| Plant cell walls are mainly composed of cellulose, hemicellulose and lignin and other substances. In addition to cellulose, xylan, a principal component of plant hemicellulose, is the second most abundant polysaccharide in nature. It is also the main renewable natural resources. With the increase of population and resource consumption, the exploitation and utilization of renewable resources had become academic issues of common concern.The filamentous ascomycete Aspergillus niger is a well-known over-producer of enzymes, in particular plant cell-wall hydrolyzing enzymes, such as cellulases and xylanases. The full-genome sequencing of A. niger had been completed. Hemicellulase and other hydrolases to degrade complex polysaccharides produced by the strain had been widely used in food, textile and pulp and paper industry and other industries. A. niger has a considerable strong xylanase activity. Filamentous fungal xylanase gene has been extensively cloned and expressed in several host systems. Studies on xylanase have been refined to the molecular level, and some research on xylanase gene regulation had been performed. However, the study mainly focuses on a single gene or several genes. Comprehensive and systematic research on xylanase regulation at the genomic and proteomic level is not very detailed, therefore it can not reflect the general view of the xylan metabolism in filamentous fungi and only a few researches on the molecular mechanism of xylanase induction had been carried out. Consequently, research on hemicellulose catabolism of filamentous fungi at the genomic level is very necessary in fundamental research and practical application. The function clarification of some novel genes will contribute to understand xylanase induction mechanism and regulation of metabolic networks or important signal transduction mechanism in A. niger. In this paper, suppression subtractive hybridization (SSH) protocol was used to study the differential gene expression under xylan induction and non-induction with A. niger as materials.Aspergillus niger and Trichoderma reesei have been extensively used as model organisms for diverse transformation and expression systems, only a few research on Penicillium chrysogenum was performed. The detailed research mainly focused on the regulation ofβ-lactam antibiotics. The filamentous fungus P. chrysogenum is well known by its ability to synthesizeβ-lactam antibiotics like benzylpenicillin and isopenicillin N as well as other secondary metabolites, and it could secrete various kinds of protease, xylanase and glucose oxidase. The strain has outstanding ability to synthesize and secrete different proteins. The content of xylanase was 40% when the strain was cultured in the medium containing of 1% xylan, indicating that the promoter of xylanase gene from P. chysogenum belonged to strong promoter and it could secrete large amounts of protein into the fermentation broth. Therefore, P. chysogenum is an excellent eukaryotic gene expression system and a potent cell factory for white biotechnology. In addition, cellulase activity of the strain is very low. These features are beneficial to the expression of alkaline xylanase gene to improve the quality of pulp and bio-bleaching. In this paper, P. chrysogenum as a host system is used to high-efficiently express the xylanase gene from alkalophilic Bacillus.The main research works in this thesis are as follows:1,Mutagenesis and optimization for the production of xylanase in A. nigerIn order to screening the mutants with higher xylanase activities, the mutation breeding of the strain NA1003 was carried out by ultraviolet and diethyl sulfate (DES)mutagenesis. A mutant strain whose xylanase activity is 6235U/ml was selected and numbered as An-19. The colonial morphology was observed by scanning electron microscope. The result showed that the conidia of the mutant strain are brown and the conidia volume and hyphal diameter become larger compared with original strain. The mutant strain grows fast and can cover the plate in 3 days. The xylanase genes from the original and mutant strain were cloned and sequenced. The sequencing results has showed that the identity of the gene is 100%, indicating that the mutation does not change the primary structure of xylanase gene. The genetic stability of mutant stain An-19 was performed and the results showed that the strain was a stable strain to produce a large number xylanase. The culture conditions of mutant strain were optimized by response surface analysis using Center Composite Design (CCD) and Design-Expert 7.0 software. The culture medium were optimized as follows:wheat bran 20 g/L, NH4NO3 10 g/L and CaCO3 20 g/L. The xylanase activity was up to 8516U/mL under the optimum conditions.2,Cloning and sequence analysis of differentially expressed genes from A. niger induced by xylanTo identify xylan-related genes in A. niger, suppression subtractive hybridization (SSH) was performed to generate a subtracted cDNA library between induced (Tester) and non-induced A. niger (Driver) using the PCR-SelectTM cDNA Subtraction Kit according to the manufacturer's protocol. PCR analysis showed that the length of inserted fragments in white bacteria clones were in the range of 250-1 000 bp. The quality of SSH library was identified to be favorable by reverse dot blotting.119 positive clones with different inserts was randomly selected and sequenced. The sequencing results were analyzed by BlastX software, indicating that the different expressional cDNA fragments are mostly relate to the xylanase gene. A series of ESTs of 41 kinds of proteins including sugar transporter, xylan hydrolymes, transcriptional factor and other functional proteins as well as unknown proteins, were isolated from the subtractive libarary.7 cDNAs encoding 4 kinds of sugar transporter including xylobiose transporter, xylose transport, arabinose transport and hexose transporter accounted for 5% of the total sequences.22 cDNAs encoding 12 kinds of xylanases including endo-xylanase A and B,β-xylosidase, arabinosidase,α-galactosidase,β-glucosidase, Xylulokinase, as well as xylitol dehydrogenase, aldose epimerase, endoglucanase C, a-glucuronidase, and ferulic acid esterase accounted for 18.4% of the total sequences. The full lengths of specific genes were cloned by searching the genomic data of A. niger. The sequence analysis of full-length genes showed that the promoter regions of enzyme-coding genes except arabinosidase bandβ-glucosidase had a specific sequence of 5'-GGCTAA, which could be specificially binded by XlnR and consequently enhance the expression of xylanase system. In addtion, XlnR not only could be used as the transricptional factor of xylanase, but also involved in the transcripton regulation of part of celluase. Six specific genes were analyzed by RT-PCR and the results revealed that six genes was expressed under the induction of xlan. RT-PCR analysis further confirm that the qulity of constructed SSH libarary was good. The construction of SSH library and cloning of differential genes have established a solid foundation for cloning xylanase-relevant genes and further studying xylanase induction mechanism from filamentous fungi.3,Purification of extracellular protease from P. chrysogenum and construction of protease-deficient strainAn extracellular alkaline serine protease from P. chrysogenum FS010 has been purified. The purification procedure involved:ammonium sulfate precipitation, DEAE ion-exchange chromatography and sephadex G-100 gel chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of about 43,000Da. The protease has a maximum activity at pH 9.0 and 35℃. PMSF and DFP are its specific inhibitors, indicating the enzyme blongs to serine protease.A TBLASTN search using PEPA from A. niger and P.janthinellum, as query identified a single orthologous gene in the genome sequence of P. chrysogenum. The gene (gene id:8313187, protein id:XP002567415.1) was named pepA. The promoter and terminator of pepA from P. chrysogenum were also cloned. The promoter region of pep A was 1099bp, and the terminator region of pep A was 563bp. To knock-out the pep A from P. chrysogenum, a deletion vector pApepA was constructed in which the complete coding region of pep A was replaced by a phleomycin resistance expression cassette. The deletion vector pApepA was amplified by PCR and transformed into the P. chrysogenum strain FS010. Three transformants were screened and shown to be deleted in the pepA gene by the PCR amplification and Southern blotting. Protease activities of supernatant from three transformants were measured and the results showed that the protease activtity of transformantΔPEP14 was the lowest, which was 80.9% lower than of control strain and the other two transformants were 61.2%和40.2% lower than of control strain, respectively. The measurement of protease activity showed that the absence of aspartic proteinase of P. chrysogenum could decrease the major extracellular protease activity of fermentation broth, but the content of total protein in fermentation broth remained stable. The genetic stability of transformantΔPEP14 was studied and the result showed that transformantΔPEP14 was a genetic stable strain with low protease activity.4,Secretion expression of an alkaline xylanase gene in protease-deficient Penicillium chrysogenumAccording to the codon usage bias in P. chrysogenum, Codon-Adaptation tool was performed to adapt the alkaline xylanase gene xyl from alkalophilic Bacillus. The adapted xyl1 sequence of CAI value was 0.99 and GC content was 60.94%, which was 61.2% higher than that of starting sequence. Blast analysis showed that the sequence similarity of the adapted xyl1 with the original xyl was only 74%.The plasmid pUC19 was used as the carbon backbone of the expreesion vector. The strong xylanase promoter (Pxyn) and terminator sequences (Txyn) from P. chrysogenum were used to express bacterial alkaline xylanase gene, resulting the expression vector pxyl-1. The expression vector pxyl-1 was amplified and transformed into the P. chrysogenum strainΔPEP14. Four transformants were screened and confirmed by PCR amplification and Southern blotting. Southern blot and RT-PCR analysis further confirmed that four transformants contained the alkaline xylanase gene xyl1 and the gene could be specifically transcribed in transformants.The enzymatic properties of recombinant xylanase produced by P. chiysogenum transformants XY35 were preliminary studied. The results confirmed that recombinant xylanase could be secreted into the culture. SDS-PAGE of the extracellular proteins indicated a molecular weight of about 41,000Da. The xylanase activity of control strain was 67.4 U/mg, whereas enzyme activity of transformant XY35 was 572.4U/mg. The content of total protein of fermented broth is 3.45mg/mL. Gel scanning analysis showed that the extracellular xylanase accounted for 15.9% of total protein.The properties of crude enzyme from Transformant XY35 and original strain were preliminary studied and the results showed that the optimal temperature and pH for recombinant xylanase was 40℃and 8.0, respectively. The optimal temperature and pH of acidic xylanase from original strainΔPEP 14 was 40℃and 6.0, respectively. Comparison of characteristics of xylanase from transformant XY35 and original strain suggested that the alkaline xylanase gene had been successfully expressed. Recombinant alkaline xylanase showed nearly 80% of its maximal activity in the pH range 3.0-9.5, indicating that the recombinant xylanase could be used for pulp pre-bleaching. The recombinant enzyme lost most of the activity when the temperature exceeds 50℃, and retaining only 40% of the maximum activity at 55℃, indicating that thermal stability of the xylanase should be further improved. The research of gene expression and regulation in P. chrysogenum could provide the necessary theoretical basis for the industrial breeding of Penicillium. Alkaline xylanase has potential applications in environmental protection, bio-pulping process and other biotechnology.
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