Tuning Promoter Strengths for Improved Synthesis and Function of Electron Conduits in Escherichia coli

Introduction of the electron transfer complex MtrCAB from Shewanella oneidensis MR-1 into a heterologous host provides a modular and molecularly defined route for electrons to be transferred to an extracellular inorganic solid. However, an Escherichia coli strain expressing this pathway En displayed limited control of MtrCAB expression and impaired cell growth. To overcome these limitations and to improve heterologous extracellular electron transfer, we used an E. coli host with a more tunable induction system and a panel of constitutive promoters to generate a library of strains that separately transcribe the mtr and cytochrome c maturation (ccm) operons over 3 orders of magnitude. From this library, we identified strains that show 2.2 times higher levels of MtrC and MtrA and that have improved cell growth. We find that a similar to 300-fold decrease in the efficiency of MtrC and MtrA synthesis with increasing mtr promoter activity critically limits the maximum expression level of MtrC and MtrA. We also tested the extracellular electron transfer capabilities of a subset of the strains using a three-electrode microbial electrochemical system. Interestingly, the strain with improved cell growth and fewer morphological changes generated the largest maximal current per cfu, rather than the strain with more MtrC and MtrA. This strain also showed similar to 30-fold greater maximal current per cfu than its ccm-only control strain. Thus, the conditions for optimal MtrCAB expression and anode reduction are distinct, and minimal perturbations to cell morphology are correlated with improved extracellular, electron transfer in E. coli.