Aristidou A A, San K Y, Bennett G N
Department of Chemical Engineering, Rice University, Houston, Texas 77251-1892, USA.
Biotechnol Prog. 1995 Jul-Aug;11(4):475-8. doi: 10.1021/bp00034a019.
Genetic and metabolic engineering provide powerful and effective tools for the systematic manipulation and fine tuning of cellular metabolic activities. In this study, successful application of such techniques to enhance recombinant protein production by reducing acetate accumulation in Escherichia coli is presented. The alsS gene from Bacillus subtilis encoding the enzyme acetolactate synthase was introduced into E. coli cells using a multicopy plasmid. This newly introduced heterologous enzyme modifies the glycolytic fluxes by redirecting excess pyruvate away from acetate to acetolactate. Acetolactate is then converted to a nonacidic and less harmful byproduct acetoin, which appears in the broth. Furthermore, comparative fermentation studies show that the reduction in acetate accumulation leads to a significant improvement of recombinant protein production. The expression of a model recombinant CadA/beta-galactosidase fusion protein, under the control of a strong pH-regulated promoter, was found to increase by about 60% for the specific protein activity (to a level of 30% of total cellular protein) and 50% in terms of the volumetric activity in a batch fermenter. In fed-batch cultivation, the engineered strain achieved a volumetric recombinant protein yield of 1.6 million units/mL (about 1.1 g/L of beta-galactosidase), which represented a 220% enhancement over the control strain. In the meantime, acetate excretion was maintained below 20 mM compared with 80 mM for the control, and the final cell density was improved by 35%.
基因工程和代谢工程为系统地操纵和微调细胞代谢活动提供了强大而有效的工具。在本研究中，展示了将这些技术成功应用于通过减少大肠杆菌中乙酸积累来提高重组蛋白产量的过程。使用多拷贝质粒将来自枯草芽孢杆菌的编码乙酰乳酸合酶的alsS基因导入大肠杆菌细胞。这种新引入的异源酶通过将过量的丙酮酸从乙酸重定向到乙酰乳酸来改变糖酵解通量。然后乙酰乳酸被转化为一种非酸性且危害较小的副产物3-羟基丁酮，其出现在发酵液中。此外，对比发酵研究表明，乙酸积累的减少导致重组蛋白产量显著提高。发现在强pH调节启动子控制下的模型重组CadA/β-半乳糖苷酶融合蛋白的表达，在分批发酵罐中，比活性提高了约60%（达到总细胞蛋白的30%水平），体积活性提高了约50%。在补料分批培养中，工程菌株的重组蛋白体积产量达到160万单位/mL（约1.1 g/L的β-半乳糖苷酶），比对照菌株提高了220%。同时，乙酸排泄维持在20 mM以下，而对照为80 mM，最终细胞密度提高了35%。
