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