A synthetic library of RNA control modules for predictable tuning of gene expression in yeast

Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we describe a novel class of RNA‐based control modules that provide predictable tuning of expression levels in the yeast Saccharomyces cerevisiae . A library of synthetic control modules that act through posttranscriptional RNase cleavage mechanisms was generated through an in vivo screen, in which structural engineering methods were applied to enhance the insulation and modularity of the resulting components. This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes. We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems. Synopsis The design of complex biological systems encoding desired functions require the development of genetic tools for the precise control of protein levels in cells (Elowitz and Leibler, 2000 ; Gardner et al , 2000 ; Basu et al , 2004 ). For example, in the design of engineered metabolic networks, the tuning of enzyme levels is often critical for overcoming metabolic burden (Jones et al , 2000 ; Jin et al , 2003 ), the accumulation of toxic intermediates (Zhu et al , 2001 ; Pfleger et al , 2006 ) and detrimental consequences associated with the redirection of cellular resources from native pathways (Alper et al , 2005b ; Paradise et al , 2008 ). Various examples of libraries of genetic control modules have been described that have been generated through the randomization of well‐characterized gene expression control elements (Basu et al , 2004 ; Pfleger et al , 2006 ; Anderson et al , 2007 ). However, most of these studies have been conducted in Escherichia coli such that there is a lack of similar tools for other cellular chassis. The budding yeast, Saccharomyces cerevisiae , is a relevant organism in industrial processes, including biosynthesis and biomanufacturing strategies (Ostergaard et al , 2000 ; Szczebara et al , 2003 ; Nguyen et