Screening And Analyzing Of Metagenomic BAC Libraries Of Yak Rumen Microbe

Rumen of ruminant animals is known as a natural reactor involved in highly efficient lignocellulose degradation. The research on rumen microbes is a subject of keen interest in terms of biofuel development. Traditionally, the studies were dependent on isolating of fibrolytic bacteria, and thereafter the lignocellulose degrading enzymes were analyzed individually. Now, using gonomic or metagenomic technologies, a variety of new genes or gene clusters associated with lignocellulose degrading enzymes could be found in rumen, based on which lignocellulose degrading mechanism could be studied and discussed. The advantage of BAC metagenomic library is that it can hold big inserted fragment, which have attracted extensive attention in recent years. More new genes and gene clusters could be achieved by constructing and screening BAC metagenomic library.The BAC metagenomic libraries of yak rumen was screened using established high-throughout screening platform for lignocellulose degrading enzymes. In this platform, lipase, xylanase, cellulase, and esterase activities of 96 clones were determined at the same time with three different substrates on the screening flat. Among 9600 BAC clones, 60 lipase positive clones, 123 xylanase positive clones, 156 cellulase positive clones, 101 esterase positive clones were screened out, with positive rate of 0.625%, 1.28%, 1.625%, 1.05%, respectively. Among them, 79 clones were found having two kinds of enzyme activities, and 11 clones having three kinds of enzyme activities. The results suggested that lots of genes and gene clusters of lignocellulose degrading enzymes existed in the BAC libraries. The sequences of 4 xylanlytic positive clones were analyzed, and lots of glycoside hydrolase and regulator genes were found, moreover, a possible new xylanase gene cluster was discovered. Partial genes of this gene cluster were cloned and expressed in E.coli, and then the protein were purified and the enzymes activities were determined. The present result verified that the ORF33 and ORF34 gene in this gene cluster, encoded endoxylanase, exoxylanase/xylosidase respectively, which were involved in degrading xylan main chain. Based on this, the function and regulation mechanism of this gene cluster could be further researched.