未培养微生物来源的纤维素酶功能基因的筛选
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摘要
纤维素是由吡喃型D-葡萄糖以β-1,4-糖苷键连接而成的直链状大分子,是地球上含量最丰富的生物质资源。随着社会经济的发展,生物质资源的利用越来越受到人们的重视。在自然界中,有很多微生物都能产生将纤维素降解转化为葡萄糖的纤维素酶,而葡萄糖可以进一步作为发酵生产包括乙醇在内的化学基产品原料;同时,纤维素酶在饲料添加剂,纺织,造纸等行业也有着广阔的应用前景。但是,已知的纤维素酶普遍存在着活力不高,效率较低,发酵生产成本比较高的缺点,限制了纤维素酶的大规模工业化应用。因此,有必要筛选新型高效纤维素酶。
目前,通过传统的微生物筛选方法所获得的产纤维素酶菌株大多为已知菌种,筛选到新菌种的几率很低。另一方面,在自然界中存在着大量的未培养微生物(＞99%),而某些特殊生境的微生物有可能产生高效降解纤维素的酶或酶系。通过提取并纯化特定环境样品中微生物的总DNA,进而构建环境基因组文库,并从构建的各种基因组文库中筛选新的纤维素酶基因,为纤维素酶的筛选提供了一条新的筛选途径。
本论文以λ噬菌体(Stratagene)为载体成功构建了四川九寨沟森林土壤、东营市某屠宰厂的牛瘤胃液、济南市动物园亚洲象的新鲜排泄物以及取自新疆喀纳斯的腐烂木材等环境的基因组文库,文库分析发现其外源插入DNA片段有很好的随机性,文库的容量达到了实验筛选的需求。通过相关纤维素酶活性筛选的方法对所构建文库进行了初步筛选,得到了5个新的内切纤维素酶基因和2个新的β-葡萄糖苷酶基因。基因序列显示,它们与GenBank中已知的纤维素基因不仅在核苷酸水平上没有明显的同源性,而且其编码产物与已知的纤维素酶在氨基酸水平上的一致性低于50%,相似性低于70%,说明筛选到的纤维素酶基因为新的纤维素基因,证明了环境基因组学研究方法在筛选新纤维素酶基因,尤其是未培养微生物来源的纤维素酶方面的可行性。同时,研究结果也表明相关的纤维素酶系广泛存在于诸如森林土壤、牛瘤胃液及大象排泄物等环境中。
对所分离纤维素酶的氨基酸序列进一步分析表明,其中四个内切纤维素酶属于糖苷水解酶第5家族,另一个内切纤维素酶属于糖苷水解酶第9家族,但它们只含有相应家族糖苷水解酶的催化结构域,而不包含CBM区,推测原因可能是含有CBM区的纤维素酶基因不能在大肠杆菌体系中正确地折叠表达,因而无法用活性筛选的方法得到这些基因。所筛选获得的两个β-葡萄糖苷酶均属于糖苷水解酶第3家族,它们均含有糖苷水解酶3C功能域和糖苷水解酶3功能域。遗传进化树分析结果表明,这些纤维素酶与所属家族的纤维素酶处于相邻的分支上。但对这些纤维素酶粗酶液的初步分析结果显示,与同家族的已知纤维素酶相比,目前尚未发现其存在某些特殊的催化特性。
总之,本论文成功构建了多个环境样品的基因组文库,并从中筛选克隆到了新的纤维素酶基因,相关结果验证了环境基因组学研究方法在筛选新纤维素酶基因,尤其是未培养微生物来源的纤维素酶方面的可行性,为进一步的大规模筛选奠定了基础;论文所建立的方法也可以用于通过环境基因组学的方法筛选其它活性产物的工作。同时,论文工作也预示了环境基因组学功能活性筛选与其它分析方法相结合在阐述复杂生境催化体系中结构组成及其变化中的重要性。
Cellulose is composed of repeating cellobiose units linked by-1,4-glucosidic bonds, and it is the most abundant biomass in the world. Cellulose can be degraded to glucose through different cellulases, which can be then fermented to ethanol and some other useful chemicals. But the fermentation cost of cellulases is too high at present, and the hydrolysis activity of the known cellulses is still not high enough to be commericialy applied. Therefore, generation of novel cellulases with high catalysis activity either through screening or through engineering is urgently needed before they could be commercilally scaled up.
The biosphere is dominated by microorganisms which play an important role in the substance cycle of the earth, yet about 99 % of these microorganisms have not been or can not be cultured by traditional methods. With the development of molecular biology and its application in microbiology, a new research field, namely environmental genomics, has been developed. Without prior isolation and cultivation of relative microorganisms from environmental samples, all the genetic materials are directly extracted and an environmental genomic library is constructed, from which enzymes such as cellulases could be screened from environmental genomic library by functional screening.
In the present research, we directly isolated the environmental DNA from different environment samples by direct cell lysis method, and purified the metagenomic DNA by Sepharose 4B. Four different environmental genomic libraries were constructed by cloning the purified enzyme-digested DNA into the ZAP Express Vector. These libraries had inserted fragments with an average size of 4.6～6.1 Kb and the total capacity of the library is about 10~8 bp which presented a good randomness of the cloned DNA. Functional screening strategy was applied to search for positive clones expressing cellulases from the constructed libraries. Five independent clones expressing endo-glucanase and two independent clones expressingβ-glucosidase were screened out from the different libraries. Correspondingly, 7 cellulase genes encoding the above five endo-glucanases and twoβ-glucosidases were retrieved and sequenced. Sequence analysis showed that all the encoded products shared less than fifty percent identity and seventy percent similarity to cellulases in the database suggesting that all of the 7 cellulase genes were novel genes. Results of doamin analysis indicated that four endo-glucanases belong to glycolsyl hydrolase family 5 (GHF 5) containing a GHF 5 catalytic domain, and one endo-glucanase belongs to glycolsyl hydrolase family 9 (GHF 9) containing a GHF 9 catalytic domain, while all theβ-glucosidases belonged to glycolsyl hydrolase family 3 (GHF 3). Phylogenetic analysis also revealed that isolated cellulases fell into the same groups as known glycolsyl hydrolase family 5 ,9 or glycolsyl hydrolase family 3 members. Unfortunately, primary characterization of the catalytic properties with crude enzyme extracts revealed no unique properties compared with known family members.
In summary, four different environmental genomic libraries were successfully constructed in our research, and several novel cellulases were screened out from those libraries. The presented results confirmed that environmental genomics is a workable method to screen novel cellulases, especially from uncultured microorganisms. The strategy and methods described here also laid the foundation for further large-scale screening of other bio-active materials. At the same time, our work indicates that functional screening combined with other analytical methods will be playing an important role in elucidating the hierarchical structure and component dynamics of some complex eco-systems.
目前,通过传统的微生物筛选方法所获得的产纤维素酶菌株大多为已知菌种,筛选到新菌种的几率很低。另一方面,在自然界中存在着大量的未培养微生物(＞99%),而某些特殊生境的微生物有可能产生高效降解纤维素的酶或酶系。通过提取并纯化特定环境样品中微生物的总DNA,进而构建环境基因组文库,并从构建的各种基因组文库中筛选新的纤维素酶基因,为纤维素酶的筛选提供了一条新的筛选途径。
本论文以λ噬菌体(Stratagene)为载体成功构建了四川九寨沟森林土壤、东营市某屠宰厂的牛瘤胃液、济南市动物园亚洲象的新鲜排泄物以及取自新疆喀纳斯的腐烂木材等环境的基因组文库,文库分析发现其外源插入DNA片段有很好的随机性,文库的容量达到了实验筛选的需求。通过相关纤维素酶活性筛选的方法对所构建文库进行了初步筛选,得到了5个新的内切纤维素酶基因和2个新的β-葡萄糖苷酶基因。基因序列显示,它们与GenBank中已知的纤维素基因不仅在核苷酸水平上没有明显的同源性,而且其编码产物与已知的纤维素酶在氨基酸水平上的一致性低于50%,相似性低于70%,说明筛选到的纤维素酶基因为新的纤维素基因,证明了环境基因组学研究方法在筛选新纤维素酶基因,尤其是未培养微生物来源的纤维素酶方面的可行性。同时,研究结果也表明相关的纤维素酶系广泛存在于诸如森林土壤、牛瘤胃液及大象排泄物等环境中。
对所分离纤维素酶的氨基酸序列进一步分析表明,其中四个内切纤维素酶属于糖苷水解酶第5家族,另一个内切纤维素酶属于糖苷水解酶第9家族,但它们只含有相应家族糖苷水解酶的催化结构域,而不包含CBM区,推测原因可能是含有CBM区的纤维素酶基因不能在大肠杆菌体系中正确地折叠表达,因而无法用活性筛选的方法得到这些基因。所筛选获得的两个β-葡萄糖苷酶均属于糖苷水解酶第3家族,它们均含有糖苷水解酶3C功能域和糖苷水解酶3功能域。遗传进化树分析结果表明,这些纤维素酶与所属家族的纤维素酶处于相邻的分支上。但对这些纤维素酶粗酶液的初步分析结果显示,与同家族的已知纤维素酶相比,目前尚未发现其存在某些特殊的催化特性。
总之,本论文成功构建了多个环境样品的基因组文库,并从中筛选克隆到了新的纤维素酶基因,相关结果验证了环境基因组学研究方法在筛选新纤维素酶基因,尤其是未培养微生物来源的纤维素酶方面的可行性,为进一步的大规模筛选奠定了基础;论文所建立的方法也可以用于通过环境基因组学的方法筛选其它活性产物的工作。同时,论文工作也预示了环境基因组学功能活性筛选与其它分析方法相结合在阐述复杂生境催化体系中结构组成及其变化中的重要性。
Cellulose is composed of repeating cellobiose units linked by-1,4-glucosidic bonds, and it is the most abundant biomass in the world. Cellulose can be degraded to glucose through different cellulases, which can be then fermented to ethanol and some other useful chemicals. But the fermentation cost of cellulases is too high at present, and the hydrolysis activity of the known cellulses is still not high enough to be commericialy applied. Therefore, generation of novel cellulases with high catalysis activity either through screening or through engineering is urgently needed before they could be commercilally scaled up.
The biosphere is dominated by microorganisms which play an important role in the substance cycle of the earth, yet about 99 % of these microorganisms have not been or can not be cultured by traditional methods. With the development of molecular biology and its application in microbiology, a new research field, namely environmental genomics, has been developed. Without prior isolation and cultivation of relative microorganisms from environmental samples, all the genetic materials are directly extracted and an environmental genomic library is constructed, from which enzymes such as cellulases could be screened from environmental genomic library by functional screening.
In the present research, we directly isolated the environmental DNA from different environment samples by direct cell lysis method, and purified the metagenomic DNA by Sepharose 4B. Four different environmental genomic libraries were constructed by cloning the purified enzyme-digested DNA into the ZAP Express Vector. These libraries had inserted fragments with an average size of 4.6～6.1 Kb and the total capacity of the library is about 10~8 bp which presented a good randomness of the cloned DNA. Functional screening strategy was applied to search for positive clones expressing cellulases from the constructed libraries. Five independent clones expressing endo-glucanase and two independent clones expressingβ-glucosidase were screened out from the different libraries. Correspondingly, 7 cellulase genes encoding the above five endo-glucanases and twoβ-glucosidases were retrieved and sequenced. Sequence analysis showed that all the encoded products shared less than fifty percent identity and seventy percent similarity to cellulases in the database suggesting that all of the 7 cellulase genes were novel genes. Results of doamin analysis indicated that four endo-glucanases belong to glycolsyl hydrolase family 5 (GHF 5) containing a GHF 5 catalytic domain, and one endo-glucanase belongs to glycolsyl hydrolase family 9 (GHF 9) containing a GHF 9 catalytic domain, while all theβ-glucosidases belonged to glycolsyl hydrolase family 3 (GHF 3). Phylogenetic analysis also revealed that isolated cellulases fell into the same groups as known glycolsyl hydrolase family 5 ,9 or glycolsyl hydrolase family 3 members. Unfortunately, primary characterization of the catalytic properties with crude enzyme extracts revealed no unique properties compared with known family members.
In summary, four different environmental genomic libraries were successfully constructed in our research, and several novel cellulases were screened out from those libraries. The presented results confirmed that environmental genomics is a workable method to screen novel cellulases, especially from uncultured microorganisms. The strategy and methods described here also laid the foundation for further large-scale screening of other bio-active materials. At the same time, our work indicates that functional screening combined with other analytical methods will be playing an important role in elucidating the hierarchical structure and component dynamics of some complex eco-systems.
引文
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[3]Lynd LR,Weimer PJ,van Zyl WH,Pretorius IS.Microbial cellulose utilization:fundamentals and biotechnology.Microbiol Mol Biol Rev 2002;66:506-77,table.
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[6]Liu J,Tsai C,Liu J,Cheng K,Cheng C.The catalytic domain of a Piromyces rhizinflata cellulase expressed in Escherichia coli was stabilized by the linker peptide of the enzyme.Enzyme Microb Technol 2001;28:582-589.
[7]Meinke A,Damude HG,Tomme P,Kwan E,Kilburn DG,Miller RC,Jr.,Warren RA,Gilkes NR.Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop.J Biol Chem 1995;270:4383-4386.
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[2]赵荣乐.纤维素酶研究进展.喀什师范学院学报.2005,26,(6):51-54.
[3]Lynd LR,Weimer PJ,van Zyl WH,Pretorius IS.Microbial cellulose utilization:fundamentals and biotechnology.Microbiol Mol Biol Rev 2002;66:506-77,table.
[4]Tomme P,Warren RA,Gilkes NR.Cellulose hydrolysis by bacteria and fungi.Adv Microb Physiol 1995;37:1-81.
[5]Srisodsuk M,Kleman-Leyer K,Keranen S,Kirk TK,Teeri TT.Modes of action on cotton and bacterial cellulose of a homologous endoglucanase-exoglucanase pair from Trichoderma reesei.Eur J Biochem 1998;251:885-892.
[6]Liu J,Tsai C,Liu J,Cheng K,Cheng C.The catalytic domain of a Piromyces rhizinflata cellulase expressed in Escherichia coli was stabilized by the linker peptide of the enzyme.Enzyme Microb Technol 2001;28:582-589.
[7]Meinke A,Damude HG,Tomme P,Kwan E,Kilburn DG,Miller RC,Jr.,Warren RA,Gilkes NR.Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop.J Biol Chem 1995;270:4383-4386.
[8]尹清强,王景林.纤维素酶制剂对奶牛产奶最和饲料效率的影响.吉林畜牧兽医.1991,(6):12-14.
[9]梁靖,纤维素酶在速溶茶中的应用研究.茶叶.2002,28(1):25-26.
[10]周正红.高孔荣.纤维素酶在食品发酵工业中的应用及其前景.广州化学.1996.(4):8-14.
[11]Amann RI,Ludwig W,Schleifer KH.Phylogenetic identification and in situ detection of individual microbial cells without cultivation.Microbiol Rev 1995;59:143-169.
[12]Schmidt TM,DeLong EF,Pace NR.Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.J Bacteriol 1991;173:4371-4378.
[13]Healy FG,Ray RM,Aldrich HC,Wilkie AC,Ingram LO,Shanmugam KT.Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic,anaerobic digester maintained on lignocellulose.Appl Microbiol Biotechnol 1995;43:667-674.
[14]Stein JL,Marsh TL,Wu KY,Shizuya H,DeLong EF.Characterization of uncultivated prokaryotes:isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.J Bacteriol 1996;178:591-599.
[15]Li X,Qin L.Metagenomics-based drug discovery and marine microbial diversity.Trends Biotechnol 2005;23:539-543.
[16] Lee SW, Won K, Lim HK, Kim JC, Choi GJ, Cho KY. Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl Microbiol Biotechnol 2004; 65: 720-726.
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