Zur Erfassung trans-wirksamer Komponenten der Sauerstoff-abhängigen Gärungsregulation der Hefe Pichia stipitis
To construct a strain for the isolation of regulatory PsADH2-mutants a reporter gene was brought under the control of the PsADH2-promotor. Following the mutagenesis of the strain, a potential PsADH2-mutant was isolated. The transformation recipient showed in well-controlled batch fermentations a behavior, which differed from the wild type strain. Under aerobic conditions a comparably high ADH activity was measured. After shifting the O2-tension to 20 % a slowed ADH induction, and under anaerobic conditions a strongly weakened ADH- and PDC-activity was measured. Under anaerobic conditions a Pasteur effect was seen, which was less pronounced in the transformation strain than in the wild type. The PsHIS3-gene was sequenced and via southern hybridization identified as a single-copy gene. The suitability of PsURA3 and ScTRP5 as reporter genes was investigated. In P. stipitis the selection of Ura3- and Trp5-auxotrophic mutants on media containing 5-FOA respecttively 5-FAA was possible. As the introduction of the Ura3-auxotrophy into the transformation strain P. stipitis PJH53 his3-1 trp5-10 did not prove possible, neither by disruption of the gene, nor by crossing Ura3-auxotrophic strains, ScTRP5 was brought under the control of the PsADH2-promotor as a reporter gene. To integrate the reporter cassette into the transformation recipient, various vector types were constructed and their transformational properties were investigated. In general, the transformation efficiency of P. stipitis could be improved by transforming linear DNA with blunt-ends, or by co-transformation of the DNA to be integrated and double-strand DNA with complementary sticky-ends. The transformation of circular, non-replicating vectors or of ends-out cassettes with sticky-ends did not lead to transformants. To achieve a stable integration of the reporter cassette, an ends-out vector was used, which was embedded into pUC19 sequences with blunt ends and which yielded similarly high transformation rates as autonomous vectors. Southern-hybridizations demonstrated that the reporter cassette integrated mainly ectopic. Through a plasmid loss test of the autonomous reporter plasmid, it was established, that ScTRP5 is expressed under the control of the PsADH2-promotor. In case of an ectopic integration of the reporter cassette or in case of the occurence of an autonomous vector in the cell the creation of an amplificate through PCR, which was only expected with homologous cassette integration, was possible. The use of PCR as the single detection method for homologous vector integration can lead to misinterpretation of transformation data. The induction of a Trp-auxotrophy was possible in the reporter strain by the use of UV-mutagenesis but not by the use of REMI of various vectors. Contrary to expectations, the selection of transformants with integrated reporter cassette proved possible even under aerobic conditions. For the selection of regulatory ADH2-mutants, this means that constitutive regulation mutants cannot be distinguished from the non-mutated original strain. However, the selection of non-activating mutants is possible.
info:eu-repo/classification/ddc/570, Pichia stipitis, Gärung, Genregulation, Sauerstoff, Biowissenschaften, Biologie, Sauerstoff-abhängige Transkriptionsregulation, HIF1 alpha, Transformationseffizienz, ends-out Kassette, Reportergen, 5-FAA
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