CRISPR-Based Construction of a BL21 (DE3)-Derived Variant Strain Library to Rapidly Improve Recombinant Protein Production.
CRISPR-Based Construction of a BL21 (DE3)-Derived Variant Strain Library to Rapidly Improve Recombinant Protein Production.
复制标题
DOI：
10.1021/acssynbio.1c00463
复制
发表时间：
2021-12
影响因子：
4.7
通讯作者：
Ziwei Li;Zixu Zhang;Yan Xu;Tianqiong Shi;Chao Ye;Xiaoman Sun;He Huang
中科院分区：
文献类型：
--
作者：
Ziwei Li;Zixu Zhang;Yan Xu;Tianqiong Shi;Chao Ye;Xiaoman Sun;He Huang
大肠杆菌BL21 （DE3）是应用最广泛的重组蛋白表达宿主。然而，并不是每个蛋白都能在BL21 （DE3）中高表达，因此往往需要针对不同的蛋白进行单独的优化策略，这既耗时又难以快速应用于工业生产。构建更多的宿主是丰富蛋白表达选择的良好选择。T7 RNAP的表达水平是pET表达系统的核心控制节点，调节其表达水平是提高难表达蛋白产量的有效途径。通过改变基因组中T7 RNAP的核糖体结合位点（RBS）序列，可以获得具有不同T7 RNAP翻译水平的多种BL21 （DE3）衍生变异宿主。本研究利用碱基编辑器和CRISPR-Cas9构建了含有不同T7 RNAP RBS序列的BL21 （DE3）衍生变异菌株库。值得注意的是，CRISPR-Cas9系统结合简并引物，单次编辑构建了理论覆盖率为87.5%的RBS文库，比使用碱基编辑器更加方便高效。变异菌株文库中目标基因的表达量为亲本菌株的28% ~ 220%。此外，我们构建了重组蛋白生产的高通量宿主筛选平台，使我们能够在3天内获得某些目标蛋白的最佳表达宿主。作为概念的证明，所有8种难以表达的蛋白的生产都得到了极大的改善，包括自溶蛋白、膜蛋白、抗菌肽和难溶蛋白。其中，葡萄糖脱氢酶在最佳寄主中的表达量比亲本菌株增加了298倍。这种策略简单有效，不需要先进的设备，在任何实验室都可以进行。
Escherichia coli BL21 (DE3) is the most widely used host for recombinant protein expression. However, not every protein can be highly expressed in BL21 (DE3), so individual optimization strategies are often required for different proteins, which is time-consuming and difficult to apply rapidly for industrial production. Constructing more hosts is a good choice to enrich protein expression selection. The expression level of T7 RNAP is the core control node of the pET expression system, so regulating its expression level is an effective way of improving the production of difficult-to-express proteins. Various BL21 (DE3)-derived variant hosts with different translation levels of T7 RNAP could be obtained by changing the ribosomal binding site (RBS) sequences of T7 RNAP in a genome. Here, a BL21 (DE3)-derived variant strain library with different RBS sequences of T7 RNAP was constructed using a base editor and CRISPR-Cas9. Notably, the CRISPR-Cas9 system combined with degenerate primers enabled the construction of an RBS library with 87.5% of the theoretical coverage in single editing, which is more convenient and efficient than the use of a base editor. The expression level of a target gene in the variant strain library ranged from 28 to 220% of the parental strain. Furthermore, a high-throughput host-screening platform for recombinant protein production was constructed, which enabled us to obtain the best expression host for certain target proteins in only 3 days. As a proof of concept, the production of all eight difficult-to-express proteins was greatly improved, including autolytic protein, membrane proteins, antimicrobial peptides, and hardly soluble proteins. Among them, the expression of glucose dehydrogenase in the best host exhibited a 298-fold increase compared to the parental strain. This strategy is simple and effective, requires no advanced equipment, and can be carried out in any laboratory.