The Escherichia coli/Corynebacterium glutamicum shuttle vector pEKEx2 is an IPTG-inducible expression vector that has been used successfully for the synthesis of numerous proteins in C. glutamicum. We discovered that the leaky gene expression observed for pEKEx2-derived plasmids relates to reduced functionality of the plasmid-encoded repressor LacI carrying a modified C-terminus, while duplicate DNA sequences in the pEKEx2 backbone contribute to plasmid instability. We constructed the pEKEx2-derivatives pPBEx2 and pPREx2, which harbor a restored lacI gene and which lack the unnecessary duplicate DNA sequences. pPREx2 in addition enables fusion of target genes to a C-terminal Strep-tag II coding region for easy protein detection and purification. In the absence of inducer, the novel vectors exhibit tight gene repression in C. glutamicum, as shown for the secretory production of Fusarium solani pisi cutinase and the cytosolic production of green fluorescent protein and C. glutamicum myo-inositol dehydrogenase. Undesired heterogeneity amongst clones expressing cutinase from pEKEx2 was attributed to the loss of a vector fragment containing the cutinase gene, which likely occurred via homologous recombination of the identical flanking DNA sequences. Such loss was not observed for pPBEx2. Using pPREx2, IolG-Strep was successfully produced and purified to homogeneity by Strep-Tactin affinity chromatography, obtaining 1.5 mg IolG with a specific activity of 27 μmol·min⁻¹·(mg protein)⁻¹ from 100 mL culture. The tight gene repression in the absence of inducer and the improved plasmid stability make expression vectors pPBEx2/pPREx2 attractive alternatives to the available molecular tools for genetic manipulation and high-level production of recombinant proteins in C. glutamicum.

的大肠杆菌/谷氨酸棒杆菌穿梭载体pEKEx2是已经成功地用于许多蛋白质在合成中的IPTG可诱导的表达载体的谷氨酸棒杆菌。我们发现，在 pEKEx2 衍生质粒中观察到的泄漏基因表达与携带修饰 C 端的质粒编码阻遏物 LacI 的功能降低有关，而 pEKEx2 主链中的重复 DNA 序列导致质粒不稳定。我们构建了 pEKEx2 衍生物 pPBEx2 和 pPREx2，它们具有恢复的lacI基因并且缺少不必要的重复 DNA 序列。此外，pPREx2 还可以将目标基因融合到 C 端 Strep-tag II 编码区，以便于蛋白质检测和纯化。在没有诱导剂的情况下，新载体在谷氨酸棒杆菌中表现出严格的基因抑制，如茄病镰刀菌角质酶的分泌产生以及绿色荧光蛋白和谷氨酸棒杆菌的胞质产生所示-肌醇脱氢酶。表达来自 pEKEx2 的角质酶的克隆之间不希望的异质性归因于包含角质酶基因的载体片段的丢失，这可能通过相同侧翼 DNA 序列的同源重组发生。pPBEx2 没有观察到这种损失。使用pPREx2，成功生产IolG-Strep，并通过Strep-Tactin亲和层析纯化至均质，从100 mL培养物中获得1.5 mg IolG，比活性为27 μmol·min ^-1 ·(mg protein) ^-1。在不存在诱导剂的情况下严密的基因抑制和改进的质粒稳定性使表达载体 pPBEx2/pPREx2 成为现有分子工具的有吸引力的替代品，用于基因操作和重组蛋白的高水平生产C. 谷氨酸。