Autocatalytic base editing for RNA-responsive translational control.

Genetic circuits that control transgene expression in response to pre-defined transcriptional cues would enable the development of smart therapeutics. To this end, here we engineer programmable single-transcript RNA sensors in which adenosine deaminases acting on RNA (ADARs) autocatalytically convert target hybridization into a translational output. Dubbed DART VADAR (Detection and Amplification of RNA Triggers via ADAR), our system amplifies the signal from editing by endogenous ADAR through a positive feedback loop. Amplification is mediated by the expression of a hyperactive, minimal ADAR variant and its recruitment to the edit site via an orthogonal RNA targeting mechanism. This topology confers high dynamic range, low background, minimal off-target effects, and a small genetic footprint. We leverage DART VADAR to detect single nucleotide polymorphisms and modulate translation in response to endogenous transcript levels in mammalian cells.

Light-Controlled Fermentations for Microbial Chemical and Protein Production.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

The Neurospora crassa Inducible Q System Enables Simultaneous Optogenetic Amplification and Inversion in Saccharomyces cerevisiae for Bidirectional Control of Gene Expression.

Bidirectional optogenetic control of yeast gene expression has great potential for biotechnological applications. Our group has developed optogenetic inverter circuits that activate transcription using darkness, as well as amplifier circuits that reach high expression levels under limited light. However, because both types of circuits harness Gal4p and Gal80p from the galactose (GAL) regulon they cannot be used simultaneously. Here, we apply the Q System, a transcriptional activator/inhibitor system from Neurospora crassa, to build circuits in Saccharomyces cerevisiae that are inducible using quinic acid, darkness, or blue light. We develop light-repressed OptoQ-INVRT circuits that initiate darkness-triggered transcription within an hour of induction, as well as light-activated OptoQ-AMP circuits that achieve up to 39-fold induction. The Q System does not exhibit crosstalk with the GAL regulon, allowing coutilization of OptoQ-AMP circuits with previously developed OptoINVRT circuits. As a demonstration of practical applications in metabolic engineering, we show how simultaneous use of these circuits can be used to dynamically control both growth and production to improve acetoin production, as well as enable light-tunable co-production of geraniol and linalool, two terpenoids implicated in the hoppy flavor of beer. OptoQ-AMP and OptoQ-INVRT circuits enable simultaneous optogenetic signal amplification and inversion, providing powerful additions to the yeast optogenetic toolkit.

Optogenetic Control of Microbial Consortia Populations for Chemical Production.

Microbial co-culture fermentations can improve chemical production from complex biosynthetic pathways over monocultures by distributing enzymes across multiple strains, thereby reducing metabolic burden, overcoming endogenous regulatory mechanisms, or exploiting natural traits of different microbial species. However, stabilizing and optimizing microbial subpopulations for maximal chemical production remains a major obstacle in the field. In this study, we demonstrate that optogenetics is an effective strategy to dynamically control populations in microbial co-cultures. Using a new optogenetic circuit we call OptoTA, we regulate an endogenous toxin-antitoxin system, enabling tunability of Escherichia coli growth using only blue light. With this system we can control the population composition of co-cultures of E. coli and Saccharomyces cerevisiae. When introducing in each strain different metabolic modules of biosynthetic pathways for isobutyl acetate or naringenin, we found that the productivity of co-cultures increases by adjusting the population ratios with specific light duty cycles. This study shows the feasibility of using optogenetics to control microbial consortia populations and the advantages of using light to control their chemical production.

Optogenetic Amplification Circuits for Light-Induced Metabolic Control.

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 23-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light duty cycles as low as ∼1%, and strong homogeneous light-induction in bioreactors of at least 5 L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

Dynamical Modeling of Optogenetic Circuits in Yeast for Metabolic Engineering Applications.

Dynamic control of engineered microbes using light via optogenetics has been demonstrated as an effective strategy for improving the yield of biofuels, chemicals, and other products. An advantage of using light to manipulate microbial metabolism is the relative simplicity of interfacing biological and computer systems, thereby enabling in silico control of the microbe. Using this strategy for control and optimization of product yield requires an understanding of how the microbe responds in real-time to the light inputs. Toward this end, we present mechanistic models of a set of yeast optogenetic circuits. We show how these models can predict short- and long-time response to varying light inputs and how they are amenable to use with model predictive control (the industry standard among advanced control algorithms). These models reveal dynamics characterized by time-scale separation of different circuit components that affect the steady and transient levels of the protein under control of the circuit. Ultimately, this work will help enable real-time control and optimization tools for improving yield and consistency in the production of biofuels and chemicals using microbial fermentations.

Optogenetic control of the lac operon for bacterial chemical and protein production.

Control of the lac operon with isopropyl ß-D-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.

Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering.

The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in Saccharomyces cerevisiae. The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible GAL1 promoter (PGAL1-S), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with PGAL1-S achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and PGAL1-S open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.

Current and future modalities of dynamic control in metabolic engineering.

Metabolic engineering aims to maximize production of valuable compounds using cells as biological catalysts. When incorporating engineered pathways into host organisms, an inherent conflict is presented between maintenance of cellular health and generation of products. This challenge has been addressed through two main modalities of dynamic control: decoupling growth from production via two-phase fermentations and autoregulation of pathways to optimize product formation. However, dynamic control can offer even greater potential for metabolic engineering through open-loop and closed-loop control modalities of the production phase. Here we review recent applications of dynamic control strategies in metabolic engineering. We then explore the potential of integrating biosensors and computer-assisted feedback control as a promising future modality of dynamic control.

Optogenetic regulation of engineered cellular metabolism for microbial chemical production.

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation with only light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g l-1 of isobutanol and 2.38 ± 0.06 g l-1 of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for the production of valuable products.