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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.
Mit Hilfe eines Finite-Volumen-Verfahrens wird die dreidimensionale reibungsbehaftete Stroemung um einen schlagenden Fluegel berechnet. Die Bewegungsparamter sind dabei vergleichbar mit den Bedingungen im Reiseflug grosser Voegel. Das numerische Verfahren wird fuer den gezeigten Anwendungsfall, indem eine Methode zur Realisierung von beliebigen periodischen Bewegungen implemetiert wird. Eine Verifizierung erfolgt durch den Vergleich mit analytischen, numerischen und experimentellen Ergebnissen aus der Literatur. Mit dem verifizierten Verfahren werden die instationaeren Groessen wie Druck und Geschwindigkeiten in der Umgebung des bewegten Fluegels berechnet. Daraus ergeben sich ferner die zeitlich gemittelten Groessen wie Schub und Wirkungsgrad des Schlagfluges. Ausserdem wird die Interaktion von Laengs- und Querwirbeln im Nachlauf des schlagenden Fluegels untersucht. Es zeigt sich ein dominanter Einfluss der pulsierenden Randwirbel aufgrund der endlichen Spannweite, waehrend die Anfahr- und Stoppwirbel, verursacht durch die Fluegelbewegung, im Reiseflug grosser Voegel nahezu vernachlaessigbar erscheinen.