Biosynthesis of tetrapetalones

The biosynthesis of tetrapetalones (tetrapetalones A, B, C, and D) in Streptomyces sp. USF-4727 was studied by feeding experiments with [1-13C] sodium propanoate, [1-13C] sodium butanoate, [carbonyl-13C] 3-amino-5-hydroxybenzoic acid (AHBA) hydrochloride, and [1-13C] glucose, followed by analysis of the 13C-NMR spectra. These feeding experiments revealed that the four tetrapetalones were polyketide compounds constructed from propanoate, butanoate, AHBA, and glucose. The tetrapetalone biosynthetic pathway was also suggested in this study. In this pathway, tetrapetalone A (1) is synthesized by polyketide synthase (PKS) using AHBA as a starter unit, then the side chain of 1 is subjected to acetoxylation to produce tetrapetalone B (2). Additionally, 1 is oxidized and transformed into tetrapetalone C (3). In a similar way, 2 is converted to tetrapetalone D (4). Therefore, the biosynthetic relationship of the four tetrapetalones was indicated.     

Biosynthesis of the isoprenoid moieties of furanonaphthoquinone I and endophenazine A in Streptomyces cinnamonensis DSM 1042

Streptomyces cinnamonensis DSM 1042 produces the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and isoprenylated phenazines, predominantly endophenazine A. However, the recently identified biosynthetic gene cluster for these compounds only contains a single gene for a mevalonate pathway enzyme, that is, a putative mevalonate kinase gene. This is in strong contrast to all Streptomyces strains examined so far, where all six genes encoding the mevalonate pathway enzymes are clustered in a single operon of 6.8 kb and, thus, raised the question about the biosynthetic origin of the isoprenoid moieties of FNQ I and endophenazine A. In this study, we investigated the incorporation of [13C2]acetate and [2-13C]glycerol into FNQ I and endophenazine A. The results unequivocally prove that the isoprenoid building blocks of both compounds are predominantly formed via the mevalonate pathway (approximately 80%) but that the MEP pathway (approximately 20%) contributes to the biosynthesis of these molecules, too. In actinomycetes, this is the first experimentally proven example of the utilization of both biosynthetic routes for the formation of one single secondary metabolite. The incorporation pattern of [2-13C]glycerol was consistent with a "reverse" prenyl transfer, that is, with the formation of a C-C bond from C-3 of GPP to the polyketide nucleus of FNQ I.     

Biosynthetic origin of BE-10988 in Streptomyces sp. BA10988

The biosynthetic origin of the tumor-inhibitory derivative, BE-10988, was studied in Streptomyces sp . BA10988 by retrobiosynthetic NMR analysis using [U-(13)C6]glucose as a precursor. The isotopologue compositions of the indole moieties of BE-10988 and tryptophan were virtually identical. This indicates that tryptophan or a closely related metabolite served as a biosynthetic precursor of BE-10988 in analogy to the biosynthetic pathway of camalexin, a structurally related phytoalexin in Arabidopsis thaliana. Labeling experiments with [U-(13)C8(15)N]indole, L-[ring-(2)H5]tryptophan, or L-[U-(13)C3(15)N]cysteine confirmed this hypothesis. However, transfer of the label from [ring-(2)H5]indole pyruvic acid, but not from the known camalexin precursor, [ring-(2)H5]indole-3-acetaldoxime, showed that plants and bacteria have evolved independent mechanisms of tryptophan modification in the biosynthesis of thiazolylindole derivatives.     

One metabolite, two pathways: convergence of polypropionate biosynthesis in fungi and marine molluscs

Structural similarity or even the identity of polyketide compounds does not necessarily imply unique biosynthesis. Feeding experiments with a (13)C labeled precursor establish that the C(3) units in 7-methyl-cyercene-1 (1) are derived from intact propionate in the marine mollusc Ercolania funerea. The same compound in the terrestrial fungus Leptosphaeria maculans/Phoma lingam is synthesized by an acetate/SAM pathway thus proving for the first time metabolic convergence of polyketide biosynthesis in eukaryotes. Traditional (1)H-(13)C NMR correlation spectroscopy has been successfully applied to estimate (13)C incorporation in biosynthetic experiments.     

Biosynthetic study of FR-900848: unusual observation on polyketide biosynthesis that did not accept acetate as origin of acetyl-CoA

The biosynthetic pathway of a potent antifungal agent, FR-900848, has been examined by administration of several ¹³C-labeled precursors to Streptoverticillium fervens HP-891. Although none of the ¹³C-labeled acetate was incorporated into FR-900848, the labeling pattern of FR-900848 derived from d-[U-¹³C₆]glucose revealed that the fatty acid backbone of FR-900848 has been biosynthesized via a polyketide pathway. These unusual results strongly show that the major pathway to provide acetyl-CoA in this microorganism is glycolysis. Feeding experiments with d-[U-¹³C₆]glucose, [1,3-¹³C₂]glycerol, and l-[Me-¹³C]methionine provided information on the biosynthetic origin of structurally unusual parts (polycyclopropane and aminonucleoside) in this antibiotic.     

Directed biosynthesis of alkaloid analogs in the medicinal plant Catharanthus roseus

Terpene indole alkaloids are plant natural products with diverse structures and biological activities. A highly branched biosynthetic pathway is responsible for the production of approximately 130 different alkaloids in Madagascar periwinkle (C. roseus) from a common biosynthetic intermediate derived from tryptamine. Although numerous biosynthetic pathways can incorporate unnatural starting materials to yield novel natural products, it was not clear how efficiently the complex, eukaryotic TIA pathway could utilize unnatural substrates to make new alkaloids. This work demonstrates that the TIA biosynthetic machinery can be used to produce novel alkaloid structures and also highlights the potential of this pathway for future metabolic engineering efforts.     

Biosynthese de la botryodiplodine,mycotoxine de penicillium roqueforti: Incorporations d'acetate[1-13C], [2-13C], [1-2 13C]et d'acide orsellinique 2-13C-carboxyle 13C], [3-4 13C]

Incorporations of [1-¹³C], [2-¹³C], [1-2¹³C]acetate and [2-¹³C, carboxyl-¹³C], [3-4¹³C]orsellinic acid into botryodiplodin indicate that this mycotoxin is biosynthesized by the polyketide pathway. Orsellinic acid is a precursor of botryodiplodin. A biosynthetic pathway, using orsellinic acid as precursor, is proposed.     

Studies on the biosynthesis of the notoamides: synthesis of an isotopomer of 6-hydroxydeoxybrevianamide E and biosynthetic incorporation into notoamide J

6-Hydroxydeoxybrevianamide E is proposed as a biosynthetic precursor to several advanced metabolites isolated from both marine-derived Aspergillus sp. and a terrestrial-derived Aspergillus versicolor. To verify the role of this reverse-prenylated indole alkaloid as an intermediate along the biosynthetic pathway, [(13)C](2)-[(15)N]-6-hydroxydeoxybrevianamide E was synthesized and fed to Aspergillus versicolor. Analysis of the metabolites showed incorporation of the intermediate only into the natural product notoamide J.     

Biosynthesis of Mikrolin

Mikrolin (8) and dechloromikrolin (9) have been shown to exist as tautomeric mixtures in solution. The structures of mono-O-trifluoroacetyl Mikrolin (10) and di-O-acetyl Mikrolin (11) have been elucidated. The products 15 to 23 from reduction of the metabolites 8 9 with Pd/C and Zn in aqueous acetic acid have been identified. The ¹³C-NMR. spectra of Mikrolin (8) and dechloromikrolin (9) and their derivatives have been completely assigned Based on the results of incorporation experiments with sodium [1-¹³C]-, [2-¹³C]- and [1, 2-¹³C]-acetate, a biosynthetic pathway is proposed for Mikrolin (8) .     

Biosynthesis of highly modified meroterpenoids in aspergillus variecolor. Incorporation of 13C-labelled acetates and methionine into anditomin and andilesin C

Incorporations of [1-¹³C]?, [2-¹³C]?, [1,2-¹³C₂]-acetates and [¹³C]-methionine into anditomin, a metabolite of $\text{Aspergillus variecolor}$, indicate its formation by a mixed polyketide-terpenoid biosynthetic pathway similar to that elucidated for andibenin; observations are made on the possible biosynthetic relationship of the $\text{A. variecolor}$ metabolites with austin and terretonin, mycotoxins recently isolated from $\text{A. ustus}$ and $\text{A. terreus}$ respectively.     

Biosynthesis of anthecotuloide,an irregular sesquiterpene lactone from Anthemis cotula L. (Asteraceae) via a non-farnesyl diphosphate route

Retrobiosynthetic analysis of the allergenic sesquiterpene lactone, anthecotuloide, suggested that this natural product could be formed either by head to head condensation of geranyl diphosphate with dimethylallyl diphosphate, or from farnesyl diphosphate (FPP), the accepted regular sesquiterpene precursor via the rearrangement of a germacranolide precursor. Isotopic labelling of anthecotuloide has now been achieved by feeding [1-(13)C]-glucose, [U-13C6]-glucose and [6,6-(2)H2]-glucose to aseptically grown plantlets of Anthemis cotula(family Asteraceae). Analysis of labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that the isoprene building blocks of this sesquiterpene are formed exclusively via the MEP terpene biosynthetic pathway. This was supported by results from an experiment using [U-13C6]-glucose. A deuterium labelling experiment using [6,6-(2)H2]-glucose supported the original proposal and showed that anthecotuloide is formed from a non FPP precursor. Isotope ratio mass spectrometry suggested that there were two pathways for sesquiterpene biosynthesis in A. cotula.     

Catalytic mechanism of S-ribosylhomocysteinase (LuxS): direct observation of ketone intermediates by 13C NMR spectroscopy

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce l-homocysteine and 4,5-dihydroxy-2,3-pentanedione (DHPD). This is a key step in the biosynthetic pathway of the type II autoinducer (AI-2) in both Gram-positive and Gram-negative bacteria. Previous studies demonstrated that LuxS contains a catalytically essential Fe2+ ion. The catalytic mechanism of LuxS was investigated using 2- and 3-13C-labeled SRH as substrate and 13C NMR spectroscopy. These studies revealed the presence of 2- and 3-keto intermediates in the catalytic pathway. The 2-keto intermediate was chemically synthesized, and its chemical and kinetic competence was demonstrated. The results support a catalytic mechanism in which the metal ion catalyzes an internal redox reaction, shifting the carbonyl group from the C-1 position to the C-3 position. Subsequent beta-elimination at the C-4 and C-5 positions releases homocysteine as a free thiol. The results also suggest that Cys-84 and Glu-57 are the possible general acids/bases for proton transfer during catalysis and that the keto intermediates are released from the enzyme active site before rebinding and completion of the reaction.     

Polyketide β-branching in bryostatin biosynthesis: identification of surrogate acetyl-ACP donors for BryR, an HMG-ACP synthase

In vitro analysis of natural product biosynthetic gene?products isolated from unculturable symbiotic bacteria is necessary to probe the functionalities of these enzymes. Herein, we report the biochemical characterization of BryR, the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) homolog implicated in β-branching at C13 and C21 of the core ring system from the bryostatin metabolic pathway (Bry). We confirmed the activity of BryR using two complementary methods, radio-SDS PAGE, and Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS). The activity of BryR depended on pairing of the native acetoacetyl-BryM3 acceptor acyl carrier protein (ACP) with an appropriate donor acetyl-ACP from a heterologous HMGS cassette. Additionally, the ability of BryR to discriminate between various ACPs was assessed using a surface plasmon resonance (SPR)-based protein-protein binding assay. Our data suggest that specificity for a protein-bound acyl group is a distinguishing feature between HMGS homologs found in PKS or PKS/NRPS biosynthetic pathways and those of primary metabolism. These findings reveal an important example of molecular recognition between protein components that are essential for biosynthetic fidelity in natural product assembly and modification.     

Notoamide E: Biosynthetic incorporation into notoamides C and D in cultures of Aspergillus versicolor NRRL 35600

Notoamide E, a short-lived secondary metabolite, has been proposed as a biosynthetic intermediate to several advanced metabolites isolated from Aspergillus versicolor. In order to verify the role of this indole alkaloid along the biosynthetic pathway, synthetic doubly ¹³C-labeled notoamide E was fed to Aspergillus versicolor. Analysis of the metabolites showed significant incorporation of notoamide E into the natural products notoamides C and D.     

On the biosynthetic origin of methoxymalonyl-acyl carrier protein, the substrate for incorporation of "glycolate" units into ansamitocin and soraphen A

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of "glycolate" units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the "glycolate" unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). d-[1,2-(13)C(2)]Glycerate is also incorporated specifically into the "glycolate" units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-(13)C(2)]glycerol were developed as part of this work.     

Studies on the formation and incorporation of streptolidine in the biosynthesis of the peptidyl nucleoside antibiotic streptothricin F

Streptothricin F (STF, 1) is a peptidyl nucleoside antibiotic produced by Streptomyces lavendulae. Studies were conducted to address the formation and timing of incorporation of the arginine-derived base streptolidine (4) during the biosynthesis of 1. [guanidino-(13)C]Streptolidine (10) was prepared by modification of an established method and used in whole-cell incorporation experiments. Analysis of the purified STF by (13)C NMR revealed a 1.9% enrichment of the guanidino carbon, confirming 4 as an advanced precursor to 1 and supporting proposals that 1 is assembled via a convergent biosynthetic pathway. To identify advanced intermediates in the conversion of L-arginine to 4, (2S,3R)-[guanidino-(13)C]capreomycidine (32) was prepared from oxazolidine aldehyde (18) via 1,1-dimethylethyl (4R,1'S)-4-(1',3'-diaminopropyl)-2,2-dimethyl-3-oxazolidinecarboxylate (30). Treatment of 30 with Br(13)CN yielded the corresponding diprotected amino alcohol, which was readily converted to 32. The STF isolated from whole-cell incorporation experiments with 32 showed no significant (13)C enrichment at the guanidino carbon. These results suggest that 32 may be an enzyme-bound intermediate, unable to enter the cell, or is not a precursor to STF.     

Rapid baseline separation of enantiomers and a mesoform of all-trans-astaxanthin, 13-cis-astaxanthin, adonirubin, and adonixanthin in standards and commercial supplements

Astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) is widely used as important colorant in the aquaculture feed industry, and as nutraceuticals in human health products. Synthetic all-trans-astaxanthin consists of a mixture of a pair of enantiomers (3R,3'R and 3S,3'S) and a mesoform (3R,3'S). A high-performance liquid chromatography (HPLC) method for direct, rapid, and baseline separation of three stereoisomers of all-trans-astaxanthin is described for the first time on an immobilized cellulosic column (Chiralpak IC). Enantiomers of two important precursors in the biosynthetic pathway of astaxanthin, adonirubin and adonixanthin, were also directly separated. In addition, the major cis form of astaxanthin (13-cis-astaxanthin) resulted from isomerization was isolated with preparative C18 separation, and the separation of all four stereoisomers of 13-cis-astaxanthin is achieved. Finally, a stereoisomeric purity test of commercial astaxanthin supplements confirmed that they were from a natural source, although their levels were quite low.     

Biosynthetic pathway of 24-membered macrolactam glycoside incednine

Incednine was isolated from Streptomyces sp. ML694-90F3 as an inhibitor of anti-apoptotic function of Bcl-2/Bcl-xL oncoproteins. The structure of incednine is quite unique with a characteristic 24-membered macrocyclic lactam aglycone and two unusual aminosugars. To understand its biosynthetic pathway, the incorporation studies were carried out with [1-¹³C]acetate, [1,2-¹³C₂]acetate, [1-¹³C]propionate, l-[¹³C₅,¹⁵N]glutamate, [1,2,3-¹³C₃]glycerol, d-[6,6-²H₂]glucose, and l-[CH₃-¹³C]methionine. As a result, acetate, propionate, and glycerol were well incorporated into the elongation units of the macrolactam moiety, which indicates that its basic skeleton could be constructed by standard polyketide synthase, whereas all atoms of the starter unit were labeled by [¹³C₅,¹⁵N]glutamate suggesting that glutamate is somehow decarboxylated and rearranged into 3-aminobutyrate as the unique starter unit. The origins of the sugar moieties and methyl groups were also clarified. Based on the incorporation pattern, a plausible biosynthetic pathway for incednine is proposed.     

Biosynthetic pathway of macrolactam polyketide antibiotic cremimycin

Biosynthetic origin of macrolactam polyketide antibiotic cremimycin was investigated by feeding experiments with [1-¹³C]acetate, [1,2-¹³C₂]acetate, [1-¹³C]propionate, succinate-d₄, and d-[6,6-²H₂]glucose. NMR analysis of the resultant isotope-enriched cremimycins showed distinctive incorporation patterns, which suggested that the aglycon of cremimycin was constructed from two propionates and eleven acetates. Thus, 3-oxononanoate was proposed as a potential polyketide intermediate, that is, aminated to be the unique nitrogen-containing moiety of cremymicin. Further, characteristic propionate biosynthetic pathway in the cremimycin-producing strain was also described.     

Studies on the biosynthesis of taxol. Synthesis Of taxa-4(20), 11(12)-diene-2alpha,5alpha-diol

The synthesis of taxa-4(20),11(12)-diene-2alpha,5alpha-diol is described. An improved procedure for the intramolecular Diels-Alder cycloaddition previously reported in our synthesis of taxa-4(5), 11(12)-diene has been utilized to prepare a taxoid with oxygenation in the B and C rings. It has been established previously that taxa-4(20),11(12)-dien-5alpha-ol is the first oxygenated intermediate on the biosynthetic pathway to Taxol. Taxa-4(20), 11(12)-diene-2alpha,5alpha-diol (5), which has been observed in a biosynthetic conversion, is a potential candidate as the second oxygenated intermediate on the Taxol biosynthetic pathway, has been prepared to probe the intermediacy of this substance.