Study Of The Expression Of Rhizomucor Miehei Lipase Gene In Filamentous Fungi

Filamentous fungi have great potential in the production and secretion of heterologous proteins. The important characteristics of fungi protein expression have gradually been acknowledged with the development of the research in fungi, including the high expression level, effective extracellular secretion, the similar modification system with higher eukaryotes and the natural activity of the expressed heterologous proteins,. Aspergillus oryzae and Aspergillus awamori are important strains for industrial application in heterologous protein production.The secretion efficiency of heterologous proteins is often not satisfactory in filamentous fungi, particularly for proteins not from filamentous fungi, where the yield is generally 2~3 order of magnitude lower than homologous protein expression. With the development of molecular genetic techniques, we can modify filamentous fungi using gene engineering methods to enhance the expression and secretion level of heterologous proteins. In addition, the low transformation efficiency of filamentous fungi limits their industrial applications.In this study, we first screened resistance markers for wild host strains, including study of influence of the pyridine thiamine (PT) to A. oryzae IFO4177, influence of hygromycin B to A. oryzae IFO4177 and A. awamori CBS115.52. The results illustrated that PT could greatly inhibit the growth of A. oryzae and the PT resistance gene (ptrA) could be used as the selection marker for wide A. oryzae host. The results also suggested that A. oryzae was not as sensitive to hygromycin B as A. awamori which could be inhibited by 100μg/mL hygromycin B, so hygromycin resistance gene could be used in wild-type transformation system for A. awamori. We also studied agrobacterium-mediated transformation for wide A. oryzae.Transformation experiments were first done in A. oryzae IFO4177, including protoplast transformation and agrobacterium-mediated transformation. Due to the lack of appropriate selection markers, there were no stable transformants detected in A. oryzae. Subsequently, we ameliorated the transformation and conducted agrobacterium-mediated transformation in A. awamori. Rhizomucor miehei lipase (pHGW-RML) was transformed into A. awamori, and the RML transformants were detected at DNA, RNA and protein level. And the lipase activity of stably positive transformants was examined.