title: Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques.
abstract: Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces.
authors.affiliation: Department of Industrial Microbiology, Centre for Synthesis and Chemical Biology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.
authors.forename: Maria
authors.initials: M
authors.lastname: Carmody
authors.forename: Barry
authors.initials: B
authors.lastname: Byrne
authors.forename: Barry
authors.initials: B
authors.lastname: Murphy
authors.forename: Ciaran
authors.initials: C
authors.lastname: Breen
authors.forename: Susan
authors.initials: S
authors.lastname: Lynch
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Flood
authors.forename: Shirley
authors.initials: S
authors.lastname: Finnan
authors.forename: Patrick
authors.initials: P
authors.lastname: Caffrey
mesh_terms: D000666:Amphotericin B / Q000096:biosynthesis* / Q000737:chemistry; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D005818:Genetic Engineering; D008958:Models, Molecular; D008968:Molecular Conformation; D011523:Protoplasts / Q000502:physiology; D015183:Restriction Mapping; D017384:Sequence Deletion; D013302:Streptomyces / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D000666:Amphotericin B