Media and strains

Escherichia coli XL-10 Gold (Agilent Technologies) was used for construction and propagation of vectors used in this work. K. phaffii X-33 strain (Invitrogen) was the selected strain as control and background strain for genetic manipulations. Bacterial strains were grown using LB broth (0.5% Yeast Extract, 1% Tryptone and 1% NaCl, pH 7.2) at 37 °C under 150 rpm agitation. LB plates were poured using 1.5% agar supplemented with 25 µg/mL zeocin for selective pressure and maintenance of plasmids pPICH-lacZ-3tc and pPICH-ku70-3tc. Yeast strains were grown on YPD medium (1% Yeast Extract, 2% Peptone and 2% Glucose) under 200 rpm agitation at 30 °C. YPD plates were poured with 2% agar and supplemented with 100 µg/mL zeocin whenever necessary. The blue and white colony assay was performed on YPD plates containing zeocin and 2% top agar X-Gal. Tetracycline was added to the medium at different concentrations ranging from 5 to 100 µM.

lacZ and ku70p expression vector construction

The backbone vector used for the cloning of the lacZ reporter gene was pPICHOLI (Mobitec). The vector was digested with BamHI and NotI restriction enzymes (New England Biolabs) to release the AOX1 promoter, ORF and terminator sequences. The resulting linearized plasmid was named pPICH. Then, lacZ reporter gene was amplified by PCR from pLG∆178 (Guarente and Mason 1983) using Phusion DNA polymerase (New England Biolabs) under the following conditions: initial denaturation at 98 °C for 1 min followed by 40 cycles of 98 °C for 30 s, 60 °C for 30 s and 72 °C for 2 min; final extension at 72 °C for 5 min. Reaction was set according to the manufacturer’s instructions manual and primers lacZ-Fw and lacZ-Rv (see Additional file 1: Table S1) were used at a final concentration of 0.25 µM. The lacZ fragment was cloned into pPICH vector using the NEBuilder HiFi DNA assembly kit (New England Biolabs) on a proportion 1:2 vector-insert with 50 ng of total DNA. The reaction was incubated at 50 °C for 60 min and subsequently used to transform chemically competent E. coli XL-10 Gold. Transformants were selected on LB plates containing zeocin and correct insertion was further confirmed by PCR and restriction analysis. The resulting vector was named pPICH-lacZ. The KU70 gene (GenBank accession number CP014717.1) was amplified by PCR from genomic DNA extracted from K. phaffii GS115 (Invitrogen). Primers ku70-Fw and Ku70-Rv were used for amplification and PCR conditions were similar to those used for lacZ amplification. The resulting vector was named pPICH-ku70. HA-tag was cloned into pPICH-ku70 via PCR with the primers described in Additional file 1: Table S1. The tetracycline aptamer was synthesized by GenOne and clone into pUC19 plasmid. The sequence was amplified by PCR, purified, digested with SphI and NdeI (New England Biolabs) and cloned into pPICH-lacZ and pPICH-ku70 plasmids. K. phaffii transformants are mentioned as TRS (tetracycline-responsive strain) Blue when transformed with pPICH-lacZ-3tc and TRS ku70 when transformed with pPICH-ku70-3tc. Both plasmids contain three sequences of the tetracycline riboswitch controlling the ORFs. Plasmid maps used for negative control without the tetracycline riboswitch are shown in Additional file 1: Fig. S1. Sequences of all plasmids used in this work are available in Additional files 3 and 4.

β-Galactosidase activity assay

Cells were grown overnight at 30 °C and 200 rpm agitation on YPD supplemented with zeocin and tetracycline. Then, cells were harvested by centrifugation at 3000×g for 5 min. An aliquot of these cells was reinoculated in fresh medium for a final concentration of 0.1 OD600nm. Cells were grown up to ~ 0.7–0.8 OD600nm and a total of 3 OD600nm were collected by centrifugation. The pellet was resuspended in 1 mL Z buffer (60 mM Na2HPO4, 10 mM KCl and 1 mM MgSO4). Cell permeabilization was performed by adding 25 µL of 0.1% SDS and 50 µL chloroform followed by 3 cycles of strong agitation for 30 s, incubation at 30 °C for 15 min and 5 min at − 80 °C. Then, reaction was started by adding 200 µL of 4 mg/mL ONPG solution at 30 °C. The reaction was stopped after 5 min when solution turned yellow by addition of 500 µL of 1 M NaCO3. Measurement of 600 nm absorbance was performed immediately and 420 nm absorbance was done after separation of cell debris by centrifugation. β-Galactosidase activity was calculated by the following formula:

$$Miller\;units= \times \left(\frac}}}* V*T}\right),$$

where T is the reaction time (min) and V is the volume (mL) collected from culture. The data used for calculations in this work are provided in the Additional file 2.

Statistical analysis

All samples were analyzed using GraphPad Prism software. Unpaired t-Test was calculated from three independent experiments with triplicates of every sample each time. Figures are representative of the results and show mean values and standard deviation.

Protein extraction

One isolated colony was inoculated in liquid YPD media containing zeocin (100 µg/mL). The culture was kept at 30 °C under 200 rpm agitation for 24 h. Cells were collected by centrifugation at 2000×g for 5 min and washed twice with sterile water. Then, a new batch was prepared by adding cells to a final concentration of 0.1 OD600nm on fresh medium at 30 °C and 200 rpm agitation until cell concentration was ~ 1.0 OD600nm. The culture was centrifuged for 10 min at 5000×g at 4 °C and the pellet was resuspended in 450 µL of ice-cold extraction buffer (50 mM Tris-HCl, 1.25% Tween 20 [pH 8.8]) and 200 μm glass beads were added. The extraction buffer was supplemented with a protease inhibitor cocktail (cOmplete Protease Inhibitor, Roche) prior to cell lysis. Protein extraction was performed by 10 cycles of 30 s under intense agitation with Precellys Lysing kit (Bertin) and 1 min of ice-cold water bath. The extract was collected with the aid of a pipette and placed in another ice-cold tube. Extraction buffer (200 µL) was added to the glass beads and vortexed to retrieve the remaining extract trapped and mixed with the volume obtained before. The whole protein extract was centrifuged at 18,000×g for 30 min at 4 °C and the supernatant containing soluble proteins was collected and stored at − 20 °C until use.

Protein concentration determination

Protein concentration was determined by using BioRad Protein Assay Dye Reagent. The standard curve was made using BSA accordingly to the concentration of the sample.

Polyacrylamide gel electrophoresis (PAGE) and Western blotting

Protein extracts were initially analyzed in SDS-PAGE (5% stacking, 12% resolving gel). Samples were boiled for 5 min in loading dye containing 100 mM Tris-HCl pH 6.8, 8 M urea, 20% glycerol, 4% SDS, 0.2% bromophenol blue and 100 mM β-mercaptoethanol prior to application. Electrophoresis was performed at room temperature at a constant current of 0.05 A. Gel content was evaluated with Coomassie Brilliant Blue G-250 staining (Merck). Every gel was prepared in duplicates as one was used for Western blotting. Protein transfer to nitrocellulose membranes (Merck Millipore) were performed at a constant current 0.15 A at room temperature for 60 min. The quality of the transfer was evaluated by dying the membrane with Ponceau S. Membrane was blocked for 1 h using 5% skim milk solution reconstituted in TBST buffer. Then, the membrane was incubated with a 1 µg/mL solution of monoclonal rabbit anti-HA antibody (SAB5600193, Sigma Aldrich) in TBST buffer supplemented with 3% BSA for 1 h at room temperature. Secondary anti-rabbit antibody conjugated with peroxidase was used for revelation after 1 h of incubation at room temperature. Revelation was performed by incubating the membrane with revealing buffer (Abcam) for 2 min in a dark chamber. Chemiluminescence was obtained on semi-automatic capture mode after 1 min of exposure on the Imager 680 (Amersham). The membrane was washed with TBST three times for 5 min each prior to every incubation step (blocking, primary and secondary antibody and revelation). Measurement of band densitometry was performed on ImageJ 1.54 using area under the peak method. Background was excluded from measurement. No editing was performed in the images presented. Contrast and brightness were adjusted for better visualization of the data. Ku70p estimated size: ~ 60 kDa.