米根霉脂肪酶基因pro-ROL和m-ROL在毕赤酵母中的密码子优化、表达和酶学性质的比较分析

Codon optimization, expression and enzymatic comparison of Rhizopus oryzae lipases pro-ROL and m-ROL in Pichia pastoris

摘要:

米根霉Rhizopus oryzae脂肪酶不仅在众多工业领域中具有良好的应用价值，其典型的分子内伴侣结构(Intramolecular chaperon) 也是研究蛋白质翻译后加工和成熟的理想材料。以R. oryzae HU3005为材料，克隆了其脂肪酶前体基因 (pro-ROL) 和成熟脂肪酶基因 (m-ROL)，并实现了其在巴斯德毕赤酵母Pichia pastoris GS115中的分泌表达。酶学性质比较分析表明：m-ROL对中等链长底物 (C10和C12) 具有更高的水解活性，而pro-ROL更倾向于短链底物 (C4)，且在pH 8.0时活性最高。再者，pro-ROL具有比m-ROL更好的温度稳定性。推测m-ROL和pro-ROL脂肪酶酶学性质的差异可能是由前序列对脂肪酶的折叠和修饰的影响而导致。为提高m-ROL的表达水平，采用重叠延伸PCR技术将基因中8个低频密码子替换为高频密码子。在摇瓶条件下，发酵72 h后，经密码子优化后的m-ROL酶活和蛋白质含量分别达到132.7 U/mL和50.4 U/mL，而初始m-ROL和pro-ROL酶活和蛋白质含量分别仅为28.7 U/mL和14.4 mg/L、29.6 U/mL和14.1 mg/L。

Abstract:

Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.