Medium, strain and plasmid
P. pastoris was cultured in YPD broth and BMY medium along with its derivatives, BMMY, BMGY, BMGMY and BMMGY. Saccharomyces cerevisiae (S. cerevisiae) was incubated in YPD broth and YPDA medium, Y2HGold in SD/Try (TAKARA) and Y187 in SD/Leu medium (TAKARA). Constructed plasmids were transformed into Escherichia coli (E. coli) DH5α and Trans110 (Trans Gen) cells, and these cells were cultured at 37 °C in Luria-Bertani (LB) medium. All formulas used are listed below. YPD (1% yeast extract, 2% tryptone, 2% glucose), BMY (1 L: 10 g yeast extract, 20 g tryptone, 3 g K2HPO4, 11.8 g KH2PO4, 13.4 g YNB, 4 × 10−4 g biotin, 10 mL glycerol), BMMY (1 L: 10 g yeast extract, 20 g tryptone, 3 g K2HPO4, 11.8 g KH2PO4, 13.4 g YNB, 4 × 10−4 g biotin, 5 mL methanol), BMGMY (BMGY plus 0.5% methanol) and BMMGY medium (BMGY plus 0.5% methanol). YPDA (YPD with the addition of 15 mL 0.2% adenine), LB (0.5% yeast extract, 1% tryptone, 1% NaCl), TB/SB (2.6 g peptone, 4.8 g yeast extract, 0.462 g KH2PO4, 2.51 g K2HPO4, 2 g glycerol). SD/Try and SD/Leu medium was purchased from TAKARA. The solid medium was prepared by adding 2 % agar powder.
Electroporation was used to transform P. pastoris. Transformation and recombinant DNA operations were performed as described previously[11]. Selection of marker-resistant colonies was performed using LB with 50 µg/mL ampicillin or kanamycin, and YPD with 0.3 mg/mL G418 or 0.1 mg/mL zeocin.
Strains, plasmids and primers
See Tables 1, 2 and 3.
Plasmid and strain construction
M1/2 expression plasmid and strain construction
Initially, the P. pastoris genome was extracted. The sequences of Mxr1-150AA (M1) and Mxr1-400AA (M2) were amplified by PCR using Pfu DNA polymerase (Thermo Scientific, Waltham, MA, USA) and the P. pastoris genome. These two sequences vary in the number of N-terminal residues: 150 amino acids (M1) and 400 amino acids (M2), respectively. Subsequently, the PCR products were digested with NdeI/XhoI, and the digested fragments were inserted into the pSVT7 plasmid. The recombinant plasmid was chemically transformed into E. coli BL21(DE3) cells.
M1/2-pGBKT7 recombinant plasmid construction
M1/2 were amplified with Pfu DNA polymerase and the P. pastoris genome was used as the template. The NdeI/XhoI-digested PCR products were ligated into the pGBKT7 plasmid. Recombinant plasmids M1- pGBKT7 and M2-pGBKT7 were transformed into S. cerevisiae following the protocol provided by TAKARA.
AD-pGADT7 recombinant plasmid construction
The general procedure was the same as above. Target genes (like tkl1) were amplification and the PCR products were double-digested with the same restriction enzymes, and the digested fragments were ligated into the appropriate plasmid. Recombinant plasmid AD-pGADT7 was transformed into S. cerevisiae using the protocol provided by TAKARA.
Gene expression studies in E. coli BL21 (DE3) cells
E. coli BL21(DE3) cells were inoculated into LB medium and grown at 30 °C, 230 rpm for 16 h. The cells were then transferred to TB/SB and cultured at 30 °C, 230 rpm for 5 h. IPTG at a final concentration of 0.5 mmol/L was added to the culture to induce protein expression. Cells were harvested 20 h later by centrifugation at 4 °C and 4000 ´ g. Then recombinant protein was extracted from the cells.
Protein extraction and purification
One hundred microliters of culture (OD600 = 3) was collected by centrifugation (4000 ´ g, 5 min, 4 °C) and resuspended in 400 mL lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 0.01% Tween-20, 1 mM PMSF, pH 8.0). Cell disruption was achieved by ultrasonic vibration with 20 % power (SONIC uibra cell). The supernatant containing the recombinant protein was collected following centrifugation (4000 ´ g, 5 min, 4 °C). The target protein was isolated using an ÄKTA purifilerTM UPC10 system.
M1/2/3 pull-down assay
His-M1/2 and His-M3 fusion proteins were immobilized to beads (DynabeadsÒ His-Tag Isolation) and washed four times with washing buffer (50 mM sodium phosphate, 300 mM NaCl, 0.01% Tween-20, pH 8.0). A total protein (or purified Tkl1p-His protein) from a yeast culture in BMMY or BMGY medium was added (Reaction solution). The mixture was incubated for 30 min and the beads washed four times with A solution (3.25 mM sodium phosphate, 70 mM NaCl, 0.01% Tween-20, pH 7.4) (Washing solution). Finally, 50 mL His elution buffer (300 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 0.01% Tween-20, pH 7.4) was added to the samples and these were stirred for 2 min. The His-tagged protein was eluted (Elution solution), and proteins that interacted with the His-tag protein were transferred to sample tubes. All mixtures were subjected to SDS-PAGE and MS analysis.
M1/2 yeast two-hybrid
The protocol from TAKARA was used. Y2HGold cells transformed with the M1 or M2-pGBKT7 recombinant plasmids (SD) were cultured in YPD medium. Y187 with the AD-pGADT7 recombinant plasmid (AD) were also cultured in YPD medium. Equal volumes of the cultures containing SD and AD were then mixed and added to 0.5 mL 2´ YPDA. The resuspended cells were mixed thoroughly. This mixture was incubated overnight (20–24 h) at 30 °C, 200 rpm. Finally, a 100 mL aliquot of the incubated mating culture was incubated in SD/Try/Leu/His/Ade medium at 30 °C for 3–5 d until visible single colonies appeared.
Dmxr1 mutant construction
The P. pastoris strain carrying the mxr1 gene deletion was constructed by homologous recombination using kan as a marker. The upstream region of the mxr1 gene was amplified initially by PCR using Pfu DNA polymerase (Thermo Scientific) and the P. pastoris genome as the template. The primers for this PCR, mxr1s-1 and mxr1s-2, included SphI and BamHI restriction sites, respectively. The 0.6-kb PCR-amplified fragment was inserted into SphI/BamHI-digested pMD™19-T plasmid (TAKARA) to create the pMXR1UP plasmid. The downstream region of the mxr1 gene was also amplified with primers mxr1x-1 and mxr1x-2, carrying restriction sites for KpnI and EcoRI, respectively. This 0.5-kb PCR fragment was inserted into pMD19-T to yield the pMXR1Down plasmid. Next, the G418 resistance gene with its own promoter and terminator (1556 bp) was amplified by PCR, using pFA6a-KanMX6 as the template and primers kan-1 and kan-2, which carried BamHI and KpnI restriction sites, respectively, and the fragment was cloned into the SphI/BamHI-digested plasmid pMXR1UP to give the pMXR1UP-Kan plasmid. This plasmid was digested with KpnI/EcoRI to generate a 2.2-kb fragment that was then inserted into KpnI/EcoRI-digested pMXR1Down plasmid, yielding a P. pastoris mxr1 deletion plasmid, pMD19-T-MXR1-del. The deletion cassette was released from pMD19-T-MXR1-del as a 2.7-kb EcoRI/SphI-digested fragment and transformed by electroporation into wild-type P. pastoris (strain X-33). G418-resistant transformants were isolated on YPD supplemented with 1 mg/mL G418. The correct integration of the deletion cassette into the genome and replacement of the mxr1 open reading frame (ORF) in the transformants was confirmed by PCR analysis and Sanger sequencing.
mxr1 and tkl overexpression strain construction
The mxr1 gene and tkl were amplified by PCR using genomic DNA as the template, and different primers. The fragment was ligated into the pMD™19-T plasmid (TAKARA) and sequenced. The recombinant plasmid and pGAPZB plasmid (Invitrogen) were then digested with PmlI/XhoI, and the mxr1 fragment was inserted into pGAPZB to yield pGM. Finally, pGM was digested with AvrII and electro-transformed into the P. pastoris X-33 Dmxr1 mutant and P. pastoris X-33 wild-type, yielding Mxr1 overexpression strains mxr1-Dmxr1 and mxr1-wt, respectively. tkl1 were amplified by PCR using genomic DNA as the template. Purified PCR products were PmlI/KpnI-digested. This was followed by ligation into pGAPZB to create pGAPZB-tkl1. Then all the above plasmids were transformed into X-33 respectively by electro-transformation.
Real-time PCR
Total RNA extraction was carried out according to the standard procedure described in the Quantscrip TR kit (TAKARA). The reaction consisted of 5´ gDNA Eraser Buffer (2 μL), gDNA Eraser (1.0 μL), total RNA (4 μL, 500 ng) and RNase-Free ddH2O (3 μL). Samples were incubated at 42 °C for 2 min. cDNA served as the template for real-time PCR. The reverse transcription system contained 5 μL of the SYBR Premix Ex Taq II (2´), both forward and reverse primers (10 μM, 0.5 μL), cDNA (1 μL) and dH2O (3 mL). Each experiment was carried out in triplicate. The transcription level was normalized to an endogenous reference gene, glyceraldehyde-3-phosphate dehydrogenase (gapdh). The data processing method was 2–DDCT [31].
Western blotting detection
Total protein samples were taken from cells cultured in YPD. Cultivating E. coli BL21 cells were transferred from 1× YPD after 16 h to 2× YPD. Cells were harvested ~6 h later and the cell pellet washed twice with ice-cold 50 mM potassium phosphate buffer (pH 7.0). An electronic oscillator was used to disrupt cells to obtain soluble proteins. Before oscillation, 200 μL of ice-cold PEBF (0.7882% Tris-HCl, 0.0585% EDTA and 2 μL of 100 mM PMSF) and glass beads were also added to ensure efficient cell disruption. The lysed cells were centrifuged at 10,000 g for 1 min, and the supernatant was collected and used for SDS-PAGE analysis.
Xylulose detection
Wild-type (X-33) cells and Tkl1p over-expression strain cells were harvested when the OD600 reached ~6.0. The harvested cells were transferred from YPD to BMMY and incubated overnight at 30 °C and 230 rpm. Total protein extraction was the same as described above. The reaction system included formaldehyde (1–7%), xylulose (20 mM), and PEB and Mxr1 proteins (25 mL). The reaction was initiated by the addition of the crude cell extract (70 mL) and terminated with 20 mL H2SO4-Na2SO3 (2 M, pH 2.0). High-performance liquid chromatography (HPLC) was performed to quantify the xylulose level. The HPLC parameters were as follows: an Aminexâ HPX-87H ion exclusion column, column temperature 60 °C, the flow rate of 0.6 mL/min and 5 mM H2SO4 as eluent [32, 33].
Formaldehyde and Methanol detection
Cells were incubated overnight in BMGY medium (0.5% methanol) and then transformed into fresh BMMY medium incubate for 12h, 30℃. Cells were centrifuged and supernatant was collected for methanol and formaldehyde detection. HPLC was used for detection followed by Zhan[11]. Formaldehyde concentration was determined via Nash [34].