As Saccharomyces cerevisiae is engineered further as a microbial factory for industrially relevant but potentially cytotoxic molecules such as ethanol, issues of cell viability arise that threaten to place a biological limit on output capacity and/or the use of less refined production conditions. Evidence suggests that one naturally evolved mode of survival in deleterious environments involves the complex, multigenic interplay between disparate stress response and homeostasis mechanisms. Rational engineering of such resistance would require a systems-level understanding of cellular behavior that is, in general, not yet available. To circumvent this limitation, we have developed a phenotype discovery approach termed global transcription machinery engineering (gTME) that allows for the generation and selection of nonphysiological traits. We alter gene expression on a genome-wide scale by selecting for dominant mutations in a randomly mutagenized general transcription factor. The gene encoding the mutated transcription factor resides on a plasmid in a strain carrying the unaltered chromosomal allele. Thus, although the dominant mutations may destroy the essential function of the plasmid-borne variant, alteration of the transcriptome with minimal perturbation to normal cellular processes is possible via the presence of the native genomic allele. Achieving a phenotype of interest involves the construction and diversity evaluation of yeast libraries harboring random sequence variants of a chosen transcription factor and the subsequent selection and validation of mutant strains. We describe the rationale and procedures associated with each step in the context of generating strains possessing enhanced ethanol tolerance.
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