Hydroxylation of ectoine and synthetic ectoine derivatives via E. coli -mediated whole-cell biotransformation

The compatible solutes ectoine and hydroxyectoine are compounds mainly produced by halophilic organisms in order to prevent the loss of intracellular water when exposed to high osmotic pressure. Furthermore, these substances were shown to stabilize enzymes and other biomolecules under several stress factors like heat, cold, desiccation or oxidation stress in vitro. Although ectoine and hydroxyectoine only differ in a single hydroxyl group, they show partly different traits regarding their stabilizing abilities. Especially hydroxyectoine, a glass-forming substance, is highly effective against heat and desiccation for biomolecules and whole cells, making it a compound of high commercial value. The industrial production of these compounds is accomplished by the natural producer Halomonas elongata. Under high saline conditions and elevated temperatures, a mixture of both substances is produced. The purification and seperation of both solutes by chromatographic methods is a high-cost process, causing a demand for cheaper production procedures. The whole-cell biotransformation of ectoine by E. coli is a very competitive method concerning production costs, as it is possible to completely convert ectoine into its hydroxylated form under low-salt conditions. E. coli is able to take up ectoine and convert it to hydroxyectoine by heterologously produced ectoine hydroxylase EctD, while the product is excreted into the medium. Neither the educt nor the product is metabolized by this organism, making it a suitable biotransformation system. \n\t \nIn this work it could be shown that the E. coli-mediated whole-cell biotransformation performance is highly dependent on the availability of 2-oxoglutarate as it is an important cofactor for ectoine hydroxylase EctD. Under N-limiting growth conditions, intracellular 2 oxoglutarate concentrations are increased in the stationary growth phase which enables the conversion of ectoine to hydroxyectoine by resting cells. Furthermore, it was shown that the hydroxylation performance is dependent on the heterologously produced ectoine hydroxylase, as well as the biotransformation strain. By the use of E. coli DH5α overproducing EctD of the psychrophilic organism Sphingopyxis alaskensis, a complete conversion of 30 mM ectoine was carried out at low salt conditions (1 % NaCl). Higher salinities do not significantly increase the hydroxylation performance. Limiting factors are enzyme stability/activity, a reduced glucose uptake rate at N-limiting conditions and, to a certain extent, a competition for uptake mechanisms in E. coli between ectoine and its hydroxylated product. \n\t \nBesides ectoine, several synthetic ectoine derivatives are recognized as substrates for the ectoine hydroxylases of H. elongata, S. alaskensis, Pseudomonas stutzeri and Acidiphilium cryptum as could be shown in vivo and in vitro. The corresponding hydroxylated synthetic ectoine derivatives were produced, purified and identified via 13C-NMR. In addition, novel (hydroxylated) synthetic ectoine derivatives were characterized regarding their ability to preserve the functionality of biomolecules like enzymes (LDH, RNase A) in vitro or whole cells when exposed to stress factors like heat, desiccation, freeze-thaw stress and osmotic pressure. Especially hydroxylated solutes proved to be highly effective against desiccation and heat stress. These (hydroxylated) synthetic ectoine derivatives might be of commercial interest for different branches like medical or skin-care industries, as they show superior stabilizing traits for certain applications when compared to their natural lead compound ectoine.