Use of an on/off tetracycline riboswitch to control protein production in Komagataella phaffii

Abstract

The methylotrophic yeast Komagataella phaffii is one of the most important microbial platforms to produce recombinant proteins. Despite its importance in the context of industrial biotechnology, the use of synthetic biology approaches in K. phaffii is hampered by the fact that few genetic tools are available for precise control of gene expression in this system. In this work, we used an RNA aptamer activated by tetracycline to modulate protein production at the translational level. Using lacZ as gene reporter, we have demonstrated significant reduction of the heterologous protein upon addition of tetracycline. Furthermore, this genetic control device was applied for the control of Ku70p. This protein is involved in non-homologous recombination and the control of its production paves the way for the development of strains exhibiting higher rates of homologous recombination.

Keywords: Aptamer; Komagataella phaffii; Riboswitch; Tetracycline; Translational control of gene expression.

Fig. 1

Map of the plasmids containing the tetracycline aptamer (tc) controlling protein production. A pPICH-lacZ-3tc episomal vector containing lacZ under control of the tc aptamer. B pPICH-ku70-3tc episomal vector containing ku70 under control of the tc aptamer. All sequences were amplified by PCR and cloned into pPICH vector by homologous recombination or via PCR

Fig. 2

Description of the on-off tetracycline regulation system on mRNA translation. On the absence of tetracycline, the ribosome binds to the mRNA and translates to a protein. When tetracycline is present, the aptamer folds itself into a tridimensional structure that prevents ribosome assembly thus reducing protein production

Fig. 3

The tetracycline aptamer hampers lacZ production in K. phaffii. Cells were incubated on YPD plates containing X-GAL for 3 days at 30 °C and 4 days at 4 °C to enhance blue color intensity. Increasing concentrations of tetracycline show a dose-dependent inhibition response, preventing X-Gal metabolism and decreasing the blue color intensity in higher concentrations. Figure is representative of three independent experiments with triplicates of each spot on every plate

Fig. 4

β-Galactosidase activity is reduced after exposure to tetracycline. The TRS Blue strain has shown to decrease protein production after 30 min of exposure in liquid media (n = 3). *Unpaired t-test shows that TRS Blue tc samples are significantly different from the other groups tested at a 95% confidence interval

Fig. 5

Reduction of Ku70p expression by influence of tetracycline. TRS ku70 strain exposure to 250 µM tetracycline for 8 h has shown approximately 40% of reduction in protein translation. Ku70p calculated expected size: ~ 60 kDa