Identification of the Verruculogen Prenyltransferase FtmPT3 by a Combination of Chemical,Bioinformatic and Biochemical Approaches

Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin‐type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O‐prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His₈‐FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. K_M values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 μM , respectively. Average turnover number (k_cat) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s^?1. FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13‐dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.     

Genome Mining of the Hitachimycin Biosynthetic Gene Cluster: Involvement of a Phenylalanine‐2,3‐aminomutase in Biosynthesis

Hitachimycin is a macrolactam antibiotic with (S)‐β‐phenylalanine (β‐Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β‐Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine‐2,3‐aminomutase (PAM), five polyketide synthases, four β‐amino‐acid‐carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)‐β‐Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.     

Sphinganine‐Like Biogenesis of (E)‐1‐Nitropentadec‐1‐ene in Termite Soldiers of the Genus Prorhinotermes

In 1974, (E)‐1‐nitropentadec‐1‐ene, a strong lipophilic contact poison of soldiers of the termite genus Prorhinotermes, was the first‐described insect‐produced nitro compound. However, its biosynthesis remained unknown. In the present study, we tested the hypothesis that (E)‐1‐nitropentadec‐1‐ene biosynthesis originates with condensation of amino acids with tetradecanoic acid. By using in vivo experiments with radiolabeled and deuterium‐labeled putative precursors, we show that (E)‐1‐nitropentadec‐1‐ene is synthesized by the soldiers from glycine or L ‐serine and tetradecanoic acid. We propose and discuss three possible biosynthetic pathways.     

Heterologous Biosynthesis of Fungal Indole Sesquiterpene Sespendole

Indole sesquiterpene sespendole, which has been isolated from the filamentous fungus Pseudobotrytis terrestris FKA‐25, is a specific inhibitor of lipid droplet synthesis in mouse macrophages. The biosynthetic pathway that involves genes encoding six enzymes (spdEMBQHJ) was elucidated through heterologous expression of spd genes in Aspergillus oryzae, biotransformation experiments, and in vitro enzymatic reactions with a recombinant protein, thereby revealing the mechanism underlying the characteristic modification on the indole ring, catalyzed by a set of prenyltransferase (SpdE)/cytochrome P450 (SpdJ) enzymes. Functional analysis of the homologous genes encoding these enzymes involved in the biosynthesis of lolitrem allowed a biosynthetic pathway for the bicyclic ring skeleton fused to the indole ring to be proposed.     

Synthesis of C‐Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies‐Based Biosynthetic Pathway

Carminic acid is a C‐glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C‐glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane‐bound enzymes.     

Biosynthesis of Colabomycin E,a New Manumycin‐Family Metabolite,Involves an Unusual Chain‐Length Factor

Colabomycin E is a new member of the manumycin‐type metabolites produced by the strain Streptomyces aureus SOK1/5‐04 and identified by genetic screening from a library of streptomycete strains. The structures of colabomycin E and accompanying congeners were resolved. The entire biosynthetic gene cluster was cloned and expressed in Streptomyces lividans. Bioinformatic analysis and mutagenic studies identified components of the biosynthetic pathway that are involved in the formation of both polyketide chains. Recombinant polyketide synthases (PKSs) assembled from the components of colabomycin E and asukamycin biosynthetic routes catalyzing the biosynthesis of “lower” carbon chains were constructed and expressed in S. aureus SOK1/5‐04 ΔcolC11–14 deletion mutant. Analysis of the metabolites produced by recombinant strains provided evidence that in both biosynthetic pathways the length of the lower carbon chain is controlled by an unusual chain‐length factor supporting biosynthesis either of a triketide in asukamycin or of a tetraketide in colabomycin E. Biological activity assays indicated that colabomycin E significantly inhibited IL‐1β release from THP‐1 cells and might thus potentially act as an anti‐inflammatory agent.     

Biosynthesis of Trehangelin in Polymorphospora rubra K07–0510: Identification of Metabolic Pathway to Angelyl‐CoA

Trehangelins are trehalose angelates discovered from endophytic actinomycete Polymorphospora rubra K07‐0510. We identified the trehangelin biosynthetic gene cluster, including genes that encode a glycoside hydrolase‐like protein (thgC), α‐amylase (thgD), 3‐ketoacyl‐ACP synthase III (thgI), 3‐ketoacyl‐ACP reductase (thgK), enoyl‐CoA hydratase (thgH) and acyl transferase (thgJ). Heterologous expression of thgH, thgI, thgJ and thgK confirmed the importance of these genes in the biosynthesis of trehangelin A. Enzymatic activity studies showed that ThgI catalyses the condensation of acetyl‐CoA and methylmalonyl‐CoA to 2‐methylacetoacetyl‐CoA (MAA‐CoA), ThgK catalyses NADPH‐dependent reduction of MAA‐CoA to 3‐hydroxy‐2‐methylbutyryl‐CoA (HMB‐CoA) and ThgH catalyses the dehydration of HMB‐CoA to angelyl‐CoA (AN‐CoA). This is the first report on the elucidation of the enzymatic formation of AN‐CoA.     

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.     

Aromatic Polyketide GTRI‐02 is a Previously Unidentified Product of the act Gene Cluster in Streptomyces coelicolor A3(2)

The biosynthesis of aromatic polyketides derived from type II polyketide synthases (PKSs) is complex, and it is not uncommon that highly similar gene clusters give rise to diverse structural architectures. The act biosynthetic gene cluster (BGC) of the model actinomycete Streptomyces coelicolor A3(2) is an archetypal type II PKS. Here we show that the act BGC also specifies the aromatic polyketide GTRI‐02 ( 1 ) and propose a mechanism for the biogenesis of its 3,4‐dihydronaphthalen‐1(2H)‐one backbone. Polyketide 1 was also produced by Streptomyces sp. MBT76 after activation of the act‐like qin gene cluster by overexpression of the pathway‐specific activator. Mining of this strain also identified dehydroxy‐GTRI‐02 ( 2 ), which most likely originated from dehydration of 1 during the isolation process. This work shows that even extensively studied model gene clusters such as act of S. coelicolor can still produce new chemistry, offering new perspectives for drug discovery.     

Five‐Membered Cyclitol Phosphate Formation by a myo‐Inositol Phosphate Synthase Orthologue in the Biosynthesis of the Carbocyclic Nucleoside Antibiotic Aristeromycin

Aristeromycin is a unique carbocyclic nucleoside antibiotic produced by Streptomyces citricolor. In order to elucidate its intriguing carbocyclic formation, we used a genome‐mining approach to identify the responsible enzyme. In silico screening with known cyclitol synthases involved in primary metabolism, such as myo‐inositol‐1‐phosphate synthase (MIPS) and dehydroqunate synthase (DHQS), identified a unique MIPS orthologue (Ari2) encoded in the genome of S. citricolor. Heterologous expression of the gene cluster containing ari2 with a cosmid vector in Streptomyces albus resulted in the production of aristeromycin, thus indicating that the cloned DNA region (37.5 kb) with 33 open reading frames contains its biosynthetic gene cluster. We verified that Ari2 catalyzes the formation of a novel five‐membered cyclitol phosphate from d ‐fructose 6‐phosphate (F6P) with NAD⁺ as a cofactor. This provides insight into cyclitol phosphate synthase as a member of the MIPS family of enzymes. A biosynthetic pathway to aristeromycin is proposed based on bioinformatics analysis of the gene cluster.     

Structural Diversification of Macrolactones by Substrate‐Flexible Cytochrome P450 Monooxygenases

The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C‐4 position, which is different from the intrinsic C‐12 hydroxylation position in the natural substrate. This is the first report of C‐4 hydroxylation activity of cytochrome P450 monooxygenase involved in the biosynthesis of 14‐membered macrolides. EryF hydroxylase from the erythromycin biosynthetic pathway of Saccharopolyspora erythraea and OleP oxidase from the oleandomycin biosynthetic pathway of Streptomyces antibioticus also showed a certain degree of plasticity towards alternative substrates. In particular, EryF and OleP were found to oxidize a 12‐membered macrolactone as an alternative substrate. These results demonstrate the potential usefulness of these enzymes to diversify macrolactones by post‐PKS oxidations.     

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4‐Oxoproline Synthase

X‐type actinomycins (Acms) contain 4‐hydroxyproline (Acm X₀) or 4‐oxoproline (Acm X₂) in their β‐pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4‐hydroxyproline‐ or 4‐oxoproline‐containing Acm halves. In turn, we show—using an artificial Acm half analogue (PPL 1) with proline in its peptide chain—their conversion into the 4‐hydroxyproline‐ and 4‐oxoproline‐containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host‐encoded ferredoxin reductase in a NADH (NADPH)‐dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4′′‐oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.     

Biosynthesis of the Fluorinated Natural Product Nucleocidin in Streptomyces calvus Is Dependent on the bldA‐Specified Leu‐tRNAUUA Molecule

Nucleocidin is one of the very few natural products known to contain fluorine. Mysteriously, the nucleocidin producer Streptomyces calvus ATCC 13382 has not been observed to synthesize the compound since its discovery in 1956. Here, we report that complementation of S. calvus ATCC 13382 with a functional bldA‐encoded Leu‐tRNA^UUA molecule restores the production of nucleocidin. Nucleocidin was detected in culture extracts by ¹⁹F NMR spectroscopy, HPLC‐ESI‐MS, and HPLC‐continuum source molecular absorption spectroscopy for fluorine‐specific detection. The molecule was purified from a large‐scale culture and definitively characterized by NMR spectroscopy and high‐resolution MS. The nucleocidin biosynthetic gene cluster was identified by the presence of genes encoding the 5′‐O‐sulfamate moiety and confirmed by gene disruption. Two of the genes within the nucleocidin biosynthetic gene cluster contain TTA codons, thus explaining the dependence on bldA and resolving a 60‐year‐old mystery.     

Synthetic Chain Terminators Off‐Load Intermediates from a Type I Polyketide Synthase

Modular biocatalysis is responsible for the generation of countless bioactive products and its mining remains a major focus for drug discovery purposes. One of the enduring hurdles is the isolation of biosynthetic intermediates in a readily‐analysed form. We prepared a series of nonhydrolysable pantetheine and N‐acetyl cysteamine mimics of the natural (methyl)malonyl extender units recruited for polyketide formation. Using these analogues as competitive substrates, we were able to trap and off‐load diketide and triketide species directly from an in vitro reconstituted type I polyketide synthase, the 6‐deoxyerythronolide B synthase 3 (DEBS3). The putative intermediates, which were extracted in organic solvent and characterised by LC‐HR‐ESI‐MS, are the first of their kind and prove that small‐molecule chain terminators can be used as convenient probes of the biosynthetic process.     

Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short‐chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C₂ to C₆. In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole‐cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane‐1,2‐diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)‐1,2‐PDO (1.0 % yield) or 9.6 mm (S)‐1,2‐PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.     

Parallel Post‐Polyketide Synthase Modification Mechanism Involved in FD‐891 Biosynthesis in Streptomyces graminofaciens A‐8890

To isolate a key polyketide biosynthetic intermediate for the 16‐membered macrolide FD‐891 ( 1 ), we inactivated two biosynthetic genes coding for post‐polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD‐892 ( 2 ), which lacks the epoxide moiety at C8–C9, the hydroxy group at C10, and the O‐methyl group at O‐25 of FD‐891, was isolated from the gfsF/gfsG double‐knockout mutant. In addition, 25‐O‐methyl‐FD‐892 ( 3 ) and 25‐O‐demethyl‐FD‐891 ( 4 ) were isolated from the gfsF and gfsG mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of 2 and 4 in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8‐C9 of 2 and 3 and subsequent hydroxylation at C10, to afford 4 and 1 , respectively. These results suggest that a parallel post‐PKS modification mechanism is involved in FD‐891 biosynthesis.     

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

FD‐891 is a 16‐membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL‐60 and Jurkat cells. We identified the FD‐891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A‐8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD‐891 including the oxidation states and α‐alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD‐891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD‐891 production. Instead, the gfsF gene‐disrupted mutant accumulated a novel FD‐891 analogue 25‐O‐methyl‐FD‐892, which lacked the epoxide and the hydroxyl group of FD‐891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25‐O‐methyl‐FD‐892 into FD‐891. In the course of the GfsF reaction, 10‐deoxy‐FD‐891 was isolated as an enzymatic reaction intermediate, which was also converted into FD‐891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD‐891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD‐891 in S. graminofaciens.     

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.