Culture of S. cerevisiae, chemicals, and materials
Saccharomyces cerevisiae s2805 was kindly purchased and provided by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) in Korea. S. cerevisiae was transformed with pYES2.0 plasmid for mock strain, and pYES2::QRPL::GFP, pYES2::QRPL::ALD6::GFP plasmid for recombinant strain. Mock and recombinant strains were cultured in SD medium (0.67 % yeast nitrogen base, 0.5 % casamino acid, and 2 % D-glucose). To induce the activation of GAL promoter on pYES2.0 plasmid, the medium was changed to SG medium (0.67 % yeast nitrogen base, 0.5 % casamino acid, and 2 % D-galactose) for 12 h. Formaldehyde (1.09 g/mL) was purchased from Sigma Chemical Co. (St. Louis, USA).
DNA isolation, manipulation, and transformation
Genomic DNA was prepared from S. cerevisiae s2805 using Wizard SV genomic DNA kit (Promega, Madison, USA), according to the manufacturer’s instructions. Plasmid DNA, such as pYES2.0 (Invitrogen, USA) and pEGFP-C1 (Clontech Laboratories, Inc., USA), was prepared from recombinant E. coli cells using an alkaline lysis technique with a QIA spin Miniprep kit (Qiagen, Germany). All of the DNA modifications were analyzed by agarose gel electrophoresis, and ligation was performed using standard procedures (Sambrook et al., 2001). The PCR experiments were carried out using a T Gradient Thermocycler (Biometra, Germany), Ex TaqTM DNA polymerase (Takara Bio Inc., Japan), and genomic DNA as the template.
The PCR products were purified using a QIA quick PCR purification kit (Qiagen, Germany). The oligonucleotides used for PCR amplification were purchased from AccuOligo (Bioneer Co., Korea). Figure 1 describes two diagrams of the transfer vector with QRPL signal peptide sequence (QRPL). The transformation to E. coli was performed by electroporation with an Electro cell Manipulator (BTX Technologies Inc., USA); and yeast transformation was performed by lithium acetate method24.
Design of transfer vector (pYES2::QRPL::GFP)
Plasmids used in this study were constructed for the expression of QRPL signal peptide sequence with green fluorescence protein (GFP) (Fig. 1 a). To amplify the QRPL gene (5’- CAA CGG TCT TTG - 3'), the primer was designed to include QRPL signal peptide sequence from CPY in the pYES2 plasmid. PCR products were prepared of the two parts of the gene, then primer R1 and F2 included QRPL with EarI restriction enzyme site. PCR via primer: Primer-F1 (5’- TTT AGC GGC CGC ATG GTG AGC AAG GGC GAG -3’) and Primer-R1 (5’- ATA CTC TTC CAA CGG TCT TTG TAC GTC GCC GTC CAG CTC -3’). The PCR product was prepared with restriction enzyme site NotI and EarI sites. In addition, Primer-F2 (5’- TTT CTC TTC CAA AGA CCG TTG AAC GGC CAC AAG TTC AGC -3’) and Primer-R2 (5’- ATT GCA TGC TTA CTT GTA CAG CTC GTC -3’) were designed with restriction enzyme site EarI sites and SphI. Underline on the primer R1 and F2 indicates QRPL sequence. The fusion PCR fragment was then cloned into the NotI and SphI site to make pYES2::QRPL::GFP. This vector was transformed into S. cerevisiae cells to make recombinant strain MBTL-Q-DJ1. In parallel, the original vector pYES2.0 was transformed into yeast to make mock strain MBTL-M-DJ.
Design of transfer vector (pYES2::QRPL::ALD6::GFP)
Plasmids used in this study were constructed for the expression of QRPL signal peptide sequence with Aldehyde dehydrogenase 6 (ALD6). The DNA containing QRPL with ALD6 was synthesized by Bioneer Co., Korea. The PCR product was inserted into the BamHI and NotI site of pYES2.0, to make pYES2::QRPL::ALD6 plasmid. After that, the GFP gene was amplified from pEGFP using PCR via primers: Primer-GFP-F (5’- ATT GCG GCC GCA GTG AGC AAG GGC GAG G -3’) and Primer-GFP-R (5’- GGT GCA TGC CTT GTA CAG CTC ATC CAT -3’). The DNA fragment of GFP was then cloned into the NotI and SphI site to make pYES2::QRPL::ALD6::GFP. This vector was transformed into S. cerevisiae to make recombinant strain MBTL-Q-DJ2 (Fig. 1 b).
Fluorescence microscopy
Recombinant and mock strains were cultured in SD and SG medium. The cells were washed with PBS buffer and stained with 100 nM Lyso-Tracker Red DND-99 (Molecular Probes, Leiden, The Netherlands) in PBS for 30 °C for 10 min. All strains were washed by PBS twice, and the intensity of protein expression observed by fluorescence microscopy (Zeiss, Germany). Fluorescent images were analyzed using the Zeiss image Browser.
Isolation of vacuole and vacuolar enzyme from recombinant yeast
Recombinant and mock strains were both cultured in SD medium. To induce pYES2.0 plasmid, cells were cultured on SG medium for 12 h. All strains were harvested after growing at OD600 0.8–1.0, then the vacuole was isolated from mock and recombinant strains.
The 0.1 M Tris-SO4 buffer (100 mM Tris-SO4 (pH 9.4), 10 mM dithiothreitol (DTT)) was mixed with harvesting cells. This mixture was incubated at 30 °C at 90 rpm for 10 min and centrifuged at 3,000 rpm for 5 min. The supernatant was discarded, and the mixed pellet exposed to Lyticase (2,000 U/mL) (Sigma, St. Louis, USA) for 1 h, to make it flexible in cell membranes. This was followed by centrifugation for 5 min, and the pellet was washed twice with Sorbitol K+ phosphate buffer (20 mM K+ phosphate buffer, 1.2 M Sorbitol). In the next step, breaking buffer (20 mM Tris-Cl (pH 7.4), 0.6 mM Sorbitol, and 1 mM phenyl methane sulfonyl fluoride (PMSF)) was mixed with pellet, and ultra-sonication applied at 40 W for 30 min (20 s on / 10 s off pulse) in the ice. The mixture was centrifuged for 10 min, and the supernatant was discarded. The pellet was mixed again with breaking buffer, and a second ultra-sonication was applied for 20 min (10 s on / 10 s off pulse). Lastly, the first centrifuge (500× g, 5 min) was made to collect supernatant, and the second centrifuge (20,000× g, was made for 30 min at 4 °C). The pellet contains vacuoles from cells 5,25,26.
The vacuolar enzyme was extracted by 0.1 % NP40 buffer containing 1 mM DTT at a ratio of 1:1. The mixture was vortexed for 10 min, followed by the reaction in ice for 30 min. After incubation, the mixture was centrifuged at 13,000 rpm at 4 °C for 10 min. The supernatant contained vacuolar enzymes that would be used in the aldehyde reduction assay.
Luminescence assay
The vacuolar enzyme was exposed to formaldehyde to evaluate the efficiency of aldehyde reduction. Formaldehyde was prepared at 62.5, 125, 250, 500, and 1,000 ppm, and then the vacuolar enzyme was exposed to the formaldehyde at room temperature for 1 h. After that, the mixture was centrifuged at 20,000× g at 4 °C for 10 min, the supernatant was collected for luminescent reaction with Vibrio fischeri at a ratio of 1:1 at 30 °C for 30 min, and the luminescence was measured by GloMax® Explorer system (Promega Co., Ltd., USA) 27,28.
Data analysis
All the data were obtained from three independent samples, run simultaneously for error analysis. The data were analyzed using Sigma Plot (SPS, Chicago, IL. USA), and the results were reported with the standard deviations. In addition, the correlation between cell viability and several experimental conditions was reported. A p-value of <0.05 was considered significant.