Harnessing bacterial electroactivity with materials science and synthetic biology

Abstract

Biotechnology has been transformed by increased cellular control over molecular products and processes. The fields of metabolic engineering and synthetic biology have standardized methods for biosynthesizing chemicals and progressed development of novel vaccines, genetic therapies, and transgenic organisms. Despite these significant advances, cellular programmability has largely stopped short of controlling the non-biological world – forward bioengineering of abiotic and inorganic processes, such as bio-electronic interfaces and material syntheses, remains a substantial challenge. To realize the benefits of engineered biology with such technologies, chassis organisms and extracellular pathways that connect genetics with inorganic transformations are required. In this work, we develop the electroactive bacterium Shewanella oneidensis and its extracellular electron transfer pathways as a genetically tunable platform to control redox-driven reactions. To engineer S. oneidensis electroactivity for redox chemistry, we accomplish two overarching objectives. For the first objective, we establish genotype-phenotype relationships between S. oneidensis and material syntheses/transformations that tune material properties and functioning. In Chapter 2, we determine that S. oneidensis activates metal catalysts involved in atom-transfer radical polymerization and that polymerization rate depends on carbon metabolism and expression of key electron transfer proteins. In Chapter 3, we show that S. oneidensis reductively precipitates palladium nanoparticles onto the cell surface and that expression of outer membrane cytochromes and availability of redox-active metabolites alters particle size and cellular localization. In Chapter 4, we demonstrate that S. oneidensis respires onto synthetic mineral analogs (metal-organic frameworks) via outer membrane cytochromes and that these bio-functionalized materials enable chromium remediation. For the second objective, we develop genetic tools to program extracellular electron flux and material formation by S. oneidensis. In Chapter 5, we use transcription/translation regulators and high throughput redox analysis to build plasmid-based logic gates in S. oneidensis that express single extracellular electron transfer proteins and exhibit inducible electroactivity responses, similar to fluorescent protein circuits. In Chapter 6, we leverage one of these S. oneidensis circuits to control radical crosslinking of a semi-synthetic hydrogel and predictably modulate gel stiffness via inducer molecule concentration. Taken together, this work provides new knowledge of S. oneidensis-controlled material transformations and presents a workflow to program extracellular electron transfer using synthetic biology