DEVELOPMENT OF PREDICTIVE STRATEGIES TO IMPROVE PLANT-BASED PRODUCTION OF HIGH-VALUE PROTEINS IN CHLOROPLAST-TRANSFORMED TOBACCO

Abstract

Global demand for high value proteins is rapidly expanding in critical areas of agriculture, biopharmaceuticals, and industrial processing. Current methods of producing these high-value proteins are cell cultures that have high capital costs, require specialized maintenance, and are relatively inflexible to changing market demands. Chloroplast-engineered high biomass crops may offer a cheap and versatile alternative; however, this technology is still largely limited to proof-of-concept experiments. Growth chamber trials demonstrated the remarkable metabolic flexibility of transplastomic Nicotiana tabacum plants to synthesize a recombinant cellulase to as much as 38% of TSP with no deleterious mutant phenotype. These engineered plants also maintained robust heterologous protein yields even when grown in the unpredictable conditions of open field cultivation, an important step towards viable marketability. In an effort to address some of the current limitations of transplastomic plants, additional growth analyses investigated ways that foreign proteins can impact the host plant, such as imposing greater resource demands, exhausting protein synthesis, altering gene expression, and enzymatically interfering with host plant metabolism. Finally, analyses of in silico and in vivo mRNA secondary structure identified a likely mechanism of function for the downstream box (DB) regulatory region to conserve the native folding conformation of the transgenic 5’UTR. Synthetically designed DBs confirmed this hypothesized mechanism for recombinant cellulase production in N. tabacum plastids with on-going work focusing on expanding applicability to other high-value proteins and host organisms.