Development of a self-cloning system for Actinomadura verrucosospora and identification of polyketide synthase genes essential for production of the angucyclic antibiotic pradimicin.
A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 10(5) CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.
Dairi
T
T
Biotechnology Research Center, Toyama Prefectural University, Kurokawa 5180, Kosugi, Toyama 939-0398, Japan. dairi@pu-toyama.ac.jp
Hamano
Y
Y
Furumai
T
T
Oki
T
T
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D000595:Amino Acid Sequence; D018943:Anthracyclines; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis*; D000935:Antifungal Agents / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics; D011523:Protoplasts; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial; D000095643:Pradimicins and Benanomicins
D018943:Anthracyclines; D000903:Antibiotics, Antineoplastic; D000935:Antifungal Agents; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; C058036:pradimicin A; D000095643:Pradimicins and Benanomicins