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
2020-04-08
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 Biodeg52:35–42 [CrossRef]
    [Google Scholar]
  2. Alvarez H. M., Steinbüchel A.. 2002; Triacylglycerols in prokaryotic microorganisms. Appl Microbiol Biotechnol60:367–376 [CrossRef][PubMed]
    [Google Scholar]
  3. Alvarez H. M., Steinbüchel A.. 2010; Physiology, biochemistry and molecular biology of triacylglycerol accumulation by Rhodococcus . In Biology of Rhodococcus pp263–290 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 Microbiol165:377–386 [CrossRef][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/Lipid99:239–246 [CrossRef]
    [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 Biotechnol7:170–181 [CrossRef][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 Biochem2:67–78
    [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 Genomics9:75 [CrossRef][PubMed]
    [Google Scholar]
  9. Baños S., Pérez-Redondo R., Koekman B., Liras P.. 2009; Glycerol utilization gene cluster in Streptomyces clavuligerus . Appl Environ Microbiol75:2991–2995 [CrossRef][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 Microbiol139:349–359 [CrossRef][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 Biotechnol131:880–886 [CrossRef][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 Interact14:599–608 [CrossRef][PubMed]
    [Google Scholar]
  14. da Silva G. P., Mack M., Contiero J.. 2009; Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol Adv27:30–39 [CrossRef][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. Microbiology161:593–610 [CrossRef][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 Bioeng94:821–829 [CrossRef][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 Technol100:356–361 [CrossRef][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. Structure6:1407–1418 [CrossRef][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 A88:5041–5045 [CrossRef][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 Bacteriol149:413–419
    [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 Technol100:3775–3778 [CrossRef][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 Biotechnol24:1703–1711 [CrossRef]
    [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 [CrossRef] [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 Microbiol136:2367–2375 [CrossRef][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 Biotechnol52:508–515 [CrossRef][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 Microbiol56:163–174 [CrossRef][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 Protoc4:363–371 [CrossRef][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 Biofuels6:134 [CrossRef][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 Biofuels8:31 [CrossRef][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 Chem274:6817–6819 [CrossRef][PubMed]
    [Google Scholar]
  31. Lin E. C. C.. 1976; Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol30:535–578 [CrossRef][PubMed]
    [Google Scholar]
  32. Marmur J.. 1961; A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol3:208–218 [CrossRef]
    [Google Scholar]
  33. Mindich L.. 1968; Pathway for oxidative dissimilation of glycerol in Bacillus subtilis . J Bacteriol96:565–566[PubMed]
    [Google Scholar]
  34. Németh A., Kupcsulik B., Sevella B.. 2003; 1.3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026. World J Microbiol Biotechnol19:659–663 [CrossRef]
    [Google Scholar]
  35. Nikel P. I., Kim J., de Lorenzo V.. 2014; Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440. Environ Microbiol16:239–254 [CrossRef][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. Biochemistry37:16565–16572 [CrossRef][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 Technol82:43–49 [CrossRef][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 Microbiol46:124–130 [CrossRef][PubMed]
    [Google Scholar]
  39. Refaat A. A.. 2009; Correlation between the chemical structure of biodiesel and its physical properties. Int J Environ Sci Technol6:677–694 [CrossRef]
    [Google Scholar]
  40. Richey D. P., Lin E. C. C.. 1972; Importance of facilitated diffusion for effective utilization of glycerol by Escherichia coli . J Bacteriol112:784–790[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 Microbiol74:6216–6222 [CrossRef][PubMed]
    [Google Scholar]
  42. Saitou N., Nei M.. 1987; The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol4:406–425[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 Combust2012:285185[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 Mikrobiol38:209–222 (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 Bacteriol178:5215–5221[PubMed]
    [Google Scholar]
  47. Spurr A. R.. 1969; A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res26:31–43[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 Evol28:2731–2739 [CrossRef][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 Res22:4673–4680 [CrossRef][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 Bacteriol189:5903–5915 [CrossRef][PubMed]
    [Google Scholar]
  51. Unger V. M.. 2000; Fraternal twins: AQP1 and GlpF. Nat Struct Biol7:1082–1084 [CrossRef][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 Bacteriol175:1087–1094[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 Microbiol35:349–355[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 Biotechnol55:547–555 [CrossRef][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. Microbiology141:1193–1198 [CrossRef][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 J65:30–36[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. Biochemistry43:362–373 [CrossRef][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. Biochemistry48:346–356[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 Bioeng114:453–6[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

Most cited this month

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