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

Abstract

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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Acknowledgments

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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Keywords

  • 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