Transient expression of sustainable food proteins in alternative plant species.

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Oxford Department of Biology / KyomeiBBSRC theme: Bioscience for sustainable agriculture and foodThe current food and medicine industry heavily relies on animal proteins that are produced with an unsustainably high carbon footprint. A sustainable alternative is presented by plant-based production systems, as this harvests light energy directly and provides an eukaryotic expression platform that is free of animal pathogens. For instance, the infiltration of leaves with Agrobacterium tumefaciens offer fast, safe and efficient way to produce proteins in plants. This 'agroinfiltration' strategy has already been used to produce interferon, growth hormones, albumin, antibodies and vaccines. Two examples of agroinfiltration-based products are the Ebola-neutralizing antibody cocktail ZMapp (PMID35000569) and the SARS-CoV-2 vaccine Covifenz (PMID35507508). Most agroinfiltration-based protein expressions are performed on the tobacco relative Nicotiana benthamiana, which is easy to grow and agroinfiltrate. However, public acceptance of proteins produced in tobacco and tobacco relatives is poor. Relations with the tobacco industry adds an additional risk, as illustrated by the WHO rejecting Covid19 vaccines produced by Medicago because of their links to the tobacco industry (PMID35379666). This project therefore explores alternative plants for efficient protein production by agroinfiltration. Indeed, transient protein expression by agroinfiltration has also been shown for e.g. pepper, lettuce, pea, lupine, soybean, rapeseed, Arabidopsis, radish and turnip (e.g. PMID10755307; PMID32775949; PMID33811500), but no comparative studies of expression levels, nor further optimization studies have been performed yet.THE PROJECT AIM is to identify alternative plant species for efficient protein expression, taking advantage of our recently established screening assays. Using our traffic light reporter system we can simultaneously report the state of Agrobacterium (with LUX); transgene expression (with GFP) and secretion efficiency (with secreted RFP). We have used the LUX/GFP reporters to study the effect of light on protein expression (PMID34369027), and we have used the traffic light reporter system to increase protein expression levels by depleting immune components and express effectors (Dodds, King & VdH, unpublished). This traffic light reporter system is robust and will facilitate the screening of hundreds of plants at various parameters using the microplate reader (Tecan) and/or laser scanner (Typhoon). This project will be in close collaboration with Kyomei (www.kyomei.co.uk), a start-up company based in Cambridge, UK, focused on production of sustainable, carbon-neutral food ingredients to enhance the taste and nutritional properties of meat alternatives. This collaborator has a vested interest in alternative plant species for transient protein expression, and is also the ideal partner to translate discoveries generated by this project.By developing alternative protein expression platforms in more relatable plant species, this project will have a clear and direct impact on sustainable food and medicine production in the UK and the world by using plants to produce food additives and medicinal proteins in a sustainable and carbon neutral way, which is a clear priority of the UK government and the BBSRC. By partnering with Kyomei, these discoveries will have an immediate impact on commercial protein expression in the UK. All findings will be published open access to ensure further applications of our discoveries.