Novel nikkomycin analogues generated by mutasynthesis in Streptomyces ansochromogenes

Background 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. Results 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. Conclusions 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.

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 [12]. 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
Mutasynthesis requires the generation of mutants that are blocked in the formation of key biosynthetic intermediate of the end-product. For this purpose, we inactivated sanL by double-crossover recombination in S. ansochromogenes 7100 ( Figure 1B). Sequence analysis revealed that sanL encodes a L-lysine 2-aminotransferase which showed 97% identity with NikC from S. tendae. The disruption of sanL was confirmed by PCR and Southern blot (data not shown). The resulting inactivated mutant was subjected to HPLC analysis to assess nikkomycin production. In comparison with nikkomycin production in the wild-type strain, no nikkomycin was detected in the fermentation medium of sanL mutant (ΔsanL) (Figure 2A). To test the antifungal activity of ΔsanL strain, the culture filtrates of ΔsanL and wild-type strains were subjected to bioassays against C. albicans and A. longipes. Culture filtrate of ΔsanL lost the ability to inhibit the growth of C. albicans and A. longipes ( Figure 2B and C). Because sanL is situated within a group of 7 genes that form an operon, inactivation of downstream genes by polar effects on transcription might have contributed to the phenotype observed. To exclude this possibility, the ΔsanL strain was complemented by pSET152::sanL in which a single copy of sanL was driven by the constitutive hrdB promoter. The resulting complementation strain (sanLc) restored nikkomycin production ( Figure 2A) and its antifungal activities ( Figure 2B and C).

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 [4]. 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 [15]. 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 wildtype 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
In order to characterize the compounds responsible for antifungal activity, we isolated and purified them from 3 L broth of ΔsanL strain in fermentation tank. The two Figure 1 Chemical structure (A) and organization of the gene cluster for nikkomycin biosynthesis (B). The peptidyl moiety (HPHT) and nucleoside moiety of nikkomycin were indicated by blue color and red color, respectively. The solid arrow shows sanL and its orientation. The 2.1 kb BglII/BstXI fragment contains sanL and its flanking sequences. The 3.1 kb BglII/BstXI fragment was used for disruption of sanL. The kanamycin resistance gene (neo) was inserted into BclI site of sanL.
purified compounds (nikkomycin Px and Pz) are white powders, soluble in water but insoluble in ethanol, ether, ethyl acetate, methanol and acetone. When the compounds were dissolved in 0.1% trifluoroacetic acid (TFA) solution, nikkomycin Px and Pz showed maximum ultraviolet absorption at 290 nm and 260 nm, respectively ( Figure 3A, B, C and D). In addition, the yields of two compounds were determined. It showed that ΔsanL strain supplemented with nicotinic acid can produce nikkomycin Px and Pz at 84 mg/L and 122 mg/L, respectively. The yield of nikkomycin Pz is close to that of nikkomycin Z (120 mg/L) in the wild-type strain, whereas the yield of nikkomycin Px is lower than that of nikkomycin X (220 mg/L) in the wildtype strain [16]. To compare their antifungal activities, five micrograms of each compound (nikkomycin X, Z, Px and Pz) were tested against C. albicans and A. longipes. Both nikkomycin Px and Pz exhibited comparatively strong inhibitory activity as nikkomycin X and Z produced by the wild-type strain ( Figure 3E and F).

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.
For nikkomycin Px ( Figure 5A), ESI-MS analysis revealed the same fragmentation pattern as nikkomycin Pz, indicating the structure similarity between these two compounds. The molecular formula was determined as   1 H and 13 C signals of the base group were finally assigned by 1 H-13 C HSQC data (Additional file 1: Figure S2B) and 1 H-13 C HMBC data (Additional file 1: Figure S2C). The nucleoside moiety in nikkomycin Px was corroborated as aminohexuronic acid with N-glycosidically  Thus, it was confirmed that nikkomycin Px shares the same nucleoside moiety with nikkomycin X, but its peptidyl moiety was determined as 4-(3′-pyridinyl)-homothreonine (equivalent to 4′′-(3′′′-pyridinyl)-homothreonine in nikkomycin Px). Chemical shifts of nikkomycin Px were summarized in Table 1.

Stability of nikkomycin Px and Pz under different pHs and temperatures
The stability of nikkomycin Px and Pz was performed. They were more stable than nikkomycin X and Z at pH from 4 to 10. Under acid conditions (pH4, 5 and 6), 60-80% nikkomycin Px and Pz were present after 24 days of incubation whereas only 0-20% nikkomycin X and Z were remained ( Figure 6A). Under neutral and alkaline conditions (pH7, 8, 9 and 10), 40% nikkomycin Px and Pz were present after 24 days of incubation while nikkomycin X and Z were completely degraded ( Figure 6A).
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.

Discussion
Owing to their low toxicity to mammals and bees, nikkomycins are considered as competitive inhibitors of chitin synthetases in fungi, insects, acarids and yeasts [3]. During the past several decades, extensive studies have been carried out to elucidate the biosynthetic pathway of nikkomycins [4,[7][8][9][10][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 [17]. 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].

Conclusions
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.

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 [11]. E. coli DH5α was used for propagating plasmids. E. coli ET12567 (pUZ8002) was used for conjugal transfer of DNA from E. coli to Streptomyces [19].
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 [16].
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) [20] 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 BglII-BstXI DNA fragment containing complete sanL was blunted and inserted into the EcoRV site of pBluescript II KS(+). The resulting plasmid was linearized with BclI and blunted with Mung bean nuclease. The kanamycin-resistance gene (neo) was obtained from pUC119::neo after digestion with BamHI and KpnI, blunted and ligated into the blunted BclI site of sanL to generate pBS-L::neo. A 3.1 kb insert of pBS-L::neo was isolated after digestion with HindIII and EcoRI, 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 [21]. 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 (Kan r ) and apramycin sensitivity (Apr s ). Kan r /Apr s strains were further verified by PCR and Southern blot analysis. The confirmed sanL disruption mutant was designated as ΔsanL.

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 [22]. 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 [5]. 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 predissolved 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 [23]. 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