Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physi
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ORIGINAL PAPER
Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences Ai-Qun Yu • Jian-Chun Zhu • Biao Zhang Lai-Jun Xing • Ming-Chun Li
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Received: 14 February 2012 / Revised: 26 May 2012 / Accepted: 18 July 2012 / Published online: 24 August 2012 Ó Springer-Verlag 2012
Abstract Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and a-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that D12-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by D15-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded D9-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.
Communicated by Reinhard Kraemer. A.-Q. Yu J.-C. Zhu L.-J. Xing M.-C. Li (&) Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin 300071, People’s Republic of China e-mail: [email protected] B. Zhang Tianjin Traditional Chinese Medicine University, Tianjin, China
Keywords P. pastoris Fad9A Fad9B Fad12 Fad15 Gene disruption Abbreviations ALA a-Linolenic acid LA Linoleic acid OA Oleic acid UFA Unsaturated fatty acid
Introduction Since the mid-1980s, many studies have shown that in a range of mammalian species, nearly all unsaturated fatty acids (UFAs) have important functions, especially those polyunsaturated fatty acids (PUFAs) that function mainly by altering membrane lipid composition, cell membrane structure, cellular metabolism, cell differentiation, DNA replication and regulation of gene expression (Takeuchi et al. 2010; Mauvoisin et al. 2010). Therefore, PUFAs are necessary for prevention and treatment of various diseases and essential for good health. In mammalian cells, PUFAs are also known to play important roles as precursors of signaling compounds as well as structural molecules (Gill and Valivety 1997; Funk 2001). Disruptions of the desaturase gene (Miyazaki et al. 2001) and alterations in the biosynthesis of PUFAs can cause various diseases, includin
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