Redirecting Primary Metabolism to Boost Production of Tyrosine-Derived Specialised Metabolites in Planta

Abstract

L-Tyrosine-derived specialized metabolites perform many important functions in plants, and have valuable applications in human health and nutrition. A necessary step in the overproduction of specialised tyrosine-derived metabolites in planta is the manipulation of primary metabolism to enhance the availability of tyrosine. Here, we utilise a naturally occurring de-regulated isoform of the key enzyme, arogenate dehydrogenase, to re-engineer the interface of primary and specialised metabolism, to boost the production of tyrosine-derived pigments in a heterologous plant host. Through manipulation of tyrosine availability, we report a 7-fold increase in the production of tyrosine-derived betalain pigments, with an upper range of 855 mg·kg−1·FW, which compare favourably to many in vitro and commercial sources of betalain pigments. Since the most common plant pathway for tyrosine synthesis occurs via arogenate, the de-regulated arogenate dehydrogenase isoform is a promising route for enhanced production of tyrosine-derived pharmaceuticals in diverse plant hosts.

The evolution of betalain biosynthesis in Caryophyllales

“…As a result, these deregulated ADHα enzymes are able to synthesize higher concentrations of tyrosine in in vitro assays relative to the canonical tyrosine‐sensitive ADHβ, with 10‐fold increases in tyrosine reported on transient assay in N. benthamiana (Lopez‐Nieves et al ., ). A subsequent study coupled the deregulated ADHα to a module comprising the complete betalain biosynthesis pathway to demonstrate that increased availability of tyrosine results in as much as a seven‐fold increase in betalain pigmentation on transient assay in N. benthamiana (Timoneda et al ., ). Together these studies imply an intimate link between the evolution of Caryophyllales‐specific deregulated ADHα, and Caryophyllales‐specific betalain biosynthesis.…”

“…An orthologue of cDOPA5GT was also characterized from the distantly related Celosia cristata (Sasaki et al, 2005), indicating that this class of enzyme is broadly conserved across Caryophyllales. Furthermore, M. jalapa cDOPA5GT has been included in multigene constructs containing the other betalain synthesis genes designed for heterologous accumulation of stable betacyanins, suggesting the importance of cDOPA5GT for betacyanin pigmentation (Polturak et al, 2016(Polturak et al, , 2017Timoneda et al, 2018).…”

Evolution of l‐DOPA 4,5‐dioxygenase activity allows for recurrent specialisation to betalain pigmentation in Caryophyllales

“…Construction of the multigene vectors containing the genes of the betalain biosynthetic pathway (DODA, BvCYP76AD1, MjcDOPA-5GT) was carried out using MoClo GoldenGate cloning following the protocol described (Engler et al, 2014;Timoneda et al, 2018) in order to produce level 2 binary vectors ( Fig. S2).…”

“…Upon transient transformation in N. benthamiana, DODA genes encoding enzymes that carry out the L-DOPA 4,5-dioxgenase reaction necessary for betalamic acid production will produce betalains. For instance, the previously characterised B. vulgaris DODA, BvDODAa1 (Hatlestad et al, 2012), exhibits high levels of L-DOPA 4,5-dioxygenase activity as inferred by a high level of betacyanin production under heterologous expression (Polturak et al, 2016;Timoneda et al, 2018). Accordingly, we use the production of betacyanin in this heterologous assay as a proxy for L-DOPA 4,5-dioxygenase activity, with the relative strength of betacyanin production indicating the relative strength of L-DOPA 4,5-dioxygenase activity between loci.…”

“…1). The core genes encoding betalain synthesis enzymes have been elucidated, primarily through heterologous assays in Saccharomyces cerevisiae and Nicotiana benthamiana, which has emerged as an essential tool for betalain research in planta (Polturak et al, 2016;Timoneda et al, 2018). The key enzymatic step in betalain biosynthesis involves conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) to betalamic acid, the central chromophore of betalain pigments.…”