Metagenomic approach to study the polyhydroxyalkanoate genes in hydrocarbon contaminated site

Plastics being xenobiotic are recalcitrant to microbial degradation. Plastic production also involves a number of harmful chemicals which pose environmental as well as human health risks. There is an obvious need to minimize the generation of plastic waste and to search for newer technologies that can play a vital role in mitigating these problems.;Research has been going on for quiet some time with the idea of developing an alternate environment friendly product. Many biodegradable plastic materials have been exploited such as: 1) PHAs (polyhydroxyalkanoates), 2) polylactides, 3) aliphatic polyesters, 4) polysaccharides, 5) co-polymers and /or blends of above. Of these, PHAs have gained more and more importance the world over due to their structural diversity and close analogy to plastics. The other advantage which PHAs have is that they are biodegradable and environmental friendly. Many microorganisms have the ability to degrade these macromolecules enzymatically.;PHAs are polyesters of various hydroxyalkanoates that are synthesized by many grampositive and gram-negative bacteria. It was in the last century (1926) when Lemoigne reported the formation of poly(3-hydroxbutyrate) (PHB) inside bacteria for the first time. Since then research in this field has developed considerably with the discovery of many more different PHAs from at least 75 different genera. This polymer is accumulated intracellularly to levels as high as 90% of the cell dry weight under conditions of nutrient stress and acts as a carbon and energy reserve.;Research over the years has focused on the use of alternative substrates, novel extraction methods, genetically enhanced species and mixed cultures with a view to make PHAs more commercially attractive. Enormous advances in molecular analysis of PHA biosynthesis genes and PHA production have been well documented. Many strategies such as homologous or heterologous gene probes, short consensus oligonucleotide hybridization or PCR techniques have been employed for identifying PHA synthase genes and other genes involved in PHA biosynthesis. In Escherichia coli, a previous non-PHA producer, many strategies such as pathway engineering has been developed to set up microbial production by recombinant DNA technology. The sequence analysis of 89 complete and 34 partially sequenced genomes was done in an attempt to identify domains of PHA genes using RPSBLAST. Computer simulations of PHA granule formation in vivo are being done to help design strategies to optimize the fermentation process and achieve higher yields of PHA.;Although PHA have been commercially developed and marketed, there are several important factors influencing the large scale commercial production. Efforts are being devoted with a renewed interest to increase PHA yield and productivity. A search is on to identify organisms capable of PHA production at higher levels.;Conventional methods for searching microbes employ ineffective laboratory method with the result that a major portion of the bacteria remain largely untapped, unknown, and uncharacterized due to lack of proper culture conditions. On the other hand, Metagenomics represents a powerful tool to access the abundant microbial diversity of native environmental samples. It helps to effectively characterize the genetic diversity present in any samples regardless of the availability of laboratory culturing techniques.;The term “metagenome” was first coined by Handelsman, 1998, as: The genomes of the total microbiota found in nature. Metagenomics, instead of removing microbes from their environment to isolate them, just directly isolates genomic DNA. This DNA is then cloned and environmental genetic libraries are constructed. Relevant clones from the libraries are sequenced or screened for the expression of gene products. Nucleotide-sequence based screening is highly efficient in comparison to enzyme-activity based screening. The success of metagenomic approach has already been demonstrated in identifying novel products ranging from small-sized genes conferring enzymatic activities such as lipases, amylases. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community.;The application of metagenomics to PHA will facilitate the trapping of those microbes that might have a better potential to produce PHA but are being missed out due to lack of proper culture techniques. This study is aimed at using metagenomics approach for bioprospecting of the PHA synthase gene in the environmental sample. Hydrocarbon contaminated sites are rich in carbon and have nutrient imbalance, providing excellent conditions for microbes to accumulate large amounts of PHA. And therefore are appropriate sources of sampling for our study. (Abstract shortened by UMI.).