Cell-cell communication in yeast using auxin biosynthesis and auxin responsive CRISPR transcription factors

Abstract

A true engineering framework for synthetic multicellular systems requires a programmable means of cellcell communication. Such a communication system would enable complex behaviors, such as pattern formation, division of labor in synthetic microbial communities, and improved modularity in synthetic circuits. However, it remains challenging to build synthetic cellular communication systems in eukaryotes due to a lack of molecular modules that are orthogonal to the host machinery, easy to reconfigure, and scalable. Here, we present a novel cell-to-cell communication system in Saccharomyces cerevisiae (yeast) based on CRISPR transcription factors and the plant hormone auxin that exhibits several of these features. Specifically, we engineered a sender strain of yeast that converts indole-3-acetamide (IAM) into auxin via the enzyme iaaH from Agrobacterium tumefaciens. To sense auxin and regulate transcription in a receiver strain, we engineered a reconfigurable library of auxin degradable CRISPR transcription factors (ADCTFs). Auxin-induced degradation is achieved through fusion of an auxin sensitive degron (from IAA co-repressors) to the CRISPR TF and co-expression with an auxin F-box protein. Mirroring the tunability of auxin perception in plants, our family of ADCTFs exhibits a broad range of auxin sensitivities. We characterized the kinetics and steady state behavior of the sender and receiver independently, and in co-cultures where both cell types were exposed to IAM. In the presence of IAM, auxin is produced by the sender cell and triggers de-activation of reporter expression in the receiver cell. The result is an orthogonal, rewireable, tunable, and arguably scalable cell-cell communication system for yeast and other eukaryotic cells.

New approaches in bioprocess‐control: Consortium guidance by synthetic cell‐cell communication based on fungal pheromones

“…As pointed out above, systems integrating multiple different species into a single device would provide the opportunity to exploit the diverse metabolic capacities of different species, and wiring them by pheromone‐based cell‐cell communication would allow for the implementation of sensor‐actor systems that fulfill increasingly complex tasks. Additionally, purely synthetic communication modules – based on plant signaling components such as isopentenyladenine or auxin – have been utilized for artificial cell‐cell communication in S. cerevisiae (Table ), representing fully orthogonal communication modes that might also be transferrable to alternative host cells to yield inter‐species communication. Furthermore, several fungal species such as S. cerevisiae and C. albicans are known to utilize quorum sensing systems, based on farnesol or aromatic alcohols, to regulate a set of metabolic traits as well as filament and biofilm formation .…”

“…Thus, designing artificial intra‐ and inter‐species communication for use in cellular consortia must include rigorous testing of crosstalk and, if necessary, switch to fully orthogonal communication modes. To this end, plant‐derived signaling molecules as well as pheromones from distantly related species might be considered (see Section ). In addition, directed evolution of pheromones and pheromone receptors might be a valuable approach to limit crosstalk and to open up new communication channels.…”

Auxin 2016: a burst of auxin in the warm south of China

“…She further showed how this synthetic pathway can be used to analyze how the organization of ARF-binding motifs in promoters influences binding and transcriptional activity, an analysis that supports the idea that it is not only binding site spacing that determines activity in response to a given ARF but also the number and orientation of sites (PierreJerome et al, 2016). Eric Klavins (Washington University, Seattle, WA, USA) further discussed how the logic underlying the auxin pathway can be used to design synthetic networks and even cell-cell communication systems (Khakhar et al, 2016). He also showed that an inducible auxin-degradable transcription factor can allow the design of a switch that controls growth in yeast.…”

Plant synthetic biology for molecular engineering of signalling and development

“…Plant hormone pathways have been engineered to allow small-molecule regulation of target proteins, including triggering their turnover 78–80 or relocalization 81 . Recently, an enzyme for auxin biosynthesis has been combined with components from the auxin response pathway to produce a robust sender–receiver system in yeast 82 . A sender strain expresses an enzyme from bacteria that converts a precursor called indole-3-acetamide into auxin.…”