Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea

• Published in: Molecular microbiology 2002-06, Vol.44 (5), p.1213-1224
• Main Authors: Cortés, Jesús, Velasco, Javier, Foster, Graham, Blackaby, Andrew P., Rudd, Brian A. M., Wilkinson, Barrie
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cloning, Molecular; DNA Primers; Molecular Sequence Data; Molecular Structure; Multienzyme Complexes - classification; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mutation; Naphthols - metabolism; Naphthoquinones - chemistry; Naphthoquinones - metabolism; Operon - genetics; Phenotype; Phylogeny; Pigments, Biological - biosynthesis; Pigments, Biological - chemistry; Pigments, Biological - genetics; Saccharopolyspora - genetics; Saccharopolyspora - metabolism
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.2002.02975.x

Summary The soluble, diffusible red‐brown pigment produced by a Saccharopolyspora erythraea‘red variant’ has been shown to contain glycosylated and polymerized derivatives of 2,5,7‐trihydroxy‐1,4‐naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8‐tetrahydroxynaphthalene (THN), which is biosynthesized in bacteria by a chalcone synthase‐like (CS‐like) type III polyketide synthase (PKS). A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8‐7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS‐like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red‐brown pigment‐producing (rpp) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector‐recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CS‐like protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS‐like protein, which is sufficient in vitro for the synthesis of THN from malonyl‐CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investigate any link between this and the modulation of erythromycin A titre, which has been observed for S. erythraea variants.