Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector.

The metagenomic approach has greatly accelerated the discovery of new enzymes by giving access to the genetic potential of microorganisms from various environments. Function-based screening depends on adequate expression of the foreign genes in the heterologous host, which can be challenging in large-insert libraries. In this study, the shuttle cosmid vector pFX583 was used for the construction and screening of a metagenomic library. This vector allows T7 RNA polymerase-directed transcription of the cloned DNA and can be used in Escherichia coli and Streptomyces lividans. The DNA used for the library construction was obtained from an enriched biomass. The library was screened for lipolytic and proteolytic activities using E. coli and S. lividans as hosts. Numerous E. coli clones with lipolytic activity were detected. Unfortunately, proteases could not be detected in both hosts. From the lipolytic activity screen, a gene coding for a new lipase was isolated, and partial characterization was conducted.

Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library.

The construction of a cosmid library from the biomass produced in an enriched Sequencing Fed-Batch Reactor allowed the isolation of a new lipase by functional screening. The open reading frame of 928 bp encoded a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa. The amino acid sequence analysis revealed the presence of the conserved pentapeptide GXSXG essential for lipase activity. Alignment with known sequences of proteins showed no more than 52% identity with different lipases, confirming the discovery of a novel gene sequence. The lipase was cloned and expressed in Streptomyces lividans and further purified by a combination of hydrophobic interaction and size-exclusion chromatography. Spectrophotometric assays with different p-nitrophenyl esters demonstrated a preference for long-length acyl chains, especially p-nitrophenylmyristate (C14). Moreover, the enzyme presented an optimal activity at 60 degrees C and at alkaline pH of 10.5.

Saccharomyces cerevisiae Boulardii Reduces the Deoxynivalenol-Induced Alteration of the Intestinal Transcriptome.

Type B trichothecene mycotoxin deoxynivalenol (DON) is one of the most frequently occurring food contaminants. By inducing trans-activation of a number of pro-inflammatory cytokines and increasing the stability of their mRNA, trichothecene can impair intestinal health. Several yeast products, especially Saccharomyces cerevisiae, have the potential for improving the enteric health of piglets, but little is known about the mechanisms by which the administration of yeast counteracts the DON-induced intestinal alterations. Using a pig jejunum explant model, a whole-transcriptome analysis was performed to decipher the early response of the small intestine to the deleterious effects of DON after administration of S. cerevisiae boulardii strain CNCM I-1079. Compared to the control condition, no differentially expressed gene (DE) was observed after treatment by yeast only. By contrast, 3619 probes-corresponding to 2771 genes-were differentially expressed following exposure to DON, and 32 signaling pathways were identified from the IPA software functional analysis of the set of DE genes. When the intestinal explants were treated with S. cerevisiae boulardii prior to DON exposure, the number of DE genes decreased by half (1718 probes corresponding to 1384 genes). Prototypical inflammation signaling pathways triggered by DON, including NF-κB and p38 MAPK, were reversed, although the yeast demonstrated limited efficacy toward some other pathways. S. cerevisiae boulardii also restored the lipid metabolism signaling pathway, and reversed the down-regulation of the antioxidant action of vitamin C signaling pathway. The latter effect could reduce the burden of DON-induced oxidative stress. Altogether, the results show that S. cerevisiae boulardii reduces the DON-induced alteration of intestinal transcriptome, and point to new mechanisms for the healing of tissue injury by yeast.