Karhunen T, Mäntylä A, Nevalainen K M, Suominen P L
Research Laboratories, Alko Ltd, Helsinki, Finland.
Mol Gen Genet. 1993 Dec;241(5-6):515-22. doi: 10.1007/BF00279893.
The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37-63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.
在一步操作中，将生物技术领域重要的丝状真菌里氏木霉的染色体纤维二糖水解酶1基因座（cbh1）替换为一个表达盒，该表达盒包含在cbh1启动子控制下的内切葡聚糖酶I cDNA（egl1）。37%至63%的转化体中缺失CBHI蛋白，这表明将线性表达盒靶向cbh1基因座是有效的。对完整的cbh1 - egl1盒在cbh1基因座处表达的研究表明，egl1 cDNA由cbh1启动子表达的效率与cbh1自身相同。此外，携带两个cbh1 - egl1表达盒拷贝的菌株产生的EG I量是宿主菌株产生的主要纤维素酶蛋白CBHI量的两倍。单拷贝转化体中egl1特异性mRNA的水平比未转化的宿主菌株中高约10倍，这表明cbh1启动子的强度比egl1启动子强约10倍。用酶联免疫吸附测定（ELISA）测得的分泌型EG I蛋白增加10倍，与egl1特异性mRNA的增加密切相关。
