Host-aware RNA-based control of synthetic microbial consortia

Microbial consortia have been utilised for centuries to produce fermented foods and have great potential in applications such as therapeutics, biomaterials, fertilisers, and biobased production. Working together, microbes become specialized and perform complex tasks more efficiently, strengthening both cooperation and stability of the microbial community. However, imbalanced proportions of microbial community members can lead to unoptimized and diminished yields in biotechnology. To address this, we developed a burden-aware RNA-based multicellular feedback control system that stabilises and tunes coculture compositions. The system consists of three modules: a quorum sensing-based communication module to provide information about the densities of cocultured strains, an RNA-based comparator module to compare the ratio of densities of both strains to a pre-set desired ratio, and a customisable growth module that relies either on heterologous gene expression or on CRISPRi knockdowns to tune growth rates. We demonstrated that heterologous expression burden could be used to stabilise composition in a two-member E. coli coculture. This is the first coculture composition controller that does not rely on toxins or syntrophy for growth regulation and uses RNA sequestration to stabilise and control coculture composition. This work provides a fundamental basis to explore burden-aware multicellular feedback control strategies for robust stabilisation of synthetic community compositions.