[Establishment of gene transduction system in geldanamycin producer - Streptomyces hygroscopicus 17997 and its application for gene disruption experiment].
Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997.
Gao
Hui-Ying
HY
Institute of Medicinal Biotechnology, CAMS & PUMC, Beijing 100050, China.
Wang
Yi-Guang
YG
Gao
Qun-Jie
QJ
Shang
Guang-Dong
GD
Sun
Gui-Zhi
GZ
Yang
Ying
Y
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics; D016227:Benzoquinones / Q000378:metabolism*; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005822:Genetic Vectors / Q000235:genetics; D047029:Lactams, Macrocyclic / Q000378:metabolism*; D005810:Multigene Family / Q000235:genetics / Q000502:physiology; D016133:Polymerase Chain Reaction; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D000429:Alcohol Oxidoreductases; C087192:polyketide synthase ketoreductase; C001277:geldanamycin