Capturing Dynamics of Adventitious and Cultured Virus Propagation in Plate and Suspension Cell Cultures Via Modeling

Biocatalysis, microbiome, microbial reduction, biomaterials, gut microbiota, synthetic biology

Biocatalysis overcomes the setbacks of traditional chemical catalysis such as high energy consumption, dangerous or harsh conditions, and abrasive regeneration requirements; however, it lacks the modularity seen in chemical systems. Potential products are often limited to known biochemical transformations and deviations in substrate scope are frequently poorly tolerated. To bridge the advantages of traditional catalysis with biocatalysis we propose extracellular electron transfer (EET) as a whole-cell biocatalytic mechanism for controlling extracellular redox reactions. EET is an anaerobic respiration process that couples carbon oxidation to the reduction of extracellular metal species. In the model electroactive bacterium Shewanella oneidensis MR-1, the key EET-protein pathway, the Mtr-pathway, can be controlled via genetic engineering and metabolism manipulation to direct electron flux from the bacteria. As a result, the electron flux can be directed towards a redox catalyst of interest to control metal-controlled synthetic transformations. A model reaction is Cu(I)-catalyzed Alkyne-Azide Cycloaddition (CuAAC), due to the strict requirement for Cu(I) over Cu(II), high degree of substrate specificity, and ability to occur in water. We found that both anaerobic and aerobic conversions were controlled via EET from actively respirating MR-1 to reduce Cu(II) to Cu(I). Due to the link between EET and the central carbon metabolism, the rate of reaction could be controlled by solely changing the carbon sources and switched between “OFF” and “ON” states. As a facultative anaerobe, the bacteria can remove oxygen from their environment and withstand greater perturbations than a traditional chemical reductant, allowing for multiple reaction cycles without regeneration. Mtr-pathway dependence was confirmed as knockouts attenuated the reaction and complementation of the proteins in their cognate knockout rescued the ability to perform the reaction. Preliminary substrate screens indicate a high degree of modularity and robust substrate and ligand scope.

First, we applied this system to enable biorthogonal CuAAC membrane labelling on live mammalian cells, indicating that this biocatalyst can be employed in robust and complex environments. Next, the system was adapted to control the formation of a 4-arm-PEG hydrogel in an engineered living materials system. Then, leveraging the high degree of genetic control, a series of two-input genetic Boolean logic gates controlling the terminal protein, MtrC, in the Mtr-pathway allowed the cells to sense dilute chemical stimuli in their environment and actuate a change in material stiffness.

Finally, the system was adapted for use in a microdroplet system to screen for EET-capable bacteria in a population. A screen analyzing a pseudo-mixed population of MR-1 (WT) and an Mtr-pathway knockout revealed that the EET-capable strain could be enriched over 200%. More complex mixed populations containing Escherichia coli Nissle 1917, Saccharomyces cerevisiae, and MR-1 revealed approximately a 400% increase in MR-1. Utilizing this high-throughput screen, bacteria harvested from mineral sediment from Lake Austin was sorted in microdroplets for individual EET-capable cells via fluorescent readout. This screen isolates bacteria to individual micro-droplets removing cellular resource competition. As bacterial consortia found in the human gut, soil, and natural bodies of water can often prove challenging to isolate and characterize in a laboratory setting, this screen allows for phenotypic screens without the requirement of batch culture.

Overall, these works display the breadth of application of EET to control CuAAC in small molecule synthesis, engineered living materials, and phenotypic microfluidic screens. Further work will include use of the microfluidic assay to study S. oneidensis mutant libraries and other mixed consortia for high-performing EET phenotypes.

Teaching Interests:

Chemical Engineering Thermodynamics (graduate and undergraduate), Chemical Engineering mass balances, Analytical techniques