Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid

Abstract

2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l−1 DHB with a molar yield of 0.15.

Advancement of Metabolic Engineering Assisted by Synthetic Biology

“…In addition to fueling biosynthesis, synthetic pathways have been built for the biosynthesis of various chemical compounds, such as succinic acid [39], 2,4-dihydroxybutyric acid [40], and maleate [41], and the engineered bio-factories have already been commercialized for the food industry.…”

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms

“…To circumvent this constraint, efforts have been directed toward creating enzymes that can perform reactions that have not been possible biologically for constructing novel metabolic pathways to produce compounds without known natural biosynthesis routes. For example, the versatile chemical, 2,4-dihydroxybutyric acid (2,4-DHB), was produced by exploiting a natural metabolic pathway involving aspartate to utilize malate, a structurally similar analog, as precursor ( Walther et al, 2017 ). By engineering the enzymes in the natural pathway, novel enzymes with previously unreported activities in nature, namely malate semialdehyde reductase, malate kinase, and malate semialdehyde dehydrogenase, were generated to create an artificial 2,4-DHB-producing pathway.…”

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant

“…On the basis of this strategy, several researchers have investigated changing substrate speci city and improving enzymatic activities against unnatural substrates. 38,39,40 In this study, using a rational enzyme design based on the formation of new hydrogen bonds and improvement of the FDC-ccMA a nity, we tailored FDC mutants for producing 1,3-butadiene. FDCs derived from Aspergillus niger and Saccharomyces cerevisiae (AnFDC and ScFDC, respectively) have prFMN and can catalyze the prFMN-dependent decarboxylation of α,β-unsaturated carboxylic acids.…”