Senda Naoko, Enomoto Toshihiko, Kihara Kenta, Yamashiro Naoki, Takagi Naosato, Kiga Daisuke, Nishida Hirokazu
Research & Development Group, Hitachi, Ltd., Kokubunji, Tokyo, Japan.
Department of Electrical Engineering and Bioscience, Waseda University, Shinjuku, Tokyo, Japan.
Synth Biol (Oxf). 2022 Nov 25;7(1):ysac029. doi: 10.1093/synbio/ysac029. eCollection 2022.
New materials with a low environmental load are expected to be generated through synthetic biology. To widely utilize this technology, it is important to create cells with designed biological functions and to control the expression of multiple enzymes. In this study, we constructed a cell-free evaluation system for multiple protein expression, in which synthesis is controlled by T7 promoter variants. The expression of a single protein using the T7 promoter variants showed the expected variety in expression levels, as previously reported. We then examined the expression levels of multiple proteins that are simultaneously produced in a single well to determine whether they can be predicted from the promoter activity values, which were defined from the isolated protein expression levels. When the sum of messenger ribonucleic acid (mRNA) species is small, the experimental protein expression levels can be predicted from the promoter activities (graphical abstract (a)) due to low competition for ribosomes. In other words, by using combinations of T7 promoter variants, we successfully developed a cell-free multiple protein synthesis system with tunable expression. In the presence of large amounts of mRNA, competition for ribosomes becomes an issue (graphical abstract (b)). Accordingly, the translation level of each protein cannot be directly predicted from the promoter activities and is biased by the strength of the ribosome binding site (RBS); a weaker RBS is more affected by competition. Our study provides information regarding the regulated expression of multiple enzymes in synthetic biology.
有望通过合成生物学产生具有低环境负荷的新材料。为了广泛应用这项技术，创建具有设计生物功能的细胞并控制多种酶的表达非常重要。在本研究中，我们构建了一个用于多种蛋白质表达的无细胞评估系统，其中合成由T7启动子变体控制。如先前报道的那样，使用T7启动子变体表达单一蛋白质显示出预期的表达水平差异。然后，我们检测了在单个孔中同时产生的多种蛋白质的表达水平，以确定是否可以从由分离的蛋白质表达水平定义的启动子活性值来预测它们。当信使核糖核酸（mRNA）种类的总和较小时，由于对核糖体的竞争较低，实验性蛋白质表达水平可以从启动子活性预测（图摘要（a））。换句话说，通过使用T7启动子变体的组合，我们成功开发了一种具有可调表达的无细胞多蛋白质合成系统。在存在大量mRNA的情况下，对核糖体的竞争成为一个问题（图摘要（b））。因此，每种蛋白质的翻译水平不能直接从启动子活性预测，并且受到核糖体结合位点（RBS）强度的影响；较弱的RBS受竞争影响更大。我们的研究提供了有关合成生物学中多种酶调控表达的信息。
