**5. Diversity of products screened from extremophilic metagenomes**

In addition to the screening for extremozymes, the extremophilic metagenomes have also been subjected to screening for identification of small molecules and secondary metabolites which could have potential pharmaceutical applications as antibiotics, antifungal, anti-inflammatory, anti-tuberculosis, anti-cancer and immunosuppressive etc. Both functional screening of metagenome library and homology search based screening approaches have been successfully used for this purpose [22, 24, 36, 40, 62]. In comparison the extremozymes, there are relatively fewer high throughput assays available for detecting metagenomic clones that can produce small molecules and/or secondary metabolites. Thus functional screening has not been used very often for metagenomic libraries with the objective of identifying novel secondary metabolites. Therefore, there is a constant need for development of innovative functional screening methods for identification of small molecules and secondary metabolites of extremophilic origin. A few discreet studies have shown examples of novel screening approaches. In one such example a novel screening method was developed with use of indicator "Chrome Azurol-S" (CAS), which undergoes chromogenic change from orange to blue in the presence of iron. This screening method was subsequently used for identification of metagenomic clones (as well as cultivable isolates) encoding siderophores (the iron chelators). In these studies gene clusters encoding novel siderophores were identified from novel uncultivable strains.

In comparison to the functional screening, the homology search screening has been more frequently used for screening of metagenomes for the extremophilic metabolites. For the homology search screening, the metagenomic sequence data is probed to identify gene(s)/gene cluster(s) containing conserved domains or sequence that are predicted to be associated with biosynthesis of a secondary metabolites of interest. The most prominent secondary metabolites identified through homology search screening of extremophilic metagenome sequences has successfully led to the identified and characterization of: (i) glycopeptide antibiotics; (ii) cyanobactins cytotoxins; (iii) type –II polyketides antibiotics and anticancer molecules; and (iv) Trans-acyltransferse (trans-AT) polyketides [89–93]. While each of these classes of small molecules/secondary metabolites have been previously identified and characterized from the cultivable microbial diversity (more specifically actinobacterial diversity), however, with use of homology search screening of the extremophilic metagenomes, a number of novel representatives of the chemical scaffolds have been successfully identified and characterized.

#### **5.1 Identification and characterization of glycopeptide**

Glycopeptides are small molecule secondary metabolites produced by diverse organisms ranging from Proteobacteria to higher plants with Actinobacteria being the single most important source. These small molecules exhibit antibacterial activity against some of the most resistant Gram-positive pathogenic bacteria [94]. Consequently, glycopeptide are molecules of great scientific and industrial significance. The assortment of glycopeptides isolated and characterized from cultivable bacterial diversity is only very limited; therefore, several studies have been carried out with the objective of widening the catalogue of the glycopeptides through exploitation of culture- independent approaches. In one such study, soil metagenome was used as the DNA template and used for amplification a gene corresponding to OxyC, an oxidation coupling enzyme which is highly conserved and catalyzes a vital intermediate reaction during synthesis of many glycopeptides. This approach

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

Using rather simple and direct readout assays (e.g. appearance of either a halo or a color), the functional screening of the metagenomic libraries have been carried out for a number of extremophilic environments. For example, in a recent study, the Antarctic desert soil metagenomic library was screened for psychrophilic esterases using agar plates based screening approach [83, 84]. The positive clone with desired activity was selected on the basis of formation of a clear halo around the metagenomic clone. The halo formation indicated tributyrin hydrolysis; and resulted in identification and characterization of a novel cold-active psychrophilic esterase. Noticeably, it was found to be only distantly related to previously reported

While the abovementioned example for isolation and characterization of a novel psychrophilic esterase clearly highlights the value of 'functional screening' of the metagenomic libraries of the extremophilic origin, yet, it is also well acknowledged that many of the extremozymes and extremophilic metabolites are not expressed from the clones of the metagenomics library and therefore, they are not amenable to identification by library screening assays [85]. Several attempts have been made to evade the apparent limitations associated with library screening approach to metagenomics. Screening and development of alternative host for functional metagenomics screening [86] and development and application of 'Reporter Vectors' has been one of the most distinct attempts in this regard. One such Reporter Vector for metagenomic library screen was developed to have productinduced gene-expression of a reporter gene. It is done by coupling of the reporter gene to a product-sensitive transcription factor; Thus upon formation of a desired product, the transcription of the reporter gene is initiated, which could be subsequently monitored through standard reporter gene assay (e.g. fluorescence) [87]. Complementation assays have also been used as a strategy for functional screening

**4.1 Functional screening of extremophilic metagenomic libraries**

and isolation of novel biocatalysts from metagenomic libraries [88].

**4.2 Homology search based screening of the extremophilic metagenome**

The alternative approach is based on direct sequencing and homology search based screening of the gene(s), protein(s) and secondary metabolites of interest. This approach generally involves DNA amplification (PCR) step as a necessary step of sequencing procedures. Even the next generation sequencing platforms (i.e. Pyrosequencing, Sequencing by Synthesis, and Ion Sequencing) involve the step for PCR amplification of the metagenomic Pool DNA [20, 53, 79]. An earlier approach for homology search based screening of metagenomic DNA used 'heterologous probe –hybridization'; however, that approach has given way to NGS approaches. With advancement in the field of genome informatics, and metagenome informatics, it is now easily feasible to detect conserved enzymatic sequence motifs in metagenomic DNA sequences including the metagenomes of extremophilic environments [24]. The most noticeable advantage of this approach over the functional screening of the metagenomic library is the inherent high throughput and flexibility to extend the scope of screening using *in silico* homology search and screening [82]. The sequence homology search based screening approach has been used with primary metagenomic sequence data as well as with the pre- existing metagenomic datasets. The homology search based screening of metagenomic sequences gets limited only in terms of the 'existing sequence databases'. In other words any novel sequence(s) with significant divergence from the previously characterized/reference sequences or not having homology gets identified as "sequence with unknown

**70**

function".

lipases.

resulted in identification of multiple predicted glycopeptide-encoding gene clusters from the soil metagenomic libraries. In the follow up studies, the novel glycopeptide synthesis related gene(s) and gene cluster(s) identified from the metagenomic DNA were transformed and heterologously expressed in a *Streptomyces* expression host [95, 96]. Such technical intervention resulted in several new derivative glycopeptide antibiotics (with methyl, sulfur and sugar substitution) were generated being synthesized.
