Optimisation of tomato fruit carotenoid content for nutritional improvement and industrial exploitation.

The colour of most fruits, flowers and vegetables is due to the presence of natural pigments such as carotenoids. This specific class of chemical molecules are also essential components of the human diet. In the body, they have a beneficial effect dissipating reactive molecular species that can damage biological structures. In addition to dietary sources, carotenoids are also synthesised chemically using petrochemical by-products. This chemical production of carotenoids generates products that are used in the health, food, feed, cosmetic and pharma industry. The production process is expensive, results in unnatural compound mixtures and has unfavourable environmental credentials. Therefore, generating renewable sources of these compounds, and others, is an important challenge that will have far reaching benefits for society.
The increase of carotenoid pigments in plants, through traditional and modern genetic intervention approaches to boost biosynthesis, has been the biotechnological focus for several decades. However, with the advent of genome sequencing and other modern technologies, it has become apparent that the degradation of these compounds is an important factor that acts to regulate levels in tissues/organs such as flowers, fruit and seeds. An important enzyme that can potentially act on carotenoids to convert them to smaller products derived from their parent molecules has been identified. In order to confirm and determine the effects of this enzyme further, we will:

(i) Use tomato varieties with altered carotenoid content and determine carotenoid levels compared to their derived apocarotenoids. This will provide information on how the plant balances its carotenoid content.
(ii) Isolate/enrich the enzyme activity and introduce precursors to ascertain the potential resulting products.
(iii) Generate and characterise plants which have been altered to prevent carotenoid degradation. Studying these plants will allow us, in crop plants, to determine the role played by carotenoid catabolism.

Collectively these data will, in the future, have societal and economic impact benefiting the consumer, welfare services and industries.

Our present experimental data indicates that enzymatic carotenoid catabolism is more prevalent than first perceived and has a major effect on the levels and types of derived apocarotenoids. In order to assess the potential of manipulating carotenoid catabolism as a means of; (i) enhancing carotenoid content in sink tissues, (ii) altering volatiles/aroma profiles and (iii) the generation of new or rare chemical entities, the OptiCar project will focus on the role of a key enzymatic candidate responsible for carotenoid catabolism.
Firstly, using modern state of the art metabolite profiling, it is intended to quantitatively determine carotenoids and apocarotenoids (including newly identified apocarotenoid glycosides) in a range of backgrounds with altered carotenoid contents.
Based on our preliminary data and published findings, the candidate gene encoding a Carotenoid Cleavage Dioxygenase-4 (CCD4) has been postulated to be the progenitor of carotenoid catabolism in ripe fruit. To test this hypothesis it is intended to perform in vitro enzyme assays where putative precursor carotenoids are introduced into the incubations and the resulting products analysed. if To validate the functionality of the CCD4 in planta it is intended to knock-out or reduce levels by fruit specific RNAi.
In addition, CRISPR technology will be used to generate mutants in CCD4 and finally we will test the ability to elevate levels of industrially high-value ketocarotenoids by down-regulating the catabolism of carotenoids. This will be performed by either combining CCD4 down-regulation with the ketocarotenoid biosynthetic genes in a multi-gene construct or by genetic crossing. Biochemical and molecular characterisation will be carried out on these transgenic plants. These data will elucidate the effects of reducing carotenoid catabolism on carotenoid content and associated sequestration mechanisms.

The OptiCar project outputs have the potential to impact on multiple industrial sectors, society, our welfare and wellbeing, humanitarian aspects as well as benefiting UK R+D skills and competitiveness. The potential industrially exploitable outputs from the project include tools and resources enabling the development of new varieties with increased nutritional and industrial carotenoids.
Industrially the applicants believe there are three routes to markets; (i) Quality traits in fruits and vegetables, (ii) renewable platforms for high-value industrial pigments (carotenoids and apocarotenoids) and (iii) chemical standards for analytical purposes. Providing a means whereby nutritional content and/or aesthetic colour can be altered or increased is aligned with the product portfolio of several multinational Agri-biotech companies and SMEs as well as the UK tomato growers. An example of a multinational with an interest in this work is Syngenta who have provided a letter of support. Thus, the procedures are in place to convert these proof of concept activities into direct commercial practice using technologies acceptable to the consumer. In addition, working in tomato means its role as a model for fleshy fruit can readily be utilised and the approaches translated to other fruits and vegetables.
The applicants have demonstrated the technical, production and economic feasibility of using plant systems as renewable sources through EU funding. The work proposed now provides the opportunity to add value to this pipeline delivering better and different products. In the case of ketocarotenoids (such as canthaxanthin and astaxanthin), there is an urgent need for them as sustainable feeds supplements. The approaches described offer a means of adding these compounds directly to the feed without chemical synthesis or solvent enrichment and because they are a feed component there is no GM material in the final product, improving the regulatory aspects of the approach. Finally, analytical standards of these compounds are highly priced and in most cases rare as no amenable sources are readily available. The biosources generated in the project will deliver a means from which these compounds can be enriched and purified to an analytical standard. Presently, in some cases, ketocarotenoids and apocarotenoids can fetch 1000 euro per 1mg. Thus, the project offers multiple routes for commercial exploitation that can benefit the UK's bioeconomy, creating wealth through licensing agreements, tax returns, new products, better products and both employment and enterprise opportunities.
The well-established health benefits of diets rich in fruits and vegetables containing health promoting phytonutrients such as carotenoids means that the outputs from the project have the opportunity to impact directly on societal aspect and welfare of the national. For example, the vast economic losses and welfare costs associated with diet related disease states are estimated to be 42 trillion or a 3% global GDP loss. The project has the potential to generate nutrient dense fruits with altered or intensified aesthetic properties. These improved quality traits will hopefully encourage the consumer to eat more fruit and vegetables, which is one of the government's policies for improving the Nation's health (www.doh.gov.uk/fiveaday). The absence of health promoting carotenoids (provitamin A) in staple crops found in low income countries has resulted in significant activities to deliver humanitarian products rich in provitamin A. These have predominantly attempted to manipulate biosynthetic pathways. The strategy postulated in OptiCar could offer an alternative approach through reduced degradation. Thus, the project outputs could be exploited to address key Millennium Development goals.
The impact of the proposed project on training and skills development both directly to the project participants and beyond, has been outlined in the pathways to impact section.