The Cloning And Expressions Of Halogenase Genes And Metagenomic Nonribosomal Peptide Synthetase Genes Of Mangrove Soil Microbes

Mangroves are unique woody plant communities growing in intertidal zones of tropical and subtropical coast. The mangrove ecosystem has the characteristics of high productivity, high return rates and high rates of decomposition which are called "three-high" feature. Microorganisms are an important biological composition in mangrove ecosystem, and play a key role in its energy and substances cycle. Because the mangrove ecosystem is in the intersection of freshwater and seawater and have both marine and terrestrial nature, the microorganisms form particular mechanisms of metabolism regulation and chemical defense and their secondary metaboites will also become a new source of natural products.First, we investigated the diversity of halogenase genes of the actinomycetes which were isolated from mangrove sediments by using two pairs of specific degenerate primers and found it was abundant in halogenase genes. Based on these results it was believed that these actinomycetes had potential ability to produce kinds of organohalogen compounds. And then three FADH₂-dependent halogenases were expressed in E.coli in order to explain the mechanism of halogenation. But they couldn't express as soluble protein in our experiments.when investigating the diversity of halogenase genes of the actinomycetes, we found a halogenase gene fragment of strain MGR072 exhibited 64% similarity with the halogenase gene asm12 of Actinosynnema pretiosum subsp. Auranticum. Then we used the primers which could amplify the AHBA synthetase gene to test the strain MGR072, and the result was positive. Based on the results above we presumed the strain MGR072 could produce a new ansamycin compound. After construction the genomic library of it, we got a 70Kb gene cluster associated with the biosynthesis of the new ansamycin compound, and preliminary studied on the cultural condition.At last, a metagenomic cosmid library was constructed with the large DNA fragments obtained from mangrove sediments, and was screened the NRPS gene by sequence-driven screening. A clone 4F11 was obtained and it maybe contains a whole NRPS gene cluster.