| Lignocellulose is the most abundant renewable biological resource on earth.Comprehensively promoting the high-value utilization of lignocellulose is one of the important ways to achieve carbon peaking and carbon neutrality.Trichoderma reesei is the most widely used cellulase industrial workhorse which has a strong ability of protein synthesis and secretion.Taking lignocellulose as its carbon source can effectively reduce the fermentation cost of T.reesei,and induce more efficient cellulases cocktails that in accordance with the degradation of certain lignocellulose materials.Considering the special structure of lignocellulose,physical or chemical pretreatment is needed to increase the accessibility of enzyme to cellulose in the process of high-value utilization of lignocellulose.However,some toxic compounds,such as acetic acid,furfural and 5-hydroxymethylfurfural,are usually produced in the pretreatment process,which severely hamper the later enzymatic hydrolysis and microbial fermentation process.How to relieve the inhibition of these harmful components in lignocellulose during microbial fermentation is one of the key problems to be solved in the process of high-value utilization of lignocellulose.Thus,it is necessary to study the utilization efficiency of T.reesei on different pretreated lignocellulose and its tolerance to inhibitors to meet the requirement of carbon peaking and carbon neutrality policy.Taking sugarcane bagasse as the research object of lignocellulosic materials,the analysis of T.reesei’s utilization efficiency of different pretreated lignocellulose and improving furfural tolerance of T.reesei were carried out in this thesis.The following results were obtained:1.Effects of different pretreated sugarcane bagasse on the growth and enzyme production of T.reeseiThis study compared the effects of acid-pretreated sugarcane bagasse(Ac-SCB),alkali-pretreated sugarcane bagasse(Ak-SCB)and unpretreated sugarcane bagasse(Un-SCB)as carbon source on the growth,extracellular protein secretion and enzyme production of T.reesei RUT-C30.The results showed that Ac-SCB severely inhibited the spore germination and mycelial growth of T.reesei,and the biomass of T.reesei on Ak-SCB increased two folds at 48 h after inoculation.Comparing to cultivated on Un-SCB,T.reesei increased extracellular protein secretion by 12.8% and filter paper activity(FPA)by 8.4% on Ac-SCB,and decreased extracellular protein secretion by4.7% and FPA by 14.2% on Ak-SCB,which may be related to the 40% protein adsorption ratio of Ak-SCB.2.Effects of different pretreated sugarcane bagasse on transcriptome and protein secretome of T.reeseiThis chapter reveals the gene expression differences in T.reesei cultivated on different pretreated sugarcane bagasse by quantitative transcription analysis,transcriptomic analysis and protein secretory analysis.Quantitative transcriptional analysis of five cellulase genes at three time point showed that the transcriptional regulation trend of T.reesei in different pretreated sugarcane bagasse was different.Transcriptome data analysis showed that when cultivated on Ac-SCB,genes involved in DNA replication,DNA replication initiation,DNA repair and DNA recombination of T.reesei were significantly up-regulated,which may be related to the stress response to DNA damage caused by furfural;oxidoreductase activity and oxygen binding related genes were significantly down regulated,which may be due to that Ac-SCB limited the utilization of oxygen in T.reesei.Ak-SCB induced down-regulation of T.reesei protease synthesis at transcriptional level which led to the reduction of the number of secreted proteases.Secretomic data showed that 111 extracellular proteins,including 36 glycoside hydrolases,5 carbohydrate esterases,5 oxidoreductases and 6 proteases were secreted by T.reesei on Un-SCB;177 extracellular proteins were secreted on Ac-SCB,including 29 glycoside hydrolases,4 carbohydrate esterases,4 oxidoreductases and 5proteases were secreted by T.reesei on Ac-SCB;130 extracellular proteins,including29 glycoside hydrolases,1 carbohydrate esterase,4 oxidoreductases and 2 proteases were secreted by T.reesei on Ak-SCB.A large amount of hemicelluloses and lignins were removed in pretreated lignocelluloses,which caused T.reesei secrete more extracellular proteins,but less proteins related to plant cell wall degradation.3.Study on the underlying mechanism of furfural tolerance in T.reeseiIn order to resolve the inhibitory effect of furfural on microbial growth and fermentation,the fully fertile T.reesei CBS series strain was used as the starting strain,and the 8 m M furfural tolerant mutant CBS3-16-1 was obtained by two rounds of ARTP mutation.To systematically study the adaptive evolution mechanism of furfural tolerance in T.reesei,the sexual reproduction technology of T.reesei was established in this study,and furfural tolerance mutants were sexually backcrossed with wild-type strains to obtain traits segregation.Furfural tolerance trait was separated and the wellseparated progeny strains were sequenced,then the furfural tolerance-related loci GL985073 which contain 172 genes in T.reesei was identified by Bulk Segregation Analysis. |
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