The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae

Microbial strains, growth media and maintenance

Molecular biology techniques

DNA fragments were amplified by PCR amplification with Phusion Hot Start II High Fidelity Polymerase (Thermo Scientific) and desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO) performed according to the manufacturers’ instructions. Diagnostic PCRs were run with DreamTaq polymerase (Thermo Scientific). Oligonucleotide primers used in this study are listed in Additional file 1. PCR products were separated by electrophoresis on 1% (w/v) agarose gels (Thermo Scientific) in TAE buffer (Thermo Scientific) and, if required, purified with a Zymoclean Gel DNA Recovery kit (Zymo Research, Irvine, CA) or a GenElute PCR Clean-Up kit (Sigma-Aldrich). Yeast or E. coli plasmids were isolated with a Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research), or a Sigma GenElute Plasmid kit (Sigma-Aldrich), respectively. A YeaStar Genomic DNA kit (Zymo Research) or an SDS/lithium acetate protocol [39] was used to isolate yeast genomic DNA. Yeast strains were transformed using the lithium acetate/polyethylene glycol method [40]. Single-colony isolates were obtained from three consecutive re-streaks on selective solid agar plates, followed by analytical PCR analysis of the relevant genotype. E. coli DH5α cultures were transformed by chemical transformation [41]. After isolation, plasmids were verified by restriction analysis and analytical PCR.

Plasmid construction

Strain construction

Gene expression cassettes were PCR amplified with oligonucleotide primers shown in Additional file 1 and genomic DNA of CEN.PK113-7D or plasmids described in Table 2. Gene knock-outs and construct integrations were introduced with a chimeric CRISPR/Cas9 editing system [44]. To enable CRISPR/Cas9 mediated editing in strain IMX080, the SpCas9 expression cassette was amplified from p414-pTEF1-cas9-tCYC1 (Addgene plasmid # 43802) and integrated into the GAL1 locus via in vivo assembly, together with the amdSYM marker, yielding strain IMX486. For overexpression of the non-oxidative pentose phosphate pathway (PPP), IMX486 and IMX581 were co-transformed with gRNA plasmid pUDE335 and repair fragments flanked with either 60 bp homologous to GRE3 or with synthetic tags [46] assisting homologous recombination of the PPP expression cassettes (gre3 _flank -pTDH3-RPE1-TagH, TagH-pPGK1-TKL1-TagI, TagI-pTEF1-TAL1-TagA, TagA-pPGI1-NQM1-TagB, TagB-pTPI1-RKI1-TagC, TagC-pPYK1-TKL2-gre3 _flank ). After counter selection of the URA3-based plasmid pUDE335, the resulting strains, IMX604 and IMX918, respectively, were co-transformed with pUDE348 and repair fragments flanked with either 60 bp homologous to GAL80 or with synthetic tags [46] (GAL80 _flank -pTPI1-araA-TagG, TagG-pTPI1-araA-TagA, TagA-pTPI1-araA-TagB, TagB-pTPI1-araA-TagC, TagC-pTPI1-araA-TagD, TagD-pTPI1-araA-TagM, TagM-pTPI1-araA-TagN, TagN-pTPI1-araA-TagO, TagO-pTPI1-araA-TagI, TagI-pPYK1-araB-TagK, TagK-pPGK1-araD-GAL80 _flank ) resulting in nine copies of araA and a single copy of araB and araD integrated in the GAL80 locus. After verification of the resulting strains IMX929 and IMX658, respectively, plasmid pUDE348 was counter selected in IMX929 to yield strain IMX928. Disruption of HXK2 in IMX658 was done by PCR amplification and transformation of the KlURA3-based deletion cassette from pUG-72 [76] to obtain strain IMX660 upon transformation and plating in solid SMA. GAL2 was disrupted in IMX660 by transformation with a KanMX cassette amplified from pUD405 with primers 944 and 945 flanked with 60 bp homologous to GAL2. Transformants were incubated for 2 h in YPE before plating on YPEG-G418, yielding strain IMX844. Expression of PcaraT in IMX658 was achieved by transforming IMX658 with the gRNA plasmid pUDE327 together with an expression cassette of PcaraT (pADH1-PcaraT-tPMA1) with flanking regions homologous to the HXK2 locus amplified with the primer pair 7660 and 7676. Counter selection of the pUDE327 and subsequent transformation of a DNA fragment derived from CEN.PK113-7D using primers 2641 and 1522 repaired uracil auxotrophy and resulted in strain IMX728. GAL2 was disrupted in IMX728 by transformation with a KanMX cassette amplified from pUD405 with primers 944 and 945 flanked with 60 bp homologous to GAL2. Transformants were incubated for 2 h in YPE before plating on YPEG-G418, yielding strain IMX869. Strains IMX1505-1509 were constructed by co-transforming pUDR245 or pUDR246 and a GAL2-flanked expression cassette (pADH1-ORF-tPMA1) amplified from pPWT111, 113, 116, 118 or 123, respectively, amplified with the primer pair 10585 and 10584. IMX1504, harbouring a knockout of GAL2, was constructed by co-transforming pUDR245 and a repair fragment based on the annealed primers 9563 and 9564. Transformation of GAL2 and PcaraT plasmids, and the pRS313-mcs plasmid (as an empty plasmid/control) into the hexose-transporter deletion strain DS68625 yielded strains DS68625-GAL2, DS68625-PcaraT, and DS68625-mcs.

Growth experiments in shake flasks

Thawed 1-mL aliquots from frozen stock cultures were used to inoculate shake flask precultures on SM-urea supplemented with either d-glucose (20 g L⁻¹), l-arabinose (20 g L⁻¹), or both sugars (both 20 g L⁻¹). These precultures were used to inoculate a second culture which was subsequently used to inoculate a third culture which was inoculated at an initial OD₆₆₀ of 0.1 and used to monitor growth. Optical densities at 660 nm were measured with a Libra S11 spectrophotometer (Biochrom, Cambridge, United Kingdom). Maximum specific growth rates (μ_max) were derived from at least four consecutive data points derived from samples taken during the exponential growth phase of each culture.

Spot plates

l-Arabinose-metabolizing S. cerevisiae strains expressing putative P. chrysogenum l-arabinose transporter genes (IMX1504-1509) were grown on SMD medium and a total number of approximately 10⁴, 10³, 10², and 10¹ cells were spotted on duplicate agar plates as described previously [47, 48] containing either 20 g L⁻¹ l-arabinose or d-glucose as carbon source (pH 6). Cell numbers were estimated from calibration curves of OD₆₆₀ versus cell counts determined with an Accuri flow cytometer (Becton–Dickinson B.V., Breda, The Netherlands), derived from exponentially growing shake flask cultures of S. cerevisiae CEN.PK113-7D on SMD medium. SMA and SMD plates were incubated at 30 °C for 97 and 41 h, respectively.

Chemostat cultivation

Aerobic carbon-limited chemostat cultures of P. chrysogenum were grown at 25 °C in 3-L turbine-stirred bioreactors (Applikon, Schiedam, The Netherlands) with a working volume of 1.8 L and a dilution rate of 0.03 h⁻¹ as described previously [49], with the exception that, in addition to cultures grown on 7.5 g L⁻¹ d-glucose, chemostat cultures were also grown on either 7.5 g L⁻¹ l-arabinose or 5.8 g L⁻¹ ethanol. Aerobic, l-arabinose-limited chemostat cultures of S. cerevisiae were grown at 30 °C in 2-L Applikon bioreactors with a working volume of 1 L and at a dilution rate of 0.05 h⁻¹. SMA (7.5 g L⁻¹ l-arabinose) supplemented with 0.15 g L⁻¹ Pluronic antifoam PE 6100 was used as culture medium for the initial batch phase and for chemostat cultivation, with the exception of the initial batch phase of strain IMX929 which was grown on 20 g L⁻¹ l-arabinose. Cultures were stirred at 800 rpm, kept at pH 5.0 by automatic addition of 2 M KOH, and sparged with 0.5 L min⁻¹ air. Upon completion of the batch phase, chemostat cultivation was initiated, ensuring a constant culture volume with an electric level sensor. When after at least five volume changes, biomass dry weight and CO₂ production varied by less than 2% over two consecutive volume changes, the culture was considered to be in steady state.

Analytical methods

Penicillium chrysogenum biomass dry weight was determined in duplicate by filtration of 10 mL culture sample over pre-weighed glass fibre filters (Type A/E, Pall Life Sciences, Hoegaarden, Belgium). After filtration, filters were washed with demineralized water and dried for 10 min at 600 W in a microwave oven (Bosch, Stuttgart, Germany) prior to reweighing. Biomass dry weight in S. cerevisiae culture samples was determined with a similar procedure using nitrocellulose filters (0.45-µm pore size; Gelman Laboratory, Ann Arbor, MI) and drying for 20 min in a microwave oven at 360 W output. Optical density (OD) of the cultures was determined at 660 nm with a Libra S11 spectrophotometer (Biochrom, Cambridge, United Kingdom). Determination of CO₂ and O₂ concentrations in the bioreactor exhaust gas and HPLC analysis of metabolite concentrations in culture supernatant samples were performed as described previously [50].

Sampling, RNA extraction, microarrays analysis, and data analysis

Samples (60 mL) from P. chrysogenum chemostat cultures were rapidly filtered over a glass fibre filter (Type A/E, Pall Life Sciences) and further processed for total RNA extraction by phenol–chloroform extraction [49]. The cRNA sample preparation (cDNA synthesis, purification, in vitro transcription, labelling, purification, fragmentation and biotinylation) was performed according to Affymetrix recommendations [31]. Eventually cRNA samples were hybridized onto custom-made P. chrysogenum GeneChip microarrays (array code DSM_PENa520255F). Data acquisition, hybridization, quantification of processed array images, and data filtering were performed using the Affymetrix GeneChip Operating Software (GCOS version 1.2). Global array normalization was performed by scaling the global fluorescence intensity of each microarray to 100. The scaling factors of the individual arrays were highly similar and ranged from 0.21 to 0.35. Subsequently, significant variations in expression were statistically estimated by comparing replicate array experiments using the Significance Analysis of Microarray software (SAM version 2.0) [51] with the multiclass setting. A false discovery rate of 1% was applied to minimize the chance of false-positive hits. Genes with an over threefold higher transcript level in arabinose-grown cultures than in d-glucose-grown cultures and a less than threefold difference in ethanol- and d-glucose-grown cultures were deemed to show arabinose-specific expression. Transcriptome data of strain DS17690 grown on d-glucose, ethanol or arabinose are accessible at NCBI Genome Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under Accession Numbers GSE12632, GSE24212 and GSE10449, respectively [49].

Analysis of sugar uptake kinetics

Uptake experiments with [¹⁴C] l-arabinose, [¹⁴C] d-xylose, or [¹⁴C] d-glucose, labelled at the first carbon atom (50–60 mCi/mmol) (ARC St. Louis, MO), were performed with S. cerevisiae hexose-transporter deletion strains (DS68625) harbouring a low copy plasmid with constitutively expressed PcaraT (pRS313-PcaraT) or GAL2 (pRS313-GAL2). The experimental workflow was carried out as described previously [45] with [¹⁴C] l-arabinose concentrations of 0.5–2000 mmol L⁻¹, [¹⁴C] d-xylose concentrations of 0.5–500 mM, or [¹⁴C] d-glucose concentrations of 0.1–500 mmol L⁻¹. Transport competition experiments were carried out in the presence of 50 mmol L⁻¹ [¹⁴C] l-arabinose and 0–500 mmol L⁻¹ d-glucose or d-xylose, and at [¹⁴C] l-arabinose concentration of 2 mmol L⁻¹ together with increasing d-glucose and xylose concentrations of 0–20 mM. Maximum biomass-specific transport rates (‘V_max’) calculated from transport assays were expressed as nmol sugar transported per milligram biomass dry weight per minute [nmol (mg biomass)⁻¹ min⁻¹]. As this V_max is influenced by the expression level of the relevant transporter, it is not solely dependent on intrinsic transporter kinetics. The impact of proton-gradient uncoupling on transport activity was determined in 200 μL synthetic medium at a [¹⁴C]-l-arabinose concentration of 2 mmol L⁻¹, by comparing transport rates upon addition of either 10 μmol L⁻¹ CCCP (0.5 µL of a stock solution dissolved in 100% DMSO), 0.5 μL DMSO (control), or 0.5 µL water.

Phylogenetic methods

Protein sequences used for generation of a phylogenetic tree were derived from NCBI (https://www.ncbi.nlm.nih.gov/) and the Saccharomyces Genome Database (https://www.yeastgenome.org/). Mafft was used to generate a CLUSTAL format alignment of all sequences, using the L-INS-i method default settings (https://mafft.cbrc.jp/alignment/server/) [52, 53]. Alignments were further processed using neighbour-joining and a 500 times bootstrap. The resulting Newick tree file was visualized and midpoint rooted in iTOL (https://itol.embl.de/) [54]. Gene accession numbers were ScGAL2: P13181, PcaraT: CAP85508, SsaraT: XP_001382755, Atstp2: OAP13698, Kmaxt1: GZ791039, Pgaxt1: GZ791040, Amlat1: AY923868, Amlat2: AY923869, Nclat-1: EAA30346, Mtlat-1: XP_003663698.

Chemostat-based transcriptome analysis of P. chrysogenum for identification of possible l-arabinose transporter genes

PcAraT: a P. chrysogenum l-arabinose transporter that can be functionally expressed in S. cerevisiae

Saccharomyces cerevisiae strains in which HXT transporter genes have been deleted and which express heterologous pathways for pentose metabolism have proven to be powerful platforms for screening and characterization of heterologous pentose transporter genes [19, 26, 56]. To enable screening for P. chrysogenum l-arabinose transporters, S. cerevisiae strains were first engineered for l-arabinose consumption. Using CRISPR/Cas9-mediated in vivo assembly [44], the overexpression cassettes for all structural genes involved in the non-oxidative pentose phosphate pathway (TAL1, NQM1, TKL1, TKL2, RKI1, RPE1) were stably integrated into the GRE3 locus, thereby inactivating synthesis of the Gre3 aldose reductase. Subsequently, nine copies of an expression cassette for overexpression of codon-optimized L. plantarum l-arabinose isomerase AraA and single copies of L. plantarum AraB (l-ribulokinase) and AraD (l-ribulose-5-phosphate-4-epimerase) expression cassettes were integrated into the GAL80 locus, using a strain construction strategy previously described for expression of a d-xylose pathway into S. cerevisiae [50]. This integration inactivated GAL80 and thereby alleviated transcriptional repression by d-glucose of GAL2, which encodes the major l-arabinose transporter in S. cerevisiae [57, 58]. The resulting strain IMX929 was able to grow in liquid media supplemented with l-arabinose as the sole carbon source and was used as a platform strain to test if any of the five selected putative P. chrysogenum transporter genes, placed under the control of the constitutive ADH1 promoter, could support l-arabinose transport in S. cerevisiae. To this end, single copies of codon-optimized expression cassettes were integrated into the GAL2 locus of the l-arabinose-metabolizing S. cerevisiae strain IMX928, a uracil auxotrophic daughter strain of IMX929, thereby inactivating the GAL2 gene. Consistent with previous studies [19, 26], inactivation of GAL2 in the l-arabinose metabolizing strain IMX928 yielded a strain (IMX1504) that was unable to grow on SMA plates (Fig. 1). All five strains in which GAL2 had been replaced by putative P. chrysogenum transporter genes (IMX1505-1509) showed vigorous growth on SMD plates. However, only strain IMX1508, which expressed the P. chrysogenum gene Pc20g01790, showed growth on l-arabinose (Fig. 1). Based on this observation, Pc20g01790 was designated PcaraT (P. chrysogenum Arabinose Transporter). A Blast-p search revealed strong homology of Pc20g01790 with the K. lactis gene HGT1, which encodes a high-affinity d-glucose and galactose transporter [59, 60].

PcaraT encodes a high-affinity, high-specificity l-arabinose transporter

The impact of the presence of d-glucose and d-xylose on l-arabinose transport by Gal2 and PcAraT was investigated in transport assays with 50 mmol L⁻¹ [¹⁴C] l-arabinose and increasing concentrations of non-radioactive d-glucose or d-xylose. In these assays, both transporters exhibited a reduced l-arabinose transport capacity in the presence of d-glucose or d-xylose (Table 4, Additional file 3). At a concentration of 100 mmol L⁻¹ (i.e. twice the concentration of l-arabinose), d-xylose and d-glucose inhibited l-arabinose uptake rate via Gal2 by 29 and 85%, respectively. In contrast, l-arabinose transport via PcAraT was less impaired at this concentration of d-xylose, and especially, d-glucose (22 and 63% inhibition, respectively). To study the transport mechanism of PcAraT, the impact of the protonophore uncoupler CCCP on transport kinetics was tested. Transport of l-arabinose via Gal2, which mediates facilitated diffusion of sugars [61], was not affected by CCCP, whilst this uncoupler completely abolished transport via PcAraT (Additional file 7). These results indicate that PcAraT mediates proton-coupled import of l-arabinose.

Functional expression of PcaraT in an l-arabinose-fermenting S. cerevisiae strain enables l-arabinose consumption in the presence of d-glucose

The ability to transport l-arabinose in the presence of d-glucose is a highly relevant characteristic in the construction of platform S. cerevisiae strains for conversion of lignocellulosic hydrolysates [8]. To investigate whether expression of PcaraT can confer this ability, a set of three strains was constructed that (i) could not metabolize d-glucose due to the deletion of HXK1, HXK2, GLK1 and GAL1 [20, 62]; (ii) (over)expressed non-oxidative PPP enzymes and the L. plantarum AraA, AraB and AraD genes to enable l-arabinose metabolism; and (iii) had different genotypes with respect to l-arabinose transport (GAL2, PcaraT/gal2Δ and gal2Δ in strains IMX660, IMX869 and IMX844, respectively). Since these ‘arabinose specialist strains’ cannot grow on d-glucose, the impact of the presence of d-glucose on l-arabinose metabolism can be directly measured via its effect on growth. As anticipated, strain IMX844 (gal2Δ) was unable to grow on synthetic medium supplemented with either 20 g L⁻¹ l-arabinose or a mix of 20 g L⁻¹ of each, l-arabinose and d-glucose. In contrast, the l-arabinose specialist strains IMX660 (GAL2) and IMX869 (PcaraT/gal2Δ) grew on synthetic medium with l-arabinose as the sole carbon source at specific growth rates of 0.240 ± 0.001 and 0.099 ± 0.001 h⁻¹, respectively (Fig. 2a). However, when 20 g L⁻¹ d-glucose was added to the l-arabinose medium, strain IMX660 (GAL2) did not show growth during a 120-h batch cultivation experiment (Fig. 2b), whilst strain IMX869 (PcaraT/gal2Δ) grew at 60% of the specific growth rate observed in the absence of d-glucose (µ = 0.057 ± 0.003 h⁻¹ versus 0.099 ± 0.001 h⁻¹, Fig. 2b). This result indicated that expression of PcAraT in strain IMX869 enabled uptake of l-arabinose in the presence of d-glucose.

Low residual substrate concentrations in chemostat cultures confirm high-affinity l-arabinose transport kinetics of PcAraT

In duplicate steady-state chemostat cultures, the biomass-specific l-arabinose consumption rate of strain IMX1508 (PcaraT) was approximately 14% higher than the one of strain IMX929 (GAL2; 0.8 ± 0.1 and 0.7 ± 0.1 mmol g⁻¹ h⁻¹), reflecting the slightly lower biomass yield of the former strain. This difference in biomass yield is close to the difference of 8.1% that, based on published estimates of the P/O ratio and proton stoichiometry of the plasma membrane ATPase in aerobic S. cerevisiae cultures (both close to 1.0, [63, 64]), would be expected if l-arabinose uptake via PcAraT occurred via symport with a single proton.