High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch

Metabolic engineering can produce a wide range of bulk and fine chemicals using renewable resources. These approaches frequently require high levels of activity from multiple heterologous enzymes. Directed evolution techniques have been used to improve the activity of a wide range of enzymes but can be difficult to apply when the enzyme is used in whole cells. To address this limitation, we developed generalizable in vivo biosensors using engineered RNA switches to link metabolite concentrations and GFP expression levels in living cells. Using such a sensor, we quantitatively screened large enzyme libraries in high throughput based on fluorescence, either in clonal cultures or in single cells by fluorescence activated cell sorting (FACS). By iteratively screening libraries of a caffeine demethylase, we identified beneficial mutations that ultimately increased the enzyme activity in vivo by 33 fold and the product selectivity by 22 fold. As aptamer selection strategies allow RNA switches to be readily adapted to recognize new small molecules, these RNA-based screening techniques are applicable to a broad range of enzymes and metabolic pathways

A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

Engineered biological systems hold promise in addressing pressing human needs in chemical processing, energy production, materials construction, and maintenance and enhancement of human health and the environment. However, significant advancements in our ability to engineer biological systems have been limited by the foundational tools available for reporting on, responding to, and controlling intracellular components in living systems. Portable and scalable platforms are needed for the reliable construction of such communication and control systems across diverse organisms. We report an extensible RNA-based framework for engineering ligand-controlled gene-regulatory systems, called ribozyme switches, that exhibits tunable regulation, design modularity, and target specificity. These switch platforms contain a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of a hammerhead ribozyme sequence. We examined two modes of standardized information transmission between these domains and demonstrate a mechanism that allows for the reliable and modular assembly of functioning synthetic RNA switches and regulation of ribozyme activity in response to various effectors. In addition to demonstrating examples of small molecule-responsive, in vivo functional, allosteric hammerhead ribozymes, this work describes a general approach for the construction of portable and scalable gene-regulatory systems. We demonstrate the versatility of the platform in implementing application-specific control systems for small molecule-mediated regulation of cell growth and noninvasive in vivo sensing of metabolite production

The Regulatory Roles of the Galactose Permease and Kinase in the Induction Response of the GAL Network in Saccharomyces cerevisiae

The GAL genetic switch of Saccharomyces cerevisiae exhibits an ultrasensitive response to the inducer galactose as well as the "all-or-none" behavior characteristic of many eukaryotic regulatory networks. We have constructed a strain that allows intermediate levels of gene expression from a tunable GAL1 promoter at both the population and the single cell level by altering the regulation of the galactose permease Gal2p. Similar modifications to other feedback loops regulating the Gal80p repressor and the Gal3p signaling protein did not result in similarly tuned responses, indicating that the level of inducer transport is unique in its ability to control the switch response of the network. In addition, removal of the Gal1p galactokinase from the network resulted in a regimed response due to the dual role of this enzyme in galactose catabolism and transport. These two activities have competing effects on the response of the network to galactose such that the transport effects of Gal1p are dominant at low galactose concentrations, whereas its catabolic effects are dominant at high galactose concentrations. In addition, flow cytometry analysis revealed the unexpected phenomenon of multiple populations in the gal1{Delta} strains, which were not present in the isogenic GAL1 background. This result indicates that Gal1p may play a previously undescribed role in the stability of the GAL network response

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

Synthetic biology: advancing biological frontiers by building synthetic systems

Advances in synthetic biology are contributing to diverse research areas, from basic biology to biomanufacturing and disease therapy. We discuss the theoretical foundation, applications, and potential of this emerging field

Higher-Order Cellular Information Processing with Synthetic RNA Devices

The engineering of biological systems is anticipated to provide effective solutions to challenges that include energy and food production, environmental quality, and health and medicine. Our ability to transmit information to and from living systems, and to process and act on information inside cells, is critical to advancing the scale and complexity at which we can engineer, manipulate, and probe biological systems. We developed a general approach for assembling RNA devices that can execute higher-order cellular information processing operations from standard components. The engineered devices can function as logic gates (AND, NOR, NAND, or OR gates) and signal filters, and exhibit cooperativity. RNA devices process and transmit molecular inputs to targeted protein outputs, linking computation to gene expression and thus the potential to control cellular function

Dynamically Reshaping Signaling Networks to Program Cell Fate via Genetic Controllers

Introduction: Engineering of cell fate through synthetic gene circuits requires methods to precisely implement control around native decision-making pathways and offers the potential to direct developmental programs and redirect aberrantly activated cell processes. We set out to develop molecular network diverters, a class of genetic control systems, to activate or attenuate signaling through a mitogen-activated protein kinase (MAPK) pathway, the yeast mating pathway, to conditionally route cells to one of three distinct fates. Methods: We used a combination of genetic elements—including pathway regulators, RNA-based transducers, and constitutive and pathway-responsive promoters—to build modular network diverters. We measured the impact of these genetic control systems on pathway activity by monitoring fluorescence from a transcriptional pathway reporter. Cell fate determination was measured through halo assays, in which mating-associated cell cycle arrest above a certain concentration of pheromone from wild-type cells results in a “halo” or cleared region around a disk saturated in pheromone. A phenomenological model of our system was built to elucidate design principles for dual diverters that integrate opposing functions while supporting independent routing to alternative fates. Results: We identified titratable positive (Ste4) and negative (Msg5) regulators of pathway activity that result in divergent cell fate decisions when controlled from network diverters. A positive diverter, controlling Ste4 through a feedback architecture, routed cells to the mating fate, characterized by pathway activation in the absence of pheromone. A negative diverter, controlling Msg5 through a nonfeedback architecture, routed cells to the nonmating fate, characterized by pathway inhibition in the presence of pheromone. When integrated into a dual-diverter architecture, the opposing functions of these positive and negative diverters resulted in antagonism, which prevented independent routing to the alternative fates. However, a modified architecture that incorporated both constitutive and feedback regulation over the pathway regulators enabled conditional routing of cells to one of three fates (wild type, mating, or nonmating) in response to specified environmental signals. Discussion: Our work identified design principles for networks that induce differentiation of cells in response to environmental signals and that enhance the robust performance of integrated mutually antagonistic genetic programs. For example, integrated negative regulators can buffer a system against noise amplification mediated through positive-feedback loops by providing a resistance to amplification. Negative feedback can play an important role by reducing population heterogeneity and mediating robust, long-term cell fate decisions. The dual-diverter configuration enables routing to alternative fates and minimizes impact on the opposing diverter by integrating differential regulatory strategies on functionally redundant genes. Molecular network diverters provide a foundation for robustly programming spatial and temporal control over cell fate