醋酸激酶
反义RNA
大肠杆菌
绿色荧光蛋白
酶
蛋白质生物合成
生物
生物化学
基因
质粒
重组DNA
感应(电子)
信使核糖核酸
化学
分子生物学
物理化学
作者
Jaoon Young Hwan Kim,Hyung Joon
摘要
Abstract A problem with the use of Escherichia coli to produce foreign proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits protein expression. Here we firstly employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). Three recombinant plasmids containing antisense genes targeting either or both of pta and ackA were constructed, and their effects on the acetate pathway and foreign protein productivity compared to control plasmid without any antisense genes were determined in E. coli BL21. Green fluorescent protein (GFP) was employed as a model foreign protein, and timing of antisense expression was controlled by using the intrinsic ackA promoter. We found that the antisense method partially reduced mRNA levels of target enzyme genes and, over time, lowered the concentration of acetate in culture media in all antisense‐regulated strains. Notably, total production of GFP was enhanced 1.6‐ to 2.1‐fold in antisense‐regulated strains, even though the degree of acetate reduction was not significantly large. It was revealed that the acetate pathway has more critical roles in cellular physiology than expected in the previous reports. When the scale of culture was increased, enhancement of protein production became larger, demonstrating that this antisense strategy can be successfully applied to practical large‐scale protein production processes. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 841–853, 2003.
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