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Metab Eng. 2014 Sep;25:72-81. doi: 10.1016/j.ymben.2014.06.004. Epub 2014 Jun 17.

Engineering E. coli for the biosynthesis of 3-hydroxy-γ-butyrolactone (3HBL) and 3,4-dihydroxybutyric acid (3,4-DHBA) as value-added chemicals from glucose as a sole carbon source.

Author information

1
Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room E17-504, Cambridge, MA 02139, USA; Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
2
Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Microbiology Graduate Program, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3
Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room E17-504, Cambridge, MA 02139, USA; Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: kljp@mit.edu.

Abstract

3-hydroxy-γ-butyrolactone (3HBL) is a versatile chiral synthon, deemed a top value-added chemical from biomass by the DOE. We recently reported the first biosynthetic pathway towards 3HBL and its hydrolyzed form, 3,4-dihydroxybutyric acid (3,4-DHBA) in recombinant Escherichia coli using glucose and glycolic acid as feedstocks and briefly described their synthesis solely from glucose. Synthesis from glucose requires integration of the endogenous glyoxylate shunt with the 3,4-DHBA/3HBL pathway and co-overexpression of seven genes, posing challenges with respect to expression, repression of the glyoxylate shunt and optimal carbon distribution between the two pathways. Here we discuss engineering this integration. While appropriate media and over-expression of glyoxylate shunt enzymes helped overcome repression, two orthogonal expression systems were employed to address the expression and carbon distribution challenge. Synthesis of up to 0.3g/L of 3HBL and 0.7g/L of 3,4-DHBA solely from glucose was demonstrated, amounting to 24% of the theoretical maximum.

KEYWORDS:

3-hydroxy-γ-buytrolactone; Glycolate; Glyoxylate shunt; Hydroxyacids; Metabolic burden; Orthogonal expression systems; T7 RNAP based expression

PMID:
24954784
DOI:
10.1016/j.ymben.2014.06.004
[Indexed for MEDLINE]

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