Selection and optimization of gene targets for the metabolic engineering of E. coli

This thesis is about identifying genetic interventions that improve the performance of targeted pathways in the metabolism of the bacterium Escherichia coli. Three case studies illustrate three disparate approaches to identifying genetic interventions: (i) combining metabolomic measurements with thermodynamic calculations to identify rate-limiting reaction steps in a target pathway; (ii) use of stoichiometric, optimization-based models of metabolism to predict target genetic interventions in silico; and (iii) the mutagenesis of promoter sequences to fine-tune the expression level of rate-limiting genes. These techniques can be classified by both the number of strain modifications created, and the number of variables measured in each. Taken together, the cases suggest that the best methods for identifying genetic interventions balance the number of strain modifications with the number of measured variables. The first case is butyrate production in recombinant E. coli. A strain of E. coli deleted for the production of lactate, ethanol, and acetate was designed to minimize competing pathways for carbon, and was unexpectedly found to exhibit oxygen auxotrophy. Expression of genes from Clostridium acetobutylicum resulted in production of 3-hydroxybutyric acid, but not butyric acid.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2009

Author dc:creator

• Fischer, Curt R., Ph. D. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• Gregory N. Stephanopoulos.

• Chemical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/51566

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/51566