Molecular Approaches For Producing High-Value Proteins In Transgenic Sugarcane Stalks

The prospect of producing high-value compounds in plants has numerous advantages, including reduced costs and environmental footprint. However, a key challenge is the ability to accumulate such products in commercially-viable quantities. A goal of this project was to develop sugarcane as an efficient recombinant expression system for production of high-value proteins. Sugarcane (Saccharum spp. hybrid) is an ideal crop for production of protein-based therapeutics, due to its fast growth, efficient photosynthetic pathway, and high biomass production capacity. Different approaches were evaluated for enhancing the expression of two recombinant proteins in sugarcane stalks, one being a mammalian enzyme with a broad-spectrum antimicrobial activity and potential use in the food, cosmetic, and agriculture industries, and the other, a plant-derived carbohydrate-binding protein with a potent anti-insecticidal activity and potential medical application. These approaches ranged from the use of promoter stacking for co-expressing the recombinant gene from multiple promoters (tissue-regulated/inducible or constitutive), to incorporation of a double terminator or plant viral 3’ untranslated regions (UTR) to the recombinant gene expression cassette for boosting transcription and stabilizing the transcript message. Strategies to increase translational activity for recombinant sequences included the use of synthetic coding sequences with codon optimization for expression in sugarcane and monocots, and insertion of plant viral 5’UTRs at the translation start site. Expression levels were also boosted through event stacking by re-transforming high-expressing transgenic lines arising from independent transformation events. These data demonstrate the feasibility of commercially producing recombinant proteins in sugarcane stalks, and developing sugarcane as a biofactory for high-value proteins.