Benchmarking genetic elements for high-level protein expression using a novel Marchantia polymorpha transient...

Marchantia polymorpha has emerged as a promising model system for investigations in plant synthetic biology. Quantitatively characterizing plant genetic elements is fundamental to achieving predictable and controlled gene expression. However, only a few genetic parts are currently available for Marchantia. Additionally, the characterization of gene expression elements still relies on stable transformation assays. Here, we developed an Agrobacterium-mediated transient expression system to rapidly evaluate genetic parts in Marchantia. The entire experimental workflow can be completed within 8 days. Using this high-throughput system, we systematically benchmarked 21 promoters, 15 terminators, and 7 signal peptides from diverse sources. We identified a truncated CaMV35S promoter variant (P_35S-3), a native terminator (T_MpAct1), and a heterologous signal peptide (SP_SdMir) as top-performing elements. Notably, the P_35S-3 promoter exhibited a 409-fold activity increase over the standard CaMV35S promoter P_35S. Utilizing this potent element, we achieved an eGFP protein yield of 319.8 μg/g fresh weight in stable transgenic lines. The reliability of this transient system was further validated by stable transformation, where the two signal peptides exhibited a relative performance consistent with the transient assays. Our transient expression system provides a rapid and efficient platform for the characterization of gene expression elements, thereby expanding the genetic toolkit for Marchantia and enhancing protein expression levels.