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Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses.

ABSTRACT: To obtain an efficient ethanologenic Escherichia coli strain, we reduced the functional space of the central metabolic network, with eight gene knockout mutations, from over 15,000 pathway possibilities to 6 pathway options that support cell function. The remaining pathways, identified by elementary mode analysis, consist of four pathways with non-growth-associated conversion of pentoses and hexoses into ethanol at theoretical yields and two pathways with tight coupling of anaerobic cell growth with ethanol formation at high yields. Elimination of three additional genes resulted in a strain that selectively grows only on pentoses, even in the presence of glucose, with a high ethanol yield. We showed that the ethanol yields of strains with minimized metabolic functionality closely matched the theoretical predictions. Remarkably, catabolite repression was completely absent during anaerobic growth, resulting in the simultaneous utilization of pentoses and hexoses for ethanol production.

SUBMITTER: Trinh CT

PROVIDER: S-EPMC2446564 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses.

Trinh Cong T CT Unrean Pornkamol P Srienc Friedrich F

Applied and environmental microbiology 20080418 12

To obtain an efficient ethanologenic Escherichia coli strain, we reduced the functional space of the central metabolic network, with eight gene knockout mutations, from over 15,000 pathway possibilities to 6 pathway options that support cell function. The remaining pathways, identified by elementary mode analysis, consist of four pathways with non-growth-associated conversion of pentoses and hexoses into ethanol at theoretical yields and two pathways with tight coupling of anaerobic cell growth ...[more]

PMID: 18424547

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Project description:BackgroundThe biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable.ResultsIn the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (> 0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol.ConclusionsThe biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.

Dataset Information

Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses.

Publications

Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses.

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