Building the Synthetic Biology Toolbox with Enzyme Variants to Expand Opportunities for Biofortification of Provitamin A and Other Health-Promoting Carotenoids

Carotenoids are a large class of
structures that are important
in human health and include both provitamin A and nonprovitamin A
compounds. Vitamin A deficiency is a global health problem that can
be alleviated by enriching provitamin A carotenoids in a range of
food crops. Suitable plants for biofortification are those with high
levels of the provitamin A biosynthetic precursor, lycopene, which
is enzymatically converted by lycopene β-cyclase (LCYB) to β-carotene,
a provitamin A carotenoid. Crops, such as citrus, naturally accumulate
high levels of provitamin A and other health-promoting carotenoids.
Such plants may have useful genes to expand the synthetic biology
toolbox for producing a range of phenotypes, including both high provitamin
A crops and crops with unique compositions of health-promoting carotenoids.
To examine enzyme variants having different activity levels, we introduced
two citrus LCYB alleles into tomato, a plant with fruit rich in lycopene.
Overexpression in tomato of the stronger allele of the citrus chromoplast-specific
lycopene β-cyclase (CsLCYb2a) produced “golden”
transgenic tomato fruits with 9.3-fold increased levels of β-carotene
at up to 1.5 mg/g dry weight. The use of the weaker allele, CsLCYb2b, also led to enhanced levels of β-carotene
but in the context of a more heterogeneous composition of carotenoids.
From a synthetic biology standpoint, these allelic differences have
value for producing cultivars with unique carotenoid profiles. Overexpression
of the citrus LCYB genes was accompanied by increased
expression of other genes encoding carotenoid biosynthetic enzymes
and increased size and number of chromoplasts needed to sequester
the elevated levels of carotenoids in the transgenic tomato fruits.
The overexpression of the citrus LCYB genes also
led to a pleiotropic effect on profiles of phytohormones and primary
metabolites. Our findings show that enzyme variants are essential
synthetic biology parts needed to create a wider range of metabolic
engineering products. In this case, strong and weak variants of LCYB
proved useful in creating dietary sources to alleviate vitamin A deficiency
or, alternatively, to create crops with a heterogeneous composition
including provitamin A and healthful, nonprovitamin A carotenoids.