1887

Abstract

Diverse and elaborate pathways for nutrient utilization, as well as mechanisms to combat unfavourable nutrient conditions make KT2440 a versatile micro-organism able to occupy a range of ecological niches. The fatty acid degradation pathway of is complex and correlated with biopolymer medium chain length polyhydroxyalkanoate (mcl-PHA) biosynthesis. Little is known about the second step of fatty acid degradation (β-oxidation) in this strain. analysis of its genome sequence revealed 21 putative acyl-CoA dehydrogenases (ACADs), four of which were functionally characterized through mutagenesis studies. Four mutants with insertionally inactivated ACADs (PP_1893, PP_2039, PP_2048 and PP_2437) grew and accumulated mcl-PHA on a range of fatty acids as the sole source of carbon and energy. Their ability to grow and accumulate biopolymer was differentially negatively affected on various fatty acids, in comparison to the wild-type strain. Inactive PP_2437 exhibited a pattern of reduced growth and PHA accumulation when fatty acids with lengths of 10 to 14 carbon chains were used as substrates. Recombinant expression and biochemical characterization of the purified protein allowed functional annotation in KT2440 as an ACAD showing clear preference for dodecanoyl-CoA ester as a substrate and optimum activity at 30 °C and pH 6.5–7.

Funding
This study was supported by the:
  • Environmental Protection Agency of Ireland (Award 2008-ET-LS1)
  • Ministry of Education, Science and Technological Development of Serbia (Award 173048)
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.078758-0
2014-08-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/8/1760.html?itemId=/content/journal/micro/10.1099/mic.0.078758-0&mimeType=html&fmt=ahah

References

  1. Alexeyev M. F. ( 1999). The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria. Biotechniques 26:824–826, 828[PubMed]
    [Google Scholar]
  2. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. ( 1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [View Article][PubMed]
    [Google Scholar]
  3. Blatny J. M., Brautaset T., Winther-Larsen H. C., Haugan K., Valla S. ( 1997). Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon. Appl Environ Microbiol 63:370–379[PubMed]
    [Google Scholar]
  4. Brandl H., Gross R. A., Lenz R. W., Fuller R. C. ( 1988). Pseudomonas oleovorans as a source of poly(beta-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977–1982
    [Google Scholar]
  5. Campbell J. W., Cronan J. E. Jr ( 2002). The enigmatic Escherichia coli fadE gene is yafH. J Bacteriol 184:3759–3764 [View Article][PubMed]
    [Google Scholar]
  6. Cerrone F., Choudhari S. K., Davis R., Cysneiros D., O’Flaherty V., Duane G., Casey E., Guzik M. W., Kenny S. T. & other authors ( 2014). Medium chain length polyhydroxyalkanoate (mcl-PHA) production from volatile fatty acids derived from the anaerobic digestion of grass. Appl Microbiol Biotechnol 98:611–620 [View Article][PubMed]
    [Google Scholar]
  7. Chen G.-Q. ( 2009). A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem Soc Rev 38:2434–2446 [View Article][PubMed]
    [Google Scholar]
  8. Choi K.-H., Kumar A., Schweizer H. P. ( 2006). A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397 [View Article][PubMed]
    [Google Scholar]
  9. Dos Santos V. A. P. M., Heim S., Moore E. R. B., Strätz M., Timmis K. N. ( 2004). Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440. Environ Microbiol 6:1264–1286 [View Article][PubMed]
    [Google Scholar]
  10. Elbahloul Y., Steinbüchel A. ( 2009). Large-scale production of poly(3-hydroxyoctanoic acid) by Pseudomonas putida GPo1 and a simplified downstream process. Appl Environ Microbiol 75:643–651 [View Article][PubMed]
    [Google Scholar]
  11. Escapa I. F., Morales V., Martino V. P., Pollet E., Avérous L., García J. L., Prieto M. A. ( 2011). Disruption of β-oxidation pathway in Pseudomonas putida KT2442 to produce new functionalized PHAs with thioester groups. Appl Microbiol Biotechnol 89:1583–1598 [View Article][PubMed]
    [Google Scholar]
  12. Fiedler S., Steinbüchel A., Rehm B. H. ( 2002). The role of the fatty acid β-oxidation multienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: molecular characterization of the fadBA operon from P. oleovorans and of the enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida. Arch Microbiol 178:149–160 [View Article][PubMed]
    [Google Scholar]
  13. Ghisla S., Thorpe C. ( 2004). Acyl-CoA dehydrogenases. A mechanistic overview. Eur J Biochem 271:494–508 [View Article][PubMed]
    [Google Scholar]
  14. Hartmann R., Hany R., Geiger T., Egli T., Witholt B., Zinn M. ( 2004). Tailored biosynthesis of olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] in Pseudomonas putida GPo1 with improved thermal properties. Macromolecules 37:6780–6785 [View Article]
    [Google Scholar]
  15. Hartmann R., Hany R., Pletscher E., Ritter A., Witholt B., Zinn M. ( 2006). Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] by Pseudomonas putida GPo1: batch versus chemostat production. Biotechnol Bioeng 93:737–746 [View Article][PubMed]
    [Google Scholar]
  16. Hazer B., Steinbüchel A. ( 2007). Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications. Appl Microbiol Biotechnol 74:1–12 [View Article][PubMed]
    [Google Scholar]
  17. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. ( 1998). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86 [View Article][PubMed]
    [Google Scholar]
  18. Hume A. R., Nikodinovic-Runic J., O’Connor K. E. ( 2009). FadD from Pseudomonas putida CA-3 is a true long-chain fatty acyl coenzyme A synthetase that activates phenylalkanoic and alkanoic acids. J Bacteriol 191:7554–7565 [View Article][PubMed]
    [Google Scholar]
  19. Kim J.-J. P., Miura R. ( 2004). Acyl-CoA dehydrogenases and acyl-CoA oxidases. Structural basis for mechanistic similarities and differences. Eur J Biochem 271:483–493 [View Article][PubMed]
    [Google Scholar]
  20. Lageveen R. G., Huisman G. W., Preusting H., Ketelaar P., Eggink G., Witholt B. ( 1988). Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 54:2924–2932
    [Google Scholar]
  21. Lewenza S., Gardy J. L., Brinkman F. S., Hancock R. E. ( 2005). Genome-wide identification of Pseudomonas aeruginosa exported proteins using a consensus computational strategy combined with a laboratory-based PhoA fusion screen. Genome Res 15:321–329 [View Article][PubMed]
    [Google Scholar]
  22. Liu Q., Luo G., Zhou X. R., Chen G. Q. ( 2011). Biosynthesis of poly(3-hydroxydecanoate) and 3-hydroxydodecanoate dominating polyhydroxyalkanoates by β-oxidation pathway inhibited Pseudomonas putida. Metab Eng 13:11–17 [View Article][PubMed]
    [Google Scholar]
  23. Lu X., Zhang J., Wu Q., Chen G.-Q. ( 2003). Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) via manipulating the fatty acid β-oxidation pathway in E. coli.. FEMS Microbiol Lett 221:97–101 [View Article][PubMed]
    [Google Scholar]
  24. Marchler-Bauer A., Bryant S. H. ( 2004). CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32:Web ServerW327–W331 [View Article][PubMed]
    [Google Scholar]
  25. Marchler-Bauer A., Anderson J. B., Chitsaz F., Derbyshire M. K., DeWeese-Scott C., Fong J. H., Geer L. Y., Geer R. C., Gonzales N. R. & other authors ( 2009). CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res 37:DatabaseD205–D210 [View Article][PubMed]
    [Google Scholar]
  26. Marchler-Bauer A., Lu S., Anderson J. B., Chitsaz F., Derbyshire M. K., DeWeese-Scott C., Fong J. H., Geer L. Y., Geer R. C. & other authors ( 2011). CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39:DatabaseD225–D229 [View Article][PubMed]
    [Google Scholar]
  27. Martin L. B., Nikodinovic J., McMahon A. M., Vijgenboom E., O'Connor K. E. ( 2008). Assessing the catalytic activity of three different sources of tyrosinase: a study of the oxidation of mono- and difluorinated monophenols. Enzyme Microb Technol 43:297–301 [View Article]
    [Google Scholar]
  28. McMahon B., Mayhew S. G. ( 2007). Identification and properties of an inducible phenylacyl-CoA dehydrogenase in Pseudomonas putida KT2440. FEMS Microbiol Lett 273:50–57 [View Article][PubMed]
    [Google Scholar]
  29. McMahon B., Gallagher M. E., Mayhew S. G. ( 2005). The protein coded by the PP2216 gene of Pseudomonas putida KT2440 is an acyl-CoA dehydrogenase that oxidises only short-chain aliphatic substrates. FEMS Microbiol Lett 250:121–127 [View Article][PubMed]
    [Google Scholar]
  30. Molloy S., Nikodinovic-Runic J., Martin L. B., Hartmann H., Solano F., Decker H., O’Connor K. E. ( 2013). Engineering of a bacterial tyrosinase for improved catalytic efficiency towards d-tyrosine using random and site directed mutagenesis approaches. Biotechnol Bioeng 110:1849–1857 [View Article][PubMed]
    [Google Scholar]
  31. Nelson K. E., Weinel C., Paulsen I. T., Dodson R. J., Hilbert H., Martins dos Santos V. A., Fouts D. E., Gill S. R., Pop M. & other authors ( 2002). Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4:799–808 [View Article][PubMed]
    [Google Scholar]
  32. O’Brien W. J., Frerman F. E. ( 1977). Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization. J Bacteriol 132:532–540[PubMed]
    [Google Scholar]
  33. Olivera E. R., Carnicero D., García B., Miñambres B., Moreno M. A., Cañedo L., Dirusso C. C., Naharro G., Luengo J. M. ( 2001). Two different pathways are involved in the β-oxidation of n-alkanoic and n-phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications. Mol Microbiol 39:863–874 [View Article][PubMed]
    [Google Scholar]
  34. Prieto M. A., de Eugenio L. I., Galán B., Luengo J. M., Witholt B. ( 2007). Synthesis and degradation of polyhydroxyalkanoates. Pseudomonas: a Model System in Biology397–428 Ramos J. L., Filloux A. Berlin: Springer; [View Article]
    [Google Scholar]
  35. Puchałka J., Oberhardt M. A., Godinho M., Bielecka A., Regenhardt D., Timmis K. N., Papin J. A., Martins dos Santos V. A. ( 2008). Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology. PLOS Comput Biol 4:e1000210 [View Article][PubMed]
    [Google Scholar]
  36. Qi Q., Steinbüchel A., Rehm B. H. A. ( 1998). Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid β-oxidation by acrylic acid. FEMS Microbiol Lett 167:89–94[PubMed]
    [Google Scholar]
  37. Rehm B. H. ( 2007). Biogenesis of microbial polyhydroxyalkanoate granules: a platform technology for the production of tailor-made bioparticles. Curr Issues Mol Biol 9:41–62[PubMed]
    [Google Scholar]
  38. Sambrook J., Russell W. D. ( 2001). Molecular Cloning a Laboratory Manual, 3rd edn. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory Press;
    [Google Scholar]
  39. Schlegel H. G., Kaltwasser H., Gottschalk G. ( 1961). [A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies]. Arch Mikrobiol 38:209–222 [View Article][PubMed]
    [Google Scholar]
  40. Stadtman E. R. ( 1957). Preparation and assay of acyl coenzyme A and other thiol esters; use of hydroxylamine. Methods Enzymol 3:931–941 [View Article]
    [Google Scholar]
  41. Steinbüchel A. ( 2001). Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathway as a successful example. Macromol Biosci 1:1–24 [View Article]
    [Google Scholar]
  42. Sun Z., Ramsay J. A., Guay M., Ramsay B. ( 2007). Increasing the yield of MCL-PHA from nonanoic acid by co-feeding glucose during the PHA accumulation stage in two-stage fed-batch fermentations of Pseudomonas putida KT2440. J Biotechnol 132:280–282 [View Article][PubMed]
    [Google Scholar]
  43. Sun Z., Ramsay J. A., Guay M., Ramsay B. A. ( 2009). Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by Pseudomonas putida KT2440. Appl Microbiol Biotechnol 82:657–662 [View Article][PubMed]
    [Google Scholar]
  44. van Duuren J. B. J. H., Puchałka J., Mars A. E., Bücker R., Eggink G., Wittmann C., Dos Santos V. A. P. M. ( 2013). Reconciling in vivo and in silico key biological parameters of Pseudomonas putida KT2440 during growth on glucose under carbon-limited condition. BMC Biotechnol 13:93 [View Article][PubMed]
    [Google Scholar]
  45. Ward P. G., O’Connor K. E. ( 2005). Bacterial synthesis of polyhydroxyalkanoates containing aromatic and aliphatic monomers by Pseudomonas putida CA-3. Int J Biol Macromol 35:127–133 [View Article][PubMed]
    [Google Scholar]
  46. Winsor G. L., Lam D. K., Fleming L., Lo R., Whiteside M. D., Yu N. Y., Hancock R. E., Brinkman F. S. ( 2011). Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 39:DatabaseD596–D600 [View Article][PubMed]
    [Google Scholar]
  47. Zinn M., Witholt B., Egli T. ( 2001). Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 53:5–21 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.078758-0
Loading
/content/journal/micro/10.1099/mic.0.078758-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error