Minas W, Bailey J E
Institut für Biotechnologie, ETH-Hönggerberg, Zürich, Switzerland.
Biotechnol Prog. 1995 Jul-Aug;11(4):403-11. doi: 10.1021/bp00034a007.
The effects on cloned amylase production of co-overexpression of prlF, a gene that appears to interact with the sec protein export machinery in Escherichia coli, was investigated by comparing three expression systems: (i) a high copy number plasmid with the Bacillus stearothermophilus alpha-amylase gene (amyS) cloned with its promoter downstream of the lac promoter; (ii) a pBR322-based vector with amyS under control of the indigenous Bacillus promoter; and (iii) a temperature-inducible vector with runaway replicon and lambda pL promoter-controlled gene expression. In addition, protease mutants (lon-) of E. coli C600 were used to evaluate the influence of the Lon protease on net enzyme formation and activity degradation during batch fermentations. Our results show that alpha-amylase synthesis occurred during exponential growth and ceased in the stationary phase. While strong promoters on high copy number plasmids severely impaired cell viability, resulting in culture lysis at mid-log phase, co-overexpression of prlF greatly improved cell viability, as well as the yield and specific production of alpha-amylase for the expression constructs considered. lon deficiency slightly increased amylase stability during the late stationary phase. However, the specific productivity of lon- strains was only about 40-60% that of the isogenic E. coli C600 equivalent.
通过比较三种表达系统，研究了prlF基因（该基因似乎与大肠杆菌中的sec蛋白输出机制相互作用）共过表达对克隆淀粉酶产生的影响：（i）一种高拷贝数质粒，其携带嗜热脂肪芽孢杆菌α-淀粉酶基因（amyS），该基因与其启动子克隆在lac启动子下游；（ii）一种基于pBR322的载体，其中amyS受本地芽孢杆菌启动子控制；（iii）一种具有失控复制子和λ pL启动子控制的基因表达的温度诱导型载体。此外，使用大肠杆菌C600的蛋白酶突变体（lon-）来评估Lon蛋白酶在分批发酵过程中对净酶形成和活性降解的影响。我们的结果表明，α-淀粉酶合成在指数生长期发生，并在稳定期停止。虽然高拷贝数质粒上的强启动子严重损害细胞活力，导致在对数中期培养物裂解，但prlF的共过表达极大地提高了细胞活力，以及所考虑的表达构建体的α-淀粉酶产量和比产量。lon缺陷在稳定期末期略微增加了淀粉酶稳定性。然而，lon-菌株的比生产力仅为同基因大肠杆菌C600等效菌株的约40-60%。
