解脂耶罗维亚酵母工程菌合成超长链脂肪酸及温度的影响

Engineering the yeast Yarrowia lipolytica to synthesize very- long-chain fatty acids and the effects of temperature on fatty acids synthesis

摘要:

【背景】解脂耶罗维亚酵母属于产油微生物，大量研究表明该酵母能够高产长链脂肪酸和油脂，但是应用该酵母合成超长链脂肪酸仍待研究。【目的】工程化解脂耶罗维亚酵母合成高值超长链脂肪酸，并研究温度对脂肪酸合成的影响。【方法】合成密码子优化的拟南芥(Arabidopsis thaliana)延长酶基因AtFAE1、非洲芥菜(Brassica tournefortii)延长酶基因BtFAE1和碎米芥属植物Cardamine graeca的延长酶基因CgKCS，分别构建质粒pYLEX1-AtFAE1、pYLEX1-BtFAE1、pYLEX1-CgKCS和pYLEX1-AtFAE1-BtFAE1-CgKCS。以解脂耶罗维亚酵母菌株Po1g为宿主，通过化学法分别转化上述4个质粒，获得工程菌Po1g-AtFAE1、Po1g-BtFAE1、Po1g-CgKCS和Po1g-AtFAE1-BtFAE1- CgKCS，比较评价超长链脂肪酸的合成。在此基础上，过表达内源二酯酰甘油酰基转移酶基因DGAT1 (diacylglycerol acyltransferase)提高产油量，并研究温度对生物量、产油、脂肪酸组成的影响。【结果】在解脂耶罗维亚酵母中3个延长酶的延长能力明显不同，AtFAE1主要催化C20:1脂肪酸的合成，BtFAE1更有利于芥酸(C22:1)的合成，而CgKCS能够催化合成神经酸(C24:1)，但是三者共表达并未提高神经酸产量。在表达CgKCS基因的菌株中过表达DGAT1基因，细胞油脂含量提高50%。温度实验表明，低温有利于解脂耶罗维亚酵母合成不饱和脂肪酸，反之，高温利于其合成饱和脂肪酸。【结论】脂肪酸延长酶基因CgKCS可直接催化C18:1脂肪酸合成C24:1的超长链脂肪酸，并且通过优化培养温度可提高不饱和脂肪酸的合成。本研究为构建超长链脂肪酸细胞工厂以及发酵优化提供理论和技术参考。

Abstract:

[Background] The yeast Yarrowia lipolytica is an oleaginous microorganism and can produce long-chain fatty acids and oils with high cell contents. However, the synthesis of very-long-chain fatty acids by this yeast remains to be investigated. [Objective] To engineer Y. lipolytica to synthesize very-long-chain fatty acids with high values and evaluate the influences of temperature on biomass, oil production, and fatty acids compositions. [Methods] The fatty acids elongation enzyme genes AtFAE1 in Arabidopsis thaliana, BtFAE1 in Brassica tournefortii and CgKCS in Cardamine graeca were codon-optimized according to the frequency of codon usage in Y. lipolytica. The plasmids pYLEX1-AtFAE1, pYLEX1-BtFAE1, pYLEX1-CgKCS and pYLEX1-AtFAE1-BtFAE1-CgKCS were constructed and used to transform the host strain Y. lipolytica Po1g, generating the yeast strains Po1g-AtFAE1, Po1g-BtFAE1, Po1g-CgKCS and Po1g-AtFAE1-BtFAE1-CgKCS. Furthermore, the DGAT1 gene encoding diacylglycerol acyltransferase was overexpressed in the strain Po1g-CgKCS to improve oil production. [Results] The results showed that the overexpression of AtFAE1, BtFAE1, and CgKCS led to the remarkably difference in the composition and content of fatty acid in Y. lipolytica. AtFAE1 mainly catalyzes the synthesis of C20:1, BtFAE1 prefers to synthesize erucic acid (C22:1), and CgKCS can catalyze the synthesis of nervonic acid (C24:1). Nevertheless, co-expression of the three genes in Y. lipolytica did not increase the production of nervonic acid. Overexpression of the gene DGAT1 in Po1g-CgKCS apparently increased the oil content by 50%. The results from the temperature experiment showed that a lower temperature benefited the synthesis of unsaturated fatty acids, while a higher temperature benefited the synthesis of saturated fatty acids in Y. lipolytica in our tests. [Conclusion] The fatty acids elongase CgKCS can directly catalyze the synthesis of the C24:1 fatty acid from the C18:1 fatty acid, and the synthesis of unsaturated fatty acids can be improved by optimizing the culture temperature. This study provides theoretical and technical guidance for the construction of cell factories for very-long-chain fatty acids and the optimization of fermentation.