Polyketide biosynthetic gene bioprospection in metagenomic DNA from Atlantic Forest soil. / Bioprospecção de genes biossintéticos de policetídeos em DNA metagenômico de solo de Mata Atlântica.

AUTOR(ES)

Karen Cristina Massini

DATA DE PUBLICAÇÃO

2009

RESUMO

The Brazilian Atlantic Forest is considered as one of the most important reservoir of biodiversity in the planet. This biome is extremely important for its richness of plant and animal species but although with their composition and interactions poorly known and unexplored from a microbiological perspective. One gram of soil can contain near 10 billion microorganisms of different species. The majority of the soil microorganisms is not cultivable in laboratory (only 0.1-10% are recovered), being necessary to use new techniques to overcome this problem. Many of the soil microorganisms are biotechnologically important for the production of bioactive compounds. The Actinobacteria phylum is abundant in soil and important economically due to the capacity of synthesize many antibiotics. Nevertheless, the Atlantic Forest microbial biodiversity has not been properly study. Few works show microbial metabolic products with potential use in industries and, still less, antimicrobials isolated from this biome. The present work searched two new methodological alternatives: one culture independent, the metagenome, to verify the presence of polyketide synthases biosynthetic genes; and the second, the culture dependent, to select potential bacteria producers of bioactive compounds. The metagenome intend the total DNA analysis of a sample, focusing in to know the genetic information of the complex microbial diversity. Several approaches were used in order to obtain DNA of high molecular weight and quality toconstruct metagenomic libraries and search for polyketide synthases (PKS) genes types I and II, that usually are organized in clusters of 30 to 100 kb. A DNA extraction method was optimized obtaining DNA of approximately 50 kb, and used for the detection of PKS gens by PCR approaches using primers based in polyketide synthases type I and II (ketosynthase ) conserved regions of Actinomycetes. The PKS I and PKSII amplicons (600-700 bp) were cloned and two metagenoic libraries were obtained (KS I and KS II). The clones were sequenced and analyzed in a phylogenetic tree. Phylogenetic analysis of PKS I genes reveled high similarities with genes of several divisions of bacterias pointing the presence of provable new genes related with the synthesis of polyketides produzrd by PKS I and hybrid PKS with non ribosomal peptides (NRPs). Polyketide type I genes showed similarity with Streptomyces and uncultered bacteria. The analysis of polyketide II genes showed high similarity with genes of Actinobacteria gruped in two main groups, one of them with possible new genes related with the production of important antibiotics. Using selective medium for uncultered bacteria, seven isolates were obtained being studied taxonomically and tested for the production of secondary metabolites with antibacterial and antifungal activities.

ASSUNTO(S)

biodiversity florestas genética microbiana genes forests genes biodiversidade microbial genetics microbiologia agentes microbianos microbiology microbial agents

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