Identification of Middle Chain Fatty Acyl-CoA Ligase Responsible for the Biosynthesis of 2-Alkylmalonyl-CoAs for Polyketide Extender Unit

Background: Fatty acyl-CoA ligases involved in polyketide biosynthesis remain uncharacterized.

Results: RevS classified in fatty acyl-AMP ligase clade was the middle chain fatty acyl-CoA ligase.

Conclusion: RevS was responsible for 2-alkylmalonyl-CoA biosynthesis through enzyme coupling with RevT carboxylase/reductase.

Significance: 2-Alkylmalonyl-CoA biosynthesis was strongly supported by the function of RevR and RevS, which utilized fatty acids derived from de novo biosynthesis and degradation products, respectively.

Abstract

Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important for the development of bioactive natural products. Reveromycin (RM) derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA carboxylase/reductase homolog. We mainly focused on the uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM derivatives, whereas reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed the middle chain fatty acyl-CoA ligase (FACL) but not the fatty acyl-AMP ligase activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty acid into 2-alkylmalonyl-CoA using purified RevS and RevT. The coupling reaction showed efficient conversion of hexenoic acid into butylmalonyl-CoA. RevS efficiently catalyzed C8–C10 middle chain FACL activity; therefore, we speculated that the acyl-CoA precursor was truncated via β-oxidation and converted into (E)-2-enoyl-CoA, a RevT substrate. To determine whether the β-oxidation process is involved between the RevS and RevT reaction, we performed the feeding experiment using [1,2,3,4-¹³C]octanoic acid. ¹³C NMR analysis clearly demonstrated incorporation of the [3,4-¹³C]octanoic acid moiety into the structure of RM-A. Our results provide insight into the role of uncharacterized RevS homologs that may catalyze middle chain FACL to produce a unique polyketide extender unit.

Introduction

Microorganisms harbor structurally diverse natural products, including polyketides, peptides, and terpenoids (1). Their secondary metabolites with strong biological activity have been utilized as antibiotics, antitumor drugs, immunosuppressants, hypercholesterolemia drugs, and insecticides (2). Unique chemical structures are linked to a variety of biological activities; therefore, understanding the biosynthetic machinery is important for future combinatorial biosynthesis (3–5).

Fatty acyl chains found in microbial natural products are responsible for biological activity and physiological function in microorganisms. For instance, daptomycin binds to the cell membranes of Gram-positive bacteria through its lipid moiety, followed by calcium-dependent insertion and oligomerization (6). Dimerization of alkyl resorcinol results in the generation of potent proteasome inhibitors, cylindrocyclophanes (7). The fatty acyl chain of mycolic acid plays an important role in membrane construction in Mycobacterium tuberculosis (8).

FAAL,² a member of the adenylate-forming enzyme superfamily (9), catalyzes ATP-dependent formation of acyl-AMP and loading onto the phosphopantetheine arm of the acyl carrier protein (ACP). FAALs are associated with the polyketide synthase (PKS) and nonribosomal peptide synthetase gene clusters, and the resulting fatty acyl-ACP is incorporated into the starter unit of the PKS and nonribosomal peptide synthetase assembly line (7, 10–21). In contrast to FAAL, fatty acyl-CoA ligase (FACL) catalyzes ATP-dependent conversion of fatty acid into fatty acyl-CoA through an acyl-AMP intermediate. The activated fatty acyl-CoA is utilized for phospholipid synthesis, energy generation, transcriptional regulation, and protein acylation (22, 23). However, involvement of FACL in the supply of available fatty acyl-CoA for PKS extender unit remains unclear. Atypical extender units are incorporated into polyketide structures through a variety of biosynthetic pathways (24–32). Among the related enzymes, crotonyl-CoA carboxylase/reductase (CCR) homologs, which catalyze reductive carboxylation of α,β-unsaturated acyl-CoA/ACP, have been extensively characterized as an essential step (Fig. 1A).

FIGURE 1.

Biosynthetic genes responsible for polyketide extender units of RM-A. A, polyketide natural products containing atypical extender units. Bold red type indicates carbon chains derived from atypical extender units. B, gene organization. Based on the presence of the revR, revS, and revT homologs, each gene cluster was categorized into four groups as follows: revR, revS, and revT homologs (panel I); revR and revT homologs (panel II); revT homolog with additional genes related to atypical extender unit biosynthesis (panel III); and revS homolog with putative propionyl-CoA carboxylase β-subunit homolog (panel IV). C, scheme of the possible 2-alkylmalonyl-CoA biosynthesis by RevR, RevS, and RevT. Both fatty acid degradation and de novo fatty acid biosynthetic pathway were predicted to be involved in 2-alkylmalonyl-CoA biosynthesis.

Reveromycin A (RM-A) is a polyketide compound produced by Streptomyces sp. SN-593 (33). It inhibited bone resorption by inducing apoptosis in osteoclasts (34). The activity was also linked to the inhibition of osteolytic bone metastasis induced by lung and prostate cancer cells (34–37). Streptomyces sp. SN-593 produces various RM derivatives, including RM-A (compound 1), -C (compound 2), -D (compound 3), and -E (compound 4) (Fig. 1A), whose structural diversity is derived from incorporation of atypical extender units such as butylmalonyl-CoA, isobutylmalonyl-CoA, pentylmalonyl-CoA, and hexylmalonyl-CoA, respectively. In our previous study, we identified compound 1 biosynthetic gene cluster possessing RevT, a CCR homolog (38). Although the presence of the revT gene strongly suggested the production of 2-alkylmalonyl-CoA for RM biosynthesis, we identified the revR and revS genes, which are associated in the same transcriptional unit (Fig. 1B) (38). Moreover, each gene homolog was also found in several biosynthetic gene clusters that produce polyketide compounds, including the atypical extender unit (Fig. 1) (27–32). Thus, we predicted an important role for the production of 2-alkylmalonyl-CoA by RevR and RevS, whose genes were annotated to be β-ketoacyl-(acyl carrier protein (ACP)) synthase III (KASIII) and adenylate-forming enzyme family, respectively.

In this study, we examined the molecular basis of 2-alkylmalonyl-CoA formation for RM biosynthesis through gene disruption, kinetic analysis, and precursor feeding experiments. Our results indicated that RevR is important for selective production of compound 1 via de novo fatty acid synthesis and that RevS may represent a novel class of FACLs associated with the fatty acid degradation process, providing insight into the dynamics of fatty acid metabolism for polyketide biosynthesis. Moreover, the identification of RevS function sheds light on unexplained RevS homologs found in other polyketide biosynthetic gene clusters.

Experimental Procedures

Chemicals and Enzymes

Ampicillin, kanamycin, chloramphenicol, and CoA were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Streptomycin, spectinomycin, thiostrepton, ATP, NADH, NADPH, tris(2-carboxyethyl)phosphine (TCEP), myokinase, pyruvate kinase, and lactate dehydrogenase were purchased from Sigma. All fatty acids used in this study were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Carumonam was purchased from Takeda Pharmaceutical Co., Ltd. (Tokyo, Japan). [1-¹³C]Hexanoic acid and [1,2,3,4-¹³C]octanoic acid were purchased from Cambridge Isotope Laboratories, Inc. (Cambridge, MA). All commercially available chemicals for chemical synthesis were used without further purification.

Analytical Methods

The ¹H and ¹³C NMR spectra of compound 1 and CoA derivatives were recorded on JEOL JNM-ECA-500 and JNM-AL-400 spectrometers (JEOL, Ltd., Tokyo, Japan). Chemical shifts (in ppm) were referenced against the residual solvent for ¹H NMR and 3-(trimethylsilyl)propionic-2,2,3,3d₄ acid sodium salt (TSP-d₄) as an external standard for ¹³C NMR. UV absorbance was measured using a Jasco V-630 BIO spectrophotometer (Jasco Co., Tokyo, Japan). Fast atom bombardment-mass spectra were obtained using a JMS-700 mass spectrometer. Electrospray ionization (ESI) mass spectra of CoA derivatives were obtained on a BioApex-II Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics Japan, Ltd., Tokyo, Japan) and Synapt G2 (Waters, MA). ESI-MS analysis of RM derivatives and RevS and RevT reaction products were performed using a Waters Alliance HPLC system equipped with a mass spectrometer (Q-TRAP, Applied Biosystems, CA).

Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/mass spectrometry (MS) analysis of acyl-ACP was performed using ultrafleXtreme^TM (Bruker Daltonics, Billerica, MA). Sinapinic acid was used as a matrix.

All chemical syntheses were carried out under a nitrogen atmosphere and monitored by TLC with 0.25-mm pre-coated Silica Gel plates 60F254 Art 5715 (Merck, Darmstadt, Germany). Visualization was achieved using UV light and 10% ethanol solution of phosphomolybdic acid, followed by heating. For column chromatography, Silica Gel 60 N (spherical, neutral, 0.04–0.05 mm; Kanto Chemical Co, Inc., Tokyo, Japan) and Cosmosil 75C18-DNP (Nacalai Tesque Inc., Kyoto, Japan) were utilized.

Bacterial Strains and Plasmids

Escherichia coli DH5α (Takara Bio Inc., Shiga, Japan) and E. coli GM2929 hsdS::Tn10 carrying pUB307-aph::Tn7 were used for general DNA manipulation and E. coli-Streptomyces sp. SN-593 conjugation, respectively (39). E. coli BL21 Star^TM (DE3) (Invitrogen) and Streptomyces lividans TK23 were to prepare the recombinant protein, and pET28b(+) (Novagen, Madison, WI) and pWHM3 (40) were used as expression vectors, respectively.

Culture Conditions

E. coli strains were grown at 37 °C in LB (1% Bacto-tryptone (BD Biosciences), 0.5% yeast extract (BD Biosciences), and 1% NaCl), LB agar Miller (Nacalai Tesque, Inc., Kyoto, Japan), or Terrific broth (TB) (1.2% (w/v) Bacto-tryptone, 2.4% (w/v) yeast extract, 0.4% (v/v) glycerol, 0.231% (w/v) KH₂PO₄, and 1.254% (w/v) K₂HPO₄). To select for plasmid-containing cells, 100 μg ml⁻¹ ampicillin, 50 μg ml⁻¹ kanamycin, 30 μg ml⁻¹ chloramphenicol, 50 μg ml⁻¹ streptomycin, or 100 μg ml⁻¹ spectinomycin were added to an LB plate. Streptomyces sp. SN-593 culture conditions were described previously (38). S. lividans TK23 cells were cultured at 28 °C in SK2 medium (1.4% (w/v) soluble starch, 0.35% (w/v) glucose, 0.35% (w/v) yeast extract, 0.21% (w/v) Bacto-peptone, 0.21% (w/v) beef extract (Remel Inc., KS), 0.014% (w/v) KH₂PO₄, and 0.042% (w/v) MgSO₄, pH 7.0).

Plasmid Construction for Gene Disruption and Complementation

The revR, revS, and revT gene disruptions were performed by PCR-targeted gene replacement using the plasmids pKD46, pKD13, and pCP20 and E. coli BW25113 (41, 42). Ampicillin-resistant pKD46 containing the λ Red-mediated recombination functions was used with the chloramphenicol-resistant pCC1FOS fosmid clone 3C11 containing the revR, revS, and revT genes. The plasmid pKD13 was used as a template for the FRT-flanked kanamycin-resistant gene cassette (42, 43).

To construct the revR, revS, and revT gene replacement plasmids, DNA fragments containing the disrupted gene were amplified by PCR using PrimeSTAR®HS DNA polymerase (Takara Bio). PCR conditions were as follows: 98 °C for 10 s, 25 cycles of 98 °C for 10 s, 62 °C for 5 s, and 68 °C for 5 min. The amplified fragments were ligated to the HindIII site of the pIM vector (38). For Southern analysis (Fig. 2), the AlkPhos Direct Labeling and Detection System (GE Healthcare, Little Chalfont, UK) was used. The primers used for gene disruptions and Southern analysis are shown in Table 1.

FIGURE 2.

Gene disruption and Southern hybridization. A, scheme of revR gene disruption and restriction enzyme map of wild-type and ΔrevR gene mutant strains. The bar shows the expected fragment size (bp) following NcoI and SalI digestion of genomic DNA. The bold bar shows the 3608-bp probe. B, Southern hybridization of genomic DNA from wild-type and revR gene disruptants. C, scheme of revS gene disruption and a restriction enzyme map of wild-type and ΔrevS gene mutant strains. The bar shows the expected fragment size (bp) after ApaLI and BalI digestion of genomic DNA. The bold bar shows the 5180-bp probe. D, Southern hybridization of genomic DNA from wild-type and revS gene disruptant. E, scheme of revT gene disruption and restriction enzyme map of wild-type and ΔrevT gene mutant strains. The bar shows the expected fragment size (bp) following EcoRI and NotI digestion of genomic DNA. The bold bar shows 5399-bp probe. F, Southern hybridization of genomic DNA from wild-type and revT gene disruptant. Closed and open triangles show DNA fragments from wild-type and mutant strains, respectively.

TABLE 1.

Primers used in this study

Underlines (6–14 and 39 bp) show restriction enzyme sites and homologous sequences to the target DNA region, respectively.

To obtain the gene complementation plasmid, the revR, revS, and revT genes were ligated into the BamHI and HindIII sites of pTYM19 with an aphII promoter (38, 44). Because the revR gene includes a BamHI site in the sequence, a silent mutation was introduced using the QuikChange protocol (Stratagene, La Jolla, CA). The revR gene inserted into the NdeI/XhoI restriction sites of the pET28b vector was amplified using PfuTurbo DNA polymerase (Stratagene) and the following primer pairs (with the mutation sites underlined): 5′-TCCAGCGACGAGTGGATACGCCGGCACTCCGGGAT-3′ and 5′-ATCCCGGAGTGCCGGCGTATCCACTCGTCGCTGGA-3′. PCRconditions were as follows: 98 °C for 30 s, 20 cycles of 98 °C for 30 s, 60 °C for 1 min, and 68 °C for 13 min. The resultant pET28b-revR-BamHI_mut vector was used as a template for the complementation plasmid (Table 1).

Plasmid Construction for Heterologous Gene Expression in E. coli

The revS (1740 bp) and revT (1332 bp) genes were amplified from the fosmid clone (PCC1FOS-3C11) using PrimeSTAR®HS DNA polymerase under the following conditions: 98 °C for 10 s and 25 cycles of 98 °C for 10 s, 62 °C for 5 s, and 68 °C for 1.5 min. The SRE2849 gene (243 bp) coding ACP of Streptomyces sp. SN-593 was amplified from PCC1FOS-4B1 under the following conditions: 98 °C for 10 s and 25 cycles of 98 °C for 10 s, 60 °C for 5 s, and 68 °C for 25 s. The Bacillus subtilis 168 phosphopantetheinyl transferase gene (sfp) (654 bp) was amplified from pUC19-sfp under the following conditions: 98 °C for 10 s and 25 cycles of 98 °C for 10 s, 62 °C for 5 s, and 68 °C for 30 s (45). The primers used for amplification are shown in Table 1. After restriction enzyme digestion, the revS, revT, SRE2849, and sfp gene fragments were inserted into the NdeI and XhoI sites of pET28b(+) or pACYCDuet-1 to construct pET28b-revS, pET28b-revT, pET28b-SRE2849, and pACYCDuet-1-sfp, respectively.

Plasmid Construction for Heterologous Gene Expression in S. lividans TK23

The β-lactamase gene in the pWHM3 vector was replaced with the aphI gene using λRed recombination to form pWK. Next, the tipA promoter (P_tipA) was inserted into the EcoRI and BamHI sites to construct pWK-P_tipA for expression in S. lividans TK23. To facilitate enzyme purification, the revT gene containing a His₈ tag coding sequence was amplified from pET28b-revT using the primers shown in Table 1. After restriction enzyme digestion, the fragment was inserted into the BamHI and HindIII sites of pWK-P_tipA to construct pWK-P_tipA-revT.

Heterologous Gene Expression and Purification of Enzymes

To obtain recombinant RevS, the expression plasmid was transformed into E. coli BL21 Star^TM (DE3) cells. The resultant transformants were grown on LB medium containing 50 μg ml⁻¹ kanamycin overnight. To 200 ml of TB containing 50 μg ml⁻¹ kanamycin, the preculture was added at an initial absorbance of 0.1 at 600 nm (A₆₀₀). Cells were cultured at 18 °C. When the A₆₀₀ reached 0.6, isopropyl β-d-1-thiogalactopyranoside was added to a final concentration of 0.5 mm. After further growth for 18 h at 18 °C, the cells were harvested by centrifugation and frozen at −80 °C.

After thawing on ice, the cells were suspended in 20 ml of extraction buffer (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, and 20% glycerol, 5 mm imidazole, 0.5 mg ml⁻¹ lysozyme, and 6.25 units ml⁻¹ benzonase (Sigma)). For stabilization of recombinant RevS, octanoic acid was added to the cell suspension at a final concentration of 1 mm. The cell suspension was sonicated 10 times on ice for 10 s with 1-min interval between each sonication treatment (UD-200, TOMY, Tokyo, Japan). Cell debris was removed by centrifugation (10,000 × g for 30 min) (SRX-201, TOMY), and then the supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA)-agarose (Qiagen, Hilden, Germany) column (2 × 4 cm) that had been equilibrated with buffer A (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, and 20% glycerol) containing 5 mm imidazole. After washing with the same buffer, non-specifically bound proteins were removed by washing with 20 ml of buffer A containing 5 mm imidazole and 0.2% Tween 20. After removing Tween 20 with 40 ml of buffer A containing 5 mm imidazole, the column was further washed with 40 ml of buffer A containing 40 mm imidazole. The His tag fusion protein was eluted with 25 ml of buffer A containing 250 mm imidazole. The eluted fraction was concentrated using an Amicon ultracentrifugal filter (Merck, Darmstadt, Germany), and the buffer was exchanged for stock buffer (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 20% glycerol) on the centrifugal filter. The purity of RevS was confirmed by SDS-PAGE. Size exclusion chromatography was performed as described previously (46).

To obtain recombinant RevT, pWK-P_tipA-revT was introduced into S. lividans TK23 using a polyethylene glycol-mediated protoplast method (47). The resulting transformants were grown in SK2 medium containing 15 μg ml⁻¹ kanamycin for 2 days, and then 1 ml of preculture was inoculated into 70 ml of SK2 medium containing 15 μg ml⁻¹ kanamycin. After 1 day of culture, thiostrepton was added at a final concentration of 50 μg ml⁻¹ to express RevT. After another 1 day of culture, the cells were harvested by centrifugation and frozen at −80 °C. Purification of RevT was performed as described for RevS. The His tag fusion protein was eluted with 5 ml of buffer A containing 250 mm imidazole and 1 mm NADPH and used for biochemical characterization. The purity of RevT was confirmed by SDS-PAGE. Size exclusion chromatography was performed using 50 mm Tris-HCl, pH 7.5, 10 mm NaHCO₃, 200 mm NaCl, and 10% glycerol.

To obtain apo-ACP, pET28b-SRE2849 was transformed into E. coli BL21 Star^TM (DE3) cells. The transformants were selected on LB medium containing 50 μg ml⁻¹ kanamycin. Heterologous expression and purification were performed as described for RevS. The fraction eluted from the Ni-NTA column was concentrated using an Amicon ultracentrifugal filter, and the buffer was exchanged for stock buffer.

To obtain holo-ACP, pET28b-SRE2849 and pACYCDuet-1-sfp were co-transformed into E. coli BL21 Star^TM (DE3) cells. Transformants were selected on LB medium containing 50 μg ml⁻¹ kanamycin and 30 μg ml⁻¹ chloramphenicol. Heterologous gene expression, Ni-NTA column chromatography, and buffer exchange were performed as described for apo-ACP. To eliminate α-N-gluconoylated holo-ACP (48) from the Ni-NTA fraction, holo-ACP was further purified using a Mono Q 5/50 column (GE Healthcare) that had been pre-equilibrated with 50 mm BisTris, pH 6.5, containing 0.2 m NaCl and 20% glycerol. After loading the Ni-NTA fraction (42.2 mg), the column was washed with equilibration buffer for 30 min at a flow rate of 0.2 ml min⁻¹ and eluted with a linear gradient of 0.2–0.5 m NaCl for 60 min. Subsequently, 400 μl of the Mono Q fraction (5.7 mg) containing holo-ACP was collected based on MALDI-TOF/MS analysis and used in the FAAL assay for RevS.

FACL Assay of RevS

For HPLC analysis of the RevS reaction product, an enzyme reaction was performed in a final reaction volume of 100 μl containing 100 mm HEPES, pH 7.5, 20% glycerol, 10 mm MgCl₂, 5 mm TCEP, 0.5 mm fatty acid, 2 mm CoA, 3 mm ATP, and 1 μm RevS. After preincubation of the reaction mixture at 25 °C for 3 min, the reaction was initiated by the addition of RevS and allowed to proceed for 60 min. The reaction was terminated by the rapid addition of 0.5 μl of formic acid and then centrifuged at 15,000 × g for 10 min at 4 °C (Kubota 3700, Kubota Co., Tokyo, Japan) to remove the protein. The supernatant was subjected to liquid chromatography (LC)/ESI-MS (Table 2).

TABLE 2.

Summary of RevS reaction products

RevS reaction products were analyzed by LC/ESI-MS as described under “Experimental Procedures” All mass spectra were collected in the ESI-negative mode.

^a Eluted by a linear gradient from 2 to 20% acetonitrile for saturated acyl-CoA.

^b Eluted by a linear gradient from 2 to 50% acetonitrile for (E)-2-enoyl-CoA.

^c Eluted by a linear gradient from 2 to 65% acetonitrile for (E)-3-enoyl-CoA.

To determine the substrate specificity of RevS, we quantified fatty acyl-CoA formation by HPLC and NADH oxidation using a spectrophotometer. Because the stoichiometry of the product amount in both assays was identical, we performed a spectrophotometric assay to determine the kinetic parameters of RevS (49). Assays were performed in a 1-ml quartz cuvette with a final volume of 200 μl containing 100 mm HEPES, pH 7.5, 20% glycerol, 10 mm MgCl₂, 5 mm TCEP, 0.05–3 mm fatty acid, 2 mm CoA, 3 mm ATP, 0.4 mm phosphoenolpyruvate, 0.4 mm NADH, 5 units of myokinase, 4.5 units of pyruvate kinase, 5.8 units of lactate dehydrogenase, and 0.5–2.5 μm RevS. Substrate and RevS concentrations were varied as follows: 0.1–2 mm heptanoic acid with 2 μm RevS; 0.1–2 mm octanoic acid with 1 μm RevS; 0.2–2 mm nonanoic acid with 1 μm RevS; 0.1–2 mm decanoic acid with 2 μm RevS; 0.2–2.2 mm (E)-2-hexenoic acid with 2 μm RevS; 0.1–2.5 mm (E)-2-heptenoic acid with 2.5 μm RevS; 0.1–3 mm (E)-2-octenoic acid with 2 μm RevS; 0.2–2.5 mm (E)-2-nonenoic acid with 1 μm RevS; 0.05–3 mm (E)-2-decenoic acid with 2 μm RevS; 0.05–3 mm (E)-2-undecenoic acid with 2 μm RevS; 0.05–3 mm (E)-3-hexenoic acid with 1 μm RevS, 0.05–3 mm (E)-3-heptenoic acid with 1 μm RevS, 0.05–3 mm (E)-3-octenoic acid with 0.5 μm RevS, 0.05–3 mm (E)-3-nonenoic acid with 0.5 μm RevS, and 0.05–3 mm (E)-3-decenoic acid with 0.5 μm RevS. The cofactor concentration varied from 0.01 to 0.1 mm for ATP and from 0.02 to 1 mm for CoA. After preincubation of the reaction mixture at 25 °C for 3 min, the reaction was initiated by adding RevS, and the initial rate of oxidation of NADH at 340 nm was measured using a spectrophotometer. The kinetic constant was calculated by a nonlinear regression fit to the Michaelis-Menten equation using SigmaPlot11 (Table 3).

TABLE 3.

Kinetic constants of RevS for fatty acids, ATP, and CoA

At least triplicate assays were performed. The data represent mean ± S.E. The k_cat/K_mvalue was calculated based on the mean value of value K_m and k_cat.

Enzyme	Substrate	Km	kcat	kcat/Km
		mm	min−1	mm−1 min−1
RevS	Hexanoic acid	0.32 ± 0.07	5.2 ± 0.3	16
	Heptanoic acid	0.26 ± 0.03	18.3 ± 0.5	70
	Octanoic acid	0.32 ± 0.04	43.5 ± 1.5	136
	Nonanoic acid	0.48 ± 2.44	44.3 ± 0.1	92
	Decanoic acid	0.31 ± 0.02	19.6 ± 0.3	64
	(E)-2-Hexenoic acid	0.59 ± 0.05	5.5 ± 0.2	9.3
	(E)-2-Heptenoic acid	0.57 ± 0.05	15.1 ± 0.4	27
	(E)-2-Octenoic acid	0.49 ± 0.07	35.8 ± 1.5	74
	(E)-2-Nonenoic acid	0.48 ± 0.04	49.5 ± 1.2	103
	(E)-2-Decenoic acid	0.30 ± 0.04	19.3 ± 0.7	65
	(E)-2-Undecenoic acid	0.075 ± 0.004	6.6 ± 0.1	88
	(E)-3-Hexenoic acid	0.95 ± 0.11	14.2 ± 0.6	15
	(E)-3-Heptenoic acid	0.47 ± 0.06	18.2 ± 0.7	39
	(E)-3-Octenoic acid	0.51 ± 0.92	41.3 ± 0.9	81
	(E)-3-Nonenoic acid	0.71 ± 1.19	59.6 ± 1.1	85
	(E)-3-Decenoic acid	0.38 ± 0.03	23.6 ± 0.6	62
	CoAa	0.05 ± 0.01	39.6 ± 0.8	861
	ATPa	0.039 ± 0.004	56.7 ± 2.2	1456
Migb	Octanoic acid	0.285	11	38
	CoAa	0.045
	ATPa	0.036
FadD13c	Palmitic acid	0.019 ± 0.005	1.66	87.3
	CoAd	0.13 ± 0.01
	ATPd	0.24 ± 0.05

^a Octanoic acid was used as a substrate.

^b Kinetic parameter was reported by Morsczeck et al. (65).

^c Kinetic parameter was reported by Khare et al. (66).

^d Palmitic acid was used as a substrate (66).

Preparation of (E)-2-nonenoyl-ACP

(E)-2-Nonenoyl-ACP was prepared from a reaction containing 100 mm HEPES, pH 7.5, 20% glycerol, 10 mm MgCl₂, 5 mm TCEP, 0.5 mm (E)-2-nonenoic acid, 0.4 mm of holo-ACP, 3 mm ATP, and 5 μm RevS in a final volume of 80 μl. After preincubation at 25 °C for 3 min, the reaction was initiated by the addition of RevS and incubated for 4 h. The reaction mixture (10 μl) was applied to Microcon Ultracel YM-10 (Millipore, MA). After buffer exchange with 50 mm Tris-HCl, pH 7.5, the formation of (E)-2-nonenoyl-ACP was confirmed based on MALDI-TOF/MS analysis. The reaction mixture (70 μl) containing (E)-2-nonenoyl-ACP was used for RevT reaction.

Enzyme Assay of RevT

To detect the RevT reaction product, the assay was performed in a final volume of 100 μl containing 100 mm Tris-HCl, pH 7.5, 10% glycerol, 10 mm NaHCO₃, 11 mm MgCl₂, 1 mm EDTA, 0.5 mm (E)-2-enoyl-CoA, 4 mm NADPH, and 1 μm RevT. After preincubation of the reaction mixture at 25 °C for 3 min, the reaction was initiated by the addition of RevT and allowed to proceed for 10–30 min. The reaction was terminated by the rapid addition of 0.5 μl of formic acid and centrifuged at 15,000 × g for 10 min at 4 °C to remove the protein. The supernatant was subjected to LC/ESI-MS analysis.

To determine the kinetic parameters of RevT, we quantified both butylmalonyl-CoA formation by HPLC and NADPH oxidation using a spectrophotometer. Because the stoichiometry of the reaction product in both assays was identical, we conducted a spectrophotometric assay. The assay was performed in a 1-ml quartz cuvette in a final volume of 200 μl containing 100 mm Tris-HCl, pH 7.5, 10% glycerol, 10 mm NaHCO₃, 11 mm MgCl₂, 1 mm EDTA, 0.5 mm (E)-2-enoyl-CoA, 0.3 mm NADPH, and 1 μm RevT. NADPH concentration was varied from 0.01–0.3 mm. The substrate concentration for (E)-2-enoyl-CoA varied from 0.1–2 mm. After preincubation of the reaction mixture at 25 °C for 3 min, the reaction was initiated by adding RevT, and NADPH oxidation at 340 nm was measured using a spectrophotometer. The kinetic constant was calculated by a nonlinear regression fit to the Michaelis-Menten equation using SigmaPlot11 (Table 4).

TABLE 4.

Comparison of the kinetic constant of RevT and CCR homologs reported

At least triplicate assays were performed. The data represent mean ± S.E. The k_cat/K_m value was calculated based on the mean value of value K_m and k_cat.

Enzyme	Substrate	Km	kcat	kcat/Km
		mm	min−1	mm−1 min−1
RevT	(E)-2-Hexenoyl-CoA	0.27 ± 0.01	28.3 ± 0.5	104
	(E)-2-Octenoyl-CoA	0.33 ± 0.04	36.2 ± 1.3	109
	NADPHa	0.12 ± 0.01	33.9 ± 1.5	282
	NADPHb	0.12 ± 0.01	41.1 ± 2.0	342
SalGc	Crotonyl-CoA	0.0207 ± 0.0042	15.4 ± 0.9	744
	Chlorocrotonyl-CoA	0.0044 ± 0.0018	23.1 ± 2.6	5250
CinFd	Crotonyl-CoA	0.95 ± 0.05	82.6 ± 4.4	86.6
	(E)-2-Octenoyl-CoA	0.23 ± 0.01	17.4 ± 1.1	75.7
SpnEe	Crotonyl-CoA	0.36 ± 0.03	0.218 ± 0.011	0.614
	Cinnamoyl-CoA	0.078 ± 0.002	0.112 ± 0.006	1.43

^a (E)-2-Hexenoyl-CoA was used as a substrate.

^b (E)-2-Octenoyl-CoA was used as a substrate.

^c Kinetic parameter was reported by Eustáquio et al. (27).

^d Kinetic parameter was reported by Quade et al. (31).

^e Kinetic parameter was reported by Chang et al. (67).

To examine the substrate specificity of RevT for (E)-2-enoyl-ACP, the reaction was performed in a final volume of 40 μl containing 100 mm Tris-HCl, pH 7.5, 10% glycerol, 10 mm NaHCO₃, 11 mm MgCl₂, 1 mm EDTA, 0.23 mm (E)-2-nonenoyl-ACP, 4 mm NADPH, and 5 μm RevT. After the reaction was allowed to proceed at 25 °C for 3 h, the reaction mixture (40 μl) was applied to Microcon Ultracel YM-10. After buffer exchange with 50 mm Tris-HCl, pH 7.5, the reaction product was analyzed by MALDI-TOF/MS.

In Vitro Reconstruction of Butylmalonyl-CoA

An enzyme coupling assay was performed for 1 h at 25 °C in 100 mm Tris-HCl, pH 7.5, buffer containing 20% glycerol, 10 mm NaHCO₃, 11 mm MgCl₂, 1 mm EDTA, 5 mm TCEP, 0.5 mm (E)-2-hexenoic acid, 2 mm CoA, 3 mm ATP, 4 mm NADPH, 1 μm RevS, and 1 μm RevT in a final volume of 100 μl. The reaction was terminated by the rapid addition of 0.5 μl of formic acid and centrifuged at 15,000 × g for 10 min at 4 °C to remove the protein. The supernatant was subjected to LC/ESI-MS analysis.

LC/ESI-MS Analysis of Enzyme Reaction Products

To analyze the reaction products of RevS, an LC/ESI-MS analysis was carried out. An Applied Biosystems Q-TRAP was connected to a Waters Alliance 2965 with a 2996 photodiode array detector and an XTerra®MS C₁₈ column (5 μm, 2.1 × 150 mm (Waters)). The HPLC conditions were as follows: 0.25 ml min⁻¹ flow rate; solvent A, water containing 40 mm ammonium acetate (pH 6.8); solvent B, acetonitrile. The conditions of the linear gradient elution were optimized to detect each RevS reaction product. The column was equilibrated with 2% solvent B. After injection of 1 μl of the sample into the equilibrated column, the column was developed using a linear gradient from 2 to 20% for saturated acyl-CoA, 2 to 50% for (E)-2-enoyl-CoA, and 2 to 65% acetonitrile for (E)-3-enoyl-CoA over 45 min. After the gradient, the column was washed with 100% solvent B for 10 min. All mass spectra were collected in the ESI-negative mode (Table 2).

To analyze the reaction products of RevT, LC/ESI-MS analysis was carried out. The HPLC conditions were as follows: 5 μm (2.1 × 150 mm) column, XTerra®MS C₁₈; 0.25 ml min⁻¹ flow rate; solvent A, water containing 40 mm ammonium acetate (pH 5); solvent B, acetonitrile. After injection of a 1-μl sample into the column that had been equilibrated with 2% solvent B, the column was developed over a linear gradient of 2–25% solvent B for 30 min and washed with 100% solvent B for 10 min. Mass spectra were collected in the ESI-negative mode.

Feeding of Labeled Precursors and Isolation of Compound 1

The ΔrevR mutants were grown in 70 ml of SY medium for 2 days at 28 °C, and then 1 ml of the preculture was inoculated into 70 ml of PV8 medium. After 2 days of culture at 28 °C, [1-¹³C]hexanoic acid or [1,2,3,4-¹³C]octanoic acid were added to the culture at a final concentration of 0.3 mm. Five days after inoculation, an equal volume of acetone was added to the broth and filtered to remove the mycelia. The filtrate was evaporated in vacuo to obtain an aqueous solution. The solution was adjusted to pH 4 using acetic acid and extracted twice with the same volume of ethyl acetate. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to yield a brown oil. 1.5 g of extract from 2 liters of [1-¹³C]hexanoic acid feeding culture and 0.5 g of extract from 1 liter of [1,2,3,4-¹³C]octanoic acid feeding culture were obtained.

The 1.5-g extract from [1-¹³C]hexanoic acid feeding was subjected to silica gel chromatography with stepwise elution from 100:0 to 0:100 with CHCl₃/methanol. Compound 1 was eluted 100:10 with CHCl₃/methanol. The fraction was separated using a Waters 600 HPLC system equipped with a 2996 photodiode array detector and a Senshu Pak Pegasil ODS column (20 × 250 mm) using an acetonitrile, 0.05% aqueous formic acid isocratic system (48/52) at a flow rate of 9 ml min⁻¹ to yield 7.7 mg of compound 1.

The 0.5-g extract from the [1,2,3,4-¹³C]octanoic acid feeding was separated on a Sephadex LH-20 column (GE Healthcare) and eluted with methanol. The fractions containing compound 1 were applied to a 12-g RediSep Rf column (Teledyne Isco, Inc., Lincoln, NE) for medium pressure liquid chromatography with linear gradient from 20:80 to 100:0 of ethyl acetate/hexane for 35 min at a flow rate 30 ml min⁻¹ to obtain 28 fractions. The fraction containing compound 1 was separated on a Senshu Pak Pegasil ODS (10 × 250 mm) column using a methanol, 0.05% aqueous formic acid isocratic system (67/33) at a flow rate of 4 ml min⁻¹ to yield 1.6 mg of compound 1.

Preparation of Butylmalonyl-CoA

Butylmalonyl-CoA was prepared using the transesterification method (50). To a stirred solution of butylmalonic acid (2.11 g, 13.2 mmol) and thiophenol (1.42 g, 12.8 mmol) in 100 ml of dry N,N-dimethylformamide was added a solution of N,N′-dicyclohexylcarbodiimide (3.20 g, 15.5 mmol) in 100 ml of N,N-dimethylformamide dropwise at 0 °C. After stirring for 2 h at 0 °C, the reaction was quenched with 50 ml of cold water, and the mixture was acidified to pH 4 using 1 m HCl. The precipitated solid was filtered off, and the filtrate was extracted three times with 200 ml of ether. The combined ether layers were dried over Na₂SO₄ and concentrated. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate; 1:1) to give monothiophenyl butylmalonate as a colorless solid (465 mg, 1.86 mmol, 14%). The R_f value was 0.1 (n-hexane/ethyl acetate; 8:3). ¹H NMR (400 MHz, CDCl₃) was 0.93 (t, J = 7.0 Hz, 3H), 1.39 (m, 4H), 2.01 (m, 2H), 3.70 (t, J = 7.6 Hz, 1H), and 7.43 (m, 5H). HRMS (fast atom bombardment) m/z calculated for C₁₃H₁₅O₃S [M − H]⁻ was 251.0742; found was 251.0739. To a stirred solution of CoA (free form, 100 mg, 130.2 mmol) in 7.6 ml of 0.1 m NaHCO₃ was added monothiophenyl butylmalonate (164 mg, 651 mmol, 5 eq) at 0 °C, and then 0.2 m NaOH solution was slowly added to increase the pH to 8. After stirring overnight, the solution was acidified to pH 5 using ∼2 ml of 0.2 m HCl. The mixture was fractionated twice with 5 ml of ether and 5 ml of ethyl acetate to remove excess thiophenyl ester and thiophenol. The resulting aqueous phase was lyophilized, and the residue (220 mg), after adjusting the pH to 7.4 with 0.1 m NaHCO₃ solution, was purified using Cosmosil column chromatography (gradient elution with water/methanol; methanol, 0–7.5%). A combined fraction was lyophilized to give butylmalonyl-CoA in amorphous powder (101 mg, 111 mmol, 85% yield based on free form). The R_f value was 0.3 (n-butanol/acetic acid/H₂O, 4:1:/2). HRMS (ESI) m/z calculated for C₂₈H₄₂N₇O₁₉P₃SNa₅⁺ [M + Na]⁺ was 1020.0952; found was 1020.0952; ; HRMS (ESI) m/z calculated for C₂₈H₄₃N₇O₁₉P₃SNa₄⁺ [M + Na]⁺ was 998.1133; found was 998.1132. For ¹H NMR (500 MHz, D₂O), δ was as follows: 0.76 (s, 3H); 0.88 (t, J = 6.9 Hz, 3H); 0.91 (s, 3H); 1.30 (m, 4H); 1.84 (m, 2H); 2.46 (t, J = 6.9 Hz, 2H); 3.07 (m, 2H); 3.38 (m, 2H); 3.48 (m, 2H); 3.57 (m, 2H); 3.87 (dd, J = 9.8, 4.6 Hz, 1H); 4.04 (s, 1H); 4.27 (brs (broad singlet), 2H); 4.62 (brs, 1H); 6.21 (d, J = 6.9 Hz, 1H); 8.31 (s, 1H); 8.59 (s, 1H). For ¹³C NMR (125 MHz, D₂O), δ was as follows: 16.00, 20.76, 23.75, 24.64, 30.88, 31.90, 31.92, 32.22, 32.31, 38.20, 38.28, 41.12, 41.19, 41.48, 66.98, 68.39, 68.42, 74.73, 74.78, 76.84, 76.84, 76.93, 76.93, 86.67, 86.73, 89.26, 121.52, 142.78, 152.31, 155.83, 158.56, 176.78, 177.59, 179.02, and 203.85.

Preparation of (E)-2-Hexenoyl-CoA and (E)-2-Octenoyl-CoA

(E)-2-Hexenoyl-CoA was also prepared using the transesterification method (50). To a stirred solution of (E)-2-hexenoic acid (1.14 g, 10 mmol), thiophenol (10 g, 9.0 mmol), and N,N-dimethyl-4-aminopyridine (20 mg) in 10 ml of dry methylene chloride was added a solution of N,N′-dicyclohexylcarbodiimide (2.48 g, 12.0 mmol) in 5 ml of dry methylene chloride at 0 °C. After stirring for 1 h at 0 °C and then for 1 h at room temperature, the precipitated solid was filtered off, and the organic layer was evaporated. The residue was purified using silica gel column chromatography (n-hexane/ethyl acetate, 10:1) to give thiophenyl (E)-2-hexenoate as an oil (1.54 g, 7.46 mmol, 74%). The R_f value was 0.3 (n-hexane/ethyl acetate; 5:1). For ¹H NMR (400 MHz, CDCl₃), δ was as follows: 0.98 (t, J = 7.2 Hz, 3H); 1.53 (tq (triplet of quartets), J = 7.2, 7.2 Hz, 1H); 2.22 (dtd (doublet of triplet of quartets), J = 7.2, 7.2, 1.2 Hz, 2H); 6.19 (dt (doublet of triplets), J = 15.6, 1.2 Hz, 1H); 6.99 (dt, J = 15.6, 7.2 Hz, 1H); and 7.44 (m, 5H). To a stirred solution of CoA (free form, 50 mg, 65.1 mmol) in 3.8 ml of 0.1 m NaHCO₃ solution was added the solution of thiophenyl (E)-2-hexenoate (67.2 mg, 325.5 mmol, 5 eq) in 3 ml of tetrahydrofuran at 0 °C. After stirring overnight at 4 °C, the reaction mixture was concentrated and fractionated twice with 5 ml of ether to remove excess thiophenyl ester and thiophenol. The resulting aqueous phase was lyophilized, and the pH of the residue was adjusted to 7.4 using 0.1 m NaHCO₃ solution before charge. The residue was purified using Cosmosil column chromatography (gradient solution with water/methanol; methanol, 0–12.5%). A combined fraction was lyophilized to give (E)-2-hexenoyl-CoA in amorphous powder (40 mg, 46.3 mmol, 71% yield based on free form). The R_f value was 0.3 (n-butanol/acetic acid/H₂O; 4:1:2). HRMS (ESI) m/z calculated for C₂₇H₄₁N₇O₁₇P₃SNa₄⁺ [M + Na]⁺ was 952.1083; found was 952.1081; HRMS (ESI) m/z calculated for C₂₇H₄₂N₇O₁₇P₃SNa₃⁺ [M + Na]⁺ was 930.1264; found was 930.1264. For ¹H NMR (500 MHz, D₂O), δ was as follows: 0.77 (s, 3H); 0.91 (t, J = 6.3 Hz, 3H); 0.91 (s, 3H); 1.48 (m, 2H); 2.20 (dq (doublet of quartets), J = 6.9, 1.7 Hz, 2H); 2.46 (t, J = 6.3 Hz, 2H); 3.07 (t, J = 6.3 Hz, 2H); 3.39 (t, J = 6.9 Hz, 2H); 3.47 (t, J = 6.9 Hz, 2H); 3.58 (dd, J = 9.8, 4.6 Hz, 1H); 3.86 (dd, J = 9.7, 4.6 Hz, 1H); 4.05 (s, 1H); 4.27 (m, 1H); 4.60 (brs, 1H), 6.21 (m, 2H); 6.98 (dt, J = 15.4, 6.9 Hz, 1H); 8.29 (s, 1H); 8.59 (s, 1H). For ¹³C NMR (125 MHz, D₂O), δ was as follows: 15.78, 20.97, 23.43, 23.73, 30.66, 36.59, 38.59, 38.26, 38.30, 41.15, 41.21, 41.54, 68.64, 68.68, 74.69, 74.73, 76.52, 76.55, 76.96, 77.35, 86.97, 87.01, 89.43, 121.51, 130.75, 142.80, 151.50, 152.32, 155.80, 158.55, 176.86, 177.59, and 196.77.

(E)-2-Octenoyl-CoA was synthesized from (E)-2-octenoic acid using the mixed anhydride method (51–53). To a stirred solution of (E)-2-octenoic acid (92.6 mg, 651 mmol) and triethylamine (81.4 mg, 813 mmol, 1.25 eq) in 20 ml of dry ether was added ethyl chloroformate (88.2 mg, 813 mmol, 1.25 eq) slowly at 0 °C. After stirring for 12 h at room temperature under an argon atmosphere, the separated solid was filtered off and washed quickly with a small amount of dry ether. The whole solution containing an ∼10-fold molar excess of CoA as a reactant (ether solution, 20 ml, 32.55 m) was stored at 0 °C under argon.

To a stirred solution of CoA (free form, 50 mg, 65.1 mmol) in 4 ml of 0.05 m Na₂CO₃ solution, 3 ml of ethyl acetate, and 3 ml of ethanol, an ether solution of 2 ml of 65.1 mmol of crude ethyl 1-oxo-oct-2-en carbonate (n-hexane/ethyl acetate; 5:3; R_f = 0.3) was added dropwise at 0 °C at pH 8.2. After stirring for 2 h at room temperature, the reaction mixture was evaporated under reduced pressure to remove the organic solvent, and the resulting aqueous phase was lyophilized. The residue, after adjusting the pH to 7.4 with 0.1 m NaHCO₃ solution before charge, was purified using Cosmosil column chromatography (gradient elution with water/methanol; methanol, 0–20%). A combined fraction was lyophilized to give (E)-2-octenoyl-CoA in amorphous powder (24 mg, 26.9 mmol, 41% yield based on free form). The R_f value was 0.3 (n-butanol/acetic acid/H₂O; 4:1:2). HRMS (ESI) m/z calculated for C₂₉H₄₅N₇O₁₇P₃SNa₄⁺ [M + Na]⁺ was 980.1396; found was 980.1393; HRMS (ESI) m/z calculated for C₂₉H₄₆N₇O₁₇P₃SNa₃⁺ [M + Na]⁺ was 958.1577; found was 958.1570. For ¹H NMR (500 MHz, D₂O), δ was as follows: 0.77 (s, 3H); 0.87 (t, J = 6.9 Hz, 3H); 0.91 (s, 3H); 1.29 (m, 4H); 1.45 (m, 2H); 2.21 (m, 2H); 2.46 (t, J = 6.9 Hz, 2H); 3.08 (t, J = 6.3 Hz, 2H); 3.39 (t, J = 6.3 Hz, 2H); 3.47 (t, J = 6.3 Hz, 2H); 3.58 (dd, J = 9.8, 5.2 Hz, 1H); 3.86 (dd, J = 9.8, 4.6 Hz, 1H); 4.05 (s, 1H); 4.26 (m, 2H); 4.60 (brs, 1H); 6.21 (m, 2H); 6.98 (dt, J = 15.4, 6.9 Hz, 1H); 8.29 (s, 1H); and 8.59 (s, 1H). For ¹³C NMR (125 MHz, D₂O), δ was as follows: 16.12, 20.98, 23.74, 24.60, 29.62, 30.66, 33.44, 34.48, 38.27, 38.31, 41.15, 41.22, 41.53, 68.63, 68.67, 74.69, 74.73, 76.51, 76.54, 76.96, 77.36, 77.38, 86.95, 86.99, 89.43, 121.50, 130.60, 142.79, 151.87, 152.30, 155.79, 158.53, 176.85, 177.58, and 196.76.

Results

Disruption of revR, revS, and revT Genes and Analysis of Metabolite Profiles

In the compound 1 gene cluster, the revR, revS, and revT genes were in the same transcriptional unit. Based on BLAST searching, revT gene belongs to the CCR homologs that are distributed in gene clusters producing polyketide compounds with atypical extender units (27–32). In addition to the revT gene, homologs of the revR and revS genes were also distributed in various polyketide gene clusters (Fig. 1, panels I–IV). Therefore, to understand the role of revR, revS, and revT genes, we conducted gene disruptions (Fig. 2, A, C, and E). After confirmation of gene disruption (ΔrevR, ΔrevS, and ΔrevT) by Southern hybridization (Fig. 2, B, D, and F), the metabolite profile of each gene disruptant was analyzed by LC/ESI-MS (Fig. 3). In the wild-type strain, compound 1 was the major product among the RM derivatives (Figs. 1A and 3A). Interestingly, ΔrevR mutants selectively decreased the production of compound 1 (Fig. 3B). Reintroduction of the revR gene under the control of the aph promoter restored compound 1 production to wild-type levels (Fig. 3C). ΔrevS mutants showed an ∼50% reduction of all RM derivatives (Figs. 1A and 3D), and the phenotype was recovered by reintroduction of the revS gene (Fig. 3E), suggesting that RevS is involved in the formation of C18 alkyl residues in RM derivatives. Additionally, ΔrevT mutants completely abolished the production of RMs (Fig. 3F), which was restored by complementation with the revT gene (Fig. 3G), indicating that RevT is essential for the production of RMs.

FIGURE 3.

Metabolite profiles of gene disruptants. HPLC analysis of ethyl acetate extract was from wild-type strain (A), ΔrevR mutant (B), and ΔrevR mutant complemented with revR gene (C). The ΔrevS mutant (D) and ΔrevS mutant complemented with revS gene (E) are shown. The ΔrevT mutant (F) and ΔrevT mutant complemented the revT gene (G). 1–4 indicate RM-A, RM-C, RM-D, and RM-E, respectively. Asterisk indicates the peak of RM-B, which was non-enzymatically converted into 5,6-spiroacetal structure from compound 1 (38).

Purification and Characterization of RevS

Based on BLAST searching, RevS belongs to the uncharacterized adenylate-forming enzyme family. Together with the gene disruption phenotype (Fig. 3, D and E), we hypothesized that RevS should function as an FACL or FAAL. We heterologously expressed His₈-tagged RevS in E. coli and purified the protein to homogeneity using Ni-NTA column chromatography (Fig. 4A). Both monomeric (65 kDa) and dimeric (123 kDa) RevS proteins were observed following gel filtration analysis (Fig. 4B). After gel filtration chromatography, we evaluated the specific activity of the monomeric and dimeric fractions. Based on the FACL assay, the specific activities of the monomer and dimer fractions of RevS were the same as that of the purified RevS before gel filtration (Fig. 4C). Therefore, the multimeric status of RevS on gel filtration chromatography was speculated to be the monomer-dimer equilibrium.

FIGURE 4.

Evaluation of FACL and FAAL activity of RevS. A, SDS-PAGE analysis of RevS using 12.5% polyacrylamide gel. Lane 1, molecular mass markers; lane 2, purified RevS (1 μg). B, size exclusion chromatography of RevS. Purified protein (0.8 mg) was analyzed on a Superdex 200 column. C, analysis of specific activity of the RevS fractions after gel filtration. The purified RevS after Ni-NTA column chromatography was fractionated by Superdex 200 chromatography (46). Two main peaks corresponding to the monomer and dimer fractions (each 4 ml) were concentrated using an Amicon ultracentrifugal filter. In the presence of 1 μm RevS, 1 mm octanoic acid, and 2 mm CoA, the specific activity was calculated using the spectrophotometric assay described under “Experimental Procedures.” Lane 1, purified RevS before gel filtration; lane 2, monomer fraction of RevS after gel filtration; lane 3, dimer fraction of RevS after gel filtration. D, SDS-PAGE analysis of purified apo- and holo-ACP using 15% polyacrylamide gel. Lanes 1 and 4, molecular mass markers; lane 2, His₈ tag purified apo-ACP (1 μg); lane 3, His₈ tag purified holo-ACP (1 μg). E, MALDI-TOF/MS analysis is shown for apo-ACP (i), holo-ACP and α-N-gluconoylated holo-ACP fraction after Ni-NTA column chromatography (ii), and purified holo-ACP after removing α-N-gluconoylated holo-ACP by Mono Q column chromatography (iii). F, FACL (open bar) and FAAL (closed bar) activities of RevS were measured using an enzyme-coupled spectrophotometric assay. 0.3 mm CoA or 0.3 mm purified holo-ACP was used to calculate specific activity of RevS.

To examine FACL activity, purified RevS was incubated with various fatty acids in the presence of ATP and CoA, and the reaction products were analyzed by LC/ESI-MS. RevS activated both saturated and unsaturated fatty acids with carbon chain lengths of C4–C11 into fatty acyl-CoAs (Table 2) but did not activate fatty acids with chain lengths of more than C13. Efficient acyl-CoA formation was observed when middle chain fatty acids (C8–C10) were used as the substrate. Moreover, to examine FAAL activity, we heterologously expressed holo-ACP in E. coli and purified the protein to homogeneity using Ni-NTA column chromatography (Fig. 4D). Because the His tag was not attached in Sfp, only holo-ACP was purified using Ni-NTA column chromatography after co-expression of Sfp and ACP in E. coli. The holo-ACP was further purified by Mono Q column chromatography, and its modification was confirmed by MALDI-TOF/MS analysis (Fig. 4E). Next, purified RevS was incubated with various fatty acids in the presence of ATP and holo-ACP. Although the FAAL and FACL activities of RevS showed the same substrate specificity, the specific activity of FAAL was significantly lower than that of FACL (Fig. 4F).

Analysis of Kinetic Properties of FACL Activity of RevS

To determine the kinetic parameters of RevS for each substrate, we performed spectrophotometric assays. The RevS reaction apparently followed Michaelis-Menten kinetics. High catalytic efficiency was observed when middle chain fatty acids (C8–C10) were used for the RevS assay (Table 3). In addition, RevS accepted both saturated and unsaturated fatty acids as substrates with the same catalytic efficiency. We also calculated kinetic constants for ATP and CoA. The K_m and k_cat values for ATP were 0.039 ± 0.004 mm and 56.7 ± 2.2 min⁻¹, respectively, giving a catalytic efficiency of 1456 min⁻¹ mm⁻¹. The K_m and k_cat values of CoA were 0.05 ± 0.01 mm and 39.6 ± 0.8 min⁻¹, respectively, giving a catalytic efficiency of 861 min⁻¹ mm⁻¹. The kinetic parameters were comparable with those for other FACLs reported previously (Table 3).

Phylogenetic Analysis of RevS

To obtain insight into the molecular evolution of RevS, we performed phylogenetic analysis of the RevS using the amino acid sequences of FACL, FAAL, and a variety of CoA ligases (Fig. 5A). Consistent with the presence of a FAAL-specific insertion motif (Fig. 5B) (14), RevS was classified in an FAAL clade. However, biochemical analyses clearly demonstrated that RevS is an FACL. Interestingly, RevS homologs such as CinT and SamR0482 associated in the biosynthetic gene cluster of cinnabaramide and stambomycin (Fig. 1B), respectively, were found in the same branch, suggesting that CinT and SamR0482 may be middle chain FACLs. These RevS homologs may have evolved from the FAAL family and were adapted for the production of fatty acyl-CoAs to efficiently convert them into 2-alkylmalonyl-CoAs for the PKS extender unit.

FIGURE 5.

Phylogenetic analysis and multiple sequence alignments of RevS. A, phylogenetic analysis of functionally annotated FACL, FAAL, and CoA ligases. Multiple sequence alignments were performed using the bootstrap method of ClustalW (63). The bootstrap tree was drawn using the neighbor-joining method in MEGA, version 6.06. B, sequence alignments of RevS with FACL and FAAL, whose crystal structures have been solved. The bold bar shows FAAL-specific insertion motif (14, 64). Accession numbers used are as follows: Mav_Mig (AAB87139) from Mycobacterium avium; Ath At4g05160 (Q9M0X9), Ath ACOS5 (AAP03019.1), Ath 4-coumarate-CoA ligase 1 (AAD47191), Ath 4-coumarate-CoA ligase 2 (AAD47192), Ath 4-coumarate-CoA ligase 3 (AAD47194), and Ath 4-coumarate-CoA ligase 4 (Q9LU36) from Arabidopsis thaliana; Sce Faa1p (P30624) and Sce Faa3p (P39002) from Saccharomyces cerevisiae S288c, Rno FACL ACS1 (NP_036952), Rno ACS4 (NP_446075), Rno ACS5 (NP_446059), Rno ACS6_v1 (P33124), Rno FATP1 (Q60714), and Rno FATP4 (Q91VE0) from Rattus norvegicus; EcoFAAL (3PBK) (64), Eco FadD (AAA23752), Eco2-amino-3-ketobutyryl-CoA ligase (1FC4_A), and Eco crotonobetaine/carnitine CoA ligase CaiC (CAA52113) from E. coli; Hsa Sa protein (BAB68363) from Homo sapiens; Rrh FadD19 (ADP09625) from Rhodococcus rhodochrous; Sam samR0482 (CAJ88192) from Streptomyces ambofaciens ATCC 23877 (30); Ssp CinT (CBW54660) from Streptomyces sp. JS360 (31); Tth FACL (Q5SKN9) from Thermus thermophilus Hb8; Tth phenylacetyl-CoA ligase (YP_004577) from T. thermophilus HB27; Mxa FtpD (YP_634753) from Myxococcus xanthus DK 1622 (17); Bxe benzoyl-CoA ligase (2V7B) from Burkholderia xenovorans Lb400; Bce phenylacetyl-CoA ligase Paak1 (2Y27_A) from Burkholderia cenocepacia; Awo caffeyl-CoA synthetase CarB (ADX43862) from Acetobacterium woodii; Bce o-succinylbenzyl-CoA ligase (ADK07438) from Bacillus cereus biovar anthracis str. CI; Mtu FadD3 (NP_218078.), Mtu FadD5 (NP_214680), Mtu FadD13 (NP_217605) (58), Mtu FadD15 (NP_216703), Mtu FadD19 (YP_177983), Mtu FadD10 (NP_214613), Mtu FadD28 (NP_217457) (18), Mtu FadD29 (NP_217466), Mtu FadD30 (NP_214918), Mtu FadD32 (NP_218318) (16), Mtu FadD33 (NP_215861), and Mtu FadD34 (YP_177686) from M. tuberculosis H37Rv; Ssp RevS (BAK64635) from Streptomyces sp. SN-593, Lma JamA (AAS98774) from Lyngbya majuscule (11); Kse KSE_65520 (YP_004908267) from Kitasatospora setae KM-6054; Cal PuwC (AIW82280) from Cylindrospermum alatosporum CCALA 988 (20); Sro DptE (AAX31555) from Streptomyces roseosporus NRRL 11379 (13); Bgl CayA (AIG53815) from Burkholderia gladioli pv. agaricicola (21); Ssp salicylyl-CoA ligase (BAC78380) from Streptomyces sp. WA46; Sma benzoyl-CoA ligase (AAF81733) from Streptomyces maritimus; Bsu YhfL (NP_388908) from B. subtilis subsp. subtilis str. 168; Lpn FAAL (3KXW) from Legionella pneumophila (64); and Set FACL (1PG4) from Salmonella enterica.

Purification and Biochemical Characterization of RevT

To determine the function of RevT, His₈ tag RevT was heterologously expressed in S. lividans TK23 and purified using Ni-NTA column chromatography. The molecular mass of the RevT was estimated to be 48 kDa by SDS-PAGE (Fig. 6A) and 178 kDa by gel filtration chromatography (Fig. 6B), suggesting that RevT exists as a tetramer. Incubation of purified RevT in the presence of (E)-2-hexenoyl-CoA, NaHCO₃, and NADPH was performed for 30 min. The time-dependent production of butylmalonyl-CoA was observed for 20 min (Fig. 6C).

FIGURE 6.

Biochemical characterization of RevT. A, SDS-PAGE analysis of RevT using 12.5% polyacrylamide gel. Lane 1, molecular mass markers; lane 2, His₈ tag purified RevT (1 μg). B, size exclusion chromatography of RevT. Purified protein (0.3 mg) was analyzed on a Superdex 200 column. C, LC/ESI-MS analysis of RevT reaction products. Synthetic standard of butylmalonyl-CoA (i), synthetic standard of (E)-2-hexenoyl-CoA (ii), and time-dependent production of butylmalonyl-CoA after 10 min (iii), 20 min (iv), and 30 min incubation (v). After heat treatment of RevT at 95 °C for 5 min, the supernatant was collected by centrifugation at 15,000 × g for 30 min at 4 °C. Heat-treated RevT was incubated for 20 min (vi).

Because the understanding of substrate specificity and kinetic properties of RevT is important for the synthesis of polyketide compounds, we also characterized the kinetic parameters of (E)-2-hexenoyl-CoA and (E)-2-octenoyl-CoA. The reaction apparently followed Michaelis-Menten kinetics. RevT catalyzed both (E)-2-hexenoyl-CoA and (E)-2-octenoyl-CoA with the same catalytic efficiency. The K_m and k_cat values of (E)-2-hexenoyl-CoA for the formation of butylmalonyl-CoA were 0.27 ± 0.01 mm and 28.3 ± 0.5 min⁻¹, respectively, giving a catalytic efficiency of 104 min⁻¹ mm⁻¹. The K_m and k_cat values of (E)-2-octenoyl-CoA for the formation of hexylmalonyl-CoA were 0.33 ± 0.04 mm and 36.2 ± 1.3 min⁻¹, respectively, giving a catalytic efficiency of 109 min⁻¹ mm⁻¹. The kinetic parameters were also evaluated for NADPH in the presence of (E)-2-hexenoyl-CoA and (E)-2-octenoyl-CoA (Table 4). The kinetic efficiency of RevT for (E)-2-octenoyl-CoA was better than that of CinF (31). Catalytic efficiency of SalG was significantly higher than that of RevT (27).

Incorporation of ¹³C-Labeled Fatty Acids into Compound 1

Biochemical analysis indicated that RevS efficiently converted fatty acids into octanoyl-CoA, nonanoyl-CoA, and decanoyl-CoA, which contain longer carbon chain than physiological RevT substrates. Therefore, the β-oxidation of fatty acyl-CoAs is essential for producing truncated (E)-2-enoyl-CoAs for RevT substrates. To understand the biosynthetic process, we performed a feeding experiment using [1,2,3,4-¹³C]octanoic acid. We hypothesized the activation of labeled fatty acid into [1,2,3,4-¹³C]octanoyl-CoA by RevS, the generation of [1,2-¹³C](E)-2-hexenoyl-CoA after β-oxidation process, and the conversion into [1,2-¹³C]butylmalonyl-CoA by RevT, resulting in assembly into the structure of compound 1 (Fig. 7A). In this experiment, we used the ΔrevR mutant for efficient incorporation of the fatty acid precursor into compound 1, because the mutants showed low yields of compound 1 (Fig. 3B). After feeding [1,2,3,4-¹³C]octanoic acid, we isolated compound 1 and analyzed its ¹³C NMR spectrum. In addition to the signals at 25.4 and 84.2 ppm assigned to C17 and C18 of compound 1, respectively, significant C–C coupling was observed. Moreover, the coupling constants were nearly the same value (J = 36 and 37 Hz) (Fig. 7B). These results suggest that octanoic acid was incorporated into compound 1 through the biosynthetic pathway predicted in Fig. 7A. We also tested whether [1-¹³C]hexanoic acid is incorporated into compound 1 (Fig. 7C). After the feeding experiment, we purified compound 1 from the ΔrevR mutant culture, and its ¹³C NMR spectrum was analyzed (Fig. 7D). A corresponding signal at 25.4 ppm assigned to C17 of compound 1 was highly enriched, suggesting that hexanoic acid is utilized for compound 1 biosynthesis.

FIGURE 7.

Feeding of labeled fatty acid and ¹³C NMR analysis. A, biosynthetic scheme of octanoic acid incorporation into compound 1. B, ¹³C NMR analysis of non-labeled compound 1 (panels i and iii) and labeled compound 1 after feeding of [1,2,3,4-¹³C]octanoic acid (panels ii and iv). C, biosynthetic scheme of hexanoic acid incorporation into compound 1. D, ¹³C NMR analysis of non-labeled compound 1 (panel i) and labeled compound 1 after feeding of [1-¹³C]hexanoic acid (panel ii).

RevS and RevT Are Responsible for Butylmalonyl-CoA Biosynthesis

Because of efficient incorporation of fatty acids into compound 1, we also investigated the successive conversion of (E)-2-hexenoic acid into (E)-2-hexenoyl-CoA by RevS and the carboxylase/reductase reaction by RevT to give butylmalonyl-CoA (Fig. 8A). As expected, (E)-2-hexenoic acid was efficiently converted into butylmalonyl-CoA in the RevS-RevT coupling reaction (Fig. 8B). This is the first report of in vitro reconstruction of 2-alkylmalonyl-CoA using physiological enzymes derived from a secondary metabolite gene cluster.

FIGURE 8.

In vitro enzyme coupling assay. A, reaction scheme of (E)-2-hexenoic acid into butylmalonyl-CoA by RevS and RevT. B, LC/ESI-MS chromatogram of butylmalonyl-CoA (panel i), (E)-2-hexenoic acid (panel ii), 1-h reaction product by RevS and RevT (panel iii), and 1-h reaction product using RevS and RevT treated at 95 °C for 5 min (panel iv). Peaks corresponding to butylmalonyl-CoA and (E)-2-hexenoic acid were shown at 258 nm (blue line) and 240 nm (yellow line), respectively. C, scheme of RevS and RevT reaction using ACP. D, analysis of RevT reaction product by MALDI-TOF/MS. After RevS reaction using (E)-2-nonenoic acid and holo-ACP, the formation of (E)-2-nonenoyl-ACP was confirmed by MALDI-TOF/MS (panel i). The RevS reaction mixture containing (E)-2-nonenoyl-ACP was further reacted in the presence and absence of RevT (panels ii and iii).

Moreover, we examined whether the biosynthetic route occurs from (E)-2-hexenoic acid to (E)-2-enoyl-ACP and then 2-alkylmalonyl-ACP (Fig. 8C). Because the FAAL activity of RevS was very low (Fig. 4F), we first prepared the FAAL reaction product, (E)-2-nonenoyl-ACP and performed the RevT reaction under optimized conditions (Fig. 6). However, we did not detect the conversion of (E)-2-nonenoyl-ACP into 2-heptylmalonyl-ACP (Fig. 8D), suggesting that RevS and RevT are responsible for the production of 2-alkylmalonyl-CoA but not 2-alkylmalonyl-ACP.

Discussion

In this study, we characterized the revR, revS, and revT genes, which are involved in 2-alkylmalonyl-CoA formation in RM biosynthesis. The revR gene disruption demonstrated that RevR was involved in the selective production of butylmalonyl-CoA, which was consistent with the high production of compound 1 in wild-type culture (Fig. 3). RevR showed 55% amino acid identity to BenQ that is reported to be crucial for providing the hexanoate PKS starter unit for benastatin biosynthesis (54, 55). In addition, it has been reported that KASIII is a determinant of the fatty acid priming unit producing straight or branched chain, as well as the even or odd number chain (56, 57). RevR is likely to select butyryl-CoA as a priming substrate to yield 3-oxo-hexanoyl-ACP. Next, the product may be converted into (E)-2-hexenoyl-ACP by β-keto processing enzymes (Fig. 1C). In this de novo fatty acid biosynthetic pathway, an unknown transacylase may be responsible for the conversion of (E)-2-hexenoyl-ACP into (E)-2-hexenoyl-CoA, as RevT did not accept (E)-2-enoyl-ACP as a substrate (Fig. 8D). The revS gene disruption and complementation analysis indicated the non-essentiality of RevS for the biosynthesis of RMs (Fig. 3, D and E). We speculate that RM biosynthesis in ΔrevS mutants was mainly supported by de novo fatty acid biosynthesis (Fig. 1C). Another possibility is the involvement of unidentified CoA ligase, which may support medium chain acyl-CoA formation. We found three genes showing 30–43% amino acid identity to RevS in the draft genome of Streptomyces sp. SN-593. Although three were not in the FACL clade of RevS (Fig. 5A), gene disruption and biochemical analysis will be essential for final conclusions.

Overall, the structure of the class I adenylate-forming enzymes is composed of large N-terminal and small C-terminal domains connected by a flexible hinge region (9). Crystal structure analysis of FAAL28 (FadD28) from M. tuberculosis revealed that FAAL has a specific insertion motif containing 22 amino acids (Fig. 5B). The insertion motif modulates C-terminal domain movement and disrupts access of the acyl-AMP intermediate to the CoA-binding motif (14). Amino acid alignments and phylogenetic analysis of adenylate-forming enzymes indicated that RevS belongs to the FAAL clade. Unexpectedly, biochemical analysis of RevS revealed FACL activity even in the presence of the FAAL-specific insertion motif (Fig. 5). Arora et al. (14) also reported that deletion of the insertion motif found in FadD28 resulted in the conversion of FAAL activity into FACL activity, indicating that FAAL originally contains CoA binding pocket. Because phylogenetic analysis suggested that FAAL was derived from the CoA ligase, the FACL activity of RevS may have re-evolved from the FAAL clade by generating access to the CoA binding pocket by optimizing the orientation of the N- and C-terminal domains. Interestingly, in contrast to RevS, FadD10, which does not possess the FAAL-specific insertion motif, from M. tuberculosis showed FAAL activity. This was explained by the orientation of its N- and C-terminal domains, which are quite different from those of other adenylate-forming family members (58). Therefore, crystal structure analysis of RevS is essential for fully understanding its biosynthetic mechanism.

Metabolite profiling of revS gene disruptants (Figs. 2 and 3) and kinetic analysis of RevS (Fig. 4 and Tables 2 and 3) also suggested the presence of available free fatty acids (C6–C10) during the late stationary phase and the close relationship between fatty acid metabolism and polyketide biosynthesis. Streptomyces typically utilizes de novo-synthesized fatty acids not only for building membrane phospholipids but also to accumulate neutral lipid storage compounds such as triacylglycerol (TAG) (59–61). TAG is one of the important carbon sources for energy production and a precursor for secondary metabolism. A link between TAG degradation and actinorhodin production has been suggested (59). Because TAG found in S. lividans consists of fatty acids ranging from C14 to C18 (59), the fatty acids derived from TAG degradation should mainly be utilized for the production of acetyl-CoA, but not as RevS substrates. Therefore, other mechanisms may generate medium chain fatty acids to support the RevS reaction. A possible mechanism is termination of fatty acid biosynthesis by chain length-specific acyl-ACP thioesterase. Interestingly, it has been reported that the seed of Cuphea palustris possessed C8- and C14-specific acyl-ACP thioesterases, CpFatB1 and CpFatB2, respectively, which is consistent with the accumulation of middle chain fatty acids in the seed (62). However, we found no homologous gene in Streptomyces sp. SN-593. Additional detailed studies are needed to understand fatty acid homeostasis during secondary metabolite biosynthesis.

In summary, 2-alkylmalonyl-CoA biosynthesis was strongly supported by the functions of RevR and RevS. These enzymes effectively utilized de novo fatty acid biosynthesis and fatty acid degradation products, respectively. Decreased supply of atypical building blocks in the polyketide assembly line is critical for secondary metabolite biosynthesis. Therefore, the presence of RevR and RevS may be a back-up system to ensure the production of atypical extender units. Our results explain why homologs of RevR and RevS are distributed in polyketide biosynthetic gene clusters, which utilize atypical extender units (Fig. 1).

Author Contributions

T. M. performed the genetic, biochemical, and feeding experiments and wrote the paper. S. T. designed all of the studies, constructed vectors, and wrote the paper. A. K. examined the assay condition for RevS. S. P. supported the isolation of gene disruptants. T. H. and T. S. synthesized butylmalonyl-CoA, (E)-2-hexenoyl-CoA, and (E)-2-octenoyl-CoA. T. N. supported LC/MS and NMR analysis. H. O. integrated this research. All authors reviewed the results and approved the final version of the manuscript.