U039821.pdf (23.2 MB)
Studies of the 3-phosphoglycerate kinase gene of Penicillium chrysogenum.
thesisposted on 2015-11-19, 08:53 authored by Isobel Claire. Hoskins
Studies of Saccharomyces cerevisiae and Aspergillus nidulans have shown that the 3-phosphoglycerate kinase gene (PGK) is highly expressed from a strong promoter (Holland and Holland 1978; Clements 1986). The aim of the project was to isolate and study the potentially strong PGK gene promoter of P. chrysogenum. The P. chrysogenum PGK gene was isolated and the promoter and terminator sequences determined. The promoter contained a 40bp pyrimidine rich region in which transcription was initiated at multiple sites. Matches to both the glycolytic box and the essential region of the Aspergillus nidulans PGK promoter were found in the promoter by sequence comparison. The expression of the PGK promoter was studied using a fusion between the PGK promoter and the Esherichia coli lacZ reporter gene. The gene fusion was transformed into P. chrysogenum and a transformed strain containing two copies of the fusion at the oliC locus was monitored for the reporter activity. The PGK promoter was up to three times more active during growth in media containing carbon sources metabolised by gluconeogenesis compared to those metabolised by glycolysis, and the rate at which the promoter activity increased in logarithmic growth was also greater. The expression of the P. chrysogenum PGK promoter in Aspergillus nidulans was investigated by transformation of the gene fusion into A. nidulans at the qutE locus. The PGK promoter retained its broad pattern of control. A second gene fusion was made between the P. chrysogenum PGK promoter and the Isopenicillin-N-Synthetase gene. A transformed P. chrysogenum strain containing one copy of the gene fusion at the oliC locus was compared to the original strain in a batch fermentation. The transformed strain had twofold higher IPNS mRNA and enzyme levels during the first three days of the fermentation. The penicillin titre was slightly increased in this period.
Date of award1991-01-01
Awarding institutionUniversity of Leicester