Reconstruction of the violacein biosynthetic pathway from Duganella sp. B2 in different heterologous hosts

Violacein is a bacteria-originated indolocarbazole pigment with potential applications due to its various bioactivities such as anti-tumor, antiviral, and antifungal activities. However, stable mass production of this pigment is difficult due to its low productivities and the instability of wild-type violacein-producing strains. In order to establish a stable and efficient production system for violacein, the violacein synthesis pathway from a new species of Duganella sp. B2 was reconstructed in different bacterial strains including Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes by using different vectors. The gene cluster that encodes five enzymes involved in the violacein biosynthetic pathway was first isolated from Duganella sp. B2, and three recombinant expression vectors were constructed using the T7 promoter or the alkane-responsive promoter PalkB. Our results showed that violacein could be stably synthesized in E. coli, C. freundii, and E. aerogenes. Interestingly, we found that there were great differences between the different recombinant strains, not only in the protein expression profiles pertaining to violacein biosynthesis but also in the productivity and composition of crude violacein. Among the host strains tested, the crude violacein production by the recombinant C. freundii strain reached 1.68 g L⁻¹ in shake flask cultures, which was 4-fold higher than the highest production previously reported in flask culture by other groups. To the best of our knowledge, this is the first report on the efficient production of violacein by genetically engineered strains.     

Biosynthesis of violacein: intact incorporation of the tryptophan molecule on the oxindole side, with intramolecular rearrangement of the indole ring on the 5-hydroxyindole side

Feeding experiments with a mixture of [2-13C]- and [indole-3-13C]tryptophans, of [3-13C]- and [indole-3-13 C]tryptophans (1:1 molar ratio) and of others have proved that the 1,2-shift of the indole ring occurred via an intramolecular process for formation of the left part (5-hydroxyindole side) of the violacein skeleton and demonstrated that the C-C bond from C2 of the indole ring to C2 of the side chain was completely retained for formation of the right part (oxindole side) during the entire biosynthetic process. Due to the involvement of transaminase, it has remained unresolved whether indolylpyruvic acid is the biosynthetic intermediate and/or from where the nitrogen atom of the pyrrolidone ring originates. An incorporation experiment with a mixture of [2-13C]- and [alpha-15N]tryptophans (1:1 molar ratio) verified that the nitrogen atom in the central ring was exclusively derived from the right-side tryptophan. Thus, all the carbon and nitrogen atoms in the right part of the violacein skeleton were constructed by intact incorporation of the tryptophan molecule, with decarboxylation probably occurring at a later biosynthetic stage.     

Biosynthesis of a trypanocide by Chromobacterium violaceum

Radio-isotope studies indicated not only that l-tryptophan can serve as carbon source for synthesis of the trypanocide, violacein by Chromobacterium violaceum (BB-78 strain) but also that isatin and indole 3-acetic acid are both important metabolic intermediates. Using 3-indolyl [2-¹⁴C] and [1-¹⁴C] acetic acid, it was found that the carboxylic carbon was not eliminated and that indole-3-acetic acid was incorporated intact into the pigment structure. N-Ethyl(5-hydroxy-indol-3-yl)-2-indolylethylamide is also an important metabolic intermediate in the violacein biosynthesis. This is the first report of a metabolic scheme for violacein synthesis which includes an intermediate other than l-tryptophan.     

Biosynthesis of Violacein: Origins of Hydrogen,Nitrogen and Oxygen Atoms in the 2-Pyrrolidone Nucleus

The biosynthetic origins of the hydrogen, nitrogen and oxygen atoms in the pyrrolidone ring of violacein'were established by an anaylses of the ¹H, ¹³C NMR and MS spectra of its isotope-enriched metabolites. Feeding experiments of [2-²H] and [3-²H₂]tryptophans have revealed that the hydrogen in the pyrrolidone ring was derived from the methylene protons at the 3-position of tryptophan. The stereochemical fate of the prochiral hydrogens was determined to be in the retention of the pro-S hydrogen by these feeding experiments using [3R-²H] and [3S”-²H]tryptophans. The incorporation experiment of [α-¹⁵N]tryptophan demonstrated that the nitrogen atom in the ring originated from the α-amino group of tryptophan. The incorporation experiment of ¹⁸O₂ gas verified that all the oxygen atoms of violacein were derived from the molecular oxygen.     

Sequence analysis and functional characterization of the violacein biosynthetic pathway from Chromobacterium violaceum

Violacein is a purple-colored, broad-spectrum antibacterial pigment that has a dimeric structure composed of 5-hydroxyindole, oxindole and 2-pyyrolidone subunits formed by the condensation of two modified tryptophan molecules. The violacein biosynthetic gene cluster from Chromobacterium violaceum was characterized by DNA sequencing, transposon mutagenesis, and chemical analysis of the pathway intermediates produced heterologously in Escherichia. coli. The violacein biosynthetic gene cluster spans eight kilobases and is comprised of the four genes, vioABCD, that are necessary for violacein production. Sequence analysis suggests that the products of vioA, vioC and vioD are nucleotide-dependent monooxygenases. Disruption of vioA or vioB completely abrogates the biosynthesis of violacein intermediates, while disruption of the vioC or vioD genes results in the production of violacein precursors.     

Changes in interhelical hydrogen bonding upon rhodopsin activation

Hydrogen bonding interactions between transmembrane helices stabilize the visual pigment rhodopsin in an inactive conformation in the dark. The crystal structure of rhodopsin has previously revealed that Glu122 and Trp126 on transmembrane helix H3 form a complex hydrogen bonding network with Tyr206 and His211 on H5, while the indole nitrogen of Trp265 on H6 forms a water-mediated hydrogen bond with Asn302 on H7. Here, we use solid-state magic angle spinning NMR spectroscopy to probe the changes in hydrogen bonding upon rhodopsin activation. The NMR chemical shifts of 15N-labeled tryptophan are consistent with the indole nitrogens of Trp126 and Trp265 becoming more weakly hydrogen bonded between rhodopsin and metarhodopsin II. The NMR chemical shifts of 15N-labeled histidine show that His211 is neutral; the unprotonated imidazole nitrogen is not coordinated to zinc in rhodopsin and becomes more strongly hydrogen bonded in metarhodopsin II. Moreover, measurements of rhodopsin containing 13C-labeled histidine show that a strong hydrogen bond between the side-chain of Glu122 and the backbone carbonyl of His211 is disrupted in metarhodopsin II. The implications of these observations for the activation mechanism of rhodopsin are discussed.     

Cloning and heterologous expression of the violacein biosynthesis gene cluster fromChromobacterium violaceum

The entire biosynthetic pathway for the synthesis of the antibiotic pigment violacein is encoded on a 14.5 kilobase (kb) fragment of theChromobacterium violaceum genome. When cloned intoEscherichia coli, pigment synthesis is strongly expressed, as it is in a wide range of Gram-negative bacteria. Transposon mutagenesis resulted in a number of different phenotypes that correlated with transposon insertion sites in the gene cluster.     

Isolation and Characterization of Two Groups of Novel Marine Bacteria Producing Violacein

Thirteen strains of novel marine bacteria producing a purple pigment were isolated from the Pacific coast of Japan. They were divided into two groups based on their 16S ribosomal RNA gene sequences, and both groups of bacteria belonged to the genus Pseudoalteromonas. The UV-visible spectrum of the pigment was identical to those of violacein, a pigment produced by several species of bacteria including Chromobacterium violaceum, an opportunistic pathogen. Further analysis of the chemical structure of the pigment by mass spectroscopy and nuclear magnetic resonance spectroscopy showed that the pigment was violacein. The high purity of violacein in the crude extract enabled us to employ simple and nonpolluting procedures to purify the pigment. Isolated bacteria may be useful as a C. violaceum substitute for the safe production of violacein.     

Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan biosynthesis and storage protein deficiency

The ability of genetic manipulation to yield greatly increased concentrations of free amino acids (FAAs) in seeds of soybean was evaluated by introduction of a feedback-insensitive mutant enzyme of tryptophan (Trp) biosynthesis into two transformation-competent breeding lines deficient in major seed storage proteins. The storage protein-deficient lines exhibited increased accumulation of certain other seed proteins as well as of FAAs including arginine (Arg) and asparagine in mature seeds. Introduction of the gene for a feedback-insensitive mutant of an α subunit of rice anthranilate synthase (OASA1D) into the two high-FAA breeding lines by particle bombardment resulted in a >10-fold increase in the level of free Trp in mature seeds compared with that in nontransgenic seeds. The amount of free Trp in these transgenic seeds was similar to that in OASA1D transgenic seeds of the wild-type cultivar Jack. The composition of total amino acids in seeds of the high-FAA breeding lines remained largely unaffected by the expression of OASA1D with the exception of an increase in the total Trp content. Our results therefore indicate that the extra nitrogen resource originating from storage protein deficiency was used exclusively for the synthesis of inherent alternative nitrogen reservoirs such as free Arg and not for deregulated Trp biosynthesis conferred by OASA1D. The intrinsic null mutations responsible for storage protein deficiency and the OASA1D transgene affecting Trp content were thus successfully combined and showed additive effects on the amino acid composition of soybean seeds.     

An in Vitro System from Maize Seedlings for Tryptophan-Independent Indole-3-Acetic Acid Biosynthesis

The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [¹⁴C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [¹⁴C]Trp nor [¹⁴C]serine substituted for [¹⁴C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.     

Identification and characterization of an indigoidine-like gene for a blue pigment biosynthesis in <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> CCM 3239

An incomplete oligoketide (PK; ‘polyketide’) gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.     

Antinematode Activity of Violacein and the Role of the Insulin/IGF-1 Pathway in Controlling Violacein Sensitivity in Caenorhabditis elegans

The purple pigment violacein is well known for its numerous biological activities including antibacterial, antiviral, antiprotozoan, and antitumor effects. In the current study we identify violacein as the antinematode agent produced by the marine bacterium Microbulbifer sp. D250, thereby extending the target range of this small molecule. Heterologous expression of the violacein biosynthetic pathway in E. coli and experiments using pure violacein demonstrated that this secondary metabolite facilitates bacterial accumulation in the nematode intestine, which is accompanied by tissue damage and apoptosis. Nematodes such as Caenorhabditis elegans utilise a well-defined innate immune system to defend against pathogens. Using C. elegans as a model we demonstrate the DAF-2/DAF-16 insulin/IGF-1 signalling (IIS) component of the innate immune pathway modulates sensitivity to violacein-mediated killing. Further analysis shows that resistance to violacein can occur due to a loss of DAF-2 function and/or an increased function of DAF-16 controlled genes involved in antimicrobial production (spp-1) and detoxification (sod-3). These data suggest that violacein is a novel candidate antinematode agent and that the IIS pathway is also involved in the defence against metabolites from non-pathogenic bacteria.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

Genetic organization of the biosynthetic gene cluster for the indolocarbazole K-252a in <Emphasis Type="Italic">Nonomuraea longicatena</Emphasis> JCM 11136

Indolocarbazole metabolite K-252a is a natural product that was previously reported as a potent protein kinase C inhibitor with in vitro and in vivo potency. From a biosynthetic viewpoint, this compound possesses structurally interesting features such as an unusual furanosyl sugar moiety, which are absent in the well-studied staurosporine and rebeccamycin. A cosmid library from genomic DNA of Nonomuraea longicatena JCM 11136 was constructed and screened for the presence of genes to be involved in the biosynthesis of indolocarbazole K-252a. Using as a probe an internal fragment of vioB, a Chromobacterium violaceum gene encoding a multifunctional enzyme that catalyzes tryptophan decarboxylation and condensation reaction in violacein biosynthesis, we isolated a DNA region that directed the biosynthesis of K-252a when introduced into the heterologous expression host Streptomyces albus. Sequence analysis of 45 kb revealed genes for indolocarbazole core formation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and generate new indolocarbazole analogues by genetic engineering.     

A novel regulatory circuit to control indole biosynthesis protects Escherichia coli from nitrosative damages during the anaerobic respiration of nitrate

Indole is a widely distributed microbial secondary metabolite. It mediates a broad range of physiological processes in both its producing and surrounding species. Yet, indole biosynthesis during the anaerobiosis of bacteria remains largely uncharacterized. Here, we find that while indole production is promoted during fermentation and anaerobic respiration of fumarate and trimethylamine N‐oxide in E. coli, its biosynthesis is repressed during anaerobic respiration of nitrate especially during exponential growth. We show that expression of the indole biosynthetic operon tnaCAB is repressed under this condition by the two component systems NarXL and NarPQ in the global regulator FNR dependent manner. During stationary growth phase of nitrate respiration, indole biosynthesis is derepressed. However, cellular indole concentration remains low. We demonstrate that this is due to the rapid conversion of indole into mutagenic indole nitrosative derivatives under this condition. Consistent with this, a supplement of exogenous indole during nitrate respiration causes elevated mutation frequencies in E. coli cells lacking the detoxifying efflux genes mdtEF, and ectopic over‐expression of tnaAB genes decreases the fitness of E. coli to this physiological condition. Together, these results suggest that indole production is tuned to the bioenergetics activities of E. coli to facilitate its adaptation and fitness.     

Combined solid state and solution NMR studies of α,ɛ-<Superscript>15</Superscript>N labeled bovine rhodopsin

Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of α,ɛ-¹⁵N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state ¹³C,¹⁵N-REDOR and HETCOR experiments of all possible ¹³C′ _i-1 carbonyl/¹⁵N _i -tryptophan isotope labeled amide pairs, and H/D exchange ¹H,¹⁵N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone ¹⁵N nuclei and partially to their bound protons. ¹H,¹⁵N chemical shift assignment was achieved for indole side chains of Trp35^1.30 and Trp175^4.65. ¹⁵N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175^4.65 at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.     

Purple-Pigmented Violacein-Producing <Emphasis Type="Italic">Duganella</Emphasis> spp. Inhabit the Rhizosphere of Wild and Cultivated Olives in Southern Spain

Bacteria have evolved mechanisms that allow them to grow and survive in highly competitive environments like soil and the rhizosphere. Using classical microbiological, physiological, and genetic analyses, we isolated and identified for the first time Duganella spp. associated with the rhizosphere of woody plants in Mediterranean environments that are able to produce violacein, a blue–purple secondary metabolite of considerable biotechnological interest. Based on physiological and biochemical characterization and phylogenetic analysis of different genes including 16S rRNA, gyrB, and vioA (implicated in the synthesis of violacein), the seven Duganella spp. strains isolated and studied were differentiated according to their host of origin (wild versus cultivated olives) and potentially might belong to new species. All the Duganella spp. strains produced violacein in vitro, with natural production levels significantly higher than that previously reported for other violacein-producing bacteria without optimizing growing conditions. The important biological, medical, and industrial applications of violacein make these bacteria good candidates for their biotechnological exploitation because low violacein yields are considered as one of the main limitations of using wild-type strains for extensive exploitation and pigment production. Independent of violacein production, purple-pigmented strains from olives showed proteolytic and lipolytic activities and a weak siderophore production. No in vitro inhibitory activity was demonstrated for bacteria or crude violacein filtrates against plant-pathogenic Gram-negative bacteria and fungi, but they did inhibit Gram-positive bacteria.     

The molecular architecture of major enzymes from ajmaline biosynthetic pathway

The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)₈ barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new alkaloid.