Construction and screening on mangrove soil metagenomic library for nonribosomal peptide synthetase gene

Abstract

Metagenomic study of unculturable bacteria may discover enzymes involved in synthesis of novel natural products, e.g. nonribosomal peptide synthetase (NRPS). Soil sample collected from Klongkone mangrove, Thailand was used for soil direct DNA extraction. Soil metagenomes was then purified and screened for nrps by PCR using A domain specific primer, MTF2/MTR. One-kb amplicons were cloned and sequenced for further analysis. Sequence alignment and phylogenetic analysis of deduced amino acid sequences of amplified A domain revealed that they were evolutionary related to NRPSs of cyanobacteria, actinobacteria and proteobacteria. These implied the novelty and diversity of nrps from mangrove soil metagenome. These metagenomes were used for metagenomic library construction in E. coli resulted in 14,000 library clones divided into 95 pools. A total of 31 pools were screened for anti-C. albicans ATCC 90028 and anti-B. subtilis (chloramphenicol resistant) activities. PCR screening of pool 30-39 of the library found A domain of nrps in pool 36 and 37. Sequence alignment and phylogenetic analysis revealed the novelty of these nrps. This suggested that mangrove soil metagenomic library harbored the novel and diverse nrps genes which might potentially lead to new drug discovery.