Biosynthesis of the antibiotic actinorhodin

The intense blue colour of the actinorhodin complex produced by Streptomyces coelicolor first attracted researchers into close study of this organism and into developing many of the genetic tools required for genetic manipulation of actinomycete bacteria. These genetic studies have reached their pinnacle with the completion of the entire 8.67 Mb genome sequence of this organism (). The gene cluster responsible for the biosynthesis of actinorhodin has long been thought of as the paradigm type II polyketide biosynthetic cluster (, ). Analysis of the products of genetic mutations and sequence similarities to genes in other biosynthetic pathways allowed enzyme activities encoded by the gene cluster to be assigned tentatively (; , 1992; , , ). How ever, difficulties in purifying these enzymes, coupled with inadequate knowledge of the pathways and lack of substrates, mean that few of these assignments have been confirmed with biochemical studies of the proteins. While the numbers of natural product gene clusters identified has increased significantly each year, relatively few protein biochemical studies are published. There are still fewer structural studies involving enzymes from polyketide biosynthetic pathways, with the effort focused mainly on the polyketide synthase enzymes or the analogous proteins from fatty acid biosynthesis: the crystal structure of malonyl-CoA acyltransferase (), the solution structure of the actinorhodin ACP (), the crystal structure of plant chalcone synthases (; ) and the crystal structure of the 6-deoxyerythronolide synthase thioesterase domain (). The erythromycin cytochrome P450 hydroxylase EryF () appears to be the only polyketide ‘tailoring’ enzyme structurally characterized to date.

MetaCyc actinorhodin biosynthesis - BioCyc …

Actinorhodin biosynthesis is negatively ..

Synthetic RNA Silencing of Actinorhodin Biosynthesis …

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification (“tailoring”) steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3β-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic β-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile β-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.

Modulation of Actinorhodin Biosynthesis in …

Heterologous expression of the actinorhodin polyketide synthase in the recombinant host Streptomyces lividans K4-114 led to the characterization of three new minor polyketides, the novel hexaketides BSM1 and BSM3 and 9'-hydroxyaloesaponarin II, in addition to known anthraquinone and aromatic octaketides. The structures of BSM1 and BSM3 imply that these compounds are derived from a C-5-reduced hexaketide intermediate, suggesting that the timing of the ketoreduction reaction in the actinorhodin biosynthetic pathway may take place during the polyketide elongation process rather than after the completion of the octaketide chain as previously suggested.

Biosynthesis of anthraquinones by interspecies cloning …

A DNA fragment from Streptomyces peucetius activates biosynthesis of actinorhodin in Streptomyces lividans and daunomycin in the original strain

Actinorhodin polyketide synthase acyl carrier protein

Actinorhodin (ACT) produced by A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification (“tailoring”) steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by VI-ORF1 reduces the 3β-keto group of a proposed bicyclic intermediate to an () secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its -acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic β-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile β-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.

hmgA , transcriptionally activated by HpdA, influences …

Among the putative AfsR2-target genes tested, the expression of a putative secreted solute binding protein, SCO6569 significantly repressed biosynthesis of actinorhodin in S.