Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora

The biosynthetic origin of 1,2,5,6-tetraoxygenated phenylphenalenones and the sequence according to which their oxygen functionalities are introduced during the biosynthesis in Wachendorfia thyrsiflora were studied using two approaches. (1) Oxygenated phenylpropanoids were probed as substrates of recombinant W. thyrsiflora polyketide synthase 1 (WtPKS1), which is involved in the diarylheptanoid and phenylphenalenone biosynthetic pathways, (2) Root cultures of W. thyrsiflora were incubated with ¹³C-labelled precursors in an ¹⁸O₂ atmosphere to observe incorporation of the two isotopes at defined biosynthetic steps. NMR- and HRESIMS-based analyses were used to unravel the isotopologue composition of the biosynthetic products, lachnanthoside aglycone and its allophanyl glucoside. Current results suggest that the oxygen atoms decorating the phenalenone tricycle are introduced at different biosynthetic stages in the sequence O-1 → O-2 → O-5. In addition, the incubation of W. thyrsiflora root cultures with ¹³C-labelled lachnanthocarpone established a direct biosynthetic precursor–product relationship with 1,2,5,6-tetraoxygenated phenylphenalenones.     

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.     

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.     

Aspergillus oryzae CsyB Catalyzes the Condensation of Two β-Ketoacyl-CoAs to Form 3-Acetyl-4-hydroxy-6-alkyl-α-pyrone

The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C₉–C₁₇ in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs.     

Evolution of metabolic diversity in polyketide-derived pyrones: Using the non-colinear aureothin assembly line as a model system

Polyketide-derived pyrones are structurally diverse secondary metabolites that are represented in all three kingdoms of life and are endowed with various biological functions. The aureothin family of Streptomyces metabolites was chosen as a model to study the factors governing structural diversity and the evolutionary processes involved. This review highlights recent insights into the non-colinear aureothin and neoaureothin modular type I polyketide synthase (PKS), aromatic starter unit biosynthesis, polyketide tailoring reactions, and a non-enzymatic polyene splicing cascade. Pyrone biosynthesis in bacteria, fungi, and plants is compared. Finally, various strategies to increase metabolic diversity of aureothin derivatives through mutasynthesis, pathway engineering, and biotransformation are presented. The unusual aureothin and neoaureothin assembly lines thus not only represent a model for PKS evolution, but provided important insights into non-canonical enzymatic processes that could be employed for the production of antitumor and antifungal agents.     

A comprehensive and engaging overview of the type III family of polyketide synthases

Customizing biosynthesis of natural products to yield biologically active derivatives has captivated scientists in the field of biosynthetic research. To substantiate this goal, there are scores of obstacles to consider. To create novel metabolites by mutating amino acid residues in wild-type enzymes, a researcher must broaden the range of the enzymes substrate tolerance and increase its turnover rate during reaction catalysis. In the past decade, numerous gene clusters responsible for the biosynthesis of notable natural products have been identified from a variety of organisms. Several genes coding for type III polyketide synthases, particularly the chalcone synthase superfamily enzymes, were recently uncovered and expressed in E. coli. Furthermore, it was observed and reported how these recombinant enzymes are capable of producing essential metabolites in vitro. Three of the type III polyketide synthases, chalcone synthase, octaketide synthase and pentaketide chromone synthase, have been characterized and their active sites subjected to rational engineering for biosynthetic production of their analogs. Because they are encoded in a single open reading frame and are post-translationally small in size, type III polyketide synthases are ideal targets for protein engineering. The relative ease with which these genes are expressed makes molecular biological manipulation to obtain mutated enzymes more procurable, ameliorating analysis of its biosynthetic pathway. In summary, time devoted to modification of biosynthetic proteins and unravelling of the detailed reaction mechanisms involved in biosynthesis will be shortened, paving the way for a much wider scope for metabolic engineers in future. This review focuses on the use of chalcone synthase, octaketide synthase and pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds.     

Structure and mechanism of chalcone synthase-like polyketide synthases

Polyketide synthases (PKS) produce an array of natural products with different biological activities and pharmacological properties by varying the starter and extender molecules that form the final polyketide. Recent studies of the simplest PKS, the chalcone synthase (CHS)-like enzymes involved in the biosynthesis of flavonoids, anthocyanin pigments, and antimicrobial phytoalexins, have yielded insight on the molecular basis of this biosynthetic versatility. Understanding the structure–function relationship in these PKS provides a foundation for manipulating polyketide formation and suggests strategies for further increasing the scope of polyketide biosynthetic diversity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 393–398. Received 14 June 2001/ Accepted in revised form 15 July 2001     

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.     

An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae). The deduced amino acid sequence of Hu. serrata polyketide synthase 1 showed 44-66% identity to those of other chalcone synthase superfamily enzymes of plant origin. Further, phylogenetic tree analysis revealed that Hu. serrata polyketide synthase 1 groups with other nonchalcone-producing type III polyketide synthases. Indeed, a recombinant enzyme expressed in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 4-methoxy-2',4',6'-trihydroxychalcone and 1,3-dihydroxy-N-methylacridone, respectively. In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate-binding pocket at the active site. Although acridone alkaloids have not been isolated from Hu. serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non-Rutaceae plant. Interestingly, Hu. serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).     

Oxytetracycline Biosynthesis

Oxytetracycline (OTC) is a broad-spectrum antibiotic that acts by inhibiting protein synthesis in bacteria. It is an important member of the bacterial aromatic polyketide family, which is a structurally diverse class of natural products. OTC is synthesized by a type II polyketide synthase that generates the poly-β-ketone backbone through successive decarboxylative condensation of malonyl-CoA extender units, followed by modifications by cyclases, oxygenases, transferases, and additional tailoring enzymes. Genetic and biochemical studies have illuminated most of the steps involved in the biosynthesis of OTC, which is detailed here as a representative case study in type II polyketide biosynthesis.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

Biosynthesis of Aliphatic Polyketides by Type III Polyketide Synthase and Methyltransferase in Bacillus subtilis

Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.Type III polyketide synthases (PKSs), represented by a plant chalcone synthase (CHS), are the condensing enzymes that catalyze the synthesis of aromatic polyketides in plants, fungi, and bacteria (2). CHS catalyzes the decarboxylative condensation of p-coumaroyl-coenzyme A (p-coumaroyl-CoA), called a starter substrate, with three malonyl-CoAs, called extender substrates, and synthesizing a tetraketide intermediate. The synthesized tetraketide intermediate was cyclized and aromatized by CHS and resulted in naringenin chalcone. Like CHS, most of the type III PKSs catalyze the condensation of a starter substrate with several molecules of an extender substrate and cyclization. There are many type III PKSs that differ in these specificities.Until recently, type III PKSs were discovered only from plants. In 1999, the first bacterial type III PKS, RppA, was discovered. RppA catalyzes the condensation of five malonyl-CoAs to synthesize 1,3,6,8-tetrahydroxynaphthalene, which is a precursor of hexahydroxyperylenequinone melanin in the actinomycete Streptomyces griseus (4). Since then, the genome projects of various bacteria have revealed that type III PKSs are widely distributed in a variety of bacteria. For example, ArsB and ArsC, both of which are type III PKSs in Azotobacter vinelandii, catalyze the synthesis of alkylresorcinols and alkylpyrones, respectively, which are essential for encystment as the major lipids in the cyst membrane (5). In S. griseus, the srs operon consisting of srsA, srsB, and srsC is responsible for the synthesis of methylated phenolic lipids derived from alkylresorcinols and alkylpyrones (6). The function of each of the operon members is that SrsA is a type III PKS responsible for the synthesis of phenolic lipids alkylresorcinol and alkylpyrones, SrsB is a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC is a hydroxylase acting on the alkylresorcinol methyl ethers. The phenolic lipids synthesized by the Srs enzymes confer resistance to β-lactam antibiotics (6). Therefore, it is suggested that phenolic lipids play an important role as minor components in the biological membrane in various bacteria. In fact, srsAB- and srsABC-like operons are distributed widely in both gram-positive and -negative bacteria (see Fig. S1 in the supplemental material). However, most of these type III PKSs have not been characterized.Bacillus subtilis is one of the best-characterized gram-positive bacteria. BcsA, which stands for bacterial chalcone synthase, was annotated as a homologue of type III PKS in B. subtilis (3). As described in this paper, however, this annotation needs correction. We renamed the gene bpsA (for Bacillus pyrone synthase). Moreover, the functional unknown gene ypbQ is located next to bpsA. YpbQ, consisting of 168 amino acid residues, contained an isoprenylcysteine carboxyl methyltransferase (ICMT) domain of the ICMT family members, which are unique membrane proteins that are involved in the posttranslational modification of oncogenic proteins (23). Therefore, the bpsA and ypbQ genes were predicted to form an operon, just like srsA and srsB in the srs operon in S. griseus. We therefore named ypbQ, a thus-far functionally unknown gene, bpsB.In this study, we characterized the functions of BpsA and BpsB by in vivo and in vitro experiments. The in vivo experiments revealed that the overexpression of bpsA in B. subtilis led to the production of triketide pyrones, and the co-overexpression of bpsA and bpsB led to the production of triketide pyrone methyl ethers. The in vitro analysis showed that BpsA produced triketide pyrones and a small amount of tetraketide pyrones and tetraketide resorcinols from long-chain fatty acyl CoA thioesters as starter substrates and malonyl-CoA as an extender substrate. Therefore, BpsA is a type III PKS that is responsible for the synthesis of alkylpyrones, and BpsB is a methyltransferase that acts on the alkylpyrones to yield alkylpyrone methyl ethers. BpsB is the first enzyme found to methylate alkylpyrones. Furthermore, we attempted to analyze the biological function of the aliphatic polyketides by disrupting the bpsA and bpsB genes, but no distinct phenotypic changes were detected under laboratory conditions.     

Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins

Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.     

Characterization of an alkylresorcinol synthase that forms phenolics accumulating in the cuticular wax on various organs of rye (Secale cereale)

Alkylresorcinols are bioactive compounds produced in diverse plant species, with chemical structures combining an aliphatic hydrocarbon chain and an aromatic ring with characteristic hydroxyl substituents. Here, we aimed to isolate and characterize the enzyme that forms the alkylresorcinols accumulating in the cuticular wax on the surface of all above‐ground organs of rye. Based on sequence homology with other type‐III polyketide synthases, a candidate alkylresorcinol synthase was cloned. Yeast heterologous expression showed that the enzyme, ScARS, is highly specific for the formation of the aromatic resorcinol ring structure, through aldol condensation analogous to stilbene synthases. The enzyme accepts long‐chain and very‐long‐chain acyl‐CoA starter substrates, preferring saturated over unsaturated chains. It typically carries out three rounds of condensation with malonyl‐CoA prior to cyclization, with only very minor activity for a fourth round of malonyl‐CoA condensation and cyclization to 5‐(2′‐oxo)‐alkylresorcinols or 5‐(2′‐hydroxy)‐alkylresorcinols. Like other enzymes involved in cuticle formation, ScARS is localized to the endoplasmic reticulum. ScARS expression patterns were found correlated with alkylresorcinol accumulation during leaf development and across different rye organs. Overall, our results thus suggest that ScARS synthesizes the cuticular alkylresorcinols found on diverse rye organ surfaces.     

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incanaR. Br. (Brassicaceae)

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.     

Biosynthesis of biphenyls and benzophenones – Evolution of benzoic acid-specific type III polyketide synthases in plants

Type III polyketide synthases (PKSs) generate a diverse array of secondary metabolites by varying the starter substrate, the number of condensation reactions, and the mechanism of ring closure. Among the starter substrates used, benzoyl-CoA is a rare starter molecule. Biphenyl synthase (BIS) and benzophenone synthase (BPS) catalyze the formation of identical linear tetraketide intermediates from benzoyl-CoA and three molecules of malonyl-CoA but use alternative intramolecular cyclization reactions to form 3,5-dihydroxybiphenyl and 2,4,6-trihydroxybenzophenone, respectively. In a phylogenetic tree, BIS and BPS group together closely, indicating that they arise from a relatively recent functional diversification of a common ancestral gene. The functionally diverse PKSs, which include BIS and BPS, and the ubiquitously distributed chalcone synthases (CHSs) form separate clusters, which originate from a gene duplication event prior to the speciation of the angiosperms. BIS is the key enzyme of biphenyl metabolism. Biphenyls and the related dibenzofurans are the phytoalexins of the Maloideae. This subfamily of the Rosaceae includes a number of economically important fruit trees, such as apple and pear. When incubated with ortho-hydroxybenzoyl (salicoyl)-CoA, BIS catalyzes a single decarboxylative condensation with malonyl-CoA to form 4-hydroxycoumarin. A well-known anticoagulant derivative of this enzymatic product is dicoumarol. Elicitor-treated cell cultures of Sorbus aucuparia also formed 4-hydroxycoumarin when fed with the N-acetylcysteamine thioester of salicylic acid (salicoyl-NAC). BPS is the key enzyme of benzophenone metabolism. Polyprenylated benzophenone derivatives with bridged polycyclic skeletons are widely distributed in the Clusiaceae (Guttiferae). Xanthones are regioselectively cyclized benzophenone derivatives. BPS was converted into a functional phenylpyrone synthase (PPS) by a single amino acid substitution in the initiation/elongation cavity. The functional behavior of this Thr135Leu mutant was rationalized by homology modeling. The intermediate triketide may be redirected into a smaller pocket in the active site cavity, resulting in phenylpyrone formation by lactonization.     

The Streptomyces peucetius dpsC Gene Determines the Choice of Starter Unit in Biosynthesis of the Daunorubicin Polyketide

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of Streptomyces peucetius, directly downstream of the genes encoding the beta-ketoacyl:acyl carrier protein synthase subunits, are two genes, dpsC and dpsD, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying dpsC and dpsD, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by dpsC and dpsD play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.     

LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.