Enhanced production of d-(−)-3-hydroxybutyric acid by recombinant Escherichia 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.