Engineering microbes and metabolic pathways for the production of ethylene glycol

a technology of metabolic engineering and ethylene glycol, which is applied in the direction of lyases, racemaces/epimerases, transferases, etc., can solve the problems of significant waste, achieve the effect of increasing the amount of 2-phosphoglycerate, increasing the amount of 3-phosphoglycerate, and reducing the flux to 2-phosphoglycera

Inactive Publication Date: 2013-11-28
MASSACHUSETTS INST OF TECH
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  • Application Information

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Benefits of technology

[0058]In some embodiments, the expression or activity in the cell of one or more phosphoglycerate mutases and/or of enolase is attenuated, thereby increasing the amount of 3-phosphoglycerate in the cell by reducing flux to 2-phosphoglycerate and/or increasi

Problems solved by technology

Additionally, the production of biodiesel results in significant amoun

Method used

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  • Engineering microbes and metabolic pathways for the production of ethylene glycol
  • Engineering microbes and metabolic pathways for the production of ethylene glycol
  • Engineering microbes and metabolic pathways for the production of ethylene glycol

Examples

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example 1

[0149]Researching E. coli for the biological production of ethylene glycol (EG) from sugars, we noted the pathway for the degradation of D-arabinose in K-12 strains. In the pathway, the pentose intermediate D-ribulose-1-phosphate is cleaved to yield dihydroxyacetone phosphate (DHAP), which enters into glycolysis, and glycolaldehyde. Glycolaldehyde is typically oxidized to glycolate by aldehyde dehydrogenase A (encoded by the aldA gene), but as in the analogous reaction of L-lactaldehyde to L-1,2-propanediol, glycolaldehyde can also be reduced by the oxidoreductase encoded by fucO. The fluxes through these reactions depend on the availability of oxygen, so we investigated the degradation of D-arabinose under aerobic and anaerobic conditions.

[0150]Cultures of E. coli K-12 MG1655 DE3 ΔendA ΔrecA (referred to as wild-type or WT) and E. coli K-12 MG1655 DE3 ΔendA ΔrecA ΔaldA (referred to as ΔaldA) were grown aerobically and anaerobically on minimal medium with 4 g / L D-arabinose as the ca...

example 2

[0151]Though we have shown that E. coli is capable of generating ethylene glycol from D-arabinose, biomass-derived sugars, such as D-xylose and D-glucose, are much more abundant and therefore are preferred substrates. Because D-xylose is a pentose, we next pursued the utilization of D-xylose for EG production through the previously established D-arabinose degradation pathway. D-tagatose 3-epimerase (DTE) from Pseudomonas cichorii is a promiscuous enzyme that has been shown to interconvert D-xylulose and D-ribulose [Izumori 1993], intermediates of the D-xylose degradation and D-arabinose degradation pathways, respectively. Therefore, DTE (encoded by the gene here referred to as dte) can provide a path by which D-xylose can yield EG, however, conversion of D-xylulose to D-xylulose-5-phosphate, catalyzed by xylulokinase (encoded by xylB), is a competing reaction. We generated E. coli K-12 MG1655 DE3 ΔendA ΔrecA ΔaldA ΔxylB (referred to as ΔaldA ΔxylB) and transformed the strain with a ...

example 3

[0153]To improve metabolism of D-xylose through the D-arabinose degradation pathway, we attempted to overexpress the relevant enzymes: D-ribulokinase (encoded by fucK), D-ribulose-phosphate aldolase (encoded by fucA), and glycolaldehyde reductase (encoded by fucO). These enzymes were overexpressed as part of an operon in conjunction with DTE; the order of the genes in the operon was varied. All of these were grown on D-xylose, analyzed for EG production, and compared against ΔaldA cultured on D-arabinose (FIG. 8). The best strain is ΔaldA ΔxylB / p10_T5T10-dte-fucA-fucO-fucK, which outperforms the D-arabinose results. When a plasmid of a lower copy number was used (p5_T5T10-dte-fucA-fucO-fucK), the results were similar (data not shown).

[0154]To maximize EG production, we next grew our best strain, ΔaldA ΔxylB / p5_T5T10-dte-fucA-fucO-fucK, in a bioreactor. The medium of the initial batch was minimal medium with 30 g / L D-xylose; when nearly all D-xylose was consumed, additional D-xylose ...

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Abstract

The invention relates to recombinant cells and their use in the production of ethylene glycol.

Description

RELATED APPLICATIONS[0001]This application claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application No. 61 / 602,322, filed Feb. 23, 2012, which is incorporated by reference herein in its entirety.GOVERNMENT INTEREST[0002]This invention was made with government support under Grant No. DE-AR0000059 awarded by the Department of Energy, Office of ARPA-E. The government has certain rights in this invention.FIELD OF THE INVENTION[0003]The invention relates to the production of ethylene glycol through recombinant gene expression and metabolic engineering.BACKGROUND OF THE INVENTION[0004]Ethylene glycol is an important organic compound commonly used as a precursor to polymers, primarily polyethylene terephthalate (PET) which comprises a significant share of the worlds polymer production. The major end uses of PET are synthetic fibers, commonly referred to as “polyester,” and plastic bottles. For both of these products, the demand is increasing. Another major use of ethylene...

Claims

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Application Information

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IPC IPC(8): C12P7/18C12N15/70
CPCC12P7/18C12N15/70C12Y207/01016C12Y101/01071C12Y102/01052C12Y207/01047C12Y401/02017C12Y501/03C12N9/0006C12N9/0008C12N9/0022C12N9/1096C12N9/1205C12N9/1229C12N9/16C12N9/88C12N9/90C12N15/52C12P2203/00C12Y102/01021C12Y207/01017C12P7/24
Inventor STEPHANOPOULOS, GREGORYPEREIRA, BRIANDE MEY, MARJANDUGAR, DEEPAKAVALOS, JOSE LUIS
Owner MASSACHUSETTS INST OF TECH
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