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Applied Microbiology and Biotechnology

, Volume 93, Issue 6, pp 2551–2561 | Cite as

Hydroxy-fatty acid production in a Pseudomonas aeruginosa 42A2 PHA synthase mutant generated by directed mutagenesis

  • Noelia Torrego-Solana
  • Ignacio Martin-Arjol
  • Mònica Bassas-Galia
  • Pilar DiazEmail author
  • Angeles ManresaEmail author
Applied microbial and cell physiology

Abstract

Pseudomonas aeruginosa 42A2 growing on waste frying oils is capable to synthesize polyhydroxyalkanoic acids (PHAs) and hydroxy-fatty acids as a result of several enzymatic conversions. In order to study the physiological role of PHA biosynthesis in P. aeruginosa with respect to the synthesis of hydroxy-fatty acids, an unmarked deletion mutant deficient for PHA biosynthesis was generated in P. aeruginosa 42A2. A combination of the sacB-based negative selection system with a cre-lox antibiotic marker recycling method was used for mutant isolation. Electron microscopy, nuclear magnetic resonance analysis, and transmission electron microscopy confirmed that PHA accumulation was completely abolished in the mutant strain. Interestingly, the new mutant strain showed higher carbon and oxygen uptake rate than the wild-type strain and higher efficiency in the conversion of oleic acid into (E)-10-hydroxy-8-octadecenic acid-octadecenoic acid.

Keywords

Pseudomonas aeruginosa PHA-negative mutant sacB-based negative selection cre-lox antibiotic recycling Hydroxy-fatty acids 

Notes

Acknowledgments

We thank Dra. MA Prieto for her assessment in the enzymatic determination for the study of the carbon flow. This work was financed by the Scientific and Technological Research Council (CICYT, Spain), grant CTQ2010-21183-C02-01/02/PPQ, by the IV Pla de Recerca de Catalunya (Generalitat de Catalunya), grant 2009SGR-819, and by the Generalitat de Catalunya to the “Xarxa de Referència en Biotecnologia” (XRB). Dra. N Torrego-Solana was a recipient of a BRD fellowship and postdoctoral contract from the University of Barcelona. I Martin-Arjol is a recipient of an APIF fellowship from the Universisty of Barcelona.

References

  1. Bassas M, Rodríguez E, Llorens J, Manresa A (2006) Poly (3-hydroxyalkanoate) produced from Pseudomonas aeruginosa 42A2 (NCBIM 40045): effect of fatty acid nature as nutrient. J Non-Cryst Solids 352:2259–2263Google Scholar
  2. Bassas M, Diaz J, Rodriguez E, Espuny MJ, Prieto MJ, Manresa A (2008) Microscopic examination in vivo and in vitro of natural and cross-linked polyunsaturated mclPHA. Appl Microbiol Biotechnol 78:587–596CrossRefGoogle Scholar
  3. Bofill C, Prim N, Mormeneo M, Manresa A, Pastor FIJ, Diaz P (2010) Differential behaviour of Pseudomonas sp 42A2 LipC, a lipase showing greater versatility than its counterpart LipA. Biochimie 92:307–316CrossRefGoogle Scholar
  4. Busquets M, Deroncele V, Vidal-Mas J, Rodriguez E, Guerrero A, Manresa A (2004) Isolation and characterization of a lipoxygenase from Pseudomonas 42A2 responsible for the biotransformation of oleic acid into (S)-(E)-10-hydroxy-8-octadecenoic acid. Antonie Leeuwenhoek 85:129–139CrossRefGoogle Scholar
  5. Christie W (ed) (2003) Lipid analysis. Oily, BridgewaterGoogle Scholar
  6. de Eugenio LI, Garcia P, Luengo JM, Sanz JM, San Roman J, Garcia JL, Prieto MA (2007) Biochemical evidence that phaZ gene encodes a specific intracellular medium chain length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442. Characterization of a paradigmatic enzyme. J Biol Chem 282:4951–4962CrossRefGoogle Scholar
  7. Erhan SM, Isbell TA (1997) Estolide production with modified clay catalysts and process conditions. JAOCS 74:249–254CrossRefGoogle Scholar
  8. Fernandez D, Rodriguez E, Bassas M, Viñas M, Solanas AM, Llorens J, Marqués AM, Manresa A (2005) Agro-industrial oily wastes as substrates for PHA production by the new strain Pseudomonas aeruginosa NCBB 40045: effect of culture conditions. Biochem Eng J 26:159–167CrossRefGoogle Scholar
  9. Guerrero A, Casals I, Busquets M, León Y, Manresa A (1997) Oxidation of oleic acid to (E)-10-hydroperoxy-8-octadecenoic acid and (E)-10-hydroxy-8-ocatadecenoic acids by Pseudomonas sp. 42A2. Biochim Biophys Acta 1347:75–81Google Scholar
  10. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580CrossRefGoogle Scholar
  11. Hocking PJ, Marcessault RH (1998) Polyhydroxyalkanoates. In: Kaplan DL (ed) Biopolymers from renewable resources. Springer, BerlinGoogle Scholar
  12. Joh YG, Brechany EY, Christie WW (1995) Characterization of wax esters in the roe oil of amber fish, Seriola aureovittata. JAOCS 72:707–713CrossRefGoogle Scholar
  13. Katsuki H, Yoshida T, Tanegashima C, Takaka T (1971) Improved direct method for determination of keto acids by 2,4-dinitrophenylhydrazine. Anal Biochem 43:349–356CrossRefGoogle Scholar
  14. Lee SY (1996) Bacterial polyhydroxyalkanoates. Biotechnol Bioeng 49:1–14CrossRefGoogle Scholar
  15. Lowry OH, Rosebrough N, Farr AL, Randall AL (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–257Google Scholar
  16. Luengo JM, García B, Sandoval A, Naharro G, Oliveira ER (2003) Bioplastics from microorganisms. Curr Op Microbiol 6:1–10CrossRefGoogle Scholar
  17. Martinez E, Hamberg M, Busquets M, Diaz P, Manresa A, Oliw EH (2010) Biochemical characterization of the oxygenation of unsaturated fatty acids by the dioxygenase and hydroperoxide isomerase of Pseudomonas aeruginosa 42A2. J Biol Chem 285:9339–9345CrossRefGoogle Scholar
  18. Marx CJ, Lidstrom ME (2002) Broad-Host-Range cre-lox System for Antibiotic Marker Recycling in Gram-Negative Bacteria. BioTech 33:1062–1067Google Scholar
  19. Ouyang SP, Liu Q, Fang L, Chen GQ (2007) Construction of pha-operon-defined knockout mutants of Pseudomonas putida KT2442 and their applications in poly(hydroxyalkanoate) production. Macromol Biosci 7:227–233CrossRefGoogle Scholar
  20. Pelaez M, Orellana C, Marques A, Busquets M, Guerrero A, Manresa A (2003) Natural estolides produced by Pseudomonas sp 42A2 grown on oleic acid: production and characterization. JAOCS 80:859–866CrossRefGoogle Scholar
  21. Prieto MA, de Eugenio LI, Galán B, Luengo JM, Witholt B (2007) Synthesis and degradation of polyhydroxyalkanoates. In: Ramos JL, Filloux A (eds) Pseudomonas: a model system in biology. Pseudomonas, vol. V. Springer, Berlin, pp 397–428Google Scholar
  22. Quenee L, Lamotte D, Polack B (2005) Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in Pseudomonas aeruginosa. Biotech 38:63–67CrossRefGoogle Scholar
  23. Rehm BHA (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15–33Google Scholar
  24. Rehm BHA, Steinbuchel A (1999) Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 25:3–19CrossRefGoogle Scholar
  25. Rehm B, Krüger N, Steinbüchel A (1998) A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis. J Biol Chem 273(37):24044–24051CrossRefGoogle Scholar
  26. Rehm B, Mitsky TA, Steinbüchel A (2001) Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and Rhamnolipid synthesis by Pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli. Appl Environ Microbiol 67(7):3102–3109CrossRefGoogle Scholar
  27. Romanov V, Merski MT, Hausinger RP (1999) Assays for allantoinase. Anal Biochem 268(1):49–53CrossRefGoogle Scholar
  28. Sambrook JRD (ed) (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  29. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proceed Nat Acad Sci USA 74:5463–5467CrossRefGoogle Scholar
  30. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagénesis in Gram-negative bacteria. BioTechnology 1:784–791Google Scholar
  31. Smith AW, Iglewski BH (1989) Transformation of Pseudomonas aeruginosa by electroporation. Nucl Acids Res 17:10509–10509CrossRefGoogle Scholar
  32. Solaiman DKY (1998) Genetic transformation of Pseudomonas oleovorans by electroporation. Biotech Tech 12:829–832CrossRefGoogle Scholar
  33. Spiekermann P, Rehm BHA, Kalscheuer R, Baumeister D, Steinbüchel A (1999) A sensitive, viable-colony count staining using Nile red for direct screening of bacteria that accumulate polyhydroxyalkaonic acids and other lipid storage compounds. Arch Microbiol 171:73–80CrossRefGoogle Scholar
  34. Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–34CrossRefGoogle Scholar
  35. Steinbuchel A, Valentin HE (1995) Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett 128:219–228CrossRefGoogle Scholar
  36. Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. J Non-Cryst Solids 325:1503–1555Google Scholar
  37. Witholt B, Kessler B (1999) Perspectives of medium chain length poly(hydroxyalkanoates), a versatile set of bacterial bioplastics. Curr Op Biotechnol 10:279–285Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Noelia Torrego-Solana
    • 1
    • 2
  • Ignacio Martin-Arjol
    • 2
  • Mònica Bassas-Galia
    • 3
  • Pilar Diaz
    • 1
    Email author
  • Angeles Manresa
    • 2
    Email author
  1. 1.Department of Microbiology, Faculty of BiologyUniversity of BarcelonaBarcelonaSpain
  2. 2.Laboratory of Microbiology, Faculty of PharmacyUniversity of BarcelonaBarcelonaSpain
  3. 3.Environmental Microbiology LaboratoryHelmholtz Center for Infection ResearchBraunschweigGermany

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