Taro Ozaki

NovQ is a prenyltransferase capable of catalyzing the addition of a dimethylallyl group to both phenylpropanoids and flavonoids

NovQ is a member of a recently identified CloQ/NphB class of prenyltransferases. Although NphB has been well characterized as a prenyltransferase with flexibility against aromatic substrates, few studies have been carried out on characterization of NovQ. Hence, in this study, we investigate the kinetics, substrate specificity and regiospecificity of NovQ. The corresponding novQ gene was cloned from Streptomyces niveus, which produces an aminocoumarin antibiotic, novobiocin. Recombinant NovQ was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme was a soluble monomeric 40-kDa protein that catalyzed the transfer of a dimethylallyl group to 4-hydroxyphenylpyruvate (4-HPP) independently of divalent cations to yield 3-dimethylallyl-4-HPP, an intermediate of novobiocin. Steady-state kinetic constants for NovQ with the two substrates, 4-HPP and dimethylallyl diphosphate, were also calculated. In addition to the prenylation of 4-HPP, NovQ catalyzed carbon–carbon-based and carbon–oxygen-based prenylations of a diverse collection of phenylpropanoids, flavonoids and dihydroxynaphthalenes. Despite its catalytic promiscuity, the NovQ-catalyzed prenylation occurred in a regiospecific manner. NovQ is the first reported prenyltransferase capable of catalyzing the transfer of a dimethylallyl group to both phenylpropanoids, such as p-coumaric acid and caffeic acid, and the B-ring of flavonoids. This study shows that NovQ can serve as a useful biocatalyst for the synthesis of prenylated phenylpropanoids and prenylated flavonoids.

Novel Tryptophan Metabolism by a Potential Gene Cluster That Is Widely Distributed among Actinomycetes

Background: Potential gene clusters involving indole prenyltransferase are widely distributed in actinomycetes. Results: One gene cluster was found to be responsible for 5-dimethylallylindole-3-acetonitrile biosynthesis. Conclusion: The biosynthetic route leading to 5-dimethylallylindole-3-acetonitrile represents a novel tryptophan metabolism. Significance: This result provides insight into the biosynthesis of prenylated indole derivatives that have been purified from actinomycetes. The characterization of potential gene clusters is a promising strategy for the identification of novel natural products and the expansion of structural diversity. However, there are often difficulties in identifying potential metabolites because their biosynthetic genes are either silenced or expressed only at a low level. Here, we report the identification of a novel metabolite that is synthesized by a potential gene cluster containing an indole prenyltransferase gene (SCO7467) and a flavin-dependent monooxygenase (FMO) gene (SCO7468), which were mined from the genome of Streptomyces coelicolor A3(2). We introduced these two genes into the closely related Streptomyces lividans TK23 and analyzed the culture broths of the transformants. This process allowed us to identify a novel metabolite, 5-dimethylallylindole-3-acetonitrile (5-DMAIAN) that was overproduced in the transformant. Biochemical characterization of the recombinant SCO7467 and SCO7468 demonstrated the novel l-tryptophan metabolism leading to 5-DMAIAN. SCO7467 catalyzes the prenylation of l-tryptophan to form 5-dimethylallyl-l-tryptophan (5-DMAT). This enzyme is the first actinomycetes prenyltransferase known to catalyze the addition of a dimethylallyl group to the C-5 of tryptophan. SCO7468 then catalyzes the conversion of 5-DMAT into 5-dimethylallylindole-3-acetaldoxime (5-DMAIAOx). An aldoxime-forming reaction catalyzed by the FMO enzyme was also identified for the first time in this study. Finally, dehydration of 5-DMAIAOx presumably occurs to yield 5-DMAIAN. This study provides insight into the biosynthesis of prenylated indoles that have been purified from actinomycetes.

5-Alkyl-1,2,3,4-tetrahydroquinolines, New Membrane-Interacting Lipophilic Metabolites Produced by Combined Culture of Streptomyces nigrescens and Tsukamurella pulmonis

Eight novel 5-alkyl-1,2,3,4-tetrahydroquinolines (5aTHQs) bearing different side chains have been isolated from a combined culture of Streptomyces nigrescens HEK616 and Tsukamurella pulmonis TP-B0596. The chemical structures including the absolute configuration were elucidated by spectroscopic analysis and total synthesis. 5aTHQs inhibited the growth of wild-type fission yeast while only weakly inhibiting the growth of several mutant strains synthesizing premature ergosterol. These results demonstrate that 5aTHQs are novel antifungals that may target cell membranes.

Cyclolavandulyl skeleton biosynthesis via both condensation and cyclization catalyzed by an unprecedented member of the cis-isoprenyl diphosphate synthase superfamily.

A cyclolavandulyl group is a C10 monoterpene with a branched and cyclized carbon skeleton. This monoterpene is rarely found in nature, and its biosynthesis is poorly understood. To determine the biosynthesis mechanism of this monoterpene, we sequenced the genome of Streptomyces sp. CL190, which produces lavanducyanin, a phenazine with an N-linked cyclolavandulyl structure. Sequencing and homology searches identified one candidate gene product that consists of only a cis-isoprenyl diphosphate synthase domain. Disruption of the gene and biochemical analysis of the recombinant enzyme demonstrated that the enzyme synthesized a cyclolavandulyl diphosphate essential for the biosynthesis of lavanducyanin. This enzyme is an unprecedented terpene synthase that catalyzes both the condensation of the C5 isoprene units and subsequent cyclization to form the cyclolavandulyl monoterpene structure.

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.

Insights into the biosynthesis of dehydroalanines in goadsporin

Dehydroalanines in goadsporin are proposed to be formed by GodF and GodG, which show slight homology to the N‐terminal glutamylation and C‐terminal elimination domains, respectively, of LanB, a class I lanthipeptide dehydratase. Although similar, separated‐type LanBs are conserved among thiopeptides and indispensable for their biosynthesis and biological activities, these enzymes had not yet been characterized. Here, we identified goadsporin B, which has unmodified Ser4 and Ser14, from both godF and godG disruptants. The godG disruptant also produced goadsporin C, a glutamylated‐Ser4 variant of goadsporin B. These results suggested that dehydroalanines are formed by glutamylation and glutamate elimination. NMR analysis revealed for the first time that the glutamyl group was attached to a serine via an ester bond, by the catalysis of LanB‐type enzymes. Our findings provide insights into the function of separated‐type LanBs involved in the biosynthesis of goadsporin and thiopeptides.

Structure of the quinoline N‐hydroxylating cytochrome P450 RauA, an essential enzyme that confers antibiotic activity on aurachin alkaloids

The cytochrome P450 RauA from Rhodococcus erythropolis JCM 6824 catalyzes the hydroxylation of a nitrogen atom in the quinolone ring of aurachin, thereby conferring strong antibiotic activity on the aurachin alkaloid. Here, we report the crystal structure of RauA in complex with its substrate, a biosynthetic intermediate of aurachin RE. Clear electron density showed that the quinolone ring is oriented parallel to the porphyrin plane of the heme cofactor, while the farnesyl chain curls into a U‐shape topology and is buried inside the solvent‐inaccessible hydrophobic interior of RauA. The nearest atom from the heme iron is the quinolone nitrogen (4.3 Å), which is consistent with RauA catalyzing the N‐hydroxylation of the quinolone ring to produce mature aurachin RE.

Genetic approaches to generate hyper-producing strains of goadsporin: the relationships between productivity and gene duplication in secondary metabolite biosynthesis

Improving the productivity of secondary metabolites is highly beneficial for the utilization of natural products. Here, we found that gene duplication of the goadsporin biosynthetic gene locus resulted in hyper-production. Goadsporin is a linear azole containing peptide that is biosynthesized via a ribosome-mediated pathway in Streptomyces sp. TP-A0584. Recombinant strains containing duplicated or triplicated goadsporin biosynthetic gene clusters produced 1.46- and 2.25-fold more goadsporin than the wild-type strain. In a surrogate host, Streptomyces lividans, chromosomal integration of one or two copies of the gene cluster led to 342.7 and 593.5 mg/L of goadsporin production. Expression of godI, a self-resistance gene, and of godR, a pathway-specific transcriptional regulator, under a constitutive promoter gave 0.79- and 2.12-fold higher goadsporin production than the wild-type strain. Our experiments indicated that a proportional relationship exists between goadsporin production per culture volume and the copy number of the biosynthetic gene cluster. Graphical Abstract Proportional relationship exists between Streptomyces antibiotic production and the copy number of the biosynthetic gene cluster.

Mycolic acid-containing bacteria activate heterologous secondary metabolite expression in Streptomyces lividans

Mycolic acid-containing bacteria activate heterologous secondary metabolite expression in Streptomyces lividans

Focused Genome Mining of Structurally Related Sesterterpenes: Enzymatic Formation of Enantiomeric and Diastereomeric Products

Heterologous expression of four clade-A bifunctional terpene synthases (BFTSs), giving di/sesterterpenes with unique polycyclic carbon skeletons such as sesterfisherol, enabled the isolation of the sesterterpenes Bm1, Bm2, Bm3, and Pb1. Their structures suggested that formation of the products occurs via various diastereomeric carbocation intermediates, allowing the proposal that clade-A BFTSs catalyze three-step cyclizations using several stereofacial combinations of allylic cation-olefin pairs in the corresponding intermediates to generate various stereoisomers.