A light tunable differentiation system for the creation and control of consortia in yeast

ARTICLE https://doi.org/10.1038/s41467-021-26129-7 OPEN A light tunable differentiation system for the creation and control of consortia in yeast 1234567890():,; Chetan Aditya 1,2,3, François Bertaux 1,2, Gregory Batt 1,2,4 ✉ & Jakob Ruess1,2,4 ✉ Artificial microbial consortia seek to leverage division-of-labour to optimize function and possess immense potential for bioproduction. Co-culturing approaches, the preferred mode of generating a consortium, remain limited in their ability to give rise to stable consortia having finely tuned compositions. Here, we present an artificial differentiation system in budding yeast capable of generating stable microbial consortia with custom functionalities from a single strain at user-defined composition in space and in time based on optogenetically-driven genetic rewiring. Owing to fast, reproducible, and light-tunable dynamics, our system enables dynamic control of consortia composition in continuous cultures for extended periods. We further demonstrate that our system can be extended in a straightforward manner to give rise to consortia with multiple subpopulations. Our artificial differentiation strategy establishes a novel paradigm for the creation of complex microbial consortia that are simple to implement, precisely controllable, and versatile to use. 1 Inria Paris, 2 rue Simone Iff, 75012 Paris, France. 2 Institut Pasteur, 28 rue du Docteur Roux, 75015 Paris, France. 3 Université de Paris, 85 Boulevard SaintGermain, 75006 Paris, France. 4These authors contributed equally: Gregory Batt, Jakob Ruess. ✉email: ; NATURE COMMUNICATIONS | (2021)12:5829 | https://doi.org/10.1038/s41467-021-26129-7 | www.nature.com/naturecommunications 1 ARTICLE T NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26129-7 he evolutionary transition from single cell to multicellular organisms marked a critical turning point in biology1. Such shift relied on optimizing fitness and productivity through division of labour and specialization2,3. The same principle can be extended to microorganisms living together to form microbial communities or consortia. Engineered microbial consortia hold enormous potential and have been hailed as the next frontier in synthetic biology4,5. Proof of concept studies have concretely established applications in bioproduction6,7, bioremediation8,9, and soil microbiome engineering10, paving the way for therapeutic applications using human microbiome engineering11,12. In the context of bioproduction, microbial consortia possess several advantages over traditional monocultures as functional specialization allows metabolic burden to be shared across different species. Diversification thus allows yields to be optimized simply through tuning consortia composition, rather than reengineering the strain itself13. Moreover, by including multiple species, toxic by-products produced by one species can be sequestered and/or metabolized by another, thereby improving the efficiency of the overall process14. Microbial consortia are typically generated by culturing two or more species together. Such co-culturing approaches rely on various inter-species interactions to ensure the co-existence of different species like mutualism15, emergent cooperation16, competitive amensalism17, and predation18. Despite considerable advances in our ability to engineer microbial consortia6,8,9,19–21 and in our understanding of community interactions15,16,18,21,22, dynamic control of consortium composition remains a key challenge in the field19. Typically, stable consortia are based on syntrophic or quorum sensing interactions that, albeit being autonomous, remain critically dependent on cell density, thus limiting the applicability for dynamic control. Additionally, scaling the consortium to include more than two species requires non-trivial considerations that may not lead to stable co-existence20. In light of these limitations, an externally controllable differentiation system could be well suited to address this challenge. In recent years, advances in biological control have come from coupling computers with growing cells carrying the engineered system, made possible by special platforms that integrate biological systems with the computer via a feedback loop23–27. The development of optogenetics, i.e. the use of light to trigger cellular processes, has contributed significantly to control applications by increasing the spatiotemporal resolution of the control signal23,24,28–38. Control of protein expression using light has been demonstrated both at the population level23,28,31 and in single cells30,33,34,37. Optogenetics has been used to control cellular processes in other contexts, for instance, signalling dynamics24, morphogenesis36, neuroscience38, bioproduction, and metabolic engineering29,35. However, control of population dynamics using optogenetics in a multispecies environment has not been demonstrated yet. Here, we present an artificial differentiation system in S. cerevisiae capable of generating a microbial consortium composed of functionally different subpopulations emerging from a single population akin to differentiation in multicellular organisms. Concretely, we achieve differentiation into genetically distinct subpopulations—henceforth referred to as species to highlight the analogy to natural microbial consortia—via recombination-based genetic rewiring that can be externally controlled via light. We demonstrate that our system shows desirable features including low background activity, high efficiency for optogenetic recombinases in budding yeast, graded response to varying light signals, absence of hysteresis, and dynamics that are fast, predictable, and tunable. The system reaches >99% differentiation after 4 h of light stimulation and can be stably maintained at any given intermediate level of differentiation for long periods of time (>48 h). 2 Owing to its fast and predictable dynamics, our differentiation system enables rapid and robust bidirectional control of a microbial consortium arising from a single strain at user-defined compositions in continuous cultures for extended periods in dynamic setups. Coupling the system to a growth arrest module allows us to control population growth rates in continuous culture in different physiological contexts. We show that our system can be extended to give rise to complex multispecies microbial consortia. We engineer two differentiation programmes that can be used to control the total number of species. Finally, we show that our system allows for spatial structuring of microbial consortia by imprinting patterns in 2D cultures with high resolution. To the best of our knowledge, this is the first report of lightdriven system for control of a microbial consortium. Results An optogenetic synthetic differentiation system in S. cerevisiae. We constructed an optogenetic differentiation system consisting of a blue light-inducible Cre recombinase under the control of a constitutively expr (...truncated)