Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

Abstract

Background Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. Results We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. Conclusions Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

Redox active plant phenolic, acetosyringone, for electrogenetic signaling

“…Importantly, we show that a single promoter can be toggled using two distinct mechanisms simply by varying the type of potential applied to cells in the presence of AS. The imposed electronic input, or redox context, defines the duration and magnitude of the biological response, thus further demonstrating the importance of context for redox activity 52,53 . Both H 2 O 2 and oxidized AS are signaling molecules present in the rhizosphere after plant infection.…”

Redox Active Plant Phenolic, Acetosyringone, for Electrogenetic Signaling

“…Importantly, we show that a single promoter can be toggled using two distinct mechanisms simply by varying the type of potential applied to cells in the presence of AS. The imposed electronic input, or redox context, defines the duration and magnitude of the biological response, thus further demonstrating the importance of context for redox activity[47,48]. Both H 2 O 2 and oxidized AS are signaling molecules present in the rhizosphere after plant infection.…”

Quorum Sensing from Two Engineers’ Perspectives

“…This was accomplished in part through autoinducer regulated cell growth rate, where HPr was upregulated in order to increase cell growth rate of one of the populations. In another example, redox molecules could be integrated with QS autoinducers to modulate gene expression in microbial cocultures in a context dependent manner [56] …”

“…In another example, redox molecules could be integrated with QS autoinducers to modulate gene expression in microbial cocultures in a context dependent manner. [56] There is evidence that QS is important in natural microbial consortia and microbiomes, including in the GI tract. In these instances, engineered cells may be able to influence QS networks to improve health outcomes.…”