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 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.
Similar content being viewed by others
References
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 43:555–562
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 77:717–722
Gao HJ, Wu Q, Chen GQ (2002) Enhanced production of D-(−)-3-hydroxybutyric acid by recombinant Escherichia coli. FEMS Microbiol Lett 213:59–65
Iimori T, Shibasaki M (1986) Simple, stereocontrolled synthesis of 1β-methylcarbapenem antibiotics from 3(R)-hydroxybutyric acid. Tetrahedron Lett 27:2149–2152
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 49:258–266
Lee SY, Lee Y (2003) Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(−)-hydroxycarboxylic acids. Appl Environ Microbiol 69:3421–3426
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 76:811–818
Rehm BHA (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15–33
Ren Q, Ruth K, Thony-Meyer L, Zinn M (2010) Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives. Appl Microbiol Biotechnol 87:41–52
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 102:529–534
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 105:17323–17327
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 113:480–486
Tokiwa Y, Ugwu CU (2007) Biotechnological production of (R)-3-hydroxybutyric acid monomer. J Biotechnol 132:264–272
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 75:3137–3145
Tung KK, Wood WA (1975) Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii. J Bacteriol 124:1462–1474
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 74:1058–1063
Ugwu CU, Tokiwa Y, Ichiba T (2011) Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata. Bioresour Technol 102:6766–6768
Vollbrecht D, Schlegel HG (1979) Excretion of metabolites of hydrogen bacteria III. D(−)-3-hydroxybutanoate. Eur J Appl Microbiol 7:259–266
Acknowledgements
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.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Matsumoto, K., Okei, T., Honma, I. et al. Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase. Appl Microbiol Biotechnol 97, 205–210 (2013). https://doi.org/10.1007/s00253-012-4104-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-012-4104-2