杜邦嗜热菌脂肪酶在黑曲霉中的异源表达

该文将杜邦嗜热菌脂肪酶TDL2在黑曲霉系统中异源表达,并考察TDL2自带信号肽和pCAMBIA质粒信号肽对脂肪酶TDL2表达的影响.利用PCR和无缝克隆技术,将脂肪酶TDL2的编码序列与质粒pCAMBIA0380线性片段拼接,分别构建利用β-葡萄糖苷酶bgl2的信号肽的重组质粒pCAMBIA-TDL2-1和利用TDL2信号肽序列的重组质粒pCAMBIA-TDL2-2.用根瘤农杆菌介导转化宿主细胞A. niger CICC 2213构建组成型黑曲霉工程菌A. niger/pCAMBIA-TDL2-1和A. niger/pCAMBIA-TDL2-2,在发酵120 h后其发酵单位分别达到18.5 U·mL^-1和38.3 U·mL^-1,实现了脂肪酶TDL2在黑曲霉中的异源表达.SDS-PAGE分析和活力测定结果表明:利用脂肪酶TDL2信号肽的工程菌A. niger/pCAMBIA-TDL2-2比利用bgl2信号肽的工程菌A. niger/pCAMBIA-TDL2-1的表达量更大、发酵单位更高.

The Heterologously Expression of Thermomyces dupontii Lipase in Aspergillus niger

In this study,the lipase TDL2 from Thermomyces dupontii is heterologously expressed in the Aspergillus niger,and the effects on the expression of lipase TDL2 of signal peptides,TDL2 and plasmid pCAMBIA,are investigated.Using PCR and seamless cloning techniques,the coding sequence of lipase TDL2 is spliced with the linear fragment of plasmid pCAMBIA0380,and the recombinant plasmid pCAMBIA-TDL2-1 using the signal peptide of β-glucosidase bgl2 and the plasmid pCAMBIA-TDL2-2 using the signal peptide of TDL2 are successfully constructed,respectively.Constitutive engineered A. niger/pCAMBIA-TDL2-1 and A. niger/pCAMBIA-TDL2-2 are successfully constructed by Agrobacterium tumefaciens-mediated transformation.The lipase TDL2 is heterologous expression of in Aspergillus niger.After 120 h of fermentation,the fermentation units of A. niger/pCAMBIA-TDL2-1 and A. niger/pCAMBIA-TDL2-2 reach 18.5 U·mL^-1 and 38.3 U·mL^-1,respectively.SDS-PAGE analysis and fermentation curve results show that A. niger/pCAMBIA-TDL2-2 with the signal peptide of lipase TDL2 has higher expression levels and fermentation unit than A. niger/pCAMBIA-TDL2-1 with the signal peptide of bgl2.