Temperature Resilience Through NadB We discovered a thermoresilient effect that occurs when the inherent nadB gene of the aspartate/kynurenine pathway is exchanged for that of a thermophilic ortholog.

Universally essential redox factor NAD+ is involved in vital cellular processes, and first to respond to stress. The Aspartate pathway is one of the main routes for NAD+ biosynthesis in microbes, in which L-aspartate oxidase is a highly temperature sensitive key bottleneck.We interchanged L-aspartate oxidase in P. putida KT2440 for a thermophilic ortholog, resulting in an exponential growth phase twice as steep upon a +10C temperature shift. A down-scaled millifluidics device was built to determine strain performance through temperature fluctuations occurring on industrial scale, in which the recombinant strain significantly outperformed thecontrols. We pinpointed the cause of this effect using MSMS-TOF and transcriptomics.The effect of stabilised NAD+ production on temperature robustness proved universal when implementing the mesophilicor thermophilic L-aspartate oxidase or aspartate pathway in E. coli, resulting in a 10-fold shorter lag phase upon a +7C temperature shift, or in S. cerevisiae, increasing strain robustness at 41C.