Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Appl Environ Microbiol. 2012 Aug;78(16):5831-8. doi: 10.1128/AEM.01147-12. Epub 2012 Jun 8.
Corynebacterium glutamicum is currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species use lac-derived promoters from Escherichia coli that exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains the L-arabinose regulator AraC, the P(BAD) promoter from the araBAD operon, and the L-arabinose transporter AraE, all of which are derived from E. coli. The level of inducible P(BAD)-based expression could be modulated over a wide concentration range from 0.001 to 0.4% L-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control of P(BAD) promoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case in E. coli, P(BAD) induction was not significantly affected in the presence of different carbon sources in C. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of the odhI gene from C. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering of C. glutamicum.
谷氨酸棒杆菌目前用于各种生物材料的工业生产。该物种中许多可用的诱导表达系统使用源自大肠杆菌的 lac 衍生启动子，其诱导表达和基础表达泄漏水平要低得多。我们开发了一种阿拉伯糖诱导表达系统，该系统包含来自大肠杆菌的阿拉伯糖调节剂 AraC、araBAD 操纵子的 P（BAD）启动子和 L-阿拉伯糖转运蛋白 AraE。诱导型 P（BAD）表达水平可在 0.001 至 0.4% L-阿拉伯糖的宽浓度范围内进行调节。该系统可紧密控制无泄漏表达的尿嘧啶磷酸核糖基转移酶的表达。当编码绿色荧光蛋白（GFP）的基因受 P（BAD）启动子控制时，流式细胞术分析表明 GFP 在整个细胞群体中呈高度均匀的表达谱。与大肠杆菌的情况不同，在谷氨酸棒杆菌中，不同碳源的存在对 P（BAD）诱导的影响不大，这使其在发酵应用中非常有用。我们使用该系统来调节谷氨酸棒杆菌的 odhI 基因的表达，该基因编码α-酮戊二酸脱氢酶的抑制剂，从而在生物素非限制条件下产生高水平的谷氨酸（高达 13.7 mM）。该系统为谷氨酸棒杆菌的分子生物学和代谢工程提供了一种有效的工具。
