Department of General Biology, Londrina State University, Celso Garcia Cid Road, PR 445, km 380, University Campus, 86057-970 Londrina, PR, Brazil.
Embrapa Soja, Carlos João Strass Highway, Acess Orlando Amaral, District of Warta, 86085-981 Londrina, PR, Brazil.
J Ind Microbiol Biotechnol. 2024 Jan 9;51. doi: 10.1093/jimb/kuae028.
Producing double-stranded RNA (dsRNA) represents a bottleneck for the adoption of RNA interference technology in agriculture, and the main hurdles are related to increases in dsRNA yield, production efficiency, and purity. Therefore, this study aimed to optimize dsRNA production in E. coli HT115 (DE3) using an in vivo system. To this end, we designed a new vector, pCloneVR_2, which resulted in the efficient production of dsRNA in E. coli HT115 (DE3). We performed optimizations in the culture medium and expression inducer in the fermentation of E. coli HT115 (DE3) for the production of dsRNA. Notably, the variable that had the greatest effect on dsRNA yield was cultivation in TB medium, which resulted in a 118% increase in yield. Furthermore, lactose induction (6 g/L) yielded 10 times more than IPTG. Additionally, our optimized up-scaled protocol of the TRIzol™ extraction method was efficient for obtaining high-quality and pure dsRNA. Finally, our optimized protocol achieved an average yield of 53.3 µg/mL after the production and purification of different dsRNAs, reducing production costs by 72%.
产生双链 RNA(dsRNA)是农业中采用 RNA 干扰技术的一个瓶颈，主要障碍与 dsRNA 产量、生产效率和纯度的提高有关。因此，本研究旨在通过体内系统优化大肠杆菌 HT115(DE3)中的 dsRNA 生产。为此，我们设计了一种新的载体 pCloneVR_2，该载体可在大肠杆菌 HT115(DE3)中高效生产 dsRNA。我们在大肠杆菌 HT115(DE3)的发酵过程中对培养基和表达诱导物进行了优化，以生产 dsRNA。值得注意的是，对 dsRNA 产量影响最大的变量是在 TB 培养基中培养，产量增加了 118%。此外，乳糖诱导(6 g/L)的产量比 IPTG 高 10 倍。此外，我们优化的 TRIzol™提取方法的放大方案可有效获得高质量和纯的 dsRNA。最后，在生产和纯化不同的 dsRNA 后，我们优化的方案平均产量为 53.3 μg/mL，生产成本降低了 72%。
