Trehalose acts as a uridine 5′‐diphosphoglucose‐competitive inhibitor of trehalose 6‐phosphate synthase in <i>Corynebacterium glutamicum</i>

<jats:p>Trehalose is a compatible solute widely distributed in nature. The most prevalent pathway for its synthesis starts from condensation of glucose 6‐phosphate (Glc6<jats:italic>P</jats:italic>) and uridine 5′‐diphosphoglucose (<jats:styled-content style="fixed-case">UDP</jats:styled-content>‐Glc) catalyzed by trehalose 6‐phosphate synthase (<jats:styled-content style="fixed-case">TPS</jats:styled-content>). A previous laboratory evolution experiment with the bacterium <jats:italic>Corynebacterium glutamicum</jats:italic> generated strains adapted to supraoptimal temperatures, and the R328H substitution of the <jats:styled-content style="fixed-case">TPS</jats:styled-content> encoded by <jats:italic>otsA</jats:italic> was shown to be associated with thermotolerance acquired by the evolved strains. In this study, we found that the OtsA:R328H substitution promotes both intra‐ and extracellular trehalose accumulation and demonstrated that build‐up of intracellular trehalose accounts for the Ots<jats:styled-content style="fixed-case">A<jats:sup>R</jats:sup></jats:styled-content><jats:sup>328H</jats:sup>‐dependent thermotolerance, using the mycobacterial trehalose‐specific transporter. Counterintuitively, characterization of the recombinant OtsA proteins revealed that the mutation downshifts the temperature optimum of OtsA. A search for the molecular basis of Ots<jats:styled-content style="fixed-case">A<jats:sup>R</jats:sup></jats:styled-content><jats:sup>328H</jats:sup>‐dependent enhancement of trehalose synthesis led to the unexpected findings that trehalose is an effective inhibitor of OtsA and that Ots<jats:styled-content style="fixed-case">A<jats:sup>R</jats:sup></jats:styled-content><jats:sup>328H</jats:sup> is highly tolerant to the trehalose‐mediated inhibition. The only available report on such feedback regulation of <jats:styled-content style="fixed-case">TPS</jats:styled-content> is for the silk moth from over 50 years ago [Murphy TA and Wyatt GR (1965) <jats:italic>J Biol Chem</jats:italic> 240, 1500–1508]. While trehalose acts as a Glc6<jats:italic>P</jats:italic>‐competitive inhibitor in the silk moth, the disaccharide was found to inhibit OtsA in a <jats:styled-content style="fixed-case">UDP</jats:styled-content>‐Glc‐competitive manner in <jats:italic>C. glutamicum</jats:italic>, suggesting independent origins of the negative feedback regulations found for the two species. We showed that overexpression of the wild‐type OtsA counteracts the trehalose‐dependent regulation and restores the evolved strain‐like phenotype to the isogenic wild‐type <jats:italic>otsA</jats:italic> revertant, demonstrating that thermotolerance conferred by Ots<jats:styled-content style="fixed-case">A<jats:sup>R</jats:sup></jats:styled-content><jats:sup>328H</jats:sup> is attributable to its feedback‐resistant property.</jats:p>