| Mineral resource is non-renewable energy. Nowadays, crazy mining and consumption made the energy crisis problem gradually be taken seriously in the last two hundred years, and pollution of the environment has become more and more serious. Looking for a kind of environment-friendly renewable energy to replace fossil fuels is imminent. Lignocellulose is renewable resource and widespread in nature. The degradation of cellulosic biomass into liquid fuel and high value-added chemicals by using cellulase is one of the most effective way to solve the energy crisis and environmental improvement. The filamentous fungus Penicillium oxalicum can produce a balanced extracellular lignocellulolytic enzyme system, which is very efficient for lignocellulose degradation. But the low level of the expression of cellulase limites the extensive application of cellulase. Therefore, how to improve the cellulase expression abilities for P. oxalicum is the main problem.The genome of P. oxalicum has been recently sequenced, which suggested that there were at least five extracellular and six intracellular putative β-glucosidase genes. These β-glucosidase can differentially produce oligosaccharides and monosaccharides during cellulose degradation process and may play a different role in the cellulase expression regulation network. To characterize their functional importance in cellulase expression, we overexpress these β-glucosidase gene separately, to analysis of their effects on the production of cellulase from P. oxalicum.The expression of β-glucosidase by P. oxalicum wild type strain is relatively low. However, the further over-expression β-glucosidase in P. oxalicum mutants with high cellulase production abilities might not facilitate their cellulase expression. Therefore, we assume to construct a high P-glucosidase yield strain.Because the β-glucosidase Bgl of Aspergillus niger showed high specific activity, we over-expressed the bgll gene in the P. oxalicum and abtained P.oxalicum mutant with β-glucosidase hyper-production.P. oxalicum mutants JU-A10-S and JU-A10-T were abtained by physical and chemical mutagenesis in our laboratory. These two mutant could highly produce cellulase, and the properties of the cellulase produced by which are quite different. Comparative analysis of JU-A10-S and JU-A10-T mutant genome data showed that there are many mutations between the two genome sequences. Therefore, we hypothesized that there might be a direct relationship between these mutation sites and the enzyme production performance of these mutant strains. In order to analyze the important properties of high yield cellulase of JU-A10-S compared with JU-A10-T, we analysis of genomic SNP sites based on these two mutant strains. In P. oxalicum wild-type strain 114-2, the genome specific regions containing these SNP sites were knocked out, separately. The relationship between these SNP sites and cellulase expression regulation were systematically analyzed.1.Homologous over-expression of β-glucosidase in P. oxalicumCellulosic biomass was degraded by cellulolytic enzymes which were mainly consisted of cellobiohydrolase, endogluconase and β-glucosidase. β-glucosidase is an important class of enzymes for cleave β-glucosidic linkages in oligosaccharide. In P. oxalicum genome, eleven putative β-glucosidase encoding genes were predicated and are classified in family 1 and family 3 of glycosyl hydrolase family. Five of which were predicted to be secreted, and the other 6 of which were predicated to be intracellular. To investigate whether the cellulolytic enzyme activities were enhanced by manipulation of p-glucosidase, eleven β-glucosidase encoding gene were constitutively expressed under the control of the strong P. oxalicum PDE02864 promotor whose expression levels were significantly high regardless of whether cellulose or glucose used as carbon source. Nine of 11 β-glucosidase genes were successfully overexpressed in P. oxalicum wild-type strain 114-2, and the resulting transformants exhibited high β-glucosidase activities as compared with their parent strain. Particularly, the CBG4 overexpress strain increased 7.7-fold after transfering to Avicel cellulose for 72 h as compared to the wild-type strain. In contrast, only CBG1and CBG4 overexpression strains showed 32% and 20% increase of filter paper activity （FPA） above the wild-type strain when grown on Avicel cellulose for 96 h, indicating that some of these β-glucosidases were facilitated to enhance the cellulolytic enzyme activities. Taken together, the data suggested an important approach for increasing total cellulase productivity in P. oxalicum.2. Over-expression of Aspergillus niger β-glucosidase gene bgll in P. oxalicum 4-1.Cellulase high-yield strains RE-10, protein secretion and expression intensity of cellulase was greatly increased compared with wild-type strain, but the expression of β-glucosidases is relatively low. Although this problem can be solved by means of the expression of β-glucosidases in the high-yield strains, but strong expression of β-glucosidases activity might lead to decrease of fiber enzyme activity. According to our previous experimental results, addition of β-glucosidases to the fermentation liquid of high-yield strains can significantly improve the conversion efficiency of cellulose. BecauseA. niger β-glucosidase Bglhas the high specific activity we over-expressed the A. niger bgll gene in the P. oxalicum 4-1 under the control of the strong promoter cbhl. The high yield β-glucosidases strain B16 was screened out. The B16 mutant strain β-glucosidases was about 170 IU/ml after shifiting to Vogel’s medium with 2% cellulose for 120 hours.the crude enzyme liquid of β-glucosidases from B16 mutant strain was added （the ratio of FPA:BG is 1:3） oand increase the conversion of cellulose by 50%.3. Analysis of JU-A10-S and JU-A10-T loci of SNP and mining of new regulatory factors of cellulase expressionJU-A10-S and JU-A10-T are cellulase hyper-producers. The genome sequence alignment analysis showed that there were no large fragment DNA differences between JU-A10-S and JU-A10-T. However, the cellulase production in JU-A10-T showed two-fold increase relative to that in JU-A10-S. In order to analyze the important properties of high yield cellulase of JU-A10-S compared with JU-A10-T, the genome specific sequences of these SNP sites were knocked out in the wild strain of P. oxalicum. The SNP locus deletion mutant markedly increased the cellulase expression when knocking out one of the SNP loci on chromosome sixth is located in the A region. However, the β-glucosidases activity was only 20% of the original strain when 1% microcrystalline cellulose as the sole carbon source. The amylase activity of A mutantis almost not detected on cellulose, and was only 50% of the starting strain on starch. The transcription levels of cellulolytic transcription factor CreA and AmyR were significantly decreased, and the XlnR expression level is up-regulated. It is difficult to detect the transcription of amylase encoding gene（15a） and the main extracellular P-glucosidases gene bgll. These results suggest that the A region of the SNP locus may be related to the upstream regulation of these transcription factors. We also found that the A homologous region had repetitive sequences, which showed clustered distribution in each chromosome on P. oxalicum. Therefore, the special distribution of this kind of sequence might be closely related to cellulase expression regulation. |
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