Studies Of The Expression Of Enzyme Gene Related To Glucose Metabolism In Thilavia Terrestris And The Construction Of Constitutive Expression System

Thilavia terrestris is a thermophilic filamentous fungus. It has the ability to produce series of enzymes for hydrolyzing lignocellulose. In addition, and it’s enzymes are well tolerated to high temperature, which show broad application prospects. Compared with the important cellulase producing strain Trichoderma reesei, it contain more abundant species of glycoside hydrolase. Currently, mesophilic filamentous fungi are usually used to express proteins, such as Trichoderma, Aspergillus species etc.. However, thermophilic fungi can produce enzymes very fast, and decompose biomass in high efficiency as well. They also produces cellulase and hemicellulose of high thermal stability, with advantages that mesophilic filamentous fungi can not match. The main purpose of this work is to optimize the cellulase production conditions of Thilavia terrestris in order to improve it’s ability to produce cellulase, then compare the relative expression levels of the genes which related with glucose metabolism in Thilavia terrestris, and screen strong constitutive promoter, and use a highly active promoter to construct Thielavia terrestris constitutive expression system, and finally express some valuable proteins, such as xylanase and GH61 family glycosidase.First we optimize the fermentation conditions of Thielavia terrestris for cellulase production. CMC-Na fermentation medium was used before optimization. By orthogonal experiment, 2.5% of wheat bran, 2.0% of soybean meal, initial p H of 4.0 is verified to be the best fermentation conditions of Thielavia terrestris after optimization. In this condition the filter paper activity was 1.39 U?m L-1, 44.8% higher than before. The results of variance analysis of orthogonal experiment showed that nitrogen source significantly affect the enzyme productivity of Thielavia terrestris. The enzymes produced by this strain was stable at 60℃~ 80℃.Then strong promoters related with gluclose metabolism was sreened. Up to 131 genes related with glucose metabolism in Thielavia terrestris was screened by RT-q PCR, and 18 genes with high transcription level were obtained. The quantitative PCR analysis showed that the transcription levels of these genes reached the peak when the glucose concentration in the medium was 10g/L. These genes included the genes of glyceraldehyde 3- phosphate dehydrogenase(gpd), pyruvate decarboxylase(pdc), aconitase(Acn A), isocitrate dehydrogenase(idh-1, idh-2), pyruvate dehydrogenase(pdh-1, pdh-2, pdh-3), pyruvate kinase(pyk), succinate dehydrogenase(sdh B, sdh C), succinyl coenzyme A synthetase(succinyl Co A synthetase), aldolase(fba-1, fba-1), glucose phosphate mutase(PGM), glycogen binding domain(gbm). Under different concentrations of glucose, the relative expression level of the gpd gene was much higher than other genes. The results also illustrated that the expression levels of glucose metabolism-related genes was affected by glucose in Thielavia terrestris. The promoter sequence of gpd(Pgpd), terminator sequence(Tgpd) of the gpd and a xylanase gene coding sequence(xyn) sequence in Thielavia terrestris were PCR amplified, and were ligated into p UC19 sequentially, resulting in the recombinant xylanase expression cassette p UC19-Pgpd-xyn-Tgpd. The expression cassette and plasmid p AN7-1, which carrying the selectable marker gene, were co-transformed into T. terrestris, and the transformants were selected on PDA agar plate containing 160μg/m L concentration of hygromycin B. The transformants were validated by PCR analysis one transformant was verified to harbor the expression cassette. The xylanase productivity of this recombinant was analyzed. However, there was no difference between the original strain and the transformant, indicating that the transformed strain could not over-express the target protein.The work have optimized the fermentation medium of enzyme production of T. terrestris, a thermophilic ascus fungi, and improved its’ cellulase productivity; analyzed the relative expression level of eighteen genes related with glucose metabolism and obtain a potentially strong promoter Pgpd, tried to constructed Thielavia terrestris constitutive expression system with the promoter. Although we have not obtained over-expression of the targate protein, but the result provides reference to the following study.