Linde Kampers

We determine the effect of exchanging the L-aspartate oxidase gene in different mesophiles for a thermophilic ortholog. In E. coli, the exchange results in a 10 times shorter lag phase when grown at 44 °C. In P. putida the generation time doubled when grown at 40 °C. To determine how widespread the connection between NAD+ availability and thermo-tolerance is, the mesophilic or thermophilic aspartate pathway was implemented in yeast, which generates NAD+ via the kynurenine pathway. Not only was S. cerevisiae able to use the aspartate pathway for NAD+ generation, but supplemented with the bacterial pathway the strain proved more robust when grown at 41 °C.

To determine what effect this increased thermo-tolerance could have on an industrial scale, Chapter 7 describes the development of a millifluidics device. A down-scaled microfluidics system was built for this purpose. When kept at room temperature, the device can expose the cells to temperature fluctuations ranging from 25 °C to 50 °C. The system was applied to monitor long-term recombinant strain performance with respect to these fluctuations. The recombinant thermo-tolerant strains were tested in this system over a three-day span. It was seen that with a temperature fluctuation regime of 50-40-30-40 °C, P. putida KT244O ∆nadB pS638 nadBBsm equipped with the thermophilic ortholog from B. smithii grows better than the other strains, including the wild type P. putida KT244O which is unburdened by a plasmid.

The discovery of the universal link between the NAD+ availability in microbes and their thermo-tolerance was patented. Appendix chapter 6 shows the patent. This invention relates to the field of molecular microbiology, metabolic engineering, and fermentation technology. In particular, this relates to microbial host cells that have been engineered for increased tolerance to temperature shifts, for increased performance at temperatures different from optimal temperature of a micro-organism and/or for changing at least one of the cardinal temperatures of a micro-organism by replacing an endogenous NAD+ biosynthesis gene by a heterologous gene encoding a corresponding enzyme with another temperature profile. With the patent, a full trajectory from lab to test to application was completed.