A small number of point mutations confer formate tolerance in Shewanella oneidensis

Journal Article · 21 May 2025 · Applied and Environmental Microbiology

DOI: https://doi.org/10.1128/aem.01968-24 · OSTI ID:2554111

Cross, Megan C. Gruenberg ; Aboulnaga, Elhussiny; TerAvest, Michaela A. ; Buan, ed., Nicole R.

ABSTRACT Microbial electrosynthesis (MES) is a sustainable approach to chemical production from CO ₂ 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 CO ₂ reduction to formate. However, we report that 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 gene encoding a predicted sodium-dependent bicarbonate transporter and a DUF2721-containing 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 putative sodium-dependent bicarbonate transporter improve formate tolerance of Zymomonas mobilis , indicating the potential of transferring this formate tolerance phenotype to other organisms. IMPORTANCE Shewanella oneidensis is a bacterium with a well-studied, efficient extracellular electron transfer pathway. This capability could make this organism a suitable host for microbial electrosynthesis using CO ₂ or formate as feedstocks. However, we report here that formate is toxic to S. oneidensis , limiting the potential for its use in these systems. In this work, we evolve several strains of S. oneidensis that 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 wild-type strain. Finally, the formate tolerance mechanism of one variant is studied using structural modeling and expression in another host. This study, therefore, presents a simple method for conferring formate tolerance to bacterial hosts.

• Sponsoring Organization: USDOE
• Grant/Contract Number: SC0018409
• OSTI ID: 2554111
• Journal Information: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 5 Vol. 91; ISSN 0099-2240
• Publisher: American Society for MicrobiologyCopyright Statement
• Country of Publication: United States
• Language: English