Genetic Modification Of Trichoderma Reesei Hyper-cellulolytic Mutant And Mining For New Glycoside Hydrolases

Trichoderma reesei is one of the important producers of biomass-degrading enzymes widely used in industrial production.Some high-yielding T.reesei mutants have been generated using classical mutagenesis.However,the high cost of manufacturing biomass-degrading enzymes is still a huge challenge for its commercial application.In addition,the genetic basis underlying their respective phenotypes is only partially understood.A comprehensive understanding of the effect of genetic alterations on cellulase production is beneficial to the development of more efficient cellulase producing strains.In T.reesei,pH is also an important factor affecting the production of cellulase.Plasma membrane H~+-ATPase plays a key role in regulating physiological processes such as intracellular pH homeostasis and nutrient uptake.However,the function of plasma membrane H~+-ATPase in T.reesei has not been studied so far.The combination of various glycoside hydrolases has been widely used in industrial production.To meet the emerging demands of the glycoside hydrolases in industrial processes,glycoside hydrolases with higher performance are immediately required for reducing the cost of industrial production.Metagenomics has become a powerful approach to directly study species diversity within microbial communities and prospect novel biocatalysts.To develop more efficient enzyme producing strains and prospect new glycoside hydrolases,the following three aspects were studied in this paper.1)A T.reesei mutant,SS-?,was isolated by multiple NTG mutagenesis on the basis of strain NG14.SS-? harboring intact Crel protein exhibited faster growth and an approximately 1.5-fold increase in CMCase activity compared to RUT-C30 in the presence of Avicel or lactose.Analysis of SS-? and RUT-C30 transcriptomes identified 1764 genes that were differentially expressed in SS-?,compared to RUT-C30 grown in Avicel,glucose,lactose,and wheat straw.On this basis,the transcriptional data of 65 cellulose-degrading enzymes,41 transcription factors and 152 transporters were further analyzed.To identify any genetic changes that had occurred in SS-?,we sequenced its genome.In total,184 SNPs and 40 indels were identified.Additionally,157 genes affected by mutations were identified.Among these genes,the majority were involved in transport,secretion,protein metabolism,and transcription.We further analyzed nine candidate genes with mutations specific to SS-II.Deletion of three of these genes significantly affected cellulase production in RUT-C30 when grown with AviceL Analysis of SS-? and RUT-C30 transcriptomes contributes to elucidating the role metabolic alterations play in cellulase formation.Genome re-sequencing revealed novel mutations that may affect cellulase production and other neglected areas.Our study provides a resource for identifying more genes involved in the production of cellulase and a solid theoretical basis for the construction of more efficient cellulase producing strains.In addition,we also constructed light-inducible system to express heterologous proteins in T.reesei.2)Two plasma membrane H~+-A-TPases of T.reesei were identified and functionally characterized using a target gene deletion strategy for the first time.We found that the gene tre76238 as plasma membrane H~+-ATPase plays a major role in T.reesei,while the gene tre78757 plays a minor role.Deletion of the gene tre78757 did not affect the phenotype of the strain,whereas deletion of the gene tre76238 impaired the ability of the strain to extrude protons from the cell.The disorder of pH homeostasis led to a continuous accumulation of cellulase in the strain when grown with glucose.Data analysis showed that transcriptional levels of the genes related to cellulase synthesis significantly increased in deletion strain Del238.Although the transcriptional level of xyrl did not increase,the EMSA analysis showed that there were other proteins that were associated with the cbhl promoter in the Del238.Three zinc finger proteins that may be involved in cellulase regulation were identified using pull-down technique and mass spectrometry.These findings provide a new insight into the regulation of cellulase production in T.reesei and a new strategy for improving the production of cellulase by regulating intracellular pH homeostasis.3)To prospect new glycoside hydrolases,soil microorganisms were enriched using cotton biomass as carbon source.To understand the decay process of cotton biomass,we characterized the glycoside hydrolases secreted by the microbial community.The results showed that physical contact between cells and substrates was necessary for efficient hydrolysis of the biomass.A representative microbial community structure was identified by 16S rRNA analysis,Cytophaga is likely to play a major role in the cotton biomass decomposition in this community.Based on the analysis of metagenomic sequences,32 dominant glycoside hydrolase families were identified,containing 2058 candidate genes,of which sixteen were successfully expressed in E.coli.These proteins showed activities on p-nitrophenyl-N-acetyl-P-D-glucosaminide,p-nitrophenyl-?-D-xylopyra noside,laminarin,p-nitrophenyl-?-D-glucopyranoside,p-nitrophenyl-?-D-glucuronide,carboxymethyl cellulose,or p-nitrophenyl-?-D-mannopyranoside,respectively.In addition,three proteins have low similarities less than 60%compared with the closest homologs.Soil microbial genome analysis provides a good strategy for mining novel biomass-degrading enzymes.These cloned glycoside hydrolases also have potential applications in biomass conversion and bioproduct production.Our research provides insight into the composition and interaction of enzymes and pathways of plant biomass degradation.