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bioRxiv · 10.1101/2024.06.28.601276

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

Cross, M. C. G.·Aboulnaga, E.·TerAvest, M. A.
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Abstract

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. ImportanceShewanella 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 CO2 or formate feedstocks. However, 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 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/601276v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@111c7a7org.highwire.dtl.DTLVardef@4acc78org.highwire.dtl.DTLVardef@b60062org.highwire.dtl.DTLVardef@d35dca_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Cross, M. C. G., Aboulnaga, E., TerAvest, M. A.. 2024-06-29. A small number of point mutations confer formate tolerance in Shewanella oneidensis. https://doi.org/10.1101/2024.06.28.601276

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