Strains, media, and culture conditions
Ogataea polymorpha cells were grown on YPD (10 g/L yeast extract, 10 g/L peptone, 20 g/L glucose) or mineral medium (6.7 g/L YNB without amino acids, 20 g/L of glucose or xylose, or 10 g/L of methanol) at 37 °C. For the NCYC495 leu1-1 strain, leucine (40 mg/L) was added to the medium. For selection of yeast transformants on YPD 0.1 g/L of nourseothricin, 0.3-0.5 g/L of geneticin, 0.075-0.1 g/L of zeocin, or 0.35 g/L of hygromycin were added. Ethanol fermentation of O. polymorpha strains was tested as described previously [22]. Fermentation experiments were performed in at least triplicate to ensure the results were reproducible. The bars in the figures indicate the ranges of the standard deviation.
Strains pex3Δ and pex6Δ O. polymorpha were kindly provided by Prof. Ida van der Klei, Groningen University, the Netherlands. S. stipitis strains—parental ku80 [31] and pex3∆ mutants were grown on YNB medium supplemented with 40 mg/mL of histidine and 2 g/L glucose or 1 g/L of oleate at 30 °C. Ethanol fermentation of S. stipitis strains was tested (as mentioned above) for O. polymorpha strains under oxygen-limited conditions (100 rpm) at 30 °C.
The E. coli DH5α strain (Φ80dlacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17(r
−K
, m
+K
), supE44, relA1, deoR, Δ(lacZYA-argF)U169) was used as a host for plasmid propagation. Strain DH5α was grown at 37 °C in LB medium as described previously [32]. Transformed E. coli cells were maintained on a medium containing 100 mg/L of ampicillin.
Molecular biology techniques
Standard cloning techniques were carried out as described [32]. Genomic DNA of O. polymorpha was isolated using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Restriction endonucleases and DNA ligase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer specifications. Plasmid isolation from E. coli was performed with the Wizard® Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). DNA fragments were separated on a 0.8% agarose (Fisher Scientific, Fair Lawn, NJ, USA) gel. Isolation of fragments from the gel was carried out with a DNA Gel Extraction Kit (Millipore, Bedford, MA, USA). PCR amplification of the fragments of interest was done with Phusion® High-Fidelity DNA Polymerase (Thermo Scientific, USA) according to the manufacturer’s specifications. PCRs were performed in GeneAmp® PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). Transformation of the yeasts O. polymorpha and S. stipitis was carried out as described previously [33].
Construction and analysis of das1Δ, tal2Δ, tal1Δ, pYNR1-TKL1 O. polymorpha mutants
Genomic DNA of O. polymorpha NCYC495 leu1-1 strain was used as a template for PCR amplification of 5′ and 3′ non-coding regions of DAS1 using primers OL207/OL208 and OL209/OL210 (Additional file 1). The resulting 5′ DAS1 (926 bp) and 3′ DAS1 (1257 bp) fragments were HindIII/EcoRI or EcoRI/XbaI digested and cloned into HindIII/XbaI-linearized vector pBluescript II KS(−). The resulting recombinant construct was named pLRDAS. Gene zeoR (1132 bp) conferring resistance to zeocin was amplified using vector pPICZB (Thermo Fisher Scientific) as a template and primers Ko64/Ko65. The obtained fragment was EcoRI digested and cloned into EcoRI-linearized plasmid pLRDAS. The constructed plasmid was designated as p∆DAS-Zeo.
The same approach was used for construction of the TAL2 deletion cassette. The 5′ (779 bp) and 3′ (1440 bp) non-coding regions of TAL2 were PCR amplified with primers OL211/OL212 and OL213/OL214. Amplified 5′ and 3′ non-coding regions were double digested with HindIII/EcoRI and EcoRI/BamHI and cloned into pBluescript II KS(−) digested with HindIII/BamHI. Selective marker gene conferring resistance to zeocin was amplified from pPICZB with Ko64/Ko65 and cloned between 5′ and 3′ non-coding regions of TAL2 as EcoRI digested fragment. The resulting plasmid was named p∆TAL-Zeo.
The constructed plasmids were transformed into O. polymorpha NCYC495 leu1-1 recipient strain using an standard electroporation method [33]. Transformants were selected on the solid YPD medium supplemented with 75 mg/L of zeocin after 4 days of incubation. The obtained transformants were examined by PCR using genomic DNA of recombinant strains as a template. Transformants with confirmed deletion of DAS1 and TAL2 were stabilized by altering cultivation in non-selective and selective media and once again examined by PCR. Fragments with predicted size were amplified using pairs of primers homologous to the sequence of selective marker and regions outside from the fragments used for recombination (Ko533/Ko534, Ko557/Ko559 for DAS1 and Ko535/Ko534, Ko557/Ko558 for TAL2) (Additional file 2).
The deletion cassette for isolation of tal1Δ mutant on the background of the wild-type strain was constructed as follows. Genomic DNA of O. polymorpha NCYC495 leu1-1 strain was used as template for isolation of 5′ and 3′ non-coding regions of TAL1 gene by PCR amplifications using primers OK74/OK75 and OK76/OK77. The obtained DNA fragments were fused by overlap PCR using primers OK74/OK77. The resulting fragment (1.8 kb) was BamHI/KpnI digested and cloned into the appropriate sites of pUC57 vector. The hphNT1 gene (1777 bp), conferring resistance to hygromycin, was amplified from plasmid pRS42H [34] as a template and primers Ko446 and Ko450. The resulting plasmid was designated as p∆TAL1-Hygr.
The constructed plasmid was transformed into O. polymorpha NCYC495 recipient strain using the electroporation method given above [33]. Transformants were selected on solid YPD medium supplemented with 0.35 g/L of hygromycin after 4 days of incubation. Obtained transformants were examined by PCR using genomic DNA of recombinant strains as a template. Transformants with confirmed deletion of TAL1 were stabilized by altering cultivation in non-selective and selective media and once again examined by PCR. Fragments with predicted size were amplified using pairs of primers homologous to the sequence of selective marker and regions outside from the fragments used for recombination (OK80/Ko450, Ko446/OK81). Additionally, primers OK99/OK100 for TAL1 ORF amplification were used (Additional file 3).
The conditional knockout mutant tkl1∆ was constructed by replacement of the endogenous promoter of TKL1 gene by regulated YNR1 promoter of nitrate reductase, repressed by ammonium sulfate as nitrogen source. Genomic DNA from O. polymorpha NCYC495 leu1-1 strain was used as a template for PCR amplification of 5′ non-coding regions; and part of the ORF of TKL1 gene using primers OK170/OK171 and OK174/OK175, respectively. The promoter of the YNR1 gene was amplified from genomic DNA from the O. polymorpha NCYC495 leu1-1 strain using primers OK172/OK173. The obtained DNA fragments were fused by overlap PCR using primers OK170 and OK175. The resulting fragment (3.6 kb) was KpnI/SphI digested and cloned into the corresponding sites of the pUC57 vector. Selective marker gene conferring resistance to hygromycin was amplified from plasmid pRS42H with primers Ko446 and Ko450 and cloned in XbaI-linearized plasmid pUC57-YNR1p_TKL1. The resulting plasmid was named pUC57-YNR1p_TKL1_ hphNT1.
The constructed plasmid was transformed into O. polymorpha NCYC495 recipient strain using the same electroporation method [33]. Transformants were selected on the solid YPD medium supplemented with 0.35 g/L of hygromycin after 4 days of incubation.
The correct replacement of TKL1 gene promoter was confirmed by PCR in obtained transformants using primers OK176/OK123 and OK172/OK102 (Additional file 4).
Construction and analysis of O. polymorpha strains with overexpression of DAS1, TAL2, TAL1, and TKL1 genes
The recombinant plasmids, pGLG61/DAS1 and pGLG61/TAL2, bearing the O. polymorpha genes DAS1 and TAL2, respectively, were constructed on the basis of the plasmid, pGLG61 [35].
Genomic DNA from O. polymorpha was used as a template for PCR amplifications of the DAS1 and TAL2 genes. Native promoters from these genes were substituted with a strong constitutive promoter, GAP1, from glyceraldehyde-3-phosphate dehydrogenase (GAP). First, the GAP1 promoter and ORF of the DAS1 gene with terminator sequence were amplified from O. polymorpha genomic DNA using the primers, Ko277/Ko278 and Ko279/Ko280. Then two fragments were fused by overlap PCR using primers, Ko277 and Ko280. The resulting fragment (2.9 kb) was digested with BamHI endonuclease and ligated with BamHI-linearized plasmid, pGLG61. The resulting recombinant construct was named pGLG61/DAS1 (Additional file 5A). At the next step, the GAP1 promoter and ORF of the TAL2 gene with terminator sequence were amplified from O. polymorpha genomic DNA using the primers, Ko405/Ko411 and Ko412/Ko414, respectively. Then two fragments were fused by overlap PCR using the primers, Ko405 and Ko414. The resulting fragment (1.8 kb) was digested with BamHI and BglII endonuclease and ligated with the BamHI/BglII-linearized plasmid, pGLG61. The resulting recombinant plasmid was named pGLG61/TAL2 (Additional file 5A). These plasmids were introduced into the genome of the O. polymorpha NCYC495 leu1-1 strain. Transformants were selected on solid YPD medium supplemented with 0.3–0.5 g/L of geneticin after 5 days of incubation. Selected transformants were stabilized by alternating cultivation in non-selective and selective media and examined by diagnostic PCR using the primers, Кo277/Ko278 or Кo279/Кo280, respectively.
For overexpression of the cytosolic transketolase and transaldolase recombinant plasmids, pTkZr and pTaZr, bearing the O. polymorpha genes (TKL1 and TAL1), respectively, were constructed. The strong constitutive GAP promoter and ORF of the TKL1 with terminator sequence were amplified by PCR from O. polymorpha chromosomal DNA using two pairs of primers, A58/A35 and Ko52/Ko53, respectively. The obtained PCR products were digested with SacI/NotI and then ligated with SacI/NotI-linearized vector pUC57, carrying the zeocin resistance gene as a selective marker (pUC57-Zr). The resulting plasmid was designated as pTkZr. For overexpression of the TAL1 gene, the DNA fragment bearing the GAP1 promoter was fused with the ORF of the TAL1 gene. This was accomplished by overlap PCR using two pairs of primers, K43/Ko77 and Ko76/Ko84. The PCR product was digested with BamHI and then ligated with BamHI-linearized vector, pUC57-Zr, resulting in the recombinant construct, pTaZr (Additional file 5B). The resulting plasmids were used for transformation of O. polymorpha NCYC495 leu1-1 strain. The transformants were selected on solid YPD medium supplemented with 0.1 g/L of zeocin after 3 days of incubation.
For simultaneous overexpression of the DAS1 and TAL2 genes, the plasmid, pUC57/DAS1/TAL2, was constructed. BamHI/PstI-restriction fragment containing GAPpr-TAL2 was isolated from the plasmid, pGLG61/TAL2, and cloned into the BamHI/PstI-linearized vector pUC57. The resulting recombinant construct was named pUC57/TAL2. The BamHI-restriction fragment containing GAPpr-DAS1 was isolated from the plasmid, pGLG61/DAS1, and cloned into the BamHI-linearized and dephosphorylated vector, pUC57/TAL2. The resulting recombinant construct was named pUC57/DAS1/TAL2. Gene natNT2, conferring resistance to nourseothricin, was amplified from the plasmid, pRS41N [33] by PCR using primers, OK42/OK43. The fragment obtained (1.3 kb) was NdeI-linearized and cloned into the appropriate site of pUC57/DAS1/TAL2. The resulting plasmid was named pUC57/DAS1/TAL2/NTC (Additional file 5C). Transformants were selected on solid YPD medium supplemented with 0.1 g/L of nourseothricin after 3 days of incubation. Selected transformants were stabilized by alternating cultivation in non-selective and selective media and examined by diagnostic PCR using the primers, Ko277/Ko278 and Ko279/Ko280, respectively.
Construction and analysis of pex3Δ S. stipitis mutants
Genomic DNA of S. stipitis ku80 strain was used as template PCR amplification of 5′ and 3′ non-coding regions of PEX3 gene using the primers, Ko770/Ko771 and Ko772/Ko773. The resulting 5′PEX3 (1682 bp) fragment was SacI/XbaI digested and cloned into SacI/XbaI-linearized vector pUC57. The resulting plasmid was named pUC57-5′PEX3. As the next step, 3′PEX3 (1499 bp) fragments were XbaI/BamHI digested and cloned into XbaI/BamHI-linearized vector pUC57-5′PEX3. The resulting plasmid was named pUC57-5′-3′PEX3. HIS3 gene (1399 bp) involved in histidine biosynthesis was amplified using vector p∆atg13Sc [36] as a template and primers KB5 and KB6. The obtained fragment was XbaI digested and subcloned into XbaI-linearized plasmid pUC57-5′-3′PEX3. As a result, the recombinant plasmid pUC57-5′-3′PEX3-HIS3 was constructed. This plasmid was PstI-linearized and transformed into S. stipitis ku80 his3-1 recipient strain using electroporation method [33]. Transformants were selected on solid YNB medium supplemented with 40 mg/L of histidine after 5 days of incubation at 30 °C. The obtained transformants were examined by PCR using genomic DNA of recombinant strains as a template. Transformants with confirmed deletion of PEX3 were stabilized by altering cultivation in non-selective and selective media and once again examined by PCR. Fragments with predicted size were amplified using pairs of primers homologous to the sequence of selective marker and regions outside from the fragments used for recombination (Ko774/KB9 and KB10/Ko775) (Additional file 6).
Fluorescence microscopy visualization of peroxisomal localization of Tal2 in O. polymorpha
For constitutive expression of peroxisomal SKL-tagged green fluorescent protein (GFP-SKL) in O. polymorpha, the DNA fragments harboring the GFP-SKL gene and the promoter ScTEF1 were PCR amplified with the primers, Ko799/Ko800, from the plasmid, p416TEF-GFP [37]. The terminator, ScCYC1, was amplified with the primers, Ko801/Ko802, from the genomic DNA of S. cerevisiae BY4742. The backbone plasmid containing KamMX4 selective marker was amplified with primers, Ko803/Ko804, from the plasmid, pCfB2055 [38]. Three PCR fragments were then Gibson assembled to generate the plasmid, pGFP-SLK. The obtained plasmid was digested with NotI and integrated into genome of O. polymorpha NCYC495 leu1-1 strain. Transformants were selected on the solid YPD medium supplemented with 0.3 g/L of geneticin after 4 days of incubation. One of the transformants was used as a recipient strain for localization studies of Tal2 protein. The DNA fragment harboring the gene coding for the red fluorescent protein (RFP) was amplified with the primers, Ko888/Ko889, from the plasmid, pDsRed2 (Clontech). The backbone plasmid containing TAL2 was amplified with the primers, Ko890/Ko891, from the plasmid, pGLG61/TAL2. Two PCR fragments were then Gibson assembled to generate the plasmid pGLG61/TAL2_RFP. This plasmid was introduced into genome of O. polymorpha wild-type strain carrying GFP-SKL. Transformants were selected on solid YNB medium after 5 days of incubation. Selected transformants were stabilized by alternating cultivation in non-selective and selective media and examined by diagnostic PCR using the primers, Ko892/Ko414. The resulting strains were grown at 37 °C in YNB medium with glucose, xylose, or methanol during 24 h; followed by microscopy analysis. Images were captured on a fluorescence microscope (Axio Imager A1; Carl Zeiss MicroImaging, Jena, Germany) coupled to a monochrome digital camera (Axio Cam MRm; Carl Zeiss MicroImaging) and processed using the AxioVision 4.5 (Carl Zeiss MicroImaging) and Adobe Photoshop CC software (Adobe Systems, Mountain View, CA).
Biochemical methods
The enzyme activity was measured directly after the preparation of cell-free extracts. Protein concentration was determined with Folin reagent [39]. The specific activities of total transketolase and transaldolase in cell extracts were determined spectrophotometrically as described before [25, 40].
All assay experiments were repeated at least twice.
Quantitative real-time PCR (qRT-PCR)
Expression of the XYL1, XYL2, XYL3, RPE1, TKL1, PDC1, ADH1, and CYC1 genes was analyzed by real-time PCR. The qRT-PCR was performed by 7500 Fast Real-Time PCR System (The Applied Biosystems, USA) with SG OneStep qRT-PCR kit (EURx Ltd., Gdansk, Poland) using gene-specific pairs of primers, RNA as a template, and ROX reference passive dye according to the manufacturer’s instructions as described previously [22]. The primers pairs used for qRT-PCR are listed in Additional file 1. Sequences of the tested genes were taken from O. polymorpha genome database (Ogataea polymorpha NCYC 495 leu1.1 v2.0-JGI Genome Portal. http://genome.jgipsf.org/Hanpo2/Hanpo2.home.html).
Analyses
The biomass was determined turbidimetrically with a Helios Gamma spectrophotometer (OD, 590 nm; cuvette, 10 mm) with gravimetric calibration. Concentrations of xylose and ethanol from fermentation in medium broth were analyzed by HPLC (PerkinElmer, Series 2000, USA) with an Aminex HPX-87H ion-exchange column (Bio-Rad, Hercules, USA). A mobile phase of 4 mM H2SO4 was used at a flow rate 0.6 mL/min and the column temperature was 30 °C. Alternatively, concentrations of ethanol in the medium were determined using alcohol oxidase/peroxidase-based enzymatic kit “Alcotest” [41]. Experiments were performed at least twice.