Advertisement

Production of Lipids and Proteome Variation in a Chilean Thraustochytrium striatum Strain Cultured under Different Growth Conditions

  • Carolina Shene
  • Marcelo Garcés
  • Daniela Vergara
  • Jhonatan Peña
  • Stéphane Claverol
  • Mónica Rubilar
  • Allison Leyton
Original Article
  • 148 Downloads

Abstract

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L−1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8–6%) and eicosapentaenoic acid (0.2–21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L−1 and 66 mg L−1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b – MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.

Keywords

Culture conditions Docosahexaenoic acid Polyunsaturated fatty acids Microbial lipids Thraustochytrium 

Notes

Acknowledgments

This research was supported by the Centre for Biotechnology and Bioengineering (CeBiB) FB-0001 and partially supported by the supercomputing infrastructure of the NLHPC (ECM-02; Powered@NLHPC). Parts of the experiments (Galaxy server) were performed at the Bordeaux Bioinformatic Center (CBIB). The authors thank the Dirección de Investigación at Universidad de La Frontera for economic support provided through GAP.

Supplementary material

10126_2018_9863_MOESM1_ESM.docx (484 kb)
ESM 1 (DOCX 484 kb)
10126_2018_9863_MOESM2_ESM.xlsx (27 kb)
ESM 2 (XLSX 27 kb)

References

  1. Afgan E, Baker D, van der Beek M, Blankerberg D, Bouvier D, Cech M, Chilton J, Clements D, Coraor N, Eberhard C, Grüning B, Guerler A, Hillman-Jackson J, Von Kuster G, Rasche E, Soranzo N, Turaga N, Taylor J, Nekrutenko A, Goecks J (2016) The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res 44:W3–W10CrossRefGoogle Scholar
  2. Aki T, Hachida K, Yoshinaga M, Katai Y, Yamasaki T, Kawamoto S, Kakizono T, Maoka T, Shigeta S, Suzuki O, Ono K (2003) Thraustochytrid as a potential source of carotenoids. J Am Oil Chem Soc 80:789–794CrossRefGoogle Scholar
  3. Bailey RB, DiMasi D, Hansen JM, Mirrasoul PJ, Ruecker CM, Veeder GT, Kaneko T, Barclay WR (2003) Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors. US Patent 6607900Google Scholar
  4. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917CrossRefGoogle Scholar
  5. Burja AM, Radianingtyas H, Windust A, Barrow CJ (2006) Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production. Appl Microbiol Biotechnol 72:1161–1169CrossRefGoogle Scholar
  6. Chang G, Luo Z, Gu S, Wu Q, Chang M, Wang X (2013) Fatty acid shifts and metabolic activity changes of Schizochytrium sp. S31 cultured on glycerol. Bioresour Technol 142:255–260CrossRefGoogle Scholar
  7. Chi Z, Liu Y, Frear C, Chen S (2009) Study of a two-stage growth of DHA-producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Appl Microbiol Biotechnol 81:1141–1148CrossRefGoogle Scholar
  8. Connor W (2000) Importance of n-3 fatty acids in health and disease. Am J Clin Nutr 71:171S–175SCrossRefGoogle Scholar
  9. Fan KW, Vrijmoed LLP, Jones EBG (2002) Physiological studies of subtropical mangrove thraustochytrids. Bot Mar 45:50–57CrossRefGoogle Scholar
  10. Gaertner A (1968) Eine methode des nachweises niederer mit pollen koderbarer pilze im meerwasser und im sediment. Veroff Inst Meeresforch Bremer Sonderb 3:75–92Google Scholar
  11. Ganuza E, Izquierdo MS (2007) Lipid accumulation in Schizochytrium G13/2S produced in continuous culture. Appl Microbiol Biotechnol 76:985–990CrossRefGoogle Scholar
  12. Garcés M, Claverol S, Alvear C, Rabert C, Bravo L (2014) Desiccation tolerance of Hymenophyllacea filmy ferns is mediated by constitutive and non-inducible cellular mechanisms. C R Biol 337:235–243CrossRefGoogle Scholar
  13. García-Ochoa F, Gomez E (2009) Bioreactor scale-up and oxygen transfer rate in microbial processes: an overview. Biotechnol Adv 27:153–176CrossRefGoogle Scholar
  14. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat Biotechnol 29:644–652CrossRefGoogle Scholar
  15. Gupta A, Barrow C, Puri M (2012) Omega-3 biotechnology. Thraustochytrids as a novel source of omega-3 oils. Biotechnol Adv 30:1733–1745CrossRefGoogle Scholar
  16. Horrocks LA, Yeo YK (1999) Health benefits of docosahexaenoic acid DHA. Pharmacol Res 40:211–225CrossRefGoogle Scholar
  17. Jain R, Raghukumar S, Tharanathan R, Bhosle NB (2005) Extracellular polysaccharide production by thraustochytrid protists. Mar Biotechnol 7:184–192CrossRefGoogle Scholar
  18. Jakobsen AN, Aasen IM, Josefsen KD, Strøm AR (2008) Accumulation of docosahexaenoic acid-rich lipid in thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P starvation and O2 limitation. Appl Microbiol Biotechnol 80:297–306CrossRefGoogle Scholar
  19. Jeh E-J, Kumaran RS, Hur B-K (2008) Lipid body formation by Thraustochytrium aureum (ATCC 34304) in response to cell age. Korean J Chem Eng 25:1103–1109CrossRefGoogle Scholar
  20. Ji XJ, Mo KQ, Ren LJ, Li GL, Huang JZ, Huang H (2015) Genome sequence of Schizochytrium sp. CCTCC M209059, an effective producer of docosahexaenoic acid-rich lipids. Genome Announc 3:e00819–e00815CrossRefGoogle Scholar
  21. Kidd PM (2007) Omega-3 DHA and EPA for cognition, behavior, and mood: clinical findings and structural-functional synergies with cell membrane phospholipids. Altern Med Rev 12:207–227PubMedGoogle Scholar
  22. Lewis TE, Nichols PD, McMeekin TA (1999) The biotechnological potential of thraustochytrids. Mar Biotechnol 1:580–587CrossRefGoogle Scholar
  23. Li ZY, Ward OP (1994) Production of docosahexaenoic acid by Thraustochytrium roseum. J Ind Microbiol 13:238–241CrossRefGoogle Scholar
  24. Ma Z, Tan Y, Cui G, Feng Y, Cui Q, Song X (2015) Transcriptome and gene expression analysis of DHA producer Aurantiochytrium under low temperature conditions. Sci Rep 5:14446CrossRefGoogle Scholar
  25. Ma Z, Tian M, Tan Y, Cui G, Feng Y, Cui Q, Song X (2017) Response mechanism of docosahexanoic producer Aurantiochytrium under cold stress. Algal Res 25:191–199CrossRefGoogle Scholar
  26. Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M (2009) Biodiesel production from oleaginous microorganisms. Renew Energy 34:1–5CrossRefGoogle Scholar
  27. Metz JG, Roessler P, Facciotti D, Levering C, Dittrich F, Lassner M, Valentine R, Lardizabal K, Domergue F, Yamada A, Yazawa K, Knauf V, Browse J (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293:290–293CrossRefGoogle Scholar
  28. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  29. Miller MR, Nichols PD, Carter CG (2007) Replacement of fish oil with thraustochytrid Schizochytrium sp. L oil in Atlantic salmon parr (Salmo salar L) diets. Comp Biochem Physiol A Mol Integr Physiol 148:382–392CrossRefGoogle Scholar
  30. Mo C, Douek J, Rinkevich B (2002) Development of a PCR strategy for thraustochytrid identification based on 18S rDNA sequence. Mar Biol 140:883–889CrossRefGoogle Scholar
  31. Plourde M, Cunnane SC (2007) Extremely limited synthesis of long chain polyunsaturates in adults: implications for their dietary essentiality and use as supplements. Appl Physiol Nutr Metab 32:619–634CrossRefGoogle Scholar
  32. Qiu X (2003) Biosynthesis of docosahexaenoic acid (DHA, 22:6-4, 7,10,13,16,19): two distinct pathways. Prostaglandins Leukot Essent Fat Acids 68:181–186CrossRefGoogle Scholar
  33. Quilodrán B, Hinzpeter I, Quiroz A, Shene C (2009) Evaluation of liquid residues from beer and potato processing for the production of docosahexaenoic acid (C22:6n-3, DHA) by native thraustochytrid strains. World J Microbiol Biotechnol 25:2121–2128CrossRefGoogle Scholar
  34. Ramagli LS, Rodríguez LV (1985) Quantitation of microgram amounts of protein in two-dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 6:559–563CrossRefGoogle Scholar
  35. Rao AV, Rao LG (2007) Carotenoids and human health. Pharmacol Res 55:207–216CrossRefGoogle Scholar
  36. Rasmussen RS, Nettleton J, Morrissey MT (2005) A review of mercury in seafood: special focus on tuna. J Aquat Food Prod Technol 14:71–100CrossRefGoogle Scholar
  37. Robles Medina A, Molina Grima E, Giménez Giménez A, Ibáñez González MJ (1998) Downstream processing of algal polyunsaturated fatty acids. Biotechnol Adv 16:517–580CrossRefGoogle Scholar
  38. Rubin E, Tanguy A, Perrigault M, Espinosa EP, Allam B (2014) Characterization of the transcriptome and temperature-induced differential gene expression in QPX, the thraustochytrid parasite of hard clams. BMC Genomics 15:245CrossRefGoogle Scholar
  39. Shene C, Leyton A, Rubilar M, Pinelo M, Acevedo F, Morales E (2013) Production of lipids and docosahexasaenoic acid (DHA) by a native Thraustochytrium strain. Eur J Lipid Sci Technol 115:890–900CrossRefGoogle Scholar
  40. Taoka Y, Nagano N, Okita Y, Izumida H, Sugimoto S, Hayashi M (2009) Extracellular enzymes produced by marine eukaryotes, thraustochytrids. Biosci Biotechnol Biochem 73:180–182CrossRefGoogle Scholar
  41. Yang JD, Wang NS (1992) Oxygen mass transfer enhancement via fermentor headspace pressurization. Biotechnol Prog 8:244–251CrossRefGoogle Scholar
  42. Yokoyama R, Honda D (2007) Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience 48:199–211CrossRefGoogle Scholar
  43. Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comp Biol 7:203–214CrossRefGoogle Scholar
  44. Zhang L, Zhao H, Lai Y, Wu J, Chen H (2013) Improving docosahexaenoic acid productivity of Schizochytrium sp. by a two-stage AEMR/shake mixed culture mode. Bioresour Technol 142:719–722CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Chemical Engineering and Center of Food Biotechnology and Bioseparations, BIORENUniversidad de La FronteraTemucoChile
  2. 2.Centre for Biotechnology and Bioengineering (CeBiB)Universidad de La FronteraTemucoChile
  3. 3.Center of Plant, Soil Interaction and Natural Resources Biotechnology. BIORENUniversidad de La FronteraTemucoChile
  4. 4.Plateforme Protéome, Centre de Génomique Fonctionnelle BordeauxUniversité Bordeaux SegalenBordeaux CedexFrance

Personalised recommendations