Identification of csypyrone B1 as the novel product of Aspergillus oryzae type III polyketide synthase CsyB

As a novel superfamily of type III polyketide synthases (PKSs) in microbes, four genes, csyA, csyB, csyC, and csyD, were found in the genome of Aspergillus oryzae, an industrially important filamentous fungus. Although orthologs of csyA, csyC, and csyD genes are present in a closely related species, Aspergillus flavus, csyB gene is unique to A. oryzae. To identify its function, we carried out overexpression of csyB gene under the control of α-amylase promoter in A. oryzae. 3-(3-Acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)propanoic acid, named csypyrone B1, was identified as a CsyB product. Feeding experiments of ¹³C-labeled acetate indicated that five acetate units were incorporated into csypyrone B1. Two possible mechanisms are proposed for the biosynthesis of cycpyrone B1: (1) condensation of succinyl-CoA with three acetyl/malonyl-CoAs, and the following pyrone ring cyclization; (2) condensation of butyryl-CoA with three acetyl/malonyl-CoAs, and the following pyrone ring cyclization and side-chain oxidation.     

Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds

Csypyrones B1, B2 and B3 are α-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-¹³C] and [2-¹³C] acetate feeding experiments. ¹³C NMR analyses of the methyl esters of the csypyrone B compounds fed with the ¹³C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus.     

Aspergillus oryzae type III polyketide synthase CsyA is involved in the biosynthesis of 3,5-dihydroxybenzoic acid

As a novel superfamily of type III polyketide synthases in microbes, four genes csyA, csyB, csyC, and csyD, were found in the genome of Aspergillus oryzae, an industrially important filamentous fungus. In order to analyze their functions, we carried out the overexpression of csyA under the control of α-amylase promoter in A. oryzae and identified 3,5-dihydroxybenzoic acid (DHBA) as the major product. Feeding experiments using ¹³C-labeled acetates confirmed that the acetate labeling pattern of DHBA coincided with that of orcinol derived from orsellinic acid, a polyketide formed by the condensation and cyclization of four acetate units. Further oxidation of methyl group of orcinol by the host fungus could lead to the production of DHBA. Comparative molecular modeling of CsyA with the crystal structure of Neurospora crassa 2′-oxoalkylresorcylic acid synthase indicated that CsyA cavity size can only accept short-chain acyl starter and tetraketide formation. Thus, CsyA is considered to be a tetraketide alkyl-resorcinol/resorcylic acid synthase.     

Cloning and characterization of a type III polyketide synthase from Aspergillus niger

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50°C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (k(cat)/K(m)) of 7.4×10(5)s(-1)M(-1) toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.     

Discovery of a novel superfamily of type III polyketide synthases in Aspergillus oryzae

Identification of genes encoding type III polyketide synthase (PKS) superfamily members in the industrially useful filamentous fungus, Aspergillus oryzae, revealed that their distribution is not specific to plants or bacteria. Among other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus), A. oryzae was unique in possessing four chalcone synthase (CHS)-like genes (csyA, csyB, csyC, and csyD). Expression of csyA, csyB, and csyD genes was confirmed by RT-PCR. Comparative genome analyses revealed single putative type III PKS in Neurospora crassa and Fusarium graminearum, two each in Magnaporthe grisea and Podospora anserina, and three in Phenarocheate chrysosporium, with a phylogenic distinction from bacteria and plants. Conservation of catalytic residues in the CHSs across species implicated enzymatically active nature of these newly discovered homologs.     

Biphenyl synthase,a novel type III polyketide synthase

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.     

Identification of a cryptic type III polyketide synthase (1,3,6,8-tetrahydroxynaphthalene synthase) from Streptomyces peucetius ATCC 27952

We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA).     

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

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.     

Identification of unique type II polyketide synthase genes in soil

Many bacteria, particularly actinomycetes, are known to produce secondary metabolites synthesized by polyketide synthases (PKS). Bacterial polyketides are a particularly rich source of bioactive molecules, many of which are of potential pharmaceutical relevance. To directly access PKS gene diversity from soil, we developed degenerate PCR primers for actinomycete type II KS(alpha) (ketosynthase) genes. Twenty-one soil samples were collected from diverse sources in New Jersey, and their bacterial communities were compared by terminal restriction fragment length polymorphism (TRFLP) analysis of PCR products generated using bacterial 16S rRNA gene primers (27F and 1525R) as well as an actinomycete-specific forward primer. The distribution of actinomycetes was highly variable but correlated with the overall bacterial species composition as determined by TRFLP. Two samples were identified to contain a particularly rich and unique actinomycete community based on their TRFLP patterns. The same samples also contained the greatest diversity of KS(alpha) genes as determined by TRFLP analysis of KS(alpha) PCR products. KS(alpha) PCR products from these and three additional samples with interesting TRFLP pattern were cloned, and seven novel clades of KS(alpha) genes were identified. Greatest sequence diversity was observed in a sample containing a moderate number of peaks in its KS(alpha) TRFLP. The nucleotide sequences were between 74 and 81% identical to known sequences in GenBank. One cluster of sequences was most similar to the KS(alpha) involved in ardacin (glycopeptide antibiotic) production by Kibdelosporangium aridum. The remaining sequences showed greatest similarity to the KS(alpha) genes in pathways producing the angucycline-derived antibiotics simocyclinone, pradimicin, and jasomycin.     

Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation.     

Kingdom-wide analysis of the evolution of the plant type III polyketide synthase superfamily

The emergence of type III polyketide synthases (PKSs) was a prerequisite for the conquest of land by the green lineage. Within the PKS superfamily, chalcone synthases (CHSs) provide the entry point reaction to the flavonoid pathway, while LESS ADHESIVE POLLEN 5 and 6 (LAP5/6) provide constituents of the outer exine pollen wall. To study the deep evolutionary history of this key family, we conducted phylogenomic synteny network and phylogenetic analyses of whole-genome data from 126 species spanning the green lineage including Arabidopsis thaliana, tomato (Solanum lycopersicum), and maize (Zea mays). This study thereby combined study of genomic location and context with changes in gene sequences. We found that the two major clades, CHS and LAP5/6 homologs, evolved early by a segmental duplication event prior to the divergence of Bryophytes and Tracheophytes. We propose that the macroevolution of the type III PKS superfamily is governed by whole-genome duplications and triplications. The combined phylogenetic and synteny analyses in this study provide insights into changes in the genomic location and context that are retained for a longer time scale with more recent functional divergence captured by gene sequence alterations.

Phylogenetic and syntenic analyses of whole genome data reveal that macroevolution of the type III polyketide synthase superfamily is mainly governed by whole-genome duplications and triplications.     

Polyketide biosynthesis beyond the type I,II and III polyketide synthase paradigms

Recent literature on polyketide biosynthesis suggests that polyketide synthases have much greater diversity in both mechanism and structure than the current type I, II and III paradigms. These examples serve as an inspiration for searching novel polyketide synthases to give new insights into polyketide biosynthesis and to provide new opportunities for combinatorial biosynthesis.     

植物类型III聚酮合酶超家族基因结构、功能及代谢产物

植物聚酮类化合物主要包括酚类、芪类及类黄酮化合物等,在植物花色、防止紫外线伤害、预防病原菌、昆虫危害以及作为植物与环境互作信号分子方面行使着重要的生物学功能。该类化合物具有显著多样的生物学活性,对人体保健及疾病治疗有显著意义。植物类型III 聚酮化合物合酶 (PKS) 在该类化合物生物合成起始反应中行使着关键作用,决定该类化合物基本分子骨架建成和代谢途径碳硫走向,为合成途径关键酶和限速酶。以查尔酮合酶为原型酶的植物类型III PKS超家族是研究系统进化和蛋白结构与功能关系的模式分子家族,目前已经分离得到14种植物类型III PKS基因,这些同祖同源基因及其表达产物既有共性,也表现出许多独特个性,这些个性赋予此类次生代谢产物结构上的多样性。以下综述了植物类型III PKS超家族基因结构、功能及代谢产物研究进展。     

Pyrone polyketides synthesized by a type III polyketide synthase from Drosophyllum lusitanicum

To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusitanicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The translated polypeptide sequence of DluHKS showed 51-67% identity with other plant type III PKSs. Recombinant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out sequential decarboxylative condensations with malonyl-CoA yielding α-pyrones from three to six acetate units. However, naphthalenes, the expected products, were not isolated. Since the main compound produced by DluHKS is a hexaketide α-pyrone, and the naphthoquinones in D. lusitanicum are composed of six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An involvement of accessory proteins in this biosynthetic pathway is discussed.     

Pentaketide resorcylic acid synthesis by type III polyketide synthase from Neurospora crassa

Type III polyketide synthases (PKSs) are responsible for aromatic polyketide synthesis in plants and bacteria. Genome analysis of filamentous fungi has predicted the presence of fungal type III PKSs, although none have thus far been functionally characterized. In the genome of Neurospora crassa, a single open reading frame, NCU04801.1, annotated as a type III PKS was found. In this report, we demonstrate that NCU04801.1 is a novel type III PKS catalyzing the synthesis of pentaketide alkylresorcylic acids. NCU04801.1, hence named 2'-oxoalkylresorcylic acid synthase (ORAS), preferred stearoyl-CoA as a starter substrate and condensed four molecules of malonyl-CoA to give a pentaketide intermediate. For ORAS to yield pentaketide alkylresorcylic acids, aldol condensation and aromatization of the intermediate, which is still attached to the enzyme, are presumably followed by hydrolysis for release of the product as a resorcylic acid. ORAS is the first type III PKS that synthesizes pentaketide resorcylic acids.     

In vitro synthesis of curcuminoids by type III polyketide synthase from Oryza sativa

Curcuminoids, major components of the spice turmeric, are used as a traditional Asian medicine and a food additive. Curcumin, a representative curcuminoid, has received a great deal of attention because of its anti-inflammatory, anticarcinogenic, and antitumor activities. Here we report a novel type III polyketide synthase named curcuminoid synthase from Oryza sativa, which synthesizes bisdemethoxycurcumin via a unique mechanism from two 4-coumaroyl-CoAs and one malonyl-CoA. The reaction begins with the thioesterification of the thiol moiety of Cys-174 by a starter molecule, 4-coumaroyl-CoA. Decarboxylative condensation of the first extender substrate, malonyl-CoA, onto the thioester of 4-coumarate results in the formation of a diketide-CoA intermediate. Subsequent hydrolysis of the intermediate yields a beta-keto acid, which in turn acts as the second extender substrate. The beta-keto acid is then joined to the Cys-174-bound 4-coumarate by decarboxylative condensation to form bisdemethoxycurcumin. This reaction violates the traditional head-to-tail model of polyketide assembly; the growing diketide intermediate is hydrolyzed to a beta-keto acid that subsequently serves as the second extender to form curcuminoids. Curcuminoid synthase appears to be capable of the synthesis of not only diarylheptanoids but also gingerol analogues, because it synthesized cinnamoyl(hexanoyl)methane, a putative intermediate of gingerol, from cinnamoyl-CoA and 3-oxo-octanoic acid.     

The first plant type III polyketide synthase that catalyzes formation of aromatic heptaketide

A cDNA encoding a novel plant type III polyketide synthase (PKS) was cloned from rhubarb (Rheum palmatum). A recombinant enzyme expressed in Escherichia coli accepted acetyl-CoA as a starter, carried out six successive condensations with malonyl-CoA and subsequent cyclization to yield an aromatic heptaketide, aloesone. The enzyme shares 60% amino acid sequence identity with chalcone synthases (CHSs), and maintains almost identical CoA binding site and catalytic residues conserved in the CHS superfamily enzymes. Further, homology modeling predicted that the 43-kDa protein has the same overall fold as CHS. This provides new insights into the catalytic functions of type III PKSs, and suggests further involvement in the biosynthesis of plant polyketides.     

Role of the active site cysteine of DpgA, a bacterial type III polyketide synthase

DpgA is a bacterial type III polyketide synthase (PKS) that decarboxylates and condenses four malonyl-CoA molecules to produce 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) in the biosynthetic pathway to 3,5-dihydroxyphenylglycine, a key nonproteinogenic residue in the vancomycin family of antibiotics. DpgA has the conserved catalytic triad of Cys/His/Asn typical of type III PKS enzymes, and has been assumed to use Cys160 as the catalytic nucleophile to create a series of elongating acyl-S-enzyme intermediates prior to the C(8) to C(3) cyclization step. Incubation of purified DpgA with [(14)C]-malonyl-CoA followed by acid quench during turnover leads to accumulation of 10-15% of the DpgA molecules covalently acylated. Mutation of the active site Cys160 to Ala abrogated detectable covalent acylation, but the C160A mutant retained 50% of the V(max) for DPA-CoA formation, with a k(cat) still at 0.5 catalytic turnovers/min. For comparison, a C190A mutant retained wild-type activity, while the H296A mutant, in which the side chain of the presumed catalytic His is removed, had a 6-fold drop in k(cat). During turnover, purified DpgA produced 1.2 equivalents of acetyl-CoA for each DPA-CoA, indicating 23% uncoupled decarboxylation competing with condensative C-C coupling. The C160A mutant showed an increased partition ratio for malonyl-CoA decarboxylation to acetyl-CoA vs condensation to DPA-CoA, reflecting more uncoupling in the mutant enzyme. The Cys-to-Ala mutant thus shows the unexpected result that, when the normal acyl-S-enzyme mechanism for this type III PKS elongation/cyclization catalyst is removed, it can still carry out the regioselective construction of the eight-carbon DPA-CoA skeleton with surprising efficiency.