Novel nikkomycin analogues generated by mutasynthesis in Streptomyces ansochromogenes
© Feng et al.; licensee BioMed Central Ltd. 2014
Received: 19 February 2014
Accepted: 4 April 2014
Published: 21 April 2014
Nikkomycins are competitive inhibitors of chitin synthase and inhibit the growth of filamentous fungi, insects, acarids and yeasts. The gene cluster responsible for biosynthesis of nikkomycins has been cloned and the biosynthetic pathway was elucidated at the genetic, enzymatic and regulatory levels.
Streptomyces ansochromogenes ΔsanL was constructed by homologous recombination and the mutant strain was fed with benzoic acid, 4-hydroxybenzoic acid, nicotinic acid and isonicotinic acid. Two novel nikkomycin analogues were produced when cultures were supplemented with nicotinic acid. These two compounds were identified as nikkomycin Px and Pz by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Bioassays against Candida albicans and Alternaria longipes showed that nikkomycin Px and Pz exhibited comparatively strong inhibitory activity as nikkomycin X and Z produced by Streptomyces ansochromogenes 7100 (wild-type strain). Moreover, nikkomycin Px and Pz were found to be more stable than nikkomycin X and Z at different pH and temperature conditions.
Two novel nikkomycin analogues (nikkomycin Px and Pz) were generated by mutasynthesis with the sanL inactivated mutant of Streptomyces ansochromogenes 7100. Although antifungal activities of these two compounds are similar to those of nikkomycin X and Z, their stabilities are much better than nikkomycin X and Z under different pHs and temperatures.
Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes and Streptomyces tendae. Acting as competitive inhibitors of chitin synthase, nikkomycins inhibit the growth of filamentous fungi, insects, acarids and yeasts . The gene cluster responsible for biosynthesis of nikkomycins has been cloned from S. ansochromogenes and S. tendae. Biosynthesis of nikkomycins has been studied extensively at the genetic, enzymatic and regulatory levels in both Streptomyces strains [4–11].
Mutasynthesis has become a useful method in the generation of new antibiotic derivatives. It can be started by blocking the biosynthesis of key biosynthetic intermediates, and a variety of alternative intermediates can then be fed to the mutant to produce novel antibiotic derivatives . This approach could expand the chemical diversity of antibiotics and produce novel compounds with improved pharmaceutical properties [13, 14].
Here we described the mutasynthesis of nikkomycins Px and Pz by feeding S. ansochromogenes ΔsanL with analogues of picolinic acid. Supplementation of the mutant strain with nicotinic acid led to production of two novel nikkomycin analogues which exhibited improved properties.
Construction of sanL inactivation mutant and its complementation
Feeding of ΔsanL strain with analogues of picolinic acid
Previous studies suggested that the picolinate-CoA ligase had broad substrate specificities in accepting picolinic acid and its analogues . Cultures of ΔsanL strain were fed with 1 mM analogues of picolinic acid (benzoic acid, 4-hydroxybenzoic acid, nicotinic acid and isonicotinic acid). Cultures supplemented with benzoic acid produced nikkomycin Bx and Bz, which are consistent with the results from S. tendae nikC mutant . When the cultures were supplemented with nicotinic acid, culture filtrates of ΔsanL strain regained the ability to inhibit the growth of C. albicans and A. longipes like the wild-type and complementary strains (Figure 2B and C). HPLC analysis revealed two distinct peaks at retention time 18.5 and 19.5 min (Figure 2A). In contrast, no obvious peak was detected at corresponding retention time in cultures supplemented with 4-hydroxybenzoic acid and isonicotinic acid except for a minor peak at retention time 22.2 min (Figure 2A). Bioassay was detected against C. albicans and A. longipes, no inhibition zone was observed (Figure 2B and C).
Isolation and bioactive assay of nikkomycin Px and Pz
Structural determination of nikkomycin Px and Pz
Structure modeling analysis indicated that nikkomycin Px and Pz may be new members of nikkomycin family incorporated with the supplemented substrate precursor, nicotinic acid, in the molecules. This assumption was further validated by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopic (NMR) analysis.
Summary of 1 H and 13 C NMR data for nikkomycin Px and Pz in D 2 O
δ (1H, mult., J)
δ (1H, mult., J)
7.53 (1H, s)
5.74 (1H, d, 8.1)
9.16 (1H, s)
7.42 (1H, d, 8.1)
5.53 (1H, d, 4.5)
5.65 (1H, d, 4.2)
4.43 (1H, dd, 4.5, 5.5)
4.29 (1H, dd, 4, 6)
4.52 (1H, t, 5.5)
4.42 (1H, t, 6)
4.25 (1H, dd, 4.0, 5.5)
4.17 (1H, dd, 3.5, 6.5)
4.74 (1H, d, 4.0)
4.73 (1H, d, 4)
4.36 (1H, d, 4)
4.35 (1H, d, 4)
2.36 (1H, m)
2.55 (1H, m)
5.37 (1H, s)
5.36 (1H, s)
0.68 (3H, d, 7)
0.67 (1H, d, 7.1)
8.7 (1H, s)
8.68 (1H, s)
8.49 (1H, d, 8.2)
8.46 (1H, d, 8.2)
7.97 (1H, dd, 8.1, 6)
7.94 (1H, dd, 8.1, 5.9)
8.62 (1H, d, 5.5)
8.60 (1H, d, 5.7)
Stability of nikkomycin Px and Pz under different pHs and temperatures
No obvious instability of the four compounds was observed at 10°C, but all of them became less stable with increasing temperatures (Figure 6B). The stability of nikkomycin Px and Pz is much better than that of nikkkomycin X and Z.
Owing to their low toxicity to mammals and bees, nikkomycins are considered as competitive inhibitors of chitin synthetases in fungi, insects, acarids and yeasts . During the past several decades, extensive studies have been carried out to elucidate the biosynthetic pathway of nikkomycins [4, 7–11]. Based on prior knowledge, novel nikkomycin analogues can be produced by generating mutant deficient in formation of biosynthetic intermediates and subsequently feeding the mutant with a variety of alternative intermediates.
Two novel nikkomycin analogues (nikkomycin Px and Pz) were generated by supplementation of S. ansochromogenes ΔsanL with nicotinic acid. Unlike nikkomycin X and Z, nikkomycin Px and Pz contain 4-(3′-pyridinyl)-homothreonine as the peptidyl moiety. As expected, the position of nitrogen in the pyridinyl ring was changed due to the difference between nicotinic acid and picolinic acid. Surprisingly, the hydroxyl group is absent in the pyridinyl ring of nikkomycin Px and Pz (Figures 4A and 5A). Previous study in our lab demonstrated that SanH and SanI are responsible for the hydroxylation of pyridinyl residue in nikkomycin X and Z . The lack of the hydroxyl group in the pyridinyl ring of nikkomycin Px and Pz indicated that SanH and SanI are unable to recognize substrate with 3′-pyridinyl ring.
Antifungal activities of nikkomycin Px and Pz are similar to those of nikkomycin X and Z (Figure 2B and C). This result indicated that incorporation of nicotinic acid into the peptidyl moiety of nikkomycins has no effect on their biological activities. However, nikkomycin Px and Pz displayed better stabilities than nikkomycin X and Z under different pHs and temperatures (Figure 6). This was attributed to the combined effect of change of nitrogen position and loss of hydroxyl group in the pyridinyl ring. Our results reinforced the proposal that the stability of nikkomycins was influenced by the peptidyl moiety of the compounds [15, 18].
Two novel nikkomycin analogues (nikkomycin Px and Pz) were generated by mutasynthesis with S. ansochromogenes ΔsanL strain. Nikkomycin Px and Pz showed comparable antifungal activity as nikkomycin X and Z. Moreover, they also displayed better stabilities than nikkomycin X and Z under different pHs and temperatures.
Materials and methods
Strains and culture conditions
Streptomyces ansochromogenes 7100 deposited at China General Microbiological Culture Collection (CGMCC) is a natural nikkomycin producer. Candida albicans and Alternaria longipes from CGMCC were used as indicator strains for nikkomycin activity bioassay . E. coli DH5α was used for propagating plasmids. E. coli ET12567 (pUZ8002) was used for conjugal transfer of DNA from E. coli to Streptomyces.
Streptomyces strains were grown on R2YE medium, mannitol soya flour (MS), minimal medium (MM) or in yeast extract-malt extract (YEME) liquid medium at 28°C. The culture conditions for nikkomycin production were essentially as described previously . In brief, spore suspensions were inoculated in liquid YEME and incubated at 28°C on a rotary shaker (220 rpm) for 48 hr as seed culture. A total of 1 ml seed culture was transferred into 100 ml SP medium (3% mannitol, 1% soluble starch, 0.75% yeast extract, and 0.5% soy peptone, pH 6.0)  for nikkomycin production. When necessary, apramycin or kanamycin was added at concentration of 50 μg/ml for R2YE, 7 μg/ml for MM and YEME.
Construction of ΔsanL and its complementation
For the construction of ΔsanL in S. ansochromogenes, a 2.1 kb Bgl II-Bst XI DNA fragment containing complete sanL was blunted and inserted into the Eco RV site of pBluescript II KS(+). The resulting plasmid was linearized with Bcl I and blunted with Mung bean nuclease. The kanamycin-resistance gene (neo) was obtained from pUC119::neo after digestion with Bam HI and Kpn I, blunted and ligated into the blunted Bcl I site of sanL to generate pBS-L::neo. A 3.1 kb insert of pBS-L::neo was isolated after digestion with Hin dIII and Eco RI, and the recovered fragment was then ligated into the same sites of pKC1139. The resulting pKC1139L::neo was confirmed by restriction digestion and then introduced into S. ansochromogenes 7100 by intergeneric conjugation from E. coli ET12567 (pUZ8002) according to standard techniques . The resulting transformants were inoculated on agar MM to form spores. Gray spores were harvested and spread on agar MM containing kanamycin as resistance selection. After incubating at 40°C for 3 days, the sanL disruption mutants were selected with colonies exhibiting kanamycin resistance (Kanr) and apramycin sensitivity (Aprs). Kanr/Aprs strains were further verified by PCR and Southern blot analysis. The confirmed sanL disruption mutant was designated as ΔsanL.
For complementation experiments, the constitutive hrdB promoter was used to drive the expression of sanL. The hrdB promoter was amplified from genomic DNA of Streptomyces coelicolor M145 with primers hrdB-pF/hrdB-pR (5′-aattagatctCCGCCTTCCGCCGGAACG-3′; 5′-GAACAACCTCTCGGAACGTTGA-3′). The coding region of sanL was amplified from genomic DNA of S. ansochromogenes 7100 with primer pair sanL-cF/sanL-cR (5′-ATGCTGACCGTGAACGGGAACTC-3′; 5′-attgaattcTCATGCCCGGGCCTCCTCG-3′). Prior to PCR amplification, hrdB-pR was phosphorylated with T4 polynucleotide kinase to facilitate subsequent ligation reactions. The hrdB promoter fragment was digested with Bgl II, and the sanL-coding region was digested with Eco RI. Both the hrdB promoter and sanL-coding fragment were ligated together with Bam HI/Eco RI double-digested pSET152. The resulting pSET152::sanL was introduced into the ΔsanL strain to generate the complementation strain sanLc.
Detection of nikkomycins and antifungal bioassays
For the analysis of nikkomycin, culture broths were centrifuged and the supernatants were filtered through a millipore membrane (pore diameter 0.22 μm). HPLC analysis was performed with Agilent 1100 HPLC system and ZORBAX SB C-18 (5 μm, 4.6 × 250 mm). Chemical reagent, mobile phase and gradient elution process were as described previously . The elution was detected with photodiode array at 260 and 290 nm for nikkomycins. Bioassays against C. albicans and A. longipes were carried out by a disk diffusion method as described previously . The procedure is essentially the same for both indicator strains except that an overnight culture of C. albicans was used while 5 day-old culture was used for A. longipes. The modified potato dextrose agar medium (20% potatoes, 2% glucose and 0.8% agar) was heated to dissolve and then cooled to 50°C before use. Cultures of indicator strains (50 μl for C. albicans and 10 ml for A. longipes) were well dispersed in 100 ml pre-dissolved medium and poured to a 15 cm plate. Oxford cups were placed onto the plates and antibiotics were then added into the Oxford cups. After incubation for 12–24 h at 28°C, the zone of inhibition was assessed.
Isolation of nikkomycin Px and Pz
The method for isolation of nikkomycin Px and Pz was similar to that for polyoxin P . Briefly, seed culture of S. ansochromogenes ΔsanL was prepared as mention above. A total of 30 ml seed culture transferred into 3 L SP supplemented with 1 mM nicotinic acid. After 5 days of fermentation, the culture broth of S. ansochromogenes ΔsanL was harvested by centrifugation and the supernatant was adjusted to pH 4.5 with acetic acid. The sample was then chromatographed on a macroporous absorption resin HP-20 (Mitsubishi) column, and the flow-through was collected and subjected to a Dowex 50WX2 (Sigma) column. The column was eluted with 0.4 N ammonia solution and fractions with antifungal activity was collected and concentrated to a small volume in vacuo. After addition of 6 volumes of cold ethanol, the precipitate was collected by centrifugation. The dried powder was subsequently dissolved in water and further purified by HPLC.
Stability determination of nikkomycin X, Z, Px and Pz
To dissect their stabilities under different pH conditions, the four antibiotics (nikkomycin X, Z, Px and Pz) were dissolved in buffers with different pH values: 0.2 M CH3COONa buffer (pH adjusted to 4.0 or 5.0 with acetic acid), 0.05 M KH2PO4 buffer (pH adjusted to 6.0 and 7.0 with NaOH), 0.05 M Tris buffer (pH adjusted to 8.0 with HCl), 0.025 M Na2CO3 buffer (pH adjusted to 9.0 and 10.0 with NaOH). All samples were incubated at 25°C for 27 days. To dissect the effect of temperature on their stabilities, antibiotics were dissolved in 0.05 M KH2PO4 buffer (pH 6.0) and incubated at 10, 20, 30, 40 and 50°C for 27 days. Residual antibiotics were quantified by the peak areas.
MS analysis and tandem mass spectrometry analysis were carried out on Triple-Quadrupole LC-MS/MS (Agilent 1260/6460) in positive mode. All NMR spectra were recorded on a Bruker Advance spectrometer (AV500 MHz).
This work was supported by grants from the Ministry of Science and Technology of China (Grant No. 2013CB734001), and the National Natural Science Foundation of China (Grant Nos. 31270110 and 31030003). We thank Drs Guomin Ai and Jinwei Ren (the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for assistance with Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR).
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