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

Abstract

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.

Coupling Cell Communication and Optogenetics: Implementation of a Light-Inducible Intercellular System in Yeast

“…Importantly, there is already a study demonstrating comparable or even higher metabolite production from optogenetic strains compared to chemical induction at the bioreactor level . Thus, the two-population systemafter improvement at different levelscan represent a tool to overcome challenges of light penetration in high-density cultures, such as recently described optogenetic amplifiers. , …”

“…77 Thus, the two-population system�after improvement at different levels�can represent a tool to overcome challenges of light penetration in high-density cultures, such as recently described optogenetic amplifiers. 78,79 Finally, autonomous (A) versions of our synthetic circuit were generated, where each cell is carrying all the building blocks related to light perception, pheromone production, and reporter expression (Figure 6A). Thus, each cell of A strains has the capacity to induce luciferase expression in response to optogenetically produced pheromone.…”

Light‐induced fermenter production of derivatives of the sweet protein monellin is maximized in prestationary Saccharomyces cerevisiae cultures

“…[ 39 ] Optogenetic circuits were developed for S. cerevisiae to optimize the production of acetoin, lactic acid and naringenin. [ 38,40,41 ] These examples highlight the current boost in usage of optogenetic tools in biotechnology approaches and demonstrate the potential of optogenetics for a broad application in industrial settings.…”

“…[35] A nec- were published that implemented optogenetic tools for biosynthesis processes. [23,[36][37][38][39][40][41] Namely, metabolic engineering with optogenetic expression tools resulted in increased ethanol and isobutanol production in S. cerevisiae. [23] Acetoin production in E. coli cells was optimized by regulating the cell division time period with optogenetic tools.…”

Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales

“…Moving beyond this two-step cultivation strategy, several recent studies showed that controlling growth versus production in a dynamic manner can further increase production yields 9,10 . Finely controlling the induction of the metabolic pathways leading to production is critical for such "dynamic regulation" 11 strategies.…”