An efficient gene transfer system for the pimaricin producer Streptomyces natalensis.
Streptomyces natalensis produces the antifungal polyene macrolide pimaricin. Genetic manipulation of its biosynthetic genes has been hampered by the lack of efficient gene transfer systems. We have developed a gene transfer system based on intergeneric conjugation from Escherichia coli. Using this approach, we managed to attain transformation efficiencies of 1 x 10(-4) exconjugants per recipient when using self-replicating vectors such as pHZ1358. The use of integrative vectors such as pSET152 or pSOK804 resulted in significantly lower efficiencies. Site-specific integration or the use of self-replicating plasmids did not affect pimaricin production or the essential functions of S. natalensis. Use of DNA methylation proficient E. coli donor strains resulted in no transformants, indicating the presence of methyl-specific restriction systems in S. natalensis. This methodology will enable easier manipulation of the genes responsible for pimaricin biosynthesis, and could prove valuable for the generation of new designer polyene macrolides with better antifungal activity and pharmacological properties. As an example of the validity of the method, we describe the introduction of Supercos-1-derived cosmid vectors into S. natalensis in order to promote gene replacements by double crossover recombination.
Instituto de Biotecnología INBIOTEC, Parque Científico de León, León, Spain.
Lorena L
LL
Enríquez
Marta V
MV
Mendes
Nuria
N
Antón
Sedef
S
Tunca
Susana M
SM
Guerra
Juan F
JF
Martín
Jesús F
JF
Aparicio
D003227:Conjugation, Genetic; D003360:Cosmids; D004926:Escherichia coli / Q000378:metabolism; D018014:Gene Transfer Techniques; D010866:Natamycin / Q000096:biosynthesis; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
D010866:Natamycin