Identification and Characterization of Phospholipids with Very Long Chain Fatty Acids in Brewer’s Yeast

Abstract

Yeast lipids and fatty acids (FA) were analyzed in Saccharomyces pastorianus from seven breweries and in the dietary yeast supplement Pangamin. GC–MS identified more than 30 FA, half of which were very-long chain fatty acids (VLCFA) with hydrocarbon chain lengths of ≥22 C atoms. Positional isomers ω-9 and ω-7 were identified in FA with C18–C28 even-numbered alkyl chains. The most abundant ω-7 isomer was cis-vaccenic acid. The structure of monounsaturated FA was proved by dimethyl disulfide adducts (position of double bonds and cis geometric configuration) and by GC–MS of pyridyl carbinol esters. Ultra-high performance liquid chromatography-tandem mass spectrometry with negative electrospray ionization identified the phospholipids phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine, with more than 150 molecular species. Wild-type unmutated brewer’s yeast strains conventionally used for the manufacture of food supplements were found to contain VLCFA.

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Abbreviations

CID:

Collision induced dissociation

DMDS:

Dimethyldisulfide

DMPtdEtn:

Dimethyl-phosphatidylethanolamine

ESI:

Electrospray ionization

ESI-MS:

Electrospray ionization mass spectrometry

FA:

Fatty acids

FAME:

Fatty acid methyl esters

FID:

Flame ionization detector

FT:

Fourier transform

GC–MS:

Gas chromatography–mass spectrometry

HESI:

Heated electrospray interface

HPLC:

High-performance liquid chromatography

IT-TIC:

Ion trap-total ion current

MMPtdEtn:

Monomethyl-phosphatidylethanolamine

PtdCho:

Phosphatidylcholine

PtdEtn:

Phosphatidylethanolamine

PtdIns:

Phosphatidylinositol

PtdSer:

Phosphatidylserine

SIM:

Selected-ion monitoring

TAG:

Triacylglycerol

HPLC:

High performance liquid chromatography

HPLC–MS2/ESI :

High performance liquid chromatography–tandem mass spectrometry negative electrospray ionization

VLCFA:

Very-long chain fatty acids

References

  1. 1.

    Nurminen T, Konttinen K, Suomalainen H (1975) Neutral lipids in the cells and cell envelope fractions of aerobic baker’s yeast and anaerobic brewer’s yeast. Chem Phys Lipids 14:15–32

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Kajiwara Y, Ogawa K, Takashita H, Omori T, Shimoda M, Wada H (1997) Intracellular fatty acid formation and alcohol acetyl transferase gene expression in brewing yeast (Saccharomyces cerevisiae) treated with heat shock. J Ferment Bioeng 84:594–598

    CAS  Article  Google Scholar 

  3. 3.

    Prasad R (1985) Lipids in the structure and function of yeast membrane. In: Rodolfo P, David K (eds) Adv Lipid Res. Elsevier, Amsterdam

    Google Scholar 

  4. 4.

    Sitepu IR, Sestric R, Ignatia L, Levin D, German JB, Gillies LA, Almada LAG, Boundy-Mills KL (2013) Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeast species. Biores Technol 144:360–369

    CAS  Article  Google Scholar 

  5. 5.

    Arous F, Azabou S, Triantaphyllidou IE, Aggelis G, Jaouani A, Nasri M, Mechichi T (2016) Newly isolated yeasts from Tunisian microhabitats: lipid accumulation and fatty acid composition. Eng Life Sci 17:226–236

    Article  Google Scholar 

  6. 6.

    Ryu BG, Kim J, Kim K, Choi YE, Han JI, Yang JW (2013) High-cell-density cultivation of oleaginous yeast Cryptococcus curvatus for biodiesel production using organic waste from the brewery industry. Biores Technol 135:357–364

    CAS  Article  Google Scholar 

  7. 7.

    Welch JW, Burlingame AL (1973) Very long-chain fatty acids in yeast. J Bacteriol 115:464–466

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Blagovic B, Rupcic J, Mesaric M, Maric V (2005) Lipid analysis of the plasma membrane and mitochondria of brewer’s yeast. Folia Microbiol 50:24–30

    CAS  Article  Google Scholar 

  9. 9.

    Bendova O, Richter V, Janderova B, Hausler J (1991) Identification of industrial yeast strains of Saccharomyces cerevisiae by fatty acid profiles. Appl Microbiol Biotechnol 35:810–812

    CAS  Article  Google Scholar 

  10. 10.

    Nagao A, Yamazaki M (1983) Lipid composition of microbial cells for animal feed. J Jpn Oil Chem Soc 32:207–212

    CAS  Article  Google Scholar 

  11. 11.

    Donaldson MS (2000) Metabolic vitamin B12 status on a mostly raw vegan diet with follow-up using tablets, nutritional yeast, or probiotic supplements. Ann Nutr Metab 44:229–234

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Li P, Gatlin DM III (2004) Dietary brewers yeast and the prebiotic Grobiotic™AE influence growth performance, immune responses and resistance of hybrid striped bass (Morone chrysops × M. saxatilis) to Streptococcus iniae infection. Aquaculture 231:445–456

    Article  Google Scholar 

  13. 13.

    Yokoyama K, Saitoh S, Ishida M, Yamakawa Y, Nakamura K, Inoue K, Taguchi R, Tokumura A, Nishijima M, Yanagida M, Setaka M (2001) Very-long-chain fatty acid-containing phospholipids accumulate in fatty acid synthase temperature-sensitive mutant strains of the fission yeast Schizosaccharomyces pombe fas2/lsd1. Biochem Biophys Acta 1532:223–233

    CAS  PubMed  Google Scholar 

  14. 14.

    Schneiter R, Brugger B, Amann CM, Prestwich GD, Epand RF, Zellnig G, Wieland FT, Epand RM (2004) Identification and biophysical characterization of a very-long-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular membranes. Biochem J 381:941–949

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Rezanka T, Matoulkova D, Kolouchova I, Masak J, Sigler K (2013) Brewer’s yeast as a new source of palmitoleic acid—analysis of triacylglycerols by LC–MS. ‎J Am Oil Chem Soc 90:1327–1342

    CAS  Article  Google Scholar 

  16. 16.

    Yu T, Zhou YJ, Wenning L, Liu Q, Krivoruchko A, Siewers V, Nielsen J, David F (2017) Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals. Nat Commun 8:1–10

    Article  Google Scholar 

  17. 17.

    Wenning L, Yu T, David F, Nielsen J, Siewers V (2016) Establishing very long-chain fatty alcohol and wax ester biosynthesis in Saccharomyces cerevisiae. Biotechnol Bioeng 114:1025–1035

    Article  PubMed  Google Scholar 

  18. 18.

    Rossler H, Rieck C, Delong T, Hoja U, Schweizer E (2003) Functional differentiation and selective inactivation of multiple Saccharomyces cerevisiae genes involved in very-long-chain fatty acid synthesis. Mol Genet Genom 269:290–298

    CAS  Google Scholar 

  19. 19.

    Toke DA, Martin CE (1996) Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. J Biol Chem 271:18413–18422

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Prasitchoke P, Kaneko Y, Bamba T, Fukusaki E, Kobayashi A, Harashima S (2007) Identification and characterization of a very long-chain fatty acid elongase gene in the methylotrophic yeast, Hansenula polymorpha. Gene 391:16–25

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Dittrich F, Zajonc D, Huhne K, Hoja U, Ekici A, Greiner E, Klein H, Hofmann J, Bessoule JJ, Sperling P, Schweizer E (1998) Fatty acid elongation in yeast–biochemical characteristics of the enzyme system and isolation of elongation-defective mutants. Eur J Biochem 252:477–485

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Aveldano MI, Bazan NG (1983) Molecular species of phosphatidylcholine, -ethanolamine, -serine, and -inositol in microsomal and photoreceptor membranes of bovine retina. J Lipid Res 24:620–627

    CAS  PubMed  Google Scholar 

  23. 23.

    Lindberg L, Santos AX, Riezman H, Olsson L, Bettiga M (2013) Lipidomic profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii reveals critical changes in lipid composition in response to acetic acid stress. PLoS One 8:e73936

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Tarasov K, Stefanko A, Casanovas A, Surma MA, Berzina Z, Hannibal-Bach HK, Ekroos K, Ejsing CS (2014) High-content screening of yeast mutant libraries by shotgun lipidomics. Mol BioSyst 10:1364–1376

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Rezanka T, Kolouchova I, Sigler K (2016) Lipidomic analysis of psychrophilic yeasts cultivated at different temperatures. Biochem Biophys Acta 1861:1634–1642

    CAS  PubMed  Google Scholar 

  26. 26.

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

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Rezanka T, Kolouchova I, Sigler K (2015) Precursor directed biosynthesis of odd-numbered fatty acids by different yeasts. Folia Microbiol 60:457–464

    CAS  Article  Google Scholar 

  28. 28.

    Christie WW and Han X (2010) Lipid analysis (fourth edition), isolation, separation, identification and lipidomic analysis. The Oily Press, Bridgwater, England (eBook ISBN: 9780857097866)

    Google Scholar 

  29. 29.

    Vancura A, Rezanka T, Marslalek J, Melzoch K, Basarová G, Kristan V (1988) Metabolism of l-threonine and fatty acids and tylosin biosynthesis in Streptomyces fradiae. FEMS Microbiol Lett 49:411–415

    CAS  Article  Google Scholar 

  30. 30.

    Dembitsky VM, Rezanka T, Bychek IA, Shustov MV (1991) Identification of fatty-acids from Cladonia lichens. Phytochemistry 30:4015–4018

    Article  Google Scholar 

  31. 31.

    Dodds ED, McCoy MR, Rea LD, Kennish JM (2005) Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact mass spectrometry. Lipids 40:419–428

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Francis GW (1981) Alkylthiolation for the determination of double-bond position in unsaturated fatty acid esters. Chem Phys Lipids 29:369–374

    CAS  Article  Google Scholar 

  33. 33.

    Casanovas A, Sprenger R, Tarasov K, Ruckerbauer D, Hannibal-Bach H, Zanghellini J, Jensen O, Ejsing C (2015) Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism. Chem Biol 22:412–425

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Klose C, Surma MA, Gerl MJ, Meyenhofer F, Shevchenko A, Simons K (2012) Flexibility of a eukaryotic lipidome—insights from yeast lipidomics. PLoS ONE 7:e35063

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Ejsing CS, Sampaio JL, Surendranath V, Duchoslav E, Ekroos K, Klemm RW, Simons K, Shevchenko A (2009) Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc Natl Acad Sci USA 106:2136–2141

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Sec P, Garaiova M, Gajdos P, Certik M, Griac P, Hapala I, Holic R (2015) Baker’s yeast deficient in storage lipid synthesis uses cis-vaccenic acid to reduce unsaturated fatty acid toxicity. Lipids 50:621–630

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Southwell-Keely PT, Lynen F (1974) The mechanism of production of 11-octadecenoic acid (vaccenic acid) by yeast. Biochim Biophys Acta 337:22–28

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Augustyn OPH, Kock JLF (1989) Differentiation of yeast species, and strains within a species, by cellular fatty acid analysis. 1. Application of an adapted technique to differentiate between strains of Saccharomyces cerevisiae. J Microbiol Methods 10:9–23

    CAS  Article  Google Scholar 

  39. 39.

    Papanikolaou S, Aggelis G (2011) Lipids of oleaginous yeasts. Part I: biochemistry of single cell oil production. Eur J Lipid Sci Technol 113:1031–1051

    CAS  Article  Google Scholar 

  40. 40.

    Hein EM, Hayen H (2012) Comparative lipidomic profiling of S. cerevisiae and four other hemiascomycetous yeasts. Metabolites 2:254–267

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    da Silveira Dos Santos AX, Riezman I, Aguilera-Romero MA, David F, Piccolis M, Loewith R, Schaad O, Riezman H (2014) Systematic lipidomic analysis of yeast protein kinase and phosphatase mutants reveals novel insights into regulation of lipid homeostasis. Mol Biol Cell 25:3234–3246

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The research was supported by the Czech Science Foundation (GACR) project P503 17-00027S and by Institutional Research Concepts RVO61388971.

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Correspondence to Tomáš Řezanka.

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Řezanka, T., Kolouchová, I., Gharwalová, L. et al. Identification and Characterization of Phospholipids with Very Long Chain Fatty Acids in Brewer’s Yeast. Lipids 52, 1007–1017 (2017). https://doi.org/10.1007/s11745-017-4294-6

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Keywords

  • Yeast
  • Very long chain fatty acids
  • Negative electrospray ionization
  • Phospholipids