Applied Microbiology and Biotechnology

, Volume 97, Issue 1, pp 205–210 | Cite as

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

Biotechnologically relevant enzymes and proteins


(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 13C 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.


Chiral synthesis Enantioselective Production rate Polyhydroxybutyrate Polyhydroxyalkanoate 



We thank J.M. Nduko for the technical assistance of HPLC analysis. E. coli strain was provided by National BioResource Project, Japan. This work was financially supported by Showa Denko K. K. (Japan). Pacific Edit reviewed the manuscript prior to submission.


  1. Arai Y, Nakashita H, Suzuki Y, Kobayashi Y, Shimizu T, Yasuda M, Doi Y, Yamaguchi I (2002) Synthesis of a novel class of polyhydroxyalkanoates in Arabidopsis peroxisomes, and their use in monitoring short-chain-length intermediates of β-oxidation. Plant Cell Physiol 43:555–562CrossRefGoogle Scholar
  2. de Roo G, Kellerhals MB, Ren Q, Witholt B, Kessler B (2002) Production of chiral R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters via hydrolytic degradation of polyhydroxyalkanoate synthesized by pseudomonads. Biotechnol Bioeng 77:717–722CrossRefGoogle Scholar
  3. Gao HJ, Wu Q, Chen GQ (2002) Enhanced production of D-(−)-3-hydroxybutyric acid by recombinant Escherichia coli. FEMS Microbiol Lett 213:59–65Google Scholar
  4. Iimori T, Shibasaki M (1986) Simple, stereocontrolled synthesis of 1β-methylcarbapenem antibiotics from 3(R)-hydroxybutyric acid. Tetrahedron Lett 27:2149–2152CrossRefGoogle Scholar
  5. Jossek R, Reichelt R, Steinbüchel A (1998) In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum. Appl Microbiol Biotechnol 49:258–266CrossRefGoogle Scholar
  6. Lee SY, Lee Y (2003) Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(−)-hydroxycarboxylic acids. Appl Environ Microbiol 69:3421–3426CrossRefGoogle Scholar
  7. Liu Q, Ouyang SP, Chung A, Wu Q, Chen GQ (2007) Microbial production of R-3-hydroxybutyric acid by recombinant E. coli harboring genes of phbA, phbB, and tesB. Appl Microbiol Biotechnol 76:811–818CrossRefGoogle Scholar
  8. Rehm BHA (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15–33CrossRefGoogle Scholar
  9. Ren Q, Ruth K, Thony-Meyer L, Zinn M (2010) Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives. Appl Microbiol Biotechnol 87:41–52CrossRefGoogle Scholar
  10. Shiraki M, Endo T, Saito T (2006) Fermentative production of (R)-(−)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli. J Biosci Bioeng 102:529–534CrossRefGoogle Scholar
  11. Taguchi S, Yamada M, Matsumoto K, Tajima K, Satoh Y, Munekata M, Ohno K, Kohda K, Shimamura T, Kambe H, Obata S (2008) A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme. Proc Natl Acad Sci U S A 105:17323–17327CrossRefGoogle Scholar
  12. Tappel RC, Wang Q, Nomura CT (2012) Precise control of repeating unit composition in biodegradable poly(3-hydroxyalkanoate) polymers synthesized by Escherichia coli. J Biosci Bioeng 113:480–486CrossRefGoogle Scholar
  13. Tokiwa Y, Ugwu CU (2007) Biotechnological production of (R)-3-hydroxybutyric acid monomer. J Biotechnol 132:264–272CrossRefGoogle Scholar
  14. Tseng HC, Martin CH, Nielsen DR, Prather KL (2009) Metabolic engineering of Escherichia coli for enhanced production of (R)- and (S)-3-hydroxybutyrate. Appl Environ Microbiol 75:3137–3145CrossRefGoogle Scholar
  15. Tung KK, Wood WA (1975) Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii. J Bacteriol 124:1462–1474Google Scholar
  16. Uchino K, Saito T, Jendrossek D (2008) Poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 is involved in mobilization of accumulated PHB in Ralstonia eutropha H16. Appl Environ Microbiol 74:1058–1063CrossRefGoogle Scholar
  17. Ugwu CU, Tokiwa Y, Ichiba T (2011) Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata. Bioresour Technol 102:6766–6768CrossRefGoogle Scholar
  18. Vollbrecht D, Schlegel HG (1979) Excretion of metabolites of hydrogen bacteria III. D(−)-3-hydroxybutanoate. Eur J Appl Microbiol 7:259–266CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Ken’ichiro Matsumoto
    • 1
  • Takehiro Okei
    • 1
  • Inori Honma
    • 1
  • Toshihiko Ooi
    • 1
  • Hirobumi Aoki
    • 1
    • 2
  • Seiichi Taguchi
    • 1
  1. 1.Division of Biotechnology and Macromolecular Chemistry, Graduate School of EngineeringHokkaido UniversitySapporoJapan
  2. 2.Corporate R&D CenterKawasakiJapan

Personalised recommendations