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Plants represent a safe and cost-effective platform for producing high-value proteins with pharmaceutical properties; however, the ability to accumulate these in commercially viable quantities is challenging. Ideal crops to serve as biofactories would include low-input, fast-growing, high-biomass species such as sugarcane. The objective of this study was to develop an efficient expression system to enable large-scale production of high-value recombinant proteins in sugarcane culms. Bovine lysozyme (BvLz) is a potent broad-spectrum antimicrobial enzyme used in the food, cosmetics and agricultural industries. Here, we report a novel strategy to achieve high-level expression of recombinant proteins using a combinatorial stacked promoter system. We demonstrate this by co-expressing  BvLz  under the control of multiple constitutive and culm-regulated promoters on separate expression vectors and combinatorial plant transformation. BvLz accumulation reached 1.4% of total soluble protein (TSP) (10.0 mg BvLz/kg culm mass) in stacked multiple promoter: BvLz  lines, compared to 0.07% of TSP (0.56 mg/kg) in single promoter: BvLz  lines. BvLz accumulation was further boosted to 11.5% of TSP (82.5 mg/kg) through event stacking by re-transforming the stacked promoter: BvLz  lines with additional  BvLz  expression vectors. The protein accumulation achieved with the combinatorial promoter stacking expression system was stable in multiple vegetative propagations, demonstrating the feasibility of using sugarcane as a biofactory for producing high-value proteins and bioproducts.

Unprecedented enhancement of recombinant protein production in sugarcane culms using a combinatorial promoter stacking system