Xu Changgeng, Zou Qin, Tian Jiheng, Li Mengyuan, Xing Baowen, Gong Julia, Wang Jiangyun, Huo Yi-Xin, Guo Shuyuan
Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, 100081 Beijing, China.
Beijing Institute of Technology (Tangshan) Translational Research Center, Tangshan Port Economic Development Zone, 063611 Hebei, China.
ACS Synth Biol. 2023 Feb 17;12(2):583-595. doi: 10.1021/acssynbio.2c00604. Epub 2023 Jan 18.
The UAG-based genetic code expansion (GCE) enables site-specific incorporation of noncanonical amino acids (ncAAs) harboring novel chemical functionalities in specific target proteins. However, most GCE studies were done in several whole-genome engineered chassis cells whose hundreds of UAG stop codons were systematically edited to UAA to avoid readthrough in protein synthesis in the presence of GCE. The huge workload of removing all UAG limited the application of GCE in other microbial cell factories (MCF) such as , which has 607 genes ended with UAG among its 4245 coding genes. Although the 257 essential genes count only 6.1% of the genes in , they transcribe 12.2% of the mRNAs and express 52.1% of the proteins under the exponential phase. Here, we engineered a strain named Bs-22 in which all 22 engineerable UAG stop codons in essential genes were edited to UAA via CRISPR/Cas9-mediated multiple-site engineering to minimize the negative effect of GCE on the expression of essential genes. Besides the process of constructing GCE-compatible was systematically optimized. Compared with wild-type (Bs-WT), the fluorescence signal of the eGFP expression could enhance 2.25-fold in Bs-22, and the production of protein tsPurple containing l-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) was increased 2.31-fold in Bs-22. We verified that all purified tsPurple proteins from Bs-22 contained Cou, indicating the excellent fidelity of the strategy. This proof-of-concept study reported efficient overexpression of ncAA-rich proteins in MCF with minimized engineering, shedding new light on solving the trade-off between efficiency and workload.
基于琥珀密码子（UAG）的遗传密码扩展（GCE）能够在特定靶蛋白中位点特异性地掺入具有新型化学功能的非标准氨基酸（ncAA）。然而，大多数GCE研究是在几种全基因组工程化底盘细胞中进行的，这些细胞中数百个UAG终止密码子被系统地编辑为UAA，以避免在GCE存在的情况下蛋白质合成过程中的通读现象。去除所有UAG的巨大工作量限制了GCE在其他微生物细胞工厂（MCF）中的应用，例如，在其4245个编码基因中有607个基因以UAG结尾。尽管257个必需基因仅占基因总数的6.1%，但它们在指数生长期转录了12.2%的mRNA并表达了52.1%的蛋白质。在这里，我们构建了一种名为Bs-22的菌株，其中通过CRISPR/Cas9介导的多位点工程将必需基因中所有22个可编辑的UAG终止密码子编辑为UAA，以尽量减少GCE对必需基因表达的负面影响。此外，构建与GCE兼容的过程也得到了系统优化。与野生型（Bs-WT）相比，Bs-22中eGFP表达的荧光信号可增强2.25倍，含l-（7-羟基香豆素-4-基）乙基甘氨酸（Cou）的蛋白tsPurple的产量在Bs-22中增加了2.31倍。我们验证了从Bs-22中纯化的所有tsPurple蛋白都含有Cou，这表明该策略具有出色的保真度。这项概念验证研究报道了在MCF中以最小化工程实现富含ncAA蛋白的高效过表达，为解决效率和工作量之间的权衡问题提供了新的思路。
