A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the β-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl β-d-thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the β-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l−1 from 20 g l−1 of glucose in Luria–Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l−1 (OD600 = 30 and 60), respectively. When protein expression was induced at 60 of OD600 and glucose was fed to the concentration of 15 g l−1, 10.3 g l−1 of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l−1h−1. Lowering glucose concentration to 5 g l−1 and induction of protein expression at 30 of OD600 significantly reduced final S3HB concentration to 3.7 g l−1, which also resulted in the decrease of the S3HB productivity to 0.05 g l−1h−1.
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Dr. Soon Ho Hong appreciates the supports from the 2007 Research Fund of University of Ulsan.
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Lee, S., Park, S.J., Lee, S.Y. et al. Biosynthesis of enantiopure (S)-3-hydroxybutyric acid in metabolically engineered Escherichia coli . Appl Microbiol Biotechnol 79, 633–641 (2008). https://doi.org/10.1007/s00253-008-1473-7
- (S)-3-hydroxybutyric acid
- Escherichia coli
- Metabolic engineering
- Fed-batch culture