Construction Of Fosmid Metagenomic Library Of Rumen Microbiota From Hu Sheep And Xylan Enzyme Gene Screening And Analysis

A fosmid metagenomic library of uncultured microorganism from Hu sheep rumen was constructed to explore and utilize the potential of the ruminants'gut ecosystem for xylan. According to the manual of fosmid library production kit supplied by manufacturer,12704 clones were acquired. The insert size of clone ranged from 17 to 55 kb, with average insert size of 30.9 kb, the capacity of this fosmid library was 393 Mb. The fosmid library clones cultured for 100 generations still showed good stability. With the Congo red stain analysis,18 clones had been acquired with the activity of xylanase.Choosing the one (HF94 cloning), sequence analysis identified 39 coding sequences (CDSs) within the clone HF94, with 19 (48.7%) showing significant similarity to sequenced genes from other organisms, fifteen resembling CDSs of conserved hypothetical proteins (38.5%) and five (12.8%) constituted coding sequences of hypothetical protein with no sequence similarity to enzymes in any database. CDSs with known function encoded proteins involved in environmental information processing, genetic information processing, translation, aminoacyl-tRNA biosynthesis, replication and repair; DNA replication, transcription, nucleotide transport and metabolism, amino acid and carbohydrate metabolism, lipid metabolism and fatty acid biosynthesis, coenzyme metabolism and biosynthesis and biodegradation of secondary metabolites, energy production and conversion, metabolism of cofactors and vitamins, pantothenate and CoA biosynthesis and cell envelope biogenesis. Sequence analysis found that it has three coding sequences (CDS6, CDS7,CDS30) which control carbohydrate metabolism.The CDS6 gene was comprised of 942 bp and it encoded a protein of 361 amino acids. This protein contained a glycosyl hydrolysis family 11 (pfam00457). CDS6 had significant similarty with endoxylanase from Neocallimastix frontalis, the highest similarity was 79%and had significant similarty with Endo-1,4-beta-xylanase from Fibrobacter succinogenes S85, the similarity was 60%. The CDS7 gene was comprised of 1166 bp and it encoded a protein of 387 amino acids. This protein contained a glycosyl hydrolysis family 16 (pfam00722). CDS7 had significant similarty with Licheninase from Fibrobacter succinogenes S85, the highest similarity was 84%. The CDS30 gene was comprised of 2973 bp and it encoded a protein of 990 amino acids. This protein contained a glycosyl hydrolysis family 79 (pfam00754). CDS30 had significant similarty with coagulation factor 5/8 type domain protein from Fibrobacter succinogenes S85, the highest similarity was 91%and had significant similarty with cellulose-binding familyⅡfrom Streptomyces flavogriseus, the similarity was 44%. The CDS5 gene encoded proteins had significant similarty with ybaK/ebsC protein from Fibrobacter succinogenes S85, the highest similarity was 98%. The CDS 12 gene encoded proteins had significant similarty with threonyl-tRNA synthetase from Fibrobacter succinogenes S85, the highest similarity was 98%. Encoding ybaK/ebsC protein and threonyl-tRNA synthetase genes were the housekeeping gene of microorganism, so clone HF94 may from Fibrobacter succinogenes。Clone HF94 of enzymatic activity was determined. The result showed its optimal pH was 4.5, and the optimal temperature was 60℃. In the optimal conditions, the enzyme activity at pH 4.5 and 60℃was 46.14 u/mL.