Selectively improving nikkomycin Z production by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding in Streptomyces ansochromogenes
© Liao et al; licensee BioMed Central Ltd. 2009
Received: 24 September 2009
Accepted: 23 November 2009
Published: 23 November 2009
Nikkomycins are a group of peptidyl nucleoside antibiotics and act as potent inhibitors of chitin synthases in fungi and insects. Nikkomycin X and Z are the main components produced by Streptomyces ansochromogenes. Of them, nikkomycin Z is a promising antifungal agent with clinical significance. Since highly structural similarities between nikkomycin Z and X, separation of nikkomycin Z from the culture medium of S. ansochromogenes is difficult. Thus, generating a nikkomycin Z selectively producing strain is vital to scale up the nikkomycin Z yields for clinical trials.
A nikkomycin Z producing strain (sanPDM) was constructed by blocking the imidazolone biosynthetic pathway of nikkomycin X via genetic manipulation and yielded 300 mg/L nikkomycin Z and abolished the nikkomycin X production. To further increase the yield of nikkomycin Z, the effects of different precursors on its production were investigated. Precursors of nucleoside moiety (uracil or uridine) had a stimulatory effect on nikkomycin Z production while precursors of peptidyl moiety (L-lysine and L-glutamate) had no effect. sanPDM produced the maximum yields of nikkomycin Z (800 mg/L) in the presence of uracil at the concentration of 2 g/L and it was approximately 2.6-fold higher than that of the parent strain.
A high nikkomycin Z selectively producing was obtained by genetic manipulation combined with precursors feeding. The strategy presented here might be applicable in other bacteria to selectively produce targeted antibiotics.
In this paper, a strain which produced high-level of nikkomyicns obtained by traditional strain improvement was chosen as the parent strain for genetic manipulation. A nikkomycin Z selectively producing strain was generated by blocking the imidazolone biosynthetic pathway of nikkomycin X. The subsequent uracil feeding further enhanced the yield of nikkomycin Z.
Construction of the nikkomycin Z selectively producing strain
In S. ansochromogenes, sanP is located in one of the operons . To exclude the potential polar effect of sanP disruption on the loss of nikkomycin X, the complementary plasmid pIM229::sanP constructed with wild type sanP under the control of the erm E* promoter was introduced into the sanPDM. The introduction of pIM229::sanP into sanPDM restored nikkomycin X production. HPLC analysis and bioassay showed that the amounts of nikkomycins produced by the complemented strain were almost the same as those produced by the parent strain (Fig. 3B). The disruption and complementation experiments suggested that sanP was a key determinant in nikkomycin X biosynthesis and disruption of sanP did not affect the production of nikkomycin Z.
Effect of precursor of nucleoside moiety on nikkomycin Z production
To further increase the yield of nikkomycin Z, the effects of different precursors on its production were investigated. Recent studies revealed that UMP was the direct precursor for the biosynthesis of the nucleoside moiety of nikkomycin Z  and UMP could be synthesized from uracil or uridine through salvage pathway. So, the effect of supplementation of varying concentration (1 g/L-3 g/L) of uracil on nikkomycin production was measured in S. ansochromogenes TH322 and sanPDM. As shown in Fig. 4B, uracil had a stimulatory effect on nikkomycin Z production in both strains and uracil supplementation led to much more nikkomycin Z production in sanPDM than that in TH322 at all concentrations tested in the study. Addition of uracil (1 g/L to 2 g/L) resulted in increased nikkomycin Z production from 450 mg/L to 800 mg/L in sanPDM or increased from 375 mg/L to 630 mg/L in TH322, whereas nikkomycin X production was decreased from 195 mg/L to 96 mg/L in TH322 (Fig. 4C). The change of biomass was observed in both strains. Addition of uracil (2 g/L) in the culture medium resulted in reduced the biomass by approximate 40% compared to that of the control (Fig. 4A). Addition of more uracil (3 g/L) resulted in significant inhibition of cell growth and reduced the final yield of nikkomycins in both strains. These results demonstrated that addition higher concentration of uracil might inhibit cell growth.
The uracil residue, nikkomycin Z production and biomass in culture medium of sanPDM with uracil supplementation
Nikkomycin Z production
300 ± 20
14.8 ± 2.2
400 ± 15
13.4 ± 1.7
480 ± 18
11.0 ± 2.2
790 ± 23
9.5 ± 1.4
0.080 ± 0.020
800 ± 18
9.1 ± 1.0
0.484 ± 0.031
Similar studies were carried out to determine the effect of uridine supplementation on nikkomycin Z production. Addition of uridine (0.5 g/L to 2 g/L) resulted in enhancing nikkomycin Z production from 320 mg/L to 400 mg/L without detectable effect on the biomass (data not shown). Thus, uridine exerted a less stimulatory effect than uracil on nikkomycin Z production.
Effect of precursor of peptidyl moiety on nikkomycin Z production
Effect of L-lysine and L-glutamate on nikkomycin Z production in sanPDM in the presence of 2 g/L uracil
Nikkomycin Z production
785 ± 10
9.9 ± 1.9
780 ± 16
10.2 ± 2.1
700 ± 40
9.2 ± 1.3
500 ± 9
5.3 ± 1.9
770 ± 30
9.7 ± 1.7
720 ± 12
9.5 ± 2.1
400 ± 23
4.8 ± 1.6
750 ± 30
9.6 ± 1.5
740 ± 24
9.8 ± 2.0
Nikkomycin Z is a potential antifungal agent with clinical significance. However, the presence of its isomer, nikkomycin X during fermentation interferes with the purification of nikkomycin Z and increases the production cost. Thus, we chose a nikkomycin high producing strain (S. ansochromogenes TH322) to perform genetic manipulation and obtained a nikkomycin Z selectively producing strain (sanPDM). sanP encoded a type II thioesterase which hydrolytically released SanO from β-OH-His-SanO to form β-OH-His (Fig. 2). Disruption of it resulted in blocking the synthesis of β-OH-His and failed production of nikkomycin Cx and nikkomycin X, whereas, disruption of sanP did not affect nikkomycin Z production because uracil, the base in nikkomycin Z, was biosynthesized via de novo or salvage pathway. These results were mainly in accordance with those studies in S. tendae . Disruption of nikP2 almost abolished the nikkomycin X production, while disruption of sanP in S. ansochromogenes resulted in eliminating nikkomycin X production. Loss of the nikkomycin X production in the fermentation would significantly improve the recovery of nikkomycin Z during large scale manufacturing process.
Feeding precursors have been proved to be a successful strategy to increase the yield of targeted antibiotics, such as feeding ornithine to enhance clavulanic acid production in S. clavuligerus . We found that addition of uracil (2 g/L) increased production of nikkomycin Z by 2-fold while suppressed nikkomycin X production by 85% in TH322. A possible explanation to these results was that UMP derived form uracil competed with imidazolone for incorporation into core of nikkomycin. A similar study on salinosporamide B in Salinispora tropica was verified, finding that addition of butyric acid-the precursor of salinosporamide B to the culture medium resulted in increased the production of salinosporamide B by approximately 3.2-fold while inhibited the production of salinosporamide A by 26% .
Uracil supplementation led to considerable increase of nikkomycin Z production, while its high concentration (more than 2 g/L) significantly inhibited cell growth and reduced the yield of nikkomycin Z. Our results were in accordance with the reports of Krishna . Uracil (2 g/L) could improve the rifamycin SV production by 505 mg/L in Amycolatopsis mediterranei MV35R, while higher concentration inhibited both the cell growth and rifamycin SV production. The reason why higher concentration of uracil sharply inhibited the cell growth was still unclear. Uridine had a slight effect on nikkomycin Z production. Uracil salvage pathway had been identified in S. grieus and S. coelicolor  and genes involving in uracil transport (uracil permase) and transformation to UMP (uracil phosphoribosyltransferase) were highly conserved in Streptomyces. However, gene encoding uridine kinase that converted uridine into UMP had not been found in Streptomyces so far. So, we speculated that the stimulatory effect of uridine on nikkomycin Z production may be due to the partial degradation of uridine to uracil and uracil acted as precursor to participate in nikkomycin Z biosynthesis.
Combining genetic manipulation with uracil feeding enabled us to selectively enhance the yield of nikkomycin Z. As nikkomycin Z could be recovered without additional separation from other related components in sanPDM, it would simplify the downstream purification process and lower the production cost.
Strains, plasmids and culture conditions
Streptomyces ansochromogenes TH322, a high-level producer of nikkomycins (300 mg/L nikkomycin Z and 600 mg/L nikkomycin X), was collected in our laboratory. S. ansochromogenes sanPDM, the nikkomycin Z selectively producing strain created in this study was deposited with the China General Microbiological Culture Collection Center (CGMCC) and assigned the accession number was CGMCC-3086. Candida albicans CGMCC No 2.2086 was used as indicator strain for nikkomycin bioassays. Escherichia coli JM109 was used as host for cloning and subcloning. E. coli ET12567 containing pUZ8002 was used for conjugal transfer according to the established protocol . Streptomyces-E. coli shuttle plasmid pKC1139 for gene disruption was provided by Prof. Keith Chater (John Innes Center, Norwich, UK)
S. ansochromogenes TH322 was grown on mannitol/soya (MS) medium at 28°C  for 6 days to form spores. For genomic DNA isolation, the S. ansochromogenes TH322 and sanP disruption mutant (sanPDM) were grown in YEME medium for 2 days. SP medium (3% mannitol, 1% soluble starch, 0.5% soy peptone and 0.8% yeast extract, pH 6.0) was used for nikkomycin production.
When necessary, antibiotics were used at the following concentrations: apramycin, 10 μg ml-1 in YEME or MS for S. ansochromogenes TH322, 100 μg ml-1 in LB for E. coli; kanamycin, 10 μg ml-1 in YEME or MS for S. ansochromogenes TH322, 100 μg ml-1 in LB for E. coli.
Primers and PCR conditions
The oligonucletide primers used to amplify the sanP were P1 (5'-GTGGCACCGCGACAGGCCG-3') and P2 (5'-TCAACTCTCATCGGCTC-3'). P3 (5'-GCGGCCAGCTACTTCCGGGAC-3') and P4 (5'-GCAGAAAGGCCGAGCGCATGT-3') were used to check the sanP disruption mutant. The PCR was performed using KOD (Toyobo, Japan): an initial denaturation at 95°C for 4 min followed by 30 cycles of amplification (95°C for 1 min, 62°C for 30 sec, and 68°C for 1 min) and additional 10 min at 68°C.
Construction of sanP disruption mutants
Disruption of sanP was performed by gene replacement via homologous recombination. For this purpose, a 2.5 kb Pst I-Sac I DNA fragment containing the complete sanP sequence was inserted into the Pst I-Sac I site of pBluescript II KS (-) to generate pBS::sanP. The kanamycin-resistance gene (neo) was isolated from pUC119::neo after digestion with Bam HI and Kpn I, blunted and inserted into the blunted Bam HI site of sanP in pBS::sanP to generate pBS::sanP::neo. A 3.5 kb Pst I-Sac I fragment from pBS::sanP::neo was inserted into the Pst I-Sac I site of pIJ2925 to give pIJ2925::sanP::neo. The resulting plasmid was then digested with Bgl II and the resulting 3.5 kb fragment was inserted into the Bam HI site of pKC1139 to generate pKC1139::sanP::neo.
The resulting plasmid pKC1139::sanP::neo was passed through E. coli ET12567/pUZ8002 and then introduced into S. ansochromogenes TH322 by conjugal transfer according to the established techniques. The resulting transformants were inoculated on MS plates to form spores. Gray spores were harvested and spread on MM plates containing kanamycin as the resistance selection. After incubating at 42°C for 3 days, the colonies that conferred kanamycin resistance (KanR) and apramycin sensitivity (AprS) were selected and further confirmed as sanP disruption mutatants by PCR using the sanPDM genomic DNA as template.
Complementation of the sanP mutant
For complementation analysis, complete sanP DNA fragment was amplified from S. ansochromogenes TH322 genomic DNA by PCR using P1 and P2 primers. The amplified fragment was subsequently inserted into the Eco RV site of pIM229 under the control of promoter of erm E* . The resulting recombinant plasmid pIM229::sanP was then integrated into the chromosomal attB site of sanP disruption mutant by conjugal transfer.
Fermentation of S. ansochromognes
Spores of S. anchromogenes TH322 or sanPDM were inoculated in YEME. The cultures were grown at 28°C on a rotary shaker (220 rpm) for 48 h and used as seed cultures. 1 ml (0.5% V/V) of seed culture was inoculated into flasks containing 50 ml of SP medium, and then fermented at 28°C on a rotary shaker (200 r.p.m) for 6 days.
Quantification of uracil in the culture filtrates
Uracil was analyzed according to the method of Gao  by HPLC using a Zorbax SB-C18 reverse-phase column at 254 nm with a 0.5 ml min-1 flow rate at 25°C. The elution profile was a linear gradient of 0%-20% solution B (A = 5 mM ammonium acetate; B = methanol) over 15 min. An aliquot of 20 μL culture filtrates was injected for HPLC analysis.
Bioassay and HPLC analysis of nikkomycins
The culture filtrates were harvested by centrifugation and the supernatant was filtered through a Minipore membrane (pore diameter 0.2 μm). Nikkomycin Z (Sigma, cat. No. N8028) was used as standard. HPLC analysis and bioassay of nikkomycins against Candida albicans were performed as described previously .
This work was supported by grants from the Ministry of Science and Technology of China (Grant nos. 2009CB118905 and 2006AA10A209) and from the Chinese Academy of Sciences (Grant no. KSCX2-YW-N-027).
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