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

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 Gluconobacter frateurii 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 otsAB 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 otsAB 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 otsAB 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 G. frateurii . IMPORTANCE The biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show that Gluconobacter frateurii , 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 G. frateurii , trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP + ratio to enhance thermotolerance.