β-葡萄糖苷酶基因过量表达提高Trichoderma reesei纤维素降解活性
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摘要
丝状真菌作为低等真核生物具有结构简单、生长迅速、操作简便的优点，同时，又具有真核基因表达和真核蛋白质翻译后修饰加工装置，因此近年来丝状真菌分子生物学逐渐成为现代分子生物学研究的一个热点。瑞氏木霉(Trichoderma reesei)是自然界中广泛分布的腐生性丝状真菌，具有完整的纤维素酶系和半纤维素酶系，用T.reesei作为工业菌株生产分解不同植物材料的酶类，已有多年历史。
T.reesei纤维素酶系包括3种酶，它们协同作用将纤维素降解成寡糖和葡萄糖。其中纤维二糖水解酶和内切葡聚糖酶协同作用降解纤维素生成寡糖，主要是纤维二糖，而β-葡萄糖苷酶继续将纤维二糖降解成葡萄糖。过量纤维二糖的存在对纤维二糖水解酶和内切葡聚糖酶会产生反馈抑制作用，从而降低整个纤维素酶系降解纤维素底物的效率。β-葡萄糖苷酶能够降解纤维二糖，从而可以解除这种反馈抑制。T.reesei中β-葡萄糖苷酶的分泌量很低，因而成为整个纤维素酶系有效降解纤维素底物生成葡萄糖的瓶颈。因此，提高β-葡萄糖苷酶的表达是增强T.reesei降解天然纤维素底物能力的一条重要途经。
本文从T.reesei中克隆得到编码胞外主要β-葡萄糖苷酶的基因bgll，将它克隆到本实验室构建的表达载体pTHP中。pTHP是在cbh1启动子的基础上缺失了包含3个潜在的葡萄糖阻遏位点的-677～-724区域，并将包含CCAAT盒和转录激活因子ACEⅡ结合位点的-620～-820片段以4拷贝的形式插入改造得到。将构建得到的重组质粒pTHB和含有pyrG基因的质粒pAB4-1共转化乳清酸核苷-5-磷酸脱羧酶(pyrG)基因缺陷的T.reesei菌株M23，从无尿嘧啶的平板上挑到96个转化子，通过PCR初步验证得到6株成功转入bgll基因的转化子。对这6株转化子摇瓶发酵并进行酶活测定，筛选得到一株β-葡萄糖苷酶酶活和滤纸酶活都有较大提高的转化子2-6，其β-葡萄糖苷酶酶活和滤纸酶活分别为出发菌株M23的2.8倍和1.48倍。半定量PCR分析结果显示转化子2-6染色体上bgll基因拷贝数增加，证明转入的bgll基因整合到T.reesei染色体上。结果表明由于转入的bgll基因整合到T.reesei染色体上提高了BGLI的表达量，从而提高了T.reesei整个纤维素酶系降解天然纤维素底物的能力。本研究进一步证实对丝状真菌工业生产菌株进行遗传操作是菌株改造的一条行之有效的途径。
为了解BGLI在T.reesei细胞内的分泌机制，本文将绿色荧光蛋白gfp基因作为报告基因与bgll基因融合，把得到的融合片段插入载体pTHP，再将重组质粒pTHR1和pTHR2转入pyrG基因缺陷的T.reesei菌株M23中，从无尿嘧啶的平板上得到约110株转化子，通过PCR初步验证得到7株gfp基因稳定遗传的转化子。将这些转化子接种到以乳糖为碳源的诱导培养基中诱导bgll／gfp融合基因的表达。经过酶活测定筛选得到一株转化子1-17，其β-葡萄糖苷酶酶活和滤纸酶活分别是出发菌株M23的3.3倍和1.28倍。半定量PCR分析结果表明转化子1-17染色体上的bgll基因拷贝数增加，证明转入的bgll基因整合到T.reesei染色体上。对其胞外蛋白进行SDS-PAGE检测，发现一条分子量约为100kD的蛋白条带，推测为BGLI／GFP融合蛋白，初步估算其产量为77.7μg／ml。对转化子进行荧光观测未检测到荧光，分析可能是因为以与BGLI融合蛋白形式存在的GFP不能形成正确的结构，因此不能发出荧光，或者由于所发出的荧光较弱而被菌体自发荧光掩盖，因此难以检测。
将bgll基因3′端加上编码六个组氨酸标签的序列并克隆到载体pTHP中，再将得到的重组质粒pTHB2分别与含有hph基因的质粒pAN7-1和含有amdS基因的质粒p3SR2共转化斜卧青霉(Penicillium decumbens)JU-A10。分别通过潮霉素抗性和乙酰胺为唯一碳源筛选转化子，共得到约40株转化子，通过PCR初步验证得到3株bgll基因稳定遗传的转化子。测定转化子的β-葡萄糖苷酶酶活和滤纸酶活，初步得到1株β-葡萄糖苷酶和滤纸酶活均有提高的转化子，为进一步的研究奠定了基础。
Filamentous fungi have the advantages of simple structure and fast growth, so it is easy to be manipulated and cultured. Being a kind of lower eukaryotes, it also has eukaryotic protein modification apparatus and the post translation modification mechanism. So molecular biological research on filamentous fungus has been one of the hotspots. Saprophytic fungus Trichoderma reesei has a long history of industrial production of different plant material hydrolyzing enzymes due to its extraordinary ability to secrete cellulases and hemicellulases.
The cellulase complex isolated from Trichoderma reseei comprises at least three different enzymes that together hydrolyze cellulose to oligosaccharides and glucose. Of these, the endoglucanases and cellobiohydrolases synergistically hydrolyze cellulose into small cellooligosaccharides, mainly cellobiose. Subsequently, cellobiose is hydrolyzed to glucose byβ-glucosidase. The accumulation of cellobiose could slow down the hydrolysis process significantly by inhibiting both endoglucanase and cellobiohydrolase activities.β-Glucosidases can break down cellobiose into glucose and then relieve the inhibition of endoglucanase and cellobiohydrolase by it. The amount ofβ-glucosidase secreted by T. reesei is insufficient for effective cellulose degradation. Therefore, construction of BGLI-overproducing strain is a valuable pathway to improve the efficiency of cellulose hydrolysis.
The bgl1 gene encoding the most important extracellularβ-glucosidase from T, reesei was inserted into a high-expression vector pTHP, which was constructed in our laboratory on the basis of cbh1 promoter by deletion of -677～-724 region containing three potential glucose repressor binding sites and insertion of 4 copies of -620～-820 region including the CCAAT box and ACEII binding sites. Then the recombinant plasmid pTHB was co-transformed with plasmid pAB4-l, which contains the orotidine-5'-phosphate decarboxylase (pyrG) gene from Aspergillus niger, into the pyrG-deficient strain T. reesei M23. 96 transforrnants were selected from the minimal media plate without uridine. Meanwhile, PCR amplification was carried out with the special primers to identify the transformants and the result showed that additional bgl1 copies had integrated into chromosome DNA of 6 transformants. Theβ-glucosidase and filter paper activities of the 6 transformants were examined and the transformant 2-6 showed the strongest increases in the rate of production of glucose from cellobiose (2.8-fold), and filter paper (1.48-fold). The analyzing of bgl1 gene copies on chromosome in transformant 2-6 by semiquantitative PCR indicated that the copies of bgl1 gene in transformant 2-6 were increased comparing to M23. Over-expression of BGLI by increasing the copies of bgl1 gene can enhance the ability of T. reesei to hydrolyze cellulose. The result indicated that the gene engineering method was a valuable pathway to reconstruct the strain for breeding.
To observe the secretory mechanism of BGLI, gfp gene was fused to bgl1 gene as a reporter. The fusion fragment was cloned into the vector pTHP. The recombinant plasmid pTHR1 and pTHR2 was co-transformed with plasmid pAB4-1 into T. reesei M23. 110 transformants were selected from the minimal media plate without uridine and 7 positive transformants were isolated by PCR amplification. Lactose was used as carbon source to induce the expression of the fusion protein. Theβ-glucosidase and filter paper activities of transformant 1-17 showed 3.3-fold and 1.28-fold increase comparing to the strain M23, respectively. According to semiquantitative PCR analysis, the copy number of bgl1 gene in transformant 1-17 was increased comparing to M23. It demonstrated that the bgl1 gene transformed into the T. reesei had integrated into its chromosome successfully. A band about 100kD was detected by SDS-PAGE analysis of protein from 1-17 and it was estimated to be BGLI/GFP fusion protein. The fluorescence of transformants was detected but nothing was found. The possible reason was that GFP couldn't fold to the correct structure in fusion form with BGLI or its fluorescence was so weak that it couldn't be observed under the background fluorescence of T. reesei.
The bgl1 gene containing a fragment coding 6×His tag at 3' end was cloned into the vector pTHP. Then the recombinant plasmid pTHB2 was co-transformed with the plasmid pAN7-1 containing hph gene or with p3SR2 containing amdS gene into Penicillium decumbens JU-A10. 40 transformants were selected by hygromycin B or acetamide. PCR amplification was carried out and the result showed that additional bgl1 copies had integrated into chromosome DNA of 3 transformants. Then the transformant, which showed enhanced β-glucosidase and filter paper enzyme activities, was selected for further research.
T.reesei纤维素酶系包括3种酶，它们协同作用将纤维素降解成寡糖和葡萄糖。其中纤维二糖水解酶和内切葡聚糖酶协同作用降解纤维素生成寡糖，主要是纤维二糖，而β-葡萄糖苷酶继续将纤维二糖降解成葡萄糖。过量纤维二糖的存在对纤维二糖水解酶和内切葡聚糖酶会产生反馈抑制作用，从而降低整个纤维素酶系降解纤维素底物的效率。β-葡萄糖苷酶能够降解纤维二糖，从而可以解除这种反馈抑制。T.reesei中β-葡萄糖苷酶的分泌量很低，因而成为整个纤维素酶系有效降解纤维素底物生成葡萄糖的瓶颈。因此，提高β-葡萄糖苷酶的表达是增强T.reesei降解天然纤维素底物能力的一条重要途经。
本文从T.reesei中克隆得到编码胞外主要β-葡萄糖苷酶的基因bgll，将它克隆到本实验室构建的表达载体pTHP中。pTHP是在cbh1启动子的基础上缺失了包含3个潜在的葡萄糖阻遏位点的-677～-724区域，并将包含CCAAT盒和转录激活因子ACEⅡ结合位点的-620～-820片段以4拷贝的形式插入改造得到。将构建得到的重组质粒pTHB和含有pyrG基因的质粒pAB4-1共转化乳清酸核苷-5-磷酸脱羧酶(pyrG)基因缺陷的T.reesei菌株M23，从无尿嘧啶的平板上挑到96个转化子，通过PCR初步验证得到6株成功转入bgll基因的转化子。对这6株转化子摇瓶发酵并进行酶活测定，筛选得到一株β-葡萄糖苷酶酶活和滤纸酶活都有较大提高的转化子2-6，其β-葡萄糖苷酶酶活和滤纸酶活分别为出发菌株M23的2.8倍和1.48倍。半定量PCR分析结果显示转化子2-6染色体上bgll基因拷贝数增加，证明转入的bgll基因整合到T.reesei染色体上。结果表明由于转入的bgll基因整合到T.reesei染色体上提高了BGLI的表达量，从而提高了T.reesei整个纤维素酶系降解天然纤维素底物的能力。本研究进一步证实对丝状真菌工业生产菌株进行遗传操作是菌株改造的一条行之有效的途径。
为了解BGLI在T.reesei细胞内的分泌机制，本文将绿色荧光蛋白gfp基因作为报告基因与bgll基因融合，把得到的融合片段插入载体pTHP，再将重组质粒pTHR1和pTHR2转入pyrG基因缺陷的T.reesei菌株M23中，从无尿嘧啶的平板上得到约110株转化子，通过PCR初步验证得到7株gfp基因稳定遗传的转化子。将这些转化子接种到以乳糖为碳源的诱导培养基中诱导bgll／gfp融合基因的表达。经过酶活测定筛选得到一株转化子1-17，其β-葡萄糖苷酶酶活和滤纸酶活分别是出发菌株M23的3.3倍和1.28倍。半定量PCR分析结果表明转化子1-17染色体上的bgll基因拷贝数增加，证明转入的bgll基因整合到T.reesei染色体上。对其胞外蛋白进行SDS-PAGE检测，发现一条分子量约为100kD的蛋白条带，推测为BGLI／GFP融合蛋白，初步估算其产量为77.7μg／ml。对转化子进行荧光观测未检测到荧光，分析可能是因为以与BGLI融合蛋白形式存在的GFP不能形成正确的结构，因此不能发出荧光，或者由于所发出的荧光较弱而被菌体自发荧光掩盖，因此难以检测。
将bgll基因3′端加上编码六个组氨酸标签的序列并克隆到载体pTHP中，再将得到的重组质粒pTHB2分别与含有hph基因的质粒pAN7-1和含有amdS基因的质粒p3SR2共转化斜卧青霉(Penicillium decumbens)JU-A10。分别通过潮霉素抗性和乙酰胺为唯一碳源筛选转化子，共得到约40株转化子，通过PCR初步验证得到3株bgll基因稳定遗传的转化子。测定转化子的β-葡萄糖苷酶酶活和滤纸酶活，初步得到1株β-葡萄糖苷酶和滤纸酶活均有提高的转化子，为进一步的研究奠定了基础。
Filamentous fungi have the advantages of simple structure and fast growth, so it is easy to be manipulated and cultured. Being a kind of lower eukaryotes, it also has eukaryotic protein modification apparatus and the post translation modification mechanism. So molecular biological research on filamentous fungus has been one of the hotspots. Saprophytic fungus Trichoderma reesei has a long history of industrial production of different plant material hydrolyzing enzymes due to its extraordinary ability to secrete cellulases and hemicellulases.
The cellulase complex isolated from Trichoderma reseei comprises at least three different enzymes that together hydrolyze cellulose to oligosaccharides and glucose. Of these, the endoglucanases and cellobiohydrolases synergistically hydrolyze cellulose into small cellooligosaccharides, mainly cellobiose. Subsequently, cellobiose is hydrolyzed to glucose byβ-glucosidase. The accumulation of cellobiose could slow down the hydrolysis process significantly by inhibiting both endoglucanase and cellobiohydrolase activities.β-Glucosidases can break down cellobiose into glucose and then relieve the inhibition of endoglucanase and cellobiohydrolase by it. The amount ofβ-glucosidase secreted by T. reesei is insufficient for effective cellulose degradation. Therefore, construction of BGLI-overproducing strain is a valuable pathway to improve the efficiency of cellulose hydrolysis.
The bgl1 gene encoding the most important extracellularβ-glucosidase from T, reesei was inserted into a high-expression vector pTHP, which was constructed in our laboratory on the basis of cbh1 promoter by deletion of -677～-724 region containing three potential glucose repressor binding sites and insertion of 4 copies of -620～-820 region including the CCAAT box and ACEII binding sites. Then the recombinant plasmid pTHB was co-transformed with plasmid pAB4-l, which contains the orotidine-5'-phosphate decarboxylase (pyrG) gene from Aspergillus niger, into the pyrG-deficient strain T. reesei M23. 96 transforrnants were selected from the minimal media plate without uridine. Meanwhile, PCR amplification was carried out with the special primers to identify the transformants and the result showed that additional bgl1 copies had integrated into chromosome DNA of 6 transformants. Theβ-glucosidase and filter paper activities of the 6 transformants were examined and the transformant 2-6 showed the strongest increases in the rate of production of glucose from cellobiose (2.8-fold), and filter paper (1.48-fold). The analyzing of bgl1 gene copies on chromosome in transformant 2-6 by semiquantitative PCR indicated that the copies of bgl1 gene in transformant 2-6 were increased comparing to M23. Over-expression of BGLI by increasing the copies of bgl1 gene can enhance the ability of T. reesei to hydrolyze cellulose. The result indicated that the gene engineering method was a valuable pathway to reconstruct the strain for breeding.
To observe the secretory mechanism of BGLI, gfp gene was fused to bgl1 gene as a reporter. The fusion fragment was cloned into the vector pTHP. The recombinant plasmid pTHR1 and pTHR2 was co-transformed with plasmid pAB4-1 into T. reesei M23. 110 transformants were selected from the minimal media plate without uridine and 7 positive transformants were isolated by PCR amplification. Lactose was used as carbon source to induce the expression of the fusion protein. Theβ-glucosidase and filter paper activities of transformant 1-17 showed 3.3-fold and 1.28-fold increase comparing to the strain M23, respectively. According to semiquantitative PCR analysis, the copy number of bgl1 gene in transformant 1-17 was increased comparing to M23. It demonstrated that the bgl1 gene transformed into the T. reesei had integrated into its chromosome successfully. A band about 100kD was detected by SDS-PAGE analysis of protein from 1-17 and it was estimated to be BGLI/GFP fusion protein. The fluorescence of transformants was detected but nothing was found. The possible reason was that GFP couldn't fold to the correct structure in fusion form with BGLI or its fluorescence was so weak that it couldn't be observed under the background fluorescence of T. reesei.
The bgl1 gene containing a fragment coding 6×His tag at 3' end was cloned into the vector pTHP. Then the recombinant plasmid pTHB2 was co-transformed with the plasmid pAN7-1 containing hph gene or with p3SR2 containing amdS gene into Penicillium decumbens JU-A10. 40 transformants were selected by hygromycin B or acetamide. PCR amplification was carried out and the result showed that additional bgl1 copies had integrated into chromosome DNA of 3 transformants. Then the transformant, which showed enhanced β-glucosidase and filter paper enzyme activities, was selected for further research.
引文
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42. Juhasz T. Egyhazi A. Reczey K. Beta-glucosidase production by Trichoderma reesei. Appl. Biochem. Biotechnol. 2005, 121-124: 243-254.
43．J．萨姆布鲁克，D．W．拉塞尔．《分子克隆实验指南》(第三版)科学出版社．2002．
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30. Fey P., Compton K. and Cox E. C. Green fluorescent protein production in the cellular slime molds polysphondylium pallidum and Dictyostelium discoideum. Gene. 1995, 165:127-130.
31. Haseloff J., Siemering K. R., Prasher D. C., et al. Removal of a cryptic intron and subcellularlocalization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA. 1997, 94: 2122-2127.
32. Haseloff J., and Amos B. GFP in plants. Trends Genet. 1995,11:328-329.
33. Sheen J., Hwang S., Niwa Y., et al. Green-fluorescent protein as a new vital marker in plant cells. Plant. 1995,8:777-784.
34. Spellig T., Bottin A. and Kahmann R. Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis. Mol. Gen. Genet. 1996, 252: 503-509.
35. Cubitt A. B., Heim R., Adams S. R., et al. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 1995, 20:448-455.
36. Stauber R. H., Horie K., Carney P., et al. Development and applications of enhanced green fluorescent protein mutants. Biotechniques. 1998,24:462-471.
37. Gura T. Structure of gene-tag protein solved. Science. 1996,273:1336-1336.
38. Cormack B. P., Bertram G., Egerton M., et al. Yeast-enhanced green fluorescent protein (yEGFP), a reporter of gene expression in Candida albicans. Microbiology. 1997, 143: 303-311.
39. Yang T. T., Cheng L. and Kain S. R. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent prptein. Nucleic Acids Res. 1997, 24:4592-4593.
40. Rekangalt, David, Verner, et al. Green fluorescent protein expression in the symbiotic basidiomycete fungus Hebeloma cylindrosporum. FEMS Microbiol. Lett. 2007, 268(1): 67-72.
41. Qu Y. B., Zhao X., Gao P. J., et al. Cellulase production from spent sulfite liquor and paper-mill waste fiber. Appl. Biochem. Biotechnol. 1991, 28-29: 363-368.
42. Juhasz T. Egyhazi A. Reczey K. Beta-glucosidase production by Trichoderma reesei. Appl. Biochem. Biotechnol. 2005, 121-124: 243-254.
43．J．萨姆布鲁克，D．W．拉塞尔．《分子克隆实验指南》(第三版)科学出版社．2002．
44. Penttila M., Nevalainen H., Ratto M., et al. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene. 1987, 61: 155-164.
45. Ausubel F. M., Brent R., Kingston R. E. Short Protocols in Molecular Biology, 3rd ed. John Wiley and Sons, Inc. Indianapolis, 1995.
46. Ruud J., Peter J. I. V., Jaap V., et al. Characterization of the Kexin-Like Maturase of Aspergillus niger. Appl. Environ. Microb. 2000, 66(1): 363-368.