21ENGBIO: Re-engineering amino acid metabolism in wheat grain

Lysine is one of the 20 amino acids used to make proteins and most animals, including humans, cannot make it, so rely on acquiring it through their diet. Unfortunately, cereal grains contain low concentrations of lysine, resulting in nutrient deficiency in humans and farm animals, such as pigs and chickens, that are dependent on cereal grain for their nutrition. This has resulted in imported soybeans taking much of the market for pig and chicken feed manufacture in the UK and EU, while in developing countries, lysine deficiency is a major cause of malnutrition in people who are reliant on cereal grains for their protein intake. Lysine deficiency does not occur in people in developed countries because they can acquire lysine from meat, but the National Food Strategy (2021) considers current levels of meat consumption to be unsustainable. Reducing our dependence on meat for lysine intake will require the development of a sustainable and readily-available global supply of plant-sourced lysine, which will be unachievable without major changes to the structure of global agricultural production and agri-food systems, unless cereals can be re-engineered to accumulate higher concentrations of lysine in their grains.
This project will use genome editing with CRISPR to produce high lysine, non-GM wheat lines. Lysine is synthesised from another amino acid, aspartate, via a multistep biochemical pathway. The key control point is a reaction catalysed by an enzyme called DHDPS. DHDPS is feedback-inhibited by lysine, which binds to the enzyme, and we will edit a wheat DHDPS gene so that the enzyme it encodes no longer binds lysine. We will do this in wheat that has already been edited and has high concentrations of aspartate in the grain, using selection agents that will enable us to identify plants containing a lysine-insensitive DHDPS. These agents include a lysine analogue that competes with lysine for incorporation into proteins, and compounds that inhibit DHDPS itself. These compounds will have to be synthesised and our team will include a synthetic chemist as well as plant molecular biologists and Rothamsted's Cereal Transformation Team, making it genuinely multidisciplinary. Crucially, the inhibitors bind DHDPS over the lysine binding site and we have designed changes that will not only render DHDPS lysine-insensitive but also make it resistant to the inhibitors.
The stacking of multiple edits to re-engineer amino acid biosynthesis in wheat grain makes the project an excellent fit for the bioengineered cells and systems theme of the call. The editing will require a technique called homology-directed repair, a technology that has been applied successfully in barley and maize but has not yet been used successfully in wheat, so very much a breakthrough technology. Overall, the project is high risk but high gain, with huge potential international impact, in developed as well as developing countries, affecting human nutritional status, animal feed manufacture, bioethanol production through improved animal feed co-product, market expansion for UK wheat grain, and an increase in availability of plant-derived lysine.

Lysine is the main limiting essential amino acid in cereal grains consumed by humans and other monogastric animals. Lysine deficiency in cereals has resulted in soybeans taking much of the market for pig and chicken feed manufacture in the UK and EU. It is also a cause of malnutrition in people in developing countries who rely on cereal grains for their protein intake, while the limited availability of plant-sourced lysine globally is a barrier to reducing meat consumption.

Lysine is synthesised from aspartate via the diaminopimelate pathway, and the key control point is the conversion of L-aspartate semialdehyde to HTPA in a reaction catalysed by the enzyme, dihydrodipicolinate synthase (DHDPS). DHDPS is feedback-inhibited by lysine, and GM solutions to the problem have used bacterial or modified plant DHDPS genes that encode lysine-insensitive enzymes.
This project will use CRISPR/Cas9 to edit a wheat DHDPS gene to produce high lysine, non-GM wheat. It will exploit a unique resource in the form of plants that have already been edited to knock out a seed-specific asparagine synthetase gene. The aspartate concentration in the grain of these plants is approximately double that of wild type plants. The project will use selective agents to identify successful editing, including the lysine analogue (AEC) that competes with lysine for incorporation into proteins, and DHDPS inhibitors that bind the enzyme over the lysine binding site.

The project is an excellent fit for the bioengineered cells and systems theme of the call, while the editing will require homology-directed repair using a DNA-repair template, which would be a breakthrough technology in wheat.

The project is high risk but high gain, with huge potential international impact, in developed as well as developing countries, affecting human nutritional status, animal feed manufacture, bioethanol production, the market for UK wheat grain, and the availability of plant-derived lysine.