Targeted Discovery And Biosynthesis Of Secondary Metabolites From Streptomyces Sp.LZ35 And S10

The emergence of new pathogenic bacteria calls the discovery of new antibiotics with diverse structure and novel biological functions.Secondary metabolites derived from microorganisms,especially Streptomyces,are an important part of natural products and one of the important sources of drugs or precursor drugs.There are abundant secondary metabolic biosynthetic gene clusters in microbial genome,which provide potential resources for the identification of novel drugs.Although a variety of microorganisms produce secondary metabolites,but most of the microorganisms are not culturable in laboratory conditions,or most of the gene clusters responsible for the biosynthesis of secondary metabolites are silent or poorly expressed in laboratory conditions.However,scientists all over the world are trying their best to "wake up" these silent gene clusters,and have developed a variety of tools to activate silent gene clusters.Based on two strains of streptomyces from soil,we aimed to focus on the following three aspects:discovery of secondary metabolites,biosynthesis of natural products,and determination of biological activity.Streptomyces sp.SR111,derived from Streptomyces sp.LZ35,is a marine actinomycetes isolated from Xiamen beach.The genome size of the strain is 11.3 Mb,which contained more than 40 secondary metabolic gene clusters.Although our team has identified the metabolites of multiple gene clusters from this strain,it still contains a large number of silent secondary metabolic gene clusters,which is worth exploring.The optimization of culture conditions is an important factor to explore the secondary metabolites of microorganisms,but this method is blind and cumbersome.Therefore,based on the optimization of culture conditions,we introduced melanin reporter gene melC as an indicator,and tried to use the strategy of 'reporter-guided medium selection'to activate silent gene clusters.We selected four silent secondary metabolic gene clusters BGC2,BGC8,BGC42,and BGC45 from strain SR111 as target gene clusters,and then constructed four indicator strains containing melC.Through the cultivation and screening on 22 kinds of fermentation medium,we screened the medium that can activate BGC8 gene cluster(hcl).A novel 26-ring macrolide,Hexacosalactone A(1),was successfully identified after a series of fermentation,extraction,and structural identification.After bioassay of Hexacosalactone A,we found that the compound had moderate activity against Proteusbacillus vulgaris CPCC 160013 and Staphylococcus aureus ATCC 25923(MIC=12.5 ?g/ml).Quinolamines are a class of meroterpenoid isolated from strain LZ35 by Zhang juanli,School of pharmacy,Shandong University.Quinolamines A-C(2-4)possess unique 6/6/6 and 6/6/5 tricyclic structures,and the biosynthesis of their tricyclic system may have a unique catalytic mechanism.Firstly,the biosynthetic gene cluster qul was traced based on the skeleton characteristics and bioinformatics analysis,and confirmed it by large fragment knockout.Then we investigated the functions of qulA,qulB,qulC,qulE,qulF and qulG by gene knockout,gene complementary,protein heterologous expression,and enzyme activity verification in vitro.Through the heterologous expression experiment,we found that the loading of C5N plays an important role in biosynthesis of tri-ring system of Quinolamines A-C(2-4).As second 6-membered ring of Quinolamines may formed by the nitrene transfer reaction catalyzed by cytochrome P450 monooxygenase QulE,therefore,we speculate that the formation of the third ring of Quinolamines A-C(2-4)may be the coupling reaction of nitrene transfer reaction.Streptomyces sp.S10 is an AHBA positive bacterium isolated from soil samples of Nanjing Botanical Garden.Through the whole genome sequencing and bioinformatics analysis,we found that strain S10 contains a novel pentaketo ansamycin synthesis gene cluster tpt.However,the gene cluster was in a state of silence or low-level expression under conventional laboratory culture conditions.We successfully activated tpt through promoter replacement strategy,and identified four novel pentaketo ansamycin compounds Tetrapetalones E-H(32-35)from the fermentation extract of mutant SP101.Tetrapetalones F and H have D-rhamnose side chain.Tetrapetalones hold the activities of lipoxygenase inhibitors and free radical scavengers,and they all exhibit a unique 6/5/7/5 tetra ring structure.In order to study the biosynthesis mechanism of tetrapetalones,we knocked out several possible post modification genes on the basis of SP101.Tetrapetalones A(31)and Tetrapetalones I-K(36-38)were accidentally detected from tpt8 and tpt12 knockout mutants,and these four compounds have Drhodinose sugar side chain.At the same time,we overexpressed tptRl and tptR2 in SP101,and constructed the mutant SP102.Tetrapetalones A and E-K were also detected in the metabolite of mutant SP102.In conclusion,we used different experimental strategies to dig and biosynthesize secondary metabolites from two Streptomyces strains,and successfully activated the hcl gene cluster of Streptomyces sp.SR11l and the tpt gene cluster of Streptomyces sp.S10.We obtained a novel macrolide compound Hexacosalactone A with antibacterial activity and eight tetrapetalones,respectively.In addition,we also studied the biosynthesis of Tetrapetalones and Quinolamines in detail.This study may provide new guidance and reference for the subsequent mining and biosynthesis of natural products from microorganisms.