A small number of point mutations confer formate tolerance inShewanella oneidensis

Abstract

Microbial electrosynthesis (MES) is a sustainable approach to chemical production from CO2and clean electricity. However, limitations in electron transfer efficiency and gaps in understanding of electron transfer pathways in MES systems prevent full realization of this technology.Shewanella oneidensiscould serve as an MES biocatalyst because it has a well-studied, efficient transmembrane electron transfer pathway. A key first step in MES in this organism could be CO2reduction to formate. However, wild-typeS. oneidensisdoes not tolerate high levels of formate. In this work, we created and characterized formate-tolerant strains ofS. oneidensisfor further engineering and future use in MES systems through adaptive laboratory evolution. Two different point mutations in a sodium-dependent bicarbonate transporter and a DUF2721-contianing protein separately confer formate tolerance toS. oneidensis. The mutations were further evaluated to understand their role in improving formate tolerance. We also show that the wild-type and mutant versions of theS. oneidensissodium-dependent bicarbonate transporter improves formate tolerance ofZymomonas mobilis, indicating the potential for the transfer of this formate tolerance phenotype to other organisms.ImportanceShewanella oneidensisis a bacterium with a well-studied, efficient extracellular electron transfer pathway. This capability could make this organism a suitable host for microbial electrosynthesis using CO2or formate feedstocks. However, formate is toxic toS. oneidensis, limiting the potential for its use in these systems. In this work, we evolve several strains ofS. oneidensisthat have improved formate tolerance and we investigate some mutations that confer this phenotype. The phenotype is confirmed to be attributed to several single point mutations by transferring the wild- type and mutant versions of each gene to the unmutated strain. Finally, the formate tolerance mechanism of one mutation is studied using structural modeling and expression in another host. This study therefore presents a simple method for conferring formate tolerance to bacterial hosts.