DNA sequence and functions of the actVI region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2).
Six open reading frames (ORFs) were identified by DNA sequencing of 5.7 kilobase pairs at the left end of the act cluster (the so-called "actVI region"), in the order: ORFB, ORFA, ORF1, ORF2, ORF3, ORF4. ORF1-4 are transcribed rightward and in the same direction as the ORFs of the actVA region which lies to the right of the actVI region, whereas ORFA and ORFB run in the opposite direction. By complementation of mutants and gene disruption of the wild type strain, the two previously genetically characterized actVI mutations were assigned to ORF1. Although disruption of ORFB and ORF4, using phi C31 derivatives, did not cause any obvious change in actinorhodin production, defects in actinorhodin synthesis were obtained by insertional inactivation of ORFA, ORF1, ORF2, or ORF3. RNA analysis within the ORF1/ORFA intergenic region showed overlapping divergent promoters, at least one of which is under the control of the actII-ORF4 gene product, the transcriptional activator of the act cluster. Data base searches with the deduced products of ORFB and ORF3 failed to show any significant similarities with other known proteins. The deduced product of ORFA strongly resembles those of genes of unknown function from Saccharopolyspora hirsuta and Streptomyces roseofulvus, located within polyketide synthase clusters. The ORF1 product strongly resembles beta-hydroxyacyl-CoA dehydrogenases of bacteria and mammals and the ORF2 and ORF4 products resemble each other and enoyl reductases from bacteria, animals, and plants, with a highly conserved cofactor-binding domain. These findings strongly suggest that the actVI region is involved in catalyzing reduction processes that determine the two stereochemical configurations at C-3/C-15 during actinorhodin biosynthesis. A scheme is proposed for the middle steps of the biosynthesis, that is formation of the pyran ring, leading to the benzoisochromanequinone structure.
Fernández-Moreno
M A
MA
Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma de Madrid, Spain.
Martínez
E
E
Caballero
J L
JL
Ichinose
K
K
Hopwood
D A
DA
Malpartida
F
F
D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D016366:Open Reading Frames; D013302:Streptomyces / Q000235:genetics*; D000095622:Benzoisochromanequinones
D000880:Anthraquinones; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; C013390:actinorhodin; D000095622:Benzoisochromanequinones