title: Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase.
abstract: (R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by β-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L(-1) (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L(-1), with a productivity of 0.22 g L(-1) h(-1). The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of (13)C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13W8, Kita-ku, Sapporo, 060-8628, Japan. mken@eng.hokudai.ac.jp
authors.forename: Ken'ichiro
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Takehiro
authors.initials: T
authors.lastname: Okei
authors.forename: Inori
authors.initials: I
authors.lastname: Honma
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ooi
authors.forename: Hirobumi
authors.initials: H
authors.lastname: Aoki
authors.forename: Seiichi
authors.initials: S
authors.lastname: Taguchi
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000378:metabolism*; D019342:Acetic Acid / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D015256:Coenzyme A-Transferases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering
chemical_list: D000105:Acetyl Coenzyme A; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D000102:Acetyl-CoA C-Acyltransferase; D015256:Coenzyme A-Transferases; D005947:Glucose; D019342:Acetic Acid; D020155:3-Hydroxybutyric Acid