Isolation and in silico characterization of novel esterase gene with ß-lactamase fold isolated from metagenome of north western Himalayas.

An esterase-producing clone Aph2 was isolated from the Apharwat soil metagenomic library, a mountain peak in NW Himalayas. ORF 2 (Est Ac) of clone Aph2 corresponds to 271 aa protein and showed 26 % sequence similarity to carboxylesterase gene of Synechococcus sp. JA-2-3B. Est Ac contains nucleophilic Ser in S68-X-X-K71 motif of ß-lactamases with Tyr Y103. The conserved sequences are common with family VIII carboxylesterase and class C ß-lactamase sequences. Phylogenetic analysis revealed that Est Ac sequence is closely related to esterase than to ß-lactamases. In silico 3D protein structure of Est Ac was generated using MODELLER software (9.10 version). Model was generated on the basis of carboxylesterase template (PDB:1CI8) of Est B (Burkholderia gladioli) and the stereochemical parameters of the model generated were satisfactory. Docking with diisopropyl-fluorophosphate confirmed catalytic activity of Ser68 present in S-X-X-K motif.

Isolation of a thioesterase gene from the metagenome of a mountain peak, Apharwat, in the northwestern Himalayas.

The soil metagenome of Apharwat (latitude 34.209° and longitude 74.368°) was explored for the presence of esterase encoding genes using a cultivation-independent approach, metagenomics. Among the various protocols tested, the method developed by Wechter was found to be the best for metagenome isolation from the soil under investigation. The purity of the isolated metagenomic DNA was not suitable for gene cloning. To improve the yield and purity of isolated metagenomic DNA, isothermal amplification of the isolated metagenomic DNA using phi (φ) polymerase in a strand displacement technique was performed. The amplified DNA was comparatively pure and the yield increased 50-fold. A metagenomic library was constructed in Escherichia coli (DH5α) using pUC19 as a vector with an average insert size ranging between 2 and 5 kb. Out of 10,000 clones generated, one clone carrying a ~1,870-bp insert hydrolysed tributyrin, indicating esterase activity. Sequence analysis revealed that the insert harboured three open reading frames (ORFs), of which ORF 3 encoded the esterase. Open reading frame 3 comprises 1,178 bp and encodes a putative 392 amino acid protein whose size correlates with most of the bacterial esterases. The esterase isolated in the present study is suggested to be a 4-methyl-3-oxoadipyl-CoA thioesterase (Accession No. JN717164.1), as it shows 60 % sequence similarity to the thioesterase gene of Pseudomonas reinekei (Accession No. ACZ63623.1) by BLAST, ClustalX and ClustalW analysis.

Metagenomics: Future of microbial gene mining.

Modern biotechnology has a steadily increasing demand for novel genes for application in various industrial processes and development of genetically modified organisms. Identification, isolation and cloning for novel genes at a reasonable pace is the main driving force behind the development of unprecedented experimental approaches. Metagenomics is one such novel approach for engendering novel genes. Metagenomics of complex microbial communities (both cultivable and uncultivable) is a rich source of novel genes for biotechnological purposes. The contributions made by metagenomics to the already existing repository of prokaryotic genes is quite impressive but nevertheless, this technique is still in its infancy. In the present review we have drawn comparison between routine cloning techniques and metagenomic approach for harvesting novel microbial genes and described various methods to reach down to the specific genes in the metagenome. Accomplishments made thus far, limitations and future prospects of this resourceful technique are discussed.