Natural Products Discovery In Streptomyces Qinglanensis 172205 Guided By Genome Sequence

Mircobial natural products (NPs) are important source of lead compounds in drug discovery. The bottleneck of natural products research on heavily investigated terrestrial microorganisms and the arising drug-resistant pathogens make the researchers focus on microorganisms in special or extreme environment for novel pharmaceutical compounds. Therefore, natural products discovery from mangrove Streptomyces is becoming a hotspot. Meanwhile, rapid development of the sequencing technology, bioinformatics and molecular biology as well as more available genome sequence in public database drive the transformation from traditional natural products discovery strategy to genome mining strategy guided by genome sequence. Hence, mining the secondary metabolites from mangrove Streptomyces by a genome-guided strategy will be significant and potential.A sequenced novel species Streptomyces qinglanensis 172205 isolated from a mangrove soil sample was investigated in this study. Through genome-guided NPs discovery strategy, the secondary metabolites of strain 172205 would be mined fully for novel compounds. Bioassays would be applied for discovering bioactive molecules. Additionally, biosynthetic genes or gene clusters of partial secondary metabolites would be located in the genome sequence for building a relative complete connection between natural products and gene clusters, thereby providing a potential strain with clear metabolite background.Firstly, we investigated the NPs-producing potential in strain 172205 by bioinformatics analysis. The comprehensive tool, antiSMASH, predicted 28 gene clusters related to secondary metabolites. We focused on some interesting clusters and predicted the structures of their corresponding products. A main product enterocin (XDB-1),15S-17,18-dehydroxantholipin (XDB-10), (3E,5E,7E)-3-methyldeca-3,5,7-triene-2,9-dione (XDB-11), qinlactone A (XDB-12), qinlactone B (XDB-13) and qinlactone C (XDB-14) were targeting isolated and identified by using the OSMAC, high expressed gene cluster deletion strategies, regulatory gene manipulation etc., while ectoine and ferrioxamine E were identified rapidly by LC-MS. Among them, compound XDB-10-XDB-14 were new compounds. Next, PreQo base (XDB-2) was isolated from the crude extract of wild type strain in No.2 medium, which was isolated from nature for the first time. New compound qinbactin (XDB-5), cinnamic acid (XDB-6), pyrrole-2-carboxylic acid (XDB-7),2,3-dihydroxybenzoyl-L-serine (XDB-8) and coproporphyrin III analogs (XDB-9) were identified from the crude extract of strain 172205?encE1 in No.2 medium. (E)-3-Methylthio-acrylic acid (XDB-15), methyl 3-hydroxy-4,5-dimethoxybenzoate (XDB-16) and methyl 4-hydroxy-3-methoxybenzoate (XDB-17) were obtained from the crude extract of strain 172205Aenc in D.O. medium. Additionally, we used a bioinformatics tool-PowerBLAST combined with antiSMASH results to locate their biosynthetic gene clusters for supplement biosynthetic gene clusters in strain 172205. Biosynthetic genes of most secondary metabolites were located in the genome successfully.Secondly, strain 172205 was proved to incorporate more exogenous starter units into enc-based analogs by feeding exogenous precursor experiment, thus being a potential microorganism for engineering unnatural enc-derived polyketide metabolites. We also found currently that enc clusters only exist in antinomycetes and have a specificity among genus and species by comparing putative enc clusters from different microorganisms using comparative genomics.Then, we proved that prolonging fermentation time and fermentation in agar plates are helpful to increase secondary metabolites diversity by observing the effects of changing fermentation conditions and adding chemical elicitors on secondary metabolites of strain 172205. Adding tannic acid in the fermentation could induce strain 172205 to produce 3,4'-di-O-methylellagic acid (XDB-3) and 3,3'-di-O-methylellagic acid (XDB-4) by biodegradation and biotransformation.Finally, all compounds were subjected to antimicrobial and anticancer bioactivity tests. The results showed that XDB-1 and XDB-10 exhibited strong growth inhibition against Staphylococcus aureus ATCC 51650 with the MIC (minimum inhibitory concentration) values of 1.5?g/mL and 0.75?g/mL, respectively. XDB-10 also had obvious growth inhibition against Candida albicans ATCC 10231 (MIC=3.13?g/mL) and strong cytotoxicity against MCF-7 and Hela with the IC50 values of 3.28 and 3.65?g/mL, respectively. XDB-6 showed moderate growth inhibition against S. aureus with the MIC value of 12.5?g/mL and obvious cytotoxicity against MCF-7 and Hela (IC50=18.50 and 7.79?g/mL). XDB-11-XDB-13 exhibited moderate cytotoxicity with the IC50 values of 40?g/mL approximately.