteins,respectively.The identification of 570 N-glycosylation sites in 316 protein groups was performed,of which the quantification of 498 sites in 278 proteins was accurately completed(fold change ratio >1.5 and p < 0.05).The number of up-regulated N-glycoproteins was 68,58,and 35 in T50/T30,T55/T30,and T55/T50,respectively.The number of down-regulated N-glycoproteins was 92,191,and 93 in T50/T30,T55/T30,and T55/T50,respectively.The analysis of subcellular prediction found that the differentially expressed proteins were mainly concentrated in the nucleus(30%),mitochondria(25%)and cytoplasm(25%).Gene Ontology(GO)enrichment-based analyses were conducted on proteome data.In the category of molecular functions,the high enrichment of many up-regulated proteins was realized in carbohydrate or polysaccharide binding and hydrolase activity,pattern binding,and unfolded protein binding.The enrichment of down-regulated proteins mostly occurred in the ribosome's structural constituents,structurally molecular activity,and oxidoreductase activity.For the category of biological processes,the enrichment of up-regulated proteins occurred in carbohydrate or polysaccharide metabolic,protein folding,cellular amino acid metabolic or biosynthetic process,and m RNA splicing.The enrichment of down-regulated proteins occurred in the translation,biosynthetic or metabolic process of peptide,amide,and protein and biosynthetic and homeostatic process of cellular macromolecule or nitrogen compound.In the category of cellular components,the high enrichment of up-regulated proteins occurred in the extracellular region,membrane or envelope,and mitochondrion.In contrast,the enrichment of down-regulated proteins mainly occurred in the ribosome and non-membrane-bounded organelle.Therefore,differentially expressed proteins could be highly correlated to carbohydrate metabolism,protein synthesis and metabolism,and protein folding,suggesting that energy metabolism and protein homeostasis play important roles in the thermophily mechanism of C.thermophilum.Analysis of differentially expressed N-glycoprotein found that most differentially expressed N-glycoproteins are distributed in the extracellular region(46%),cytoplasm(18%)and nucleus(11%).The cluster analysis were conducted on N-glycoproteome data.In GO functional enrichment,the biological process analysis showed that up-regulated glycoproteins of T55/T50 were mostly enriched in the glycoprotein metabolic macromolecule glycosylation and glycoprotein biosynthetic process.Up-regulated glycoproteins of T50/T30 and T55/30 were enriched in macromolecule biosynthetic and catabolic process.In molecular function,up-regulated glycoproteins of T55/T50 were mostly enriched in unfolded protein binding,and up-regulated glycoproteins of T50/T30 and T55/T30 were enriched in pattern binding,hydrolase activity,carbohydrate binding,and others.The KEGG pathway analysis of differentially expressed N-glycoprotein revealed five important pathways in response to temperature changes—cell cycle,autophagy,N-glycan biosynthesis,m RNA monitoring pathway,and protein processing in the endoplasmic reticulum.Therefore,it was shown that N-glycosylation can participate in the high temperature adaptability of C.thermophilum by regulating the synthesis and metabolism of glycoproteins and protein processing in the endoplasmic reticulum.Three differentially expressed N-glycoproteins Ct GLU1,Ct MAN1 and Ct GLU2 were selected to study the effect of N-glycosylation on protein activity and thermal stability.The N248 glycosylation site of protein Ct GLU1 was differentially modified,with the difference folds of 0.445,0.75 and 0.334 in T50/T30,T55/T50 and T55/T30,respectively.the N286 glycosylation site of protein Ct MAN1 was differentially modified,with the difference folds in T50/T30,T55/T50 and T55/T30,respectively.The N261 glycosylation site of protein Ct GLU2 was differentially modified,with fold differences of 5.213,1.681 and 8.764 in T50/T30,T55/T50 and T55/T30,respectively.The expression vectors p PIC9K/Ct GLU1,p PIC9K/Ct MAN1 and p PIC9K/Ct GLU2,and the N-glycosylation deletion mutants expression vectors p PIC9K/Ct GLU1-M,p PIC9K/Ct MAN1-M and p PIC9K/Ct GLU2-M were constructed.The vector was transferred to Pichia pastoris,methanol-induced expression,ammonium sulfate precipitation and protein purification were performed to obtain the proteins Ct GLU1,Ct MAN1 and Ct GLU2 and the corresponding N-glycosylation deletion proteins Ct GLU1-M,Ct MAN1-M and Ct GLU2-M.The enzyme activities of Ct GLU1-M,Ct MAN1-M and Ct GLU2-M were reduced by 18%,10% and 17%,respectively.Ct GLU1 has lost its enzyme activity at 70?,but Ct GLU1-M still retains nearly 40% of its enzyme activity but the Tm value has not changed.Ct MAN1-M and Ct GLU2-M have a thermal stability of about 15% at70?,and Tm value decreased by 5.1? and 7.4? respectively.The results showed that the protein Ct GLU1 can improve thermal stability through deglycosylation,and the proteins Ct MAN1 and Ct GLU2 can improve thermal stability through glycosylation.C.thermophilum regulates N-glycosylation to affect the thermal stability and activity of specific proteins,which is also one of the strategies of thermophily.Four differentially expressed glycosyltransferases Ct GT1,Ct GT2,Ct GT3 and Ct GT4 related to N-glycosylation modification were further selected to study the effect of N-glycosylation on the high temperature adaptability of C.thermophilum.The difference multiples of Ct GT1 at T50/T30,T55/T50 and T55/T30 were 0.66,1.16 and 0.76,respectively.The difference multiples of Ct GT2 at T50/T30,T55/T50 and T55/T30 were 0.59,1.11 and0.66,respectively.The difference multiples of Ct GT3 at T50/T30,T55/T50 and T55/T30 were1.74,0.90 and 1.57,respectively.The difference multiples of Ct GT4 at T50/T30,T55/T50 and T55/T30 were 1.64,1.37 and 2.24,respectively.Overexpression strains OCt GT1,OCt GT2,OCt GT3 and OCt GT4 were constructed respectively,and all of them were highly expressed by q PCR verification.At 55?,the growth rate of OCt GT2 and OCt GT3 was significantly higher than that of the wild type,110% and 122% of wild strains respectively.The thermal stability of the cellulase secreted and expressed by OCt GT3 was also higher than that of the wild type.The gene knockout strains ?Ct GT1 and ?Ct GT4 and their reverted strains were constructed,and it was found that the gene Ct GT1 had little effect on C.thermophilum,while the gene Ct GT4 could significantly affect the growth of the strain and the thermostability of the cellulase secreted and expressed.The growth rate of ?Ct GT4 at 50? and 55? is only72% and 48% of that of the wild strain,and the morphology of the strain changes significantly.The gene knockout strains ? Ct GT2 and ? Ct GT3 were not obtained in this study.It is preliminarily speculated that the genes Ct GT2 and Ct GT3 may play an important role in C.thermophilum.The results showed that glycosyltransferase can affect the growth and high temperature adaptability of C.thermophilum.,which further proves that N-glycosylation is involved in the high temperature adaptability of C.thermophilum..All together,energy metabolismthe and the efficient protein homeostasis pathway plays a key role in the thermophily of C.thermophilum.N-glycosylation is involved in protein homeostasis by affecting related proteins,and C.thermophilum regulates N-glycosylation to affect the thermal stability and activity of specific proteins,which is also one of the strategies of thermophily.