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The microaerobic synthesis of 3-hydroxybutyric acid by the  Escherichia coli  strain BOX3.1 ∆4 P L - atoB  P L - tesB  (MG1655  lacI   Q  , ∆ ackA-pta , ∆ poxB , ∆ ldhA , ∆ adhE , ∆ fadE , P L -SD  phi10  - atoB , P  trc- ideal-4 -SD  phi10  - fadB , P L -SD  phi10  - tesB ), which was previously directly engineered for the biosynthesis of the target compound from glucose through the reversed fatty acid β-oxidation pathway, was studied. A target product yield of 0.12 mol/mol was achieved. Inactivation of the nonspecific YciA thioesterase gene in the strain led to an increase in the yield of 3-hydroxybutyric acid to 0.15 mol/mol. For the optimization of biosynthesis of target product the strain MG∆4 P L - tesB  (MG1655 ∆ ackA-pta , ∆ poxB , ∆ ldhA , ∆ adhE , P L -SD  phi10  - tesB ) was engineered, and the genes encoding key enzymes of fatty acid β-oxidation were overexpressed in the strain from the plasmid pMW118m- atoB - fadB . The level of microaerobic synthesis of 3-hydroxybutyric acid by the strain MG∆4 P L - tesB  (pMW118m- atoB - fadB ) achieved in primary evaluation conditions reached 0.35 mol/mol. Inactivation in the strain of the gene of nonspecific thioesterase YciA led to only minor decrease in acetate byproduction. Further inactivation in the strain of gene encoding nonspecific thioesterase YdiI had virtually no effect on the level of synthesis of side products. Cultivation of the constructed strain MG∆4 P L - tesB  ∆ yciA  (pMW118m- atoB - fadB ) in bioreactor under the controlled conditions ensured achievement of a yield of 3‑hydroxybutyric acid amounting to 0.75 mol/mol.

applied biochemistry and microbiology

Optimization of (S)-3-Hydroxybutyric Acid Biosynthesis from Glucose through the Reversed Fatty Acid β-Oxidation Pathway by Recombinant Escherichia coli Strains