Discovery of an Unusual Fatty Acid Amide from the ndgR_yo Gene Mutant of Marine-Derived Streptomyces youssoufiensis

Abstract

NdgR_yo, an IclR-like regulator, was selected as the target gene to activate new secondary metabolites in the marine-derived Streptomyces youssoufiensis OUC6819. Inactivation of the ndgR_yo gene in S. youssoufiensis OUC6819 led to the accumulation of a new fatty acid amide (1), with an unusual 3-amino-butyl acid as the amine component. Moreover, its parent fatty acid (2) was also discovered both in the wild-type and ΔndgR_yo mutant strains, which was for the first time isolated from a natural source. The structures of compounds 1 and 2 were elucidated by combination of LC-MS and NMR spectroscopic analyses. This study demonstrated that the ndgR_yo homologs might serve as a target for new compound activation in Streptomyces strains.

1. Introduction

Marine Streptomyces have evolved unique abilities to adapt to the marine environment, which ensures their survival in extreme habitats (e.g., low temperature, high pressure, and poor nutrients) and provides a variety of novel secondary metabolites [1]. With the increase in the number of sequences deposited in microbial genome databases, an increasing number of secondary metabolite biosynthetic gene clusters have been disclosed; however, the majority of them are silent or barely expressed under ordinary laboratory conditions [2]. Thus, activation of silent gene clusters has become an effective strategy for natural product discovery, attracting more and more scientists to this research field.

Secondary metabolism of Streptomyces is controlled by a complicated and elaborate regulatory network [3,4]. The precursors for the biosynthesis of secondary metabolites are usually derived from primary metabolism. Manipulation of the regulators in central metabolism has a far-reaching impact on the production of secondary metabolites [5]. The IclR-like global regulator, ndgR, is a representative of this metabolism that is involved in amino acid metabolism and conserved among Streptomyces species as well as other actinomycetes [6]. Disruption of ndgR in Streptomyces coelicolor led to defective differentiation and enhanced actinorhodin production in minimal media containing certain amino acids [7]. In Streptomyces clavuligerus, deletion of areB, a homolog of ndgR, resulted in increased production of clavulanic acid and cephamycin C [8].

In our effort to discover novel natural products from the marine-derived Streptomyces youssoufiensis OUC6819 by genome mining [9,10], the ndgR_yo gene was selected as a target for compound activation. The disruption of the ndgR_yo gene caused accumulation of a new fatty acid amide (1) that shares similar UV spectrum with its parent fatty acid (2) present in the wild-type strain (Figure 1). Herein, we describe the isolation, structure elucidation as well as biological evaluation of compounds 1 and 2 from the ΔndgR_yo mutant of S. youssoufiensis OUC6819.

2. Results and Discussion

The ndgR_yo gene was identified from the S. youssoufiensis OUC6819 genome using the local BlastP program. NdgR_yo harbors a Helix-Turn-Helix motif at the N-terminus, and displays 57% identity to the NdgR from S. coelicolor (NP_629686.1). A positive cosmid, pWLI551, was obtained through genome library screening (Table S2). The ΔndgR_yo mutant was obtained using a PCR-targeting strategy, as described in the Section 3.4. The fermentation broths of the wild-type and the ΔndgR_yo mutant strains were extracted with ethyl acetate, and were subsequently subjected to HPLC analysis (Figure 2). The newly accumulated compound 1 in the ΔndgR_yo mutant showed similar UV spectra with that of 2 (Figure 2), indicating they might belong to the same compound class. Large scale fermentation of the ΔndgR_yo mutant resulted in the isolation of compounds 1 and 2, followed by identification by NMR spectroscopy.

Compound 1 was isolated as a yellow oil. The molecular formula of 1 was established as C₂₂H₃₅NO₄ (five degrees of unsaturation), as determined by HR-ESIMS data (m/z 378.2654 [M + H]⁺, calcd 378.2644) (Figure S1). The structure of 1 was determined from the 1D and 2D NMR (COSY, HSQC, HMBC, and NOESY) data (Figures S2–S7). The ¹H and HSQC spectra of 1 disclosed six methyl groups (δ_H 1.64, 1.66, 1.78, 0.83, 1.63, and 1.21), three methylenes (δ_H 2.95, 2.82, 2.54), three methines (δ_H 2.71, 3.72, and 4.26), and five olefinic protons (δ_H 5.36, 5.57, 6.12, 5.33, and 5.46). The COSY spectrum established five spin systems of H-2 (δ_H 2.95)/H-3 (δ_H 5.36), H-5 (δ_H 2.82)/H-6 (δ_H 5.57)/H-7 (δ_H 6.12), H-9 (δ_H 5.33)/H-10 (δ_H 2.71)/H-11 (δ_H 3.72)/H-17 (δ_H 0.83), H-13 (δ_H 5.46)/H-14 (δ_H 1.64), and H-2′ (δ_H 2.54)/H-3′ (δ_H 4.26)/H-4′ (δ_H 1.21) (Figure 3). The HMBC correlations from H-13 and H-9 to the hydroxylated carbon C-11 (δ_C 82.3), from H-11 to C-12 (δ_C 136.7), from H-7 to C-9 (δ_C 134.4), from H-6 to C-8 (δ_C 133.7), from H-2 to C-4 (δ_C 138.4) and a carbonyl carbon C-1 (δ_C 172.4), from H-5 to C-3 (δ_C 117.3), from H-3′ to C-1, and from H-2′ to C-1′ (δ_C 173.2) established the main carbon chain of 1 (Figure 3). The HMBC correlations from the methyl protons H-15 (δ_H 1.66) to C-5 (δ_C 42.5), H-16 (δ_H 1.78) to C-9, H-17 to C-11, and H-18 (δ_H 1.63) to C-13 (δ_C 121.4), together with the COSY correlations of H-13/H-14 and H-3′/H-4′, confirmed the location of six methyl groups (Figure 3). The ¹³C chemical shifts of C-3′ (δ_C 42.2) and C-1, together with the HR-ESIMS data of 1, revealed the presence of an amide group. Moreover, the configurations of the four double bonds were confirmed to be E by NOESY correlations between H-3/H-5, H-5/H-7, H-7/H-9, H-11/H-13, H-2/H-15, H-6/H-16, and H-10/H-16 (Figure 4). The relative configuration between H-10 and H-11 was proposed to be trans by the large coupling constant value of 8.4 Hz. Then, the absolute configurations of C-10 and C-11 were determined by comparison of the experimental ECD spectra of 1 and 2 (Figure S15) with calculated ECD spectra of the (2E,4E,8E)-7-methoxy-4,6,8-trimethyldeca-2,4,8-triene moiety reported in the literature [11], which showed high agreement with the 10R, 11R calculated model. Thus, compound 1 was identified as 3-((3E,6E,8E,10R,11R,12E)-11-hydroxy-4,8,10,12-tetramethyltetradeca-3,6,8,12-tetraenamido) butanoic acid, a new branched-chain fatty acid amide with 3-amino-butyl acid as the amine component. The ¹H and ¹³C chemical shifts of compound 1 were shown in

Table 1. ¹H (600 MHz) and ¹³C (150 MHz) NMR chemical shifts of 1 and 2 in CD₃OD.

Position	1		2
	δH (J in HZ)	δC	δH (J in HZ)	δC
1		172.4		174.9
2	2.95 (2H, d, 7.2)	35.0	3.06 (2H, d, 7.2)	33.0
3	5.36 (1H, t, 6.6)	117.3	5.39 (1H, t, 6.6)	116.7
4		138.4		137.7
5	2.82 (2H, d, 7.2)	42.5	2.82 (2H, d, 7.2)	42.5
6	5.57 (1H, dt, 15.6, 7.2)	124.4	5.56 (1H, dt, 15.6, 7.2)	124.4
7	6.12 (1H, d, 15.6)	136.7	6.13 (1H, d, 15.6)	136.7
8		133.7		133.7
9	5.33 (1H, d, 9.0)	134.4	5.33 (1H, d, 9.6)	134.4
10	2.71 (1H, m)	36.1	2.71 (1H, m)	36.1
11	3.72 (1H, d, 8.4)	82.3	3.72 (1H, d, 7.8)	82.3
12		136.7		136.7
13	5.46 (1H, q, 6.6)	121.4	5.47(1H, q, 6.0)	121.4
14	1.64 (3H, d, 6.6)	11.6	1.64 (3H, d, 6.0)	11.7
15	1.66 (3H, s)	15.0	1.66 (3H, s)	15.0
16	1.78 (3H, m)	11.6	1.78 (3H, m)	11.7
17	0.83 (3H, d, 6.6)	16.6	0.83 (3H, d, 7.2)	16.7
18	1.63 (3H, s)	9.7	1.63 (3H, s)	9.7
1′		173.2
2′	2.54 (1H, dd, 15.6, 6.0) 2.42 (1H, dd, 15.6, 6.0)	40.0
3′	4.26 (1H, m)	42.2
4′	1.21 (3H, d, 6.6)	18.8

.

Compound 2 was isolated as a yellow oil. The molecular formula of 2 was established as C₁₈H₂₈O₃ (five degrees of unsaturation), as determined by HR-ESIMS data (m/z 310.2397 [M + NH₄]⁺, calcd 310.2382) (Figure S8). The structure of 2 was determined from the 1D and 2D NMR (COSY, HSQC, HMBC, and NOESY) data (Figures S9–S14). According to the ¹H and ¹³C NMR data (Table 1), compound 2 lacked the 3-amino-butyl acid moiety compared to 1. Moreover, the NOESY correlations (H-3/H-5, H-5/H-7, H-7/H-9, H-11/H-13, H-2/H-15, H-6/H-16, and H-10/H-16) as well as the experimental ECD spectrum revealed that compound 2 displays the same absolute configurations with 1. Thus, compound 2 was identified to be the parent fatty acid of 1, named (3E,6E,8E,10R,11R,12E)-11-hydroxy-4,8,10,12-tetramethyltetradeca-3,6,8,12-tetraenoic acid, which is available from Aurora Fine Chemicals in the United States. Noticeably, this is the first time that compound 2 has been isolated from a natural source.

In the antibacterial activity evaluation of compounds 1 and 2, neither of them showed obvious inhibitory effects, in the range of concentrations tested, against the multi-drug resistant (MDR) strains, including Enterococcus faecalis CCARM 5172, Enterococcus faecium CCARM 5203, Staphylococcus aureus CCARM 3090, Escherichia coli CCARM 1009, and Salmonella typhimurium CCARM 8250. Some branched-chain oleic acid derivatives exhibited growth inhibition against MCF-7 and HT-29 cells [12]. Therefore, we also tested the cytotoxicity of compounds 1 and 2 against these two cell lines, but they showed null activity up to the concentration of 50 µM (data not shown).

Fatty acid amides are a class of compounds formed from a fatty acid and an amine that play an important role in intracellular signaling, many of which in nature have ethanolamine as the amine component [13,14,15]. Inactivation of the ndgR_yo gene in S. youssoufiensis OUC6819 led to the isolation of a new fatty acid amide and (1) and its parent branched-chain fatty acid (2). The 3-amino-butyl acid moiety in 1 is likely to come from l-glutamate [16]. As the IclR-like global regulator, NdgR, is generally involved in amino acid metabolism [6], we proposed that ndgR_yo in S. youssoufiensis OUC6819 might contribute to the generation of the unusual branched-chain fatty acid amide (1) with an amino acid as the amine component.

3. Materials and Methods

3.1. General Experimental Procedures

1D (¹H and ¹³C) and 2D (COSY, HSQC, HMBC, and NOESY) NMR spectra were recorded on Bruker Avance III 600 spectrometers at 298 K. The mixing time used for the NOESY spectrum was 142 ms. Chemical shifts were reported with reference to the respective solvent peaks and residual solvent peaks (δ_H 3.31 and δ_C 49.0 for CD₃OD). HR-ESIMS data were obtained on a Q-TOF Ultima Global GAA076 MS spectrometer. HPLC was performed on an Agillent 1260 Infinity apparatus equipped with a diode array detector (DAD).

3.2. Bacterial Strains and Culture Conditions

Escherichia coli DH5α served as the host for general subcloning [17]. Escherichia coli ET12567/pUZ8002 was used as the cosmid donor host for E. coli-Streptomyces intergenic conjugation [18]. Escherichia coli BW25113/pIJ790 was used for λRED-mediated PCR-targeting [19]. The S. youssoufiensis OUC6819 was isolated from reed rhizosphere soil collected from the mangrove conservation area of Guangdong province, China [9]. E. coli strains were routinely cultured in Luria–Bertani (LB) liquid medium at 37 °C, 200 rpm, or LB agar plate at 37 °C. Streptomyces strains were grown at 30 °C on R2YE medium for sporulation and ISP4 for conjugation, and were cultured in tryptic soy broth (TSB) medium for genomic DNA preparation. Fermentation medium consists of 1% soluble starch, 2% glucose, 4% corn syrup, 1% yeast extract, 0.3% beef extract, 0.05% MgSO₄·7H₂O, 0.05% KH₂PO₄, 0.2% CaCO₃, and 3% sea salt, pH = 7.0.

3.3. DNA Isolation and Manipulation

Plasmid extractions and DNA purifications were carried out using standardized commercial kits (OMEGA, Bio-Tek, Guangzhou, China). PCR reactions were carried out using Pfu DNA polymerase (TIANGEN, Beijing, China). Oligonucleotide synthesis and DNA sequencing were performed by TSINGKE company (Qingdao, China).

3.4. Gene Inactivation

Positive cosmids harboring the ndgR_yo gene were screened against the genomic library of S. youssoufiensis OUC6819 by using PCR with primers listed in Table S1. One cosmid, pWLI551 (Table S2), was obtained and then confirmed by DNA sequencing in TSINGKE company (Qingdao, China). The amplified aac(3) IV-oriT resistance cassette from pIJ773 was transformed into E. coli BW25113/pIJ790 containing pWLI551 to replace an internal region of the target gene, the PCR primers are listed in Table S3. The mutant cosmid was constructed and introduced into S. youssoufiensis OUC6819 by conjugation from E. coli ET12567/pUZ8002 according to the reported procedure, using S. youssoufiensis OUC6819 ultrasonic fragmented mycelia as acceptors [20]. The desired mutants were selected by the apramycin-resistant and kanamycin sensitive phenotype, and were confirmed by PCR (Figure S16), using the primers listed in Table S4.

3.5. Isolation and Purification of the Compounds

The fermentation broth (50 mL) of the S. youssoufiensis OUC6819 strains was extracted twice with an equal volume of ethyl acetate, and subsequently subjected to the HPLC analysis. Analytical HPLC was performed on a YMC-Pack ODS-A column (5 μm, 4.6 × 150 mm) developed with a linear gradient from 20% to 100% B/A in 45 min (phase A: H₂O; phase B: 100% acetonitrile) at the wavelength of 220 nm. The culture broth (15 L) of a scaled-up culture of the ΔndgR_yo mutant was extracted with ethyl acetate and evaporated at room temperature, which was partitioned between 90% methanol and n-hexane to remove nonpolar components. Compounds 1 (3 mg) and 2 (10 mg) were obtained by separation of the methanol layer with a linear gradient from 70% to 90% B at a flow rate of 2.0 mL/min using a YMC-Pack ODS-A column (5 μm, 120 Å, 250×10 mm; wavelength 220 nm).

Compound 1: Yellow oil; [α] _D −8.1 (c 0.1, MeOH); CD (MeOH) λ_max (Δε) 202.5 (+5.23), 234.5 (−3.99) nm; ¹H and ¹³C NMR data, see Table 1; HR-ESIMS m/z 378.2654 [M + H]⁺ (calcd for C₂₂H₃₆NO₄, 378.2644).

Compound 2: Yellow oil; [α] _D −2.8 (c 0.1, MeOH); CD (MeOH) λ_max (Δε) 200.5 (+4.66), 231.5 (−3.51) nm; ¹H and ¹³C NMR data, see Table 1; HR-ESIMS m/z 310.2397 [M + NH₄]⁺ (calcd for C₁₈H₃₂NO₃, 310.2382).

3.6. Nucleotide Sequence Accession Number

The nucleotide sequence of ndgR_yo in this paper has been deposited in the GenBank database, and the accession number is MH252211.

4. Conclusions

A new fatty acid amide (1) with an unusual 3-amino-butyl acid as the amine component, together with its parent fatty acid (2) were isolated from the ΔndgR_yo mutant strain of S. youssoufiensis OUC6819. Compounds 1 and 2 displayed neither inhibitory effects against the five MDR bacterial strains, nor cytotoxicity against MCF-7 and HT-29 cancer cell lines. This study demonstrated that the ndgR_yo homologs might serve as a target for activation of structurally novel secondary metabolites in the Streptomyces strains.