1887

Abstract

We analysed the ability of five different rhodococcal species to grow and produce triacylglycerols (TAGs) from glycerol, the main byproduct of biodiesel production. and grew fast on glycerol, whereas and exhibited a prolonged lag phase of several days before growing. only exhibited poor growth on glycerol. DSMZ 43060 and F7 produced 3.9–4.3 g cell biomass l and 28.4–44.6 % cellular dry weight (CDW) of TAGs after 6 days of incubation; whereas PD630 and RHA1 produced 2.5–3.8 g cell biomass l and 28.3–38.4 % CDW of TAGs after 17 days of growth on glycerol. Genomic analyses revealed two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2) amongst rhodococci. Those species that possessed cluster 1 () ( and ) exhibited fast growth and lipid accumulation, whereas those that possessed cluster 2 () (, and ) exhibited delayed growth and lipid accumulation during cultivation on glycerol. Three glycerol-negative strains were complemented for their ability to grow and produce TAGs by heterologous expression of from PD630. In addition, we significantly reduced the extension of the lag phase and improved glycerol assimilation and oil production of PD630 when expressing from . The results demonstrated that rhodococci are a flexible and amenable biological system for further biotechnological applications based on the reutilization of glycerol.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000232
2016-02-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/162/2/384.html?itemId=/content/journal/micro/10.1099/mic.0.000232&mimeType=html&fmt=ahah

References

  1. Alvarez H. M. 2003; Relationship between β-oxidation pathway and the hydrocarbon-degrading profile in actinomycetes bacteria. Int Biodeter Biodeg 52:35–42 [View Article]
    [Google Scholar]
  2. Alvarez H. M., Steinbüchel A. 2002; Triacylglycerols in prokaryotic microorganisms. Appl Microbiol Biotechnol 60:367–376 [View Article][PubMed]
    [Google Scholar]
  3. Alvarez H. M., Steinbüchel A. 2010; Physiology, biochemistry and molecular biology of triacylglycerol accumulation by Rhodococcus . In Biology of Rhodococcus pp 263–290 Edited by Alvarez H. M. Heidelberg: Springer; [CrossRef]
    [Google Scholar]
  4. Alvarez H. M., Mayer F., Fabritius D., Steinbüchel A. 1996; Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630. Arch Microbiol 165:377–386 [View Article][PubMed]
    [Google Scholar]
  5. Alvarez H. M., Kalscheuer R., Steinbüchel A. 1997; Accumulation of storage lipids in species of Rhodococcus and Nocardia and effect of inhibitors and polyethyleneglycol. Fett/Lipid 99:239–246 [View Article]
    [Google Scholar]
  6. Alvarez M. F., Medina R., Pasteris S. E., Strasser de Saad A. M., Sesma F. 2004; Glycerol metabolism of Lactobacillus rhamnosus ATCC 7469: cloning and expression of two glycerol kinase genes. J Mol Microbiol Biotechnol 7:170–181 [View Article][PubMed]
    [Google Scholar]
  7. Alvarez H. M., Silva R. A., Herrero O. M., Hernández M. A., Villalba M. S. 2013; Metabolism of triacylglycerols in Rhodococcus species: insights from physiology and molecular genetics. J Mol Biochem 2:67–78
    [Google Scholar]
  8. Aziz R. K., Bartels D., Best A. A., DeJongh M., Disz T., Edwards R. A., Formsma K., Gerdes S., Glass E. M., other authors. 2008; The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9:75 [View Article][PubMed]
    [Google Scholar]
  9. Baños S., Pérez-Redondo R., Koekman B., Liras P. 2009; Glycerol utilization gene cluster in Streptomyces clavuligerus . Appl Environ Microbiol 75:2991–2995 [View Article][PubMed]
    [Google Scholar]
  10. Beijer L., Nilsson R. P., Holmberg C., Rutberg L. 1993; The glpP and glpF genes of the glycerol regulon in Bacillus subtilis . J Gen Microbiol 139:349–359 [View Article][PubMed]
    [Google Scholar]
  11. Chatzifragkou A., Makri A., Belka A., Bellou S., Mavrou M., Mastoridou M., Mystrioti P., Onjaro G., Aggelis G., other authors. 2011; Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy 36:1097–1080
    [Google Scholar]
  12. Ciapina E. M. M., Melo W. C., Santa Anna L. M. M., Santos A. S., Freire D. M. G., Pereira N. Jr 2006; Biosurfactant production by Rhodococcus erythropolis grown on glycerol as sole carbon source. Appl Biochem Biotechnol 131:880–886 [View Article][PubMed]
    [Google Scholar]
  13. Cornelis K., Ritsema T., Nijsse J., Holsters M., Goethals K., Jaziri M. 2001; The plant pathogen Rhodococcus fascians colonizes the exterior and interior of the aerial parts of plants. Mol Plant Microbe Interact 14:599–608 [View Article][PubMed]
    [Google Scholar]
  14. da Silva G. P., Mack M., Contiero J. 2009; Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol Adv 27:30–39 [View Article][PubMed]
    [Google Scholar]
  15. Dávila Costa J. S., Herrero O. M., Alvarez H. M., Leichert L. 2015; Label-free and redox proteomic analyses of the triacylglycerol-accumulating Rhodococcus jostii RHA1. Microbiology 161:593–610 [View Article][PubMed]
    [Google Scholar]
  16. Dharmadi Y., Murarka A., Gonzalez R. 2006; Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering. Biotechnol Bioeng 94:821–829 [View Article][PubMed]
    [Google Scholar]
  17. Easterling E. R., French W. T., Hernandez R., Licha M. 2009; The effect of glycerol as a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis . Bioresour Technol 100:356–361 [View Article][PubMed]
    [Google Scholar]
  18. Feese M. D., Faber H. R., Bystrom C. E., Pettigrew D. W., Remington S. J. 1998; Glycerol kinase from Escherichia coli and an Ala65 → Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation. Structure 6:1407–1418 [View Article][PubMed]
    [Google Scholar]
  19. Flaherty K. M., McKay D. B., Kabsch W., Holmes K. C. 1991; Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein. Proc Natl Acad Sci U S A 88:5041–5045 [View Article][PubMed]
    [Google Scholar]
  20. Forage R. G., Lin C. C. 1982; dha System mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418. J Bacteriol 149:413–419
    [Google Scholar]
  21. Galan M. I., Bonet J., Sire R., Reneaume J. M., Pleşu A. E. 2009; From residual to useful oil: revalorization of glycerine from the biodiesel synthesis. Bioresour Technol 100:3775–3778 [View Article][PubMed]
    [Google Scholar]
  22. Gouda M. K., Omar S. H., Aouad L. M. 2008; Single cell oil production by Gordonia sp. DG using agroindustrial wastes. World J Microbiol Biotechnol 24:1703–1711 [View Article]
    [Google Scholar]
  23. Herrero O. M., Alvarez H. M. 2015; Whey as a renewable source for lipid production by Rhodococcus strains: physiology and genomics of lactose and galactose utilization. Eur J Lipid Sci Technol [View Article] [Epub ahead of print]
    [Google Scholar]
  24. Holmberg C., Beijer L., Rutberg B., Rutberg L. 1990; Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD). J Gen Microbiol 136:2367–2375 [View Article][PubMed]
    [Google Scholar]
  25. 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 [View Article][PubMed]
    [Google Scholar]
  26. Keating L. A., Wheeler P. R., Mansoor H., Inwald J. K., Dale J., Hewinson R. G., Gordon S. V. 2005; The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth. Mol Microbiol 56:163–174 [View Article][PubMed]
    [Google Scholar]
  27. Kelley L. A., Sternberg M. J. 2009; Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371 [View Article][PubMed]
    [Google Scholar]
  28. Kurosawa K., Wewetzer S. J., Sinskey A. J. 2013; Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production. Biotechnol Biofuels 6:134 [View Article][PubMed]
    [Google Scholar]
  29. Kurosawa K., Radek A., Plassmeier J. K., Sinskey A. 2015; Improved glycerol utilization by a triacyglycerol-producing Rhodococcus opacus strain for renewable fuels. Biotechnol Biofuels 8:31 [View Article][PubMed]
    [Google Scholar]
  30. Lagrée V., Froger A., Deschamps S., Hubert J. F., Delamarche C., Bonnec G., Thomas D., Gouranton J., Pellerin I. 1999; Switch from an aquaporin to a glycerol channel by two amino acids substitution. J Biol Chem 274:6817–6819 [View Article][PubMed]
    [Google Scholar]
  31. Lin E. C. C. 1976; Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol 30:535–578 [View Article][PubMed]
    [Google Scholar]
  32. Marmur J. 1961; A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218 [View Article]
    [Google Scholar]
  33. Mindich L. 1968; Pathway for oxidative dissimilation of glycerol in Bacillus subtilis . J Bacteriol 96:565–566[PubMed]
    [Google Scholar]
  34. Németh A., Kupcsulik B., Sevella B. 2003; 1.3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026. World J Microbiol Biotechnol 19:659–663 [View Article]
    [Google Scholar]
  35. Nikel P. I., Kim J., de Lorenzo V. 2014; Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440. Environ Microbiol 16:239–254 [View Article][PubMed]
    [Google Scholar]
  36. Ormö M., Bystrom C. E., Remington S. J. 1998; Crystal structure of a complex of Escherichia coli glycerol kinase and an allosteric effector fructose 1,6-bisphosphate. Biochemistry 37:16565–16572 [View Article][PubMed]
    [Google Scholar]
  37. Papanikolaou S., Aggelis G. 2002; Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single-stage continuous culture. Bioresour Technol 82:43–49 [View Article][PubMed]
    [Google Scholar]
  38. Papanikolaou S., Muniglia L., Chevalot I., Aggelis G., Marc I. 2003; Accumulation of a cocoa-butter-like lipid by Yarrowia lipolytica cultivated on agro-industrial residues. Curr Microbiol 46:124–130 [View Article][PubMed]
    [Google Scholar]
  39. Refaat A. A. 2009; Correlation between the chemical structure of biodiesel and its physical properties. Int J Environ Sci Technol 6:677–694 [View Article]
    [Google Scholar]
  40. Richey D. P., Lin E. C. C. 1972; Importance of facilitated diffusion for effective utilization of glycerol by Escherichia coli . J Bacteriol 112:784–790[PubMed]
    [Google Scholar]
  41. Rittmann D., Lindner S. N., Wendisch V. F. 2008; Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum . Appl Environ Microbiol 74:6216–6222 [View Article][PubMed]
    [Google Scholar]
  42. Saitou N., Nei M. 1987; The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425[PubMed]
    [Google Scholar]
  43. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  44. Schlagermann P., Gottlicher G., Dillschneider R., Rosello-Sastre R., Posten C. 2012; Composition of algal oil and its potential as biofuel. J Combust 2012:285185 [CrossRef]
    [Google Scholar]
  45. 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 (in German) [CrossRef]
    [Google Scholar]
  46. Schweizer H. P., Po C. 1996; Regulation of glycerol metabolism in Pseudomonas aeruginosa: characterization of the glpR repressor gene. J Bacteriol 178:5215–5221[PubMed]
    [Google Scholar]
  47. Spurr A. R. 1969; A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43 [CrossRef]
    [Google Scholar]
  48. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 2011; mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 [View Article][PubMed]
    [Google Scholar]
  49. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 [View Article][PubMed]
    [Google Scholar]
  50. Titgemeyer F., Amon J., Parche S., Mahfoud M., Bail J., Schlicht M., Rehm N., Hillmann D., Stephan J., other authors. 2007; A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis . J Bacteriol 189:5903–5915 [View Article][PubMed]
    [Google Scholar]
  51. Unger V. M. 2000; Fraternal twins: AQP1 and GlpF. Nat Struct Biol 7:1082–1084 [View Article][PubMed]
    [Google Scholar]
  52. Voegele R. T., Sweet G. D., Boos W. 1993; Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator. J Bacteriol 175:1087–1094[PubMed]
    [Google Scholar]
  53. Vogt B., Berker R., Mayer F. 1995; Improved contrast by a simplified post-staining procedure for ultrathin sections of resin-embedded bacterial cells: application of ruthenium red. J Basic Microbiol 35:349–355 [CrossRef]
    [Google Scholar]
  54. Voss I., Steinbüchel A. 2001; High cell density cultivation of Rhodococcus opacus for lipid production at a pilot-plant scale. Appl Microbiol Biotechnol 55:547–555 [View Article][PubMed]
    [Google Scholar]
  55. Wehtje C., Beijer L., Nilsson R. P., Rutberg B. 1995; Mutations in the glycerol kinase gene restore the ability of a ptsGHI mutant of Bacillus subtilis to grow on glycerol. Microbiology 141:1193–1198 [View Article][PubMed]
    [Google Scholar]
  56. Xu J. Y., Zhao X. B., Wang W. C., Du W., Liu D. H. 2012; Microbial conversion of biodiesel by product glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production. Biochem Eng J 65:30–36 [CrossRef]
    [Google Scholar]
  57. Yeh J. I., Charrier V., Paulo J., Hou L., Darbon E., Claiborne A., Hol W. G., Deutscher J. 2004; Structures of enterococcal glycerol kinase in the absence and presence of glycerol: correlation of conformation to substrate binding and a mechanism of activation by phosphorylation. Biochemistry 43:362–373 [View Article][PubMed]
    [Google Scholar]
  58. Yeh J. I., Kettering R., Saxl R., Bourand A., Darbon E., Joly N., Briozzo P., Deutscher J. 2009; Structural characterizations of Glycerol Kinase: unraveling phosphorylation-induced long-range activation. Biochemistry 48:346–356 [CrossRef]
    [Google Scholar]
  59. Yen H. W., Yang Y. C., Yu Y. H. 2012; Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis . J Biosci Bioeng 114:453–6 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000232
Loading
/content/journal/micro/10.1099/mic.0.000232
Loading

Data & Media loading...

Supplements

Supplementary Data

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