Identification and Characterization of a Welwitindolinone Alkaloid Biosynthetic Gene Cluster in the Stigonematalean Cyanobacterium Hapalosiphon welwitschii

The identification of a 36 kb welwitindolinone (wel) biosynthetic gene cluster in Hapalosiphon welwitschii UTEX B1830 is reported. Characterization of the enzymes responsible for assembling the early biosynthetic intermediates geranyl pyrophosphate and 3‐((Z)‐2′‐isocyanoethenyl)indole as well as a dedicated N‐methyltransferase in the maturation of N‐methylwelwitindolinone C isothiocyanate solidified the link between the wel pathway and welwitindolinone biosynthesis. Comparative analysis of the ambiguine and welwitindolinone biosynthetic pathways in two different organisms provided insights into the origins of diverse structures within hapalindole‐type molecules.     

Gene Discovery in Gelsemium Highlights Conserved Gene Clusters in Monoterpene Indole Alkaloid Biosynthesis

Genome mining is a routine technique in microbes for discovering biosynthetic pathways. In plants, however, genomic information is not commonly used to identify novel biosynthesis genes. Here, we present the genome of the medicinal plant and oxindole monoterpene indole alkaloid (MIA) producer Gelsemium sempervirens (Gelsemiaceae). A gene cluster from Catharanthus roseus, which is utilized at least six enzymatic steps downstream from the last common intermediate shared between the two plant alkaloid types, is found in G. sempervirens, although the corresponding enzymes act on entirely different substrates. This study provides insights into the common genomic context of MIA pathways and is an important milestone in the further elucidation of the Gelsemium oxindole alkaloid pathway.     

Reevaluation of the violacein biosynthetic pathway and its relationship to indolocarbazole biosynthesis

The biosynthetic pathways for violacein and for indolocarbazoles (rebeccamycin, staurosporine) include a decarboxylative fusion of two tryptophan units. However, in the case of violacein, one of the tryptophans experiences an unusual 1-->2 shift of the indole ring. The violacein biosynthetic gene cluster was previously reported to consist of four genes, vioABCD. Here we studied the violacein pathway through expression of vio genes in Escherichia coli and Streptomyces albus. A pair of genes (vioAB), responsible for the earliest steps in violacein biosynthesis, was functionally equivalent to the homologous pair in the indolocarbazole pathway (rebOD), directing the formation of chromopyrrolic acid. However, chromopyrrolic acid appeared to be a shunt product, not a violacein intermediate. In addition to vioABCD, a fifth gene (vioE) was essential for violacein biosynthesis, specifically for production of the characteristic 1-->2 shift of the indole ring. We also report new findings on the roles played by the VioC and VioD oxygenases, and on the origin of violacein derivatives of the chromoviridans type.     

Identification and Characterization of the Streptazone E Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08

Streptazone derivatives isolated from Streptomyces species are piperidine alkaloids with a cyclopenta[b]pyridine scaffold. Previous studies indicated that these compounds are polyketides, but the biosynthetic enzymes responsible for their synthesis are unknown. Here, we have identified the streptazone E biosynthetic gene cluster in Streptomyces sp. MSC090213JE08, which encodes a modular type I PKS and tailoring enzymes that include an aminotransferase, three oxidoreductases, and two putative cyclases. The functions of the six tailoring enzymes were analyzed by gene disruption, and two putative biosynthetic intermediates that accumulated in particular mutants were structurally elucidated. On the basis of these results, we propose a pathway for the biosynthesis of streptazone E in which the two putative cyclases of the nuclear transport factor 2–like superfamily are responsible for C?C bond formation coupled with epoxide ring opening to give the five‐membered ring of streptazone E.     

Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Thienodolin (THN) features a tricyclic indole‐S‐hetero scaffold that encompasses two unique carbon–sulfur bonds. Although its biosynthetic gene cluster has been recently identified in Streptomyces albogriseolus, the essential enzymes for the formation of C?S bonds have been relatively unexplored. Here, we isolated and characterized a new biosynthetic gene cluster from Streptomyces sp. FXJ1.172. Heterologous expression, systematic gene inactivation, and in vitro biochemical characterization enable us to determine the minimum set of genes for THN synthesis, and an aminotransferase (ThnJ) for catalyzing the downstream conversion of tryptophan chlorination. In addition, we evaluated (and mainly excluded) a previously assumed pivotal intermediate by feeding experiments. With these results, we narrowed down four enzymes (ThnC–F) that are responsible for the two unprecedented C?S bond formations. Our study provides a solid basis for further unraveling of the unique C?S mechanisms.     

Biochemical Investigations of Two 6‐DMATS Enzymes from Streptomyces Reveal New Features of L‐Tryptophan Prenyltransferases

Two putative prenyltransferase genes, SAML0654 and Strvi8510, were identified in Streptomyces ambofaciens and Streptomyces violaceusniger, respectively. Their deduced products share 63 % sequence identity. Biochemical investigations with recombinant proteins demonstrated that L ‐tryptophan and derivatives, including D ‐tryptophan, 4‐, 5‐, 6‐ and 7‐methyl‐dl ‐tryptophan, were well accepted by both enzymes in the presence of DMAPP. Structural elucidation of the isolated products revealed regiospecific prenylation at C‐6 of the indole ring and proved unequivocally the identification of two very similar 6‐dimethylallyltryptophan synthases (6‐DMATS). Detailed biochemical investigations with SAML0654 proved L ‐tryptophan to be the best substrate (K_m 18 μm, turnover 0.3 s^?1). Incubation with different prenyl donors showed that they also accepted GPP and catalyzed the same specific prenylation. Utilizing GPP as a prenyl donor has not been reported for tryptophan prenyltransferases previously. Both enzymes also catalyzed prenylation of some hydroxynaphthalenes; this has not previously been described for bacterial indole prenyltransferases. Interestingly, SAML0654 transferred prenyl moieties onto the unsubstituted ring of hydroxynaphthalenes.     

Cloning and Heterologous Expression of the Aurachin RE Biosynthesis Gene Cluster Afford a New Cytochrome P450 for Quinoline N‐Hydroxylation

Aurachin RE is a prenylated quinoline antibiotic that was first isolated from the genus Rhodococcus. It shows potent antibacterial activity against a variety of Gram‐positive bacteria. Here we have identified a minimal biosynthesis gene cluster for aurachin RE in Rhodococcus erythropolis JCM 6824 by using random transposon mutagenesis and heterologous production. The Rhodococcus aurachin (rau) gene cluster consists of genes encoding cytochrome P450 (rauA), prenyltransferase, polyketide synthase, and farnesyl pyrophosphate synthase, as well as others including genes involved in regulation and transport. Markerless gene disruption of rauA resulted in the complete loss of aurachin RE production and in the accumulation of a new aurachin derivative lacking the N‐hydroxy group. When the recombinant RauA was expressed in Escherichia coli, it catalyzed N‐hydroxylation of the derivative to form aurachin RE. This study establishes the biosynthetic pathway of aurachin RE and provides experimental evidence for the role of P450 RauA in catalyzing N‐hydroxylation of the quinoline ring, which is indispensable for the antibacterial activity of aurachin RE.     

Biosynthetic Machinery of Diterpene Pleuromutilin Isolated from Basidiomycete Fungi

The diterpene pleuromutilin is a ribosome‐targeting antibiotic isolated from basidiomycete fungi, such as Clitopilus pseudo‐pinsitus. The functional characterization of all biosynthetic enzymes involved in pleuromutilin biosynthesis is reported and a biosynthetic pathway proposed. In vitro enzymatic reactions and mutational analysis revealed that a labdane‐related diterpene synthase, Ple3, catalyzed two rounds of cyclization from geranylgeranyl diphosphate to premutilin possessing a characteristic 5–6–8‐tricyclic carbon skeleton. Biotransformation experiments utilizing Aspergillus oryzae transformants possessing modification enzyme genes allowed the biosynthetic pathway from premutilin to pleuromutilin to be proposed. The present study sets the stage for the enzymatic synthesis of natural products isolated from basidiomycete fungi, which are a prolific source of structurally diverse and biologically active terpenoids.     

Post‐translational Modification in Microviridin Biosynthesis

Cyanobacteria are prolific producers of bioactive natural products that mostly belong to the nonribosomal peptide and polyketide classes. We show here how a linear precursor peptide of microviridin K, a new member of the microviridin class of peptidase inhibitors, is processed to become the mature tricyclic peptidase inhibitor. The microviridin (mvd) biosynthetic gene cluster of P. agardhii comprises six genes encoding microviridin K, an apparently unexpressed second microviridin, two RimK homologues, an acetyltransferase, and an ABC transporter. We have over‐expressed three enzymes of this pathway and have demonstrated their biochemical function in vitro through chemical degradation and mass spectrometry. We show that a prepeptide undergoes post‐translational modification through cross‐linking by ester and amide bond formation by the RimK homologues MvdD and MvdC, respectively. In silico analysis of the mvd gene cluster suggests the potential for widespread occurrence of microviridin‐like compounds in a broad range of bacteria.     

Biosynthesis of Hygrocins,Antitumor Naphthoquinone Ansamycins Produced by Streptomyces sp. LZ35

Hygrocins are naphthoquinone ansamycins with significant antitumor activities. Here, we report the identification and characterization of the hygrocin biosynthetic gene cluster (hgc) in Streptomyces sp. LZ35. A biosynthetic pathway is proposed based on bioinformatics analysis of the hgc genes and intermediates accumulated in selected gene disruption mutants. One of the steps during the biosynthesis of hygrocins is a Baeyer–Villiger oxidation between C5 and C6, catalyzed by luciferase‐like monooxygenase homologue Hgc3. Hgc3 represents the founding member of a previously uncharacterized family of enzymes acting as Baeyer–Villiger monooxygenases.     

Biosynthetic and Functional Color–Scent Associations in Flowers of Papaver nudicaule and Their Impact on Pollinators

Despite increasing evidence for biosynthetic connections between flower pigments and volatile compounds, examples of such relationships in polymorphic plant species remains limited. Herein, color–scent associations in flowers from Papaver nudicaule (Papaveraceae) have been investigated. The spectral reflectance and scent composition of flowers of four color cultivars was determined. We found that pigments and volatiles occur in specific combinations in flowers of P. nudicaule. The presence of indole in the bouquets is strongly associated with the occurrence of yellow pigments called nudicaulins, for which indole is one of the final biosynthetic precursors. Whereas yellow flowers emit an excess of indole, orange flowers consume it during nudicaulin production and lack the substance in their bouquet. By using the honeybee, Apis mellifera, evaluations were made on how color and scent affect the discrimination of these flowers by pollinators. Honeybees were able to discriminate artificial odor mixtures resembling those of the natural flower odors. Bees trained with stimuli combining colors and odors showed an improved discrimination performance. The results indicate that the indole moiety of nudicaulins and emitted indole might be products of the same biochemical pathway. We propose that conserved pathways account for the evolution of color–scent associations in P. nudicaule and that these associations positively affect flower constancy of pollinators.     

A Unique Amino Transfer Mechanism for Constructing the β‐Amino Fatty Acid Starter Unit in the Biosynthesis of the Macrolactam Antibiotic Cremimycin

Cremimycin is a 19‐membered macrolactam glycoside antibiotic based on three distinctive substructures: 1) a β‐amino fatty acid starter moiety, 2) a bicyclic macrolactam ring, and 3) a cymarose unit. To elucidate the biosynthetic machineries responsible for these three structures, the cremimycin biosynthetic gene cluster was identified. The cmi gene cluster consists of 33 open reading frames encoding eight polyketide synthases, six deoxysugar biosynthetic enzymes, and a characteristic group of five β‐amino‐acid‐transfer enzymes. Involvement of the gene cluster in cremimycin production was confirmed by a gene knockout experiment. Further, a feeding experiment demonstrated that 3‐aminononanoate is a direct precursor of cremimycin. Two characteristic enzymes of the cremimycin‐type biosynthesis were functionally characterized in vitro. The results showed that a putative thioesterase homologue, CmiS1, catalyzes the Michael addition of glycine to the β‐position of a non‐2‐enoic acid thioester, followed by hydrolysis of the thioester to give N‐carboxymethyl‐3‐aminononanoate. Subsequently, the resultant amino acid was oxidized by a putative FAD‐dependent glycine oxidase homologue, CmiS2, to produce 3‐aminononanoate and glyoxylate. This represents a unique amino transfer mechanism for β‐amino acid biosynthesis.     

Biosynthesis of the Fungal Organophosphonate Fosfonochlorin Involves an Iron(II) and 2-(Oxo)glutarate Dependent Oxacyclase

The fungal metabolite Fosfonochlorin features a chloroacetyl moiety that is unusual within known phosphonate natural product biochemistry. Putative biosynthetic genes encoding Fosfonochlorin in Fusarium and Talaromyces spp. were investigated through reactions of encoded enzymes with synthetic substrates and isotope labelling studies. We show that the early biosynthetic steps for Fosfonochlorin involve the reduction of phosphonoacetaldehyde to form 2-hydroxyethylphosphonic acid, followed by oxidative intramolecular cyclization of the resulting alcohol to form (S)-epoxyethylphosphonic acid. The latter reaction is catalyzed by FfnD, a rare example of a non-heme iron/2-(oxo)glutarate dependent oxacyclase. In contrast, FfnD behaves as a more typical oxygenase with ethylphosphonic acid, producing (S)-1-hydroxyethylphosphonic acid. FfnD thus represents a new example of a ferryl generating enzyme that can suppress the typical oxygen rebound reaction that follows abstraction of a substrate hydrogen by a ferryl oxygen, thereby directing the substrate radical towards a fate other than hydroxylation.     

Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP‐free intermediate towards the final metabolite. However, the timing of ACP off‐loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone‐2‐carboxylic acid‐N‐acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)‐DNPA, and the product of ActIV reaction was characterized as an ACP‐free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC‐TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.     

Two pathways for pyrrole formation in coumermycin A(1) biosynthesis: the central pyrrole moiety is formed from L-threonine

Coumermycin A₁ is an aminocoumarin antibiotic produced by Streptomyces rishiriensis. It contains three pyrrole rings, that is, two terminal 5‐methyl‐pyrrole‐2‐carboxyl moieties and a central 3‐methylpyrrole‐2,4‐dicarboxylic acid moiety. The biosynthesis of the terminal pyrrole moieties has been elucidated previously. However, the biosynthetic precursors of the central pyrrole moiety have remained unknown, and none of the genes or enzymes involved in its formation has been identified. We now show that five genes, contained in a contiguous 4.7 kb region within the coumermycin biosynthetic gene cluster, are required for the biosynthesis of this central pyrrole moiety. Each of these genes was deleted individually, resulting in a strong reduction or an abolishment of coumermycin production. External feeding of the central pyrrole moiety restored coumermycin production. One of these genes shows similarity to L ‐threonine kinase genes. Feeding of [U‐¹³C,¹⁵N]L ‐threonine and ¹³C NMR analysis of the resulting compound unequivocally proved that threonine was incorporated intact into the central pyrrole (19 % enrichment) to provide the heterocyclic nitrogen as well as four of the seven carbons of this moiety. Therefore, this pyrrole is formed via a new, hitherto unknown biosynthetic pathway. A hypothesis for the reaction sequence leading to the central pyrrole moiety of coumermycin A₁ is presented.     

Characterization of Enzymes Catalyzing Transformations of Cysteine S‐Conjugated Intermediates in the Lincosamide Biosynthetic Pathway

Lincosamides such as lincomycin A, celesticetin, and Bu‐2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l ‐proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5′‐phosphate‐dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S‐conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C?S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu‐2545. The substrates used in these studies are the β‐anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved.     

Molecular Genetics of Plant Sterol Backbone Synthesis

Sterols, which are biosynthesized via the cytoplasmic mevalonate (MVA) pathway, are important structural components of the plasma membrane and precursors of steroid hormones in both vertebrates and plants. Ergosterol and cholesterol are the major sterols in yeast and vertebrates, respectively. In contrast, plants produce a wide variety of phytosterols, which have various functions in plant development. Although the general biosynthetic pathway to plant sterols has been defined, the details of the biochemical, physiological, and developmental functions of genes involved in the biosynthetic network and their regulation are not well understood. Molecular genetic analyses are an effective approach to use when studying these fascinating problems. Since three enzymes, 3-hydroxy-3-methylglutaryl CoA reductase, farnesyl diphosphate synthase, and lanosterol synthase, have been functionally characterized in planta, we reviewed recent progress on these enzymes. Arabidopsis T-DNA and transposon insertion mutants are now widely available. The use of molecular genetics, molecular biology, and bioorganic chemical approaches on these mutants, as well as inhibitors of the MVA pathway, should help us to understand plant sterol biosynthesis comprehensively.