The Preliminary Study On The Function Of N-glycosylation-related Enzymes Of Penicillium Oxalicum In Protein Synthesis And Secretion

The degradation and conversion of lignocellulosic biomass to produce liquid fuels and compounds are important alternative ways to ensure the sustainable development of human society.Penicillium oxalicum can produce a complete lignocellulose-degrading enzyme system that degrades plant cell wall polysaccharides,effectively hydrolyzing lignocellulose into soluble sugars,and is used in the production of biofuels and chemical productsProtein glycosylation plays an important role in living organisms,such as immune protection,signal transduction regulation,protein translation regulation,protein degradation,cell wall synthesis,etc.Exploring the biological function of the N-glycosylation in cellulose-degrading fungi will deepen the understanding of the functions of glycosylation-related enzymes and substrate selectivity,which will be helpful to summarize the basic state and general rules of glycosylation modification of many enzyme components in the extracellular secretory protein group of fungi,made the route of manually adjusting the glycosylation structure gradually become feasible,and provide new ideas for increasing the yield of cellulose and producing cellulose with specific N-sugar chains.This paper takes the important cellulose degrading enzyme production fungus Penicillium oxalicum as the research object,using genetics,glycomics and secretomics research methods,focusing on the biological function of several N-glycosylation modifying enzymes,The effect of N-glycosylation modification on cellulase synthesis was confirmed,and the mechanism of N-glycosylation modification enzyme involved in cellulase synthesis was preliminary exploredMajor advances in this thesis are as follows1.The role of the ? subunit of ?-1,3-glucosidase ??Gls???in the growth state,the synthesis of the N-oligosaccharide chains of secreted protein and the synthesis of cellulases.Etc.The ?gls?? strain was obtained by homologous recombination.It was observed that compared with the parent strain WT,the amylase activity increased 5.23 times higher than WT,the protein concentration increased by 80.1%,and the ability to degrade microcrystalline cellulose was enhanced.Next,the structural analysis of the N-oligosaccharide chains of the extracellular protein revealed that the total glycosylation level of the extracellular protein of ?gls?? had changed significantly,the overall preference was for high mannose,and Glc1Man9GlcNAc2 which is a special N-oligosaccharide chain with high abundance and large proportion appeared Through secretomics analysis,we found that the knockout of ?Gls?? significantly affected the types and relative contents of extracellular proteins,the secretion of glycoside hydrolases such as ?-amylase,cellulose degrading enzymes such as?-1,4-glucanase,and cellulose degrading auxiliary enzymes such as cellulose monooxygenase Cel61A had increased.These indicated that the destruction of the N-glycosylation enzyme Gls? affected the process of N-glycosylation modification and changed the process of nascent glycoproteins.The specific extracellular?-glucosidase?BGL1?was purified,and it was found that the N-oligosaccharide chains of BGL1 produced by ?gls?? was slightly longer than that of BGL1 produced by WT,and the ability of degradation of p-Nitrophenyl-?-D-galactopyranoside?pNPG?increased by 65.3%,and the stability at 70? also increased,but the stability at pH 7.0 decreased,indicating that the change of N-oligosaccharide chains of BGL1 protein affected the enzyme activity and stability of the cellulase2.The role of the three ?-1,2-mannosidases?Mns3,Mns2,Mnsl?in the growth state,the synthesis of the N-oligosaccharide chains of secreted protein and the synthesis of cellulases.Etc.The ?mns3,?mns2,?mns1,and OEmns3 casstte were constructed and transformed P.oxalicum 1 14-2 by homologous integration.The knockout of the mnsl gene affected the normal growth of the strain under cellulose culture conditions,the extracellular protein concentration decreased by 42.4%,the ability to degrade microcrystalline cellulose was severely reduced,and the FPase activity was only 4.60%of WT.The knockout of mns3 and mns2 enhanced the strains ability to degrade microcrystalline cellulose.The FPase activity and amylase activity of ?mns3 increased by 1.07 times and more than 1.98 times respectively,the extracellular protein concentration increased by 1.29 times.The FPase activity and amylase activity of ?mns2 increased by 1.14 times and 2.71 times respectively,the extracellular protein concentration increased by 1.23 times.When analyzing the structure of the N-oligosaccharide chains of the extracellular protein,the extracellular protein produced by the Amns3 strain only existed in two high mannose types,Man8GlcNAc2 and Man9GlcNAc2,and the total glycosylation level of extracellular protein of ?mns2 did not change significantly.Through secretomics analysis,the knockout of mns3 and mns2 also significantly affected the types and relative contents of extracellular proteins,and the secretion of many glycoside hydrolases including?hemi?cellulases of ?mns3 and ?mns2 had increased,indicating Mns3 and Mns2 are involved in the process of N-glycosylation modification which indirectly or directly affected the maturation and secretion of?hemi?cellulases.Next,the site-specific N-glycosylation analysis indicated that ?wns3-BGL1 was biased towards the high-mannose type.The enzymatic properties of BGL1 in ?mns2 and ?mns3 did not change significantly.The above research suggests that Mns3 and Mns2 have different functions in the synthesis of N-oligosaccharide chains of secreted protein and the synthesis of cellulases3.Three glycoside hydrolase family 12?GH12?enzymes display diversity in substrate specifcities and synergistic action between each other.PDE₀6439?PoCel12A?,PDE₀2886?PoCel12B?,and PDE₀9014?PoCel12C?are genes that encode GH12 enzymes in P.oxalicum.Recombinant enzymes?named rCel12A,rCel12B and rCel12C?demonstrate existing diversity in the substrate specifcities.PoCel12A is typical GH12 enzymes that belong to fungal subfamilies 12-1,rCel12A is a non-typical endo-?1-4?-?-glucanase.PoCel12B is typical GH12 enzymes that belong to fungal subfamilies 12-2,rCel12B possesses a signifcantly high activity against xyloglucan.A specifc activity of rCel12B toward xyloglucan?239 ?mol/min/mg?is the second-highest value known.rCel12B is a typical xyloglucan-specifc endo-P-1,4-glucanase.PoCel12C contains a low-complexity region?LCR?domain,it is a non-typical?hemi?cellolase.A synergistic action toward phosphate-expanded cellulose?PASC?among rCel12A and rCel12B is observed.