1887

Abstract

The oleaginous bacterium strain PD630 serves as a model organism to investigate the metabolism of storage triacylglycerols (TAGs) in bacteria. The key enzyme catalysing the last step of TAG biosynthesis in bacteria is a promiscuous acyltransferase (Atf), exhibiting acyl-CoA acyltransferase activity to both diacylglycerols (DGAT activity) and fatty alcohols (wax ester synthase, WS activity). An 800 bp PCR product was obtained from chromosomal DNA of strain PD630 by using degenerate primers designed from conserved stretches of Atf proteins of strain ADP1 and mc155. The fragment was used as a probe on a strain PD630 gene library, resulting in the identification of a 3948 bp chromosomal DNA fragment containing the complete gene. An disruption mutant of strain PD630 exhibited a TAG-leaky phenotype and accumulated up to 50 % less fatty acids than the wild-type, with significantly reduced oleic acid content when cultivated in the presence of gluconate or oleic acid. Whereas DGAT activity was drastically reduced in comparison to the wild-type, WS activity remained almost unchanged in the mutant. RT-PCR analysis of gluconate-grown cells of strain PD630 showed that there is expression of under conditions of TAG synthesis. To identify additional Atfs in strain PD630, PCR employing non-degenerate primers deduced from RHA1 sequence data was used. This yielded nine additional -homologous genes exhibiting 88–99 % sequence identity to the corresponding strain RHA1 enzymes. Besides Atf1 only Atf2 exhibited high DGAT and/or WS activity when heterologously expressed in .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/016568-0
2008-08-01
2019-10-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/8/2327.html?itemId=/content/journal/micro/10.1099/mic.0.2008/016568-0&mimeType=html&fmt=ahah

References

  1. 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.[CrossRef]
    [Google Scholar]
  2. Alvarez, H. M. & Steinbüchel, A. ( 2002; ). Triacylglycerols in prokaryotic microorganisms. Appl Microbiol Biotechnol 60, 367–376.[CrossRef]
    [Google Scholar]
  3. Alvarez, H. M., Mayer, F., Fabritius, D. & Steinbüchel, A. ( 1996; ). Formation of intracytoplasmatic lipid inclusions by Rhodococcus opacus strain PD630. Arch Microbiol 165, 377–386.[CrossRef]
    [Google Scholar]
  4. Alvarez, H. M, Kalscheuer, R. & Steinbüchel, A. ( 1997a; ). Accumulation of storage lipids in species of Rhodococcus and Nocardia and effect of inhibitors and polyethylene glycol. Fett 99, 239–246.[CrossRef]
    [Google Scholar]
  5. Alvarez, H. M., Pucci, O. H. & Steinbüchel, A. ( 1997b; ). Lipid storage compounds in marine bacteria. Appl Microbiol Biotechnol 47, 132–139.[CrossRef]
    [Google Scholar]
  6. Alvarez, H. M., Kalscheuer, R. & Steinbüchel, A. ( 2000; ). Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126. Appl Microbiol Biotechnol 54, 218–223.[CrossRef]
    [Google Scholar]
  7. Alvarez, H. M., Silva, R. A., Cesari, A. C., Zamit, A. L., Peressutti, S. R., Reichelt, R., Keller, U., Malkus, U., Rasch, C. & other authors ( 2004; ). Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to dehydration. FEMS Microbiol Ecol 50, 75–86.[CrossRef]
    [Google Scholar]
  8. Anderson, A. J. & Dawes, E. A. ( 1990; ). Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54, 450–472.
    [Google Scholar]
  9. Barksdale, L. & Kim, K. S. ( 1977; ). Mycobacterium. Bacteriol Rev 41, 217–372.
    [Google Scholar]
  10. Bredemeier, R., Hulsch, R., Metzger, J. O. & Berthe-Corti, L. ( 2003; ). Submersed culture production of extracellular wax esters by the marine bacterium Fundibacter jadensis. Mar Biotechnol (NY) 5, 579–583.[CrossRef]
    [Google Scholar]
  11. Bryn, K., Jantzen, E. & Bovre, K. ( 1977; ). Occurrence and patterns of waxes in Neisseriaceae. J Gen Microbiol 102, 33–43.[CrossRef]
    [Google Scholar]
  12. Bullock, W. O., Fernandez, J. M. & Stuart, J. M. ( 1987; ). XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. Biotechniques 5, 376–379.
    [Google Scholar]
  13. Christiansen, K. ( 1978; ). Triacylglycerol synthesis in lipid particles from baker's yeast (Saccharomyces cerevisiae). Biochim Biophys Acta 530, 78–90.[CrossRef]
    [Google Scholar]
  14. Cserzö, M., Wallin, E., Simon, I., von Heijne, G. & Elofsson, A. ( 1997; ). Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng 10, 673–676.[CrossRef]
    [Google Scholar]
  15. Daniel, J., Deb, C., Dubey, V. S., Sirakova, T. D., Abomoelak, B., Morbidoni, H. R. & Kolattukudy, P. E. ( 2004; ). Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture. J Bacteriol 186, 5017–5030.[CrossRef]
    [Google Scholar]
  16. Fixter, L. M., Nagi, M. N., McCormack, J. G. & Fewson, C. A. ( 1986; ). Structure, distribution and function of wax esters in Acinetobacter calcoaceticus. J Gen Microbiol 132, 3147–3157.
    [Google Scholar]
  17. Friedrich, B., Hogrefe, C. & Schlegel, H. G. ( 1981; ). Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus. J Bacteriol 147, 198–205.
    [Google Scholar]
  18. Hohn, B. & Collins, J. ( 1980; ). A small cosmid for efficient cloning of large DNA fragments. Gene 11, 291–298.[CrossRef]
    [Google Scholar]
  19. Jäger, W., Schäfer, A., Kalinowski, J. & Pühler, A. ( 1995; ). Isolation of insertion elements from Gram-positive Brevibacterium, Corynebacterium and Rhodococcus strains using the Bacillus subtilis sacB gene as a positive selection marker. FEMS Microbiol Lett 126, 1–6.[CrossRef]
    [Google Scholar]
  20. Kalscheuer, R. & Steinbüchel, A. ( 2003; ). A novel bifunctional wax ester synthase/acyl-CoA : diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278, 8075–8082.[CrossRef]
    [Google Scholar]
  21. Kalscheuer, R., Arenskötter, M. & Steinbüchel, A. ( 1999; ). Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids). Appl Microbiol Biotechnol 52, 508–515.[CrossRef]
    [Google Scholar]
  22. Kalscheuer, R., Wältermann, M., Alvarez, H. M. & Steinbüchel, A. ( 2001; ). Preparative isolation of lipid inclusion bodies from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins. Arch Microbiol 177, 20–28.[CrossRef]
    [Google Scholar]
  23. Kalscheuer, R., Uthoff, S., Luftmann, H. & Steinbüchel, A. ( 2003; ). In vitro and in vivo biosynthesis of wax diesters by an unspecific bifunctional wax ester synthase/acyl-CoA : diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter calcoaceticus ADP1. Eur J Lipid Sci Technol 105, 578–584.[CrossRef]
    [Google Scholar]
  24. Kalscheuer, R., Luftmann, H. & Steinbüchel, A. ( 2004; ). Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase. Appl Environ Microbiol 70, 7119–7125.[CrossRef]
    [Google Scholar]
  25. Kalscheuer, R., Stölting, T. & Steinbüchel, A. ( 2006a; ). MicroDiesel: Escherichia coli engineered for fuel production. Microbiology 152, 2529–2536.[CrossRef]
    [Google Scholar]
  26. Kalscheuer, R., Stöveken, T., Luftmann, H., Malkus, U., Reichelt, R. & Steinbüchel, A. ( 2006b; ). Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters. Appl Environ Microbiol 72, 1373–1379.[CrossRef]
    [Google Scholar]
  27. Kalscheuer, R., Stöveken, T., Malkus, U., Reichelt, R., Golyshin, P. N., Sabirova, J. S., Ferrer, M., Timmis, K. N. & Steinbüchel, A. ( 2007; ). Analysis of storage lipid accumulation in Alcanivorax borkumensis: evidence for alternative triacylglycerol biosynthesis routes in bacteria. J Bacteriol 189, 918–928.[CrossRef]
    [Google Scholar]
  28. Katavic, V., Reed, D. W., Taylor, D. C., Giblin, E. M., Barton, D. L., Zou, J., MacKenzie, S. L., Covello, P. S. & Kunst, L. ( 1995; ). Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity. Plant Physiol 108, 399–409.[CrossRef]
    [Google Scholar]
  29. Leman, J. ( 1997; ). Oleaginous microorganisms: an assessment of the potential. Adv Appl Microbiol 43, 195–243.
    [Google Scholar]
  30. Marmur, J. ( 1961; ). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.[CrossRef]
    [Google Scholar]
  31. McLeod, M. P., Warren, R. L., Hsiao, W. W., Araki, N., Myhre, M., Fernandes, C., Miyazawa, D., Wong, W., Lillquist, A. L. & other authors ( 2006; ). The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proc Natl Acad Sci U S A 103, 15582–15587.[CrossRef]
    [Google Scholar]
  32. Olukoshi, E. R. & Packter, N. M. ( 1994; ). Importance of stored triacylglycerols in Streptomyces: possible carbon source for antibiotics. Microbiology 140, 931–943.[CrossRef]
    [Google Scholar]
  33. Overhage, J., Priefert, H., Rabenhorst, J. & Steinbüchel, A. ( 1999; ). Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene. Appl Microbiol Biotechnol 52, 820–828.[CrossRef]
    [Google Scholar]
  34. Packter, N. M. & Olukoshi, E. R. ( 1995; ). Ultrastructural studies of neutral lipid localization in Streptomyces. Arch Microbiol 164, 420–427.[CrossRef]
    [Google Scholar]
  35. Pelicic, V., Reyrat, J. M. & Gicquel, B. ( 1996; ). Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors. Mol Microbiol 20, 919–925.[CrossRef]
    [Google Scholar]
  36. Quandt, J. & Hynes, M. F. ( 1993; ). Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene 127, 15–21.[CrossRef]
    [Google Scholar]
  37. Ramakrishnan, L., Tran, H. T., Federspiel, N. A. & Falkow, S. ( 1997; ). A crtB homolog essential for photochromogenicity in Mycobacterium marinum: isolation, characterization, and gene disruption via homologous recombination. J Bacteriol 179, 5862–5868.
    [Google Scholar]
  38. Ratledge, C. ( 1989; ). Biotechnology of oils and fats. In Microbial Lipids, pp. 567–650. Edited by C. Ratledge & S. G. Wilkinson. London: Academic Press.
  39. Russell, N. J. & Volkman, J. K. ( 1980; ). The effect of growth temperature and wax ester compositions in the psychrophilic bacterium Micrococcus cryophilus ATCC 15174. J Gen Microbiol 118, 131–141.
    [Google Scholar]
  40. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  41. Schlegel, H. G., Kaltwasser, H. & Gottschalk, G. ( 1961; ). Ein Submersverfahren zur Kultur Wasserstoff oxydierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38, 209–222 (in German).[CrossRef]
    [Google Scholar]
  42. Simon, R., Priefer, U. & Pühler, A. ( 1983; ). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1, 784–791.[CrossRef]
    [Google Scholar]
  43. Sirakova, T. D., Dubey, V. S., Deb, C., Daniel, J., Korotkova, T. A., Abomoelak, B. & Kolattukudy, P. E. ( 2006; ). Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress. Microbiology 152, 2717–2725.[CrossRef]
    [Google Scholar]
  44. Stahmann, K. P., Kupp, C., Feldmann, S. D. & Sahm, H. ( 1994; ). Formation and degradation of lipid bodies found in the riboflavin-producing fungus Ashbya gossypii. Appl Microbiol Biotechnol 42, 121–127.[CrossRef]
    [Google Scholar]
  45. Steinbüchel, A. ( 1991; ). Polyhydroxyalkanoic acids. In Biomaterials, Novel Materials, pp. 123–213. Edited by D. Byrom. Basingstoke: Macmillan.
  46. Steinbüchel, A. & Valentin, H. E. ( 1995; ). Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett 128, 219–228.[CrossRef]
    [Google Scholar]
  47. Stöveken, T., Kalscheuer, R., Malkus, U., Reichelt, R. & Steinbüchel, A. ( 2005; ). The wax ester synthase/acyl coenzyme A : diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 187, 1369–1376.[CrossRef]
    [Google Scholar]
  48. Uthoff, S., Stöveken, T., Weber, N., Vosmann, K., Klein, E., Kalscheuer, R. & Steinbüchel, A. ( 2005; ). Thio wax ester biosynthesis utilizing the unspecific bifunctional wax ester synthase/acyl-CoA : diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1. Appl Environ Microbiol 71, 790–796.[CrossRef]
    [Google Scholar]
  49. Voss, I. & Steinbüchel, A. ( 2001; ). High cell density cultivation of Rhodococcus opacus for lipid production at a pilot scale. Appl Microbiol Biotechnol 55, 547–555.[CrossRef]
    [Google Scholar]
  50. Wältermann, M. & Steinbüchel, A. ( 2005; ). Neutral lipid-bodies in prokaryotes: recent insights into structure, formation and relationships to eukaryotic lipid depots. J Bacteriol 187, 3607–3619.[CrossRef]
    [Google Scholar]
  51. Wältermann, M. & Steinbüchel, A. ( 2006; ). Wax ester and triacylglycerol inclusions. In Inclusions in Prokaryotes (Microbiology Monographs, vol. 1), pp. 137–166. Edited by J. M. Shively & A. Steinbüchel. Heidelberg: Springer.
  52. Wältermann, M., Luftmann, H., Baumeister, D., Kalscheuer, R. & Steinbüchel, A. ( 2000; ). Rhodococcus opacus PD630 as a source of high-value single cell oil? Isolation and characterization of triacylgycerols and other storage lipids. Microbiology 146, 1143–1149.
    [Google Scholar]
  53. Wältermann, M., Stöveken, T. & Steinbüchel, A. ( 2006; ). Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA : diacylglycerol acyltransferases. Biochimie 89, 230–242.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/016568-0
Loading
/content/journal/micro/10.1099/mic.0.2008/016568-0
Loading

Data & Media loading...

Primers used for amplification and cloning of genes from PD630 [ PDF] (43 kb) Global multiple alignment of deduced Atf sequences from PD630. Positions of degenerate oligonucleotide primers and of the putative active site are underlined. [ PDF] (28 kb)

PDF

Primers used for amplification and cloning of genes from PD630 [ PDF] (43 kb) Global multiple alignment of deduced Atf sequences from PD630. Positions of degenerate oligonucleotide primers and of the putative active site are underlined. [ PDF] (28 kb)

PDF
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