(152d) Tunable Promoters for the Synthesis and Analysis of Functional Gene Networks

Synthetic biology aims to increase biological knowledge through engineering design and synthesis of biological systems. Whether the design target is an artificial gene network or a metabolite overproducing microbial strain, robust and reliable molecular tools are critical to the design effort. Because in many cases transcript expression levels are key design parameters in a synthetic biological system, we sought to develop promoter-based tools for the precise and reliable manipulation of gene expression.

Through random mutagenesis of known constitutive and regulatory promoters, we created constitutive promoter libraries in both Escherichia coli and Saccharomyces cerevisiae. In both organisms, mutants of specified strength were isolated through flow cytometry-based screens, and subjected to a battery of analyses for verifying mutant promoter functionality. We focused our efforts on a subset of the promoter mutants for which reporter gene expression was constant across several culture media, and was well correlated to transcript abundance. We demonstrated the utility of these tools by optimizing levels of gene expression for lycopene production in E. coli and for glycerol production in S. cerevisiae.

We also developed tools for robust control of gene regulation. Mutants of an oxygen-responsive yeast promoter were isolated which induce faster and to a higher level in response to conditions of moderate oxygen depletion. The ease with which even large-scale industrial fermentors can achieve moderate oxygen depletion suggests that these tools may find application at industrial scales, unlike other inducible systems for gene expression.