S104: Synthetic control of transcription: from hybrid promoters to promoter engineering to synthetic operon design

Synthetic biology provides the means of designing novel parts to aid in the controlled, tunable, and regulated expression of individual genes, circuits, and pathways.  Here, we describe recent advances in synthetic control of transcription by hybrid promoters in both Saccharomyces cerevisiae and the nonconventional yeast, Yarrowia lipolytica.  These synthetic promoters are comprised of two modular components—the enhancer element and the core promoter element.  We demonstrate that upstream activating sequences can serve as “synthetic transcriptional amplifiers” that can be used either individually or in tandem to tune and regulate gene expression.  By utilizing such an approach, we can create libraries with ranges of over 400 fold in mRNA level, create the strongest known constitutive promoters, and synthetically impart strength and regulation traits to promoter elements.  Second, we will discuss the synthetic design of expression cassettes and operons.  Importantly, we measure and model the impact of 5’ UTR regions on translational control to design synthetic cassettes that have sustained, high-level expression regardless of cloning strategy.  Finally, we discuss the impact of genetic context for synthetic parts—a particularly important aspect for characterization and predicable function.  By synthetically altering upstream regions, terminator components, and promoter structure, defined and reproducible function can be obtained.  Collectively, these results demonstrate novel approaches to synthetically alter and control gene transcription—a central goal of metabolic engineering and synthetic biology efforts.  Thus, we conclude with specific applications of these tools for metabolic pathway engineering.