Integrated Biotechnology Laboratory, School of Life Sciences, Anhui University, Hefei, 230601, China.
Microb Biotechnol. 2014 Jul;7(4):360-70. doi: 10.1111/1751-7915.12127. Epub 2014 Apr 30.
In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1-84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l(-1).
在这项研究中，利用构建渗漏菌株的策略，从大肠杆菌中敲除 mrcA、mrcB、pal 和 lpp 基因（作为对照），采用单基因和/或双基因缺失方法。然后，构建表达外源目的蛋白的重组菌株，包括 Trx-hPTH（与人甲状旁腺激素 1-84 融合伴侣硫氧还蛋白）和瑞替普酶，并测试渗漏菌株的分泌效率。最后，在摇瓶或发酵罐中，在基础培养基（改良 R 培养基）和复杂培养基（Terrific Broth 培养基）中进行这些重组渗漏菌株的目标蛋白发酵实验。结果表明，所得渗漏菌株在遗传上是稳定的，并且与原始菌株相比，在复杂培养基中的生长曲线相似，双基因缺失菌株（高达 88.9%/mrcA lpp-pth）向改良 R 培养基分泌目的蛋白的水平高于单基因缺失菌株（高达 71.1%/lpp-pth）和宿主大肠杆菌 JM109（DE3）（接近零）。在发酵罐中，Trx-hPTH 的最高胞外产量可达 680 mg/L。
