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

Jackson, Mark A.; Chan, Lai Yue; Harding, Maxim D; Craik, David J.; Gilding, Edward K.

doi:10.1101/2021.12.09.472022

Cited by 2 publications

(1 citation statement)

References 62 publications

(74 reference statements)

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“…The RNA-seq data comparing gene expression between wild-type and ΔAEP N. benthamiana are available through the National Center for Biological Information-Sequence Read Archive BioProject PRJNA784697. Data used to create figures and perform analyses are available online in the Dryad repository: https://doi.org/10.5061/dryad.k6djh9w88 ; Jackson et al (2022) .…”

Section: Data Availabilitymentioning

confidence: 99%

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

Jackson

Chan

Harding

et al. 2022

Journal of Experimental Botany

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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.

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Section: Data Availabilitymentioning

confidence: 99%

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

Jackson

Chan

Harding

et al. 2022

Journal of Experimental Botany

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Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA)

De-Thier,

Pyati,

Bell

et al. 2023

Microb Cell Fact

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Background New bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it’s insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA). Results We report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose–response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins. Conclusions Our results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies.

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

Cited by 2 publications

References 62 publications

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

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

Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA)

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