| In our group, since 1998, we have isolated about 800 marine actinomyces fromJiaozhou Bay, and found 12 novel compounds from four strains. Selected marineactinomyces strain M045 and M048 which produced novel anti-tumor antibiotics,strain M095 which produced holomycin, and M097 with new anthraquinonecompounds as materials, we described their genetic transformation system, whichwould pave the way for strain's genetic transformation and combinatorialbiosynthesis.(i) Genetic transformation system was established for strain M045 using intergenericconjugation. The heterologous cloning and expression of the Allophycocyanin gene(apc) originating from cyanobacterium Anacystis nidulans UTEX625 in this strainwas used to demonstrate the availability of the transformation system. A segment ofPKS gene, which had high identity with actinomycin synthetase, was cloned by PCRand Genome Walker. This gene was interrupted by gene disruption, but the mutantstrain was not obtained. So reverse genetic was applied to clone the gene clustergoverning the biosynthesis of antibiotic chinikomycin. The Fosmid library have beenconstructed, and the screening of fosmid library by southern blotting is going on.(ii) Two methods including PEG-mediated protoplasts transformation andintergeneratic conjugation were investigated for strain M048, and the transformationfrequency was 10-4exconjugants /recipient. As strain M048 came from marine with ahigh salt environment, the osmotic press for maintaining protoplast was different fromStreptomyces lividans, which came from terrestrial. The conditions influencingprotoplasts formation and transformation for these strains were optimized, and theoptimal concentration of sucrose was 0.4 M.As plasmid pIJ8600 integrated on the chromosome of M048, we analysedanti-microbial activities, TLC and HPLC-MS. The results showed the crude extractfrom M048/pIJ8600 could inhibit strongly seven testing strains compared with thewild type, and TLC and HPLC-MS were also different. These results suggested thatthe integration of pIJ8600 changed the biosynthesis of some compounds, andaccumulated some compounds with strong anti-microbial activities, such asbafilomycin.A partial of PKS gene was cloned from strain M048, and this gene was interruptedby gene disruption. The results showed that anti-microbial activities of M048 mutantstrain were enhanced, and HPLC was changed, too. It indicated the disruption of thisPKS gene blocked some compounds biosynthesis and produced morechandranaimycin C or produced other compounds which had strong activity.(iii) Tranformants of strain M095 were obtained using conjugal transfer. There was nodifference between strain M095 and M095/pIJ8600, suggesting that the attachmentsite (attB) of this strain was neutral. Using PCR and Genome Walker, a segment ofglucosyl-transferase gene was cloned, and this gene was disrupted to validate itsfunction. The anti-microbial activities and HPLC analysis demonstrated this genemight participate in the biosynthesis of some compounds.(iv) Exconjugants obtained for strain M097 using conjugal transfer. Allophycocyaningene (apc) was expressed in this strain, and its production was purified. In vitroactivity test revealed that the purified recombinant APC had effective scavengingabilities on hydroxyl radicals. These results demonstrated the heterologous gene couldeffectively expressed in actinomycetes and could folded accurately, which proved thevalidity of the genetic transformation system for marine actinomyces. Biologicalactivity of M097/pIJ8600 had no difference compared with the wild type, whichindicated the attachment site of this strain was neutral.The thesis introduced gene engineering into marine actinomyces for the first time.We built the genetic transformation system for four marine actinomyces. This systemwould pave the way for reforming these strains. The analysis of biological activity forpartial PKS gene disrupted strain indicated the possibility using this transformationsystem to manipulate some compounds biosynthesis.
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