Abstract:
The market value of molecular biology enzymes is growing rapidly, due to increasing applications in the Biotechnology industry. Such nucleic acid manipulating enzymes include polymerases, ligases, nucleases, phosphatases, methylases and topoisomerases. In this study, we analysed soils from the Kogelberg Biosphere Reserve that is situated in area of high plant endemism within the Cape Floral Region. These soils are characterised by an acidic pH and are typically oligotrophic and yet support a unique vegetation type termed fynbos (‘fine bush’). We carried out high throughput nucleic acid sequence analysis of bacterial 16S rRNA gene library and a fosmid library prepared from a soil suspension that was size-selected (0.22 m) to enrich for viruses. Sequence data were assembled and analysed using the following bioinformatics (CLC genomics workbench, MetaVir, VIROME, MG-RAST, RAST and QIIME). Based on analysis of the 16S rRNA gene marker, there was a high level of bacterial diversity that was dominated by 5 bacterial taxonomic groups; namely: Actinobacteria, Proteobacteria, Acidobacteria, Planctomycetes and Bacteroidetes. The analysis of viral diversity using sequence data from PolB, PolB2, terL and T7gp17 gene markers revealed many bacteriophages with several members of the order Caudovirales; such as Siphoviridae, Podoviridae and Myoviridae. A combination of sequence- and functional- based screening approaches was used to screen for open reading frames (ORFs) encoding nucleic acid manipulating enzymes. A total of nine (9) ORFs (sequence identify < 60) were identified and belonged to the following enzyme classes: three ligases (RNALig2, RNALig3 and DNAlig), three DNA polymerases (PolB, PolA1 and PolA2), and three Nucleases (Restriction endonuclease (RE), Homing endonuclease (HNHc) and Endonuclease 7 (E7)). Various attempts were made to recombinantly express the identified ORFs, including the use of different expression vectors (pET20b(+), pET28a(+), pET30b(+) and pMAL-C5X) and host strains (E. coli BL21 DE3, BL21 DE3 pLysS, and BL21 AI cells) as well as trying various cultivation and induction conditions. A successful expression strategy was achieved only with DNAlig gene fused to a maltose-binding affinity tag using the pMAL-C5X expression vector. The purified recombinant DNALig protein was subsequently purified and assayed for activity. The purified DNALig protein showed an ATP-dependent DNA ligation activity and could actively ligate both restricted blunt-ended and sticky-ended restricted DNA molecule. Through the use of high-throughput next generation nucleic acid sequencingcoupled with sequence- and function- based screening methods, this study was able to highlight the value of analysing the soil metavirome for the discovery of novel nucleic acid manipulating enzymes for biotechnology research and development.
