A Single-Nucleotide Insertion in a Drug Transporter Gene Induces a Thermotolerance Phenotype in Gluconobacter frateurii by Increasing the NADPH/NADP ⁺ Ratio via Metabolic Change

Abstract

Thermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adapted strain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The Δ strain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The Δ mutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP ratio was higher in strain Wild-G, and much higher in the Δ strain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance in The biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show that, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP ratios and reductions in ROS levels. We concluded that in , trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP ratio to enhance thermotolerance.

Mutations in degP and spoT Genes Mediate Response to Fermentation Stress in Thermally Adapted Strains of Acetic Acid Bacterium Komagataeibacter medellinensis NBRC 3288

“…Such stress tolerance may be related to the generation of reactive oxygen species (ROS). Microbial cells generate a large amount of ROS, especially when grown at growth-limiting high temperatures ( Davidson et al, 1996 ; Matsushita et al, 2016 ; Chang et al, 2017 ; Nantapong et al, 2019 ), and thermally adapted strains have been shown to generate less ROS than the wild-type strain when grown at higher growth temperatures ( Matsumoto et al, 2018 , 2020 ). Acetic acid fermentation may similarly generate ROS due to structural damage to the cell membrane caused by acetic acid and/or ethanol.…”

“…Experimental evolution is one of the means of obtaining thermotolerant microorganisms. Thermal adaptation has been previously achieved in various microorganisms, including Escherichia coli ( Rudolph et al, 2010 ; Rodriguez-Verdugo et al, 2014 ), Saccharomyces cerevisiae ( Caspeta et al, 2014 ), Acetobacter pasteurianus ( Azuma et al, 2009 ; Matsutani et al, 2013 ; Matsumoto et al, 2020 ), Komagataeibacter oboediens ( Taweecheep et al, 2019 ), and Gluconobacter frateurii ( Hattori et al, 2012 ; Matsumoto et al, 2018 ). Furthermore, the mechanism associated with thermotolerance was also analyzed using these strains.…”

Trehalose alleviates high‐temperature stress in Pleurotus ostreatus by affecting central carbon metabolism

“…In addition, trehalose regulates the PPP in pathogenic fungi during infection [ 29 ]. Moreover, trehalose mediates thermotolerance in Gluconobacter frateurii ; research has shown that trehalose does not contribute to thermotolerance directly but that a metabolic change including increased carbon flux to the PPP is instead the key factor [ 30 ]. However, the studies above reflect the role of trehalose in a single metabolic pathway, and few reports have addressed the regulatory effects of trehalose in multiple metabolic pathways and in redirecting metabolic flux.…”

Microbial Stress: From Model Organisms to Applications in Food, Microbiotechnology and Medicine

“…Such stress tolerance may be related to the generation of reactive oxygen species (ROS). Microbial cells generate a large amount of ROS, especially when grown at growth-limiting high temperatures Matsushita et al, 2016;Chang et al, 2017;Nantapong et al, 2019), and thermally adapted strains have been shown to generate less ROS than the wild-type strain when grown at higher growth temperatures (Matsumoto et al, 2018.…”

“…Experimental evolution is one of the means of obtaining thermotolerant microorganisms. Thermal adaptation has been previously achieved in various microorganisms, including Escherichia coli (Rudolph et al, 2010;Rodriguez-Verdugo et al, 2014), Saccharomyces cerevisiae (Caspeta et al, 2014), Acetobacter pasteurianus (Azuma et al, 2009;Matsutani et al, 2013;, Komagataeibacter oboediens (Taweecheep et al, 2019), and Gluconobacter frateurii (Hattori et al, 2012;Matsumoto et al, 2018). Furthermore, the mechanism associated with thermotolerance was also analyzed using these strains.…”