Two independently evolved natural mutations additively deregulate TyrA enzymes and boost tyrosine production in planta.

l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure-function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.

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

The evolution of l-DOPA 4,5-dioxygenase activity, encoded by the gene DODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that l-DOPA 4,5-dioxygenase activity evolved via a single Caryophyllales-specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear. To address this, we functionally characterised 23 distinct DODA proteins for l-DOPA 4,5-dioxygenase activity, from four betalain-pigmented and five anthocyanin-pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of l-DOPA 4,5-dioxygenase activity. We find that low l-DOPA 4,5-dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated l-DOPA 4,5-dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro-synteny. In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated l-DOPA 4,5-dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.

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

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.

On-line stable isotope gas exchange reveals an inducible but leaky carbon concentrating mechanism in Nannochloropsis salina.

Carbon concentrating mechanisms (CCMs) are common among microalgae, but their regulation and even existence in some of the most promising biofuel production strains is poorly understood. This is partly because screening for new strains does not commonly include assessment of CCM function or regulation despite its fundamental role in primary carbon metabolism. In addition, the inducible nature of many microalgal CCMs means that environmental conditions should be considered when assessing CCM function and its potential impact on biofuels. In this study, we address the effect of environmental conditions by combining novel, high frequency, on-line (13)CO2 gas exchange screen with microscope-based lipid characterization to assess CCM function in Nannochloropsis salina and its interaction with lipid production. Regulation of CCM function was explored by changing the concentration of CO2 provided to continuous cultures in airlift bioreactors where cell density was kept constant across conditions by controlling the rate of media supply. Our isotopic gas exchange results were consistent with N. salina having an inducible "pump-leak" style CCM similar to that of Nannochloropsis gaditana. Though cells grew faster at high CO2 and had higher rates of net CO2 uptake, we did not observe significant differences in lipid content between conditions. Since the rate of CO2 supply was much higher for the high CO2 conditions, we calculated that growing cells bubbled with low CO2 is about 40 % more efficient for carbon capture than bubbling with high CO2. We attribute this higher efficiency to the activity of a CCM under low CO2 conditions.