Recumbency Penicillium Cellulase Genes

As the fossil fuels are being consumed more and more,the traditional energy, which is based on coal and petroleum,is at the danger of being used up.In order to ensure the energy supply and the economic development,every country of the world begins to look for a new substitutable energy.Cellulose is the most ample renewable energy,taking up 40 percent of all the biomass on the earth.Thus,using the cellulose sufficiently will be an excellent way to relieve the energy crisis,which is vital for the sustainable development of human society.Most of cellulose in the nature is degradated by microorganisms,of which the filamentous fungus plays an important role.Researchers are interested in cellulases secreted from filamentous fungi by the reason that the filamentous fungi secrete the cellulases out of cell and the cellulases can be harvested easily.It is essential to industrialization.The wild strain directly screened from the nature generally secretes a little quantity of cellulases that is far from practical industrial production.As a result, the wild strain must be artificially reconstructed in order to obtain the improved strain. Mutagenesis and appropriate screening methods are good strategies to hit the target and nowadays many improved mutants play a vital role in the industrialization of celllulases. Our laboratory screened a derepression mutant Penicillium decumbens JU-A10 which can secrete five or six times FPA activities compared with the wild strain 114-2 in the same cultural condition.Now,the JU-A10 has been applied to industry to produce a large quantity of cellulase.Up to now,researchers can not elucidate clearly the regulatory mechanisms involved in cellulase secretion and the mutagenesis mechanisms of the improved strain, which impedes further artificial reconstruction of the wild strain.This work compared the wild strain with the mutant,mainly focusing on three fields:1 comparing the extracelhlar cellulase compenents of the wild strain 114-2 and mutant JU-A10 through SDS-PAGE,PAGE,cellulases activity staining and specific celluases activity.In the same cultural conditon,the mutant JU-A10 can secret more quantity of extracellular protein than the wild strain 114-2.When the glucose is the sole carbon source in the culture,the average of extracellular protein concentration of the 114-2 is 0.11 mg/ml and the JU-A10 is 0.18 mg/ml;When the microcrystalline cellulose is the sole carbon source,the average of extracellular protein concentration of the 114-2 is 0.24 mg/ml and the JU-A10 is 1.10 mg/ml.When the glucose is the sole carbon source in the culture,from the SDS-PAGE analysis of extracellular protein,we can see JU-A10 secrets more kinds of proteins most of which are cellulases through activity staining. When the microcrystalline cellulose is the sole carbon source,the SDS-PAGE spectrums of extracellular protein of the two strains don't differ clearly.From the activity staining analysis of cellulase components,we can see the endocellulases andβ-glucosidase of JU-A10 are identical to those of 114-2,while the cellobiohydrolases are different in the two strains.From the specific activity analysis of cellulase components,we can see the specific endocellulases,FPA and cellobiohydrolases of two strain don't change clearly,while the specificβ-glucosidase of 114-2 is much higher than that of JU-A10.As a result,from the experiment data we can summarize two conclusions:(1)The mutation effects on the transcription regulation of JU-A10 and the JU-A10 secrets more cellulases than those of wild strain 114-2.(2)The quantity ofβ-glucosidase secreted by JU-A10 is the same as the wild strain 114-2 which indicates the nonidentity of the trgulation mechanism betweenβ-glucosidase and other cellulase components.2 Cloning the cbh1 encoding exoglueanaseⅠfrom wild strain 114-2 and JU-A10 and comparing the 2 kb upstream sequence of cbh1 of the two strains.ExoglucanaseⅠis the most important cellulases component.The exoglucanaseⅠgene(cbh1)was first cloned from Penicillium decumbens by a modified thermal interlaced-PCR(TAIL-PCR)and RT-PCR.The total length of cbh1 was 1500 bp (GenBank Accession No.EF397602).It contained 2 introns and encoded 453 amino acids.The deduced amina acid sequence revealed that CBHⅠhas a modular structrue with a predicted molecular mass of 46-kDa and consisting of a fungal type carbohydrate binding module aeparated from a catalytic domain by a threonine rich linker region.the putative gene product is homologous to fungal cellobiohydrolases in Familiy 7 of the glycosyl hydrolases.The upstream sequence(2 kb)of cbh1 gene was also cloned and sequenced.It contained two putative binding sites for the carbon catabolite repressor CREⅠand two putative binding sites for cellulases transcriptional regulator ACEⅠ. Compared the upstream sequence of the wild strain 114-2 with that of mutant JU-A10, four single base mutations were detected on the upstream sequences(1.9 kb)of cbh1 gene.The result suggests that the evidently enhanced cellobiohydrolase activity of the mutant is not due to cbh1 protein-coded sequence.The true reason maybe refer to single base mutations of the upstream sequence that effect the transcription regulation of mutant JU-A10.As a result,the secretion of CBHⅠinhanced.3 The cbh1 gene of Penicillium decumbens expression in Pichia pastorisThis work constrcts a recombinatant plasmid pPIC9K-cbh1 and transform it into methylotrophic yeast(Pichia pastoris)for expressing CBHⅠprotein.We obtained the postive strains on MD plates without histidine.The expressed protein has been evaluated by SDS-PAGE and the molecular mass was 66 kDa.The expressed protein can breake down the substance pNPC,which seggests that the cbh1 gene we cloned is fight.4.Cloning the bgl1 encodingβ-Glueosidase from wild strain 114-2Theβ-Glucosidase gene(bgl1)was first cloned from Penicillium decumbens by a modified thermal interlaced-PCR(TAIL-PCR)and self-formed adaptor-PCR (SEFA-PCR).The total length of cbh1 was 2963 bp.It contained 5 introns and encoded 861 amino acids.The putative gene product is homologous to fungal cellobiohydrolases in Familiy 3 of the glycosyl hydrolases(GenBank Accession No.EU700488).