Engineering of de novo pathways for biosynthesis of glutathione analogues in Escherichia coli

dc.contributor.advisorGeorgiou, Georgeen
dc.contributor.committeeMemberMolineux, Ian J.en
dc.contributor.committeeMemberAppling, Dean R.en
dc.contributor.committeeMemberMullins, Charles B.en
dc.contributor.committeeMemberMaynard, Jenniferen
dc.creatorVeeravalli, Karthiken 2010en
dc.description.abstractThe low molecular weight (L.M.W.) thiol redox couple formed by γ-L-glutamyl-L-cysteinyl glycine, also called glutathione (reduced and oxidized), is present in most eukaryotes and a few species of bacteria. Glutathione plays a role in numerous cellular processes by providing a means of shuttling electrons to different enzymatic systems. As a result, thiol-dependent redox metabolic processes are highly coupled. Due to tight coupling of redox reactions, it is difficult to understand how changes in the concentration of glutathione would affect a specific glutathione-dependent process. Interestingly, only a small subset of bacteria encode the canonical enzyme for the biosynthesis of glutathione, namely γ-glutamyl cysteine synthetase (gshA gene product). The mechanisms by which glutathione-dependent processes are carried out in bacteria which do not have the genes for biosynthesis of glutathione or other L.M.W. thiols is not well understood. A genetic selection to restore a glutathione-dependent phenotype in E. coli, lacking the gene involved in first step of glutathione biosynthesis (gshA), was used to address how bacteria lacking gshA might substitute for glutathione. Genetic and biochemical analyses of the E. coli mutants isolated in the selection revealed a de novo pathway for biosynthesis of γ-glutamyl cysteine, the product formed normally by GshA. Additionally we found that the unnatural analogue of glutathione, γ-glutamyl homocysteine could also be formed by this pathway. Bioinformatic analysis suggested that bacteria lacking gshA may use these de novo pathways for biosynthesis of γ-glutamyl cysteine or γ-glutamyl homocysteine, which could serve as potential substitutes for glutathione. The engineering of de novo biosynthetic pathways for γ-glutamyl cysteine and γ-glutamyl homocysteine provided us a strategy for engineering a pathway for biosynthesis of another unnatural analogue of glutathione, β-aspartyl cysteine. Both γ-glutamyl homocysteine and β-aspartyl cysteine could potentially be used as orthologus redox couples in E. coli operating in parallel to glutathione to shuttle electrons to specific pathways which may thus be decoupled from glutathione availability. Glutathione-dependent enzymes that can use orthologous redox couples instead are biochemically isolated from network of other redox reactions in the cell and could be used to direct metabolic fluxes to specific pathways with high efficiencies. Towards this end, we show that glutathione transferase, a glutathione-dependent enzyme, can be engineered to use analogous thiols like γ-glutamyl cysteine as cofactors.en
dc.description.departmentChemical Engineeringen
dc.subjectThiol-redox buffersen
dc.subjectOrthologous pathwaysen
dc.subjectGamma-glutamyl cysteineen
dc.subjectGlutathione Transferaseen
dc.subjectE. colien
dc.subjectB-aspartyl cysteineen
dc.subjectBeta-aspartyl cysteineen
dc.subjecty-Glutamyl cysteineen
dc.subjecty-Glutamyl homocysteineen
dc.titleEngineering of de novo pathways for biosynthesis of glutathione analogues in Escherichia colien
dc.type.genrethesisen Engineeringen Engineeringen of Texas at Austinen of Philosophyen

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