Construction of a synthetic metabolic pathway for the production of 2,4-dihydroxybutyric acid from homoserine

Thomas Walther,Florence Calvayrac,Yoann Malbert,Ceren Alkim,Clémentine Dressaire,Hélène Cordier,Jean Marie François

doi:10.1016/j.ymben.2017.12.005

Abstract

2,4-dihydroxybutyrate (DHB) is a precursor for the chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate. Since no annotated metabolic pathway exists for its microbial production from sugar, we have conceived a two-step synthetic metabolic pathway which converts the natural amino acid homoserine to DHB. The pathway proceeds through the homoserine transaminase-catalyzed deamination of homoserine to obtain 2-oxo-4-hydroxybutyrate (OHB), and continues with the reduction of OHB to DHB, which is catalyzed by an OHB reductase enzyme. We identified homoserine transaminase and OHB reductase activity in several candidate enzymes which act on sterically cognate substrates, and improved OHB reductase activity of lactate dehydrogenase A of Lactococcus lactis by structure-based enzyme engineering. Fed-batch cultivation of a homoserine-overproducing Escherichia coli strain which expressed homoserine transaminase and OHB reductase enzymes resulted in the production of 5.3g/L DHB at a yield of 0.1g/g.

Highlights

The sulfur-containing essential amino acid (L)-methionine and its analogue 2-hydroxy-4(methylthio)butyrate (HMTB) are important supplements in poultry diets (Dilger and Baker, 2007; Sauer et al, 2008)
While most of the 20 proteinogenic amino acids are produced by fermentation processes from renewable sugar, the production of methionine and HMTB is still dominated by petroleum-based syntheses (Faurie et al, 2003; Leuchtenberger et al, 2005; Willke, 2014)
Direct cost-efficient microbial synthesis of methionine or its analogue HMTB from sugar is hampered by both the high metabolic cost of sulfur incorporation (Kromer et al, 2006) and the comparatively low solubility of methionine which limits product concentrations at physiological temperatures and near neutral pH to approximately 50 g/L (Fuchs et al, 2006). To tackle these technological problems, we have recently proposed a two-stage process which includes the fermentative production of DHB from sugar (Walther et al, 2017), and the subsequent carbonconserving chemical incorporation of methanethiol to produce HMTB (Deck et al, 2009)

Summary

Introduction

The sulfur-containing essential amino acid (L)-methionine and its analogue 2-hydroxy-4(methylthio)butyrate (HMTB) are important supplements in poultry diets (Dilger and Baker, 2007; Sauer et al, 2008). While most of the 20 proteinogenic amino acids are produced by fermentation processes from renewable sugar, the production of methionine and HMTB is still dominated by petroleum-based syntheses (Faurie et al, 2003; Leuchtenberger et al, 2005; Willke, 2014) This is mainly due to the very high metabolic cost of incorporating sulfur which requires the NADPH-dependent reduction of the standard sulfur source sulfate to sulfide (Krömer et al, 2006). An elegant two-stage industrial production process for (L)-methionine was recently developed in which the precursors Oacetylhomoserine or O-succinyl-homoserine are produced by optimized microorganisms through the fermentation of sugars (Hong et al, 2014; Shim et al, 2016). It was proposed that acetate or succinate, which is released during the enzymatic conversion of the precursor, can be recycled as substrate or be commercialized (Shim et al, 2016)

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Conclusion