Repurposing geminiviruses for engineering gene circuits in plants: a copper-inducible BeYDV-derived expression system for biomanufacturing

The need for sustainable and scalable platforms for biomolecule production has fuelled plant-based biomanufacturing. However, sustained recombinant protein expression in plants often leads to cytotoxicity and reduced yields. A key challenge is the development of inducible and efficient gene expression systems that enable precise transcriptional activation. Plant synthetic biology provides powerful tools to address this limitation by enabling the design of synthetic gene circuits. Here, we present CuBe, a bean yellow dwarf virus (BeYDV)-derived gene amplification circuit, stably integrated into Nicotiana benthamiana and activated by copper sulphate (CuSO₄), a widely used and cost-effective agricultural input. CuBe combines a copper-responsive transcriptional activator with a replicative vector—harbouring a gene of interest—and replication-associated proteins (Rep/RepA), both derived from the geminivirus BeYDV. We demonstrate the functionality of CuBe by achieving high levels of eGFP and an antibody upon CuSO₄ treatment. This system exhibits minimal background expression, a high dynamic range, and compatibility with various activation methods, including post-harvest and hydroponic applications. These results highlight the potential of N. benthamiana lines carrying CuBe as a scalable platform for biomanufacturing. More broadly, this work underscores the potential of synthetic biology in repurposing plant-associated microbes for biotechnological applications.