Down-regulation of acetate pathway through antisense strategy in Escherichia coli:: Improved foreign protein production
Down-regulation of acetate pathway through antisense strategy in Escherichia coli:: Improved foreign protein production
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DOI：
10.1002/bit.10735
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发表时间：
2003-09-30
影响因子：
3.8
通讯作者：
Cha, HJ
中科院分区：
文献类型：
--
作者：
Kim, JYH;Cha, HJ
使用大肠杆菌生产外源蛋白的一个问题是，尽管内源性产生的乙酸盐在生理上是不可或缺的，但它抑制蛋白质表达。在这里，我们首先采用了反义RNA策略作为一个精心设计的代谢工程工具，部分阻断两个主要的乙酸途径酶，磷酸转乙酰酶（PTA）和乙酸激酶（ACK）的生物合成。构建了三个含有pta和ackA反义基因的重组质粒，并在E. coli BL21。采用绿色荧光蛋白（GFP）作为模型外源蛋白，并通过使用内在ackA启动子来控制反义表达的时间。我们发现，反义方法部分降低了靶酶基因的mRNA水平，并且随着时间的推移，降低了所有反义调节菌株培养基中乙酸盐的浓度。值得注意的是，GFP的总产量在反义调节的菌株中提高了1.6至2.1倍，即使乙酸盐减少的程度并不显着大。结果表明，乙酸途径在细胞生理中的作用比以前的报道中预期的更重要。当培养规模增加时，蛋白质产量的提高变得更大，表明这种反义策略可以成功地应用于实际的大规模蛋白质生产过程。(C)2003 Wiley Periodicals，Inc.
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. (C) 2003 Wiley Periodicals, Inc.