Thermal stress-based pathway engineering and bioprocess optimization of Corynebacterium glutamicum for de novo hydroxyectoine production

Background: Hydroxyectoine is a high-value compatible solute with applications in cosmetics, healthcare, and biotechnology. Its microbial production has traditionally relied on halophilic organisms requiring high-salinity cultivation, motivating the development of non-halophilic production hosts. Corynebacterium glutamicum is an attractive platform because of its industrial robustness and high metabolic capacity through the aspartate-family amino acid pathway. Results: A synthetic hydroxyectoine pathway was constructed by combinatorially varying the predicted translation initiation rates of four translational units comprising ask, ectAB, ectC, and ectD. A library of C. glutamicum transformants was screened at 40{degrees}C using hydroxyectoine-associated thermoprotection as a functional selection principle. Fast-growing variants generally displayed increased hydroxyectoine formation and higher hydroxyectoine-to-ectoine ratios than a strain carrying the native pathway configuration. The selected combination reached a hydroxyectoine-to-ectoine ratio of 4.7 and was transferred into the lysine-producing strain DM1729SL in order to increase precursor availability and hydroxyectoine production. Carbon-limited fed-batch cultivations showed that both temperature and dissolved oxygen influenced production. The best performance was obtained at 30{degrees}C and 50% relative dissolved oxygen, yielding 12.9 g/L hydroxyectoine with a yield of 0.161 g/g glucose and a volumetric productivity of 0.26 g/L/h after glucose depletion. A subsequent hypoosmotic downshock increased extracellular hydroxyectoine recovery to 14.5 g/L. Conclusions: Thermal stress-based combinatorial pathway balancing enabled efficient de novo hydroxyectoine production in C. glutamicum. Integration of precursor-enhanced host metabolism, controlled oxygen supply, fed-batch cultivation, and osmotic downshock resulted in a substantial improvement over previously reported de novo hydroxyectoine production in this host and establishes C. glutamicum as a promising platform for further hydroxyectoine process development.