Implementation of Synthetic Metabolic Pathways for the Production of 2,4-Dihydroxybutyric Acid

Implementation of Synthetic Metabolic Pathways for the Production of 2,4- Dihydroxybutyric Acid

Thomas Walther1-4, Clément Auriol1-4, Romain Irague1-4, Christopher M. Topham1-4, Clémentine Dressaire1-4, Audrey Baylac1-4, Florence Calvayrac1-4, Hélène Serrano-Bataille1-4, Julie Fredonnet1-4, Hélène Cordier1-4, Isabelle André1-4, Marc Maestracci5, Robert Huet5, Magali Remaud-Simeon1-4, Jean Marie François1-4

1Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, 31077 Toulouse, France ;

2INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France ; 3CNRS, UMR5504, Toulouse, France ; 4TWB, 3 rue des Satellites, Canal Biotech Building 2, 31400 Toulouse, France; 5Adisseo SA, Antony Parc II, 10 place General de Gaulle, 92160 Antony, France.

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, petrochemical synthesis of DHB is not economically viable, and no natural metabolic pathways exist for the biochemical production of DHB. We have therefore conceived three synthetic metabolic pathways for the synthesis of DHB starting from the natural metabolites malate or
homoserine.
(I)*

ATP ADP

malate

CoASH, ATP ADP, Pi

(IV)

glyoxylate

Acetyl CoA

(V)
malyl-Pi
malyl-CoA

NAD(P)H NAD(P)+, Pi

(II)*
malate

(VI)*

NAD(P)H NAD(P)+, CoASH

(VII)* (VIII)*

semialdehyde

NAD(P)H

2-oxogluta glutamate rate

NAD(P)H NAD(P)+

(III)*

NAD(P)+

homoserine
2-oxo-4- hydroxybutyrate

2,4-dihydroxybutyrate

(DHB)

Synthetic metabolic pathways for the biosynthesis of 2,4-dihydroxybutyric acid. (*) Previously unreported enzymatic activities. (I) malate kinase, (II) malate semialdehyde dehydrogenase, (III) malate semialdehyde reductase, (IV) malyl-CoA synthetase, (V) malyl-CoA-lyase, (VI) malyl-CoA reductase, (VII) homoserine transaminase, (VIII) 2-oxo-4-hydroxybutyrate reductase.

The two pathways departing from malate proceed via activation of malate by phosphorylation or acylation to yield malylphosphate or malyl-CoA, respectively. These compounds are then subject to two successive rounds of reduction to yield DHB via the common intermediate malate semialdehyde.
Synthesis of DHB from homoserine requires deamination of homoserine to yield 2-oxo-4- hydroxybutyrate followed by the reduction of the latter to obtain DHB.
The pathways employ several previously unreported enzymatic activities. Those activities were obtained by rational design based on structural and mechanistic knowledge, and/or by screening of candidate enzymes acting on sterically similar cognate substrates.
Each of the individual pathways has its particular thermodynamic and physiological requirements which were met by rational strain design. The pathways were expressed in genetically optimised Escherichia coli strains, and production of DHB from glucose was demonstrated.