The authors describe a library of synthetic RNA control elements that provide programmable post-transcriptional regulation of gene expression in yeast. This toolkit is then used to study endogenous regulation of the ergosterol biosynthetic pathway.
Rnt1p hairpins can act as effective posttranscriptional gene regulatory elements in the yeast Saccharomyces cerevisiae.Modification of the cleavage efficiency box (CEB) region of an Rnt1p hairpin can modulate Rnt1p cleavage rates, and thus the resulting gene regulatory activities of the hairpin control elements.A library of Rnt1p hairpins can act as a set of synthetic control modules that provide predictable tuning of gene expression over a wide range of expression levels.The Rnt1p-based control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators, including feedback control around endogenous proteins.
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 al, 2004; Veen and Lang, 2004; Ro et al, 2006; Hawkins and Smolke, 2008). The majority of existing methods for tuning gene expression in yeast are through transcriptional control mechanisms in the form of inducible and constitutive promoter systems (Hawkins and Smolke, 2006; Nevoigt et al, 2006; Nevoigt et al, 2007). RNA-based control modules based on posttranscriptional mechanisms may offer an advantage in that they can be coupled to any promoter of choice, providing for enhanced control strategies and finer resolution tuning of protein expression levels. Although posttranscriptional control elements, such as internal ribosome entry sites and AU-rich elements, have been applied to regulate heterologous gene expression in yeast (Vasudevan and Peltz, 2001; Zhou et al, 2001; Lautz et al, 2010), these control elements have exhibited substantial variability in activity and have not been engineered as synthetic libraries exhibiting a wide range of predictable gene regulatory activities.
RNase III enzymes are a class of enzymes that cleave double-stranded RNA. The S. cerevisiae RNase III enzyme, Rnt1p, exhibits a number of unique features that allow it to recognize very specific RNA hairpin substrates that harbor a consensus AGNN tetraloop sequence. Despite extensive characterization of this enzyme and its demonstrated role in processing non-coding RNA and mRNA, neither natural nor synthetic Rnt1p substrates have been used to control gene expression levels in yeast. Therefore, we developed a genetic control system based on directed Rnt1p processing of a target transcript. Specifically, Rnt1p hairpins were immediately flanked by a clamp sequence (that insulates the hairpin structure from surrounding sequences) and placed downstream of a gene of interest, where they direct cleavage and thus inactivate the transcript, resulting in rapid transcript degradation. We validated this Rnt1p-based control system with two Rnt1p hairpins based on previous in vitro studies and demonstrated that Rnt1p hairpins can act as gene control modules in yeast.
Previous in vitro studies had identified three key regions in Rnt1p hairpins: the cleavage efficiency box (CEB), the binding stability box and the initial binding and positioning box (Lamontagne et al, 2003). The CEB region affects the processing of the hairpin stem by Rnt1p, such that nucleotide (nt) modifications in this region are expected to specifically modulate the cleavage rate. We created an Rnt1p hairpin library by randomizing the CEB region (12 nt). This library was placed downstream of a fluorescent reporter protein and a cell-based screening assay was used to identify functional members of the library that resulted in lowered fluorescence levels. The functional Rnt1p hairpin library comprises 16 unique sequences that span a large gene regulatory range—from 8 to 85% (Figure 3A)—and are fairly evenly distributed across this range. The negative controls for each sequence (constructed by mutating the required consensus tetraloop sequence) demonstrated that the majority of gene knockdown observed from each hairpin is due to Rnt1p processing (Figure 3B). A correlation analysis on the transcript and protein levels for each library hairpin construct indicated a strong positive correlation and a strong preservation of rank order between the two in vivo regulatory measurements (Figure 3C). Characterization of the hairpin library in a different genetic context supported the broader utility of these control modules for providing predictable gene control.
We applied the Rnt1p control modules to titrating a key enzyme component of the endogenous ergosterol biosynthesis network—the ERG9 genetic target. Squalene synthase, encoded by the ERG9 gene, is responsible for catalyzing the conversion of two molecules of farnesyl pyrophosphate to squalene, the first precursor in the ergosterol biosynthetic pathway in S. cerevisiae (Poulter and Rilling, 1981; Figure 6A). We integrated several members of the Rnt1p hairpin library downstream of the native ERG9 gene to cover the regulatory range of the library (Figure 6B). A strong positive correlation and preservation of rank order was observed between the ERG9 transcript levels and their yEGFP3 counterparts (Figure 6C). However, ERG9 expression levels did not fall below ∼40%, regardless of the Rnt1p hairpin strength, indicating that a previously identified endogenous feedback mechanism associated with the native ERG9 promoter acts to maintain ERG9 expression levels at that threshold value. In addition, most strains exhibited high relative ergosterol levels and growth rates, except for two strains harboring synthetic Rnt1p hairpins that resulted in the lowest expression levels, which exhibited a significant reduction in the amount of ergosterol produced and growth rate (Figure 6D and E). Our studies indicate that the endogenous feedback mechanism can be acting to increase ERG9 expression levels to the desired set point in the slow-growing strains, but the perturbations introduced in these strains may result in other impacts on the pathway that inhibit the endogenous control systems from restoring cellular growth to wild-type rates. These studies support the unique ability of the synthetic Rnt1p hairpin library to systematically titrate pathway enzyme levels by introducing precise perturbations around major control points while maintaining native cellular control strategies acting through transcriptional mechanisms.
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.