解脂耶氏酵母脂肪酶基因lip1的克隆、密码子优化及功能表达

Gene cloning,codon optimization and functional expression of Yarrawia lipolytica lipase Lip1

【目的】克隆解脂耶氏酵母(Yarrawia lipolytica)脂肪酶基因lip1,并通过密码子优化,首次实现其在毕赤酵母(Pichia pastoris)中的诱导型和组成型表达。【方法】通过PCR扩增Y.lipolytica脂肪酶基因lip1,根据P.pastoris密码子偏爱性,运用重叠延伸PCR合成改造后基因MLip1,将其分别克隆至诱导型分泌载体pPIC9K和新构建的组成型分泌载体pG AP9K上,电转至P.pastoris GS115中,G418抗性筛选得到高拷贝转化重组子,摇瓶发酵,以对硝基苯酚酯为底物检测脂肪酶水解活力,并对表达产物进行酶学性质分析。【结果】从Y.lipolytica中成功克隆了脂肪酶基因lip1(GenBank:Z50020),全长1461bp,编码486个氨基酸残基,无内含子,无信号肽;密码子优化后基因表达水平相对于出发基因有较大提高,且组成型表达优于诱导型;Lip1酶学性质分析表明,其最适底物为pNPB(C4),45℃下Tris-Hcl缓冲液pH8.5时最大水解酶活为8.07U/mL。【结论】Y.lipolytica脂肪酶基因lip1的克隆丰富了脂肪酶基因资源,其高效基因工程菌的构建为将来实现规模化生产奠定了技术基础。

[Objective]To implement inducible and constitutive over-expression of Yarrowia lipolytica lipase gene lip1 in Pichia pastoris using codon optimization.[Methods]We cloned Y.lipolytica lipase gene lip1 according to codon bias of P.pastoris,and optimized lip1 using overlap extension PCR synthesis.Then,we cloned the original and optimized genes into the induced vector pPIC9K and newly built constitutive carrier pGAP9K,and electrotransformated the resultant expression plasmids into P.pastoris GS115.Through G418 resistance screening,high copy transformatants were selected and fermented in shake flasks.P-nitrophenyl palmitate （pNPP） was used as substrates for assay the activities of the expressed lipase,and the characteristics of the lipase were further examined.[Results]We successfully cloned lipase gene lip1 from Y.lipolytica,nucleotide sequence revealed that the open reading frame （ORF） had 1461 nucleotides,encoding 486 amino acids,without any intron or any signal peptide.SDS-PAGE analysis and fermentation result showed that the optimized gene had a higher expression level than the original one,and the constitutive expression was superior to the inducible expression.Preliminary analysis showed that the optimal substrate of Lip1 was p-nitrophenyl butyrate （C4）,the optimum temperature and pH was 45℃ and 8.5,respectively.[Conclusion]Y.lipolytica lipase gene lip1 can be over-expressed through both inducible and constitutive expressions using codon optimization,which lays a solid foundation to further study Y.lipolytica lipase family,and also provides an important prerequisite for scale production and industrial application of the lipase.