The clavulanic acid biosynthetic cluster of Streptomyces clavuligerus: genetic organization of the region upstream of the car gene.
The genetic organization of the region upstream of the car gene of the clavulanic acid biosynthetic gene cluster of Streptomyces clavuligerus has been determined. Sequence analysis of a 12.1 kb region revealed the presence of 10 ORFs whose putative functions, according to database searches, are discussed. Three co-transcriptional units are proposed: ORF10-11, ORF12-13 and ORF15-16-17-18. Potential transcriptional terminators were identified downstream of ORF11 (fd) and ORF15. Targeted disruption of ORF10 (cyp) gave rise to transformants unable to produce clavulanic acid, but with a considerably higher production of cephamycin C. Transformants inactivated at ORF14 had a remarkably lower production of clavulanic acid and similar production of cephamycin C. Significant improvements of clavulanic acid production, associated with a drop in cephamycin C biosynthesis, were obtained with transformants of S. clavuligerus harbouring multiple copies of plasmids carrying different constructions from the ORF10-14 region. This information can be used to guide strain improvement programs, blending random mutagenesis and molecular cloning, to optimize the yield of clavulanic acid.
Mellado
Encarnación
E
Área de Biotecnologı́a, Antibióticos SA, Avenida de Antibióticos 59-61, 24009 León, Spain1.
Lorenzana
Luis Miguel
LM
Área de Microbiologı́a, Facultad de Biologı́a, Universidad de León, 24071 León, Spain2.
Rodrı Guez-Sáiz
Marta
M
Área de Biotecnologı́a, Antibióticos SA, Avenida de Antibióticos 59-61, 24009 León, Spain1.
Dı Ez
Bruno
B
Área de Biotecnologı́a, Antibióticos SA, Avenida de Antibióticos 59-61, 24009 León, Spain1.
Liras
Paloma
P
Área de Microbiologı́a, Facultad de Biologı́a, Universidad de León, 24071 León, Spain2.
Barredo
José Luis
JL
Área de Biotecnologı́a, Antibióticos SA, Avenida de Antibióticos 59-61, 24009 León, Spain1.
D001483:Base Sequence; D002513:Cephamycins / Q000096:biosynthesis; D019818:Clavulanic Acid / Q000096:biosynthesis*; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D023061:Gene Order; D005798:Genes, Bacterial / Q000235:genetics*; D015698:Genomic Library; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014169:Transformation, Bacterial
D002513:Cephamycins; D019818:Clavulanic Acid