Towards engineering microbial consortia using RNA-based genetic controllers

In nature, quorum sensing is a mechanism used by microbes to communicate and coordinate behaviours at the population level. Over the last two decades, synthetic biologists have used the unique property of quorum sensing to sense population density for coordinating cellular behaviours of single and mixed cultures. This gave rise to the development of multicellular biosynthesis systems for metabolic engineering and of spatially distributed systems for synthetic biology. However, robustly controlling the composition of multicellular systems remains a challenge and limits its wide adoption by the metabolic engineering community. Current strategies for controlling synthetic microbial communities vastly rely on engineer- ing metabolic dependencies between microbial species in a process called syntrophy. While syntrophy guarantees the survival of all strains in the coculture, it does not provide a way to control community composition. Existing genetic circuits that can dynamically control community composition often impose too much burden on their hosts for division of labour to be a viable solution to improve yields and titers of valuable metabolic products. Here we investigate the potential of using RNA-based gene circuits to reduce the cost of express- ing heterologous genes for the control of community composition in a two-member E. coli coculture. In this work, we present the development of three genetic modules that rely on RNA species to detect changes in population density and to regulate growth rate when community composition becomes unstable. Together, the modules work in concert to stabilise community composition around a ratio set by the intrinsic properties of the circuit’s genetic components. We identify the key parameters of the circuits that enable tuning of the composition ratio. We characterise the cost of expressing each module of the genetic controller by measuring its impact on the host growth rate and on consumption of free cellular resources. Together these findings highlight the importance of developing host-aware circuits to control community composition so as to enable their wide adoption by metabolic engineers.Open Acces

Burden-driven feedback control of gene expression

Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable

Synthetic microbe-to-plant communication channels

Abstract Plants and microbes communicate to collaborate to stop pests, scavenge nutrients, and react to environmental change. Microbiota consisting of thousands of species interact with each other and plants using a large chemical language that is interpreted by complex regulatory networks. In this work, we develop modular interkingdom communication channels, enabling bacteria to convey environmental stimuli to plants. We introduce a “sender device” in Pseudomonas putida and Klebsiella pneumoniae, that produces the small molecule p-coumaroyl-homoserine lactone (pC-HSL) when the output of a sensor or circuit turns on. This molecule triggers a “receiver device” in the plant to activate gene expression. We validate this system in Arabidopsis thaliana and Solanum tuberosum (potato) grown hydroponically and in soil, demonstrating its modularity by swapping bacteria that process different stimuli, including IPTG, aTc and arsenic. Programmable communication channels between bacteria and plants will enable microbial sentinels to transmit information to crops and provide the building blocks for designing artificial consortia