Zur Erfassung trans-wirksamer Komponenten der Sauerstoff-abhängigen Gärungsregulation der Hefe Pichia stipitis
type: CreativeWork
description: 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 a…
abstract: 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 a…
author: Schruff, Barbara Susanne
language: ger
keywords: 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
date published: 2005
date modified: 2026-07-30T19:42:48Z
provider: RWTH Aachen University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Publikationsserver der RWTH Aachen University