N-Acetylglucosamine Utilization by Saccharomyces cerevisiae Based on Expression of Candida albicans NAG Genes

Strains and media. C. albicans BWP17 and S. cerevisiae BY strains were used (Table 1). Cells were grown either in rich medium (yeast extract-peptone-dextrose [YPD]; 2% peptone, 2% glucose, 1% yeast extract) or in minimal medium at 30°C: complete synthetic medium (CSM); 2% glucose, 6.7 g/liter yeast nitrogen base [YNB] with ammonium sulfate and without amino acids, 0.79 g/liter CSM) or synthetic defined medium (2% glucose, 6.7 g/liter YNB, with ammonium sulfate and without amino acids). Minimal media were supplemented with the strain-specific requirements for amino acids.

Regulation of gene expression was done using the MET3 promoter. Activation of MET3p-controlled gene expression was achieved by growing cells in media lacking methionine and cysteine as described previously (7). Minimal media for GlcNAc utilization contained 6.7 g/liter YNB with ammonium sulfate without amino acids, 0.1% to 10% GlcNAc and 2 g/liter asparagine as nitrogen sources, and glutamate and other amino acids to complement auxotrophies. Addition of G418 (200 μg/ml) was used for plasmid selection in S. cerevisiae. For plasmid construction and amplification Escherichia coli DH5α served as a host.

Disruption of ScGNA1.Since ScGNA1 encodes an essential gene, the EUROSCARF collection does not contain a deletion strain for this gene. We used PCR-based gene targeting to disrupt GNA1 in a strain that harbors the NAG3 or NAG4 and the NAG5 genes. Transformants were generated using NATMX2 as a selectable marker gene, which confers resistance to the antibiotic nourseothricin (cloNAT; used at 200 μg/ml). Correct integration was verified by PCR. Primers were obtained from biomers.net and are listed in Table 2 .

Molecular biology techniques.Standard molecular tools were used (24). Yeast transformation was done using the lithium acetate method (10). Bacterial transformation was done by electroporation. Plasmids were purified according to the manufacturers' instructions (Promega or Qiagen). DNA sequencing was performed by MWG-Biotech AG (Ebersberg, Germany). PCRs were performed using standard programs, e.g., 95°C for 5 min, 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 2 min, and 72°C for 5 min. Taq polymerase was used. All enzymes were supplied by New England Biolabs.

Plasmid constructs.A divergent promoter construct harboring the Ashbya gossypii MET3 promoter and the Candida maltosa LEU2 promoter was constructed using pFA vector plasmids (25). To this end pFA-NATMX3-AgMET3p was cleaved with SmaI/BglII to eliminate NATMX3. pFA-CmLEU2 was cleaved with BamHI/PmeI to yield CmLEU2, which was inserted into the SmaI/BglII sites of the former plasmid.

The C. albicans NAG3 and NAG4 open reading frames (ORFs) were amplified with short terminator regions from genomic DNA and cloned as 1,767-bp and 1,906-bp fragments, respectively, into pRS-ScMET3prom or pRS-AgTEFprom (7) to generate plasmids 804 (pRS-ScMET3p-NAG3; 7,608 bp) and 805 (pRS-ScMET3p-NAG4; 7,747 bp) and plasmids C258 (pRS-AgTEFp-NAG3; 7,448 bp) and C259 (pRSAgTEFp-NAG4; 7,587 bp), respectively. Plasmid 805 contains only the XhoI site, and plasmid C259 contains an EcoRI site instead (see Fig. 2, below).

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FIG. 2.

Plasmid constructs for expression of C. albicans NAG genes in S. cerevisiae. We used the pRS vector series as a plasmid backbone. Thus, all plasmids also harbored the CEN6/ARSH4 cassette for replication and plasmid distribution in S. cerevisiae. Expression of the NAG3 and NAG4 genes was achieved by using either the A. gossypii TEF or MET3 promoters as indicated. For construction details see Materials and Methods.

The C. albicans NAG5 ORF was amplified from genomic DNA and cloned via yeast in vivo recombination into pRS415-kanMX. In this way the kanR ORF was replaced with the CaNAG5 ORF, placing CaNAG5 under the control of the A. gossypii TEF promoter-generating plasmid, plasmid 363 (8,097 bp).

The C. albicans NAG1 and NAG2 ORFs with their terminators were amplified from genomic DNA and cloned as 916-bp BglII/XhoI and 1,392-bp BamHI/SacII fragments into pRS415. In vivo recombination via yeast was used to fuse the NAG genes with the divergent AgMET3prom-CmLEU2prom. This placed NAG1 under the control of the Candida maltosa LEU2 promoter and NAG2 under the control of the A. gossypii MET3 promoter. This cassette was then cloned next to CaNAG5 cleaved with SmaI/XhoI as a PmeI/XhoI fragment, generating plasmid C236 (pRS415-NAG1-NAG2-NAG5; 10,846 bp).

Fermentations.Fermentations were done in bench-scale tall-tube reactors containing 90 ml minimal medium 1.7 g/liter YNB without (NH₄)₂SO₄, 2 g/liter asparagine, glutamate, histidine, and uracil, 0.5 g/liter MgSO₄, 0.5 g/liter KH₂PO₄, 1 g/liter K₂HPO₄ containing 10% GlcNAc, with or without glucose (5 g/liter). G418 was added to ensure plasmid maintenance when necessary. An enzymatic ethanol assay (K-ETOH; Megazyme) was used to measure the ethanol content of the cultures. Ethanol was measured regularly over 2 weeks according to the manufacturer's protocol.

GlcNAc metabolism in S. cerevisiae and C. albicans.GlcNAc serves several purposes in fungal cells. It is used as UDP-N-acetyl-d-glucosamine in providing GlcNAc molecules for N-linked glycosylation, in GPI-anchored proteins and, of course, as a building block for chitin. In S. cerevisiae chitin is a component of the cell wall and is found enriched at the bud neck, forming the bud scars. UDP-GlcNAc is synthesized by way of four consecutive steps from fructose-6-phosphate. The four corresponding genes that take part in this process, GFA1, GNA1, PCM1/AGM1, and QRI1/UAP1, are all essential and conserved (Fig. 1). S. cerevisiae lacks genes for chitin catabolism. However, they can be found in various other organisms, including C. albicans. S. cerevisiae conceptually lacks four enzymatic activities for the degradation of chitin, which are a GlcNAc permease/transporter, a GlcNAc kinase, a deacetylase, and a deaminase (Fig. 1). Interestingly, genes involved in chitin catabolism are clustered within a 10-kb fragment in C. albicans. Although several of the genes in that cluster have been characterized in C. albicans (see reference 3), the functions of the putative NAG3 and NAG4 transporters have not been analyzed in detail. Nevertheless, we hypothesized that within this cluster all genes necessary for the utilization of GlcNAc are present, and we chose to express these genes in S. cerevisiae.

C. albicans NAG3 or -4 and NAG5 can complement the loss of S. cerevisiae GNA1.The CaNAG3 and CaNAG4 open reading frames coding for putative GlcNAc transporters were placed under the control of the ScMET3 promoter. The CaNAG5 ORF, encoding a GlcNAc kinase, was put under the control of the Ashbya gossypii TEF promoter (Fig. 2). This was done to ensure the expression of the ORFs in S. cerevisiae, since the C. albicans genes are either constitutively expressed or induced upon growth in GlcNAc-containing medium. The corresponding Nag3p, Nag4p, and Nag5p should provide the S. cerevisiae cells with GlcNAc-6-phosphate, which is also the product of Gna1p (Fig. 1). Since we were unable to delete GNA1 in a haploid BY4742 background, as expected, we first transformed the NAG3- or 4- and the NAG5-containing plasmids into BY4742 and subsequently went on to delete ScGNA1 in this strain. We were able to delete ScGNA1 in strains carrying either NAG3 or NAG4 and NAG5. However, the gna1 deletion strain became dependent on the addition of GlcNAc to the growth medium and failed to grow on glucose-based media (Fig. 3). These results suggested that both Nag3p and Nag4p are functionally redundant as GlcNAc transporters and confirmed the function of Nag5p as a GlcNAc kinase. Upon nonselective growth in the presence of GlcNAc, both plasmids were maintained. This suggests that S. cerevisiae encodes/expresses neither GlcNAc transporter nor GlcNAc kinase activities. On the other hand, this result confirmed the successful expression of two of the four enzymatic activities required for chitin catabolism in S. cerevisiae. Furthermore, we noticed that strains expressing NAG3 or -4 and NAG5 became sensitive to elevated concentrations of GlcNAc and were unable to grow on solid or in liquid media containing >10 mM GlcNAc (data not shown, but see Fig. 4, below).

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FIG. 3.

Expression of NAG3 or -4 and NAG5 in S. cerevisiae can functionally replace ScGNA1. Transformants of the respective strains were streaked onto CSM minimal medium containing glucose and relevant amino acids and G418 for plasmid selection. The plate in the upper panel was supplemented with 0.5 g/liter (2 mM) GlcNAc, while the plate in the lower panel was without GlcNAc. The plates were incubated for 4 days at 30°C prior to photography. The parental strain (BY4742) grew under both conditions.

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FIG. 4.

Relief of GlcNAc sensitivity in S. cerevisiae by expression of the four NAG genes. Strains harboring the indicated genes on freely replicating plasmids were grown on CSM minimal medium supplemented with 12 mM GlcNAc. Note that strains harboring only a GlcNAc transporter (NAG3 or NAG4 when either MET3 promoter driven or TEF promoter driven) or a GlcNAc kinase (NAG5) were not sensitive to GlcNAc; strains expressing only a GlcNAc transporter and a GlcNAc kinase (NAG3 and NAG5 or NAG4 and NAG5) became GlcNAc sensitive at the concentration of GlcNAc used, a sensitivity that was repressed in a strain that encodes all four NAG genes. Plates were incubated for 4 days at 30°C and then photographed.

Coexpression of NAG1 and NAG2 suppresses GlcNAc sensitivity of S. cerevisiae strains carrying NAG3 or -4 and NAG5.To express CaNAG1 and CaNAG2 in S. cerevisiae we cloned these ORFs in the CaNAG5-containing plasmid. Here, CaNAG1 was placed under the control of the Candida maltosa LEU2 promoter, and CaNAG2 was placed under the control of the A. gossypii MET3 promoter (Fig. 2). This plasmid, carrying NAG1, NAG2, and NAG5, was then cotransformed into S. cerevisiae BY4742 together with a NAG4-containing plasmid. We observed that S. cerevisiae strains carrying the NAG3 or -4 and NAG5 genes were sensitive to >10 mM GlcNAc in the medium and failed to proliferate. The expression of NAG1 and NAG2 (together with NAG3 and NAG5) should provide the catabolic protein set that is needed to convert GlcNAc-6-phosphate into fructose-6-phosphate. We hypothesized that this should decrease the pool of GlcNAc-6-phosphate in the cells and suppress the GlcNAc sensitivity. All transformants were obtained on glucose media. Upon transfer to selective plates containing 12 mM GlcNAc (2.65 g/liter), strains carrying only NAG3, NAG4, or NAG5 were able to grow. Thus, the expression of only a GlcNAc transporter or a GlcNAc kinase did not result in GlcNAc sensitivity. This indicates that S. cerevisiae lacks GlcNAc transporter and GlcNAc kinase functions under these conditions. Upon expression of both a GlcNAc transporter and a GlcNAc kinase, cells became sensitive to elevated levels of GlcNAc. Notably, strains expressing all four NAG genes were able to grow in the presence of 12 mM GlcNAc, demonstrating the functional expression of these four genes in S. cerevisiae (Fig. 4).

Utilization of GlcNAc as the sole carbon source by S. cerevisiae.Evident functional expression of the four NAG genes in S. cerevisiae suggested that expression would be sufficient to support growth of S. cerevisiae on minimal media containing GlcNAc as the sole carbon source. To test this, S. cerevisiae was grown in both liquid and solid minimal media containing 2% GlcNAc but no glucose. In both cases we observed that expression of the four NAG genes was sufficient to sustain growth of S. cerevisiae on GlcNAc as the carbon source (Fig. 5). Growth, however, was slower than that of wild-type C. albicans on these media.

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FIG. 5.

Utilization of GlcNAc as the sole carbon source in S. cerevisiae. The indicated strains were streaked onto minimal medium plates containing either 2% glucose or 2% (approximately 8 mM) GlcNAc as the sole carbon source or were grown in liquid minimal medium containing 2% GlcNAc as the sole carbon source. Plates were incubated for 4 days at 30°C and then photographed (A); liquid cultures were grown for 4 days at 30°C, centrifuged, and photographed (B).

Ethanol production based on GlcNAc.Uptake of GlcNAc and its catabolism generates fructose-6-phosphate, which can be used as a fermentable carbon source. Therefore, we sought to determine whether growth on GlcNAc also confers the ability to ferment GlcNAc and produce ethanol with this S. cerevisiae strain that expressed the four NAG genes. To this end we used S. cerevisiae BY4743 as a control strain and compared it to a BY4743 strain that was cotransformed with the NAG gene-containing plasmids. The strains harboring the NAG genes were able to grow in GlcNAc medium as described above and also fermented GlcNAc, as evidenced by the ethanol production. The ethanol yield obtained after 11 days of fermentation was low, at around 3 g/liter, and rather slow, as it continued for up to 2 weeks (Fig. 6).

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FIG. 6.

Fermentation of GlcNAc by S. cerevisiae. Ethanol production by S. cerevisiae strains utilizing GlcNAc was monitored over 2 weeks and is plotted in grams/liter, including standard deviations. Two independent strains were tested in multiple experiments.