SummaryThe growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant‐based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large‐scale production due to high operational costs and batch‐to‐batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)‐derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis‐acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper‐inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast‐derived CUP2‐Gal4 copper‐responsive transcriptional activator for Rep/RepA expression, and (iv) a copper‐inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO4 application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti‐SARS‐CoV‐2 antibody upon copper treatment. Notably, the system is functional in post‐harvest applications, a strategy with high potential impact for large‐scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost‐effective and scalable production of recombinant proteins in plants.

中文翻译：

---

CuBe：一种基于双子病毒的铜调节表达系统，适用于收获后激活


摘要对生物分子生产可持续平台的需求不断增长，推动了植物基生产系统的发展。农化渗透是当前的行业标准，具有多项优势，但由于运营成本高且批次间可变性，在大规模生产方面面临限制。或者，在这里，我们描述了 CuBe 系统，这是一种新型豆黄矮病毒 （BeYDV） 衍生的条件复制表达平台，在本氏烟草中稳定转化并被硫酸铜 （CuSO4） 激活，硫酸铜是一种廉价且广泛使用的农业投入。CuBe 系统利用集成到植物基因组中的四个遗传模块的合成电路：（i） 含有目标基因 （GOI） 的复制载体，两侧是用于双子病毒复制的顺式作用元件，并且新排列以仅在形成环状复制子时进行转基因转录，（ii） 铜诱导型 Rep/RepA 蛋白对复制子形成至关重要， （iii） 用于 Rep/RepA 表达的酵母衍生的 CUP2-Gal4 铜响应性转录激活剂，以及 （iv） 铜诱导型 Flp 重组酶，以最大限度地减少基础 Rep/RepA 表达。CuSO4 应用触发系统激活，导致染色体外复制子的形成、GOI 的表达和所需重组蛋白的积累。我们证明了 CuBe 系统在本氏奈瑟菌植物中的功能，该植物在铜处理后表达高水平的 eGFP 和抗 SARS-CoV-2 抗体。值得注意的是，该系统在收获后应用中发挥作用，这种策略对大规模生物制造具有很高的潜在影响。 这项工作将 CuBe 系统作为农杆菌渗透的有前途的替代方案，可在植物中经济高效且可扩展地生产重组蛋白。