Study of the Potential of the Reversal of the Fatty-Acid Beta-Oxidation Pathway for Stereoselective Biosynthesis of ( S

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UCERS, BIOLOGY, SELECTION, AND GENE ENGINEERING

Study of the Potential of the Reversal of the Fatty-Acid Beta-Oxidation Pathway for Stereoselective Biosynthesis of (S)-1,3-Butanediol from Glucose by Recombinant Escherichia coli Strains A. Yu. Gulevicha, *, A. Yu. Skorokhodovaa, and V. G. Debabova aKurchatov Institute National Research Center, State Research Institute for Genetics and Selection of Industrial Microorganisms

(Kurchatov Institute NRC—GOSNIIgenetika), Moscow, 117545 Russia *e-mail: [email protected] Received August 1, 2019; revised August 20, 2019; accepted August 20, 2019

Abstract—The possible contribution of collateral enzymes to the formation of the key precursor metabolite, 3-hydroxybutyryl-CoA, has been evaluated in a recombinant Escherichia coli strain engineered for 1,3-butanediol biosynthesis from glucose via the inverted fatty-acid beta-oxidation pathway. Inactivation of the 3-hydroxyadipyl-CoA dehydrogenase gene, paaH, did not prevent 1,3-butanol biosynthesis during anaerobic glucose utilization by a strain with an intact, essential gene, fabG. This gene encodes 3-ketoacyl-ACP reductase, which can catalyze the conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. The subsequent inactivation in the strain of the fadB gene, which encodes (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase of the fatty-acid beta oxidation led to the cessation of 1,3-butanediol synthesis. The respective diol was also not found among the products secreted by the strain possessing the intact fabG and paaH genes upon the individual deletion of the fadB gene. It was established that the collateral enzymes did not participate in the formation of 3-hydroxybutyryl-CoA in the studied strains, and the respective CoA derivative was synthesized solely by the (S)-specific enzyme of the fatty-acid beta-oxidation pathway. The results indicate that reversal of the fatty-acid beta oxidation pathway can ensure the enantioselective biosynthesis of the (S)-stereoisomer of 1,3-butanediol in engineered E. coli strains. Keywords: 1,3-butanediol, fatty-acid beta oxidation, Escherichia coli, glucose, metabolic engineering, stereoisomer DOI: 10.1134/S0003683820080049

INTRODUCTION The current strategies for sustainable development require a consistent but steady transition from petrochemical processes in the organic synthesis of useful compounds to corresponding processes based on microbial biotechnology [1]. Among the products of industrial biotechnology, increased attention is attracted by substances that can serve as flexible building blocks in the subsequent synthesis of a wide range of high value–added chemicals. Compounds with optically active centers play a special role among these building blocks due to their promise as chiral synthons. For instance, (R)- and (S)-stereoisomers of 3hydroxycarboxylates, and 1,3-diols are convenient synthons for the obtainment of various macrolide and beta-lactam antibiotics and the needed agricultural pesticides and insecticides [2, 3]. The enantioselective Abbreviations: ACP—acyl carrier pr