Precise electronic control of redox reactions inside Escherichia coli using a genetic module

Abstract

18Microorganisms regulate the redox state of different biomolecules to precisely control 19 biological processes. These processes can be modulated by electrochemically coupling 20 intracellular biomolecules to an external electrode, but current approaches afford only 21 limited control and specificity. Here we describe specific electrochemical control of the 22 Microorganisms accomplish important biological functions such as conserving energy, 32 regulating gene expression, and powering biosynthesis using different redox-active 33 biomolecules. To enable control of these processes in any microorganism, researchers have 34 coupled the redox state of these biomolecules to an external electrode using membrane-35 permeable, small molecule redox mediators 1-5 , redox polymers 6 , and membrane-36 intercalated nanostructures 7,8 . These approaches can allow cells to produce electrical 37 current or consume it, resulting in either oxidation or reduction of intracellular redox species, 38 respectively. Bioelectrochemical devices can then be used to drive biosynthetic reactions 39 1,4,5 , perform bioelectronic sensing 9 , actuate gene expression 3 , and modulate cellular growth 40 10,11 within the microorganism of interest. Despite these accomplishments, these strategies 41 Results 75E. coli consume current using mtr and native oxidoreductases 76 We first sought to determine if the Mtr pathway could allow cathodic electrons to 77 directly enter a heterologous host upon addition of an electron acceptor. Since E. coli has 78 two MK-linked fumarate reductases, FrdABCD and SdhABCD, we hypothesized that the Mtr 79 pathway in E. coli could deliver cathodic electrons via these native proteins to fumarate 80 ( Figure 1A). To probe the specific role of the Mtr pathway, we compared the behavior of 81 several strains: E. coli expressing only the cytochrome c maturation (ccm) genes (abbrev. 82Ccm-E.coli) 17 , E. coli expressing ccm and mtrCAB (abbrev. Mtr-E. coli) 15 , and E. coli 83 expressing ccm and cymAmtrCAB (abbrev. CymAMtr-E. coli) 17 . The ccm genes are required 84 to make cytochromes c in the C43(DE3) parental background. 85To prepare E. coli for cathodic conditions, individual strains were first grown 86 aerobically, incubated in potentiostatic-controlled bioreactors under anaerobic, anodic 87 conditions (∆V=+200 mV Ag/AgCl ) for at least one day. Under these conditions, the CymAMtr-88 E. coli strain produced a significant steady-state current, while Ccm-E. coli and Mtr-E. coli 89 produced much lower currents (Figure 2A), reinforcing that CymA is important for current 90 production [16][17][18] . As anaerobic conditions were maintained, the electrode bias was then 91 switched to cathodic conditions (∆V= -560 V Ag/AgCl ), fumarate was added, and current 92 consumption was measured. In the absence of E. coli, neither current consumption nor 93 fumarate reduction was observed (Figure S1A). Likewise, the Ccm-E. coli strain did not 94 consume significant levels of current (Figure 2B). In contrast, both the Mtr-E. coli and t...

Tailored extracellular electron transfer pathways enhance the electroactivity of Escherichia coli

“…Electrochemical measurments were carried out in a single chambered cell with M9-lactate medium [40] as the electrolyte, a Pt wire counter electrode, an Ag/AgCl reference electrode and a 1 x 1.1 x 0.5 cm graphite felt (Alfa Aesar) electrode connected to a Pt wire as the anode.…”

Engineering Wired Life: Synthetic Biology for Electroactive Bacteria

“…E. coli strains engineered with the S. oneidensis CymA-Mtr EET pathways can also take up electrons from electrodes and reduce a range of electron acceptors. − An intriguing possibility would be to connect the reducing power provided through direct extracellular electron uptake to a fully synthetic CO 2 fixation pathway in E. coli.…”