Development Of The Genetic Manipulation System For Amycolatopsis Orientalis HCCB 10007 And Studies On The Function Of Genes Involved In The Grixazone Biosynthesis Of Streptomyces Griseus

Vancomycin-resistant Enterococcus faecium (VRE) was first reported in 1980s, and then the number of VRE has been increasing. People are afraid that methicillin-resistant Staphylococcus aureus (MRSA) would be resistant to vancomycin finally. Under this circumstance, it is necessary to do research and development on novel glycopeptide antibiotics for treating serious infections caused by VRE. With the increasing knowledge about the biosynthesis of some glycopeptide antibiotics including vancomycin, reprogramming the biosynthetic pathway in vivo, based on the established DNA transformation system for producing strains, will be one of the efficient ways to develop novel compounds.The conditions for protoplast preparation from A. orientalis HCCB 10007 were optimized. The results showed that about 1.58xl07/ml of protoplasts were formed when the mycelia were incubated in TSB medium containing 2% glycin, and treated with 1 mg/ml lysozyme for 30 min at 28 °C. The frequency of protoplast regeneration was 2.5%. PCR identification confirmed that the Amy cola tops is medi terranei-E. coli shuttle plasmid pRLAM containing apramycin resistance gene was introduced into A. orientalis HCCB 10007 by PEG-assisted transformation of protoplasts. This result paved the way of gene-disruption and/or gene-replacement in A. orientalis HCCB 10007.The apramycin resistance gene was inserted into the different site of the gt/E to construct plasmids with different sizes of the homologous DNA for gene crossover. These plasmids were introduced into the protoplast of A. orientalis HCCB 10007, but the real disruptants by double-crossover were not obtained. The upstream gene of gtfE, gtfD, was cloned from the genomic DNA of A. orientalis HCCB 10007. The gtfD can be used to lengthen the homologous DNA of the constructed plasmids for gene disruption, while it will be another target gene for disruption based on the established gene disruption and /or replacement system.Streptomyces produces a wide variety of secondary metabolites including antibiotics, immunomodulators, and displays a complex morphological differentiation, which makes the genus to be of industrial importance and one of the model prokaryotes for studying multicellular differentiation. Some regulatory steps for morphological differentiation and secondary metabolism share common genes or metabolites. A-factor(2-isocapryloyl-3R-hydroxyrri'ethyl-y-butyrolactone) acts as a switch for aerial mycelium formation and secondary metabolite formation in Streptomyces griseus.The production of a diffusible yellow pigment, grixazone, is one of the phenotypes triggered by A-factor. To reveal the whole pathway and the regulation mechanism of grixazoneproduction, the griA, griB, and griJ involved in the biosynthesis pathway were studied.The plasmids pUC-griA-Kmr and pUC-AgriB::Kmr were constructed by in-frame deletion of griA and insertional inactivation of griB respectively, for both of which the apramycin resistance gene was used as selectable marker. Two plasmids were introduced into 5. griseus IFO13350 by PEG-assisted transformation of protoplasts. The correct gene disruptants obtained via homologous crossover were confirmed by southern hybridization. The griA can produce grixazone but not griB Km. The plasmid for griB complementation was constructed with the E. coli-Streptomyces shuttle plasmid pKU209, which was introduced into the griB Kmr. The result showed that the grixazone production can be restored partially, which indicated that griB was essential for grixazone production. A homology search suggested that GriB is a transmembrane efflux protein.The plasmid for gril disruption was constructed by cloning two non-contiguous restriction fragments carrying the 5' and 3' ends of griJ into plasmid pUC19, and then apramycin resistance gene was used as selective marker. The recombinant plasmid was introduced into S. griseus IFO13350 by PEG-assisted transformation of protoplasts, griJ was obtained by single crossover and confirmed by southern hybridization. griJ failed to produce grixazone and gene...