Characterisation of two bifunctional cellulase-xylanase enzymes isolated from a bovine rumen metagenome library.

Ruminant digestive tract microbes hydrolyse plant biomass, and the application of metagenomic techniques can provide good coverage of their glycosyl hydrolase enzymes. A metagenomic library of circa 70,000 fosmids was constructed from bacterial DNA isolated from bovine rumen and subsequently screened for cellulose hydrolysing activities on a CMC agar medium. Two clones were selected based on large clearance zones on the CMC agar plates. Following nucleotide sequencing, translational analysis and homology searches, two cellulase encoding genes (cel5A and cel5B) belonging to the glycosyl hydrolyse family 5 were identified. Both genes encoded pre-proteins of about 62 kDa, containing signal leader peptides which could be cleaved to form mature proteins of about 60 kDa. Biochemical characterisation revealed that both enzymes showed alkaline pH optima of 9.0 and the temperature optima of 65 °C. Substrate specificity profiling of the two enzymes using 1,4-ß-D-cello- and xylo-oligosaccharides revealed preference for longer oligosaccharides (n ≥ 3) for both enzymes, suggesting that they are endo-cellulases/xylanases. The bifunctional properties of the two identified enzymes render them potentially useful in degrading the ß-1,4 bonds of both the cellulose and hemicellulose polymers.

Low-coverage sequencing in a deep intercross of the Virginia body weight lines provides insight to the polygenic genetic architecture of growth: novel loci revealed by increased power and improved genome-coverage.

Genetic dissection of highly polygenic traits is a challenge, in part due to the power necessary to confidently identify loci with minor effects. Experimental crosses are valuable resources for mapping such traits. Traditionally, genome-wide analyses of experimental crosses have targeted major loci using data from a single generation (often the F2) with individuals from later generations being generated for replication and fine-mapping. Here, we aim to confidently identify minor-effect loci contributing to the highly polygenic basis of the long-term, bi-directional selection responses for 56-d body weight in the Virginia body weight chicken lines. To achieve this, a strategy was developed to make use of data from all generations (F2-F18) of the advanced intercross line, developed by crossing the low and high selected lines after 40 generations of selection. A cost-efficient low-coverage sequencing based approach was used to obtain high-confidence genotypes in 1Mb bins across 99.3% of the chicken genome for >3,300 intercross individuals. In total, 12 genome-wide significant, and 30 additional suggestive QTL reaching a 10% FDR threshold, were mapped for 56-d body weight. Only 2 of these QTL reached genome-wide significance in earlier analyses of the F2 generation. The minor-effect QTL mapped here were generally due to an overall increase in power by integrating data across generations, with contributions from increased genome-coverage and improved marker information content. The 12 significant QTL explain >37% of the difference between the parental lines, three times more than 2 previously reported significant QTL. The 42 significant and suggestive QTL together explain >80%. Making integrated use of all available samples from multiple generations in experimental crosses are economically feasible using the low-cost, sequencing-based genotyping strategies outlined here. Our empirical results illustrate the value of this strategy for mapping novel minor-effect loci contributing to complex traits to provide a more confident, comprehensive view of the individual loci that form the genetic basis of the highly polygenic, long-term selection responses for 56-d body weight in the Virginia body weight chicken lines.

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library.

AIMS: The aim of this study was to demonstrate the application of environmental sample pre-enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s). METHODS AND RESULTS: A positive recombinant clone (UVCL29), conferring an esterase phenotype was identified from a shotgun gene library. The complete sequence of the 3.0 kb DNA insert from the pUVCL29 recombinant plasmid was obtained using primer-walking strategies. Nucleotide sequence analysis revealed a complete 945 bp open reading frame (ORF1). Translational analysis of the ORF1 showed a protein of 314 amino acids (named EstAM) with a predicted molecular weight of 34 kDa. EstAM's primary structure showed a classical (-G-D-S-A-G-) motif, corresponding with the generally conserved (G-x-S-x-G) esterase signature motif. Identity searches indicated that EstAM has high sequence similarity with esterases from family IV. EstAM was successfully expressed in Escherichia coli in a biologically active form. Partial purification was achieved using a one-step Pro-PurTM IMAC column. Biochemical characterization revealed that EstAM has a temperature optimum of 40 degrees C. CONCLUSION: Based on its substrate profile, EstAM was classified as a carboxylesterase because of its preference for short p-nitrophenyl ester substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is a demonstration of the successful application of environmental sample pre-enrichment technology in accessing novel esterases from a mining environment.