Engineering xylose induction in Vibrio natriegens for biomanufacturing applications.

Xylose is an abundant, inexpensive and readily available carbohydrate common in minimally processed feedstocks such as seaweed and algae. While a wide variety of marine microbes have evolved to utilize seaweed and algae, only a few currently have the requisite characteristics and genetic engineering tools necessary to entertain the use of these underutilized feedstocks. The rapidly growing Gram-negative halophilic bacterium Vibrio natriegens is one such chassis. In this study, we engineered and tested xylose induction in V. natriegens as a tool for scalable bioproduction applications. First, we created a sensing construct based on the xylose operon from Escherichia coli MG1665 and measured its activity using a fluorescent reporter and identified that cellular import plays a key role in induction strength and that expression required the XylR transcription factor. Next, we identified that select deletions of the promoter region enhance gene expression, limiting the effect of carbohydrate repression when xylose is used as an inducer in the presence of industrially relevant carbon sources. Lastly, we used the optimized constructs to produce the biopolymer melanin using seawater mimetic media. One of these formulations utilized a nori-based seaweed extract as an inducer and demonstrated melanin yields comparable to previously optimized methods using a more traditional and costly inducer. Together, the results demonstrate that engineering xylose induction in V. natriegens can provide an effective and lower cost option for timed biosynthesis in scalable biomanufacturing applications using renewable feedstocks.

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

To probe signal propagation and genetic actuation in microbial consortia, we have coopted the components of both redox and quorum sensing (QS) signaling into a communication network for guiding composition by "programming" cell lysis. Here, we use an electrode to generate hydrogen peroxide as a redox cue that determines consortia composition. The oxidative stress regulon of Escherichia coli, OxyR, is employed to receive and transform this signal into a QS signal that coordinates the lysis of a subpopulation of cells. We examine a suite of information transfer modalities including "monoculture" and "transmitter-receiver" models, as well as a series of genetic circuits that introduce time-delays for altering information relay, thereby expanding design space. A simple mathematical model aids in developing communication schemes that accommodate the transient nature of redox signals and the "collective" attributes of QS signals. We suggest this platform methodology will be useful in understanding and controlling synthetic microbial consortia for a variety of applications, including biomanufacturing and biocontainment.

Rapid, high-titer biosynthesis of melanin using the marine bacterium Vibrio natriegens.

Melanin is one of the most abundant natural biomolecules on Earth. These macromolecular biopolymers display several unique physical and chemical properties and have garnered interest as biomaterials for various commercial and industrial applications. To this end, extensive research has gone into refining methods for the synthesis and extraction of melanin from natural and recombinant sources. In this study, we developed and refined a procedure using a recombinant microbial system for the biosynthesis of melanin using the tyrosinase enzyme Tyr1 and tyrosine as a substrate. Using the emergent microbial chassis organisms Vibrio natriegens, we achieved maximal yields of 7.57 g/L, and one of the highest reported volumetric productivities of 473 mg L-1 h-1 with 100% conversion rates in an optimized, minimally defined medium. Additionally, we identified and investigated the use of a native copper responsive promoter in V. natriegens for stringent regulation of heterologous protein expression as a cost effective alternative to traditional IPTG-based induction. This research represents a promising advancement towards a green, rapid, and economical alternative for the biomanufacture of melanin.

Redox-enabled electronic interrogation and feedback control of hierarchical and networked biological systems.

Microelectronic devices can directly communicate with biology, as electronic information can be transmitted via redox reactions within biological systems. By engineering biology's native redox networks, we enable electronic interrogation and control of biological systems at several hierarchical levels: proteins, cells, and cell consortia. First, electro-biofabrication facilitates on-device biological component assembly. Then, electrode-actuated redox data transmission and redox-linked synthetic biology allows programming of enzyme activity and closed-loop electrogenetic control of cellular function. Specifically, horseradish peroxidase is assembled onto interdigitated electrodes where electrode-generated hydrogen peroxide controls its activity. E. coli's stress response regulon, oxyRS, is rewired to enable algorithm-based feedback control of gene expression, including an eCRISPR module that switches cell-cell quorum sensing communication from one autoinducer to another-creating an electronically controlled 'bilingual' cell. Then, these disparate redox-guided devices are wirelessly connected, enabling real-time communication and user-based control. We suggest these methodologies will help us to better understand and develop sophisticated control for biology.

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

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.

Enhanced electrochemical measurement of ß-galactosidase activity in whole cells by coexpression of lactose permease, LacY.

Whole-cell biosensing links the sensing and computing capabilities of microbes to the generation of a detectable reporter. Whole cells enable dynamic biological computation (filtered noise, amplified signals, logic gating etc.). Enzymatic reporters enable in situ signal amplification. Electrochemical measurements are easily quantified and work in turbid environments. In this work we show how the coexpression of the lactose permease, LacY, dramatically improves electrochemical sensing of ß-galactosidase (LacZ) expressed as a reporter in whole cells. The permease facilitates transport of the LacZ substrate, 4-aminophenyl ß-d-galactopyranoside, which is converted to redox active p-aminophenol, which, in turn, is detected via cyclic voltammetry or chronocoulometry. We show a greater than fourfold improvement enabled by lacY coexpression in cells engineered to respond to bacterial signal molecules, pyocyanin and quorum-sensing autoinducer-2.

Electronic signals are electrogenetically relayed to control cell growth and co-culture composition.

There is much to be gained by enabling electronic interrogation and control of biological function. While the benefits of bioelectronics that rely on potential-driven ionic flows are well known (electrocardiograms, defibrillators, neural prostheses, etc) there are relatively few advances targeting nonionic molecular networks, including genetic circuits. Redox activities combine connectivity to electronics with the potential for specific genetic control in cells. Here, electrode-generated hydrogen peroxide is used to actuate an electrogenetic "relay" cell population, which interprets the redox cue and synthesizes a bacterial signaling molecule (quorum sensing autoinducer AI-1) that, in turn, signals increased growth rate in a second population. The dramatically increased growth rate of the second population is enabled by expression of a phosphotransferase system protein, HPr, which is important for glucose transport. The potential to electronically modulate cell growth via direct genetic control will enable new opportunities in the treatment of disease and manufacture of biological therapeutics and other molecules.

Interactive Materials for Bidirectional Redox-Based Communication.

Emerging research indicates that biology routinely uses diffusible redox-active molecules to mediate communication that can span biological systems (e.g., nervous and immune) and even kingdoms (e.g., a microbiome and its plant/animal host). This redox modality also provides new opportunities to create interactive materials that can communicate with living systems. Here, it is reported that the fabrication of a redox-active hydrogel film can autonomously synthesize a H2 O2 signaling molecule for communication with a bacterial population. Specifically, a catechol-conjugated/crosslinked 4-armed thiolated poly(ethylene glycol) hydrogel film is electrochemically fabricated in which the added catechol moieties confer redox activity: the film can accept electrons from biological reductants (e.g., ascorbate) and donate electrons to O2 to generate H2 O2 . Electron-transfer from an Escherichia coli culture poises this film to generate the H2 O2 signaling molecule that can induce bacterial gene expression from a redox-responsive operon. Overall, this work demonstrates that catecholic materials can participate in redox-based interactions that elicit specific biological responses, and also suggests the possibility that natural phenolics may be a ubiquitous biological example of interactive materials.

Mediated Electrochemical Probing: A Systems-Level Tool for Redox Biology.

Biology uses well-known redox mechanisms for energy harvesting (e.g., respiration), biosynthesis, and immune defense (e.g., oxidative burst), and now we know biology uses redox for systems-level communication. Currently, we have limited abilities to "eavesdrop" on this redox modality, which can be contrasted with our abilities to observe and actuate biology through its more familiar ionic electrical modality. In this Perspective, we argue that the coupling of electrochemistry with diffusible mediators (electron shuttles) provides a unique opportunity to access the redox communication modality through its electrical features. We highlight previous studies showing that mediated electrochemical probing (MEP) can "communicate" with biology to acquire information and even to actuate specific biological responses (i.e., targeted gene expression). We suggest that MEP may reveal an extent of redox-based communication that has remained underappreciated in nature and that MEP could provide new technological approaches for redox biology, bioelectronics, clinical care, and environmental sciences.

Redox Electrochemistry to Interrogate and Control Biomolecular Communication.

Cells often communicate by the secretion, transport, and perception of molecules. Information conveyed by molecules is encoded, transmitted, and decoded by cells within the context of the prevailing microenvironments. Conversely, in electronics, transmission reliability and message validation are predictable, robust, and less context dependent. In turn, many transformative advances have resulted by the formal consideration of information transfer. One way to explore this potential for biological systems is to create bio-device interfaces that facilitate bidirectional information transfer between biology and electronics. Redox reactions enable this linkage because reduction and oxidation mediate communication within biology and can be coupled with electronics. By manipulating redox reactions, one is able to combine the programmable features of electronics with the ability to interrogate and modulate biological function. In this review, we examine methods to electrochemically interrogate the various components of molecular communication using redox chemistry and to electronically control cell communication using redox electrogenetics.

A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks.

There is a growing interest in mediating information transfer between biology and electronics. By the addition of redox mediators to various samples and cells, one can both electronically obtain a redox "portrait" of a biological system and, conversely, program gene expression. Here, we have created a cell-based synthetic biology-electrochemical axis in which engineered cells process molecular cues, producing an output that can be directly recorded via electronics-but without the need for added redox mediators. The process is robust; two key components must act together to provide a valid signal. The system builds on the tyrosinase-mediated conversion of tyrosine to L-DOPA and L-DOPAquinone, which are both redox active. "Catalytic" transducer cells provide for signal-mediated surface expression of tyrosinase. Additionally, "reagent" transducer cells synthesize and export tyrosine, a substrate for tyrosinase. In cocultures, this system enables real-time electrochemical transduction of cell activating molecular cues. To demonstrate, we eavesdrop on quorum sensing signaling molecules that are secreted by Pseudomonas aeruginosa, N-(3-oxododecanoyl)-l-homoserine lactone and pyocyanin.

A redox-based electrogenetic CRISPR system to connect with and control biological information networks.

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.

Homologous Quorum Sensing Regulatory Circuit: A Dual-Input Genetic Controller for Modulating Quorum Sensing-Mediated Protein Expression in E. coli.

We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.

Redox-Based Synthetic Biology Enables Electrochemical Detection of the Herbicides Dicamba and Roundup via Rewired Escherichia coli.

Synthetic biology is typically exploited to endow bacterial cells with new biosynthetic capabilities. It can also serve to create "smart" bacteria such as probiotics that detect and treat disease. Here, we show how minimally rewiring the genetic regulation of bacterial cells can enable their ability to recognize and report on chemical herbicides, including those routinely used to clear weeds from gardens and crops. In so doing, we demonstrate how constructs of synthetic biology, in this case redox-based synthetic biology, can serve as a vector for information flow mediating molecular communication between biochemical systems and microelectronics. We coupled the common genetic reporter, ß-galactosidase, with the E. coli superoxide response regulon promoter pSoxS, for detection of the herbicides dicamba and Roundup. Both herbicides activated our genetic construct in a concentration dependent manner. Results indicate robust detection using spectrophotometry, via the Miller assay, and electrochemistry using the enzymatic cleavage of 4-aminophenyl ß-d-galactopyranoside into the redox active molecule p-aminophenol. We found that environmental components, in particular, the availability of glucose, are important factors for the cellular detection of dicamba. Importantly, both herbicides were detected at concentrations relevant for aquatic toxicity.