TY - JOUR A1 - Eilert, Eva A1 - Kranz, Andreas A1 - Hollenberg, Cornelis P. A1 - Piontek, Michael A1 - Suckow, Manfred T1 - Synthesis and release of the bacterial compatible solute 5-hydroxyectoine in Hansenula polymorpha JF - Journal of biotechnology N2 - 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 (2.8 g L⁻¹ culture supernatant, 365 μmol/g dcw) in which almost 100% conversion of ectoine to 5-hydroxyectoine without necessity of high salinity were achieved. Y1 - 2013 UR - http://www.ncbi.nlm.nih.gov/pubmed/23467000 U6 - https://doi.org/10.1016/j.jbiotec.2013.02.005 VL - 167 IS - 2 SP - 85 EP - 93 PB - Elsevier ER - TY - JOUR A1 - Eilert, Eva A1 - Rolf, Theresa A1 - Heumaier, Andreas A1 - Hollenberg, Cornelis P. A1 - Piontek, Michael A1 - Suckow, Manfred T1 - Improved processing of secretory proteins in Hansenula polymorpha by sequence variation near the processing site of the alpha mating factor prepro sequence JF - Journal of biotechnology N2 - The literature as well as databases are ambiguous about the exact start of human interleukin-6 (IL-6)--three possibilities for the initiation of the mature protein are described. These three variants of IL-6, different in the exact initiation of the mature protein (A28, P29, or V30), were expressed in Hansenula polymorpha using the Saccharomyces cerevisiae MFα prepro sequence instead of the homologous pre sequence. All three IL-6 variants were secreted but the processing by the Kex2 protease showed significant differences. V30-IL-6 showed correctly processed material but also a molecule species of higher molecular weight indicating incomplete processing of the MFα pro peptide. P29-IL-6 did not yield any correctly processed IL-6, instead only the unprocessed pro form was found in the culture supernatant. Only A28-IL-6 led to 100% correctly processed material. N-terminal sequencing of this material revealed a start at V30--obviously the first two amino acids (Ala28-Pro29) have been removed by a so far unknown protease. Thus expression of both A28-IL-6 and V30-IL-6 as MFα prepro fusion proteins resulted in the very same mature V30-IL-6, however, the ratio of correctly processed molecules was significantly higher in the case of A28-IL-6. The expression of an MFα prepro-interferon α-2a (IFNα-2a) fusion protein in H. polymorpha leads to about 50% correctly processed molecules and 50% misprocessed forms which contain part of the pro peptide at the N-termini. The insertion of A28 and P29 of IL-6 between the pro peptide and the start of the mature IFNα-2a led to correct processing and elimination of all high molecular weight isoforms observed in earlier experiments. Y1 - 2013 UR - http://www.ncbi.nlm.nih.gov/pubmed/22982399 U6 - https://doi.org/10.1016/j.jbiotec.2012.08.024 VL - 167 IS - 2 SP - 94 EP - 100 PB - Elsevier ER - TY - PAT A1 - Suckow, Manfred A1 - Eilert, Eva A1 - Piontek, Michael T1 - Verfahren zum Herstellen eines Ziel-Proteins im Verbund mit einem heterologen Ferritin-Protein unter Verwendung von Hefe-Wirtszellen N2 - Klassifikationen IPC C12N1/16; C12N15/81; C12R1/645; CPC C12N15/81 (EP); C12P21/02 (EP); C07K2319/01 (EP); C07K2319/35 (EP); Y1 - 2013 UR - https://worldwide.espacenet.com/patent/search?q=pn%3DEP2484766B1 ER -