Systematics-guided bioprospecting for bioactive microbial natural products.

Advances in the taxonomic characterization of microorganisms have accelerated the rate at which new producers of natural products can be understood in relation to known organisms. Yet for many reasons, chemical efforts to characterize new compounds from new microbes have not kept pace with taxonomic advances. That there exists an ever-widening gap between the biological versus chemical characterization of new microorganisms creates tremendous opportunity for the discovery of novel natural products through the calculated selection and study of organisms from unique, untapped, ecological niches. A systematics-guided bioprospecting, including the construction of high quality libraries of marine microbes and their crude extracts, investigation of bioactive compounds, and increasing the active compounds by precision engineering, has become an efficient approach to drive drug leads discovery. This review outlines the recent advances in these issues and shares our experiences on anti-infectious drug discovery and improvement of avermectins production as well.

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library.

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K(m) of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0-60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0-10.0 showing the highest activity at pH 9.0.

Construction and preliminary analysis of a deep-sea sediment metagenomic fosmid library from Qiongdongnan Basin, South China Sea.

Preliminary characterization of the microbial phylogeny and metabolic potential of a deep-sea sediment sample from the Qiongdongnan Basin, South China Sea, was carried out using a metagenomic library approach. An effective and rapid method of DNA isolation, purification, and library construction was used resulting in approximately 200,000 clones with an average insert size of about 36 kb. End sequencing of 600 individual clones from the fosmid library generated 1,051 sequences with an average sequence length of 619 bp. Phylogenetic ascription indicated that this library was dominated by Bacteria, predominantly Proteobacteria, though Planctomycetes were also relatively abundant. Sulfate-reducing and anaerobic ammonium-oxidizing bacteria, which play important roles in the cycling of sedimentary nutrients, were abundant in the library. Cluster of orthologous groups category analysis showed that most of the genes contained in the end sequences were related to metabolism, and with cellular processes and signaling. Functional groups assigned by SEED (subsystems-based annotations) highlighted the existence of 'one-carbon' metabolism within this community as well as identifying functional genes involved in methanogenesis. Furthermore, diverse genes involved in the biodegradation of xenobiotics were found using Kyoto Encyclopedia of Genes and Genomes metabolic pathway analysis.

Novel lipolytic genes from the microbial metagenomic library of the South China Sea marine sediment.

Metagenomic cloning is a powerful tool for the discovery of novel genes and biocatalysts from environmental microorganisms. Based on activity screening of a marine sediment microbial metagenomic library, a total of 19 fosmid clones showing lipolytic activity were identified. After subcloning, 15 different lipolytic genes were obtained; their encoded proteins showed 32-68% amino acid identity with proteins in the database. Multiple sequence alignment and phylogenetic tree analysis demonstrated that most of these predicted proteins are new members of known families of bacterial lipolytic enzymes. However, two proteins, FLS18C and FLS18D, could not be assigned to any known family, thus probably representing a novel family of the bacterial lipolytic enzyme. The activity assay results indicated that most of these lipolytic enzymes showed optimum temperature for hydrolysis at 40-50 degrees C with p-nitrophenol butyrate as a substrate. The lipolytic gene fls18D was overexpressed, and the resulting protein FLS18D was characterized as an alkaline esterase. Furthermore, the whole sequence of fosmid pFL18 containing FLS18C and FLS18D was shotgun sequenced, and a total of 26 ORFs on it were analyzed and annotated.