Cloning And Characterization Of Meilingmycin Biosynthetic Genes

There are two insecticidal antibiotics, nanchangmycin and meilingmycin, produced inStreptomyces nanchangensis NS3226, which was isolated from the rhizosphere soil of a teaplant, Thea oleosa, in Jiangxi Agricultural University. The nanchangmycin biosyntheticgene cluster had been cloned and characterized. But only portion of meilingmycinbiosynthetic gene cluster has been cloned in previously studies. This study aims to clonethe complete meilingmycin biosynthetic gene cluster and elucidates the meilingmycinbiosynthetic mechanism. We hope to pave the way for producing novel insecticidalcompounds by combinatorial biosynthesis.Five meilingmycins A-E, with A as the major components, were isolated from S.nanchangensis NS3226. Through NMR characterization, meilingmycins A-E were provedto be identical with reported milbemycin α11, α13, α14, β1, and β9, respectively.Sequencing of a previously cloned103-kb region identified three modular type Ipolyketide synthases genes putatively encoding for the last11elongation steps, threepost-modification genes and one transcriptional regulatory gene for meilingmycinbiosynthesis. However, the expected loading module and the first two elongation moduleswere still missing. In meilingmycin, the presence of a methyl group at C24and a hydroxylgroup at C25suggested that the elongation module1contains amethylmalonyl-CoA-specific AT domain (ATp) and a ketoreductase (KR) domain. Basedon the conserved motifs of ATp and KR domains, a pair of primers were designed for PCR amplification, and the1.40-kb expected fragment was amplified, which showed significanthomology with module1of avermectin PKS. A PKS meiA1gene encoding one loadingand two elongation modules, with a downstream C5-O-methyltransferase gene meiD, wassubsequently localized55kb apart from previously sequenced region, and its deletionabolished meilingmycin production. A series of deletions within the55-kb inter-clusterregion ruled out its involvement in meilingmycin biosynthesis. Furthermore, gene deletionof meiD eliminated meilingmycin D and E with methyl on C5.Gene deletion and complementation of meiR proved that MeiR is a positivetranscriptional regulator in meilingmycin biosynthetic pathway. An adpA homologousgene adpAnwas cloned from S. nanchangensis NS3226, and its deletion abolishedmeilingmycin production whereas nanchangmycin still produced in the mutant.The DH7domain in meilingmycin biosynthetic gene cluster was mutated viasite-directed mutation and the mutant was unable to produce meilingmycin. However aseries of new compounds seemed to be13-hydroxyl-meilingmycins accumulated in themutant. aveBI-aveBVIII responsible for the biosynthesis of oleandrose in avermectinbiosynthetic gene cluster were cloned and introduced into the mutated strain, but we can’tdetect the expected compounds with oleandrose attached to the13-hydroxyl-meilingmycins.By means of gene deletion and complementation, nanM was confirmed as amethyltransferase gene in nanchangmycin biosynthetic gene cluster in S. nanchangensisNS3226, which is responsible for the methylation of L-rhodinose at carbon4’. The newcompound demethyl-nanchangmycin was isolated from nanM deletion mutant HYL14andidentified by NMR. By the same method, nanG5was confirmed as a glycosyltransferase innanchangmycin biosynthetic gene cluster, which catalyzed the attachment of a deoxysugarmoiety to the nanchangmycin aglycone at carbon19. The new compounddeglycosyl-nanchangmycin was isolated from nanG5deletion mutant HYL6and identifiedby NMR. The bioassay revealed that the activity of demethyl-nanchangmycin and deglycosyl-nanchangmycin were both lower than that of nanchangmycin against Bacilluscereus1126. Thus, the4’-O-methylation of L-rhodinose and glycosylation at C-19arenecessary for the activity of nanchangmycin.A new strategy for narrowing down screening of antibiotic biosynthetic gene clusterfrom genomic library by PCR was carried out for cloning antibiotics biosynthetic genecluster. Cosmids containing target gene were identified by narrow-down PCR using themixed DNA of96cosmids in one plate, the mixed DNA of12cosmids in each lane and thesingle clone as templates gradually. Comparing with traditional method of in situ colonyhybridization, this method is easier, faster and more accurate.Deletion of sti on Streptomyces plasmid pIJ101made its derivative pHZ1358anefficient vector for gene disruption and replacement. Here, we further optimized pHZ1358by the construction of a derivative plasmid pJTU1278, in which a cassette carryingmultiple cloning sites and a lacZ selection marker was introduced for convenient plasmidconstruction in E. coli. Furthermore, we also deleted oriT region of the pJTU1278togenerate a vector pJTU1289, which can be used specifically for PCR-targeted replacement.The efficient application of these vectors was demonstrated by the deletion of a geneinvolved in avermectin biosynthetic in S. avermitilis.