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

Abstract

SummaryThe 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 pigmentproducing (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 CSlike 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 investi-

Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338

“…Detoxification is a key function for soil bacteria, and S. erythraea like the previously sequenced streptomycetes has numerous genes for transporters mediating resistance to various heavy metals, as well as a substantial cohort (36) of cytochrome P450 enzymes. Some of these have known roles in specific hydroxylation steps during biosynthesis of erythromycin and other secondary metabolites [21][22][23] , but others are predicted to play a role in selective oxidation and detoxification of organic materials.…”

“…The gene clusters for erythromycin (ery) 121 , and for a second modular polyketide synthase (PKS) of unknown function (pke) 22 , have been previously analyzed, as has the gene (SACE_1243, rppA) for a type III PKS, which generates the reddish pigment typical of S. erythraea 23 .…”

Crystal Structure of a Bacterial Type III Polyketide Synthase and Enzymatic Control of Reactive Polyketide Intermediates

“…These additional residues may facilitate in vivo protein-protein interactions with upstream and downstream enzymes, including the P450 or ORF3 proteins encoded next to the THNS gene in several gene clusters obtained from Streptomyces species (2,6). The elimination of this C-terminal extension was previously shown to have no detrimental effect upon the in vitro activity of S. coelicolor THNS (8).…”

“…Whereas type I PKSs and their fatty acid synthase ancestors have been intensely studied, their three-dimensional domain organization remains obscure due to their size and complexity. In stark contrast, bacteria use a structurally simple type III PKS architecturally organized as a homodimeric condensing enzyme (ϳ40 kDa) to synthesize THN from five coenzyme A (CoA)-tethered malonyl units, without the use of additional enzymes or catalytic domains (5)(6)(7)(8).…”

Discovery of Unusual Biaryl Polyketides by Activation of a SilentStreptomyces venezuelaeBiosynthetic Gene Cluster

“…They are multifunctional enzymes that catalyse elongation of diverse acyl‐CoA starter units with one or more malonyl‐CoA extender units to form poly‐β‐ketoacyl‐CoA intermediates that undergo a range of cyclisation reactions to form diverse aromatic products 4, 7, 11. This type of PKS is exemplified by RppA, which assembles 1,3,6,8‐tetrahydroxynaphthalene (THN) in several Streptomyces species 12, 13, 14, 15…”