A hybrid cytcmaturation system enhances the bioelectrical performance of engineeredEscherichia coliby improving the rate-limiting step

Abstract

18Bioelectronic devices can use electron flux to enable communication between biotic components 19 and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes 20 by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe 21 inefficient electrical performance, which we hypothesize is due to the limited compatibility of the 22 E. coli cytochrome c maturation (ccm) systems with MR-1 cyt c. Here we test whether the 23 bioelectronic performance of E. coli can be improved by constructing hybrid ccm systems with 24 parts from both E. coli and S. oneidensis MR-1. Our results show that the hybrid CcmH can 25 increase cytochrome c expression and the stoichiometry of protein components also shifted. 26 Electrochemical measurements show that the electron flux per cell increased 48% with the hybrid 27 system. Additionally, the electrical response doubled with addition of exogenous flavin, which 28 gives us a quantitative understanding of the bottleneck step in this electron conduit. These results 29 demonstrate that this hybrid ccm system can enhance the bioelectrical performance of the cyt c 30 expressing Escherichia coli, allowing to build more efficient bioelectronic devices. 31 32 KEYWORDS 33 Microbial electrochemistry, microbial fuel cell, bioelectronic sensor, synthetic biology, 34 cytochrome c maturation 35 36

Tailored extracellular electron transfer pathways enhance the electroactivity of Escherichia coli

“…The postive effect of STC expression on EET confirms that electron delivery across CymA in the inner membrane was limiting EET [31] . Since reduction of STC improves turnover of CymA, the system was limited in electron transfer from CymA to MtrA.…”

“…Future bioengineering approaches to enhance EET could therefore target accumulation of NADH and ubiquinol instead of the Mtr pathway, to increase electron delivery to the inner membrane. Modification of the ccm machinery was previously shown to increase CymA expression levels [31] , and could be coupled with the here reported improved electron transfer pathway to further enhance the electroactivity of engineered E. coli.…”

“…In addition, heterologous expression of STC alongside CymA and MtrA was previously shown to improve reduction of a soluble electron acceptor, as compared to expression of other periplasmic cytochromes from S. oneidensis MR-1 [17] . As electron transfer through CymA may be limiting electron transfer rates in engineered E. coli [31] , its oxidation by STC for rapid turnover makes this approach especially attractive to increase EET. In a second approach, we aimed to combine cytochromes from both E. coli and S. oneidensis MR-1 to leverage the native ability of E. coli for electron delivery to the periplasm in a hybrid EET pathway.…”

A living conductive marine biofilm engineered to sense and respond to small molecules

“…This is an important step in the search for biosensors: they should ideally be able to function in environmental sample matrices. In contrast, E. coli engineered to express the Mtr and CymA proteins have used cells grown aerobically on rich medium and preinduced to measure the increase in current 9, 41 or additional engineering to immobilize cells at the electrode 42 . Inducible current during biofilm formation has been demonstrated in S. oneidensis with a refactored native promoter for trimethylamine N-oxide (TMAO) 6 , with the DAPG sensor used here 16 , and with sensors for IPTG, Tc, and arabinose 7 .…”

“…E. coli engineered to express the MtrCAB conduit and CymA showed a catalytic wave centered near 0.210 V SHE (0 V Ag/AgCl) 10 . More recently, a redox potential of 0.051 V SHE was reported using differential pulse voltammetry (DPV) for an improved strain of E. coli expressing a modified version of the ccm operon 41 .…”