Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes.
A Streptomyces pristinaespiralis strain, which produces a streptogramin antibiotic pristinamycin II (PII) as a mixture of two biologically active molecules PIIB and PIIA, was genetically engineered to produce exclusively PIIA. The snaA,B genes, which encode a PIIA synthase that performs oxidation of the precursor (PIIB) to the final product (PIIA), were integrated in the chromosome of S. pristinaespiralis using an integrative derivative of the pSAM2 genetic element from Streptomyces ambofaciens. Integration was due to site-specific recombination at the attB site of S. pristinaespiralis, and no homologous recombination at the snaA,B locus was observed. The attB site of S. pristinaespiralis was sequenced and found to overlap the 3' end of a pro-tRNA gene. The integrants were stable in industrial conditions of pristinamycin production and showed no decrease in PII biosynthesis. Western blot analysis showed a constant production of the PIIA synthase in the overall fermentation process due to expression of the cloned snaA,B genes from the constitutive ermE promoter. This allows the complete conversion of the PIIB form into PIIA.
Sezonov
G
G
Laboratoire de Biologie et Génétique Moléculaire, URA CNRS 2225, Orsay, France. sezonov@igmors.u-psud.fr
Blanc
V
V
Bamas-Jacques
N
N
Friedmann
A
A
Pernodet
J L
JL
Guérineau
M
M
D001287:Attachment Sites, Microbiological / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D014769:Virginiamycin / Q000096:biosynthesis* / Q000737:chemistry
D004269:DNA, Bacterial; D014769:Virginiamycin