Synthetic Microbial Metabolism Refactoring for the Production of a Chemical Synthon, 2,4-Dihydroxybutyric Acid

Clément Auriol^1-4 ;Audrey Baylac^1-4, Romain Irague^1-4, Christopher Topham^1-4, Clémentine Dressaire^1-4, Jean Marie François^1-4 and Thomas Walther^1-4

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.

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. Synthetic metabolic pathways for the production of DHB have been developed that employ the previously unreported enzymatic activities malate kinase, malate semialdehyde dehydrogenase and DHB dehydrogenase [1] whereas the second pathway employs malate synthetase or malyl-CoA lyase, previously unreported malyl-CoA reductase, and malate semialdehyde reductase activities [2]. Most of these enzymes were obtained either by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically similar cognate substrates, or they were identified by screening of natural enzymes and further improved by rational design. As the two pathways depart from malate or glyoxylate, metabolic engineering of E. coli for optimizing these DHB precursors was carried out that differed from previous reported works [3]. Finally, the synthetic pathways were expressed in the genetically optimized E. coli strains, and significant in vivoproduction of DHB was obtained.

[1] Walther, T., Cordiez, H., Topham Ch., Andre I, Remaud-Simeon, M., Huet, R. & François, J. (2012)  A novel method of production of 2,4-dihydroxybutyric. Patent WO2012/056318A1

(2]Walther, T., Cordier, H., Dressaire, C. François, J. (2013). A production of 2,4-dihydroxybutyric by malyl-CoA pathway . Patent WO2013/160762A1

[3] Zhang X, Wang X, Shanmugam KT, Ingram LO (2011) L-malate production by metabolically engineered Escherichia coli. Appl Environ Microbiol 77:427-434.