Bioprocess and Biosystems Engineering

, Volume 42, Issue 1, pp 71–81 | Cite as

Fermentation performance and metabolomic analysis of an engineered high-yield PUFA-producing strain of Schizochytrium sp.

  • Lingjun Geng
  • Shenglan Chen
  • Xiaoman Sun
  • Xuechao Hu
  • Xiaojun Ji
  • He Huang
  • Lujing RenEmail author
Research Paper


The ω-3/long-chain polyunsaturated fatty acids (LC-PUFAs) play an important role in human health, but they cannot be synthesized in sufficient amounts by the human body. In a previous study, we obtained an engineered Schizochytrium sp. strain (HX-RS) by exchanging the acyltransferase (AT) gene, and it was able to co-produce docosahexaenoic acid and eicosapentaenoic acid. To investigate the mechanism underlying the increase of PUFA content in HX-RS, the discrepancies of fermentation performance, key enzyme activities and intracellular metabolites between HX-RS and its wild-type parent strain (WTS) were analyzed via fed-batch fermentation in 5-L bioreactors. The results showed that the cell dry weight (CDW) of HX-RS was higher than that of the WTS. Metabolomics combined with multivariate analysis showed that 4-aminobutyric acid, proline and glutamine are potential biomarkers associated with cell growth and lipid accumulation of HX-RS. Additionally, the shift of metabolic flux including a decrease of glyceraldehyde-3-phosphate content, high flux from pyruvate to acetyl-CoA, and a highly active glycolysis pathway were also found to be closely related to the high PUFA yield of the engineered strain. These findings provide new insights into the effects of exogenous AT gene expression on cell proliferation and fatty acid metabolism.


Polyunsaturated fatty acids Schizochytrium sp. Fed batch Metabolomics 



This work was financially supported by the Outstanding Youth Foundation of Jiangsu Natural Science Foundation (BK20160092), the National Natural Science Foundation of China (No. 21878151), the Program for Innovative Research Teams in Universities of Jiangsu Province (2015) and Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTE1829).


  1. 1.
    Lee JH, O’Keefe JH, Lavie CJ, Marchioli R, Harris WS (2008) Omega-3 fatty acids for cardioprotection. Mayo Clin Proc 83, 324–332CrossRefGoogle Scholar
  2. 2.
    Brett MT, Müller-Navarra DC, Persson J (2009) Crustacean zooplankton fatty acid composition. In: Martin Kainz Michael T, Brett Michael T (eds) Lipids in aquatic ecosystems, Arts. Springer, New York, US, pp 115–146CrossRefGoogle Scholar
  3. 3.
    Costa LG, Manzo L (2006) Contaminants in fish: risk–benefit considerations. Toxicol Lett 164:367–374Google Scholar
  4. 4.
    Barclay W, Weaver C, Metz J, Hansen J, Cohen Z, Ratledge C (2010) Development of a docosahexaenoic acid production technology using Schizochytrium: historical perspective and update. In: Cohen Z, Ratledge C (eds) Single cell oils, Academic Press and AOCS Press. Urbana, USA, pp 75–96CrossRefGoogle Scholar
  5. 5.
    Kiy T, Rüsing M, Fabritius D (2005) Production of docosahexaenoic acid by the Marine microalga, Ulkenia sp. In: Cohen Z, Ratledge C (eds) Single cell oils. AOCS, Champaign, pp 36–52Google Scholar
  6. 6.
    Wynn JP, Behrens PW, Sundararajan A, Hansen J, Apt K (2005) Production of single cell oils by dinoflagellates. In: Cohen Z, Ratledge C (eds) Single cell oils. AOCS, ChampaignGoogle Scholar
  7. 7.
    . Ren LJ, Chen SL, Geng LJ, Ji XJ, Xu X, Song P, Gao S, Huang H (2018) Exploring the function of acyltransferase and domain replacement in order to change the polyunsaturated fatty acid profile of Schizochytrium sp. Algal Res 29:193–201CrossRefGoogle Scholar
  8. 8.
    Heath C, Kiss R (2007) Cell culture process development: advances in process engineering. Biotechnol Prog 23:46–51CrossRefGoogle Scholar
  9. 9.
    Ren LJ, Ji XJ, Huang H, Qu L, Feng Y, Tong QQ, Ouyang PK (2010) Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp. Appl Microbiol Biotechnol 87:1649–1656CrossRefGoogle Scholar
  10. 10.
    Xia JM, Wu XJ, Yuan YJ (2007) Integration of wavelet transform with PCA and ANN for metabolomics data-mining. Metabolomics 3:531–537CrossRefGoogle Scholar
  11. 11.
    Li J, Ren LJ, Sun GN, Qu L, Huang H (2013) Comparative metabolomics analysis of docosahexaenoic acid fermentation processes by Schizochytrium sp. under different oxygen availability conditions. Omics-a J Integr Biol 17:269–281CrossRefGoogle Scholar
  12. 12.
    Yu XJ, Sun J, Sun YQ, Zheng JY, Wang Z (2016) Metabolomics analysis of phytohormone gibberellin improving lipid and DHA accumulation in Aurantiochytrium sp. Biochem Eng J 112:258–268CrossRefGoogle Scholar
  13. 13.
    Hasunuma T, Sanda T, Yamada R, Yoshimura K, Ishii J, Kondo A (2011) Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Microb Cell Fact 10:2CrossRefGoogle Scholar
  14. 14.
    Yu S, Huang D, Wen J, Li S, Chen Y, Jia X (2012) Metabolic profiling of a Rhizopus oryzae fumaric acid production mutant generated by femtosecond laser irradiation. Biores Technol 114:610–615CrossRefGoogle Scholar
  15. 15.
    Ren L, Hu X, Zhao X, Chen S, Wu Y, Li D, Yu Y, Geng L, Ji X, Huang H (2017) Transcriptomic analysis of the regulation of lipid fraction migration and fatty acid biosynthesis in Schizochytrium sp. Sci Rep 7:3562–3571CrossRefGoogle Scholar
  16. 16.
    Ren LJ, Sun GN, Ji XJ, Hu XC, Huang H (2014) Compositional shift in lipid fractions during lipid accumulation and turnover in Schizochytrium sp. Bioresour Technol 157:107–113CrossRefGoogle Scholar
  17. 17.
    Ding MZ, Zhou X, Yuan YJ (2010) Metabolome profiling reveals adaptive evolution of Saccharomyces cerevisiae during repeated vacuum fermentations. Metabolomics 6:42–55CrossRefGoogle Scholar
  18. 18.
    Strelkov S, Von EM, Schomburg D (2004) Comprehensive analysis of metabolites in Corynebacterium glutamicum by gas chromatography/mass spectrometry. Biol Chem 758:853–861Google Scholar
  19. 19.
    Takeda Y, Suzuki F, Inoue H (1969) [27] ATP citrate lyase (citrate-cleavage enzyme). Methods Enzymol 13:153–160CrossRefGoogle Scholar
  20. 20.
    Wynn JP, Ratledge C (1997) Malic Enzyme is a major source of NADPH for lipid accumulation by Aspergillus Nidulans. Microbiology 143:253–257CrossRefGoogle Scholar
  21. 21.
    Ling X, Guo J, Liu X, Zhang X, Wang N, Lu Y, Ng IS (2015) Impact of carbon and nitrogen feeding strategy on high production of biomass and docosahexaenoic acid (DHA) by Schizochytrium sp. LU310. Biores Technol 184:139–147CrossRefGoogle Scholar
  22. 22.
    Yan J, Cheng R, Lin X, You S, Li K, Rong H, Ma Y (2013) Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium. Appl Microbiol Biotechnol 97:1933–1939CrossRefGoogle Scholar
  23. 23.
    Los DA, Mironov KS, Allakhverdiev SI (2013) Regulatory role of membrane fluidity in gene expression and physiological functions. Photosynth Res 116:489–509CrossRefGoogle Scholar
  24. 24.
    Papanikolaou S, Sarantou S, Komaitis M, G (2004) Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media. J Appl Microbiol 97:867–875CrossRefGoogle Scholar
  25. 25.
    Nikawa JI, Yamashita S (1997) Phosphatidylinositol synthase from yeast 1. Biochim Et Biophys Acta 1348:173CrossRefGoogle Scholar
  26. 26.
    Song X, Tan Y, Liu Y, Zhang J, Liu G, Feng Y, Cui Q (2013) Different impacts of short-chain fatty acids on saturated and polyunsaturated fatty acid biosynthesis in Aurantiochytrium sp. SD116. J Agric Food Chem 61:9876–9881CrossRefGoogle Scholar
  27. 27.
    Ratledge C (2004) Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie 86:807–815CrossRefGoogle Scholar
  28. 28.
    Chen Z, Zheng Z, Yi C, Wang F, Niu Y, Li H (2016) Intracellular metabolic changes in Saccharomyces cerevisiae and promotion of ethanol tolerance during the bioethanol fermentation process. Rsc Ad 6:105046–105055CrossRefGoogle Scholar
  29. 29.
    Yu XJ, Sun J, Zheng JY, Sun YQ, Wang Z (2016) Metabolomics analysis reveals 6-benzylaminopurine as a stimulator for improving lipid and DHA accumulation of Aurantiochytrium sp. J Chem Technol Biotechnol 91:1199–1207CrossRefGoogle Scholar
  30. 30.
    Cui M, Lin Y, Zu Y, Efferth T, Li D, Tang Z (2015) Ethylene increases accumulation of compatible solutes and decreases oxidative stress to improve plant tolerance to water stress in Arabidopsis. J Plant Biol 58:193–201CrossRefGoogle Scholar
  31. 31.
    Berney M, Weimar MR, Heikal A, Cook GM (2012) Regulation of proline metabolism in mycobacteria and its role in carbon metabolism under hypoxia. Mol Microbiol 84:664–681CrossRefGoogle Scholar
  32. 32.
    Balázs R, Machiyama Y, Hammond BJ, Julian T, Richter D (1970) The operation of the gamma-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro. Biochem J 116:445CrossRefGoogle Scholar
  33. 33.
    Kumar S, Punekar NS (1997) The metabolism of 4-aminobutyrate (GABA) in fungi. Mycol Res 101:403–409CrossRefGoogle Scholar
  34. 34.
    Cheng JS, Cui SF, Ding MZ, Yuan YJ (2013) Insights into the roles of exogenous glutamate and proline in improving streptolydigin production of Streptomyces lydicus with metabolomic analysis. J Ind Microbiol Biotechnol 40:1303–1314CrossRefGoogle Scholar
  35. 35.
    Wang X, Jin M, Balan V, Jones AD, Li X, Li BZ, Dale BE, Yuan YJ (2014) Comparative metabolic profiling revealed limitations in xylose-fermenting yeast during co-fermentation of glucose and xylose in the presence of inhibitors. Biotechnol Bioeng 111:152–164CrossRefGoogle Scholar
  36. 36.
    Ren LJ, Feng Y, Li J, Qu L, Huang H (2013) Impact of phosphate concentration on docosahexaenoic acid production and related enzyme activities in fermentation of Schizochytrium sp. Bioprocess Biosyst Eng 36:1177–1183CrossRefGoogle Scholar
  37. 37.
    Ling X, Guo J, Zheng C, Ye C, Lu Y, Pan X, Chen Z, Ng IS (2015) Simple, effective protein extraction method and proteomics analysis from polyunsaturated fatty acids-producing micro-organisms. Bioprocess Biosyst Eng 38:2331–2341CrossRefGoogle Scholar
  38. 38.
    Siniossoglou S (2013) Phospholipid metabolism and nuclear function: roles of the lipin family of phosphatidic acid phosphatases. Biochim Biophys Acta 1831:575–581CrossRefGoogle Scholar
  39. 39.
    Yamashita S, Nikawa J (1997) Phosphatidylserine synthase from yeast. Biochim Et Biophys Acta 1348:228CrossRefGoogle Scholar
  40. 40.
    Ding MZ, Tian HC, Cheng JS, Yuan YJ (2009) Inoculum size-dependent interactive regulation of metabolism and stress response of Saccharomyces cerevisiae revealed by comparative metabolomics. J Biotechnol 144:279–286CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Lingjun Geng
    • 2
  • Shenglan Chen
    • 2
  • Xiaoman Sun
    • 2
  • Xuechao Hu
    • 1
    • 2
  • Xiaojun Ji
    • 1
    • 2
  • He Huang
    • 1
    • 3
    • 4
  • Lujing Ren
    • 1
    • 2
    Email author
  1. 1.Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM)NanjingChina
  2. 2.College of Biotechnology and Pharmaceutical EngineeringNanjing Tech UniversityNanjingPeople’s Republic of China
  3. 3.School of Pharmaceutical SciencesNanjing Tech UniversityNanjingPeople’s Republic of China
  4. 4.State Key Laboratory of Materials-Oriented Chemical EngineeringNanjing Tech UniversityNanjingPeople’s Republic of China

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