Strains, media, and culture conditions
O. polymorpha cells were grown on YPD (10 g/L yeast extract, 10 g/L peptone, 20 g/L glucose) or mineral (6.7 g/L YNB without amino acids, 20 g/L of glucose or xylose) media 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 or 0.2 g/L of zeocin were added. Alcoholic fermentation of O. polymorpha cells was tested at 45 oC as described previously  in medium with 10 % xylose or mixtures of glucose and xylose with ratios 7 % : 3 %, 5 % : 5 %, or 3 % : 7 %. The fermentation experiments were performed in at least triplicate to ensure the reproducibility of results. Bars in the figures indicate ranges of the standard deviation.
For electrochemical measurements, cells were taken after 24h cultivation under fermentation conditions with 10 % xylose or mixture of glucose and xylose (7 %: 3 %). The cells were pelleted, washed with 5 mM sodium phosphate buffer in 95 mM NaCl, pH 7.0 and starved with or without 2-deoxyglucose for 1 h.
E. coli DH5α strain (Φ80dlacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17(rK−, mK+), supE44, relA1, deoR, Δ(lacZYA-argF)U169) was used as a host for plasmid propagation. DH5α cells were grown at 37 °C in LB medium as described previously . Transformed E. coli cells were maintained on a medium containing 100 mg/L of ampicillin.
Construction O. polymorpha strains with engineered endogenous Hxt1 or S. cerevisiae Hxt7 and Gal2 transporters
O. polymorpha genome database (https://mycocosm.jgi.doe.gov/Hanpo2/ Hanpo2.info.html) was used for retrieval of HXT1 gene sequence. The open reading frame of HXT1 gene along with its own gene terminator was amplified from O. polymorpha NCYC495 genomic DNA using primers OK161/OK162 (Additional file 4: Table S1). The native promoter of this gene was substituted by a strong constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase (GAP1).
The promoter of GAP1 gene was amplified from O. polymorphaNCYC495 genomic DNA using primers OK159/OK160. The resulting fragments were fused by overlap PCR using primers OK159/OK162. The amplified fragment (2.6 kb) was XbaI/SphI digested and cloned into the corresponding sites of the pUC57/zeo vector, carrying selective marker gene conferring resistance to zeocin. The constructed plasmid was named pUC19/zeo/HXT1 (Additional file 3: Fig. S3) and used as a template for the construction of modified versions of Hxt1.
One version of Hxt1 was engineered by substitution of asparagine to alanine at position 358 to increase the specific xylose uptake rate and decrease affinity to glucose. Primers OK163/OK164 and OK165/OK162 were used for amplification of N- and C-fragments of HXT1 gene. The resulting fragments were fused by overlap PCR using primers OK163/OK162. The amplified fragment (1.6 kb) was NotI/SphI digested and cloned into the corresponding sites of the pUC19/zeo/HXT1 vector instead of the native version of HXT1gene. The constructed plasmid was named pUC19/zeo/HXT1_N358A (Additional file 3: Fig. S3).
The predicted targets of ubiquitination were identified in the sequence of HXT1 gene using UbPred program software http://www.ubpred.org. The N-terminally located lysine residues at positions 8, 9 and 30 identified as potentially involved in ubiquitination were replaced by arginine residues. Primers OK159/OK167 and OK166/OK162 were used for amplification of GAP1 promoter with N-fragment and C-fragments of HXT1 gene, respectively. The resulting fragments were fused by overlap PCR using primers OK159/OK162. The amplified fragment (2.6 kb) was XbaI/SphI digested and cloned into the corresponding sites of the pUC19/zeo vector. The constructed plasmid was named pUC19/zeo/HXT1_K (Additional file 3: Fig. S3).
The modified version of HXT1 with all mentioned modifications was obtained by PCR from the plasmid pUC19/zeo/HXT1_K using primers ОК163/ОК164 and ОК165/ОК162. The resulting fragments were fused by overlap PCR using primers OK163/OK162. The amplified fragment (1.6 kb) was NotI/SphI digested and cloned into the corresponding sites of the pUC19/zeo/HXT1 vector instead of the native version of HXT1 gene. The constructed plasmid was named pUC19/zeo/HXT1_N358A_K (Additional file 3: Fig. S3).
Primers OK161/ OK216 were used for amplification of the corresponding fragment (1.6 kb) from the plasmid pUC19/zeo/HXT1_N358A_K. The resulting fragment was NotI-digested and cloned into the corresponding site of the pUC19_prGAP_NTC . The constructed plasmid was named pNTC_HXT1_N358A_K (Additional file 3: Fig. S3).
The S. cerevisiae genome database (https://www.yeastgenome.org/) was used for retrieval of HXT7 and GAL2 gene sequences. The modified version of Hxt7 was constructed by replacement of asparagine residue at position 370 for serine. Primers OK203/OK204 and OK205/OK217 were used for amplification of N- and C-fragments of HXT7 gene from a genomic DNA of S. cerevisiae BY4742. The resulting fragments were fused by overlap PCR using primers OK203/OK217. The amplified fragment (1.7 kb) was XbaI digested and cloned into the corresponding site of the pUC19_prGAP_NTC. The constructed plasmid was named pNTC_HXT7_N370A (Additional file 3: Fig. S3).
Primers OK207/OK208 and OK209/OK218 were used for amplification of N- and C-fragments of GAL2 gene from a genomic DNA of S. cerevisiae BY4742. The resulting fragments were fused by overlap PCR using primers OK207/OK218. The amplified fragment (1.7 kb) was XbaI digested and cloned into the corresponding site of the pUC19_prGAP_NTC. The constructed plasmid was named pNTC_GAL2_N376F (Additional file 3: Fig. S3).
Visualization of membrane localization of Hxt1 in O. polymorpha by fluorescence microscopy
The DNA fragment harboring the gene coding for the green fluorescent protein (GFP) was amplified using primers Ko819 and Ko820 from the plasmid pGFP-SLK . The backbone plasmids containing HXT1 or HXT1_N358A_K was amplified with the primers Ko821/Ko822 from the plasmids pUC19/zeo/HXT1 or pUC19_prGAP_NTC_HXT1_ N358A_K. Two PCR fragments were then Gibson assembled to generate the plasmids pUC19/zeo/HXT1_GFP or pUC19_prGAP_NTC_HXT1_ N358A_K_GFP. These plasmids were introduced into the genome of O. polymorpha hxt1Δ mutant. Transformants were selected on solid YPD medium supplemented with 0.2 g/L of zeocin after 3 days of incubation. Selected transformants were stabilized by alternating cultivation in non-selective and selective media and examined by diagnostic PCR using primers, OK161/Ko820. The resulting strains were grown at 37 °C in YNB medium with xylose during 96 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).
Quantitative real‑time PCR (qRT‑PCR)
Expression of the HXT1, HXT7 and GAL2 genes was analyzed by real-time PCR. The qRT-PCR was performed by 7500 Fast RealTime PCR System (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 . The pairs of primers used for qRT-PCR are listed in Additional file 4: Table S1. Sequences of the tested genes were taken from O. polymorpha genome database.
The optical density (OD) of cell suspensions for biomass determination was measured using a «Helios-λ» spectrophotometer at λ 590 and λ 663 nm for O. polymorpha transformation . Concentrations of xylose and ethanol from fermentation in the 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” . Experiments were performed at least twice. Measurements of the extracellular acidification
The extracellular acidification was determined by pH electrode (Hanna Instruments HI1131B), measuring pH from 0 to 13 at -5 to 100 °C. The software NT-MDT Nova 850 and the computer program LabChart were used to monitor changes in the concentrations of hydrogen ions in the test solutions. Measurements were performed in 9 mL of 5 mM sodium phosphate-75 mM NaCl buffer, pH 7. At the beginning of every experiment, the signal was allowed to settle before the cells were added into the test solution. Once the signal has settled, 2% D-xylose or 2% D-glucose were added. At the end of the experiment, the known concentration of NaOH was added for calibration.