Developing Genetic Tools Of Chaetomium Thermophila And Heterologous Expression Of Its Enzyme Genes

Thermophilic filamentous fungi have excellent enzyme gene resources and fermentation characteristics,but they are rarely studied and developed.Many biomass-degrading thermophilic fungi can secrete a variety of biomass-degrading enzymes with excellent thermal stability,including cellulase,protease,amylase,laccase,chitinase,lipase,and esterase.The development of these enzymes into enzyme preparation products has a very wide range of application values in the fields of breeding,planting,environmental protection,food and health products.This thesis takes four thermophilic fungi isolated from high-temperature compost soil as the research object.First,the lignocellulose degradation ability is evaluated,and it is found that Chaetomium thermophilum has strong cellulose degradation ability.In order to further increase the cellulase expression of C.thermophila,this thesis then explored the genetic manipulation system of C.thermophila,and its main extracellular enzyme component cellobiohydrolase(CBH1)Carried out heterologous expression and separation and purification.The specific experimental results are as follows:1.Study on the growth and enzyme-producing properties of four thermophilic fungi isolated from high-temperature compostingHigh-temperature compost is rich in high-temperature-resistant microorganisms that can degrade biomass.In this thesis,four thermophilic fungi isolated from high-temperature compost were identified and evaluated for their cellulose degradation ability.Through ITS molecular identification,the four strains are:C.thermophilum,Thermomyces lanuginosus,Rhizopus microsporus and Myceliophthora thermophila;According to literature search,in recent years,thermostable thermophilic enzymes have been obtained from C.thermophila,which has caused a wide range of people,but there are still few studies on C.thermophila.T.lanuginosus is a thermophilic fungus that is widely distributed and can produce thermostable lipase with industrial value.R.microsporum is a plant rhizosphere fungus,which has certain research in agricultural production.M.thermophila is a thermophilic fungus widely used in industry at present,and the research on its protein secretion and metabolites is relatively mature.The growth status and fermentation enzyme production of four thermophilic fungi were studied.The growth temperature of the four strains was 45?,and they grew faster on PDA medium,and the colonies could reach 8.50 cm within 5 days;C.thermophilum can secrete up to 0.25 mg/mL of extracellular protein,and its filter paper enzyme activity can reach 1.07 U/mL;M.thermophila can secrete up to 0.07 mg/mL of extracellular protein,and its filter paper enzyme activity can reach 0.204 U/mL;Under the test conditions of this paper,T.lanuginosus and R.microsporus microsporus hardly secrete protein;comparing with the the filamentous fungus industrial production strain-Trichoderma reesei wild-type strain QM6a.The study found that the amount of extracellular protein secretion and expression of C.thermophila is relatively high,and its filter paper enzyme activity,AZO-CMC enzyme activity and AZO-XYLAN enzyme activity are all higher than those of T.lanuginosus,R.microsporum,M.thermophila and T.reesei QM6a.This result shows that among the four thermophilic fungi,C.thermophila has a strong ability to degrade lignocellulose and has great development potential.2.Analysis of growth and enzyme production of C.thermophila under different substrate conditionsThrough the above research,it is found that C.thermophila has a strong cellulose degradation ability compared with the other three thermophilic bacteria.Therefore,C.thermophila was used as the experimental object to analyze its biomass accumulation,protein secretion,and cellulase and hemicellulase activities under different substrate conditions.The results showed that when microcrystalline cellulose(Avicel),bagasse,and tea dregs were used as substrates,they had a degradation effect on the three substrates;the maximum extracellular secretion on Avicel was 252 ?g/mL,and the filter paper enzyme activity can reach 0.820 U/mL,but the overall amount of exocrine protein is lower;reach 0.820 U/mL,but the overall amount of exocrine protein is lower;compared with the wild-type strain QM6a of T.reesei,the enzyme activities of AZO-CMC and AZO-XYLAN are higher than those of T.reesei QM6a;In addition,the main component of the exocrine protein of C.thermophila is cellobiohydrolase I(CBHI),and its extracellular secretion ratio,expression level and activity are relatively high.3.Establishment of genetic manipulation system of C.thermophilaC.thermophila has a relatively low amount of external secretion protein.In order to further improve its protein secretion ability through genetic modification,the genetic manipulation system of C.thermophila was explored.Because the commonly used resistance selection markers for filamentous fungi,such as hygromycin,are easily inactivated under high temperature,they cannot play a role in selection.Therefore,this paper uses terbinafine,which is more stable at high temperature,as a resistance screening marker.It is known from the literature that the overexpression of the erg1 gene in the ergosterol biosynthetic pathway can make filamentous fungi resistant to terbinafine.In this paper,the cDNA sequence of the erg1 gene of C.thermophila was inserted between the Actin promoter and the Tgpd terminator to construct the expression vector p Ac tin-erg 1-Tgpd.The p Ac tin-erg 1-Tgpd was transformed into C.thermophila by means of protoplast transformation to make it resistant to terbinafine for screening.The results showed that protoplasts were produced in a large amount after about 1.5 hours of lysis;the terbinafine concentration was explored,and the growth of protoplasts was significantly inhibited when the concentration of terbinafine was 16?g/mL;After the protoplast transformation,no transformant growth was observed in the screening medium,and the genetic transformation test was unsuccessful.In the later stage,other transformation methods will be tried for subsequent transformation studies.4.Heterologous expression and protein purification of CBHI gene of C.thermophileSince the genetic transformation system of C.thermophila was not successfully constructed,in order to further increase the expression of its enzyme,an attempt was made to express its enzyme gene heterologously.From the above studies,it is found that the main component of the exocrine protein of C.thermophila is CBH1,indicating that its CBH1 plays a key role in the degradation of cellulose.In order to further try to increase the expression level of C.thermophila enzyme genes in other systems,this thesis tried to heterologous expression and purification in E.coli and P.pastoris.In the heterologous expression of E.coli,the pBRla plasmid was used as the vector in this study,and the cbh1 gene cDNA sequence of C.thermophila was inserted between the araC promoter and the TrrnB terminator to construct two expression vectors respectively.The plasmid pRB la-sp-cbhl with the signal peptide of C.thermophila itself and the plasmid pRBla-cbhl without the signal peptide of C.thermophila itself.The pRB1a-cbh1 and pRB1a-sp-cbh1 were transformed into E.coli BW25113 for heterologous expression.The results showed that the CBH1 protein formed inclusion bodies in E.coli cells and could not be successfully expressed.Considering the shortcomings of prokaryotic expression that a large number of inclusion bodies may be formed,and the protein cannot be folded correctly and secreted to the outside of the cell,this paper tried to heterologously express the CBHI of C.thermophila in P.pastoris.Based on the pPIC9K plasmid vector,the cDNA sequence of the Cbh1 gene of C.thermophila was inserted between the Aox1 promoter and the terminator to construct the pPIC9K-cbh1 expression vector.The pPIC9K-cbh1 was transformed into P.pastoris GS115 for heterologous expression.The results showed that CBHI protein can be expressed in P.pastoris;the enzyme activity of CBHI was determined by the 4-Methylumbelliferyl ?D-cellobioside method,and the enzyme activity was 1.84 U/ml;afterwards,it was fermented and purified to obtain the purified protein concentration.It is 222 ?g/mL,its enzyme activity is 12.21 U/mL,and its specific activity is 54.95 U/mg.