Mining a South African deep mine metagenome for the discovery of novel biocatalysts

[摘要] English: The construction and screening of gene libraries prepared from DNA directly isolated fromenvironmental samples is a recent and powerful tool for the discovery of new enzymes ofbiotechnological interest (Gabor et al., 2004). Standard methods based on the screeningof isolated microorganisms are inherently limited to the tiny fraction of cultivable microbialspecies (<1%); environmental gene banks in principle provide access to the entiresequence space present in nature (Handelsman et al., 1998). Environmental librariesallow the screening of functional classes of genes from thousands of organisms andresearch in this area will provide an essential backdrop for understanding evolution andbiochemical pathways (Rajendran et al., 2008). The metagenomics approach has beenshown to be an efficient method for obtaining novel biocatalysts and useful genes fromuncultured microorganisms from diverse environments. Before proceeding to the metagenome analysis, we constructed genomic libraries from aSouth African deep mine isolate Geobacillus thermoleovorans GE-7. The library wasscreened for lipolytic activity on LB tributyrin (TLB). Active clones were sequenced using454 technology, and the sequencing results revealed the presence of the lipA and GDSLlipases, of which the latter has not yet been characterized in this organism. This familydisplays the characteristic G-D-S-L motif instead of the conventional G-X-S-X-G (Xdenotesany amino acid) motif. GDSL lipases are hydrolytic enzymes with multifunctionalproperties such as broad substrate specificity and regiospecificity. They have potential foruse in the hydrolysis and synthesis of ester compounds that are of interest inpharmaceutical, food, biochemical and the biological sector (Akoh et al., 2004; Lämmle etal., 2007). In addition, genes associated with fatty acid degradation, different glycolyticactivities, lipolytic activity, spore germination, proper protein folding, antibiotic resistanceand the cell wall were also identified in the active clones. Some of the genes identifiedmay also aid in understanding how this organism had adapted to the environment fromwhich it was isolated from.Biofilm collected from the Beatrix gold mine was selected for the metagenomic studies.We performed a diversity assessment of the biofilm by cloning and sequencing of the 16S(bacterial and archaeal) and 18S eukaryotic ribosomal DNA. Phylogenetic assessment ofthe bacterial clones indicated that clonal sequences were affiliated with at least 5 phyla ofthe domain bacteria.Sequences allocated to the phyla that are present in the bacteriallibrary, have been reported to be isolated from various environments including marineenvironments e.g. the Sargasso Sea and sea floor basalts, sub-surface ground water,limestone caves, tar pits, alkaliphilic hot springs, mine drainage sites and biofilms (Choand Giovanni, 2003; Kim and Crowley, 2007; Ikner et al., 2007; and Stepanauskas andSieracki, 2007). According to the rarefaction analysis, of the 37 clones analyzed 29different OTUs were observed. However, the rarefaction results indicate that only a portionof the richness in the bacterial community (at the ³97% sequence identity level) wassurveyed with the 16S clones sequenced as the curve did not reach an asymptote.Sequence data obtained from the archaeal clonal library showed sequence identities withthat of uncultured archaea present in the database, in particular a single unculturedarchaeal library from a marine sample. This could be attributed to the fact that there islimited sequence data on archaea present in the databases because only 4 taxonomicgroups of the domain are known thus far (Baker et al., 2003).The metagenome was screened by the sequenced-based approach for cytochrome P450monooxygenases, in particular the CYP153 family (terminal hydroxylases and long chainalkane degraders). Cloning and sequencing of the CYP153 PCR products, revealed thepresence of this family of enzymes in the metagenome P450's are involved in a plethora ofmetabolic processes, both anabolic and catabolic, and collectively interact with anenormous variety of substrates (De Mot and Parret, 2002). CYP's in bacteria are involvedin the biosynthesis of secondary metabolites such as antibiotics and in the utilization ofhydrophobic low molecular weight compounds such as alkanes and aromatics (Kubota etal., 2005). The biocatalytic production of the anticancer drug perillyl alcohol from limonenehas been reported to involve a CYP153 cytochrome from Mycobacterium sp. therebyindicating the potential application of this enzyme in the clinical setting (Urlacher andEiben, 2006).For the function-based approach, both small and large-insert metagenomic libraries wereconstructed. The libraries were screened for lipolytic, amylase, protease as well asantibacterial and antibiotic resistant genes. Only lipolytic active clones were obtained.Sequence analysis of selected TLB active clones revealed the presence of three differentlipolytic enzymes (isochorismatase, sulfatase and phosholipase, patatin family protein).Only the phospholipase, patatin protein was further characterized. Sequence analysisrevealed the presence of the classical esterase motif (G-X-S-X-G) and conservedaspartatic acid and histidine residues in the patatin. According to phylogenetic analysisphospholipase, patatin proteins constitute a new family of lipolytic enzymes, since they donot form part of any of the eight classes representative of lipolytic enzymes. The patatinwas heterologously expressed in E. coli. Biochemical analysis of the partially purifiedprotein showed that the enzyme had a preference for shorter carbon chained substrates,indicating that patatin displays esterase rather than lipase activity and functioned optimallyat 30°C and pH 8 which was expected considering that the enzyme was isolated from amesophilic environment.

[发布机构] University of the Free State