Applied Microbiology and Biotechnology

, Volume 97, Issue 7, pp 2859–2866 | Cite as

Growth condition optimization for docosahexaenoic acid (DHA) production by Moritella marina MP-1

  • Kumar B. Kautharapu
  • John Rathmacher
  • Laura R. Jarboe
Biotechnological products and process engineering

Abstract

The marine organism Moritella marina MP-1 produces the polyunsaturated fatty acid docosahexaenoic acid (DHA). While the basic metabolic pathway for DHA production in this organism has been identified, the impact of growth conditions on DHA production is largely unknown. This study examines the effect of supplemental carbon, nitrogen and salts, growth temperature and media composition and pH on DHA and biomass production and the fatty acid profile. The addition of supplemental nitrogen significantly increased the overall DHA titer via an increase in biomass production. Supplemental glucose or glycerol increased biomass production, but decreased the amount of DHA per biomass, resulting in no net change in the DHA titer. Acidification of the baseline media pH to 6.0 increased DHA per biomass. Changes in growth temperature or provision of supplemental sodium or magnesium chloride did not increase DHA titer. This organism was also shown to grow on defined minimal media. For both media types, glycerol enabled more DHA production per biomass than glucose. Combination of these growth findings into marine broth supplemented with glycerol, yeast extract, and tryptone at pH 6.0 resulted in a final titer of 82 ± 5 mg/L, a nearly eightfold increase relative to the titer of 11 ± 1 mg/L seen in the unsupplemented marine broth. The relative distribution of other fatty acids was relatively robust to growth condition, but the presence of glycerol resulted in a significant increase in myristic acid (C14:0) and decrease in palmitic acid (C16:0). In summary, DHA production by M. marina MP-1 can be increased more than fivefold by changing the growth media. Metabolic engineering of this organism to increase the amount of DHA produced per biomass could result in additional increases in titer.

Keywords

Moritella marina MP-1 Polyunsaturated fatty acids Membrane fluidity Polyketide Marine broth 

References

  1. Alder J, Watson R (2007) Fisheries globalization: fair trade or piracy? In: Taylor WW, Schechter MG, Wolfson LG (eds) Globalization: effects on fisheries resources. Cambridge University Press, New York, pp 47–74Google Scholar
  2. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8):911–917CrossRefGoogle Scholar
  3. Busch B, Hertweck C (2009) Evolution of metabolic diversity in polyketide-derived pyrones: using the non-colinear aureothin assembly line as a model system. Phytochem 70(15–16):1833–1840. doi:10.1016/j.phytochem.2009.05.022 CrossRefGoogle Scholar
  4. Calderone CT (2008) Isoprenoid-like alkylations in polyketide biosynthesis. Nat Prod Rep 25(5):845–853. doi:10.1039/b807243d CrossRefGoogle Scholar
  5. Campbell CD, Vederas JC (2012) Biosynthesis of lovastatin and related metabolites formed by fungal iterative PKS enzymes. Biopolym 93(9):755–763. doi:10.1002/bip.21428 CrossRefGoogle Scholar
  6. Chapkin RS, Seo JM, McMurray DN, Lupton JR (2008) Mechanisms by which docosahexaenoic acid and related fatty acids reduce colon cancer risk and inflammatory disorders of the intestine. Chem Phys Lipids 153(1):14–23. doi:10.1016/j.chemphyslip.2008.02.011 CrossRefGoogle Scholar
  7. Connor SL, Zhu N, Anderson GJ, Hamill D, Jaffe E, Carlson J, Connor WE (2000) Cheek cell phospholipids in human infants: a marker of docosahexaenoic and arachidonic acids in the diet, plasma, and red blood cells. Am J Clin Nutr 71(1):21–27Google Scholar
  8. Delong EF, Yayanos AA (1986) Biochemical function and ecological significance of novel bacterial lipids in deep-sea prokaryotes. Appl Environ Microbiol 51(4):730–737Google Scholar
  9. Hites RA, Foran JA, Carpenter DO, Hamilton MC, Knuth BA, Schwager SJ (2004) Global assessment of organic contaminants in farmed salmon. Science 303(5655):226–229CrossRefGoogle Scholar
  10. Hur Byung-Ki CD-W, Kim H-J, Park C-I, Suh H-J (2002) Effect of culture conditions on growth and production of docosahexaenoic acid (DHA) using Thraustochytrium aureum ATCC 34304. Biotechnol Bioprocess Eng 7(1):6CrossRefGoogle Scholar
  11. Jenke-Kodama H, Sandmann A, Muller R, Dittmann E (2005) Evolutionary implications of bacterial polyketide synthases. Mol Biol Evol 22(10):2027–2039. doi:10.1093/molbev/msi193 CrossRefGoogle Scholar
  12. Jenkins DJA, Sievenpiper JL, Pauly D, Sumaila UR, Kendall CWC, Mowat FM (2009) Are dietary recommendations for the use of fish oils sustainable? Can Med Assoc J 180(6):633–637. doi:10.1503/cmaj.081274 CrossRefGoogle Scholar
  13. Jeong YS, Song SK, Lee SJ, Hur BK (2006) The growth and EPA synthesis of Shewanella oneidensis MR-1 and expectation of EPA biosynthetic pathway. Biotechnol Bioprocess Eng 11(2):127–133CrossRefGoogle Scholar
  14. Kautharapu KB, Jarboe L (2012) Genome sequence of psychrophilic deep sea bacterium Moritella marina MP-1. J Bacteriol 194:6296–6297Google Scholar
  15. Kramer JK, Fellner V, Dugan ME, Sauer FD, Mossoba MM, Yurawecz MP (1997) Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids. Lipids 32(11):1219–1228CrossRefGoogle Scholar
  16. Lagarde M (2008) Docosahexaenoic acid: nutrient and precursor of bioactive lipids. Eur J Lipid Sci Technol 110(8):673–678. doi:10.1002/ejlt.200800087 CrossRefGoogle Scholar
  17. Lee SJ, Jeong YS, Kim DU, Se JW, Hur BK (2006) Eicosapentaenoic acid (EPA) biosynthetic gene cluster of Shewanella oneidensis MR-1: cloning, heterologous expression, and effects of temperature and glucose on the production of EPA in Escherichia coli. Biotechnol Bioprocess Eng 11(6):510–515CrossRefGoogle Scholar
  18. Li JJ, Huang CJ, Xie D (2008) Anti-obesity effects of conjugated linoleic acid, docosahexaenoic acid, and eicosapentaenoic acid. Mol Nutr Food Res 52(6):631–645. doi:10.1002/mnfr.200700399 CrossRefGoogle Scholar
  19. 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(5528):290–293CrossRefGoogle Scholar
  20. Morita N, Ichise N, Yumoto I, Yano Y, Ohgiya S, Okuyama H (2005a) Cultivation of microorganisms in the cultural medium made from squid internal organs and accumulation of polyunsaturated fatty acids in the cells. Biotechnol Lett 27(13):933–941. doi:10.1007/s10529-005-7187-3 CrossRefGoogle Scholar
  21. Morita N, Nishida T, Tanaka M, Yano Y, Okuyama H (2005b) Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria. Biotechnol Lett 27(6):389–393. doi:10.1007/s10529-005-1532-4 CrossRefGoogle Scholar
  22. Nagano N, Taoka Y, Honda D, Hayashi M (2009) Optimization of culture conditions for growth and docosahexaenoic acid production by a marine thraustochytrid, Aurantiochytrium limacinum mh0186. J Oleo Sci 58(12):623–628CrossRefGoogle Scholar
  23. Napier JA (2002) Plumbing the depths of PUFA biosynthesis: a novel polyketide synthase-like pathway from marine organisms. Trends Plant Sci 7(2):51–54CrossRefGoogle Scholar
  24. Nishida C, Uauy R, Kumanyika S, Shetty P (2004) The joint WHO/FAO expert consultation on diet, nutrition and the prevention of chronic diseases: process, product and policy implications. Public Health Nutr 7(1A):245–250. doi:10.1079/PHN2003592 Google Scholar
  25. Nishida T, Hori R, Morita N, Okuyama H (2010) Membrane eicosapentaenoic acid is involved in the hydrophobicity of bacterial cells and affects the entry of hydrophilic and hydrophobic compounds. FEMS Microbiol Lett 306:91–96. doi:10.111/j.1574-6968.2010.01943x CrossRefGoogle Scholar
  26. Shen B (2003) Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr Opin Chem Biol 7(2):285–295. doi:10.1016/s1367-5931(03)00020-6 CrossRefGoogle Scholar
  27. Siddiqui RA, Harvey K, Stillwell W (2008) Anticancer properties of oxidation products of docosahexaenoic acid. Chem Phys Lipids 153(1):47–56. doi:10.1016/j.chemphyslip.2008.02.009 CrossRefGoogle Scholar
  28. Stevens PJC, Baker EA, Anderson NH (1988) Factors affecting the foliar absorption and redistribution of pesticides. 2. Physicochemical properties of the active ingredient and the role of the surfactant. Pestic Sci 24(1):31–53CrossRefGoogle Scholar
  29. Usui K, Hiraki T, Kawamoto J, Kurihara T, Nogi Y, Kato C, Abe F (2012) Eicosapentaenoic acid plays a role in stabilizing dynamic membrane structure in the deep-sea piezophile Shewanella violacea: a study employing high-pressure time-resolved fluorescence anisotropy measurement. Biochim Biophys Acta 1818:574–583. doi:10.1016/j.bbamem.2011.10.010 CrossRefGoogle Scholar
  30. Valenzuela A (2009) Docosahexaenoic acid (DHA), an essential fatty acid for the proper functioning of neuronal cells: their role in mood disorders. Grasas Y Aceites 60(2):203–212. doi:10.3989/gya.085208 CrossRefGoogle Scholar
  31. Wall R, Ross RP, Fitzgerald GF, Stanton C (2010) Fatty acids from fish: the anti-inflammatory potential of long-chain omega-3 fatty acids. Nutr Rev 68(5):280–289. doi:10.1111/j.1753-4887.2010.00287.x CrossRefGoogle Scholar
  32. Wang F, Xiao X, Ou HY, Gai YB, Wang FP (2009) Role and regulation of fatty acid biosynthesis in the response of Shewanella piezotolerans WP3 to different temperatures and pressures. J Bacteriol 191(8):2574–2584. doi:10.1128/jb.00498-08 CrossRefGoogle Scholar
  33. Wanner BL (1994) Gene expression in bacteria using TnphoA and TnphoA’ elements to make and switch phoA gene, lacZ (op) and lacZ (pr) fusions. In: Adolph KW (ed) Methods Mol Genet. vol 3. Academic, Orlando, pp 291–310Google Scholar
  34. Wolosin JM, Ginsburg H, Lieb WR, Stein WD (1978) Diffusion within egg lecithin bilayers resembles that within soft polymers. J Gen Physiol 71(1):92–100CrossRefGoogle Scholar
  35. Yang Ge LY, Gao M (2011) Effects of pH and aeration on the production of docosahexaenoic acid by Thraustochytrium Aureum in controlled batch fermentor cultures. Adv Mater Res 5:183–185. doi:10.4028/www.scientific.net/AMR.183-185.50 Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Kumar B. Kautharapu
    • 1
  • John Rathmacher
    • 2
  • Laura R. Jarboe
    • 1
  1. 1.Chemical and Biological EngineeringIowa State UniversityAmesUSA
  2. 2.Metabolic Technologies IncAmesUSA

Personalised recommendations