Uncultured Microbial Sources Cellulase Screening Of Functional Genes

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.