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 - Hollenberg, Cornelis P. A1 - Piontek, Michael A1 - Suckow, Manfred T1 - The use of highly expressed FTH1 as carrier protein for cytosolic targeting in Hansenula polymorpha JF - Journal of biotechnology N2 - The iron storage protein ferritin is a member of the non-heme iron protein family. It can store and release iron, therefore it prevents the cell from damage caused by iron-dioxygen reactions as well as it provides iron for biological processing. To study whether the human ferritin heavy chain (FTH1) can be expressed in Hansenula polymorpha, we integrated an expression cassette for FTH1 and analyzed the protein expression. We found very efficient expression of FTH1 and obtained yields up to 1.9 g/L under non-optimized conditions. Based on this result we designed a FTH1-PTH fusion protein to successfully express the parathyroid hormone fragment 1-34 (PTH) for the first time intracellular in H. polymorpha. Y1 - 2012 UR - http://www.ncbi.nlm.nih.gov/pubmed/22212821 U6 - https://doi.org/10.1016/j.jbiotec.2011.12.014 VL - 159 IS - 3 SP - 172 EP - 176 PB - Elsevier ER - TY - THES A1 - Eilert, Eva T1 - New applications of heterologous gene expression in Hansenula polymorpha for protein and metabolite production N2 - The methylotrophic yeast Hansenula polymorpha has been established as a highly efficient expression system. Strong promoters derived from the methanol metabolism pathway and mitotically stable high-copy integration have made this organism very effective for heterologous protein expression. To improve the processing of secretory proteins targeted by the MFα prepro sequence, variations in the amino acid composition near the processing site of Kex2p were investigated (Manuscript I). The insertion of Ala-Pro between MFα prepro sequence and protein of interest leads to an improved processing of the signal sequence in case of interleukin-6 (IL-6) and interferon-α 2a (IFNα-2a). Modeling of the transition sequences confirmed a better exposition of the Kex2p-cleavage site in dependency of Ala-Pro insertions. Analysis of the secreted proteins revealed activity of an Ala-Pro specific endoprotease. Partial removal of the N-terminal Ala-Pro dipeptide was also detected during IFNα-2a processing. The putative protease responsible for the processing of Ala-Pro dipeptides was identified by gene deletion. Significant similarities with Saccharomyces cerevisiae Ste13p were identified. To generally improve productivity in the H. polymorpha system new fusion protein strategies were investigated (Manuscript II). The recombinantly strongly expressed cytosolic ferritin heavy chain (FTH1) was analyzed with regard to its potential as fusion partner for difficult-to-express target proteins. A fragment of the parathyroid hormone (PTH) was chosen to explore whether fusion to FTH1 enhances PTH stability. We could show that the expression level of this fusion protein was at least of the magnitude of that of FTH1 without fusion partner. The solubility of the fusion protein was increased in comparison to FTH1 alone and no degradation of PTH occurred when fused C-terminally to FTH1. Ectoine and 5-hydroxyectoine belong to the family of compatible solutes which are known to contribute to the adaptation of the cell to osmotic stress by mediation of a constant turgor. H. polymorpha was engineered to express the enzymes of the H. elongata 5-hydroxyectoine biosynthesis pathway (Manuscript III). Expression of the enzymes EctB, EctA, EctC and EctD in H. polymorpha resulted in production of 5-hydroxyectoine in gram per liter scale with almost full conversion of ectoine to 5-hydroxyectoine. We showed that 5-hydroxyectoine synthesis was influenced by feeding of methanol during cultivation. Furthermore, 5-hydroxyectoine synthesis was downregulated by the addition of methionine, lysine or threonine indicating the existence of a feedback regulated aspartate kinase in H. polymorpha. N2 - Die methylotrophe Hefe Hansenula polymorpha hat sich als effizientes System zur heterologen Genexpression etabliert. Besondere Merkmale dieses Systems sind die starken Promotoren des Methanolstoffwechsels und die Fähigkeit zur mitotisch stabilen Integration einer Expressionskassette in einer hohen Kopienzahl. Am Beispiel der MF α prepro Sequenz sollte die Prozessierung sekretorisch exprimierter Proteine verbessert werden (Manuskript I). Hierfür wurden Veränderungen in der Nähe der Kex2p Erkennungssequenz durchgeführt. Durch das Einfügen von Ala-Pro zwischen MF_ prepro Sequenz und Zielprotein konnte die Prozessierung im Fall von Interleukin-6 (IL-6) und Interferon- α 2a (IFN α -2a) verbessert werden. Eine Modellierung dieses Sequenzübergangs zeigte eine verbesserte Präsentation der Kex2p-Schnittstelle. Nach Analyse des sekretierten IL-6 wurde außerdem die Aktivität einer Ala-Pro spezifischen Endoprotease festgestellt. Auch im Falle von IFN α -2a konnte eine partielle Abspaltung des N-terminalen Ala-Pro-Dipeptids festgestellt werden. Ein Protease, welche mutmaßlich an der Prozessierung dieser Ala-Pro-Dipeptide beteiligt ist, wurde per Gendeletion identifiziert. Diese Protease wies signifikante Ähnlichkeiten mit der S. cerevisiae Ste13p auf. Zur allgemeinen Verbesserung der Ausbeute bei der Produktion heterologer Proteine wurden neue Fusionsprotein-Strategien untersucht (Manuskript II). Hierfür wurde das rekombinant cytosolisch stark exprimierte Protein Ferritin (schwere Kette, FTH1) im Hinblick auf das Potenzial als Fusionspartner für schwierig zu exprimieren Zielproteine analysiert. Ein Fragment des Parathormons (PTH) wurde gewählt, um zu untersuchen, ob die Fusion beider Proteine die Stabilität von PTH erhöht. Wir konnten zeigen, dass die Expression dieses Fusionsproteins mindestens in der Größenordnung des FTH1 ohne Fusionspartner war. Die Löslichkeit des Fusionsproteins wurde gegenüber FTH1 allein erhöht und es trat kein Abbau des PTH in Fusion mit FTH1 auf. Ectoin und 5-Hydroxyectoin gehören zur Familie der kompatiblen Solute, die hauptsächlich zur Anpassung der Zelle bei osmotischem Stress durch Anpassung des Turgors beitragen. Ein H. polymorpha-Stamm wurde entwickelt, welcher die Enzyme der 5-Hydroxyectoin-Biosynthese aus H. elongata exprimiert (Manuskript III). Die Expression der Enzyme EctB, EctA, EctC und EctD in H. polymorpha resultierte in der Produktion von 5-Hydroxyectoin im Gramm pro Liter Bereich. Es fand außerdem eine nahezu vollständige Umwandlung von Ectoin in 5-Hydroxyectoin statt. Wir konnten zeigen, dass die 5-Hydroxyectoin-Synthese durch Zugabe von Methanol während der Kultivierung beeinflusst wurde. Desweiteren wurde die 5-Hydroxyectoin-Synthese durch die Zugabe von Methionin, Lysin oder Threonin herunterreguliert, was andeutet, dass in H. polymorpha eine Feedback-regulierte Aspartatkinase existiert. T2 - Neue Ansätze der heterologen Genexpression in Hansenula polymorpha zur Protein- und Metabolitenproduktion Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:hbz:061-20130527-092604-7 PB - Institut für Molekulare Mykologie, Institut für Mikrobiologie CY - Düsseldorf 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 - Eilert, Eva A1 - Suckow, Manfred A1 - Piontek, Michael T1 - A method for producing ectoine or a derivative thereof and a yeast cell for use as a host cell in such a method N2 - Klassifikationen IPC C12N15/81; C12N15/82; C12P17/12; C12P7/42; CPC C12N15/815 (EP); C12P17/12 (EP); Y1 - 2014 UR - https://depatisnet.dpma.de/DepatisNet/depatisnet?action=pdf&docid=EP000002743350A1&xxxfull=1 ER - TY - PAT A1 - Kraemer, Marco A1 - Svetlichny, Vitaly A1 - Eilert, Eva T1 - Method for producing a fermentation product: Angemeldet durch DIREVO Industrial Biotechnology GmbH N2 - Prioritätsdaten: EP15191039A·2015-10-22; US201562245054P·2015-10-22 Bibliographische IPC: C12P7/06; C12P7/14; Y1 - 2017 UR - https://depatisnet.dpma.de/DepatisNet/depatisnet?action=pdf&docid=WO002017067698A1&xxxfull=1 ER -