| In nature, a larger amount of plant biomass is formed annually, making it an attractive potential as a renewable source of energy and basic chemicals. With the intensification of fossil fuels shortage and environment pollution, effective use of these renewable resource is an excellent way to reduce consumption of fossil fuels, alleviate the energy crisis, and protect environment. Fungi and bacteria are the main organisms of cellulose degradation and have evolved complex, multi-component enzyme systems, to mediate conversion of cellulose to glucose. Since fungi produce large amount of extracellular cellulases, they have been widely studied and utilized for industrial application. However, low efficiency and high cost of cellulase is the bottleneck in industrial application. Understanding the composition of cellulase system and regulation of cellulase expression are important for improvement of cellulase production and enzymes activity.Current research regarding fungi lignocellulase-degrading enzymes is mainly around Trichoderma and Aspergillus. Many achievements about composition of cellulase system and regulation of cellulase expression have been obtained by analysis of genome, transcriptome and proteome. However, few research regarding the composition of cellulase system and regulation of cellulase expression in other filamentous fungi is available, especially in Penicillium. In the thesis, we analyze the composition of extracellular enzymes system of P. decumbens. We also compare the transcription of lignocellulose-degrading enzyme genes and the extracellular proteome in wild strain and mutant strain grown on different culture conditions. Moreover, we study the function of two genes which may lead to the overproduction of cellulase in mutant strain. These studies would be helpful to deepen our understanding of composition of cellulase system and regulation of cellulase expression in P. decumbens, and helpful to directly renovate the strain at genetic level. The main results of the thesis are as follows: 1. Cloning of cel5A and cel7B encoding endoglucanases from P. decumbens, and characterization of two recombinant endoglucanasesTwo endoglucanase genes{cel7B and cel5A) were cloned from P. decumbens using degenerate PCR and TAIL-PCR. DNA sequencing results showed that cel7B had an open reading frame of 1425 bp without introns and encoded a polypeptide of 474 amino acids. cel5A was composed of 1549 bp containing 3 introns and encoded a polypeptide of 411 amino acids The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of cel7B gene in its chromosomal DNA. The expression level of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that two genes were coordinately expressed and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimal active at 60?and pH 4.0. The recombinant Cel7B showed more than 8-fold,30-fold,5-fold higher enzyme activity towards carboxymethyl cellulose, barely?-glucan and PASC respectively in comparison with that of Cel5A. However, their activities toward pNPC and Avicel were minor difference. Compared to Cel5A, Cel7B is a strict endoglucanase.2. Transcription analysis of lignocellulolytic enzymes of P. decumbens and its mutant strain cultured on different carbon sourcesTranscription levels of six important lignocellulolytic enzymes genes (cel5A, cel6A, cel7A, cel7B, xynl0A, and xynl1A) from both strains were detected on different carbon sources (glucose, sorbose, lactose, cellobiose, cellulose, and cellulose-wheat bran), by means of real-time quantitative polymerase chain reaction. For both strains, the six genes are coordinately regulated at transcriptional level. Glucose and cellobiose repressed whereas cellulose and cellulose-wheat bran induced expression of six genes in both strains. Lactose induced expression of six genes in wild strain but show no such inductive effect in mutant strain, which may be due to the destroy of lactose transport or lactose metabolic pathway. On glucose repression condition, the mutant JU-A10-S appeared obviously derepressed. Expression levels of all genes tested in the mutant strain JU-A10-S were substantially higher than those in wild type strain 114-2 on all carbon sources, which may be partially caused by the mutation of creA gene.3. Analysis of composition of extracellular enzymes system of P. decumbens and comparative analysis of the secretome of wild strain and mutant strain using 2D-DIGEThe protein composition of the extracellular proteome of the wild strain P. decumbens 114-2 and its mutant strain JU-A10-T grown on glucose and cellulose-wheat bran was explored using 2D-DIGE. A total of 38 proteins were identified in the extracellular proteome. The majority of these extracellular proteins are biomass degradation enzymes involved in degradation of cellulose, hemicellulose, protein, starch, pectin and chitin. For wild strain grown on glucose, three basal cellulase were identified in extracellular proteome.Pectase, amylase and protease PepA with high abundance were also detected. For wild strain grown on cellulose-wheat bran, the amount and content of cellulase and hemicellulase were significantly increased, but no pectase were detected, suggesting that expression of pectase and (hemi-)cellulase synthesis are regulated by different system. Compared to wild strain, the mutant strain appeared obviously derepressed on glucose condition. The amount of cellulases and hemicellulases were significantly increased and more types of cellulases and hemicellulases were detected. However, compared to wild strain, expression of amylases was repressed in mutant strain. Compared to wild strain, the mutant strain appeared overproduction on cellulose-wheat bran condition: extracellular protein concentration, cellulase and xylanase activities were significantly increased. The amount of several cellulases and hemicelluases were significantly increased, while others were almost decreased, suggesting that there are some difference in regulation of cellulase and hemicellulase expression. In addition, in the extracellular proteome of mutant stain JU-A10-T, only trace amylase was detected and no protease were detected, suggesting that some genetic variations affecting the related regulatory system.4. Deletion of?-1,2-mannosidase gene and serine protease kinase gene in P. decumbensThe deletion vector of a-1,2-mannosidases gene (mns1) and serine protease kinase gene (pk1) was constructed using fusion PCR and nest PCR, and transformed into P. decumbens. Deletion of mns1 and pk1 had no effect on morphological characteristics, such as colony diameter, colour of spores, volume of spores, and width of mycelia. Compared to the original strain, the extracellular protein concentration and enzymes activities were significantly increased in mnsl deletion stain. MNS1 may be involved in degradation of misfolded protein in fungi.The extracellular protein concentration and enzymes activities were also significantly increased in pkl deletion strain compared to the original strain. The function of PK1 was still unknown, and further studies are needed to confirm the function of PK1 in signal transduction. |
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