Biosearch Italia S.p.A., Via R. Lepetit, 34, 21040 Gerenzano (VA), Italy
Correspondence
Flavia Marinelli
fmarinelli{at}biosearch.it
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ABSTRACT |
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INTRODUCTION |
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GE2270 is a novel potent antibiotic inhibiting protein synthesis in Gram-positive bacteria (Goldstein et al., 1993; Selva et al., 1991
). It is the natural precursor of clinical candidate BI-K0376 specifically active against Propionibacterium acnes, including isolates resistant to currently available therapies (Jabes et al., 2002
). GE2270 was discovered from a targeted screen for elongation factor Tu binding antibiotics applied to 34 000 actinomycetes (Selva et al., 1997
). It is characterized by a highly modified peptide backbone consisting of six thiazole rings, one pyridine ring and one oxazoline ring (Fig. 1
) (Colombo et al., 1995
; Kettenring et al., 1991
; Tavecchia et al., 1994
). It is thus structurally related to other thiazolylpeptide antibiotics such as thiostrepton and nosiheptide, which also consist of highly modified peptides with several thiazole rings and which inhibit bacterial protein synthesis. GE2270, and the closely related molecules amythiamicin (Shimanaka et al., 1995
) and GE37468 (Stella et al., 1995
), act as selective inhibitors of bacterial elongation factor Tu (Anborgh & Parmeggiani, 1991
; Heffron & Jurnak, 2000
; Hogg et al., 2002
; Landini et al., 1996
), whereas thiostrepton and related molecules inhibit bacterial protein synthesis by binding to the L11 protein in the 23S ribosomal complex (Cameron et al., 2002
; Porse et al., 1998
).
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In the biosynthesis of GE2270, as for other thiazolylpeptide antibiotics, the thiazole rings derive from one molecule of cysteine and the carboxy group of the adjacent amino acid, leading to the formation of unsubstituted rings; the pyridine ring is derived from the head to head condensation of two serine residues; and the oxazoline ring results from cyclization of serine (De Pietro et al., 2001; Mocek et al., 1993a
, b). Labelling studies on GE2270 component A biosynthesis showed that the methyl groups at ring E (R2), the N-methyl group of asparagine (R3) and the methylene and O-methyl groups at ring D (R1) (Fig. 1
) are derived from a cellular methyl donor (De Pietro et al., 2001
). It was discovered that in P. rosea [2-13C]glycine and [3-13C]serine are effective sources of methylating equivalents; the results of isotopic enrichment are consistent with the existence of a pathway whereby glycine is converted to serine by methylation, and C-2 of glycine can generate methyl groups (De Pietro et al., 2001
).
Vitamin B12 (cobalamin) is a coenzyme involved in carbon skeletal rearrangements and it acts as a cofactor for several enzymes by catalysing methyl transfer reactions, including the methionine synthase (Matthews, 2001). This enzyme transfers a methyl group from N5-methyl tetrahydrofolate to homocysteine as the final step in methionine synthesis. The methyl group of methionine may be derived from serine (or glycine) via N5,N10-methylenetetrahydrofolate, i.e. the folate branch of the methionine pathway (Blanco et al., 1998
). Methionine, via its activated form, S-adenosyl-L-methionine, is considered as the general source of methyl groups in secondary metabolism (Roth et al., 1996
). O-, N- and C-methyltransferases that use S-adenosyl-L-methionine as methyl donor are ubiquitous in the biosynthesis of secondary metabolites such as staurosporine (Yang et al., 1999
), citreamicin (Pearce et al., 1991
), avermectins (Schulman et al., 1986
), erythromycin (Haydock et al., 1991
), avilamycin (Weitnauer et al., 2002
), daunomycin (Connors & Strohl, 1993
), FK-506 (Shafiee et al., 1994
), tylosin (Kreuzman et al., 1988
; Seno & Baltz, 1981
) and A10255 (Favret et al., 1992
), the latter being a thiopeptide antibiotic belonging to the same class as GE2270. Much less is known about different methyltransferases involved in the biosynthesis of other antibiotics such as fortimicin, bialaphos and fosfomycin that seem to utilize methylcobalamin as a direct methyl donor (Kamigiri et al., 1992
; Kuzuyama et al., 1992
, 1995
) and not as a prosthetic group. Different components of the above described methylation pathways i.e. vitamin B12, commercially available as cyanocobalamin, methylcobalamin, cobalt ion, methionine and folic acid were assayed in P. rosea fermentations to investigate their effect on GE2270 antibiotic complex production.
Among inhibitors of methylation reactions, sinefungin, a natural nucleoside structurally analogous to S-adenosyl-L-methionine, acts as a specific inhibitor of S-adenosyl-L-methionine-dependent methyltransferases (Barbes et al., 1990; Borchardt et al., 1979
). Sinefungin was found to inhibit methyltransferases involved in the biosynthesis of secondary metabolites, causing the accumulation of de-methylated antibiotics, as seen for avermectins (Schulman et al., 1985
, 1987
), citreamicin (Pearce et al., 1991
), tylosin (Kreuzman et al., 1988
), pradimicins (Kakinuma et al., 1993
; Saitoh et al., 1995
) and staurosporine (Yang et al., 1999
). S-Adenosyl-L-homocysteine, which is the common nucleoside reaction product, is often also employed to inhibit S-adenosyl-L-methionine-dependent methyltransferases in antibiotic biosynthesis (Bauer et al., 1988
; Connors & Strohl, 1993
; Kreuzman et al., 1988
; Shafiee et al., 1994
). Other methylation inhibitors are aminopterin (4-aminofolate), amethopterin (10-methylaminofolate), D-methionine and DL-ethionine. Aminopterin and amethopterin are structural analogues of folic acid and affect the biosynthesis of tetrahydrofolate derivatives, which act as coenzymes of one-carbon group transferases (Brown & Williamson, 1982
). Sulfamethizole, as for the other sulfonamides, competes with p-aminobenzoate, which is the biosynthetic precursor of folic acid (Brown & Williamson, 1982
). Aminopterin has been used to direct biosynthesis toward demethylated metabolites in the monensin producer Streptomyces cinnamoniensis (Pospisil & Zima, 1987
) and citreamicin in Micromonospora citrea (Pearce et al., 1991
). D-methionine and ethionine act as antimetabolites of L-methionine. The addition of DL-ethionine, D-methionine and aminopterin to the tetracycline producers yields a number of demethylated tetracycline precursors (Miller et al., 1964
).
In this paper, we investigate the effect of coenzymes and inhibitors involved in the methylation pathway on the production of GE2270 antibiotic with the aim of better understanding its biosynthetic pathway and of modulating its complex composition towards the accumulation of desired metabolites.
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METHODS |
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Flask fermentation.
Frozen cultures in D/Seed medium were maintained at -80 °C as a working cell bank and 5 ml was used to inoculate 500 ml Erlenmeyer flasks containing 100 ml medium C. The inoculated flasks were incubated for 48 h at 28 °C with shaking at 200 r.p.m. Then 5 ml aliquots were transferred to other shake flasks containing the same medium with added methylation inhibitors and/or vitamins. Fermentations were carried out for 7 days at 28 °C with shaking at 200 r.p.m. Biomass was measured as packed mycelium volume after centrifugation at 1200 g for 10 min in a 10 ml graduated conical glass tube. Production of the antibiotic and its complex composition were measured by HPLC as described below. Data represent the mean values of three replicas of each fermentation experiment and each experiment was repeated at least twice. Standard deviation and coefficient of variation (CV) were estimated.
Bioreactor fermentations.
Two millilitres of the working cell bank was inoculated into six 500 ml Erlenmeyer flasks containing 100 ml D/Seed medium. After 72 h at 28 °C with shaking at 200 r.p.m., the cultures were transferred to Chemap Braun CF 3000 fermenters, containing 14 l medium C. Fermentations were carried out at 28 °C, stirred at 900 r.p.m. with an aeration rate of 0·5 l air (l culture)-1 min-1. Biomass was measured as packed mycelium volume after centrifugation at 1200 g for 10 min in a 10 ml graduated conical glass tube. Production of the antibiotic and its complex composition were measured by HPLC as described below.
Media composition.
Vegetative medium D/Seed had the following composition (g l-1): soluble starch (Cerestar), 20; polypeptone (BBL), 5; yeast extract (Costantino), 3; meat extract (Costantino), 2; soybean meal (Cargill), 2; calcium carbonate (Carlo Erba), 1. Medium was prepared in distilled water and the pH was adjusted to 7·0 before sterilization at 121 °C for 20 min.
Fermentation medium C had the following composition (g l-1): glucose (Baker), 10; soluble starch (Cerestar), 35; hydrolysed casein (Costantino), 5; yeast extract (Costantino), 8; meat extract (Costantino), 3·5; soybean meal (Cargill), 3·5; calcium carbonate (Carlo Erba), 2. Medium was prepared in distilled water and the pH was adjusted to 7·2 before sterilization at 121 °C for 30 min. All media were supplemented with 0·03 % (v/v) Hodag AFM-5 (Prodotti Gianni) as antifoaming agent.
HPLC analysis of the GE2270 complex.
One volume of fermentation culture was mixed with 2 vols acetonitrile and shaken at room temperature for 20 min. After centrifugation at 15 000 g for 10 min, supernatant was collected and injected in an analytical HPLC apparatus (1082 Hewlett Packard) equipped with a UV detector at 254 nm. For the analysis of GE2270 component A (HPLC method 1), the Brownlee RP-18 column (4·6x220 mm, 5 µm) was eluted with a mobile phase composed of 20 mM NaH2PO4/ acetonitrile (9 : 1) as eluent A and 20 mM NaH2PO4/acetonitrile (3 : 7) as eluent B. A linear gradient from 45 to 75 % (v/v) eluent B in 20 min was run with a flow of 1·5 ml min-1.
To better resolve the various GE2270 metabolites (HPLC method 2), the Bakerbond C-8 column (4·6x220 mm, 5 µm) was eluted with a mobile phase composed of 2·5 g ammonium formate l-1/tetrahydrofuran/acetonitrile (80 : 10 : 10) as eluent A and 2·5 g ammonium formate l-1/tetrahydrofuran/acetonitrile (20 : 40 : 40) as eluent B. The separation was accomplished by a linear gradient from 20 to 35 % (v/v) eluent B in 20 min with a flow of 1·5 ml min-1.
In both methods, a solution of 1 mg pure GE2270 A ml-1 was used as external standard (STD). Relative production of each GE2270 metabolite (GE2270 m) was calculated as follows:
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GE2270 total complex production was calculated as the sum of the concentrations of all the GE2270 metabolites, identified as previously reported (Colombo et al., 1995; Selva et al., 1995
).
All the HPLC chromatograms reported in this paper were recorded using the same value of attenuation.
Methylation inhibitors and cofactors.
Sinefungin, S-adenosyl-L-homocysteine, amethopterin (metotrexate), aminopterin, sulfamethizole, DL-ethionine, D-methionine, vitamin B12 (cyanocobalamin), methylcobalamin and folic acid were obtained from Sigma. Sinefungin, vitamin B12 and methylcobalamin were dissolved in water; amethopterin, aminopterin and folic acid in NaOH aqueous solution; DL-ethionine and D-methionine in acidified water; s-adenosyl-L-homocysteine in dimethylformamide and then in water. All the solutions were sterilized by filtration through 0·22 µm Millipore filters before addition to the cultures.
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RESULTS |
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Effect of methylation inhibitors
Table 3 shows the effect of different methylation inhibitors added to P. rosea fermentations. Sinefungin, a structural analogue of S-adenosyl-L-methionine, markedly changed GE2270 complex composition, as shown by HPLC profiles of fermentation broths in Fig. 4
(a) and (b). Concomitant improvements in the analysis (second HPLC method described in Methods) allowed better identification of the demethylated components. As shown in Fig. 4(a)
, in standard fermentation conditions there is a significant production of components E, D1 and D2, a lower amount of B1 and C1 and traces of C2 and B2 (see also Fig. 1
and Table 4
). Component T, which differs from all the others by the oxidation of the oxazoline ring, is the only one eluting after component A and it is produced in almost undetectable trace amounts. As shown in Fig. 4(b)
, in the presence of sinefungin, component A was strongly reduced and component D2 became the main component in the antibiotic complex. Total complex productivity was only slightly reduced, but the ratio between D2, which differs from A only in the free hydroxyl group on the D ring (Fig. 1
), to A rose from 0·24 to 5·51 (Table 3
).
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S-Adenosyl-L-homocysteine, which is the nucleoside product of S-adenosyl-L-methionine-dependent methylations, did not produce significant changes in GE2270 complex composition and total productivity (data not shown), when assayed at the same concentrations as sinefungin (see below and Table 4). Even if it is likely that higher concentrations of S-adenosyl-L-homocysteine inhibit the methyltransferase converting D2 into A component, these results point out a significantly different sensitivity of the enzyme to sinefungin and to S-adenosyl-L-homocysteine.
Table 4 shows a more detailed investigation of the effect of increasing amounts of sinefungin on the different measurable components of the GE2270 complex. A dose-dependence was found between the increased concentration of sinefungin and the selective production of component D2.
When sinefungin was added at 60 µg ml-1 (0·158 mM), the D2/A ratio was 24-fold enhanced in comparison to the fermentation in standard conditions: D2 composed more than 70 % of the complex, whereas A composed merely 10 %. Addition of higher concentrations of sinefungin than those reported in Table 4 interfered with growth and caused a reduction of GE2270 productivity (data not shown).
Other detectable factors in the GE2270 complex showed some fluctuations when sinefungin was supplemented, but, as shown in Table 4, none of them was significantly reduced, except for the disappearance of component B1. Component B1 bears the same methylated group at the D ring as does component A, but it is not N-methylated at the asparagine moiety (Fig. 1
). A concomitant accumulation of the demethylated component E was not detected, suggesting that this methylation is not a bottleneck in the biosynthetic flux towards the synthesis of GE2270 component D2 and then A. However, we cannot exclude a chromatographic limit of the detection method, which hampered a more precise quantitative analysis and resolution of minor factors in the GE2270 complex.
These results, together with those of vitamin B12, indicate that methylation of the hydroxy group at ring D is a late methylation reaction in GE2270 biosynthesis and it is catalysed by an S-adenosyl-L-methionine methyltransferase.
Combined effect of vitamin B12 and sinefungin on D2 biosynthesis
As described above, addition of vitamin B12 to P. rosea favoured the production of component A, whereas sinefungin supplementation stopped conversion of component D2 to A. When vitamin B12 and sinefungin were simultaneously added to the fermentation broths, maximum volumetric productivity of D2 was almost doubled, as reported in Fig. 5. These data show that vitamin B12 has two quite different effects on GE2270 biosynthesis: one is on the late methylation reaction converting D2 into A, and the other is a stimulating action on antibiotic backbone synthesis.
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DISCUSSION |
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In addition to the selective production of component A, the twofold increase in GE2270 total antibiotic yield achieved by the addition of vitamin B12 to the fermentation of P. rosea represents an innovative and industrially relevant aspect of this study (Rizzo & Gastaldo, 1999). In previous studies on production of methylated metabolites in fortimicin (Yamamoto et al., 1977
), coumermycin (Claridge et al., 1966
) and A10255 (Favret & Boeck, 1992
) antibiotic complexes, no similar improvement of total productivity was stimulated by vitamin B12 or correlated molecules. However, vitamin B12 was previously identified as the compound needed to restore thienamycin production (Chen et al., 1993
) in biosynthetic studies by blocked mutants of Streptomyces cattleya.
Confirmation of the type and the role of the methyltransferase involved in component A formation resulted from the use of methylation inhibitors. Sinefungin, a natural nucleoside structurally analogous to S-adenosyl-L-methionine, strongly enhanced the accumulation of component D2, which differs from component A only in the lack of a methyl group at ring D. Thus methylation of D2 into A is a late step in the GE2270 biosynthetic pathway and the responsible O-methyltransferase, being specifically inhibited by sinefungin, is an S-adenosyl-L-methionine methyltransferase. Mass balance of this reaction is in agreement with the proposed pathway because the amount of D2 metabolite that accumulated corresponded to the amount of A component lost. Sinefungin did not provoke other marked changes in complex composition, suggesting that different methyltransferases are involved in the addition of the other one-carbon units. It is interesting to note that component B1, which bears the same methoxy group at ring D as does factor A, but which is not N-methylated at the asparagine moiety, disappeared in the presence of sinefungin. A concomitant accumulation of component E was not detected, suggesting that this methylation is not a bottleneck in the biosynthetic flux towards the synthesis of GE2270 component D2 and then A.
All of the other methylation inhibitors screened failed to enhance production or give rise to new metabolites, nor was the biosynthesis of GE2270 blocked. A complete understanding of the effect of these inhibitors would require testing over a greater range of concentrations and generally a deeper investigation. For those inhibitors, such as structural analogues of folate and of p-aminobenzoate, or methionine antimetabolites, which showed little or no effect, our work cannot rule out other explanations for their partial or total inefficacy, such as the impermeability of P. rosea cells to some of them or toxicity for others. The different activity of methylation inhibitors on secondary metabolism has been reported (Miller et al., 1964; Pearce et al., 1991
; Pospisil & Zima, 1987
). In the case of staurosporine biosynthesis, among the different methyltransferase inhibitors assayed, sinefungin was the only one able to produce a demethyl derivative and it was shown to specifically inhibit the O-methyltransferase, but not the N-methyltransferase, even if both the O- and N-methyl groups of staurosporine derived from methionine (Yang et al., 1999
). Sinefungin was also the most active and the least toxic methyltransferase inhibitor in citreamicin-producing fermentations, but it stopped only one of the two O-methyltransferases involved in the modification of two adjacent hydroxy groups (Pearce et al., 1991
). Sinefungin was added to parental or mutant strains of Actinomadura spp. and Streptomyces avermitilis to understand biosynthesis and to produce new analogues of pradimicin and avermectin, respectively (Kakinuma et al., 1993
; Saitoh et al., 1995
; Schulman et al., 1985
). Sinefungin differs from S-adenosyl-L-methionine and s-adenosyl-L-homocysteine in the side chain: an ornithine residue is linked to the 5' position of adenosine through a carboncarbon bond (Barbes et al., 1990
). Different susceptibilities to sinefungin and to S-adenosyl-L-homocysteine were previously reported for methyltransferases involved in primary and secondary metabolism (Barbes et al., 1990
; Borchardt et al., 1979
). In staurosporine production, 3'-O-methyltransferase was selectively inhibited by sinefungin and not by S-adenosyl-L-methionine (Yang et al., 1999
). The two rate-limiting O-methyltransferases which catalyse the penultimate and the terminal steps in tylosin biosynthesis (Bauer et al., 1988
; Kreuzman et al., 1988
) are sensitive differentially to inhibition by adenine-containing compounds and particularly to sinefungin. Sinefungin is a potent inhibitor for macrocin O-methyltransferase, but a poor inhibitor for demethylmacrocin methyltransferase. The inhibition efficiency appears to be governed by the side chain of the nucleosides (Kreuzman et al., 1988
).
One surprising result was the effect of the simultaneous addition of vitamin B12 and sinefungin to P. rosea cultures. Once the last methylation reaction converting D2 into A was selectively inhibited by sinefungin, it clearly appeared that vitamin B12 exerted a major stimulatory effect on earlier steps of GE2270 biosynthesis. Further studies are needed to understand how vitamin B12 acts in the synthesis of GE2270. Recent investigations (De Pietro et al., 2001) by adding labelled amino acid precursors have shown that peptide skeleton formation in GE2270 is very similar to that of other thiazolylpeptides (Mocek et al., 1993a
, b
). The assembly of the amino acids is achieved by the well known multienzyme complex peptidyl synthetase. Neither the assembly, nor the biosynthesis of the amino acids constituting the chain appears to involve reactions known to be dependent on cobalamins. However, the pattern of 13C-labelled precursor incorporation revealed the existence in P. rosea of an uncommon pathway by which serine, the precursor of the several cysteine units present in GE2270, originates from glycine by the addition of a one-carbon unit. This differs from the common pattern by which glycine originates from serine with simultaneous generation of methylating equivalents. Furthermore, the results reported by De Pietro and co-workers give clear evidence that all the one-carbon units participating in GE2270 formation derive from carbon 2 of glycine. This requires the cleavage of glycine possibly with the formation of CO2, NH+4 and 13C-methylenetetrahydrofolate, as proposed in the biosynthesis of A10255 by Streptomyces gardneri (Favret et al., 1992
). The observation that the methylene group of glycine is an effective source of methylating equivalents has also been made during the biosynthesis of the thiopeptide sulfomycin by Streptomyces arginensis (Fate et al., 1996
). It is tempting to suggest that the enhancing effect of vitamin B12 on GE2270 antibiotic production can be explained by assuming that cobalamins act as coenzymes in one of these poorly studied reactions.
From a more applied point of view, the work reported in this paper allowed the production of industrially valuable levels of two different GE2270 metabolites as starting points for the synthesis of interesting semi-synthetic derivatives (Lociuro et al., 1997). In particular, the GE2270 component D2 was used as a starting scaffold bearing a free hydroxy group, which represents a new handle for chemical modifications.
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ACKNOWLEDGEMENTS |
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Received 28 November 2002;
revised 20 February 2003;
accepted 3 March 2003.
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