The Role Of Carbon Metabolism Repressor (cre) In The Regulation Of Cellulase Produced By Rhizopus Stolonifer

Cellulase, is a complex enzyme, work as biological catalysis when degrade the cellulose and widely found in nature. Generally, strains mainly used for the production of cellulose are filamentous fungi, it produce exoglucanase、 endoglucanse and β-glycosidase. The dual mechanism of three enzymes combination of positive and negative regulator regulate multiple genes transcription and coding. Cellulase gene expression is affected by carbon source, glucose, which inhibits the expression of these genes. CRE protein is a common carbon metabolic inhibitor exist in filamentous fungi. Currently, removing carbon catabolite repression in expression of cellulose and enhancing expression of cellulose genes have become an important trend in research.The laboratory collected and isolated a stain of high yield cellulose named after Rhizopus stolonifer TP-02, the study basis on the original strain. A carbon metabolism repressor gene was extracted from the genomic,an antibiotic resistance gene hygB was inserted into the cre, which was transform into germinal spores by electroporation, finally obtain the mutant △CRE. And Study on characteristics of cellulose enzyme gene transcription regulation. Main research result are follows:(1)According to the published gene sequence, design degenerate primers, take the RhizopusstoloniferTP-02 chromosome as a template to amplification and get length of the sequence was 1284bp, which code 428 amino acids. The genes of Rhizopus stolonifer and Aspergillus has high similarity.(2) Taking pRS303H plasmid as a template, clone an antibiotic resistance gene fragments. According to bioinformatics software, analyzing the integration site of hygB gene and cre gene and cloning downstream of the cre homologous sequences, in which hygromycin work as a selection marker. Construct the fusion gene cre-HygB by overlapping PCR, the full-length of gene was 2310 bp. The fusion gene was transformed into the germinal spores of R.stolonifer TP-02 by electroporation, and then a mutant △CRE was screened by the hygromycin resistance. The screened sporodochium were passaged on the plate containing the hygromycin, and selected the strain that passageed over 10 generations.(3)The chromosomal DNA was extracted from transformants, Verify the screening of strains with hygromycin resistance by the primers of resistance gene.Using CRE homology region primers of the upstream and downstream to amplify the full length, the results showed that target band is single,and the size is uniform.PCR validation results show, △CRE strain was successfully constructed. To further verify the copy number of △CRE strains exogenous fragment in the chromosome when the homologous recombination was occurred. Extracting the total RNA from this 5 strains’ transformants, synthesizing the cDNA single-strand by reverse transcription kit, and using the RT-PCR, taking the SYBR as fluorescent dye, to analysis. The results showed that there was a strain which was not existed the copy number of original strain CRE gene, the homologous recombination is successful, and obtained the △CRE strains.(4)In order to analyze the function of cre gene in Rhizopus stolonifer, we compared the original TP-02 strain and the mutant △CRE strain by fermentation experiments. Selected different concentrations of carbon source for fermentation, under 2% concentration of glucose, filter paper activity of △CRE strain increase by 33% compared with Rhizopus stolonifer strain; under 3% concentration of glucose, filter paper activity of mutant strain increase by 21.8%.With the increasing of the sugar concentration, this phenomenon of the peak enzyme activity appeared hysteresis was indicated that weak the CRE gene could significantly reduced the inhibitory effect of CRE on the expression of cellulase. RT-qPCR was utilized to analyze the transcription features of cellulases secreted by the mutant. It was found that the transcription of eg, bg, cbhl and cbh2 are improved than the wild type, the values are 48.75%,26%, 5.6% and 38.6%, respectively. Those three mainly types enzymes’ synthetic metabolism were closely related to carbohydrates in the process of Rhizopus stolonifer producted cellulose by using glucose. The cellulase related genes expression was up-regulated by the CRE gene knockout, and it had the regulatory specificity. Different sugar concentrations would affect the production of key enzymes in the culture process.The disruption of the carbon metabolism repressor CRE could lift its inhibition effect for cellulose transcriptional regulation, weak the carbon metabolism repression effect of cellulase production process, and make the cellulase gene express efficiently.It improved the yield of cellulase, and provided a powerful guarantee for the industrial production.