Gewinnung von Gärungsmutanten der Hefe Pichia stipitis durch zufällige Integrationsmutagenese

摘要

Plants are live wires. Through the photosynthesis the tamed strength of the sunlight imply in plants. Every time fuel-prices arise, plant material gets more strongly into the view of the industrial society depended on mobility. Additionally the reinforced utilization of regenerative sources of energy can contribute to the reduction of the output of greenhouse-gases. Ethanol from renewable primary products belongs to the regenerative sources of energy, which can be used above all in the traffic-area. The costs for the ethanol production could be drastically reduced by the utilization of agricultural and forestry residues as raw material. The pentose xylose is the second most abundant sugar in this material. The respiratory yeast Pichia stipitis examined in this dissertation is one of the best naturally occurring xylose fermenting organism known. However, P. stipitis shows a low ethanol tolerance and a low ethanol volumetric production rate in comparison to Saccharomyces cerevisiae. Moreover, the induction of the fermentation with P. stipitis takes place through a lowering of the oxygen-concentration in the medium. These physiological qualities impede the commercial utilization of P. stipitis since they would mean a cost-intensive bio processing. The exact mechanism of the induction of the fermentation in P. stipitis is not known. To be able to construct a P. stipitis or S. cerevisiae strain, which can be use efficiently in the production of ethanol from renewable biomass, the regulation-mechanism of the fermentation must be cleared up. In this dissertation an efficient integration mutagenesis was developed for P. stipitis to generate putative fermentation mutants for further examinations. This system leads to accidental, marked and ectopic integrations by means of integrative transformation of a linear vector. The application of a linearized integrative vector in an optimized transformation leads to a 50- to 300-fold increase of the transformation frequency. Also the application of a linearized autonomous-replicating vector leads in comparison to the circular autonomous-replicating vector to a three to sextuple increase of the transformation frequency. In many works, a method called REMI (restriction enzyme-mediated integration) led to an increase of the transformation-harvest. It should be investigated in this work whether the addition of a restriction enzyme increases the transformation frequency and/or has influence on the site or the frequency of the integration. The addition of the restriction enzymes BamHI, EcoRI, HincII, KpnI, PstI, SacI and XbaI increased the transformation frequency. The dimension of the increase dependents on the enzyme, its started quantity and the vector-quantity. The restriction enzyme SmaI shows no increase of the transformation rate. The presence of a restriction enzyme during the transformation doesn't lead to integration into the corresponding recognition-sequence in P. stipitis. The integrations in the chromosomal DNA occurred at micro-homologies to the terminal flanks of the linear vector. The majority of events integrated into single site, independently from presence or absence of a restriction enzyme. Further P. stipitis selection marker were executed in this work by complementation experiments. This led to the sequences from the genes PsTRP1, PsTRP2 and PsADE5. All nearer examined putative fermentation mutants showed that it is difficult to find fermentation mutants because of the complex regulation of the ethanol production in P. stipitis. Growth and fermentation seem to be joined together narrow. Many of the putative fermentative mutants showed growth-defects. The sequences of mutated genes of three different mutants were analyzed. It is about the genes PsPTC1, PsINP53 and PsHSP90. All three genes are involved in the mediation of stress answers and the mutants showed pleiotrope effects. The mutation in the PsHSP90 gene influences the ethanol production and possibly the ethanol reassimilation. Still, it possibly has effects on the function of a sugar transporter and on the activity of the enzyme xylitol dehydrogenase, a component of the xylose utilization. The mutant P. stipitis hsp90 is not able to produce ethanol from xylose. The mutation in the PsPTC1 gene leads to a fast formation of oblong cells and pseudo hyphal. The mutation in the PsPTC1 gene has an inferior influence on the fermentation-behaviour in a clone, which builds pseudo hyphal, as in a clone, which forms round single cells. The mutation in the PsINP53 gene leads to a reduced ethanol formation rate and probably a reinforced ethanol reassimilation. Reason for this could be a diminution of the intracellular phosphatidylinositol concentration.