Lydia Dankmeyer

Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene

Pseudomonas alcaligenes secretes a lipase with a high pH optimum, which has interesting properties for application in detergents. The expression of the lipase is strongly dependent on the presence of lipids in the growth medium such as soybean oil. The promoter of the gene was characterized and found to have resemblance to sigma54 controlled promoters, which are known to be tightly regulated. The transcription start was mapped precisely downstream of a sequence with close similarity to the -12/-24 consensus sequence of sigma54 controlled promoters. Interestingly, a hyperproducer mutant strain was isolated and found to have a C to T mutation in the -12/-24 promoter consensus region. In addition an Upstream Activating Sequence (UAS) with homology to sigma54 UAS consensus sequences was identified. It was demonstrated that an increase of the distance from the UAS to the transcription start or the deletion of the UAS results in significantly lower expression levels of lipase. A systematic mutational analysis of the UAS sequence has resulted in a variant with an increased lipase expression.

Acetate–glycerol cometabolism: Cultivating Schizosaccharomyces pombe on a non-fermentable carbon source in a defined minimal medium

The growth of the fission yeast Schizosaccharomyces pombe on glucose and glycerol was monitored on-line in shake flasks and microtiter plates. The Edinburgh Minimal Medium 2 was improved by doubling its concentrations, improving its buffer and increasing its sulphur and iron concentrations additionally. By growing S. pombe on mixed carbon sources, it was shown that glycerol and glucose complement one another. Several tests were performed to establish the cultivation of S. pombe with non-fermentable glycerol as the main carbon source in minimal medium. Interestingly, a synergistic effect of glycerol and acetate was discovered which can significantly improve the growth of the fission yeast on glycerol. S. pombe showed optimal respiration activity, growth, and product formation by co-utilizing 20g/L glycerol and 2.5g/L sodium acetate.