Systematics-based Study Of Secondary Metabolite Genes And Products Of Mangrove Streptomyces

Resources and strategies are very important for microbial natural products discovery. Mangrove is a typical tropical ecosystem located at the transition area between the land and the sea, over the past several years, many novel actinobacteria species and new compounds have been identified and derived from this environment, which has proven to be a productive source of new natural products.In this study,77 Streptomyces strains isolated from mangrove before were classified based on their 16S rRNA gene sequences, and detected by PCR for genes associated with secondary-metabolite biosynthesis. Three primer sets were employed to specifically target ketosynthase-methyl-malonyl-CoA transferase (KS-AT) domains and P-ketoacyl synthase (KSa) domains associated with type I and II polyketide synthase (PKSs), and adenylation domains associated with nonribosomal peptide synthases (NRPSs). PCR products yielded by 40 strains were cloned, their DNA sequences were determined and analyzed by BLAST search. Diversity of secondary-metabolite biosynthesis genes in 40 strains were analysed. Phylogenetic tree constructed from 16S rRNA gene sequences represented the evolution of these strains, and phylogenetic analysis was used to investigate whether their PKS and NRPS genes are related to the evolution and the isolation location of the Streptomyces strains. Compounds produced by fermentation medium 3 (FM3) were examined by high performance liquid chromatography (HPLC), and then analyzed the difference among different strains. After sequencing the gemone of Streptomyces sp. 061316 and Streptomyces qinglanensis 172205, gene clusters associated with secondary-metabolite biosynthesis were identified, annotated and analyzed by antiSMASH. Based on the genome sequences of 10 actinobacteria species (including other 8 strains genome sequencd by our lab), the clones amplified from these strains were located in the genome to assessed the PCR-based approach. Streptomyces qinglanensis 172205, from which no compounds have been isolated before, was incubated in 8 fermentation media in order to determine which kind of medium was optimal to produce the secondary metabolites, and two media was used to test the production of compounds in Kijanimicin and Tetronomycin classes which were predicted by antiSMASH.77 Streptomyces strains were divided into 15 clades. NRPS sequences were found to be extensively distributed among 77 Streptomyces strains (79.22%), and PKS I sequences were detected in 64.94% of the analyzed strains, while 59.74% for PKS II sequences. PKS and NPRS genes couldn’t be detected in 5 strains. A total of 62 unique KS-AT domain sequences of PKS I gene were derived from 27 positive strains by cloning, and more than 50% of these sequences shared low homology (49%-84%) with sequences available in the GenBank, the results indicate that these strains can produce variety of polyketide compounds and some of them may be novel.40 unique KSa domain sequences were also obtained from 27 positive strains, and the results of BLASTx search show that these strains commonly possess 1-2 of type II PKS biosynthetic pathways. The diversity of NRPS gene is the highest one, approximately 118 unique adenylation domain sequences were cloned from 34 positive strains, and 26 sequences shared higher than 85% identity with their top BLAST matches which are associated with the known pathways. The phylogenetic analyses of KS and AT domain from PKS I genes indicate that the KS and AT domains are clustered based on the function of KS domain and specificity of substrate, respectively. The phylogenetic trees of KS and AT domain didn’t represent the evolution of the streptomyces strains. The phylogenetic analysis of KSa domain shows that there are two kinds of PKS II genes, one is responsible for antibiotic synthesis, while the other is involved in the biosynthesis of spore pigments. The KSa sequences for the synthesis of aromatic polyketides can be divided into different clusters which have the similar structure. The spore pigment-producing KSa sequences were correlated with the phylogenetic position of the streptomyces strains. The phylogenetic analysis of NRPS gene is similar to AT domain of PKS I gene. What’s more, strains isolated from different location may possess similar PKS or NRPS genes.The HPLC analysis of products yieled by streptomyces strains indicates that strains in different caldes can produce diverse secondary metabolites, and strains with highly similar 16S rRNA gene sequences (higher than 99%) may produce similar compounds.Streptomyces sp.061316 completed genome sequence show that the genome contains 7.3 Mb (71.42% GC content), and the results predicted by antiSMASH reveal that its genome included 25 secondary-metabolite biosynthetic pathways representing 12 different classes. The genome of Streptomyces qinglanensis 172205 contains 6.2 Mb (72.68% GC content), and its genome contained 21 secondary-metabolite biosynthetic clusters representing 9 different classes. All of the results indicate that both of the strains possess the great potential to produce diverse secondary metabolites. Comparing the secondary metabolites biosynthetic gene sequences obtained by PCR products cloning and genome sequnencing of 10 strains, led to the detection of about 31% of targrted type I PKS pathways,92% of type II PKS pathways, and 51% of NRPSs. These results indicated that the type II PKS pathways gene primers we used in this study were satisfied for PCR-cloning approach, but not for the primers of type I PKS and NRPS.The products analysis of Streptomyces qinglanensis 172205 extracted from 8 media reveal that media FM9, FM17and FM19 are optimal to produce the secondary metabolites. The predicted compounds kijanimicin and tetronomycin couldn’t be detected from the products fermented with media E1 and AlBFe+C by LC-MS.This study indicates that Streptomyces from mangrove possess abundant secondary-metabolite biosynthetic genes. PCR-cloning detection for diversity analysis of biosynthetic genes and genome sequences prediction can provide valuable information for the discovery of novel compounds that one strain may produce.