Enzymatic formation of an aromatic dodecaketide by engineered plant polyketide synthase

Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase (PKS) that catalyzes iterative condensations of eight molecules of malonyl-CoA to produce the C₁₆ aromatic octaketides SEK4 and SEK4b. On the basis of the crystal structures of OKS, the F66L/N222G double mutant was constructed and shown to produce an unnatural dodecaketide TW95a by sequential condensations of 12 molecules of malonyl-CoA. The C₂₄ naphthophenone TW95a is a product of the minimal type II PKS (whiE from Streptomyces coelicolor), and is structurally related to the C₂₀ decaketide benzophenone SEK15, the product of the OKS N222G point mutant. The C₂₄ dodecaketide naphthophenone TW95a is the first and the longest polyketide scaffold generated by a structurally simple type III PKS. A homology model predicted that the active-site cavity volume of the F66L/N222G mutant is increased to 748 Å³, from 652 Å³ of the wild-type OKS. The structure-based engineering thus greatly expanded the catalytic repertoire of the simple type III PKS to further produce larger and more complex polyketide molecules.     

Flavanone synthase from cell suspension cultures of Haplopappus gracilis and comparison with the synthase from parsley

Flavanone synthase was isolated and purified ca 62-fold from cell suspension cultures of Haplopappus gracilis. The enzyme preparation catalysed the formation of naringenin from 4-coumaryl-CoA and malonyl-CoA with a pH optimum of ca 8. The same enzyme was also capable of synthesizing eriodictyol from caffeyl-CoA and malonyl-CoA; in this case the pH optimum lay between 6.5 and 7. The homogeneous flavanone synthase from cell suspension cultures of parsley showed the same dependence of the pH optimum on the nature of the cinnamyl-CoA. It can be concluded that both naringenin and eriodictyol are natural products of the synthase reaction.     

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis

1. [¹⁴C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO₃⁻. In a cell-free yeast preparation addition of HCO₃⁻ stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of β-hydroxy-β-methylglutaryl-CoA formed from [2-¹⁴C]- and [1,3-¹⁴C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-¹⁴C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-¹⁴C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.     

Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves

Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C_18:0 and C_16:0 were synthesized in equal proportion. With the epidermal FAS system when either acetyl-CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts.

The properties of malonyl-CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the K_m values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that β-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell.

     

Biphenyl synthase from yeast-extract-treated cell cultures of<Emphasis Type="Italic"> Sorbus aucuparia</Emphasis>

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The biphenyl aucuparin accumulated in Sorbus aucuparia L. cell cultures in response to yeast extract treatment. Incubation of cell-free extracts from challenged cell cultures with benzoyl-CoA and malonyl-CoA led to the formation of 3,5-dihydroxybiphenyl. This reaction was catalysed by a novel polyketide synthase, which will be named biphenyl synthase. The most efficient starter substrate for the enzyme was benzoyl-CoA. Relatively high activity was also observed with 2-hydroxybenzoyl-CoA but, instead of the corresponding biphenyl, the derailment product 2-hydroxybenzoyltriacetic acid lactone was formed.Abbreviations BIS biphenyl synthase - BPS benzophenone synthase - DTT dithiothreitol     

Enzymatic formation of intermediates in the biosynthests of ajmalicine: Strictosidine and cathenamine

Pre-purified enzymes isolated from Catharanthus roseus suspension cultures synthesize strictosidine and cathenamine from tryptamine and secologanin. Whereas strictosidine showed metabolic activity, cathenamine accumulates during the cell-free incubations in the absence of reduced pyridine nucleotides. In the presence of δ-d-gluconolactone (0.1 M), strictosidine accumulates in a yield of ca 50%. Optimum conditions for its accumulation in crude extracts were found to be at pH 4.1, 0.25 mM tryptamine and 1.25 mM secologinin. Strictosidine synthase is stable for more than 1.5 months at 4°. The optimum conditions for the enzymatic synthesis of cathenamine are 1.54 mM tryptamine and 7.7 mM secologanin at pH 7.5. In the presence of NH₄⁺ the formation of the latter alkaloid decreases due to the synthesis of unidentified compounds.     

A microsomal fatty acid synthetase coupled to acyl-CoA reductase in Euglena gracilis

Microsomal particles from dark-grown Euglena gracilis incorporated malonyl-CoA into fatty acids and fatty alcohols in the presence of acetyl-CoA, NADH, NADPH, and ATP with an optimum pH of 8.0. Schmidt degradation of the individual fatty acids derived from [l,3-¹⁴C]malonyl-CoA showed that the microsomal fatty acid synthesis was a de novo type. Detailed analysis of the products formed in the absence of various cofactors showed that the role of ATP was specifically in the formation of fatty alcohols and that fatty acid reduction specifically required NADH.The major aliphatic chains synthesized by the microsomes were C₁₆, C₁₈, and C₁₄ in both the acyl portions and alcohols. Although relative concentrations of acetyl-CoA and malonyl-CoA influenced the chain length distribution of products, C₁₆remained the major product in both the alcohol and the acid fractions. Effects of NADPH and NADH concentrations on malonyl-CoA incorporation suggested that the two reductive steps involved in the microsomal fatty acid synthesis have different pyridine nucleotide specificity. The apparent K_m for malonyl-CoA was 4.2 × 10^?4m. Based on the experimental results a mechanism is suggested by which carbon is channeled into wax esters under conditions of nutritional abundance in dark-grown E. gracilis.     

Localization of ATPase protein in sarcoplasmic reticulum membrane.

The substrate specificity of an extensively purified flavanone synthase from light-induced cell suspension cultures of Petroselinum hortense was investigated. p-Coumaroyl-CoA was found to be the only efficient substrate for flavanone synthesis, producing naringenin (5,7,4′-trihydroxyflavanone). Besides 4-hydroxy-6[4-hydroxystyryl]2-pyrone (F. Kreuzaler and K. Hahlbrock (1975) Arch. Biochem. Biophys.169, 84–90) two further release products of the synthase reaction in vitro were identified as 4-hydroxy-5,6-dihydro-6(4-hydroxyphenyl)2-pyrone and p-hydroxybenzalacetone. The apparent K_m values for malonyl-CoA and p-coumaroyl-CoA in the reaction leading to naringenin, and for p-coumaroyl-CoA in the reaction leading to the styrylpyrone derivative were 35, 1.6, and 2.6 μm, respectively. With caffeoyl-CoA as substrate only a very small amount of eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) was formed besides relatively large amounts of the corresponding styrylpyrone, dihydropyrone, and benzalacetone derivatives. No flavanone formation was observed with feruloyl-CoA as substrate, but again appreciable amounts of the three types of short-chain release products were formed. No reaction at all took place with cinnamoyl-CoA, p-methoxycinnamoyl-CoA, isoferuloyl-CoA, or p-hydroxybenzoyl-CoA.None of the styrylpyrone, dihydropyrone, and benzalacetone derivatives has been detected in the cell cultures in vivo. The present results suggest that naringenin is the only natural product of the synthase reaction and that further substitution in the B-ring of the flavonoids occurs in parsley at or after the flavanone stage. The nature of the smaller release products is consistent with the assumption of a stepwise addition of acetate units from malonyl-CoA to the acyl moiety of the starter molecule, p-coumaroyl-CoA.     

Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering

Escherichia coli only maintains a small amount of cellular malonyl-CoA, impeding its utility for overproducing natural products such as polyketides and flavonoids. Here, we report the use of various metabolic engineering strategies to redirect the carbon flux inside E. coli to pathways responsible for the generation of malonyl-CoA. Overexpression of acetyl-CoA carboxylase (Acc) resulted in 3-fold increase in cellular malonyl-CoA concentration. More importantly, overexpression of Acc showed a synergistic effect with increased acetyl-CoA availability, which was achieved by deletion of competing pathways leading to the byproducts acetate and ethanol as well as overexpression of an acetate assimilation enzyme. These engineering efforts led to the creation of an E. coli strain with 15-fold elevated cellular malonyl-CoA level. To demonstrate its utility, this engineered E. coli strain was used to produce an important polyketide, phloroglucinol, and showed near 4-fold higher titer compared with wild-type E. coli, despite the toxicity of phloroglucinol to cell growth. This engineered E. coli strain with elevated cellular malonyl-CoA level should be highly useful for improved production of important natural products where the cellular malonyl-CoA level is rate-limiting.     

Crystal structure of the acyltransferase domain of the iterative polyketide synthase in enediyne biosynthesis

Biosynthesis of the enediyne natural product dynemicin in Micromonospora chersina is initiated by DynE8, a highly reducing iterative type I polyketide synthase that assembles polyketide intermediates from the acetate units derived solely from malonyl-CoA. To understand the substrate specificity and the evolutionary relationship between the acyltransferase (AT) domains of DynE8, fatty acid synthase, and modular polyketide synthases, we overexpressed a 44-kDa fragment of DynE8 (hereafter named AT_DYN10) encompassing its entire AT domain and the adjacent linker domain. The crystal structure at 1.4 Å resolution unveils a α/β hydrolase and a ferredoxin-like subdomain with the Ser-His catalytic dyad located in the cleft between the two subdomains. The linker domain also adopts a α/β fold abutting the AT catalytic domain. Co-crystallization with malonyl-CoA yielded a malonyl-enzyme covalent complex that most likely represents the acyl-enzyme intermediate. The structure explains the preference for malonyl-CoA with a conserved arginine orienting the carboxylate group of malonate and several nonpolar residues that preclude α-alkyl malonyl-CoA binding. Co-crystallization with acetyl-CoA revealed two noncovalently bound acetates generated by the enzymatic hydrolysis of acetyl-CoA that acts as an inhibitor for DynE8. This suggests that the AT domain can upload the acyl groups from either malonyl-CoA or acetyl-CoA onto the catalytic Ser⁶⁵¹ residue. However, although the malonyl group can be transferred to the acyl carrier protein domain, transfer of the acetyl group to the acyl carrier protein domain is suppressed. Local structural differences may account for the different stability of the acyl-enzyme intermediates.     

Anaerobic degradation of malonatevia malonyl-CoA bySporomusa malonica,Klebsiella oxytoca,andRhodobacter capsulatus

Anaerobic decarboxylation of malonate to acetate was studied withSporomusa malonica, Klebsiella oxytoca, andRhodobacter capsulatus. WhereasS. malonica could grow with malonate as sole substrate (Y=2.0 g·mol^–1), malonate decarboxylation byK. oxytoca was coupled with anaerobic growth only in the presence of a cosubstrate, e.g. sucrose or yeast extract (Y _s =1.1–1.8 g·mol malonate^–1).R. capsulatus used malonate anaerobically only in the light, and growth yields with acetate and malonate were identical. Malonate decarboxylation in cell-free extracts of all three bacteria was stimulated by catalytic amounts of malonyl-CoA, acetyl-CoA, or Coenzyme A plus ATP, indicating that actually malonyl-CoA was the substrate of decarboxylation. Less than 5% of malonyl-CoA decarboxylase activity was found associated with the cytoplasmic membrane. Avidin (except forK. oxytoca) and hydroxylamine inhibited the enzyme completely, EDTA inhibited partially. InS. malonica andK. oxytoca, malonyl-CoA decarboxylase was active only after growth with malonate; malonyl-CoA: acetate CoA transferase was found as well. These results indicate that malonate fermentation by these bacteria proceedsvia malonyl-CoA mediated by a CoA transferase and that subsequent decarboxylation to acetyl-CoA is catalyzed, at least withS. malonica andR. capsulatus, by a biotin enzyme.Abbreviations CoASH Coenzyme A - EDTA ethylenediamine tetraacetate     

Enzymatic synthesis of aromatic compounds in higher plants. Formation of bis-noryangonin (4-hydroxy-6[4-hydroxystyryl]2-pyrone) from p-coumaroyl-CoA and malonyl-CoA.

Cell-free extracts from light-induced cell suspension cultures of Petroselinum hortense catalyzed, in the presence of mercaptoethanol or dithioerythritol, the formation of bisnoryangonin from p-coumaroyl-CoA and malonyl-CoA. Radioactivity from the ³H- and ¹⁴C-labeled acyl moieties of p-coumaroyl-CoA and malonyl-CoA, respectively, was incorporated into the product at a molar ratio of 1:2. This result supports earlier conclusions from experiments in vivo favoring a mechanism of synthesis for the pyrone ring of bisnoryangonin according to the “acetate rule.”Bis-noryangonin could not be detected in cultured Petroselinum hortense cells in vivo. Our present results suggest that the styrylpyrone derivative formed in vitro is an artificial product of the first enzyme of the flavonoid pathway, flavanone synthetase. In the course of a 300-fold purification of this enzyme, the bis-noryangonin-synthesizing activity was always associated with the flavanone synthetase activity. The concentration of certain thiol reagents, such as mercaptoethanol or dithioerythritol, the ionic strength of the buffer, and the degree of purity of the enzyme preparation had a pronounced, differential effect on the amounts of flavanone and styrylpyrone formed by the flavanone synthetase. A possible explanation for the mechanism of formation of the artificial product, bis-noryangonin, is discussed.     

Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: Release of the products as free fatty acids

In marine bacteria and some thraustochytrids (marine stramenopiles) long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are produced de novo by PUFA synthases. These large, multi-domain enzymes carry out the multitude of individual reactions required for conversion of malonyl-CoA to the final LC-PUFA products. Here we report on the release of fatty acids from the PUFA synthase found in Schizochytrium, a thraustochytrid that has been developed as a commercial source for DHA-enriched biomass and oil. Data from in vitro activity assays indicate that the PUFAs are released from the enzyme as free fatty acids (FFAs). Addition of ATP and Mg²⁺ to in vitro assays facilitates appearance of radiolabel from ¹⁴C-malonyl-CoA in a triacylglycerol fraction, suggesting the involvement of acyl-CoA synthetases (ACS). Furthermore, addition of triascin C, an inhibitor of ACSs, to the assays blocks this conversion. When the Schizochytrium PUFA synthase is expressed in Escherichia coli, the products of the enzyme accumulate as FFAs, suggesting that the thioesterase activity required for fatty acid release is an integral part of the PUFA synthase.     

A novel 4-hydroxycoumarin biosynthetic pathway

Coumarin forms in melilotoside (trans-ortho-coumaric acid glucoside)-containing plant species upon cell damage. In moldy melilotoside-containing plant material, trans-ortho-coumaric acid is converted by fungi to 4-hydroxycoumarin, two molecules of which spontaneously combine with formaldehyde to give dicoumarol. Dicoumarol causes internal bleeding in livestock and is the forerunner of the warfarin group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from elicitor-treated Sorbus aucuparia cell cultures. The encoded isoenzymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter substrate and catalyzed a single decarboxylative condensation with malonyl-CoA to give 4-hydroxycoumarin. When elicitor-treated S. aucuparia cell cultures were fed with the N-acetylcysteamine thioester of salicylic acid, 4-hydroxycoumarin accumulated in the culture medium. Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted in the formation of 3,5-dihydroxybiphenyl which is the precursor of the phytoalexins of the Maloideae.     

Alternative pathways of xanthone biosynthesis in cell cultures of Hypericum androsaemum L.

The biosynthesis of xanthones was studied in cell cultures of Hypericum androsaemum L. We have detected a new benzophenone synthase, for which the preferred substrate is benzoyl-CoA, itself supplied by 3-hydroxybenzoate:coenzyme A ligase. The stepwise condensation of benzoyl-CoA with three molecules of malonyl-CoA, catalyzed by benzophenone synthase, yields 2,4,6-trihydroxybenzophenone. This intermediate is subsequently converted by benzophenone 3′-hydroxylase, a cytochrome P450 monooxygenase. These biosynthetic steps, leading to the formation of 2,3′,4,6-tetrahydroxybenzophenone, represent an alternative pathway to that recently proposed for cell cultures of Centaurium erythraea [Peters et al., Planta (1997) in press].     

Characterization of recombinant human acetyl-CoA carboxylase-2 steady-state kinetics

Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, a key metabolite in the fatty acid synthetic and oxidation pathways. The present study describes the steady-state kinetic analysis of a purified recombinant human form of the enzyme, namely ACC2, using a novel LC/MS/MS assay to directly measure malonyl-CoA formation. Four dimensional matrices, in which bicarbonate (HCO₃^?), ATP, acetyl-CoA, and citrate were varied, and global data fitting to appropriate steady-state equations were used to generate kinetic constants. Product inhibition studies support the notion that the enzyme proceeds through a hybrid (two-site) random Ter Ter mechanism, one that likely involves a two-step reaction at the biotin carboxylase domain. Citrate, a known activator of animal forms of ACC, activates both by increasing k_cat and k_cat/K_M for ATP and acetyl-CoA.     

The effect of diphenylamine on carotenogenesis in Phycomyces blakesleeanus

The presence of diphenylamine (DPA) during growth of mutant strains of Phycomyces blakesleeanus caused the expected inhibition of the formation of unsaturated carotenes, and the accumulation of phytoene in all cases. Cell extracts from DPA-grown cultures incubated with [2-¹⁴C]mevalonic acid, also exhibited these effects. Inhibition of in vitro carotenogenesis was similarly shown by extracts from normally-grown mycelia, incubated with DPA. Removal of DPA from mycelia or from cell extracts, resulted in the formation of unsaturated carotenes. The ratio of 15-cis-all-trans-phytoenes from mycelia grown ± DPA was only marginally altered, but, in vitro, the presence of DPA caused a significant increase in the formation of the all-trans isomer in the C5 strain. These results indicate that DPA acts by post-translational regulation of enzymic activities.     

Life by a new decarboxylation-dependent energy conservation mechanism with Na as coupling ion

We report here a new mode of ATP synthesis in living cells. The anaerobic bacterium Propionigenium modestum gains its total energy for growth from the conversion of succinate to propionate according to: succinate + H₂O → propionate + HCO₃^- (G^o' = -20.6 kJ/mol). The small free energy change of this reaction does not allow a substrate-linked phosphorylation mechanism, and no electron transport phosphorylation takes place. Succinate was degraded by cell-free extracts to propionate and CO₂ via succinyl-CoA, methyl-malonyl-CoA and propionyl-CoA. This pathway involves a membrane-bound methylmalonyl-CoA decarboxylase which couples the exergonic decarboxylation with a Na⁺ ion transport across the membrane. The organism also contained a membrane-bound ATPase which was specifically activated by Na⁺ ions and catalyzed and transport of Na⁺ ions into inverted bacterial vesicles upon ATP hydrolysis. The transport was abolished by monensin but not by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone. Isolated membrane vesicles catalyzed the synthesis of ATP from ADP and inorganic phosphate when malonyl-CoA was decarboxylated and malonyl-CoA synthesis from acetyl-CoA when ATP was hydrolyzed. These syntheses were sensitive to monensin which indicates that Na⁺ functions as the coupling ion. We conclude from these results that ATP synthesis in P. modestum is driven by a Na⁺ ion gradient which is generated upon decarboxylation of methylmalonyl-CoA.     

Effects of acetoacetyl-CoA synthase expression on production of farnesene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Efficient production of sesquiterpenes in Saccharomyces cerevisiae requires a high flux through the mevalonate pathway. To achieve this, the supply of acetyl-CoA plays a crucial role, partially because nine moles of acetyl-CoA are necessary to produce one mole of farnesyl diphosphate, but also to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA and, therefore, represents a potential target to increase the flux through the mevalonate pathway. This study investigates the effect of acetoacetyl-CoA synthase on growth as well as the production of farnesene and compares different homologs regarding their efficiency. While plasmid-based expression of nphT7 did not improve final farnesene titers, the construction of an alternative pathway, which exclusively relies on the malonyl-CoA bypass, was detrimental for growth and farnesene production. The presented results indicate that the overall functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain CL190.