Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences
Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences
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DOI：
10.1007/s00203-012-0835-9
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发表时间：
2012-08
影响因子：
2.8
通讯作者：
Aiqun Yu;Jian-Chun Zhu;Biao Zhang;L. Xing;Mingchun Li
中科院分区：
文献类型：
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作者：
Aiqun Yu;Jian-Chun Zhu;Biao Zhang;L. Xing;Mingchun Li
不饱和脂肪酸(UFAs)是毕赤酵母GS115膜脂的主要组成成分，包括油酸(OA，C18：1n-9)、亚油酸(LA，C18：2n-6)和丙氨酸-亚麻酸(α-linolens acid，ALA，C18：3n-3)。为了在分子水平上阐明不饱和脂肪酸的生物合成途径，并研究它们在该菌株生长发育中的可能作用，我们通过同源重组的方法对该菌株进行了去饱和酶基因的修饰。对相应突变体的脂肪酸组成进行了气相色谱分析，证实Fad12编码的∆12-去饱和酶负责LA的形成，而ALA是由Fad15编码的∆15-去饱和酶合成的。Fad9A和Fad9B的同时缺失是致命的，补充OA可以恢复生长，这表明Fad9A和Fad9都编码了将SA转化为OA的∆9去饱和酶。表型分析表明，野生型和Fad15突变体的生长速度几乎相同，而Fad12突变体的生长速度比这两个菌株慢得多。此外，OA与GS115的耐冷性和耐酒精性呈正相关，而LA和ALA不影响GS115的耐冷性和耐酒精性。此外，我们还发现GS115对高浓度甲醇的耐受性与这三种不饱和脂肪酸无关。
Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids inPichia pastorisGS115. 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 ∆12-desaturase encoded byFad12was responsible for the formation of LA, and ALA was synthesized by ∆15-desaturase encoded byFad15. Simultaneous deletion ofFad9AandFad9Bwas lethal and supplementation of OA could restore growth, indicating that possibly bothFad9AandFad9Bencoded ∆9-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type andFad15mutant grew at almost the same rate,Fad12mutant 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.