Sun Xiao-Man, Zhang Zi-Xu, Wang Ling-Ru, Wang Jing-Gang, Liang Yan, Yang Hai-Feng, Tao Rong-Sheng, Jiang Yu, Yang Jun-Jie, Yang Sheng
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, Jiangsu, China.
HuaRui Biotechnology Company, Huzhou, Zhejiang, China.
Biotechnol Bioeng. 2021 Jan;118(1):153-163. doi: 10.1002/bit.27558. Epub 2020 Sep 18.
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.
大肠杆菌BL21(DE3)是一种优良且广泛应用于重组蛋白生产的宿主菌。许多变体宿主菌是从BL21(DE3)发展而来的，但提高特定蛋白的表达水平在生物技术领域仍然是一项重大挑战。在本研究中，我们发现当BL21(DE3)过表达葡萄糖脱氢酶（一种重要的工业酶）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也观察到了这种现象。这使得无法通过延长发酵时间进一步提高所产酶的活性，不利于降低工业酶的生产成本。对膜结构的分析和信使核糖核酸表达分析表明，细胞在自溶期可能经历了一种程序性细胞死亡（PCD）形式。然而，阻断BL21(DE3)中三种已知的PCD途径并不能完全缓解自溶。因此，我们试图通过控制重组蛋白的表达速度来开发一种抗自溶的高效表达宿主菌。为了找到更合适的蛋白表达速率，对高强度启动子lacUV5和低强度启动子lac进行洗牌和重组，得到启动子变体lacUV5-1A和lac-1G。结果表明，lac启动子只需改变一个碱基，将+1位置的A变为G，即可得到抗自溶的改良宿主菌BL21(DE3-lac1G)。结果，43小时时的葡萄糖脱氢酶活性从37.5大幅提高到452.0 U/ml。在放大发酵中，新宿主菌在43小时时能够以89.55 U/ml/h的高速率生产模型酶，而使用BL21(DE3)时仅为3 U/ml/h。重要的是，BL21(DE3-lac1G)也成功提高了其他10种酶的产量。本研究构建的工程化大肠杆菌菌株通过抑制细胞自溶方便地优化了重组蛋白的过表达，在工业应用中显示出巨大潜力。
