The hypothetical dithiolopyrrolone biosynthetic pathway includes a final step of pyrrothine nucleus acylation. The presence of an enzymatic activity catalysing this reaction was investigated in Saccharothrix algeriensis NRRL B‐24137. To understand the effect exerted by organic acids on the level of dithiolopyrrolone production, their influence on enzymatic expression was studied. Methods and Results: The transfer of acetyl‐CoA or benzoyl‐CoA on pyrrothine was assayed in the cell‐free extract of Sa. algeriensis NRRL B‐24137. This study reports the presence of an enzymatic activity catalysing this reaction that was identified as either pyrrothine N‐acetyltransferase or N‐benzoyltransferase. The stimulation of benzoyl‐pyrrothine (BEP) production by addition of benzoic acid at 1·25 mmol l−1 into the culture medium was demonstrated, and results showed that under the same conditions of growth, pyrrothine N‐benzoyltransferase specific activity was doubled. This study shows that BEP production is enhanced in the presence of benzoic acid partly because of an induction of pyrrothine N‐benzoyltransferase.





Baziard, Geneviève

Bijeire, Laurent

Chorin, Anne-Claire

Lebrihi, Ahmed

Mathieu, Florence

Monje, Marie-Carmen

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    OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible Any correspondence concerning this service should be sent  to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr This is an author’s version published in: http://oatao.univ-toulouse.fr/23174  To cite this version:  Chorin, Anne-Claire and Bijeire, Laurent and Monje, Marie-Carmen  and Baziard, Geneviève and Lebrihi, Ahmed  and Mathieu, Florence  Expression of pyrrothineN-acyltransferase activities inSaccharothrix algeriensisNRRL B-24137: new insights into dithiolopyrrolone antibiotic biosynthetic pathway. (2009) Journal of Applied Microbiology, 107 (5). 1751-1762. ISSN 1364-5072 Official URL : https://doi.org/10.1111/j.1365-2672.2009.04496.x Open  Archive  Toulouse  Archive  Ouverte was recovered by filtration. The 1H NMR spectrum,obtained on a Bruker DPX 300 MHz spectrometer, confirmed that the precipitate is pyrrothine hydrochloride:dH (DMSO, d6): 7Æ41 (1H, s, C=CH) 3Æ9 5Æ3 (1H, br s,NH3+) 3Æ27 (3H, s, CH3).Cell free extract preparationSaccharothrix algeriensis NRRL B 24137 was grown inSSM or SSM supplemented with organic acids asdescribed in the section ‘Culture conditions’. The biomasswas recovered by centrifugation of culture broth at5000 g for 15 min (4K15; Sigma), then washed twice withphysiological water (0Æ9% NaCl) and once with lysisbuffer (Tris HCl 50 mmol l 1, pH 8). Wet cells werefinally recovered by filtration on 0Æ2 lm membrane filters(Advantec, Dublin, Ireland). Wet cells (0Æ6 g) werere suspended in 1 ml of lysis buffer and transferred to aFast Protein Blue tube (MP Biomedicals, Irvine, CA,USA). Two disruption cycles (30 s, 5 m s 1) were carriedout in a Fast Prep disruptor (MP Biomedicals). The lysing matrix was discarded, and then the sample was centrifuged at 10 000 g for 30 min (centrifuge 1 15K; Sigma)to remove the cell debris. The supernatant constitutingthe soluble cell free extract of Sa. algeriensis NRRLB 24137 was used immediately for the assay of pyrrothineN acyltransferase activity. A sample was frozen at )80Cfor further protein assays. Samples were always kept inice and centrifugations were carried out at 4C.Pyrrothine N acyltransferase assayPyrrothine N acyltransferase activity was assayed in a finalvolume of 100 ll. The reaction mixture contained 80 ll ofcell free extract in Tris HCl buffer (50 mmol l 1, pH 8),10 ll of acetyl or benzoyl coenzyme A at 5 mmol l 1 inbidistilled water and 10 ll of pyrrothine hydrochoride at2Æ5 mmol l 1 in methanol. The reaction mixture wasincubated at 30C for 5 and 10 min. The reaction wasstopped by adding fresh 2Æ5% w ⁄ v trichloroacetic acid(Fisher). The cell free extract was properly diluted withTris HCl buffer (50 mmol l 1, pH 8) before assay toobserve a product formation linear in time within10 min.Enzymatic activity was identified as either acetyltransferase or benzoyltransferase activity according to the acylgroup donor used during the assay, i.e. acetyl CoA andbenzoyl CoA, respectively. A unit of enzyme is definedas the enzyme activity producing 1 lmol of thiolutin(acetyl pyrrothine) or BEP per minute. Specific enzymaticactivity was expressed in lU mg 1 of protein.Enzymatic activity was also expressed in mU g 1 ofDCW. A value of 162 mg of proteins per gram of drycells was used to achieve conversion from U mg 1 of proteins to U g 1 of DCW. Protein content in dry cells wasdetermined as follow. DCW and intracellular protein concentration were determined every 24 h, for 7 days, in aculture of Sa. algeriensis NRRL B 24137 on SSM. The plotof proteins in mg l 1 vs DCW in g l 1 indicated a linearcorrelation between the two parameters with a slope of162 mg of proteins per gram of DCW (R2 = 0Æ91).HPLC analysis for dithiolopyrrolone quantificationDithiolopyrrolones were quantified by HPLC (Bio tekInstruments, Milan, Italy). The analytical column wasProntoSIL 120 5 C18 SH, 150 · 4Æ6 mm (Bishoff Chromatography, Leonberg, Germany) fitted with a guard columnof 10 · 4 mm, and detection was achieved with a diodearray detector (UV vis 545 V; Bio tek instruments).For detection and quantification of thiolutin and BEPproduced in the culture medium, analyses were performed as described by Bouras et al. (2006a) with aninjection volume of 80 ll.For detection and quantification of dithiolopyrrolonesformed during enzymatic assay, analyses were performedunder the following chromatographic conditions. Sampleswere analysed by linear gradient elution using a mixtureof methanol ⁄bidistilled water (solvent A ⁄ solvent B) asmobile phase and a flow rate of 0Æ8 ml min 1. Columntemperature was maintained at 30C and injectionvolume was 40 ll. UV detection of antibiotics was carriedout at 390 nm. For the acetyltransferase assay, elution wascarried out with a linear gradient from 30% A to 47% Ain 17 min. Pyrrothine and thiolutin retention times (Rt)were 7 and 12Æ3 min, respectively. For the benzoyltransferase assay, the mobile phase composition was 30% A,reached 45% A in 15 min, 100% A in 35 min and wasfinally kept at this value for 2 min. Pyrrothine and BEPretention times (Rt) were 8 and 29 min, respectively.Both antibiotics were quantified using a thiolutinstandard calibration curve. Indeed, the molar absorbtivityvalues at 390 nm of pyrrothine derivatives are close. e390is in the range of 8317 9333 mol 1 l cm 1 as describedby Lamari et al. (2002b).The BEP chemical structure was confirmed by comparison of the UV spectrum obtained with those mentionedby Bouras et al. (2008). The UV spectrum of BEP yieldedkmax in nm (relative absorbance): 231 (1), 309 (0Æ45), 401(0Æ68).Replication of experiments and statistical analysisData of growth and dithiolopyrrolone production represent the average of triplicate flasks, and error bars denotestandard errors. Values of acyltransferase specific activitiesCundliffe, E. (2006) Antibiotic production by actinomycetes:the janus faces of regulation. J Ind Microbiol Biotechnol 33,500 506.De la Fuente, A., Lorenzana, L.M., Martin, J.F. and Liras, P.(2002) Mutants of Streptomyces clavuligerus with disruptions in different genes for clavulanic acid biosynthesisproduce large amounts of holomycin: possible crossregulation of two unrelated secondary metabolic pathway.J Bacteriol 184, 6559 6565.Demain, A.L. and Zhang, J. (1998) Cephalosporin C production by Cephalosporium acremonium: the methionine story.Crit Rev Biotechnol 18, 283 294.Ellis, J.E., Fried, J.H., Harrison, I.T., Rapp, E. and Ross, C.H.(1977) Synthesis of holomycin and derivatives. J Org Chem42, 2891 2893.Fazeli, R.M., Cove, J.H. and Baumberg, S. (1995) Physiologicalfactors affecting streptomycin production by Streptomycesgriseus ATCC 12475 in batch and continuous culture.FEMS Microbiol Lett 126, 55 61.Furumai, T., Takeda, K. and Okanishi, M. (1982) Function ofplasmids in the production of aureothricin. I. Eliminationof plasmids and alteration of phenotypes caused by protoplast regeneration in Streptomyces kasugaensis. J Antibiot(Tokyo) 35, 1367 1373.Guo, Y., Chen, G. and Bin, L. (2008) Novel dithiolopyrrolonesand their therapeutical applications. PatentWO2008038175. 3 April 2008.Hagio, K. and Yoneda, N. (1974) Total syntheses of holomycin,thiolutin and aureothricin. Bull Chem Soc Jpn 47, 1484 1489.Hjelmgaard, T., Givskov, M. and Nielsen, J. (2007) Expedienttotal synthesis of pyrrothine natural products and analogs.Org Biomol Chem 5, 344 348.Kice, J.L. (1968) Electrophilic and nucleophilic catalysis of thescission of the sulfur sulfur bond. Acc Chem Res 1, 58 64.Krupinski, V.M., Robbers, J.E. and Floss, H.G. (1976)Physiological study of ergot: induction of alkaloidsynthesis by tryptophan at enzymatic level. J Bacteriol125, 158 165.Lamari, L., Zitouni, A., Boudjella, H., Badjin, B., Sabaou, N.,Lebrihi, A., Lefebvre, G., Seguin, E. et al. (2002a) Newdithiolopyrrolones antibiotics from Saccharothrix sp. SA233: I. Taxonomy, fermentation, isolation and biologicalactivities. J Antibiot (Tokyo) 55, 696 701.Lamari, L., Zitouni, A., Dob, T., Sabaou, N., Lebrihi, A.,Germain, P., Seguin, E. and Tillequin, F. (2002b) Newdithiolopyrrolone antibiotics from Saccharothrix sp. SA233. II. Physiochemical properties and structureelucidation. J Antibiot (Tokyo) 55, 702 707.Lebrihi, A., Lefebvre, G. and Germain, P. (1988) A study onthe regulation of cephamycin C and expandase biosynthesis by Streptomyces clavuligerus in continuous and batchculture. Appl Microbiol Biotechnol 28, 39 43.Li, J., Chen, G. and Webster, J.M. (1995) Antimicrobialmetabolites from a bacterial symbiont. J Nat Prod 58,1081 1086.Li, B., Lyle, M.P.A., Chen, G., Li, J., Hu, K., Tang, L.,Alaoui Jamali, M.A. and Webster, J. (2007) Substituted6 amino 4H [1,2]dithiolo[4,3 b]pyrrol 5 ones: synthesis,structure activity relationships, and cytotoxic activity onselected human cancer cell lines. Bioorg Med Chem 15,4601 4608.Martin, J.F. and Demain, A.L. (1980) Control of antibioticbiosynthesis. Microbiol Rev 44, 230 251.Mc Inerney, B.V., Gregson, R.P., Lacey, M.J., Akhurst, R.J.,Lyons, G.R., Rhodes, S.H., Smith, D.R., Engelhardt, L.M.et al. (1991) Biologically active metabolites fromXhenorhabdus spp., part I dithiolopyrrolone derivativeswith antibiotic activity. J Nat Prod 54, 774 784.McDermott, J.F., Lethbridge, G. and Bushell, M.E. (1993)Estimation of the kinetic constants and elucidation oftrends in growth and erythromycin production in batchand continuous cultures of Saccharopolyspora erythraeausing curve fitting techniques. Enzyme Microb Technol 15,657 663.McIntyre, J.J., Bull, A.T. and Bunch, A.W. (1996) Vancomycinproduction in batch and continuous culture. BiotechnolBioeng 49, 412 420.Minamigushi, K., Kumagai, H., Masuda, T., Kawada, M.,Ishizuka, M. and Takeushi, T. (2001) Thiolutin, an inhibitor of huvec adhesion to vitronectin, reduces paxillin inhuvecs and suppresses tumor cell induced angiogenesis. IntJ Cancer 93, 307 316.Nguyen, K.T., Nguyen, L.T. and Be˘hal, V. (1995a) The induction of valine deshydrogenase activity from Streptomycesby l valine is not repressed by ammonium. Biotechnol Lett17, 31 34.Nguyen, K.T., Nguyen, L.T. and Be˘hal, V. (1995b) Theavermictin producing Streptomyces avermitilis possessesan inducible valine deshydrogenase. Biotechnol Lett 17,151 156.Nimi, O., Kubota, H. and Sugiyama, M. (1981) Effect ofarginine on gramicidin S biosynthesis by Bacillus brevi.J antibiot (Tokyo) 35, 615 621.Ninomiya, Y.T., Yamada, Y., Shirai, H., Onitsuka, M., Suhara,Y. and Maruyama, H.B. (1980) Biochemically activesubstances from microorganisms. V. Pyrrothines, potentplatelet aggregation inhibitors of microbial origin. ChemPharm Bull (Tokyo) 28, 3157 3162.Oliva, B., O’Neill, A., Wilson, J.M., O’Hanlon, P.J. andChopra, I. (2001) Antimicrobial properties and mode ofaction of the pyrrothine holomycin. Antimicrob AgentsChemother 45, 532 539.Ortmann, R., Matern, U. and Grisebach, H. (1974) NADPHdependent formation of thymidine diphosphodihydrostreptose from thymidine diphospho d glucose in a cell freesystem from Streptomyces griseus and its correlation withstreptomycin biosynthesis. Eur J Biochem 43, 265 271.Pamboukian, C.R.D. and Facciotti, M.C.R. (2004) Production ofthe antitumoral retamycin during continuous fermentationsof Streptomyces olindensis. Process Biochem 39, 2249 2255.

Expression of pyrrothineN-acyltransferase activities inSaccharothrix algeriensisNRRL B-24137: new insights into dithiolopyrrolone antibiotic biosynthetic pathway

International audienceThe hypothetical dithiolopyrrolone biosynthetic pathway includes a final step of pyrrothine nucleus acylation. The presence of an enzymatic activity catalysing this reaction was investigated in Saccharothrix algeriensis NRRL B‐24137. To understand the effect exerted by organic acids on the level of dithiolopyrrolone production, their influence on enzymatic expression was studied. Methods and Results: The transfer of acetyl‐CoA or benzoyl‐CoA on pyrrothine was assayed in the cell‐free extract of Sa. algeriensis NRRL B‐24137. This study reports the presence of an enzymatic activity catalysing this reaction that was identified as either pyrrothine N‐acetyltransferase or N‐benzoyltransferase. The stimulation of benzoyl‐pyrrothine (BEP) production by addition of benzoic acid at 1·25 mmol l−1 into the culture medium was demonstrated, and results showed that under the same conditions of growth, pyrrothine N‐benzoyltransferase specific activity was doubled. This study shows that BEP production is enhanced in the presence of benzoic acid partly because of an induction of pyrrothine N‐benzoyltransferase.

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Expression of pyrrothineN-acyltransferase activities inSaccharothrix algeriensisNRRL B-24137: new insights into dithiolopyrrolone antibiotic biosynthetic pathway

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