Development of tomato genotypes with enhanced xanthophyll content in ripe fruit

Zeaxanthin is a high-value carotenoid used as a food supplement and a colourant in
foods and animal feed in order to improve the flavour and appearance of the products.
Its nutraceutical value is derived from its role in lowering the risk of developing certain
age-related eye conditions, particularly age-related macular degeneration (AMD), which
has been concluded from large clinical trials. The present limited availability of plant
renewable sources for zeaxanthin and its complex chemical synthesis, has encouraged
engineering of the existing biosynthetic pathways in plants to create new biosources of
zeaxanthin and related xanthophylls.
A good plant-based candidate for a novel source of zeaxanthin is tomato. The ripe fruit
of tomato accumulate high amounts of the red pigment lycopene from which zeaxanthin
can be derived. Even though the tomato genome encodes all functional enzymes of the
carotenoid biosynthetic pathway necessary for the synthesis of zeaxanthin, the tomato
fruit only accumulate trace amounts of this compound. Previously, a tomato line
accumulating high levels of β-carotene, a carotenoid compound derived from lycopene,
has been identified. Zeaxanthin can be generated from β-carotene in a series of two
hydroxylation steps catalysed by β-carotene hydroxylase. Through an introduction of an
additional copy of tomato β-carotene hydroxylase into the high β-carotene background,
a new line accumulating zeaxanthin in its ripe fruit has been generated. Although, the
levels of zeaxanthin in this new tomato line were too low to consider it a novel plant
source, it was shown that the de novo synthesis and accumulation of previously
unencountered xanthophylls in tomato fruit is possible to achieve.
A strategy for the enhancement of zeaxanthin content was developed in which the
generated line was crossed with the high-pigment 3 (hp3) tomato mutant in order to
reduce epoxidation of zeaxanthin into violaxanthin. This led to a 26-fold improvement
in the fruit zeaxanthin content which was high enough to provide the daily
recommended dose of zeaxanthin in one fruit serving. Thus, a potential new, renewable
tomato source of zeaxanthin has been generated.
With the use of the new gene editing technology CRISPR/Cas9, alternative mutations in
the gene encoding zeaxanthin epoxidase were generated with the potential to use the
novel variants for further enhancement of zeaxanthin content.