University of Sciences and Technology of China, Hefei, 230026, P R China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, P R China.
Microb Cell Fact. 2020 Jul 3;19(1):136. doi: 10.1186/s12934-020-01393-2.
Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that the system increased the BL21's defense ability against phage infection. Furthermore, the targeted loci on phage genome played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 h of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% of the un-infected control, which was significantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21's to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection.
大肠杆菌 BL21 可以说是蛋白质工业生产中最常用的宿主，而工业发酵常常受到噬菌体感染的困扰。CRISPR/Cas 系统由 gRNA 引导，切割特定的 DNA 盒，从而可以开发出高效的可编程噬菌体防御系统。在这项工作中，我们构建了针对 T7 噬菌体基因组多个位置的 CRISPR/Cas 系统，发现该系统提高了 BL21 抵抗噬菌体感染的防御能力。此外，噬菌体基因组上的靶向位置起着关键作用。为了更好地控制 CRISPR/Cas9 的表达，我们测试了各种模式，优化后的菌株 BL21(pT7cas9, pT7-3gRNA, prfp)在噬菌体感染后 4 小时的 OD 值显著提高，达到 2.0，与未感染噬菌体的对照培养物相似。尽管在稍后的时间点，CRISPR/Cas9 系统的防御能力不如早期明显。在噬菌体存在的情况下，工程菌株的活细胞数仅比未感染的菌株低一个数量级，这进一步证明了 CRISPR/Cas9 噬菌体防御系统的有效性。最后，在噬菌体攻击下，工程 BL21 菌株表达的 RFP 蛋白约为未感染对照的 60%，明显高于亲本 BL21。在这项工作中，我们成功构建了可编程的 CRISPR/Cas9 系统，提高了大肠杆菌 BL21 抵抗噬菌体感染的能力，并创建了一个抗性蛋白表达宿主。这项工作为保护工业大肠杆菌菌株免受噬菌体感染提供了一种简单可行的策略。
