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

English: The construction and screening of gene libraries prepared from DNA directly isolated from environmental samples is a recent and powerful tool for the discovery of new enzymes of biotechnological interest (Gabor et al., 2004). Standard methods based on the screening of isolated microorganisms are inherently limited to the tiny fraction of cultivable microbial species (<1%); environmental gene banks in principle provide access to the entire sequence space present in nature (Handelsman et al., 1998). Environmental libraries allow the screening of functional classes of genes from thousands of organisms and research in this area will provide an essential backdrop for understanding evolution and biochemical pathways (Rajendran et al., 2008). The metagenomics approach has been shown to be an efficient method for obtaining novel biocatalysts and useful genes from uncultured microorganisms from diverse environments. Before proceeding to the metagenome analysis, we constructed genomic libraries from a South African deep mine isolate Geobacillus thermoleovorans GE-7. The library was screened for lipolytic activity on LB tributyrin (TLB). Active clones were sequenced using 454 technology, and the sequencing results revealed the presence of the lipA and GDSL lipases, of which the latter has not yet been characterized in this organism. This family displays the characteristic G-D-S-L motif instead of the conventional G-X-S-X-G (Xdenotes any amino acid) motif. GDSL lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity. They have potential for use in the hydrolysis and synthesis of ester compounds that are of interest in pharmaceutical, food, biochemical and the biological sector (Akoh et al., 2004; Lämmle et al., 2007). In addition, genes associated with fatty acid degradation, different glycolytic activities, lipolytic activity, spore germination, proper protein folding, antibiotic resistance and the cell wall were also identified in the active clones. Some of the genes identified may also aid in understanding how this organism had adapted to the environment from which 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 of the bacterial clones indicated that clonal sequences were affiliated with at least 5 phyla of the domain bacteria.Sequences allocated to the phyla that are present in the bacterial library, have been reported to be isolated from various environments including marine environments 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 (Cho and Giovanni, 2003; Kim and Crowley, 2007; Ikner et al., 2007; and Stepanauskas and Sieracki, 2007). According to the rarefaction analysis, of the 37 clones analyzed 29 different OTUs were observed. However, the rarefaction results indicate that only a portion of the richness in the bacterial community (at the ³97% sequence identity level) was surveyed with the 16S clones sequenced as the curve did not reach an asymptote. Sequence data obtained from the archaeal clonal library showed sequence identities with that of uncultured archaea present in the database, in particular a single uncultured archaeal library from a marine sample. This could be attributed to the fact that there is limited sequence data on archaea present in the databases because only 4 taxonomic groups of the domain are known thus far (Baker et al., 2003).The metagenome was screened by the sequenced-based approach for cytochrome P450 monooxygenases, in particular the CYP153 family (terminal hydroxylases and long chain alkane degraders). Cloning and sequencing of the CYP153 PCR products, revealed the presence of this family of enzymes in the metagenome P450’s are involved in a plethora of metabolic processes, both anabolic and catabolic, and collectively interact with an enormous variety of substrates (De Mot and Parret, 2002). CYP’s in bacteria are involved in the biosynthesis of secondary metabolites such as antibiotics and in the utilization of hydrophobic low molecular weight compounds such as alkanes and aromatics (Kubota et al., 2005). The biocatalytic production of the anticancer drug perillyl alcohol from limonene has been reported to involve a CYP153 cytochrome from Mycobacterium sp. thereby indicating the potential application of this enzyme in the clinical setting (Urlacher and Eiben, 2006).For the function-based approach, both small and large-insert metagenomic libraries were constructed. The libraries were screened for lipolytic, amylase, protease as well as antibacterial and antibiotic resistant genes. Only lipolytic active clones were obtained. Sequence analysis of selected TLB active clones revealed the presence of three different lipolytic enzymes (isochorismatase, sulfatase and phosholipase, patatin family protein).Only the phospholipase, patatin protein was further characterized. Sequence analysis revealed the presence of the classical esterase motif (G-X-S-X-G) and conserved aspartatic acid and histidine residues in the patatin. According to phylogenetic analysis phospholipase, patatin proteins constitute a new family of lipolytic enzymes, since they do not form part of any of the eight classes representative of lipolytic enzymes. The patatin was heterologously expressed in E. coli. Biochemical analysis of the partially purified protein showed that the enzyme had a preference for shorter carbon chained substrates, indicating that patatin displays esterase rather than lipase activity and functioned optimally at 30°C and pH 8 which was expected considering that the enzyme was isolated from a mesophilic environment.