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Applied and Environmental Microbiology, May 2009, p. 3137-3145, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02667-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate{triangledown}

Hsien-Chung Tseng,1 Collin H. Martin,1,2 David R. Nielsen,1 and Kristala L. Jones Prather1,2*

Department of Chemical Engineering,1 Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, Massachusetts 021392

Received 21 November 2008/ Accepted 17 March 2009

Synthetic metabolic pathways have been constructed for the production of enantiopure (R)- and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter–1 and 2.08 g liter–1, respectively, in shake flask cultures within 2 days. The NADPH/NADP+ ratio was found to be two- to three-fold higher than the NADH/NAD+ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.

* Corresponding author. Mailing address: Department of Chemical Engineering, 77 Massachusetts Avenue, Room 66-458, Cambridge, MA 02139. Phone: (617) 253-1950. Fax: (617) 258-5042. E-mail: kljp{at}mit.edu

{triangledown} Published ahead of print on 20 March 2009.

Applied and Environmental Microbiology, May 2009, p. 3137-3145, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02667-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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