Study On Engineering Erythromycin And Antimycin Polyketide Carbon Scaffold

Polyketides from microorganisms and plant which are comprise of a large and structurally diverse family of bioactive natural products,are still indispensable for drug discovery.The unique chemical structure and biosynthetic mechanisms,particularly polyketides,elucidation of their biosynthetic machineries laying the genetic and biochemical foundation of novel analogues that are generated by rational engineering of biosynthetic pathways.Erythromycin is one of the macrolide antibiotics that widely used clinically for the treatment of Gram-positive bacteria infection.Due to its characteristics of hypoallergenicity and good drugability,it plays a very important role in the clinical antibacterial drugs and also shows the great potential in the development of new drugs.Method of combinatorial biosynthesis technology based on the transformation of biosynthetic pathways has become the most effective way to obtain novel erythromycin-like compounds,because of the the highly complex structure of erythromycin and the limitation of traditional chemical modification.Our objection is to introduce novel side chain into erythromycin polyketone carbon scaffold by AT-swapping.To establish a gene transfer system in S.erythraea HL3168,a highly productive producer of erythromycin,several methods including PEG-mediated protoplasts transformation,conjugal transfer were investigated,and the genetic system of S.erythraea HL3168 was finally established.The eryAⅢ gene deletion mutant was obtained by using CRISPR/Cas-mediated gene editing on the basis of the developed genetic system.In our preliminary study,we have constructed the gene library of S.erythraea HL3168,then a cosmid(pDEBS-AntEV350G)including the 6-dEB biosynthetic gene cluster and highly selective CCR was obtained by screening,post-repairing and PCR-targeting(AntEV350G).The integrity of this cosmid was confirmed by fermentation.We replaced DEBS-AT5 with SpnD-AT.Then we constructed different cosmids for swapping of AT domain.These cosmids will contain the sequence of the 6-dEB biosynthetic gene cluster with the AT-Swapping sequence of different other pathways,respectively:1)only the sequence of AT domain;2)the sequence of KS/AT linker and AT domain and post linker;3)the sequence of KS/AT linker and AT domain;4)the sequence of AT domain and post linker;Next,we transferred these AT-swapping cosmids into a mutant strain with deletion of eryAⅢ.We can then feed different precursors which can be recognized by the AT domains that replace the wildtype setting in the mutant strains.Subsequently,we aim to isolate new products from fermentation and structurally elucidate those.We hope to find the best effective replacement method and introduce different kinds of functional groups into the carbon scaffold of Erythromycin and other polyketides and thus develop a multiplex approach for diversity-oriented biosynthesis of a library of erythromycin-like compounds.The biosynthetic gene cluster of natural Antimycin include a hybrid polyketide synthase/nonribosomal peptide synthetase(PKS-NRPS)assembly line.Natural Antimycin also showed remarkable biological activity.In our preliminary study,we have uncovered and characterized a first aromatic CoA-linked extender unit benzylmalonyl-CoA from the biosynthetic pathways of splenocin and enterocin.We aim to further develop the new method of introducing other amino acids into the polyketone carbon scaffold on the basis of previous research.The mutant strain with deletion of fadB(enoyl-CoA hydratase)was constructed by blocking the β-oxidation pathway in Streptomyces sp.NRRL 2288.Then we fed 5-chloro-pentanoic acid into wildtype and mutant,the results showed that the yield of isopropyl chloride substituted Antimycin analogues was significantly improved in mutant strain with deletion of fadB.It also further confirmed that 5-chloro-pentanoic acid can not be subsequently degraded after forming an α,β-unsaturated acyl-CoA in the system which the β-oxidation pathway was blocked,and it would be beneficial to be transformed into the corresponding extension unit catalyzed by CCR protein.We also tentatively attempted to feed the corresponding decarboxylation degradation products of lysine,ornithine,and methionine.All of theses provide the basis for the subsequent construction of recombinant strains with enhanced ligase and enoyl reductase,and also provide guidance for crystal structure-based optimization of catalytic activity of AntE,especially for those substrates which are inactive or poor active.