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

Abstract

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, highbiomass 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 highvalue proteins and bioproducts. Recombinant proteins are currently being produced in cultured cell-based systems in mammals, microbes (bacteria and yeast), insects and plants, as well as in transgenic animals (reviewed by Demain and Vaishnav) 1. Transgenic plants constitute an attractive system for expression and production of a variety of proteins and biomolecules due to their efficient eukaryotic protein synthesis, high scalability, relatively low production costs and environmental footprint 2-4. However, selecting suitable hosts and expression vectors are key considerations since protein accumulation is determined by expression levels. Important factors to consider when selecting a plant-based production platform include biomass yield per hectare, recombinant protein yield per unit biomass, ease of transformation, scalability and safety 5. Sugarcane

Plant-based expression platforms to produce high-value metabolites and proteins

“…The cauliflower mosaic virus 35S promoter (CaMV 35S), a strong constitutive promoter in either single or multiple copies, is widely used in dicot plants, whereas the maize ubiquitin-1 (Ubi-1) promoter is used to express therapeutic proteins in monocots (Phakham et al, 2021;Mirzaee et al, 2022). Recently, Damaj et al (2020) reported a combinatorial stacked promoter system to enhance the expression of recombinant bovine lysozyme (BvLz). BvLz is a potent broad-spectrum antimicrobial enzyme used in the food, cosmetic, and agricultural industries.…”

“…BvLz is a potent broad-spectrum antimicrobial enzyme used in the food, cosmetic, and agricultural industries. Combinatorial plant transformation and co-expression of BvLz under the control of various constitutive and culm-regulated promoters yielded high levels of expression (up to 11.5% of total soluble protein) in sugarcane culms (Damaj et al, 2020). Similarly, Padilla et al (2020) successfully enhanced the expression of recombinant Galanthus nivalis L. (snowdrop) agglutinin (GNA) in sugarcane and energy cane.…”

Genetic Manipulation of Biosynthetic Pathways in Mint

“…Some interesting work has been done in the area of promoters/terminators to increase expression and reduce instances of gene silencing. Transcriptional transgene silencing has been avoided by Damaj et al (2020) who used sugarcane plants to demonstrate the potential of promoter stacking; that is, transforming a plant with multiple genetic constructs where the same coding sequence is under control of different promoters. Using this approach, the researchers managed to improve expression of a recombinant enzyme by up to 147 fold using five different promoters.…”

Sugarcane Rhizosphere Bacteria Community Migration Correlates with Growth Stages and Soil Nutrient

“…[ 3 ]. With the sharp increase in demand for sugar, more and more people are beginning to study how to increase sugarcane production in order to balance the easily growing demand [ 4 , 5 , 6 ].…”