Elucidation of the first step in betalain biosynthesis allows the heterologous production of betalain pigments in plants

Betalains are tyrosine-derived red-violet and yellow pigments found in plants only of the Caryophyllales order, which hold both scientific and economic values. Their pH in-dependence and high stability make them a natural pigment of choice for food industries. Their strong antioxidant activities have prompted research into their potential health-promoting properties and led to commercialization of a variety of betalain-based dietary supplements. While the biosynthetic process of many natural colorants is well understood, many questions remain open with regards to biosynthesis of betalains. Transcriptome analysis of the betalain-producing plants red beet (Beta vulgaris) and four o'clocks (Mirabilis jalapa) led us to the identification of a novel betalain-related cytochrome P450-type gene, CYP76AD6, which catalyzes the first step in the betalain biosynthetic pathway, namely the 3-hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine (L-DOPA) [1]. L-DOPA formation in red beet was found to be redundantly catalyzed by CYP76AD6 together with a known betalain-related enzyme, CYP76AD1. Gene silencing assays and recombinant expression in Nicotiana benthamiana and yeast cells revealed that while CYP76AD1 catalyzes both L-DOPA formation and its subsequent conversion to cyclo-DOPA, CYP76AD6 uniquely exhibits only tyrosine hydroxylase activity. The new findings enabled us to engineer stable betalain production through heterologous expression of three genes taking part in the fully decoded betalain biosynthetic pathway, namely CYP76AD1 and DOPA 4, 5-dioxygenase (BvDODA1) from red beet, and cyclo-DOPA-5-O-glucosyltransferase from four o'clocks (cDOPA5GT). High-quantity betalain production was achieved in a number of plant species, including tobacco (135 mg/kg fresh weight, leaf tissue) tomato (200 mg/kg, fruit) and eggplant (120 mg/kg, fruit). These betalain-producing transgenic plants offer an exceptional opportunity to study for example the health-promoting properties of betalains.

Keywords: Betalains, pigments, L-DOPA, plant metabolic engineering, secondary metabolism.

References:

[1] Polturak G, Breitel D, Grossman N, Sarrion-Perdigones A, Weithorn E, Pliner M, Orzaez D, Granell A, Rogachev I, Aharoni A. Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants. New Phytol 2016; 210: 269 – 283

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