Overexpression of arogenate dehydratase reveals an upstream point of metabolic control in phenylalanine biosynthesis

Abstract

Out of the three aromatic amino acids, the highest flux in plants is directed towards phenylalanine, which is utilized to synthesize proteins and thousands of phenolic metabolites contributing to plant fitness. Phenylalanine is produced predominantly in plastids via the shikimate pathway and subsequent arogenate pathway, both of which are subject to complex transcriptional and post-transcriptional regulation. Previously, it was shown that allosteric feedback inhibition of arogenate dehydratase (ADT), which catalyzes the final step of the arogenate pathway, restricts flux through phenylalanine biosynthesis. Here, we show that in petunia (Petunia hybrida) flowers, which typically produce high phenylalanine levels, ADT regulation is relaxed, but not eliminated. Moderate expression of a feedback-insensitive ADT increased flux towards phenylalanine, while high overexpression paradoxically reduced phenylalanine formation. This reduction could be partially, but not fully, recovered by bypassing other known metabolic flux control points in the aromatic amino acid network. Using comparative transcriptomics, reverse genetics, and metabolic flux analysis, we discovered that transcriptional regulation of the D-ribulose-5-phosphate 3-epimerase gene in the pentose phosphate pathway controls flux into the shikimate pathway. Taken together, our findings reveal that regulation within and upstream of the shikimate pathway shares control over phenylalanine biosynthesis in the plant cell.

Revisiting the dual pathway hypothesis of Chorismate production in plants

“…In addition to how the different pathway would derive precursors from, and provide substrate for, upstream and downstream metabolic processes, the two pathways would likely differ in their interactions with other co-localized pathways. This has already been observed with a metabolic interaction of the cytosolic Phe pathway with Trp-dependent auxin biosynthesis in which shared intermediates of the pathways create cross-talk [ 63 , 66 , 67 ]. While some such interactions could be beneficial, such as when contributing to metabolic coordination, other interactions may unproductive or even detrimental.…”

“…This would ensure that growth-related production of AAAs and their derivatives is restricted during periods when insufficient precursors are being produced to sustain other cellular processes. This effect would be compounded with the previously reported sensitivity of the DAHPS reaction rate to substrate pool sizes [ 62 , 63 ]. Any cytosolic shikimate pathway would not be exposed to the same diurnal redox variation, and thus would be better suited to non-growth related processes, such as stress-response metabolite production, that need not be intrinsically linked to photosynthetic precursor supply.…”

“…Due to the plethora of metabolites derived from, and biological processes dependent on, the shikimate pathway, this pathway and its downstream branches are frequent targets for agricultural enhancement. Previous metabolic engineering strategies, which have almost exclusively targeted the plastidial pathways, have had mixed success in modulating phytochemical production [ 63 , 67 ]. An active parallel pathway, or even individual steps, presents new targets for engineering, increasing our biotechnological toolbox.…”

“…An active parallel pathway, or even individual steps, presents new targets for engineering, increasing our biotechnological toolbox. Furthermore, due to the interconnected nature of metabolism, any engineering attempt is subject to contextual constraints that limit the expected outcome, as has previously been reported in AAA production [ 63 , 66 ]. Utilization of plant compartmentalization in biotechnology approaches is a promising method for optimizing metabolic engineering [ 70 ].…”

Strong Feedback Inhibition of Key Enzymes in the Morphine Biosynthetic Pathway from Opium Poppy Detectable in Engineered Yeast

“…In plants, the biosynthesis of aromatic amino acids (i.e., L-tryptophan, L -phenylalanine, and L-tyrosine) is regulated by feedback inhibition. , Overexpression of natural enzyme variants less sensitive or insensitive to feedback inhibition (i.e., anthranilate synthase, arogenate dehydrogenase, and arogenate dehydratase) has been used to increase aromatic amino acid production. − In engineered yeast, efforts have focused on reducing the feedback regulation of tyrosine hydroxylase by exploiting plant and nonplant enzyme variants and mutants to increase the formation of L-DOPA and improve alkaloid titers. ,, A similar approach could be considered to reduce the product inhibition of 4′OMT and SalAT/THS2. However, the opium poppy SalAT showed superior performance in an engineered yeast devoid of THS2 compared with SalAT from Papaver bracteatum and Papaver orientale .…”