Protein engineering strategies to optimise recombinant product synthesis and accumulation in Nicotiana benthamiana.

In plant molecular farming, recombinant products include traditional protein targets, such as antibodies, antigens, and enzymes, but also increasingly refers to high-value peptides, and small molecules produced by heterologous enzymatic pathways. The efficient accumulation and purification of recombinant products requires stable expression and localisation to permissive subcellular environments. Here we discuss innovation in protein engineering and complementary subcellular engineering strategies to tailor Nicotiana benthamiana for recombinant production of proteins, peptides, and small molecules. Three broad themes emerge: (i) targeting proteins via translational fusion of signal sequences can be effectively paired with engineered subcellular environments or synthetic organelles to improve protein folding and stability; (ii) fusion partners can be used to increase accumulation, facilitate purification, or sometimes both, in the case of identified multifunctional protein domains; (iii) co-expression with heterologous enzymes to perform orthogonal post-translational modification can enable the synthesis of unique peptide products. Together, these are some of the strategies that can increase recombinant expression, facilitate complex maturation requirements, shield the product from host cell metabolic processes, or protect the host from toxic product accumulation. The future of protein engineering strategies in N. benthamiana will benefit from advances in other recombinant production systems, genome-wide synthetic biology approaches, and developments in computational protein design.