Creation of Hansenula polymorpha fatty acid auxotrophic mutants for genetic study of fatty acid biosynthesis
Keywords:
fatty acid auxotrophic mutant, Hansenula polymorpha, monounsaturated fatty acid (MFA), polyunsaturated fatty acid (PFA), saturated fatty acid (SFA)Abstract
Three groups of Hansenula polymorpha fatty acid auxotrophic mutants were screened by their fatty acid requirement for growth. The mutants are saturated fatty acid auxotrophic mutants (Sfa-), monounsaturated fatty acid auxotrophic mutants (Mfa-) and polyunsaturated fatty acid auxotrophic mutants (Pfa-) which required saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids, respectively. All Sfa- mutants could grow on the media supplemented with C14:0 but not grow on the media with neither C16:0 nor C18:0. By fatty acid analysis, two of the Sfa- mutants, S7 and S16, showed a significant difference in the fatty acid composition. S7 clearly defected in the production of C18:2Δ9,12 and C18:3Δ9,12,15, while S16 significantly accumulated medium-chain saturated fatty acids, C12:0 and C14:0. Mfa- and Pfa- mutants could grow on the media supplemented with monounsaturated fatty acids better than supplemented with polyunsaturated fatty acids. The mutants obtained in this study could be used as tools for studying in genes involved in the fatty acid synthesis and biological effect of fatty acids.References
Anamnart S, Tolstorukov I, Kaneko Y, Harashima S (1998) Fatty acid desaturation in methylotrophic yeast Hansenula polymorpha strain CBS1976 and unsaturated fatty acid auxotrophic mutants. J Ferment Bioeng 85: 476-482.
Bilinski T, Litwinska J, Blaszczynski M, Bajus A (1989) Superoxide dismutase deficiency and the toxicity of the products of auto-oxidation of polyunsaturated fatty acids in yeast. Biochim Biophys Acta 1001:102-106.
Cross GAM (1987) Eukaryotic protein modification and membrane attachment via phosphatidyl inositol. Cell 48: 179–181.
Duronio RJ, Rudnick DA, Johnson RL, Linder ME, Gordon JI (1991) Myristic acid auxotrophy caused by mutation of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. J Cell Biol 266: 10498–10504.
Gleeson MA, Sudbery PE (1988) Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4: 293–303.
Fujimori K, Anamnart S, Nakagawa Y, Sugioka S, Ohta D, Oshima Y, Yamada Y, Harashima S (1997) Isolation and characterization of mutations affecting expression of the Δ9-fatty acid desaturation gene, OLE1, in Saccharomyces cerevisiae. FEBS Lett 413: 226-230.
Laoteng K, Ruenwai R, Tanticharoen M, Cheevadhanarak S (2005) Genetic modification of essential fatty acids biosynthesis in Hansenula polymorpha. FEMS Microbiol Lett 245: 169-178.
Lepage G, Roy CC (1984) Improved recovery of fatty acid through direct trans-esterification without prior extraction or purification. J Lipid Res 25: 1391-1396.
Orme T, McIntyre J, Lynen F, Kuhn L, Schweizer E (1972) Fatty acid elongation in a mutant of Saccharomyces cerevisiae deficient in fatty acid synthethase. Eur J Biochem 24: 407–415.
Oshiro T, Aiba H, Mizuno T (2003) A defect in a fatty acyl-CoA synthase gene, lcf1+, results in a decrease in viability after entry into the stationary phase in fission yeast. Mol Gen Genomics 269: 437–442.
Prasitchoke P, Kaneko Y, Bamba T, Fukusaki E, Kobayashi A, Harashima S (2008) The essential fatty acid myristate causes severe growth retardation in Hpelo disruptants of the yeast Hansenula polymorpha. Arch Microbiol 189: 297–304.
Rakpuang W (2009) Growth temperatures and various concentrations of richnoleic acid affect fatty acid
composition in two strains of Hansenula polymorpha. Pakistan J of Biol Sci 12: 986-990.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. A Laboratory Manual, 2nd ed CSHL Press, Cold Spring Harbor Laboratory Press, New York.
Sangwallek J, Kaneko Y, Tsukamoto T, Marui M, Sugiyama M, Ono H, Bamba T, Fukusaki E, Harashima S (2014) Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding Δ12 and Δ15-fatty acid desaturates in Hansenula polymorpha. Gene 533: 110-118.
Schweizer E, Bolling (1970) A Saccharomyces cerevisiae mutant defective in saturated fatty acid biosynthesis. Proc Natl Acad Sci 67: 660-666.
Sooksai S, Chewchanlertfa P, Kaneko Y, Harashima S, Laoteng K (2013) Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis. Mol Biol Rep 40: 4935-4945.
Tong F, Black PN, Bivins L, Quackenbush S, Ctrnacta V, DiRusso CC (2006) Direct interaction of Saccharomyces cerevisiae Faa1p with the Omi/HtrA protease orthologue Ynm3p alters lipid homeostasis. Mol Gen Genomics 275: 330–343.
Towler DA, Adams SP, Eubanks SR, Towery DS, Jackson Machelski E, Glaser L, Gordon JI (1987) Purification and characterization of yeast myristoyl CoA:protein N-myristolyltransferase. Proc Natl Acad USA 84: 2708–2712.
