Electrogenetic Signal Transmission and Propagation in Coculture to Guide Production of a Small Molecule, Tyrosine

Abstract

There are many strategies to actuate and control genetic circuits, including providing stimuli like exogenous chemical inducers, light, magnetic fields, and even applied voltage, that are orthogonal to metabolic activity. Their use enables actuation of gene expression for the production of small molecules and proteins in many contexts. Additionally, there are a growing number of reports wherein cocultures, consortia, or even complex microbiomes are employed for the production of biologics, taking advantage of an expanded array of biological function. Combining stimuli-responsive engineered cell populations enhances design space but increases complexity. In this work, we co-opt nature’s redox networks and electrogenetically route control signals into a consortium of microbial cells engineered to produce a model small molecule, tyrosine. In particular, we show how electronically programmed short-lived signals (i.e., hydrogen peroxide) can be transformed by one population and propagated into sustained longer-distance signals that, in turn, guide tyrosine production in a second population building on bacterial quorum sensing that coordinates their collective behavior. Two design methodologies are demonstrated. First, we use electrogenetics to transform redox signals into the quorum sensing autoinducer, AI-1, that, in turn, induces a tyrosine biosynthesis pathway transformed into a second population. Second, we use the electrogenetically stimulated AI-1 to actuate expression of ptsH, boosting the growth rate of tyrosine-producing cells, augmenting both their number and metabolic activity. In both cases, we show how signal propagation within the coculture helps to ensure tyrosine production. We suggest that this work lays a foundation for employing electrochemical stimuli and engineered cocultures for production of molecular products in biomanufacturing environments.

Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis

“…Next (Figure 1B), we placed K31-LysisE cells in a previously developed electrogenetic induction chamber (VanArsdale et al, 2022) to determine if localized, time-dependent generation of H 2 O 2 could induce a reduction in population density. These chambers catalytically produce H 2 O 2 at different rates (measured H 2 O 2 results are shown in Figure S2).…”

“…We expected this relay could be modulated through LasI production. To test this, we generated K31 and K33 transmitter constructs based upon the design in Figure 1A and a pre-existing OxyR-LasI construct with an SsrA degradation tag LAA on the C-terminus of LasI to reduce its lifetime; we have used this pre-existing construct in previous transmitter-receiver configurations (Stephens et al, 2021; VanArsdale et al, 2022) (Figure 2B, scheme). Both K31-LasI-LAA and K33-LasI-LAA variants were expected to produce less LasI than the P oxyS construct.…”

“…to activate genetic responses than that expected to be generated in the chamber volume at the same charge (Terrell et al, 2021). Moreover, our previous work also showed that electrogenetically-stimulated cells appeared to express more GFP than when provided an equivalent bolus of H2O2 to the medium (Terrell et al, 2021;VanArsdale et al, 2022). In all cases, our data reinforce the notion that cells in a mixed system transiently come in contact with the electrode, experience a higher H2O2 concentration than in the bulk solution, and respond more quickly and to a greater extent than predicted based on the bulk H2O2 level (VanArsdale et al, 2022).…”

“…Moreover, our previous work also showed that electrogenetically-stimulated cells appeared to express more GFP than when provided an equivalent bolus of H2O2 to the medium (Terrell et al, 2021;VanArsdale et al, 2022). In all cases, our data reinforce the notion that cells in a mixed system transiently come in contact with the electrode, experience a higher H2O2 concentration than in the bulk solution, and respond more quickly and to a greater extent than predicted based on the bulk H2O2 level (VanArsdale et al, 2022). Here, the net effect was equivalent to a ~2 to 4-fold increase in H2O2, suggesting a similar enhancement in signal transmission.…”

“…Cells were induced by the electrogenetic method previously described (VanArsdale et al, 2022). Briefly, catalytic formation of hydrogen peroxide was controlled by biasing a 2-mm gold disk electrode paired with a 1-cm long platinum wire counter electrode and an Ag/AgCl reference electrode.…”