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Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.

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References

  • Batard Y, Hehn A, Nedelkina S, Schalk M, Pallett K, Schaller H, Werck-Reichhart D (2000) Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems. Arch Biochem Biophys 379:161–169

    Article  CAS  Google Scholar 

  • Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    Article  CAS  Google Scholar 

  • Branduardi P, Smeraldi C, Porro D (2008) Metabolically engineered yeasts: ‘potential’ industrial applications. J Mol Microbiol Biotechnol 15:31–40

    Article  CAS  Google Scholar 

  • Braun S, Matuschewski K, Rape M, Thoms S, Jentsch S (2002) Role of the ubiquitin-selective CDC48UFD1/NPL4 chaperone (segregase) in ERAD of OLE1 and other substrates. EMBO J 21:615–621

    Article  CAS  Google Scholar 

  • Broadwater JA, Whittle E, Shanklin J (2002) Desaturation and hydroxylation. Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity. J Biol Chem 277:15613–15620

    Article  CAS  Google Scholar 

  • Collart MA, Oliviero S (1993) Preparation of yeast RNA. In: Ausubel FM, Brent R, Kingston RE et al (eds) Current protocols in molecular biology. Greene, New York, pp 13.12.1–13.12.5

    Google Scholar 

  • Czabany T, Athenstaedt K, Daum G (2007) Synthesis, storage and degradation of neutral lipids in yeast. Biochim Biophys Acta 1771:299–309

    Article  CAS  Google Scholar 

  • Dyer JM, Mullen RT (2001) Immunocytological localization of two plant fatty acid desaturases in the endoplasmic reticulum. FEBS Lett 494:44–47

    Article  CAS  Google Scholar 

  • Dyer JM, Chapital DC, Cary JW, Pepperman AB (2001) Chilling-sensitive, post-transcriptional regulation of a plant fatty acid desaturase expressed in yeast. Biochem Biophys Res Commun 282:1019–1025

    Article  CAS  Google Scholar 

  • Dyer JM, Chapital DC, Kuan JW, Mullen RT, Pepperman AB (2002a) Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds. Appl Microbiol Biotechnol 59:224–230

    Article  CAS  Google Scholar 

  • Dyer JM, Chapital DC, Kuan J-CW, Mullen RT, Turner C, McKeon TA, Pepperman AB (2002b) Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity. Plant Physiol 130:2027–2038

    Article  CAS  Google Scholar 

  • Dyer JM, Stymne S, Green AG, Carlsson AS (2008) High-value oils from plants. Plant J 54:640–655

    Article  CAS  Google Scholar 

  • Dumas B, Brocard-Masson C, Assemat-Lebrun K, Achstetter T (2006) Hydrocortisone made in yeast: Metabolic engineering turns a unicellular microorganism into a drug-synthesizing factory. Biotechnol J 1:299–307

    Article  CAS  Google Scholar 

  • Flis K, Hinzpeter A, Edelman A, Kurlandzka A (2005) The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p. Biochem J 390:655–664

    Article  CAS  Google Scholar 

  • Fritze CE, Anderson TR (2000) Epitope tagging: general method for tracking recombinant proteins. Methods Enzymol. 327:3–16

    Article  CAS  Google Scholar 

  • Gietz RD, Woods RA (1994) High efficiency transformation in yeast. In: Johnston JA (ed) Molecular genetics of yeast: practical approaches. Oxford University Press, New York, pp 121–134

    Google Scholar 

  • Hehn A, Morant M, Werck-Reichhart D (2002) Partial recoding of P450 and P450 reductase cDNAs for improved expression in yeast and plants. Methods Enzymol 357:343–351

    Article  CAS  Google Scholar 

  • Hoffman M, Wagner M, Abbadi A, Fulda M, Feussner I (2008) Metabolic engineering of ω3-very long chain polyunsaturated fatty acid production by an exclusively acyl-CoA-dependent pathway. J Biol Chem 283:22352–22362

    Article  Google Scholar 

  • Kamisaka Y, Noda N, Tomita N, Kimura K, Kodaki T, Hosaka K (2006) Identification of genes affecting lipid content using transposon mutagenesis in Saccharomyces cerevisiae. Biosci Biotechnol Biochem 70:646–653

    Article  CAS  Google Scholar 

  • Kamisaka Y, Tomita N, Kimura K, Kainou K, Uemura H (2007) DGA1 (diacylglycerol acyltransferase gene) overexpression and leucine biosynthesis significantly increase lipid accumulation in the Δsnf2 disruptant of Saccharomyces cerevisiae. Biochem J 408:61–68

    Article  CAS  Google Scholar 

  • Kajiwara S, Shirai A, Fujii T, Toguri T, Nakamura K, Ohtaguchi K (1996) Polyunsaturated fatty acid biosynthesis in Saccharomyces cerevisiae: expression of ethanol tolerance and the FAD2 gene from Arabidopsis thaliana. Appl Environ Microbiol 62:4309–4313

    Article  CAS  Google Scholar 

  • Kozak M (1987) An analysis of 5’-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15:8125–8148

    Article  CAS  Google Scholar 

  • Matsuda O, Sakamoto H, Hashimoto T, Iba K (2005) A temperature-sensitive mechanism that regulates post-translational stability of a plastidial ω-3 fatty acid desaturase (FAD8) in Arabidopsis leaf tissues. J Biol Chem 280:3597–3604

    Article  CAS  Google Scholar 

  • McCammon MT, Veenhuis M, Trapp SB, Goodman JM (1990) Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae. J Bacteriol 172:5816–5827

    Article  CAS  Google Scholar 

  • McCartney AW, Dyer JM, Dhanoa PK, Kim PK, Andrews DW, McNew JA, Mullen RT (2004) Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini. Plant J 37:156–173

    Article  CAS  Google Scholar 

  • Orlean P (1990) Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae. Mol Cell Biol 10:5796–805

    Article  CAS  Google Scholar 

  • Porro D, Sauer M, Branduardi P, Mattanovich D (2005) Recombinant protein production in yeasts. Mol Biotechnol 31(3):245–259

    Article  CAS  Google Scholar 

  • Reed DW, Schäfer UA, Covello PS (2000) Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae. Plant Physiol 122:715–720

    Article  CAS  Google Scholar 

  • Shanklin J, Whittle E, Fox BG (1994) Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry 33:12787–12794

    Article  CAS  Google Scholar 

  • Tang GQ, Novitzky WP, Carol Griffin H, Huber SC, Dewey RE (2005) Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation. Plant J 44:433–446

    Article  CAS  Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354

    Article  CAS  Google Scholar 

  • Uauy R, Dangour AD (2006) Nutrition in brain development and aging: role of essential fatty acids. Nutr Rev 64:24–33

    Article  Google Scholar 

  • Veen M, Lang C (2004) Production of lipid compounds in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 63:635–646

    Article  CAS  Google Scholar 

  • Verstrepen KJ, Chambers PJ, Pretorius IS (2006) The development of superior yeast strains for the food and beverage industries: challenges, opportunities, and potential benefits. In: Querol A, Fleet GH (eds) The yeast handbook: yeasts in food and beverages. Springer, Berlin, pp 399–444

    Chapter  Google Scholar 

  • Walenga RW, Lands WE (1975) Effectiveness of various unsaturated fatty acids in supporting growth and respiration in Saccharomyces cerevisiae. J Biol Chem 250:9121–9129

    CAS  PubMed  Google Scholar 

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Acknowledgments

The authors wish to thank Dorselyn Chapital and Rae Kuan for excellent technical assistance. This work was supported by the United States Department of Agriculture, Current Research Information System project no. 6435-41000-083-00D to JMD, the Louisiana Board of Regents Governor’s Biotechnology Initiative (to JBO’Q and JMD), and a grant from the Natural Sciences and Engineering Research Council (NSERC grant no. 217291) to RTM.

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  1. Jami B. O’Quin

    Present address: Covance Inc., 150 4th Ave. North, Nashville, TN, 37219, USA

Authors and Affiliations

  1. United States Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA, USA

    Jami B. O’Quin

  2. Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada

    Robert T. Mullen

  3. United States Department of Agriculture, Agricultural Research Service, US Arid-Land Agricultural Research Center, 21881 North Cardon Lane, Maricopa, AZ, 85238, USA

    John M. Dyer

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  1. Jami B. O’Quin
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  2. Robert T. Mullen
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  3. John M. Dyer
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Correspondence to John M. Dyer.

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O’Quin, J.B., Mullen, R.T. & Dyer, J.M. Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells. Appl Microbiol Biotechnol 83, 117–125 (2009). https://doi.org/10.1007/s00253-008-1826-2

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  • DOI: https://doi.org/10.1007/s00253-008-1826-2

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