DOI QR코드

DOI QR Code

Fermentation Process Development of Recombinant Hansenula polymorpha for Gamma-Linolenic Acid Production

  • Khongto, B. (School of Bioresources and Technology, King Mongkut's University of Technology Thonburi) ;
  • Laoteng, K. (Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology, National Sciences and Technology Development Agency at King Mongkut's University of Technology Thonburi) ;
  • Tongta, A. (School of Bioresources and Technology, King Mongkut's University of Technology Thonburi)
  • Received : 2010.03.01
  • Accepted : 2010.08.12
  • Published : 2010.11.28

Abstract

Development of the strain and the fermentation process of Hansenula polymorpha was implemented for the production of ${\gamma}$-linolenic acid ($GLA,\;C18:3{\Delta}^{6,9,12}$), an n-6 polyunsaturated fatty acid (PUFA) that has been reported to possess a number of health benefits. The mutated ${\Delta}^6$-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without the utilization of methanol, a high-cell-density culture of the yeast recombinant carrying the ${\Delta}^6$-desaturase gene was then achieved by fed-batch fermentation under glycerol-limited conditions. As a result, high levels of the ${\Delta}^6$-desaturated products, octadecadienoic acid ($C18:2{\Delta}^{6,9}$), GLA, and stearidonic acid ($C18:4{\Delta}^{6,9,12,15}$), were accumulated under the derepression conditions. The GLA production was also optimized by adjusting the specific growth rate. The results show that the specific growth rate affected both the lipid content and the fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates tested, the highest GLA concentration of 697 mg/l was obtained in the culture with a specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor ${\Delta}^6$-desaturase gene was similar to that of blackcurrant oil, with both containing similar proportions of n-3 and n-6 essential fatty acids.

Keywords

References

  1. Cahoon, E. B., J. M. Shockey, C. R. Dietrich, S. K. Gidda, R. T. Mullen, and J. M. Dyer. 2007. Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: Solving bottlenecks in fatty acid flux. Curr. Opin. Plant Biol. 10: 236-244. https://doi.org/10.1016/j.pbi.2007.04.005
  2. Castillo, M. L. R. D., G. Dobson, R. Brennan, and S. Gordon. 2004. Fatty acid content and juice characteristics in black currant (Ribes nigrum L.) genotypes. J. Agric. Food Chem. 52: 948-952. https://doi.org/10.1021/jf034950q
  3. Certik, M. and S. Shimizu. 1999. Biosynthesis and regulation of microbial polyunsaturated fatty acid production. J. Biosci. Bioeng. 87: 1-14. https://doi.org/10.1016/S1389-1723(99)80001-2
  4. d'Anjou, M. C. and A. J. Daugulis. 1997. A model-based feeding strategy for fed-batch fermentation of recombinant Pichia pastoris. Biotechnol. Tech. 11: 865-868. https://doi.org/10.1023/A:1018449930343
  5. Das, U. N. 2004. From bench to the clinic: $\gamma$-linolenic acid therapy of human gliomas. Prostaglandins Leukot. Essent. Fatty Acids 70: 539-552. https://doi.org/10.1016/j.plefa.2003.12.001
  6. Domínguez, A., E. Fermiñán, M. Sánchez, F. J. González, F. M. Pérez-Campo, S. García, et al. 1998. Non-conventional yeasts as hosts for heterologous protein production. Int. Microbiol. 1: 131-142.
  7. Fan, Y. Y. and R. S. Chapkin. 1998. Importance of dietary γ- linolenic acid in human health and nutrition. J. Nutr. 128: 1411-1414.
  8. Gellissen, G., G. Kunze, C. Gaillardin, J. M. Cregg, E. Berardi, M. Veenhuis, and I. van der Klei. 2005. New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - A comparison. FEMS Yeast. Res. 5: 1079-1096. https://doi.org/10.1016/j.femsyr.2005.06.004
  9. Gill, I. and R. Valivety. 1997. Polyunsaturated fatty acids, Part 1: Occurrence, biological activities and applications. Trends Biotechnol. 15: 401-409. https://doi.org/10.1016/S0167-7799(97)01076-7
  10. Guillou, H., S. D'andrea, V. Rioux, S. Jan, and P. Legrand. 2004. The surprising diversity of Δ6-desaturase substrates. Biochem. Soc. Trans. 32: 86-87. https://doi.org/10.1042/BST0320086
  11. Hartner, F. S. and A. Glieder. 2006. Regulation of methanol utilization pathway gene in yeasts. Microb. Cell Fact. 5: 39. https://doi.org/10.1186/1475-2859-5-39
  12. Jacob, Z. 1993. Yeast lipid biotechnology. Adv. Appl. Microbiol. 39: 185-212. https://doi.org/10.1016/S0065-2164(08)70596-3
  13. Kiel, J. A. K. W., I. Keizer-Gunnink, T. Krause, M. Komori, and M. Veenhuis. 1995. Heterologous complementation of peroxisome function in yeast: The Saccharomyces cerevisiae PAS3 gene restores peroxisome biogenesis in a Hansenula polymorpha per9 disruption mutant. FEBS Lett. 377: 434-438. https://doi.org/10.1016/0014-5793(95)01385-7
  14. Kim, S. K., Y. H. Noh, J.-R. Koo, and H. S. Yun. 2010. Effect of expression of genes in the sphingolipid synthesis pathways on the biosynthesis of ceramide in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 20: 356-362.
  15. Laoteng, K., R. Ruenwai, M. Tanticharoen, and S. Cheevadhanarak. 2005. Genetic modification of essential fatty acids biosynthesis in Hansenula polymorpha. FEMS Microbiol. Lett. 245: 169- 178. https://doi.org/10.1016/j.femsle.2005.03.006
  16. Lepage, G. and C. C. Roy. 1984. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J. Lipid Res. 25: 1391-1396.
  17. Mekhedov, S., O. M. de Ilarduya, and J. Ohlrogge. 2000. Toward a functional catalog of the plant genome: A survey of genes for lipid biosynthesis. Plant Physiol. 122: 389-401. https://doi.org/10.1104/pp.122.2.389
  18. Na-Ranong, S., K. Laoteng, P. Kittakoop, M. Tanticharoen, and S. Cheevadhanarak. 2005. Substrate specificity and preference of Δ6-desaturase of Mucor rouxii. FEBS Lett. 579: 2744-2748. https://doi.org/10.1016/j.febslet.2005.04.010
  19. Na-Ranong, S., K. Laoteng, P. Kittakoop, M. Tanticharoen, and S. Cheevadhanarak. 2006. Targeted mutagenesis of a fatty acid Δ6-desaturase from Mucor rouxii: Role of amino acid residues adjacent to histidine-rich motif II. Biochem. Biophys. Res. Commun. 339: 1029-1034. https://doi.org/10.1016/j.bbrc.2005.11.115
  20. Napier, J. A., R. Haslam, M. V. Caleron, L. V. Michaelson, F. Beaudoin, and O. Sayanova. 2006. Progress towards the production of very long-chain polyunsaturated fatty acid in transgenic plants: Plant metabolic engineering comes of age. Physiol. Plant 126: 398-406. https://doi.org/10.1111/j.1399-3054.2006.00603.x
  21. Nookaew, I., M. C. Jewett, A. Meechai, C. Thammarongtham, K. Laoteng, S. Cheevadhanarak, J. Nielsen, and S. Bhumiratana. 2008. The genome-scale metabolic model iIN800 of Saccharomyces cerevisiae and its validation: A scaffold to query lipid metabolism. BMC Syst. Biol. 2: 71. https://doi.org/10.1186/1752-0509-2-71
  22. Ratledge, C. and J. P. Wynn. 2002. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv. Appl. Microbiol. 51: 1-51. https://doi.org/10.1016/S0065-2164(02)51000-5
  23. Ratledge, C. 2004. Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie 86: 807-815. https://doi.org/10.1016/j.biochi.2004.09.017
  24. Rattray, J. B. M. and J. E. Hambleton. 1980. The lipid components of Candida boidinii and Hansenula polymorpha grown on methanol. Can. J. Microbiol. 26: 190-195. https://doi.org/10.1139/m80-029
  25. Ren, H. and J. Yuan. 2005. Model-based specific growth rate control for Pichia pastoris to improve recombinant protein production. J. Chem. Technol. Biotechnol. 80: 1268-1272. https://doi.org/10.1002/jctb.1321
  26. Sayanova, O., F. Beaudoin, L. V. Michaelson, P. R. Shewry, and J. A. Napier. 2003. Identification of Primula fatty acid Δ6- desaturases with n-3 substrate preferences. FEBS Lett. 27188: 1-5.
  27. Schenk, J., K. Balazs, C. Jungo, J. Urfer, C. Wegmann, A. Zocchi, I. W. Marison, and U. von Stockar. 2007. Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris $Mut^{+}$ strain. Biotechnol. Bioeng. 99: 368-377.
  28. Shioya, S. 1992. Optimization and control in fed-batch bioreactors, pp. 111-142. In A. Fiechter (ed.). Advances in Biochemical Engineering/Biotechnology. Springer-Verlag, Berlin, Germany.
  29. Simopoulos, A. P. 2002. The importance of ratio of omega-6/ omega-3 essential fatty acids. Biomed. Pharmacother. 56: 365- 379. https://doi.org/10.1016/S0753-3322(02)00253-6
  30. Sohn, J.-H., M. Y. Beburov, E.-S. Choi, and S.-K. Rhee. 1993. Heterologous gene expression and secretion of the anticoagulant hirudin in methylotrophic yeast Hansenula polymorpha. J. Microbiol. Biotechnol. 3: 65-72.
  31. Stearns, T., H. Ma, and D. Botstein. 1990. Manipulating yeast genome using plasmid vectors. Methods Enzymol. 185: 280- 297. https://doi.org/10.1016/0076-6879(90)85025-J
  32. Swaaf, M. E. D., L. Sijtsma, and J. T. Pronk. 2003. High-celldensity fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii. Biotechnol. Bioeng. 81: 666-672. https://doi.org/10.1002/bit.10513
  33. Wan, X., Y. Zhang, P. Wang, F. Huang, H. Chen, and M. Jiang. 2009. Production of gamma-linolenic acid in Pichia pastoris by expression of a delta-6 desaturase from Cunninghamella echinulata. J. Microbiol. Biotechnol. 19: 1098-1102. https://doi.org/10.4014/jmb.0902.071
  34. Weydemann, U., P. Keup, M. Piontek, A. W. M. Strasser, J. Schweden, G. Gellissen, and Z. A. Janowicz. 1995. High-level secretion of hirudin by Hansenula polymorpha - authentic processing of three different preprohirudins. Appl. Microbiol. Biotechnol. 44: 377-385 https://doi.org/10.1007/BF00169932
  35. Zhang, W., J. Sinha, L. A. Smith, M. Inan, and M. M. Meagher. 2005. Maximization of production of secreted recombinant proteins in Pichia pastoris fed-batch fermentation. Biotechnol. Prog. 21: 386-393.
  36. Zhou, X.-R., S. Robert, S. Singh, and A. Green. 2006. Heterologous production of GLA and SDA by expression of Echium plantagineum $\Delta6$-desaturase gene. Plant Sci. 170: 665- 673. https://doi.org/10.1016/j.plantsci.2005.10.021

Cited by

  1. Production of γ-linolenic acid using a novel heterologous expression system in the oleaginous yeast Lipomyces kononenkoae vol.33, pp.10, 2010, https://doi.org/10.1007/s10529-011-0651-3
  2. Evening primrose (Oenothera biennis) Δ6 fatty acid desaturase gene family: cloning, characterization, and engineered GLA and SDA production in a staple oil crop vol.37, pp.6, 2010, https://doi.org/10.1007/s11032-017-0682-0
  3. Synthetic biology approaches for the production of plant metabolites in unicellular organisms vol.68, pp.15, 2010, https://doi.org/10.1093/jxb/erx119
  4. Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production vol.7, pp.None, 2019, https://doi.org/10.3389/fbioe.2019.00094