| Streptomyces is a genus of Gram-positive, filamentous mycelium-forming bacterium which widely distributed in terrestrial habitats,especially soil. Unlike most other bacteria, streptomyces colonies aremulticultural, and differentiated organisms exhibiting temporal and spatialcontrol of gene expression, morphogenesis, metabolism and the flux ofmetabolites. This genus is also well known by its ability to produce a groupof natural products, such as antibiotics, parasiticides, herbicides, antitumoragents and immunosuppressnts. Streptomyces bingchenggensis wasisolated from a soil sample collected in Harbin, China, and known as idealand potential organism used for industrial-scale production of the milbemycins A3and A4, which are a family of macrocyclic lactones usedthroughout the world to treat parasitic helminthes and insect pests of manand animals. In addition to the milbemycins A3and A4, we had identifiedten novel milbemycin analogs, including four milbemycins α, twomilbemycins β and four seco-milbemycins from the S.bingchenggensis.And there are also two novel cycle pentapeptides, bingchamides A and Band one new macrolide compound, ST906, have been isolated fromS.bingchenggensis. Furthermore, another insecticidal antibiotic, thepolyether ionophore nanchangmycin can also be produced by this strain.The complete genome sequence of S. bingchenggensis was obtaind byIllumina Solexa sequecncing technology, The annotation and the predictionof secondary metabolite geneclusters of S. bingchenggensis were predictedby Glimmer software and some database on web. Preliminary analyses of the S.bingchenggensis genome sequence revealed at least23gene clustersfor the biosynthesis of polyketides, terpenes and nonribosomallysynthesized peptides. we found S.bingchenggensis genome maybe thelargest streptomyces genome known to date. And we also found somereasons for genome expansion in S.bingchenggensis. First, lower GCcontent of several regions indicates there were acquired by horizontal genetransfer. Second, a mass of multigene families reflects that gene duplicationis one of the major actuating forces for expansion. Third, many transposasegenes are found in the sub-terminal invert repeat regions, suggesting a highfrequency of insertions here. So the complete genome sequence ofS.bingchenggensis will help to identify the gene clusters associated withthe milbemycins and other secondary metabolites, and shed new insightsinto their biosynthesis. Additionally, it also provides a platform for rational development of strains for titer improvement of milbemycins using thepathway-specific and non pathway-specific regulators.The glutarimide-containing polyketides cycloheximide, streptimidone,9-methylstreptimidone and migrastatin family are potent inhibitors ofhuman tumor cell migration and thus represent novel leads for anticancerdrug discovery. Cycloheximide is an inhibitor of protein biosynthesis ineukaryotic organisms, it can interfer the translocation step in proteinsynthesis (combined with60S ribosome) thus blocking translationalelongation. So it is widely used in biomedical research to inhibit proteinsynthesis in eukaryotic cells studied in vitro. But so far, the biosyntheticpathway of cycloheximide is unknown, then we use streptomyces sp. YIM56141as a research object, and got the sequence of cycloheximidgenecluster,13genes in this cluster were choosed to do the knockout experiments, when we knock out chxB, chxC, chxD, chxE genes, neither ofthese two compounds were produced. And according to the functionalanalysis, these genes related to the synthesis of polyketide chain, so wespeculated that the6molecules malonyl-CoA were binding to thepolyketide synthase ChxE to form the polyketide chain with the help ofChxB (Acyltransferases).1molecules of glutamine is involved in thesynthesis of the glutarmide moiety with the help of ChxD (asparaginesynthetase). After that the TE will hydrolysis the polyketide chain on ChxEto form the intermediate. Then the tailing enzymes will do the followingwork, however when we knockouted the chxJ and chxI, we couldn`t foundany products, maybe the intermediate is not stable. Sequence comparationresult showed that the ChxJ is a carbosylic acid reductase, so the hydroxylgroup at C14could be reducted by this enzyme. And the ChxI is a P450 reductase, it can oxidized the hydroxyl group at C8to form the keto group.After this, the electron cloud density at C9position is increased, thenattacking the C14position as nucleophilic reaction, finally the actiphenol isformed, and the phenatic acid A is the hydrolysis product of actiphenol.The formation of cycloheximide is a little complex. With the fermentationresults of mutants, ChxG which is a NADH-flavin dependentoxidoreductase may has two function, first it can reducted the double bondbetween C12and C13, after that, the cyclohexane ring will formed notbenzene ring. Then the double bond at C14and C9in cyclohexane ringwill also be reducted by ChxG. And the Cycloheximide H1is formed.ChxH as a ketoreductase, can catalyzed the ketone group at C8to hydroxylgroup, to form the final product cycloheximide. This results answered onequestion about one gene cluster can produce two different compound, cycloheximid (with cyclohexane group) and actiphenol (with phenylgroup), also provided a good model to do some research about theglutarimide polyketide synthese.The Cycloheximide H dehydrogenase (ChxH) is shown to be the lastmodification enzyme in the generation of cycloheximide in Streptomycessp. YIM56141. In this study, we demonstrated that ChxH is a NADP(H)dependent reductase which catalyzed the reversible reduction ofcycloheximide H1. The maximal catalytic efficiency (kcat/Km) of theforward reaction was3.36-fold higher than the reverse reaction, give theresult why the cycloheximide is the final product. We also test severalsimilar substrates, and found ChxH activity was not observed withactiphenol and compounds just has one keto group like cyclohexyl ethylketone, suggesting that two keto group is important for enzyme recognition and catalysis. The reductive reaction is believed to be initiated by thetransfer of a proton from the Tyr-156hydroxyl to the carbonyl moiety onthe substrate followed by hydride transfer to C8of the cycloheximide H1.A proton relay is formed and involves the2’OH of the ribose, the Lys-160side chain, and a water molecule bound to the backbone carbonyl of Asn-116. The Ser-143also facilitates catalysis by stabilizing the substratewithin the active site.In order to study the function of the gene cluster, we use heterologousexpression, and this is also an important basis of the combination ofbiological development. The success of a gene clusterexpressed in a heterologous host, not only to improve the yield of thecompound, but also to obtain a number of new compounds. In thisexperiment, the heterologous expression vector of cycloheximide gene cluster was constructed successfully, then the vector and the chxA genewere introducted to the S. lividans K4-114heterologous host. After thefermentation, we found the cycloheximide but no actiphenol. This resultshowed that the heterologous expression is complex, and we need moreexperiment to verify the result. But this preliminary results give a goodopportunity for the heterologous expression or combinatorial biosynthesisof the glutarimide compounds.After comparison of three geneclusters, iso-MGS, LTM, CHX genecluster, we found that the regulator mgsA and its homologous gene chxA isexisted in iso-MGS gene cluster and CHX gene cluster respectively. Butthere is no similar regulator in LTM gene cluster. And in our previousexperiment, iso-MGS cann`t produced when the mgsA was knockout innative strain; and in heterologous host which can produce the iso-MGS compound, we cann`t found the expression of mgsA by RT-PCR. Thesefindings give us a good chance to study how the gene clusters is expressedin heterologous host. Therefore, we constructed the chxA and mgsAregulation gene expression plasmids pUWL201PW-chxA, pKC1139-chxA,pUWL201PW-mgsA, pKC1139-mgsA. And these vectors were introducedinto the corresponding heterologous host and the wild-type strains. Afterthe fermentation and HPLC analysis, we found that the yield of iso-MGS isincreased between20%-96%, indicating that the regulation of mgsA is notapparent in iso-MGS gene cluater. For LTM producer, the introduction ofmgsA cause the silence of LTM gene cluster but may activate othermetabolite pathway. And when introduced the chxA gene, the yield of LTMis improved about one time. Due to the correlation of these three genecluster, this discovery provides us an important clue to study the regulation mechanism of this class of compounds. But there are also manyexperiments need to do, such as isolation and identiy the new compound,RT-PCR and so on.In summary, the whole genome of streptomyces bingchenggensiswas obtained successfully, the milbemycins biosynthetic gene cluster andother secondary metabolite gene clusters were predicted based on theanalysis of genome. Through a series of molecular methods such as geneknockout, complement, we found the biosynthetic mechanism ofcycloheximid and actiphenol, we also constructed a heterologousexpression system, and do some research about the regulator mgsA andchxA. |
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