Metaolic Engineering of Esherichia Coli for the Conversion

8/3/2019 Metaolic Engineering of Esherichia Coli for the Conversion

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Metabolic engineering ofEsherichia coli

for the conversion of renewable

resources to Bio-fuels


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Metabolic Engineering Metabolic engineering offers an alternative

approach in which synthetic pathways are

engineered into user friendly hosts for theproduction of fuel molecules.

These hosts could be readily manipulated toimprove the production efficiency.

This review summarizes recent progress in theengineering ofEscherichia coli to produceadvanced biofuels.


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The pfl gene encoding pyruvate formate-lyase

(PFL) was selected as the integration site for threereasons.

PFL represents a competing branch point for the

diversion of pyruvate away from homo-ethanolproduction in ethanologenic E. coli.

This gene is expressed at very high levels in E. coli

during anaerobic growth from at least sixpromoters .


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Genetic Improvement of Escherichia

coli for Ethanol Production:

Zymomonas mobilis genes for pyruvatedecarboxylase (pdc) and alcohol dehydrogenase II

(adhB) were integrated into the Escherichia colichromosome within or near the pyruvate formate-lyase gene (pfl).

High levels of PDC and ADHII are required for

fermentation in Z. mobilis , and similarly highlevels are essential for ethanol production byrecombinant E. coli


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MATERIALS AND METHODSBacterial strains and media;

E. coli strains and plasmids used in this study.

Luria agar containing 2% glucose and appropriateantibiotics was used for the selection of recombinants.Expression of Z. mobilis ADHII in E. colirecombinants was screened with aldehyde indicator

plates .


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Genetic procedures and recombinant techniques.

Standard procedures were used for plasmid

preparations, restriction enzyme digestions, ligations,transformations, and agarose gel electrophoresis.

E. coli TC4 was used as the host for all plasmidconstructions.


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Inactivation of pfl by chromosomal integration of Z.mobilis pdc and adhB genes.

Z. mobilis pdc and adhB were integrated into E. coli ATCC

11303 by using a derivative of the integration vectorpMAK705.

pLOI543, containing a temperature-conditional pSC101replicon. pLOI510, containing an excisable Sall fragment

which includes cat and Z. mobilis pdc and adhB betweenflanking regions of an E. coli pfl gene.


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Fermentation experiments. Fermentationswere carried out in Luria broth supplemented with10% (wt/vol) glucose or 8% (wt/vol) xylose.

A Jenco model 3671 pH controller was used tomaintain a pH of 6.0 by the addition of base.

Batch fermentations were carried out in at 30C and

were stirred continuously by a 1.25-in. (3.97-cm)star-shaped magnetic bar (100 rpm).


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Hydrogen production.

PFL cleaves pyruvate to produce acetyl CoA and

formate. In E. coli, formate is rapidly degraded toproduce a mixture of carbon dioxide and hydrogen.

Hydrogen produced during incubation was measuredby gas chromatography with a thermal conductivity

detector.


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Enzyme activity.

Heat treatment was used to inactivate competingative

enzymes which complicate measurements of PDCactivity in recombinant E. coli.


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Acknoledgement


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REFERANCE KAZUYOSHI OHTA, D. S. BEALL, J. P. MEJIA, K. T. SHANMUGAM, AND L. 0.

INGRAM*Genetic Improvement of Escherichia coli for Ethanol Production:Chromosomal Integration of Zymomonas mobilis Genes Encoding PyruvateDecarboxylase and Alcohol Dehydrogenase Ilt . APPLIED AND ENVIRONMENTALMICROBIOLOGY, Apr. 1991, p. 893-900 Vol. 57, No. 4


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Metaolic Engineering of Esherichia Coli for the Conversion

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