malonyl-coenzyme-a and 1-3-6-8-tetrahydroxynaphthalene

Heterologous hosts are important platforms for engineering natural product biosynthesis. Escherichia coli is such a host widely used for expression of various biosynthetic enzymes. While numerous studies have been focused on optimizing the expression conditions for desired functional proteins, this work describes how supplement of exogenous nutrients into the fermentation broth influences the formation of natural products in E. coli. A type III polyketide synthase gene stts from Streptomyces toxytricini NRRL 15443 was heterogeneously expressed in E. coli BL21(DE3). This enzyme uses five units of malonyl-CoA to generate a polyketide 1,3,6,8-tetrahydroxynaphthalene, which can be spontaneously oxidized into a red compound flaviolin. In this work, we manipulated the fermentation broth of E. coli BL21(DE3)/pET28a-stts by supplying different nutrients including glucose and sodium pyruvate at different concentrations, from which six flaviolin derivatives 1-6 were produced. While addition of glucose yielded the production of 1-4, supplement of sodium pyruvate into the induced broth of E. coli BL21(DE3)/pET28a-stts resulted in the synthesis of 5 and 6, suggesting that different nutrients may enable E. coli to generate different metabolites. These products were purified and structurally characterized based on the spectral data, among which 2-6 are novel compounds. These molecules were formed through addition of different moieties such as acetone and indole to the flaviolin scaffold. The concentrations of glucose and sodium pyruvate and incubation time affect the product profiles. This work demonstrates that supplement of nutrients can link certain intracellular metabolites to the engineered biosynthetic pathway to yield new products. It provides a new approach to biosynthesizing novel molecules in the commonly used heterologous host E. coli.

Topics: Acyltransferases; Biosynthetic Pathways; Culture Media; Escherichia coli; Fermentation; Glucose; Malonyl Coenzyme A; Naphthols; Naphthoquinones; Polyketides; Pyruvic Acid; Streptomyces

Streptomyces coelicolor RppA (Sc-RppA), a bacterial type III polyketide synthase, utilizes malonyl-CoA as both starter and extender unit substrate to form 1,3,6,8-tetrahydroxynaphthalene (THN) (therefore RppA is also known as THN synthase (THNS)). The significance of the active site Tyr(224) for substrate specificity has been established previously, and its aromatic ring is believed to be essential for RppA to select malonyl-CoA as starter unit. Herein, we describe a series of Tyr(224) mutants of Sc-RppA including Y224F, Y224L, Y224C, Y224M, and Y224A that were able to catalyze a physiological assembly of THN, albeit with lower efficiency, challenging the necessity for the Tyr(224) aromatic ring. Steady-state kinetics and radioactive substrate binding analysis of the mutant enzymes corroborated these unexpected results. Functional examination of the Tyr(224) series of RppA mutants using diverse unnatural acyl-CoA substrates revealed the unique role of malonyl-CoA as starter unit substrate for RppA, leading to the development of a novel stericelectronic constraint model.

Topics: Amino Acid Substitution; Bacterial Proteins; Malonyl Coenzyme A; Naphthols; Polyketide Synthases; Streptomyces coelicolor; Substrate Specificity; Tyrosine

RppA is a type III polyketide synthase (PKS) that catalyzes condensation of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene (THN). In Streptomyces antibioticus IFO13271 and several other Streptomyces species, an open reading frame, named momA, is present as a neighbor of rppA. MomA belonged to the "cupin" superfamily because it contained a set of two motifs that is responsible for binding one equivalent of metal ions. MomA catalyzed monooxygenation of the THN produced from malonyl-CoA by the action of RppA to form flaviolin. In addition, it used several polyketides as substrates and formed the corresponding quinones. MomA required redox-active transition metal ions (Ni(2+), Cu(2+), Fe(3+), Fe(2+), Mn(2+), and Co(2+)) for its activity, whereas it was inhibited by a redox-inert transition metal ion (Zn(2+)). MomA neither possessed any flavin prosthetic group nor required nicotinamide cofactors for monooxygenation, which shows that MomA as a member of the cupin superfamily is a novel monooxygenase. Consistent with the catalytic property of MomA, WhiE-ORFII showing similarity in amino acid sequence to MomA and containing a cupin domain also catalyzed monooxygenation of THN. whiE-ORFII is located immediately upstream of the "minimal PKS" gene within the whiE type II PKS gene cluster for biosynthesis of a gray spore pigment in Streptomyces coelicolor A3(2), and a number of whiE-ORFII homologues are present in the biosynthetic gene cluster for polyketides of type II in various Streptomyces species. These findings show that a novel class of quinone-forming monooxygenases is involved in modification of aromatic polyketides synthesized by PKSs of types II and III.

Topics: Amino Acid Sequence; Blotting, Southern; Catalysis; Chromatography, Gel; Chromatography, High Pressure Liquid; Databases as Topic; Dimerization; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Hydrogen-Ion Concentration; Kinetics; Malonyl Coenzyme A; Mixed Function Oxygenases; Models, Chemical; Models, Genetic; Molecular Sequence Data; Multigene Family; Naphthols; Open Reading Frames; Oxidation-Reduction; Plasmids; Polyketide Synthases; Quinones; Recombinant Proteins; Sequence Homology, Amino Acid; Streptomyces; Streptomyces lividans; Temperature; Time Factors

The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, (14)C-labeled acetyl CoA and/or (14)C-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of (14)C label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.

Topics: Aspergillus oryzae; Cell-Free System; Colletotrichum; Electrophoresis, Polyacrylamide Gel; Fungal Proteins; Malonyl Coenzyme A; Multienzyme Complexes; Naphthols; Naphthoquinones; Recombinant Proteins; Sodium Dodecyl Sulfate

Chalcone synthases, which biosynthesize chalcones (the starting materials for many flavonoids), have been believed to be specific to plants. However, the rppA gene from the Gram-positive, soil-living filamentous bacterium Streptomyces griseus encodes a 372-amino-acid protein that shows significant similarity to chalcone synthases. Several rppA-like genes are known, but their functions and catalytic properties have not been described. Here we show that a homodimer of RppA catalyses polyketide synthesis: it selects malonyl-coenzyme-A as the starter, carries out four successive extensions and releases the resulting pentaketide to cyclize to 1,3,6,8-tetrahydroxynaphthalene (THN). Site-directed mutagenesis revealed that, as in other chalcone synthases, a cysteine residue is essential for enzyme activity. Disruption of the chromosomal rppA gene in S. griseus abolished melanin production in hyphae, resulting in 'albino' mycelium. THN was readily oxidized to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin), which then randomly polymerized to form various coloured compounds. THN formed by RppA appears to be an intermediate in the biosynthetic pathways for not only melanins but also various secondary metabolites containing a naphthoquinone ring. Therefore, RppA is a chalcone-synthase-related synthase that synthesizes polyketides and is found in the Streptomyces and other bacteria.

Topics: Acyltransferases; Catalysis; Cloning, Molecular; Escherichia coli; Furans; Malonyl Coenzyme A; Molecular Sequence Data; Mutagenesis, Site-Directed; Naphthols; Streptomyces griseus