Control Of Aureofuscin Biosynthesis In Streptomyces Aureofuscus To Construct Gene-engineering Strains And Purify Aureofuscin

Aureofuscin is a tetraene macrolides antifungal antibiotic produced by Streptomyces aureofuscus which was isolatated from the soil in China. The chemical constitution of Aureofuscin is similar to Natamycin. They exhibit high activity against human pathogens including mycetes, yeast fungus and mycelial fungus, but they do not exhibit high activity against bacterium. The clinical efficacy of Aureofuscin surpass amphotericin B in treating keratomycosis, fungus-caused dermatoses and colpitis mycotica. Aureofuscin was discovered on chemical constitution and initial pharmacodynamic tests in1975, but not lucubrating constantly and industrialization development. In view of the chemical constitution and bioactivity of Aureofuscin similar to Natamycin which is mainly depended on the import, the research and development of Aureofuscin possessing the China independent intellectual property right shows the splendiferous applying perspective and market potential on the antifungal agents and food preservative, also possesses active meaning on promoting development of new drugs trituration, food antiseptic and fermentation industry.Aureofuscin is a natural antiseptic with market demands. But the primary cause limited its production is the low fermentation output. In this article a systematic investigation was carried out to enhance the Aureofuscin -producing by over-expression of the certain gene in Aureofuscin biosynthesis gene cluster in the linear response and fermentation condition optimization were adopted for high Aureofuscin-producing.1) The HPLC method of Aureofuscin detection in fermentation broth was developed. The optimum determination conditions were detection wavelength: 303nm, mobile phase: methanol: water: 60:40, flow speed: 1.00mL/min, keeping time: 6.957min, log-linear regression equation Y=8E+07X -381894, R~2=0.9998. The minimal detectability was 0.25μg/mL when the signal noise ratio was 3. Averaged recovery was 100.09%,100.90%,102.41% and averaged RSD was 0.18%,1.53%,1.29%.2) A pimM-homologene in Aureofuscin biosynthesis gene cluster was discovered, named AURJ3M.Sequence analysis indicated that the sequence of the cloned AURJ3M gene is the 95% and the amino acid sequence is the 97% similarity as the published PimM gene in Natamycin biosynthesis gene cluster. The AURJ3M was registed in GenBank, and the Accession number was EU697915.3) The high-copy-number plasmid pBJPIM, containing the S.natalensis pimM gene under the promoter PermE , was constructed as follows. This fragment was ligated with pSET152, resulting in plasmid pBJPIM. It was introduced into wild-type S.aureofuscus SYAU0709 via E.coli-Streptomyces conjugation. The Aureofuscin yield improved 3-4 times in the transformants. Results showed that an extra copy of the pimM gene was existed in the recombinant strains after the integration of a single copy of recombinant plasmid pBJPIM into the chromosome of S.aureofuscus SYAU0709. Overexpression of the pimM gene causes overproduction of Aureofuscin in wild-type strain.4) The high-copy-number plasmid pBJJ3M, containing the S.aureofuscus AURJ3M gene under the promoter PermE , was constructed as follows. This fragment was ligated with pSET152, resulting in plasmid pBJJ3M. It was introduced into wild-type S.aureofuscus SYAU0709 via E.coli-Streptomyces conjugation. The Aureofuscin yield improved 5-6 times in the transformants. Results showed that an extra copy of the AURJ3M gene was existed in the recombinant strains after the integration of a single copy of recombinant plasmid pBJJ3M into the chromosome of S.aureofuscus SYAU0709. Overexpression of the AURJ3M gene causes overproduction of Aureofuscin in wild-type strain. The recombinant strains have the stable heritage.5) Base on the above optimum condition, the batch fermentation was performed in 5-liter fermentor. And the optimal fermentation conditions were that 4L/min ventilatory capacity, 220r/min rotational speed, dissolves oxygen not be lower than 20%, fermentation period 84h and pH should control nearby 5.5, and the yield of Aureofuscin is 3.85mg/mL.6) The Aureofuscin purification using X-5 macroporous resin was studied. X-5 resin possessed higher absorption and desorption rate. The time of adsorption and desorption is respectively 8h and 5h. Sample pH is 6.0 and flowed pH is 7.0. Speed of adsorption and desorption is 1.0 BV/h and 1ml/min. The mecrocrystalline of Aureofuscin was obtained.