Forward and inverse metabolic engineering strategies for improving polyhydroxybyrate production

Forward metabolic engineering (FME) is a rational approach to cellular engineering, relying on an understanding of the entire metabolic network to direct perturbations for phenotype improvement. Conversely, inverse metabolic engineering (IME) uses a global, combinatorial approach to identify genetic loci that are important for a given phenotype. These two approaches complement each other in a strain improvement program. FME and IME approaches were applied to poly-3-hydroxybutyrate (PHB)production in Synechocystis PCC6803 [IME] and recombinant E. coli [FME] in this thesis.IME was appropriate for Synechocystis, where metabolic regulation of the native PHB pathway was not well understood. A high throughput screening method was established by developing a staining protocol that quantitatively related nile red fluorescence to PHB content, while maintaining cell viability for both organisms. This was combined with fluorescence activated cell sorting (FACS) to screen for high PHB mutants. A Synechocystis insertion mutagenesis library was screened to identify gene disruptions that increased PHB. Two gene disruptions in proline biosynthesis and an unknown function were identified and characterized.An analogous IME study in E. coli did not find increased PHB mutants, but suggested an FME approach on the PHB pathway. Systematic overexpression of the pathway revealed phaB, acetoacetyl-CoA reductase, limited PHB flux. Beyond this, whole operon overexpression led to even higher PHB fluxes.In a nitrogen-limited chemostat, PHB flux did not change with dilution rate. Unlike prior pleiotropic perturbations, these systematic experiments could clearly conclude that the flux control is in the PHB pathway. At high PHB flux, growth rate was extremely hindered and was accompanied by PHB plasmid genetic instability and rapid PHB productivity loss.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Tyo, Keith E. J

Advisor dc:contributor.advisor

• Gregory 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/1721.1/46022

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/46022