Cloning Of Kitasamycin Biosynthesis Gene Cluster And Its Construction Of Genetic Engineering Strains

Kitasamycin is a family of14antibiotics with a16-membered lactone attached withtwo deoxyhexoses, mycarose and mycaminose. The differences of kitasamycins focus onthe different acyl substituent in the carbon4of mycarose moiety and the alternation of ahydroxyl and an acetyl group on carbon3of macrolide ring. Kitasamycins have strongactivities against Gram positive and Gram negative bacteria, mycoplapsma, leptospira,rickettsia et al.To get an insight into the biosynthesis of kitasamyicns and the regulation of itsproductivity, the biosynthetic gene cluster of kitasamycins from Streptomyces kitasatoensis1-9F, the kitasamycins producer, was cloned and characterized. With a pair of primers forthe gene of dTDP-glucose synthase, the cosmid6E8was screened from the genomiclibrary of S. kitasatoensis1-9F. With chromosome walking, overlapped cosmids10F3and17G6were screened, and the sequence of3cosmids was analyzed by bioinformatic tools,and there are51open-read-frames (ORFs) in97kb DNA fragment. Orf1-6and Orf39-51should not be involved in the biosynthesis of kitasamycins. KitA is aNDP-hexose-4-ketoreductase. KitB is a NDP-4-keto-6-deoxyhexose-2,3-reductase. KitCis a NDP-hexose-3-C-methyltransferase. KitD is a methoxymalonate biosynthesis protein.KitE is acyl-CoA dehydrogenase. KitF is a putative acyl carrier protein. KitG is a3-hydroxyacyl-CoA dehydrogenase. KitH is a probable glycosyltransferase. KitI is aNDP-hexose-3,5-epimerase. KitJ is a transaldolase. KitK and KitL are transcriptionalregulatory proteins. KitM is a4-O-propionyl transferase. KitN is a probable NDP-hexosedehydratase. KitO is a dTDP-glucose-4,6-dehydratase. KitP is a probable NDP-glucosesynthase. KitQ is a NDP-hexose aminotransferase. KitR is a NDP-hexose isomerase. KitTis a putative reductase. KitU1to KitU5are complete ORFs of type I polyketide synthasewith7modules involved in the biosynthesis of kitamycin's aglycon. With the putativefunctions of the genes in the cluster, the biosynthetic pathways of2deoxyhexoses andmethoxymalonyl-ACP were proposed.The genes of S-adenosylmethionine (SAM) synthetase (metK), transcriptionalactivator AdpA (adpA) from Streptomyces coelicolor, hemoglobin from Viteoscilla (vgb),KitL and KitK (kitK-kitL) were cloned and ligated with the expressing vector which can be integrated into the chromosome of Streptomyces sp and be stable for generations. The4engineering strains of S. kitasatoensis of were achieved by conjugation with E. coliharboring the metK, adpA, vgb and kitK-kitL expressing plasmids, respectively. The titersof kitasamycins of4engineering strains were higher than the wide type's, and the highestone is1.4-fold compared to1-9F. For5generations of propagation of the engineeringstrains, the titers of kitasamycin were kept at high level. These strains are suitable forindustry.