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Synthesis and biotransformation of 2-alkyl-4(1H)-quinolones by recombinant Pseudomonas putida KT2440


2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the “Pseudomonas quinolone signal” (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 μM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C3- to C13-saturated and C7:1 to C13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and β carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C5-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in >90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.

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  1. Bredenbruch F, Nimtz M, Wray V, Mott M, Müller R, Häussler S (2005) Biosynthetic pathway of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines. J Bacteriol 187:3630–3635

  2. Bredenbruch F, Geffers R, Nimtz M, Buer J, Häussler S (2006) The Pseudomonas aeruginosa quinolone signal (PQS) has iron-chelating activity. Environ Microbiol 8:1318–1329

  3. Cao H, Krishnan G, Goumnerov B, Tsongalis J, Tompkins R, Rahme LG (2001) A quorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism. Proc Natl Acad Sci USA 98:14613–14618

  4. Carl B, Arnold A, Hauer B, Fetzner S (2004) Sequence and transcriptional analysis of a gene cluster of Pseudomonas putida 86 involved in quinoline degradation. Gene 331:177–188

  5. Coleman JP, Hudson LL, McKnight SL, Farrow JM 3rd, Calfee MW, Lindsey CA, Pesci EC (2008) Pseudomonas aeruginosa PqsA is an anthranilate-coenzyme A ligase. J Bacteriol 190:1247–1255

  6. Davis RW, Botstein D, Roth JR (1980) A manual for genetic engineering. Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

  7. Debitus C, Guella G, Mancini I, Waikedre J, Guemas JP, Nicolas JL, Pietra F (1998) Quinolones from a bacterium and tyrosine metabolites from its host sponge, Suberea creba from the Coral Sea. J Mar Biotechnol 6:136–141

  8. Déziel E, Lépine F, Milot S, He J, Mindrinos MN, Tompkins RG, Rahme LG (2004) Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication. Proc Natl Acad Sci USA 101:1339–1344

  9. Déziel E, Gopalan S, Tampakaki AP, Lépine F, Padfield KE, Saucier M, Xiao G, Rahme LG (2005) The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl-L-homoserine lactones. Mol Microbiol 55:998–1014

  10. Diggle SP, Cornelis P, Williams P, Cámara M (2006a) 4-Quinolone signalling in Pseudomonas aeruginosa: old molecules, new perspectives. Int J Med Microbiol 296:83–91

  11. Diggle SP, Lumjiaktase P, Dipilato F, Winzer K, Kunakorn M, Barrett DA, Chhabra SR, Cámara M, Williams P (2006b) Functional genetic analysis reveals a 2-alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria. Chem Biol 13:701–710

  12. Diggle SP, Matthijs S, Wright VJ, Fletcher MP, Chhabra SR, Lamont IL, Kong X, Hider RC, Cornelis P, Cámara M, Williams P (2007) The Pseudomonas aeruginosa 4-quinolone signal molecules HHQ and PQS play multifunctional roles in quorum sensing and iron entrapment. Chem Biol 14:87–96

  13. Eiden F, Wendt R, Fenner H (1978) Chinolyliden-derivate. Arch Pharm (Weinheim, Germany) 311:561–568

  14. Farrow JM 3rd, Sund ZM, Ellison ML, Wade DS, Coleman JP, Pesci EC (2008) PqsE functions independently of PqsR-Pseudomonas quinolone signal and enhances the rhl quorum-sensing system. J Bacteriol 190:7043–7051

  15. Gallagher LA, McKnight SL, Kuznetsova MS, Pesci EC, Manoil C (2002) Functions required for extracellular quinolone signaling by Pseudomonas aeruginosa. J Bacteriol 184:6472–6480

  16. Grant SG, Jessee J, Bloom FR, Hanahan D (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87:4645–4649

  17. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

  18. Hays EE, Wells IC, Katzman PA, Cain CK, Jacobs FA, Thayer SA, Doisy EA, Gaby WL, Roberts EC, Muir RD, Carroll CJ, Jones LR, Wade NJ (1945) Antibiotic substances produced by Pseudomonas aeruginosa. J Biol Chem 159:725–750

  19. Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P, Cámara M (2011) Quinolones: from antibiotics to autoinducers. FEMS Microbiol Rev 35:247–274

  20. Hodgkinson JT, Galloway WRJD, Saraf S, Baxendale IR, Ley SV, Ladlow M, Welch M, Spring DR (2011) Microwave and flow syntheses of Pseudomonas quinolone signal (PQS) and analogues. Org Biomol Chem 9:57–61

  21. Iwasaki K, Uchiyama H, Yagi O, Kurabayashi T, Ishizuku K, Takamura Y (1994) Transformation of Pseudomonas putida by electroporation. Biosci Biotechnol Biochem 58:851–854

  22. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176

  23. Leisinger T, Margraff R (1979) Secondary metabolites of the fluorescent pseudomonads. Microbiol Rev 43:422–442

  24. Lépine F, Milot S, Déziel E, He J, Rahme LG (2004) Electrospray/mass spectrometric identification and analysis of 4-hydroxy-2-alkylquinolines (HAQs) produced by Pseudomonas aeruginosa. J Am Soc Mass Spectrom 15:862–869

  25. Long RA, Qureshi A, Faulkner DJ, Azam F (2003) 2-n-Pentyl-4-quinolinol produced by a marine Alteromonas sp. and its potential ecological and biogeochemical roles. Appl Environ Microbiol 69:568–576

  26. McGlacken GP, McSweeney CM, O’Brien T, Lawrence SE, Elcoate CJ, Reen FJ, O’Gara F (2010) Synthesis of 3-halo-analogues of HHQ, subsequent cross-coupling and first crystal structure of Pseudomonas quinolone signal (PQS). Tetrahedron Lett 51:5919–5921

  27. Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, Martins dos Santos VA, Fouts DE, Gill SR, Pop M, Holmes M, Brinkac L, Beanan M, DeBoy RT, Daugherty S, Kolonay J, Madupu R, Nelson W, White O, Peterson J, Khouri H, Hance I, Chris Lee P, Holtzapple E, Scanlan D, Tran K, Moazzez A, Utterback T, Rizzo M, Lee K, Kosack D, Moestl D, Wedler H, Lauber J, Stjepandic D, Hoheisel J, Straetz M, Heim S, Kiewitz C, Eisen JA, Timmis KN, Düsterhöft A, Tümmler B, Fraser CM (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4:799–808

  28. Ortori CA, Dubern JF, Chhabra SR, Cámara M, Hardie K, Williams P, Barrett DA (2011) Simultaneous quantitative profiling of N-acyl-L-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signaling molecules using LC-MS/MS. Anal Bioanal Chem 399:839–850

  29. Pesci EC, Milbank JBJ, Pearson JP, McKnight S, Kende AS, Greenberg EP, Iglewski BH (1999) Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 96:11229–11234

  30. Pustelny C, Albers A, Büldt-Karentzopoulos K, Parschat K, Chhabra SR, Cámara M, Williams P, Fetzner S (2009) Dioxygenase-mediated quenching of quinolone-dependent quorum sensing in Pseudomonas aeruginosa. Chem Biol 16:1259–1267

  31. Rampioni G, Pustelny C, Fletcher MP, Wright VJ, Bruce M, Rumbaugh KP, Heeb S, Cámara M, Williams P (2010) Transcriptomic analysis reveals a global alkyl-quinolone-independent regulatory role for PqsE in facilitating the environmental adaptation of Pseudomonas aeruginosa to plant and animal hosts. Environ Microbiol 12:1659–1673

  32. Ritter C, Luckner M (1971) Zur Biosynthese der 2-n-Alkyl-4-hydroxychinolinderivate (Pseudane) bei Pseudomonas aeruginosa. Eur J Biochem 18:391–400

  33. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

  34. Schertzer JW, Boulette ML, Whiteley M (2009) More than a signal: non-signaling properties of quorum sensing molecules. Trends Microbiol 17:189–195

  35. Schertzer JW, Brown SA, Whiteley M (2010) Oxygen levels rapidly modulate Pseudomonas aeruginosa social behaviours via substrate limitation of PqsH. Mol Microbiol 77:1527–1538

  36. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:784–791

  37. Somanathan R, Smith KM (1981) Synthesis of some 2-alkyl-4-quinolone and 2-alkyl-4-methoxyquinoline alkaloids. J Heterocyclic Chem 18:1077–1079

  38. Vial L, Lépine F, Milot S, Groleau MC, Dekimpe V, Woods DE, Déziel E (2008) Burkholderia pseudomallei, B. thailandensis, and B. ambifaria produce 4-hydroxy-2-alkylquinoline analogues with a methyl group at the 3 position that is required for quorum-sensing regulation. J Bacteriol 190:5339–5352

  39. Wells IC (1952) Antibiotic substances produced by Pseudomonas aeruginosa. Syntheses of Pyo Ib, Pyo Ic, and Pyo III. J Biol Chem 196:331–340

  40. Xiao G, Déziel E, He J, Lépine F, Lesic B, Castonguay MH, Milot S, Tampakaki AP, Stachel SE, Rahme LG (2006) MvfR, a key Pseudomonas aeruginosa pathogenicity LTTR-class regulatory protein, has dual ligands. Mol Microbiol 62:1689–1699

  41. Zhang YM, Frank MW, Zhu K, Mayasundari A, Rock CO (2008) PqsD is responsible for the synthesis of 2,4-dihydroxyquinoline, an extracellular metabolite produced by Pseudomonas aeruginosa. J Biol Chem 283:28788–28794

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We thank Prof. Dr. Alexander Steinbüchel for access to the mass spectrometer, Dr. Heinrich Luftmann, Organic Chemistry Institute, University of Münster, for helpful advice on the interpretation of mass spectra, Henrike Niederholtmeyer for construction of P. putida KT2440 pBBR-pqsH, Almut Kappius for technical assistance and Alex Truman for AQ synthesis. This work was supported by the German Research Foundation (DFG, grant FE 383/16-1 to SF) and by the Biotechnology and Biological Sciences Research Council (BBSRC, grant BB/F014392/1).

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Correspondence to Susanne Fetzner.

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Niewerth, H., Bergander, K., Chhabra, S.R. et al. Synthesis and biotransformation of 2-alkyl-4(1H)-quinolones by recombinant Pseudomonas putida KT2440. Appl Microbiol Biotechnol 91, 1399–1408 (2011). https://doi.org/10.1007/s00253-011-3378-0

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  • 2-Alkyl-4(1H)-quinolone
  • 2-alkenyl-4(1H)-quinolone isomers
  • 2-alkyl-3-hydroxy-4(1H)-quinolone
  • 2-heptyl-4(1H)-quinolone
  • Pseudomonas putida
  • Pseudomonas quinolone signal