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The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630

  • Applied Microbial and Cell Physiology
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Abstract

Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327–2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25–30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.

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References

  • Alvarez HM (2010) Biotechnological production and significance of triacylglycerols and wax esters. In: Timmis KN (ed) Microbiology of hydrocarbons, oils, lipids, and derived compounds, vol 3. Springer, Heidelberg, pp 2995–3002

    Chapter  Google Scholar 

  • Alvarez HM, Steinbüchel A (2002) Triacylglycerols in prokaryotic microorganisms. Appl Microbiol Biotechnol 60:367–376

    Article  CAS  Google Scholar 

  • Alvarez HM, Steinbüchel A (2010) Physiology, biochemistry and molecular biology of triacylglycerol accumulation by Rhodococcus. In: Alvarez HM (ed) Biology of Rhodococcus. Microbiology monographs series. Springer, Heidelberg, pp 263–290

    Google Scholar 

  • Alvarez HM, Mayer F, Fabritius D, Steinbüchel A (1996) Formation of intracytoplasmic lipid inclusion by Rhodococcus opacus PD630. Arch Microbiol 165:377–386

    Article  CAS  Google Scholar 

  • Alvarez HM, Kalscheuer R, Steinbüchel A (1997) Accumulation of storage lipids in species of Rhodococcus and Nocardia and effect of inhibitors and polyethylene glycol. Fett-Lipid 99:239–246

    Article  CAS  Google Scholar 

  • Alvarez AF, Alvarez HM, Kalscheuer R, Wältermann M, Steinbüchel A (2008) Cloning and characterization of a gene involved in triacylglycerol biosynthesis and identification of additional homologous genes in the oleogenous bacterium Rhodococcus opacus PD630. Microbiology 154:2327–2335

    Article  CAS  Google Scholar 

  • Arabolaza A, Rodriguez E, Altabe S, Alvarez H, Gramajo H (2008) Multiple pathways for triacylglycerol biosynthesis in Streptomyces coelicolor. Appl Environ Microbiol 74:2573–2582

    Article  CAS  Google Scholar 

  • Arabolaza A, D'Angelo M, Comba S, Gramajo H (2010) FasR, a novel class of transcriptional regulator, governs the activation of fatty acid biosynthesis genes in Streptomyces coelicolor. Mol Microbiol 78(1):47–63

    CAS  Google Scholar 

  • Barksdale L, Kim KS (1977) Mycobacterium. Bacteriol Rev 41:217–372

    CAS  Google Scholar 

  • Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly(b-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977–1982

    CAS  Google Scholar 

  • Daniel J, Deb C, Dubey VS, Sirakova T, Abomoelak MHR, Kolattukudy PE (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

    Article  CAS  Google Scholar 

  • Dhiman RK, Schulbach MC, Mahapatra S, Baulard AR, Vissa V, Brennan PJ, Crick DC (2004) Identification of a novel class of ω, E, E-farnesyl diphosphate synthase from Mycobacterium tuberculosis. J Lipid Res 45:1140–1147

    Article  CAS  Google Scholar 

  • Duncombe WG (1963) The colorimetric micro-determination of long chain fatty acids. Biochem J 88:7

    CAS  Google Scholar 

  • Gouda MK, Omar SH, Aouad LM (2008) Single cell oil production by Gordonia sp. DG using agro-industrial wastes. World J Microbiol Biotechnol 24:1703–1711

    Article  CAS  Google Scholar 

  • Gregory MA, Till R, Smith MC (2003) Integration site for Streptomyces phage phiBT1 and development of site specific integrating vectors. J Bacteriol 185:5320–5323

    Article  CAS  Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Google Scholar 

  • Hänisch J, Wältermann M, Robenek H, Steinbüchel A (2006a) The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins. Microbiology 152:3271–3280

    Article  Google Scholar 

  • Hänisch J, Wältermann M, Robenek H, Steinbüchel A (2006b) Eukaryotic lipid body proteins in oleogenous actinomycetes and their targeting to intracellular triacylglycerol inclusions: impact on models of lipid body biogenesis. Appl Environ Microbiol 72:6743–6750

    Article  Google Scholar 

  • Hernández MA, Alvarez HM (2010) Glycogen formation by Rhodococcus species and the effect of inhibition of lipid biosynthesis on glycogen accumulation in Rhodococcus opacus PD630. FEMS Microbiol Lett 312:93–99

    Article  Google Scholar 

  • Hernández MA, Mohn WW, Martínez E, Rost E, Alvarez AF, Alvarez HM (2008) Biosynthesis of storage compounds by Rhodococcus jostii RHA1 and global identification of genes involved in their metabolism. BMC Genomics 12:600

    Article  Google Scholar 

  • Holder JW, Ulrich JC, DeBono AC, Godfrey PA, Desjardins CA, Zucker J, Zeng Q, Leach ALB, Ghiviriga I, Dancel C, Abeel T, Gevers D, Kodira CD, Desany B, Affourtit JP, Birren BW, Sinskey AJ (2011) Comparative and functional genomics of Rhodococcus opacus PD630 for biofuels development. PLoS Genet 7(9):e1002219. doi:10.1371/journal.pgen.1002219

    Article  CAS  Google Scholar 

  • Holtzapple E, Schmidt-Dannert C (2007) Biosynthesis of isoprenoid wax ester in Marinobacter hydrocarbonoclasticus DSM 8798: identification and characterization of isoprenoid coenzyme A synthetase and wax ester synthases. J Bacteriol 189(10):3804–3812

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Kalscheuer R, Stoveken T, Malkus U, Reichelt R, Golyshin PN, Sabirova JS, Ferrer M, Timmis KN, Steinbüchel A (2007) Analysis of storage lipid accumulation in Alcanivorax borkumensis: evidence for alternative triacylglycerol biosynthesis routes in bacteria. J Bacteriol 189:918–928

    Article  CAS  Google Scholar 

  • Leman J (1997) Oleaginous microorganisms: an assessment of the potential. Adv Appl Microbiol 43:195–243

    Article  CAS  Google Scholar 

  • Li Q, Du W, Liu D (2008) Perspectives of microbial oils for biodiesel production. Appl Microbiol Biotechnol 80:749–756

    Article  CAS  Google Scholar 

  • Nakashima N, Tamura T (2004) Isolation and characterization of a rolling-circle-type plasmid from Rhodococcus erythropolis and application of the plasmid to multiple-recombinant-protein expression. Appl Environ Microbiol 70:5557–5568

    Article  CAS  Google Scholar 

  • Olukoshi ER, Packter NM (1994) Importance of stored triacylglycerols in Streptomyces: possible carbon source for antibiotics. Microbiology 140:931–943

    Article  CAS  Google Scholar 

  • Pelicic V, Jackson M, Reyrat JM, Jacobs WR Jr, Gicquel B, Guilhot C (1997) Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 94:10955–10960

    Article  CAS  Google Scholar 

  • Ratledge C (1989) Biotechnology of oils and fats. In: Ratledge SG, Wilkinson (eds) Microbial lipids. Academic, London, pp 567–650

    Google Scholar 

  • Salzman V, Mondino S, Sala C, Cole ST, Gago G, Gramajo H (2010) Transcriptional regulation of lipid homeostasis in mycobacteria. Mol Microbiol 78(1):64–77

    CAS  Google Scholar 

  • Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein Submersverfahren zur Kultur Wasserstoff oxydierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38:209–222

    Article  CAS  Google Scholar 

  • Triccas JA, Parish T, Britton WJ, Giquel B (1998) An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol Lett 167:151–156

    Article  CAS  Google Scholar 

  • Van der Geize R, Hessels GI, Van Gerwen R, Vrijbloed JW, Van der Meijden P, Dijkhuizen L (2000) Targeted disruption of the kstD gene encoding a 3-ketosteroid Δ1-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1. Appl Environ Microbiol 66:2029–2036

    Article  Google Scholar 

  • 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 

  • Wawrik B, Harriman BH (2010) Rapid, colorimetric quantification of lipid from algal cultures. J Microbiol Methods. doi:10.1016/j.minet.2010.01.016

Download references

Acknowledgments

The authors thank W.W. Mohn for the provision of pTip-QC2 vector. This study was financially supported by the SCyT of the University of Patagonia San Juan Bosco, the Agencia Comodoro Conocimiento (MCR), Oil m&s and PICT-2008-1640. H.M. Alvarez is a career investigator and M.A. Hernández a scholarship holder of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

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Authors and Affiliations

  1. Centro Regional de Investigación y Desarrollo Científico Tecnológico, Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia, San Juan Bosco Km 4-Ciudad Universitaria 9000, Comodoro Rivadavia (Chubut), Argentina

    Martín A. Hernández & Héctor M. Alvarez

  2. Microbiology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina

    Ana Arabolaza, Eduardo Rodríguez & Hugo Gramajo

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  1. Martín A. Hernández
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  2. Ana Arabolaza
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  3. Eduardo Rodríguez
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  4. Hugo Gramajo
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  5. Héctor M. Alvarez
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Correspondence to Héctor M. Alvarez.

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Hernández, M.A., Arabolaza, A., Rodríguez, E. et al. The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630. Appl Microbiol Biotechnol 97, 2119–2130 (2013). https://doi.org/10.1007/s00253-012-4360-1

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  • DOI: https://doi.org/10.1007/s00253-012-4360-1

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