High frequency transformation of the amphotericin-producing bacterium Streptomyces nodosus
Abstract
This study has investigated DNA transformation in the Amphotericin-producing organism Streptomyces nodosus. Amphotericin B is an antifungal drug with severe side effects in humans and the availability of structural variants would aid investigations into the mode of action and cytotoxity of the drug. Analogs of related polyketide drugs have been rapidly made by genetic engineering of biosynthetic genes; however, this requires the introduction of foreign DNA into the host. Protocols for protoplast formation and regeneration were established; however, preparations were recalcitrant to DNA uptake. Electroporation-mediated methodologies also were not successful. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies of 5 x 10(-5) exconjugants generated per recipient. Use of DNA methylation-impaired E. coli donor strains resulted in 100-fold higher transformation efficiencies, indicating that DNA methylation recognition systems are operable in the organis...m. This methodology will enable genetic and biochemical analysis of the gene cluster responsible for making Amphotericin B. (C) 2003 Elsevier B.V. All rights reserved.
Keywords:
conjugation / electroporation / protoplasts / Streptomyces nodosus / transformation / Streptomyces genetics / amphotericin / polyketideSource:
Journal of Microbiological Methods, 2003, 55, 1, 273-277Publisher:
- Elsevier Science Bv, Amsterdam
DOI: 10.1016/S0167-7012(03)00160-X
ISSN: 0167-7012
PubMed: 14500018
WoS: 000185956000029
Scopus: 2-s2.0-0142028153
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Institution/Community
Inovacioni centar / Innovation CentreTY - JOUR AU - Nikodinović-Runić, Jasmina AU - Barrow, KD AU - Chuck, JA PY - 2003 UR - https://cherry.chem.bg.ac.rs/handle/123456789/155 AB - This study has investigated DNA transformation in the Amphotericin-producing organism Streptomyces nodosus. Amphotericin B is an antifungal drug with severe side effects in humans and the availability of structural variants would aid investigations into the mode of action and cytotoxity of the drug. Analogs of related polyketide drugs have been rapidly made by genetic engineering of biosynthetic genes; however, this requires the introduction of foreign DNA into the host. Protocols for protoplast formation and regeneration were established; however, preparations were recalcitrant to DNA uptake. Electroporation-mediated methodologies also were not successful. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies of 5 x 10(-5) exconjugants generated per recipient. Use of DNA methylation-impaired E. coli donor strains resulted in 100-fold higher transformation efficiencies, indicating that DNA methylation recognition systems are operable in the organism. This methodology will enable genetic and biochemical analysis of the gene cluster responsible for making Amphotericin B. (C) 2003 Elsevier B.V. All rights reserved. PB - Elsevier Science Bv, Amsterdam T2 - Journal of Microbiological Methods T1 - High frequency transformation of the amphotericin-producing bacterium Streptomyces nodosus VL - 55 IS - 1 SP - 273 EP - 277 DO - 10.1016/S0167-7012(03)00160-X ER -
@article{ author = "Nikodinović-Runić, Jasmina and Barrow, KD and Chuck, JA", year = "2003", abstract = "This study has investigated DNA transformation in the Amphotericin-producing organism Streptomyces nodosus. Amphotericin B is an antifungal drug with severe side effects in humans and the availability of structural variants would aid investigations into the mode of action and cytotoxity of the drug. Analogs of related polyketide drugs have been rapidly made by genetic engineering of biosynthetic genes; however, this requires the introduction of foreign DNA into the host. Protocols for protoplast formation and regeneration were established; however, preparations were recalcitrant to DNA uptake. Electroporation-mediated methodologies also were not successful. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies of 5 x 10(-5) exconjugants generated per recipient. Use of DNA methylation-impaired E. coli donor strains resulted in 100-fold higher transformation efficiencies, indicating that DNA methylation recognition systems are operable in the organism. This methodology will enable genetic and biochemical analysis of the gene cluster responsible for making Amphotericin B. (C) 2003 Elsevier B.V. All rights reserved.", publisher = "Elsevier Science Bv, Amsterdam", journal = "Journal of Microbiological Methods", title = "High frequency transformation of the amphotericin-producing bacterium Streptomyces nodosus", volume = "55", number = "1", pages = "273-277", doi = "10.1016/S0167-7012(03)00160-X" }
Nikodinović-Runić, J., Barrow, K.,& Chuck, J.. (2003). High frequency transformation of the amphotericin-producing bacterium Streptomyces nodosus. in Journal of Microbiological Methods Elsevier Science Bv, Amsterdam., 55(1), 273-277. https://doi.org/10.1016/S0167-7012(03)00160-X
Nikodinović-Runić J, Barrow K, Chuck J. High frequency transformation of the amphotericin-producing bacterium Streptomyces nodosus. in Journal of Microbiological Methods. 2003;55(1):273-277. doi:10.1016/S0167-7012(03)00160-X .
Nikodinović-Runić, Jasmina, Barrow, KD, Chuck, JA, "High frequency transformation of the amphotericin-producing bacterium Streptomyces nodosus" in Journal of Microbiological Methods, 55, no. 1 (2003):273-277, https://doi.org/10.1016/S0167-7012(03)00160-X . .