Prior efforts in metabolic engineering have attempted to improve strain properties through the modification of components of localized pathways and certainly using gene-by-gene strategies. To this end, many successful examples have been reported that were based on re-designing regulatory networks, metabolite balancing systems, and rational or combinatorial gene deletion and amplification approaches. However, these approaches lack a cell-wide perspective and mostly fail in eliciting desired phenotypes dependent on simultaneous multiple gene modifications. A central reason is the vast size of the space of possible combinations of multiple-gene modifications combined with limited transformation capacity needed to probe this space. As a result, a huge fraction of the phenotype space that depends on multiple gene interactions has been largely unexplored.

Here, we present a method that departs from the traditional gene-phenotype mapping approach and regards the phenotype as the manifestation of a particular transcriptional profile. Hence, the method attempts to elicit new phenotypes by manipulating directly the transcriptome of a cell through engineering of specially selected global regulators. This approach allows modulation of simultaneous multiple gene expression at the highest level with profound implications for phenotype improvement of prokaryotic and eukaryotic cells alike. Specifically, we modify the sigma factors of Escherichia coli and TFIID components in Saccharomyces cerevisiae to elicit a multigenic reprogramming of the transcriptome using an approach termed global Transciption Machinery Engineering (gTME). Results using gTME for these two cellular systems will be presented in the context of several proof-of-concept studies including increased cellular tolerances (as increased ethanol tolerance), metabolite (lycopene) overproduction, and multiple phenotypes (including multiple tolerance phenotypes). In each case, the tool of global Transcription Machinery Engineering (gTME) outperformed traditional approaches, exceeding, in a matter of weeks, benchmarks achieved through decades of research.

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