Institute for Innovation, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan.
Institute for Innovation, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan; Research and Development Center for Precision Medicine, University of Tsukuba, 1-2 Kasuga, Tsukuba-shi, Ibaraki 305-8550, Japan.
J Biosci Bioeng. 2019 Sep;128(3):255-263. doi: 10.1016/j.jbiosc.2019.03.003. Epub 2019 May 7.
In recent years, it has been shown that recombinant RNA molecules have a great potential in mRNA therapy and as novel agricultural pesticides. We developed a fundamental system for efficient production of target RNA molecules in Corynebacterium glutamicum, composed of a strong promoter named F1 and a terminator derived from corynephage BFK20 in a high-copy number plasmid vector. As a target model RNA for overexpression, we designed and used an RNA molecule [designated U1A*-RNA, ∼160 nucleotides (nt) long] containing a stem/loop II (SL-II, hairpin-II) structure from U1 small nuclear RNA (snRNA), which binds to U1A protein, forming a U1 sn-ribonucleoprotein, which is essential in the pre-mRNA splicing process. C. glutamicum strains harboring the U1A*-RNA expression plasmid were cultured and the total RNA was analyzed. We observed prominent expression of RNA corresponding to the U1A*-RNA transcript along with lower expression of a 3'-region-truncated form of the transcript (∼110 nt) in an rnc (encoding RNase III)-deficient strain. We also found that the produced U1A*-RNA bound to the U1A RNA-binding domain protein, which was separately prepared with C. glutamicum. In a batch cultivation using a fermentor, the total accumulated amount of the target RNA reached about 300 mg/L by 24 h. Thus, our results indicated that our system can serve as an efficient platform for large-scale preparation of an RNA of interest.
近年来，研究表明重组 RNA 分子在 mRNA 治疗和新型农业杀虫剂方面具有巨大的潜力。我们开发了一种在谷氨酸棒杆菌中高效生产目标 RNA 分子的基本系统，该系统由一个名为 F1 的强启动子和一个源自 corynephage BFK20 的终止子组成，位于高拷贝数质粒载体中。作为过表达的目标 RNA 模型，我们设计并使用了一种 RNA 分子[命名为 U1A*-RNA，约 160 个核苷酸（nt）长]，其中包含 U1 小核 RNA（snRNA）的茎环 II（SL-II，发夹-II）结构，该结构与 U1A 蛋白结合，形成 U1 snRNP，这是前体 mRNA 剪接过程所必需的。携带 U1A*-RNA 表达质粒的谷氨酸棒杆菌菌株被培养，总 RNA 被分析。我们观察到与 U1A*-RNA 转录物相对应的 RNA 明显表达，同时在 rnc（编码 RNase III）缺陷型菌株中观察到转录物的 3'-区域截断形式（约 110 nt）的表达降低。我们还发现产生的 U1A*-RNA 与分别用谷氨酸棒杆菌制备的 U1A RNA 结合域蛋白结合。在使用发酵罐的分批培养中，目标 RNA 的总积累量在 24 小时内达到约 300mg/L。因此，我们的结果表明，我们的系统可以作为大规模制备感兴趣的 RNA 的有效平台。
