Metabolic Investigation and Auxiliary Enzyme Modelization of the Pyrrocidine Pathway Allow Rationalization of Paracyclophane-Decahydrofluorene Formation.

Fungal paracyclophane-decahydrofluorene-containing natural products are complex polycyclic metabolites derived from similar hybrid PKS-NRPS pathways. Herein we studied the biosynthesis of pyrrocidines, one representative of this family, by gene inactivation in the producer Sarocladium zeae coupled to thorough metabolic analysis and molecular modeling of key enzymes. We characterized nine pyrrocidines and analogues as well as in mutants a variety of accumulating metabolites with new structures including rare cis-decalin, cytochalasan, and fused 6/15/5 macrocycles. This diversity highlights the extraordinary plasticity of the pyrrocidine biosynthetic gene cluster. From accumulating metabolites, we delineated the scenario of pyrrocidine biosynthesis. The ring A of the decahydrofluorene is installed by PrcB, a membrane-bound cyclizing isomerase, on a PKS-NRPS-derived pyrrolidone precursor. Docking experiments in PrcB allowed us to characterize the active site suggesting a mechanism triggered by arginine-mediated deprotonation at the terminal methyl of the substrate. Next, two integral membrane proteins, PrcD and PrcE, each predicted as a four-helix bundle, perform hydroxylation of the pyrrolidone ring and paracyclophane formation, respectively. Modelization of PrcE highlights a topological homology with vitamin K oxido-reductase and the presence of a disulfide bond. Our results suggest a previously unsuspected coupling mechanism via a transient loss of aromaticity of tyrosine residue to form the strained paracyclophane motif. Finally, the lipocalin-like protein PrcX drives the exo-cycloaddition yielding ring B and C of the decahydrofluorene to afford pyrrocidine A, which is transformed by a reductase PrcI to form pyrrocidine B. These insights will greatly facilitate the microbial production of pyrrocidine analogues by synthetic biology.

Combinatory Library of Microorganisms in the Selection of Reductive Activity Applied to a Ketone Mixture: Unexpected Highlighting of an Enantioselective Oxidative Activity.

Biocatalytic processes are increasingly used in organic synthesis for the preparation of targeted molecules or the generation of molecular diversity. The search for the biocatalyst is often the bottleneck in the development of the process. We described a combinatorial approach for the selection of active strains from a library of microorganisms. In order to show the potential of the method we applied it to a mixture of substrates. We were able to select yeast strains capable of producing enantiopure alcohol from corresponding ketones with very few tests and highlight tandem reaction sequences involving several microorganisms. We demonstrate an interest in the kinetic study and the importance of incubation conditions. This approach is a promising tool for generating new products.

Cadaverine regulates biotin synthesis to modulate primary root growth in Arabidopsis.

Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3-BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8-diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3-BIO1 activity. Furthermore, cadaverine-treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl-coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.

Bioconversion of antifungal viridin to phytotoxin viridiol by environmental non-viridin producing microorganisms.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.

Biosynthesis of azaphilones: a review.

Covering up to 2020 Azaphilones are fungal polyketide pigments bearing a highly oxygenated pyranoquinone bicyclic core; they are receiving a great deal of increasing research interest for their applications in the agroalimentary, dyeing, cosmetic, printing and pharmaceutical industries. Their biosynthetic pathways are not fully elucidated; however, thanks to recent genomic approaches combined with the increasing genome sequencing of fungi, some of these pathways have been recently unveiled. This is the first review on the biosynthesis of azaphilonoids adressed from a genomic point of view.

Molecular crosstalk between the endophyte Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum - Modulation of lipoxygenase activity and beauvericin production during the interaction.

Plants comprise many asymptomatic fungal endophytes with potential roles of plant protection against abiotic and biotic stresses. Endophytes communicate with their host plant, with other endophytes and with invading pathogens but their language remains largely unknown. This work aims at understanding the chemical communication and physiological interactions between the fungal endophyte Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum. Oxylipins, common means of communication, such as 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), had been shown in our earlier studies to be overproduced during dual culture between the two fungal antagonists. On the other hand, the mycotoxin beauvericin was reduced in the interaction zone. The present work addresses the mechanisms underlying these changes. Hydroperoxy oxylipins are produced by lipoxygenases and P. variabile contains two lipoxygenase genes (pvlox1 and pvlox2), whereas pvlox2, but not pvlox1, is specifically up regulated during the interaction and none of the F. oxysporum lox genes vary. Heterologous expression of pvlox2 in yeast shows that the corresponding enzyme PVLOX2 produces 13-HPODE and, therefore, is most likely at the origin of the overproduced 13-HPODE during the interaction. Compellingly, beauvericin synthase gene beas expression is induced and beauvericin amounts increase in F. oxysporum mycelium when in contact with P. variabile. 13-HPODE, however, does not affect beas gene expression. Beauvericin, indeed, inhibits P. variabile growth, which counteracts and biotransforms the mycotoxin leading to reduced amounts in the interaction zone which allows further expansion of the endophyte. In order to study the interaction between the protagonists in planta, we set up an in vitro tripartite interaction assay, including the model plant Arabidopsis thaliana. F. oxysporum rapidly kills A. thaliana plants, whereas P. variabile provides up to 85% reduction of plant death if present before pathogen attack. Future studies will shed light on the protection mechanisms and the role of oxylipins and beauvericin degradation herein with the long-term aim of using endophytes in plant protection.

A Reactive Eremophilane and Its Antibacterial 2(1H)-Naphthalenone Rearrangement Product, Witnesses of a Microbial Chemical Warfare.

Two sesquiterpenes, 4-epi-microsphaeropsisin (1) and a dihydrofurano-2(1H)-naphthalenone (variabilone, 2) which represents a new skeleton, were isolated from endophytic fungus Paraconiothyrium variabile. Reactivity studies showed that eremophilane 1 is a precursor of 2 through acid-promoted methyl 1,2-migration and aromatization. An electrophilic intermediate of this transformation was intercepted by N-acetylcysteamine, a biomimetic nucleophile. Only compound 2 was antibacterial against endophytic bacterium Bacillus subtilis (coisolated with P. variabile), suggesting a role in the microbial competition in plants.

Dihydroanatoxin-a Is Biosynthesized from Proline in Cylindrospermum stagnale PCC 7417: Isotopic Incorporation Experiments and Mass Spectrometry Analysis.

LC-MS and GC-MS analytical conditions have been developed to detect the cis- and trans-epimers (relative configuration of the carbon bearing the acetyl or propionyl group) of dihydroanatoxin-a and dihydrohomoanatoxin-a, in biological samples. These compounds epimerize under acidic conditions, yielding a major species that was tentatively assigned as the cis-epimer. Cylindrospermum stagnale PCC 7417 was definitively shown to produce dihydroanatoxin-a (1.2 mg/g dried cells). Oscillatoria sp. PCC 9107, Oscillatoria sp. PCC 6506, and C. stagnale PCC 7417, which produce anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a, respectively, were cultivated in the presence of isotopically labeled proline, and the toxins were extracted. Interpretation of the GC-MS electron ionization mass spectra of these labeled anatoxins showed that they are all biosynthesized from proline and that the positions of the labels in these molecules are identical. These data and the fact that the ana cluster of genes is conserved in these cyanobacteria suggest that dihydroanatoxin-a is formed by the reduction of either anatoxin-a or its precursor in a specific step involving AnaK, an F420-dependent oxido-reductase whose gene is found in the ana gene cluster in C. stagnale PCC 7417. This is the first report of a cyanobacterium producing dihydroanatoxin-a, suggesting that other producers are present in the environment.

Talaroketals A and B, unusual bis(oxaphenalenone) spiro and fused ketals from the soil fungus Talaromyces stipitatus ATCC 10500.

Two novel oxaphenalenone dimers, talaroketals A () and B (), were isolated from the soil fungus Talaromyces stipitatus. Their structures and absolute configurations were determined on the basis of spectroscopic analyses, X-ray diffraction experiments and electronic circular dichroism. Compound () features a rare benzannulated 5,6-spiroketal ring system within the dimeric bis(oxaphenalenone) skeleton while the parent compound () harbors a fused bicyclic furano-pyran moiety. These two compounds may biogenetically result from the reaction of duclauxin with a dihydrofuran derivative of botryodiplodin. Additionally, talaroketals A () and B () display modest antimicrobial activity against Staphylococcus aureus.

Unexpected talaroenamine derivatives and an undescribed polyester from the fungus Talaromyces stipitatus ATCC10500.

Chemical investigation of the fungus Talaromyces stipitatus ATCC 10500, whose genome has been sequenced, led to the isolation of four undescribed talaroenamines B-E along with the known talaroenamine A. Their structures were elucidated on the basis of spectroscopic studies including mass spectrometry, extensive 2D NMR and electronic circular dichroism (ECD). Interestingly, talaroenamine A had previously been isolated from the strain of T. stipitatus Δtrop C, a strain knocked out for the gene encoding a non-heme Fe(II)-dependent dioxygenase catalyzing the oxidative ring expansion leading to the tropolone, but never from a wild-type strain. All talaroenamines were evaluated for their antiplasmodial activity and Talaroenamine D exhibited the best inhibition against the chloroquine-resistant Plasmodium falciparum (FcB1 strain) without noticeable toxicity on HeLa and preadipose cell lines. In the course of the chemical investigation of T. stipitatus, an undescribed polyester was also isolated and its absolute configuration was determined by hydrolysis and transesterification followed by gas chromatography on chiral column.

Structure of the prolyl-acyl carrier protein oxidase involved in the biosynthesis of the cyanotoxin anatoxin-a.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to 1-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a. AnaB was crystallized in its holo form and its three-dimensional structure was determined by X-ray diffraction at 2.8 Šresolution. AnaB is a homotetramer and its fold is very similar to that of the acyl-CoA dehydrogenases (ACADs). The active-site base of AnaB, Glu244, superimposed very well with that of human isovaleryl-CoA dehydrogenase, confirming previous site-directed mutagenesis experiments and mechanistic proposals. The substrate-binding site of AnaB is small and is likely to be fitted for the pyrrolidine ring of proline. However, in contrast to ACADs, which use an electron-transport protein, AnaB uses molecular oxygen as the electron acceptor, as in acyl-CoA oxidases. Calculation of the solvent-accessible surface area around the FAD in AnaB and in several homologues showed that it is significantly larger in AnaB than in its homologues. A protonated histidine near the FAD in AnaB is likely to participate in oxygen activation. Furthermore, an array of water molecules detected in the AnaB structure suggests a possible path for molecular oxygen towards FAD. This is consistent with AnaB being an oxidase rather than a dehydrogenase. The structure of AnaB is the first to be described for a prolyl-ACP oxidase and it will contribute to defining the structural basis responsible for oxygen reactivity in flavoenzymes.

A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid.

7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20 nM to 50 µM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy.

Synthesis, configuration assignment, and simultaneous quantification by liquid chromatography coupled to tandem mass spectrometry, of dihydroanatoxin-a and dihydrohomoanatoxin-a together with the parent toxins, in axenic cyanobacterial strains and in environmental samples.

We have synthesized cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a using a short synthetic route. The relative configuration of N-tert-butoxycarbonyl-cis-dihydroanatoxin-a was determined by X-ray crystallography, while that of N-tert-butoxycarbonyl-trans-dihydroanatoxin-a was confirmed by epimerization leading to the cis-diastereoisomer. The relative configuration of N-tert-butoxycarbonyl-trans- and cis-dihydrohomoanatoxin-a was inferred from their NMR spectra. Using an optimized LC-MS/MS analytical method and pure standards we have simultaneously determined anatoxin-a, homoanatoxin-a and their dihydroderivatives in axenic strains of cyanobacteria and in environmental samples from the Tarn River, France. However, in these analytical conditions, the cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a could not be separated. In axenic strains, the dihydroderivatives represented less than 3% of the total toxin content, while in field samples dihydroanatoxin-a represented from 17% to 90% of the total toxin content. Thus, the reduction of anatoxin-a to dihydroanatoxin-a is predominant in the environment. The ratio of anatoxin-a concentration over that of homoanatoxin-a in axenic strains was variable, and among the eight strains studied we found three exclusive anatoxin-a producers and five producers of homoanatoxin-a and anatoxin-a, the latter representing from 0.5% to 2.0% of the total. In the strains studied, we have amplified by PCR, and sequenced the region of anaG coding for the methylation domain proposed to be responsible for the formation of homoanatoxin-a. The sequences showed at least 88% identity and we could not relate the toxin profile of the strains to the sequence of the methylation domain.

Unprecedented anthranilate priming involving two enzymes of the acyl adenylating superfamily in aurachin biosynthesis.

Biosynthesis of many polyketide-derived secondary metabolites is initiated by incorporating starter units other than acetate. Thus, understanding their priming mechanism is of importance for metabolic engineering. Insight into the loading process of anthranilate into the biosynthetic pathway for the quinoline alkaloids aurachins has been provided by the sequencing of a partial biosynthetic gene cluster in the myxobacterium Stigmatella aurantiaca. The cluster encodes a predicted aryl:CoA ligase AuaE that was hypothesized to activate and transfer anthranilate to the acyl carrier protein AuaB. However, gene inactivation and in vitro experiments described here contradicted this model. Aided by the genome sequence of S. aurantiaca, we identified an additional aryl:CoA ligase homologue, AuaEII, encoded in a different gene operon, which is additionally required for anthranilate priming. We report the characterization of both enzymes and the elucidation of a novel non-acetate priming strategy in thio-templated biosynthetic machineries.

Insights into the reaction mechanism of the prolyl-acyl carrier protein oxidase involved in anatoxin-a and homoanatoxin-a biosynthesis.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins as well as the in-vitro reconstitution of the first steps of this biosynthesis. We now report experimental evidence supporting the proposed reaction mechanism of AnaB, a flavoprotein homologous to acyl-CoA dehydrogenase. AnaB catalyzes the two-electron oxidation of prolyl-AnaD, which is proline linked to the acyl carrier protein holo-AnaD, to dehydroprolyl-AnaD using oxygen as the second substrate. AnaB is thus an oxidase. By using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we have identified and characterized dehydroprolyl-AnaD, the AnaB product. We estimated an apparent catalytic constant of 1 min(-1) for AnaB catalysis. We synthesized several deuterium-labeled prolines and enzymatically transformed them into their corresponding prolyl-AnaD. These deuterium-labeled prolyl-AnaDs were oxidized in the presence of AnaB, and the deuterium labeling in the remaining substrate and in the product was determined by LC-MS/MS. The data supported a reaction mechanism starting with a rapid enolization followed by a slow oxidation to give the conjugated imine, which in turn was isomerized to pyrroline-5-carboxyl-AnaD. We also showed that cis- and trans-4-fluoro-L-prolyl-AnaD and 3,4-dehydro-L-prolyl-AnaD were transformed into pyrrole-2-carboxyl-AnaD by AnaB. Thus, the 4-fluoro-analogues experienced a ß-elimination supporting the AnaB-catalyzed aza-allylic isomerization. We identified by sequence alignment the AnaB active site base, Glu244. We produced, purified, and characterized the E244A AnaB mutant, which is inactive, supporting the catalytic role of E244 as a base.

Pyridoxal-5'-phosphate-dependent enzymes involved in biotin biosynthesis: structure, reaction mechanism and inhibition.

The four last steps of biotin biosynthesis, starting from pimeloyl-CoA, are conserved among all the biotin-producing microorganisms. Two enzymes of this pathway, the 8-amino-7-oxononanoate synthase (AONS) and the 7,8-diaminopelargonic acid aminotransferase (DAPA AT) are dependent on pyridoxal-5'-phosphate (PLP). This review summarizes our current understanding of the structure, reaction mechanism and inhibition on these two interesting enzymes. Mechanistic studies as well as the determination of the crystal structure of AONS have revealed a complex mechanism involving an acylation with inversion of configuration and a decarboxylation with retention of configuration. This reaction mechanism is shared by the homologous 5-aminolevulinate synthase and serine palmitoyltransferase. While the reaction catalyzed by DAPA AT is a classical PLP-dependent transamination, the inactivation of this enzyme by amiclenomycin, a natural antibiotic that is active against Mycobacterium tuberculosis, involves the irreversible formation of an adduct between PLP and amiclenomycin. Mechanistic and structural studies allowed the complete description of this unique inactivation mechanism. Several potent inhibitors of these two PLP-dependent enzymes have been prepared and might be useful as starting points for the design of herbicides or antibiotics. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

The genome sequence of the cyanobacterium Oscillatoria sp. PCC 6506 reveals several gene clusters responsible for the biosynthesis of toxins and secondary metabolites.

We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites.

Biosynthesis of cylindrospermopsin and 7-epicylindrospermopsin in Oscillatoria sp. strain PCC 6506: identification of the cyr gene cluster and toxin analysis.

Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 microg/mg and 0.29 microg/mg, respectively) and cylindrospermopsin (0.10 microg/mg and 0.11 microg/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 +/- 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA.

In vitro reconstitution of the first steps of anatoxin-a biosynthesis in Oscillatoria PCC 6506: from free L-proline to acyl carrier protein bound dehydroproline.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins in cyanobacteria and proposed a biosynthetic scheme starting from L-proline and involving three polyketide synthases for which the starter would be (S)-1-pyrroline-5-carboxylate bound to an acyl carrier protein, AnaD. We now report the in vitro reconstitution of the first steps of this biosynthesis in Oscillatoria PCC 6506. We identified in PCC 6506 the gene coding for an Sfp-like phosphopantetheinyl transferase and purified the gene product, OsPPT, that catalyzed the transfer of the phosphopantetheinyl arm to the serine 41 of AnaD. The pure adenylation protein AnaC loaded L-proline on holo-AnaD and was specific for L-proline (K(m) = 0.97 mM, k(cat) = 68 min(-1)) among the 20 natural amino acids. Among six close structural analogues of L-proline, including (S)-1-pyrroline-5-carboxylate, we only found 3,4-dehydro-L-proline to be an alternate substrate for AnaC (K(m) = 1.5 mM, k(cat) = 29 min(-1)). The putative prolyl-AnaD dehydrogenase, AnaB, purified to homogeneity as a histidine-tagged protein, showed an absorption spectrum characteristic of FAD-containing proteins. It oxidized prolyl-AnaD to dehydroprolyl-AnaD as shown by tryptic digestion of the protein followed by liquid chromatography coupled to tandem mass spectrometry. Alignment of the amino acid sequence of this dehydrogenase with related enzymes showed that AnaB belongs to the acyl-CoA dehydrogenase superfamily and thus probably catalyzes an alpha-beta-dehydrogenation of the thioester-bound proline followed by an aza-allylic isomerization to yield (S)-pyrroline-5-carboxyl-AnaD, the proposed starter for the subsequent polyketide synthase, AnaE.

Evidence that biosynthesis of the neurotoxic alkaloids anatoxin-a and homoanatoxin-a in the cyanobacterium Oscillatoria PCC 6506 occurs on a modular polyketide synthase initiated by L-proline.

Anatoxin-a and homoanatoxin-a are potent neurotoxins produced by cyanobacteria such as Oscillatoria PCC 6506. Sequencing of the genome of this strain is underway, and we have identified a 29 kb DNA fragment containing a sequence called ks2 that we previously showed to be specific to Oscillatoria cyanobacteria producing anatoxin-a and homoanatoxin-a. Bioinformatic analysis of this 29 kb fragment revealed a cluster of genes, which were annotated. The function assigned to the products of eight contiguous genes, from anaA to anaH, provides a clue to the biosynthesis of anatoxin-a and homoanatoxin-a. Proline is first loaded on an acyl carrier protein and its five-membered cycle oxidized to the pyrroline oxidation state. This activated ring is then successively loaded on three polyketide synthase modules for elongation, reduction, cyclization, and methylation. The final step is the hydrolysis of the thioester with subsequent decarboxylation. GC-MS and NMR analyses of homoanatoxin-a produced by PCC 6506 using labeled precursors confirm that proline is very likely the starter of these polyketide synthases. Using specific PCR amplifications, we have also shown that the anaC, anaE, anaF, and anaG genes are always present in the genome of cyanobacteria producing anatoxin-a and homoanatoxin-a and absent in nonproducing strains. Histidine-tagged AnaC was purified to homogeneity and showed to catalyze the loading of proline on purified histidine-tagged AnaD that had been previously transformed into its holo form using the Bacillus subtilis Sfp phosphopantetheinyl transferase. All of these data provide strong evidence that we have successfully identified the gene cluster responsible for the production of anatoxin-a and homoanatoxin-a in Oscillatoria PCC 6506.