Evaluation of the tetracycline promoter system for regulated gene expression in Kluyveromyces marxianus.
A tetracycline repressible promoter system designed for Saccharomyces cerevisiae was evaluated for use in Kluyveromyces marxianus. A plasmid was constructed containing the Escherichia coli beta-glucuronidase (gus) gene cloned downstream of the yeast tet-off promoter, the tetR-VP16 activator protein gene, and the URA3 gene for selection. The tet-off promoter-gus construct was integrated into the chromosomal DNA and tested under varying growth conditions in complex medium. The repressors tetracycline and doxycycline were both found to be effective for inhibiting gene expression. Doxycycline levels of 0.5 microg/mL or greater were sufficient to nearly completely suppress Gus synthesis. For most transformants, the induction ratio was approximately 2,000-fold. The tet-off promoter was effective at 30, 37, and 42 degrees C, although the overall Gus activity was highest at 37 degrees C. During exponential growth, little product was formed; expression increased dramatically in late exponential and early stationary phase. The promoter thus shows promise for protein synthesis following cell growth. No inducer is required and the repressor is only needed to prevent expression during the seed culture.
Pecota
Douglas C
DC
Department of Chemical Engineering and Materials Science, University of California, Irvine, California 92697-2575, USA.
Da Silva
Nancy A
NA
D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics; D007716:Kluyveromyces / Q000235:genetics*; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013696:Temperature; D013752:Tetracycline / Q000494:pharmacology*; D013997:Time Factors
D004247:DNA; D005966:Glucuronidase; D013752:Tetracycline; D004318:Doxycycline