Synthesis and release of the bacterial compatible solute 5-hydroxyectoine in Hansenula polymorpha

► Hansenula polymorpha was engineered to express the enzymes of the Halomonas elongata derived 5-hydroxyectoine biosynthesis pathway. ► Expression of EctB, EctA, EctC and EctD in H. polymorpha led to production of 5-hydroxyectoine in gram per liter scale. ► 5-Hydroxyectoine synthesis was influenced by feeding of methanol during cultivation. Ectoine and 5-hydroxyectoine belong to the family of compatible solutes which are known to mainly contribute to the adaptation of the cell to osmotic stress by mediation of a constant turgor. In addition the cell's essential functions are maintained under stress conditions like high salinity, heat or aridity stress. Hansenula polymorpha was engineered to catalyze the transformation of monomeric substrates to 5-hydroxyectoine. For this purpose four genes encoding the enzymes of the 5-hydroxyectoine biosynthesis pathway of Halomonas elongata, EctA, EctB, EctC, and EctD, were inserted into the genome of H. polymorpha. Subsequently the syntheses of ectoine and 5-hydroxyectoine were analyzed and optimized. We showed that H. polymorpha is a suitable system for recombinant 5-hydroxyectoine synthesis in gram per liter scale (2.8gL−1 culture supernatant, 365μmol/g dcw) in which almost 100% conversion of ectoine to 5-hydroxyectoine without necessity of high salinity were achieved.