Functional Analysis Of Genes In The Biosynthesis Of Avermectin And Ansamitocin

Avermectins are16-membered macrocyclic polyketides with potentantiparasitic activities, produced by Streptomyces avermitilis. Avermectinand its derivatives have been widely used in livestock industry andagriculture.Upstream of the avermectin biosynthetic gene cluster, there is theavtAB operon （sav₉33, sav₉34） encoding the ABC transporterATP-binding subunit （AvtA） and transmembrane subunit （AvtB）.According to bioinformatic analysis, AvtAB is most likely related withavermectin export. Inactivation of avtAB had no effect, but increasing thecopies of avtAB, using a high-copy-number plasmid in S. avermitilis,enhanced avermectin production about two-fold both in the wild-type andin a high-yielding producer strain on agar plates. In liquid industrialfermentation medium, the overall productivity of avermectin B1a in theengineered high-yielding producer was improved for about50%, from3.3to4.8g/L. In liquid YMG medium, the ratio of intracellular toextracellular accumulation of avermectin B1a was dropped from6:1to4.5:1in response to the high copies of avtAB. Additionally, theoverexpression of avtAB increased the concentration of avtAB mRNA30-500folds, but did not cause any increased expression of theavermectin biosynthetic genes detected by real-time RT-PCR analysis.We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production in the cell.Moreover, the AvtAB transporter seemed to be specific for avermectinsbecause the production of oligomycin A, other macrolide produced by S.avermitilis, was not affected. According to phylogenetic analysis of ABCexporters located in antibiotic biosynthetic gene clusters, AvtAB is highlyhomologous to the multidrug efflux pump of calcium-dependentantibiotic （CDA） produced by S. coelicolor. However, CDA productionwas not increased by overexpression of avtAB in S. coelicolor.The gene aveE, encoding an enzyme with the consensus sequence ofP450monooxygenase, was identified in the avermectin biosynthetic genecluster. It is suggested that furan ring closure at C6-C8a is catalyzed byAveE. To clarify the function of AveE, we inactivated aveE and separated6,8a-seco-6,8a-deoxy-5-oxoavermectin B1a aglycon, the predictedsubstrate of AveE, from the mutant. Complementation of the mutant withaveE and meiE from the meilingmycin biosynthetic gene cluster onlyrestored the production of avermectin A components, suggesting aputative demethylation function of AveE on the C5O-methyl group. Invitro characterization of AveE showed detectable activity for theconversion of6,8a-seco-6,8a-deoxy-5-oxoavermectin B1a aglycon into5-oxoavermectin B1a aglycon, verifying its function for the furan ringclosure.Double bonds placed at atypical positions, mainly shifted from α-β toβ-γ-positions, are found in the structures of various polyketides.Ansamitocins, produced by Actinosynnema pretiosum ssp. auranticumATCC31565, are maytansinoids of microbial origin. The dehydratase（DH） domains of Asm-module2and module3would be involved in theformation of the shifted double bonds of ansamitocin P-3. Site-directedmutations of active site residues of AsmDH2and AsmDH3resulted in mutants that lack the ability to produce full-length ansamitocinderivatives, suggesting that downstream KS3and KS4, acting asgatekeepers, accept only the natural intermediates for further processing.Additionally, fusion of the erythromycin thioesterase （EryTE） domain atthe end of Asm-module2could not release the assembled triketide chainfrom the PKS. Engineered Asm-module2was expressed in E. coli andpurified. Incubation of Asm-module2with unnatural substrate2-methyl-3-phenylpropanoic-SNAC resulted in several productsincluding α-methylhydrocinnamic acid, suggesting that Asm-module2had catalytic activity.