Diversity Of Xylanase Genes In Different Environments And Heterologous Expression Of Novel Xylanase Gene Cloned Directly From Metagenomic DNA

Xylan is one of the major components of plant cell wall, and is the second most abundant renewable material after celluose in nature. A large variety of cooperatively acting enzymes are required for complete hydrolysis of xylan because of its complex structure. Among them, endo-1,4-?-D-xylanase (EC 3.2.1.8) is a crucial enzyme that cleaves the ?-1,4 backbone of xylan. Xylanases are widely distributed in microorganisms, thus the latter can degrade xylan, utilize it as energy source and consequently play an important role in carbon cycle on earth. Xylanases have been extensively studied because of their great applications in animal feed, baking, pulp and paper, biofuel, and other industries. So far most xylanases are from cultured microorganisms, which is limited since vast majority of microorganisms are uncultivable. How to retrieve novel xylanases from those uncultured microbes is a hot point in the xylanase study.Xylanases are mainly confined into glycoside hydrolase (GH) family 10 and 11. Based on the sequence alignments of GH 10 and 11 xylanases, two conserved regions in close proximity to active sites or substrate binding sites were identified, respectively. According to the principles of COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP), two sets of primers, X10-F/X10-R and X11-F/X11-R, were designed. The primer sets were verified to be specific and efficient by successful amplification of objective gene fragments from xylan-degrading bacteria and fungi of various taxa, and used to study the diversity of xylanases in environments.By using the metagenomic DNA of rhizosphere soil of snow lotus, glacier soil, hot spring soil, farmland soil, pond sediment, and Mangrove soil as templates, X10-F/X10-R or X11-F/X11-R were used to amplify objective gene fragments. Except for the hot spring soil that had only GH 10 xylanase gene fragments, gene fragments of both GH 10 and 11 were amplified from other soils (about 260 bp for GH 10 and about 210 bp for GH 11). PCR products were purified and used to construct 11 clone libraries. A total of 2115 positive clones were randomly picked up from 11 libraries and sequenced. Of them, 1075 sequences were confirmed to be GH 10 xylanase gene fragments, and 684 belong to GH 11. After removing the redundant sequences, 490 distinct xylanase gene fragments (368 GH 10 and 122 GH 11) shared similarities of below 95%. Based on BLASTx analysis, 75% of the distinct fragments had similarities of below 80% with known xylanases and 54% shared similarities of below 65% with known ones. This result suggested that there are abundant xylanases in environments and most of them are novel. Based on phylogenesis, diversity and abundance analysis, these xylanase gene fragments are different in distribution and diversity, and vary in predominance. Thus the predominant xylan-degrading microorganisms in each soil environment are assumed to be different. This variance might be related to the soil factors, such as pH, oxygen content, temperature, organic matter content and so on. The vast majority of GH 10 xylanase gene fragments are from bacteria, and more GH 11 sequences are related to fungi. Moreover, the diversity of GH 11 xylanase in these soil environments is much lower than that of GH 10. The variance of diversity and distribution of GH 10 and 11 xylanases may imply their different roles in the xylan degradation in nature.Rumen harbors immensely diverse microorganisms and is a special environment in which the microbial-mediated hydrolysis of plant cell wall polysaccharides is highly efficient. By using the same method described above, a total of 173 distinct xylanase gene fragments, 107 GH 10 and 66 GH 11, were obtained from the metagenomic DNA of goat and sheep rumen. Sequence analysis showed that the similarities of these fragments with known sequences are much lower than those of soil environments with known sequences, which suggested that numerous novel xylanases exist in the rumen microenvironment. Further gene diversity, abundance and phylogenetic analysis indicated that xylanase genes of different GH families are from different taxa. GH 10 rumen xylanases are mostly distributed in noncellulolytic microorganisms, and GH 11 xylanase genes in cellulolytic microorganisms, implying their different roles in xylan degradation. Both rumens harbored similar xylan-degrading microbial communities based on the sequence comparison analysis. Each rumen has different predominant xylan-degrading microorganisms and harbors unique xylanase producers however, implying that some xylan-degrading microorganisms might be host specific.Based on the sequences of 10 xylanase gene fragments obtained from goat rumen, 7 full-length xylanase genes were cloned directly from the metagenomic DNA using a modified TAIL-PCR method. Of them, xynGR40,xynGR67,xynGR77,xynGR112 and xynGR117 encode GH 10 xylanases, and xynR8 and xynR127 are GH 11 xylanase-encoding genes. Sequence analysis showed that these genes have low identities (45–75%) with known xylanases, suggesting their novelty. Except for XynR8 that has no putative signal peptide, all other xylanases were predicted to have signal peptides and can secrete into the environment surroundings. Domain analysis indicated that XynGR40,XynGR77,XynGR117 and XynR127 are multi-domain proteins, and XynGR77 and XynR127have special structures. Thus this study provides valuable materials to study the relationships of xylanase structure and function.Four xylanase genes,xynGR40,xynGR67, xynR8 and xynR127 were expressed in Escherichia coli BL21 (DE3), the recombinant proteins were purified and characterized. Like those xylanases from rumen microenvironments that have high catalytic activities, these four recombinant xylanases had higher specific activity towards xylan than most xylanases. The pH optima ranged from 5.5 to 6.5, which are close to the pH of rumen fluid (5.8). The temperature optima varied from 30°C (XynGR40) to 55°C (XynR8). XynGR40 had low temperature activity, retaining about 10% of the activity even at 0°C. Analysis of the amino acid composition, hydrogen bonds, and salt bridges and structure comparison with thermophilic xylanases indicated that XynGR40 is a cold active xylanase. XynGR40 is the first cold active xylanase from rumen microenvironments. Its special characteristics and structures make XynGR40 a good material to study the relationship of xylanase structure and function of. Moreover, XynGR40 is thermostable at mesophilic temperatures, has high catalytic efficiency at low temperatures, is resistant to most ions, and produces simple hydrolysis products, thus has great potential for industrial applications.In summary, this study developed a rapid and efficient culture-independent molecular method to explore the diversity and distribution of xylanase genes in complex environments. By using this method, a large number of novel xylanase gene fragments were obtained from special microenvironments. The distribution of these genes is environment specific. Full-length xylanase genes can be obtained directly from metagenomic DNA using a modified TAIL-PCR method based on the sequences of some fragments. Our study draws an insight into the diversity and distribution of xylanases in various environments, which is very meaningful to understand their roles in xylan degradation in nature. Moreover, this study also provides a new way to obtain novel xylanase genes from uncultivated microorganisms.