Development of a high-performance synthetic promoter for plant-based bioproduction

Abstract

The study developed a synthetic FM promoter through domain shuffling of pararetroviral promoters, achieving 4-fold higher activity than CaMV35S in plants. It enhanced recombinant protein yields, demonstrated by effective scytovirin production against Chikungunya, proving FM's utility in plant synthetic biology and molecular farming. Plant synthetic biology requires high-performance constitutive promoters to maximise recombinant protein yields. In this study, we developed a synthetic promoter (FM) through strategic intermolecular domain shuffling of key regulatory regions derived from the full-length transcript promoters of Figwort mosaic virus (FMV) and Mirabilis mosaic virus (MMV). Functional characterization in transient systems demonstrated that this promoter drives exceptionally strong expression of reporter genes across three model plant species: Nicotiana tabacum, Nicotiana benthamiana, and Petunia × atkinsiana. Quantitative β-glucuronidase (GUS) assays revealed that the FM promoter exhibits 4.0-fold higher activity than the conventional CaMV35S promoter in tobacco, and also outperformed the modified CaMV35S2 promoter by 2.0-fold. These results were further validated in stable transgenic lines of N. tabacum and A. thaliana, where qRT-PCR and histochemical staining consistently showed superior transgene expression relative to CaMV35S controls. Through systematic mutagenesis analysis of the FM promoter, we identified that the as-1, G-Box, and ABRE cis-elements are critical for its high activity. We further demonstrated the promoter's compatibility with orthogonal regulation systems by enhancing FM-driven expression using CRISPR-dCas9/VP64 synthetic transcriptional activation. To evaluate biotechnological applications, an antiviral peptide scytovirin (SVN) was expressed under the control of the FM promoter in transgenic N. tabacum plants. In vitro antiviral assays against Chikungunya virus (CHIKV) confirmed that the plant-produced SVN retained biological activity and significantly reduced viral titers by 60%. These results collectively demonstrate the FM as a compact, high-performance synthetic promoter, making it especially valuable for plant molecular farming.

Keywords: CRISPR activation; Cis regulatory elements; Plant molecular farming; Plant pararetroviruses; Scytovirin; Synthetic biology.