Cross, M. C. G.; Aboulnaga, E.; TerAvest, M. A.

Microbial electrosynthesis (MES) is a sustainable approach to chemical production from CO2 and 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 oneidensis could 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 CO2 reduction to formate. However, wild-type S. oneidensis does not tolerate high levels of formate. In this work, we created and characterized formate-tolerant strains of S. oneidensis for 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 to S. 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 the S. oneidensis sodium-dependent bicarbonate transporter improves formate tolerance of Zymomonas mobilis, indicating the potential for the transfer of this formate tolerance phenotype to other organisms.