Metabolic engineering tolerance to amino acid related stress in Escherichia coli

Metabolic engineering seeks to discover genetic alterations for the directed improvement of industrially important strains. As such, it draws upon lessons from natural evolution to give insight into possible strategies for creating genetic diversity. Chapter 2 explores this idea in much greater detail through investigating antibiotic resistance mechanism.;Chapter 3 describes the effects that amino acid content of the recombinant protein has on the metabolic burden response. The results of this chapter indicate that overexpression of a recombinant protein containing a large portion of non-abundant amino acids may lead to lower translation efficiency. These results along with the transcriptional profiling results indicate that the primary sequence of the recombinant protein alone can strongly influence "metabolic burden" phenotypes. This insight may be useful for the further development of metabolic engineering strategies designed for selection of mutants more tolerant to amino acid related deficiencies.;Chapter 4 describes a comparative study of the costs and benefits associated with increasing tolerance to amino acid analogs using three different strain engineering techniques---chemical mutagenesis, gene duplication, and insertional mutagenesis. Interestingly, increased tolerance to aspartic acidβ hydroxamate independent of strain engineering technique was relatively large compared to other tested hydroxamates, whereas increased tolerance to glutamic acidγ hydroxamate was not achieved.;As documented in Chapter 5, using SCALEs we investigated the effects of gene overexpression to increase growth rate of E. coli when grown in the presence of aspartic acid anti-metabolites. The gene enrichment patterns look very different for each anti-metabolite tested with a few regions of overlap indicating that selection is specific. This indicates that the choice of anti-metabolite plays a critical role in determining the outcome of the selection strategy.;The research efforts described in this dissertation indicate the need for thoughtful consideration of target characteristics when initiating a strain engineering effort directed at engineering anti-metabolite or other forms of chemical tolerance. Our results provide insight for prioritizing among possible genetic engineering techniques, which will be useful for future metabolic engineering efforts directed at developing strains for industrially relevant applications.