Larsen Gitte G, Appel Karen F, Wolff Anne-Mette, Nielsen Jens, Arnau José
Department of Fungal Biotechnology, Biotechnological Institute, Kogle Allé 2, 2970, Hørsholm, Denmark.
Curr Genet. 2004 Apr;45(4):225-34. doi: 10.1007/s00294-003-0484-2. Epub 2004 Jan 20.
The promoter of the Mucor circinelloides gpd1 gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd1P) was recently cloned and used for the production of recombinant proteins, such as the Aspergillus niger glucose oxidase 1 (GOX). This represents the first example of the application of a strong and regulated promoter from this fungus for recombinant protein production. The original 741-bp gpd1P promoter fragment conferred hexose-dependent expression of GOX in M. circinelloides. To understand the regulatory mechanisms involved in gpd1P-driven expression and to develop improved promoter fragments, deletion derivatives of gpd1P were constructed. These derivatives were fused to the A. niger gox1 gene and used to construct strains containing a single copy of the expression cassette. GOX activity was detected in strains containing the full-length gpd1P and also in strains containing a 713-bp or a 361-bp derivative. Expression levels for the 361-bp derivative were high and comparable, regardless of the carbon source used. This promoter represents a useful derivative for constitutive heterologous gene expression in M. circinelloides.
编码甘油醛-3-磷酸脱氢酶（gpd1）的卷枝毛霉gpd1基因的启动子（gpd1P）最近被克隆，并用于生产重组蛋白，如黑曲霉葡萄糖氧化酶1（GOX）。这是首次将来自该真菌的强且可调控的启动子应用于重组蛋白生产的实例。原始的741bp gpd1P启动子片段赋予了GOX在卷枝毛霉中依赖己糖的表达。为了了解gpd1P驱动表达所涉及的调控机制并开发改进的启动子片段，构建了gpd1P的缺失衍生物。这些衍生物与黑曲霉gox1基因融合，并用于构建含有单拷贝表达盒的菌株。在含有全长gpd1P的菌株以及含有713bp或361bp衍生物的菌株中均检测到了GOX活性。无论使用何种碳源，361bp衍生物的表达水平都很高且相当。该启动子是卷枝毛霉中组成型异源基因表达的有用衍生物。
