Tsheten Sherpa ^1,2 Nrisingha Dey ^1*

• ¹ Institute of Life Sciences (ILS), Bhubaneswar, India
• ² Regional Centre for Biotechnology (RCB), Faridabad, Haryana, India

With the advancement of plant synthetic biology, complex genetic engineering circuits are being developed, which require more diverse genetic regulatory elements (promoters) to operate. Constitutive promoters are widely used for such gene engineering projects, but the list of strong, constitutive plant promoters with strength surpassing the widely used promoter, the CaMV35S, is low. In this work, we attempted to increase the constitutive promoter library by developing efficient synthetic promoters suitable for high-level gene expression. To do that, we selected three strong pararetroviral-based promoters from Mirabilis mosaic virus (MMV), Figwort mosaic virus (FMV), and Horseradish latent virus (HRLV) and rationally designed and combined their promoter elements. We then tested the newly developed promoters in Nicotiana benthamiana and found a highly active tri-hybrid promoter, MuasFuasH17 (MFH17). We further used these promoter elements in generating random mutant promoters by DNA shuffling techniques in an attempt to change/ improve the MFH17 promoter. We also evaluated the activity of the MFH17 promoter in Oryza sativa seedlings and studied the effect of as-1 elements present in it. Finally, we tested the efficacy and tissue specificity of the MFH17 promoter in vivo by developing transgenic Nicotiana tabacum and Arabidopsis thaliana plants and found it highly constitutive and efficient in driving the gene throughout the plant tissues. Overall, we conclude that this tripartite synthetic promoter MFH17 is a strong, highly constitutive, and dual-species (dicot and monocot) expressing promoter, which can be a valuable addition to the constitutive plant promoter library for plant synthetic biology.