Enhanced production of d-(−)-3-hydroxybutyric acid by recombinantEscherichia coli

Abstract

Wild‐type bacteria including Escherichia coli normally do not produce extracellular d‐(−)‐3‐hydroxybutyric acid (3HB). To produce extracellular chiral 3HB, a new pathway for synthesis of 3HB was constructed by simultaneous expression of genes of β‐ketothiolase (phb A), acetoacetyl‐CoA reductase (phb B), phosphor‐transbutyrylase (ptb) and butyrate kinase (buk) in E. coli strain DH5α. E. coli DH5α containing any one of the four plasmids pBHR69, pUCAB, p68CM or pKKAB that harbor the phb A and phb B genes produced small amounts of 3HB, ranging from 75 to 400 mg l−1, while E. coli DH5α harboring p68CMPTK containing genes of phb A, phb B, ptb and buk increased the 3HB concentration to 1.4 g l−1 in shake flasks supplemented with LB broth and 20 g l−1 glucose. 3HB production was further improved to over 2 g l−1 in shake flasks when E. coli DH5α hosted two plasmids simultaneously that separately contained phb A and phb B in one plasmid while ptb and buk in the other. A batch fermentation run in a 5‐l fermenter produced approximately 5 g l−1 3HB after 24 h. A fed‐batch process increased 3HB production to 12 g l−1 after 48 h of fermentation.

Thermophilic Coenzyme B 12 -Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of ( R )-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA

“…Thus, the complete route from the common metabolite acetyl-CoA via acetoacetyl-CoA and (R)-3-hydroxybutyryl-CoA to 2-hydroxyisobutyryl-CoA would be introduced. As already demonstrated previously (26), E. coli cultures expressing phaAB readily accumulated 3-hydroxybutyric acid. A concentration of 3-hydroxybutyric acid of up to 17.7 mM formed within 8 days of feeding pCDFDuet-1 [phaA-phaB]-transformed strain BL21(DE3) with gluconic acid as the main carbon source (Fig.…”

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives

“…It was also reported that R-HAs can be produced by recombinant organisms directly, without going through the PHA hydrolysis process (Chen and Wu 2005b; Gao et al 2002; Zhao et al 2003). For example, recombinant E. coli HB101 harboring PHB precursor genes phaA and phaB of R. eutropha was able to produce more than 1 g/L of 3HB monomer extracellularly after 48 h of fermentation (Wu et al 2003).…”

Asymmetric Synthesis of α-Unsubstituted β-Hydroxy Acids

“…The degradation then starts with a depolymerization by depolymerase. All these enzymes are expressed in single recombinant bacteria to produce (R)-3-hydroxybutyric acid [38,39]. Fermenting baker's yeast can also reduce a number of aliphatic 3-oxo-carboxylic acids to the corresponding (R)--hydroxy acids with high ee [40].…”