双氧水对米赫根毛霉脂肪酶在米曲霉中重组表达的调控研究

Regulation of the Recombinant Expression of a Rhizomucor miehei Lipase Gene in Aspergillus oryzae by Hydrogen Peroxide

摘要:

米赫根毛霉脂肪酶RML是具有广泛应用价值的重要微生物脂肪酶。本研究对双氧水调控米曲霉RML转化子ONL1表达脂肪酶进行了研究。荧光定量PCR和SDS-PAGE表明，在转化子培养过程中，维持培养体系中10 mmol/L双氧水2 h，使转化子ONL1的脂肪酶活力提高5倍。在双氧水处理过程中，RML的翻译未受影响，其表达水平的提高源于双氧水对其转录水平的调控。因此，双氧水调控melO启动子控制的外源基因的表达体现在转录水平。由于双氧水易挥发，导致其效果低于延长培养时间。为了在较短培养时间内获得高酶活，应在培养液中持续添加双氧水（10 mmol/L）。基于米曲霉转化子脂肪酶活力的分析和qPCR检测，确定以下策略可用于提高米曲霉中RML的表达水平：脂肪酶RML基因的密码子优化、信号肽序列优化、连续分批培养。

Abstract:

Rhizomucor miehei lipase (RML) is an important microbial lipase with a wide range of industrial applications. The effect of hydrogen peroxide (H2O2) on RML expression in A. oryzae transformant ONL1 was explored in this study. The results of quantitative fluorescence polymerase chain reaction (QF-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the RML activity of the cultivated A. oryzae ONL1 transformants was increased by five-fold after treatment with 10 mM H2O2 for two hours. The H2O2 treatment did not appear to affect the translation of RML mRNA, with the elevated expression of RML being attributed to the H2O2 regulation of RML transcription. Therefore, hydrogen peroxide might regulate melO promoter-controlled heterologous gene expression at the transcriptional level. Because of the highly volatile nature of H2O2, a prolonged cultivation time led to a lower effect on RML activity. High RML activity within a short cultivation time could be achieved by the continuous addition of H2O2 in the to maintain the concentration of 10 mM in culture medium. The results of the RML activity assay and QF-PCR analysis highlighted the following strategies for the regulation of melO promoter-controlled RML expression in A. oryzae: codon optimization of RML gene sequence, signal peptide optimization, and repeated batch culture.