Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membrane structure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high‐ and low‐strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5‐1A and lac‐1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in the improved host BL21 (DE3‐lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale‐up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3‐lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.

中文翻译：

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T7 RNA 聚合酶转录的下调通过抑制自溶增强了大肠杆菌 BL21 (DE3) 中基于 pET 的重组蛋白的产生。

大肠杆菌BL21 (DE3) 是一种优秀且广泛用于重组蛋白生产的宿主。许多变异宿主是从 BL21 (DE3) 发展而来的，但提高特定蛋白质的表达仍然是生物技术的主要挑战。在这项研究中，我们发现当 BL21 (DE3) 过表达葡萄糖脱氢酶 (GDH) 时，会诱导严重的细胞自溶。随后，我们在其他 10 种重组蛋白的表达中观察到了这种现象。这阻碍了通过延长发酵时间进一步提高生产酶活性，不利于工业酶生产成本的降低。膜结构分析和信使 RNA 表达分析表明，细胞在自溶期间可能会经历一种程序性细胞死亡 (PCD)。然而，阻断 BL21 (DE3) 中三个已知的 PCD 通路并没有完全缓解自溶。因此，我们试图通过控制重组蛋白表达的速度来开发抗自溶的强表达宿主。为了找到更合适的蛋白质表达率，高强度和低强度启动子 lacUV5 和 lac 被改组并重组，以产生启动子变体 lacUV5-1A 和 lac-1G。结果表明，lac启动子只需要改变一个碱基，+1位的A变成了G，得到了抗自溶的改良宿主BL21（DE3-lac1G）。结果，43 小时时的 GDH 活性从 37.5 大大增加到 452.0 U/ml。在放大发酵中，新宿主能够在 43 小时以 89.55 U/ml/h 的高速率生产模型酶，与使用 BL21 (DE3) 仅达到 3 U/ml/h 相比。重要的是，BL21 (DE3-lac1G) 还成功地提高了 10 种其他酶的产量。设计的本研究构建的大肠杆菌菌株通过抑制细胞自溶来方便地优化重组蛋白的过表达，显示出巨大的工业应用潜力。