Mechanistic investigation of a D to N mutation in DAHP synthase that dictates carbon flux into the shikimate pathway in yeast

Abstract

Abstract3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key enzyme in the shikimate pathway for the biosynthesis of aromatic compounds. L-Phe and L-Tyr bind to the two main DAHPS isoforms and inhibit their enzyme activities, respectively. Synthetic biologists aim to relieve such inhibitions in order to improve the productivity of aromatic compounds. In this work, we reported a point mutant of yeast DHAPS, Aro3D154N, which retains the wild type enzyme activity but converts it highly inert to the inhibition by L-Phe. The Aro3 crystal structure along with the molecular dynamics simulations analysis suggests that the D154N mutation distant from the inhibitor binding cavity may reduce the binding affinity of L-Phe. Growth assays demonstrated that substitution of the conserved D154 with asparagine suffices to relieve the inhibition of L-Phe on Aro3, L-Tyr on Aro4, and the inhibitions on their corresponding homologues from diverse yeasts. The importance of our discovery is highlighted by the observation of 29.1% and 43.6% increase of yield for the production of tyrosol and salidroside respectively upon substituting ARO3 with ARO3D154N. We anticipate that this allele would be used broadly to increase the yield of various aromatic products in metabolically diverse microorganisms.

In situ monitoring of the shikimate pathway: a combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging

“…This pathway is present only in plants and microbes, like bacteria and yeast, but never in animals. 1,2 It not only produces aromatic amino acids but also many other important aromatic intermediates and aromatic biomolecules such as chorismate, dehydroshikimate, folates, salicylic acid, resveratrol, flavonoids, dopamine, and alkaloids. These molecules have a wide range of applications in developing value-added aromatic compounds for the food, agrochemical, fuel additive, cosmetic, dye, pharmaceutical and other industries (Fig.…”

“…1). 2,3 Over 20% of all fixed carbon flows through this pathway. 4 The shikimate pathway also plays an important role mediating the production of antiviral drugs such as oseltamivir.…”

In situ monitoring of the Shikimate pathway: A combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging.

“…This pathway is present only in plants and microbes like bacteria and yeast but never in animals. 1,2 The pathway not only secretes aromatic amino acids but many other important aromatic intermediates and aromatic biomolecules such as chorismite, dehydroshikimate, folates, salicylic acid, resveratrol, flavonoids, dopamine, and alkaloids. These molecules have a wide range of applications in developing value-added aromatic compounds for food, agrochemical, fuel additive, cosmetics, dye, pharmaceutical and other industries, Figure 1.…”

“…These molecules have a wide range of applications in developing value-added aromatic compounds for food, agrochemical, fuel additive, cosmetics, dye, pharmaceutical and other industries, Figure 1. 2,3 Over 20% of all the fixed carbon flows through this pathway. 4 Shikimate pathway also plays an important intermediate in the production of antiviral drugs such as oseltamivir.…”