Rational domestication of a plant-based recombinant expression system expands its biosynthetic range

Abstract

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

Intracellular Application of an Asparaginyl Endopeptidase for Producing Recombinant Head-to-Tail Cyclic Proteins

“…Heat-treated linear mDHFR showed no activity, while the cyclic mDHFR retained a level of activity proportionate to the estimated fraction of folded protein: 58% loss in activity vs. 55% of protein unfolded. To the best of our knowledge, this is the first intracellular application of AEP catalysis in a live recombinant organism outside of plant species. , Recombinant E. coli systems offer fast growth rate, durability, ease of use, and low cost, and therefore in situ cyclization by OaAEP1-C247A in live E. coli represents a major step to improve access and usability of this powerful biocatalyst for protein modification. Notably, the linear control and cyclic protein were obtained in comparable yields (3.5 mg for cyclic mDHFR vs. 3.2 mg linear mDHFR), suggesting that our intracellular cyclization system was highly effective and that cyclic protein production in this study was limited by the abundance of substrate rather than enzyme activity.…”

Techno-Functions and Safety Concerns of Plant-Based Peptides in Food Matrices

“…Controlled production, the potential for customization Regulatory concerns, ethical considerations. Leite et al, 60 Jackson et al, 61 Hung et al 62 Microwave-assisted extraction The plant material is heated with microwaves to promote the release of peptides.…”

“…With this method, genomes encoding particular peptide sequences are inserted into microorganisms, causing them to produce the required peptides throughout their cultivation. Therefore, the peptide characteristics can be managed and customized. − …”

“…Longer peptides may increase stability or solubility, whereas shorter peptides may demonstrate higher bioavailability and functionality. PBPs have the potential for post-translational modifications such as glycosylation or phosphorylation, which might impact their stability, solubility, and interactions with cell receptors . PBPs may interact differently with particular targets depending on their conformational flexibility.…”

“…PBPs have the potential for post-translational modifications such as glycosylation or phosphorylation, which might impact their stability, solubility, and interactions with cell receptors. 61 PBPs may interact differently with particular targets depending on their conformational flexibility. Furthermore, peptides with highly flexible structures display a variety of functions compared to those with rigid structures.…”