Repressible promoters - a novel tool to generate conditional mutants in Pichia pastoris.
BACKGROUND
Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production.


RESULTS
Here we present five novel repressible Pichia pastoris promoters of different strength: PSER1, PMET3, PTHR1, PPIS1 and PTHI11. eGFP was expressed under the control of each of these promoters and its fluorescence could be successfully decreased in liquid culture by adding different supplements. We also expressed the essential genes with different native promoter strength, ERO1 and PDI1, under the control of two of the novel promoters. In our experiments, a clear down-regulation of both repressible promoters on transcriptional level could be achieved. Compared to the transcript levels of these two genes when expressed under the control of their native promoters, only ERO1 was significantly down-regulated.


CONCLUSIONS
Our results show that all of the novel promoters can be used for repression of genes in liquid culture. We also came to the conclusion that the choice of the repressible promoter is of particular importance. For a successful repression experiment it is crucial that the native promoter of a gene and the repressible promoter in its non-repressed state are of similar strength.
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 18, 1190, Vienna, Austria. diethard.mattanovich@boku.ac.at
Marizela
M
Delic
Diethard
D
Mattanovich
Brigitte
B
Gasser
D015536:Down-Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010843:Pichia / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C021370:homoserine kinase; D017855:Transferases (Other Substituted Phosphate Groups); C478389:PIS1 protein, S cerevisiae