Isolation Of AHBA-positive Actinomycetes And Natural Products Mining And Biosynthesis Of Two Positive Strains

As drug resistance has become one of the major threats to public health,the continuous development of new drugs without or with weak cross-resistance to known antibiotics is a long-term need.Actinomycetes have been favored by natural product chemists as the main source of small molecule drugs.For decades,although the isolation methods of actinomycetes have not changed fundamentally,the paradigm of discovering new natural products have gradually changed from the traditional activity-oriented to genome-oriented.Now,when a natural product is discovered,it is usually linked to a biosynthetic gene cluster,which will facilitate the subsequent biosynthetic study.Biosynthetic knowledge will,in turn,promote rational discovery of more natural products.3-amino-5-hydroxyl benzoic acid(3,5-AHBA)is a unique natural product building block derived from aminoshikimate pathway,and usually acts as the starter unit of ansamycins represented by rifamycin(antituberculous)and maytansine(anti-tumor).Occasionally,it also participates in the biosynthesis of non-ansamycins like mitomycin,an antitumor agent.Due to the potent bioactivity of 3,5-AHBA pharmacophore,the targeted search for new natural products containing 3,5-AHBA moiety is of great significance for the development of small molecule drugs.In this dissertation,we carried out a serial of researches,including:selective isolation of soil actinomycetes,screening of AHBA synthase gene positive strain,genome mining of two prioritized strains,and biosynthesis of mining products.779 actinomycetes were isolated from 55 Chinese soil samples,which served as an abundant source of actinomycetes,through selective isolation and deduplication.Out of which 276 strains(35.4%)were classified as rare actinomycetes.Through the screening of both conservative and specific AHBA synthase genes by PCR,we obtained a total of 54 AHBA synthase gene-positive bacteria(S001-S054).Some of the them failed to revive after storage,then actually we finally obtained 44 AHBA-positive strains,in which SO 10 contains two AHBA synthase genes.We selected two strains with the highest novelty,S001 and S006,to conduct further genome mining.Genome survey showed that the genome of Streptomyces sp.S006 is about 8.07 Mb in size.The AHBA synthase gene of S006 is located in a typical ansamycin gene cluster,and we have failed to activate the gene cluster by constitutive overexpression of pathway specific regulator.Further analysis based on amide synthase shows that its product is likely to be the known compound rubradirin,although the AHBA synthase sequence of this gene cluster is novel.So,we skipped this gene cluster and turned to a type I-type III hybrid PKS gene cluster named vemS006,which has a loading PKS show significant similarity with the loading PKS of ansamycins.By overexpressing the LAL family regulatory gene vemR,we successfully activated vemS006 and isolated two compounds,venemycin(2)and its new derivative 2-chloro-venemycin(1),through scaled fermentation.There are two flavin-dependent halogenase(FDH)genes,vemJ/K,and one flavin reductase(FR)gene,vemL,located within vemS006 cluster.We used CRISPR/cas9 technology to knock out vemJ and vemK.But the attempts to knockout vemK failed,and the biosynthesis of compound 1 was not affected after vemJ knockout.Therefore,we changed our mind and tried to elucidate the halogenation of venemycin by heterologous expression of VemJ/K/L and subsequent biochemical study.A series of in vitro assays showed that VemK is responsible for the halogenation of venemycin(2).To our surprise,VemK could still efficiently catalyze the halogenation reaction in the absence of the flavin reductase VemL.Like most FDHs,VemK could catalyze the chlorination(kcat=0.209±0.007 min-1,Km=0.616±0.075 ?M,kcat/Km=0.3397 ?M-1.min-1)and bromination(kcat=0.139±0.004 min-1,Km=0.515±0.058 ?M-1,kcat/Km=0.2707 ?M-1.min-1),but chlorine is preferred.Surprisingly,although VemJ cannot directly catalyze the halogenation of venemycin(2),its presence could increase the catalytic activity of VemK to some extent.Since VemK is a novel type of one-component haloenzymes,we attempted to find out the hidden domain with FR function through homology modeling and molecular docking.Site-directed mutagenesis experiments revealed that two amino acid residues T315 and R317 are likely to play a key role in recycling of FAD/FADH2 by binding NADH.Streptomyces sp.SOOlwith a 10.43Mb genome size containing 27 typical secondary metabolism gene clusters is isolated from soil collected from Laoshan,Qingdao.Multiple activation strategies were used to manipulate these gene clusters that may encode new natural products in this bacterium,but directional activation was not realized.Interestingly,when we overexpressed the regulatory genes in sml or sm5,or inserted a constitutive promoter in sm25,we accidentally activated sm8 and identified its product SRT-A1(5).From the biosynthetic point of view,SRT-A1 might be formed by dimerization of 1,3,6,8-tetrahydroxynaphthalene(THN)derivatives,which is a product of type ? PKS.Deletion of sm8-P450 abolished the biosynthesis of SRT-A1 and accumulated an intermediate whose molecular weight was 194.0796(2 heavier than THN molecular weight)which indicated that P450 might participate in the dimerization.During the exploration of SRT-A1,we also isolated an aromatic polyketide SRT-A7 with novel carbon skeleton from sm3.In summary,we established a Culture Collection of 779 actinomycetes,of which 44 AHBA synthase gene-positive strains were identified.After prioritized,Streptomyces sp.S001 and S006 were selected for further genome mining.We identified venemycin(2)and its new derivative 2-chloro-venemycin(1)from S006,and identified new compounds SRT-A1(5)and SRT-A7(6)from S001.we identified and characterized a novel single-component flavin dependent halogenase VemK,which is the first single-component FDH from Streptomyces and the first pyrone FDH ever discovered.In addition,we also carried out some preliminary studies on the two new compounds produced by sm3 and sm8 gene cluster in Streptomyces sp.S001.After all,actinomycetes,even some streptomyces,still have amounts of secondary metabolic potential to be explored,but how to effectively release these biosynthetic potentials is still challenging.The success rate of targeted discovery of natural products in our current study is low.In the future,more effective activation methods need to be developed and applied for further work.