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Isoprenoid Diphosphate Concentration in Yeast saccharomyces cerevisiae

Isoprenoid Diphosphate Concentration in Yeast saccharomyces cerevisiae


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Isoprenoid Diphosphate(IPP) is an important metabolites which is precursor in lot of secondary metabolites like Dolichol diphosphate, ubiquinone, prenylated proteins and carotenoid (not synthesized in s cerevisiae).

What is the average concentration of IPP present in yeast per gm ?


Although this information doesn't provide a direct answer to your question, I hope that it sets the scene for what is achievable in metabolic engineering from IPP. It should also provide a jumping off point for further literature research.

This is a fairly recent review of metabolic engineering of relevant pathways in various microbial systems, including Saccaharmoyes:

Ajikumar PK et al. (2008) Terpenoids: Opportunities for Biosynthesis of Natural Product Drugs Using Engineered Microorganisms. Molecular Pharmaceutics 5:167-190

The review refers to:

Ro, D. et al. (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940-943.

In this work, the introduction of a pathway-specific gene led to the production of 4.4 mg L-1 of product. This was increased to 153 mg L-1 by several manipulations including:

Increasing flux through the pathway by expressing a modified HMG CoA reductase

Decreasing flux into sterol synthesis by downregulation of the ERG9 gene combined with expression of a dominant negative allele of the transcription factor Upc2.

UPDATE

After posting my answer I found this paper:

Huang, B. et al. (2011) Metabolite target analysis of isoprenoid pathway in Saccharomyces cerevisiae in response to genetic modification by GC-SIM-MS coupled with chemometrics. Metabolomics 7:134-146

The paper includes a detailed discussion of methodologies, which I won't go into here.

Although there are no measurements of IPP, in Table 7 they present this result: geranyl pyrophosphate 32 ng ml-1 in a 48 h culture, A600 ~ 2.3. ERG9 disruption increased this to 56 ng ml-1

As far as I can tell these values are presented as ng ml-1 original culture. Using 1 U of OD600 corresponds to 0.41 g of dry cells liter−1 taken from here I calculate a value for GPP of 34 µg g-1 dry weight in the wild-type strain.


High-level recombinant production of squalene using selected Saccharomyces cerevisiae strains

For recombinant production of squalene, which is a triterpenoid compound with increasing industrial applications, in microorganisms generally recognized as safe, we screened Saccharomyces cerevisiae strains to determine their suitability. A strong strain dependence was observed in squalene productivity among Saccharomyces cerevisiae strains upon overexpression of genes important for isoprenoid biosynthesis. In particular, a high level of squalene production (400 ± 45 mg/L) was obtained in shake flasks with the Y2805 strain overexpressing genes encoding a bacterial farnesyl diphosphate synthase (ispA) and a truncated form of hydroxyl-3-methylglutaryl-CoA reductase (tHMG1). Partial inhibition of squalene epoxidase by terbinafine further increased squalene production by up to 1.9-fold (756 ± 36 mg/L). Furthermore, squalene production of 2011 ± 75 or 1026 ± 37 mg/L was obtained from 5-L fed-batch fermentations in the presence or absence of terbinafine supplementation, respectively. These results suggest that the Y2805 strain has potential as a new alternative source of squalene production.

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ISOPRENOID BIOSYNTHESIS

FIG 2 Metabolic routes for the production of isoprenoid-based biofuels in a living system. The mevalonate (MVA) pathway is from acetyl-CoA, and the deoxyxylulose 5-phosphate (DXP) pathway is from glyceraldehyde-3-phosphate and pyruvate. IPP, isoprenyl diphosphate DMAPP, dimethylallyl diphosphate IDI, isoprenyl diphosphate isomerase nudiX, nucleoside diphosphate hydrolases GPP, geranyl pyrophosphate FPP, farnesyl pyrophosphate GPPS, geranyl pyrophosphate synthase FPPS, farnesyl pyrophosphate synthase PS, pinene synthase FS, farnesene synthase BS, bisabolene synthase PPS, endogenous pyrophosphatase.

Downstream isoprenoid-based biofuel products.

FIG 3 Pathway optimization for the enhanced production of isoprenoid-based biofuels from prokaryotic hosts. Open and filled arrows indicate coexpression and fusion protein expression, respectively. Genes in uppercase letters are heterologous, whereas genes in lowercase letters are endogenous/homologous. The solid-line box shows conventional upstream pathways (MVA and DXP pathways), whereas the dashed-line box shows novel pathways via decoupling of the conventional MVA pathway. atoB, acetyl-CoA acetyltransferase gene HMGS, hydroxymethylglutaryl-CoA synthase HMGR, hydroxymethylglutaryl-CoA reductase MK, mevalonate kinase PMK, phosphomevalonate kinase PMD, mevalonate diphosphate decarboxylase dxs, deoxyxylulose-5-phosphate synthase gene ispC, deoxyxylulose-5-phosphate reductoisomerase gene ispD, 2-C-methylerythritol-4-phosphate cytidyltransferase gene ispF, 2-C-methylerythritol-2,4-cyclodiphosphate synthase gene ispG, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene IPP, isoprenyl diphosphate DMAPP, dimethylallyl diphosphate idi/IDI, isoprenyl diphosphate isomerase nudF, B. subtilis ADP ribose pyrophosphatase gene nudiX, nucleoside diphosphate hydrolases ispA, farnesyl pyrophosphate synthase (FPPS) gene GPPS, geranyl pyrophosphate synthase PS, pinene synthase AFS, farnesene synthase BIS, bisabolene synthase OLETJE, Jeotgalicoccus sp. fatty acid decarboxylase.

Results

Over-expression of SRT1 suppresses the growth and glycosylation defects of rer2

Srt1p exhibits 30% identity with Rer2p in amino acid sequence. Structurally, Srt1p (343 amino acids) is larger than Rer2p (286 amino acids) and has an extra stretch of about 30 amino acids at the N-terminus, which is relatively hydrophobic. The disruption of RER2 results in a severe growth defect, whereas the disruption of SRT1 causes no obvious growth phenotype ( Sato et al. 1999 ). On the other hand, the over-expression of SRT1 was able to recover the growth defect of Δrer2 almost to wild-type levels (Fig. 1A). The double mutant of Δrer2 Δsrt1 is inviable ( Sato et al. 1999 ). These genetic observations suggest that Rer2p and Srt1p share redundant functions, but that Rer2p provides the major activity required for cell growth. We also tested whether the glycosylation defect of Δrer2 was remedied by SRT1. Total cell lysates were prepared and the glycosylation state of carboxypeptidase Y (CPY), a vacuolar glycoprotein ( Stevens et al. 1982 ), was analysed by immunoblotting with an anti-CPY antibody. As shown in Fig. 1B, the underglycosylation phenotype of Δrer2 was almost completely restored by SRT1 on a multicopy plasmid. SRT1 on a single-copy plasmid was not enough to suppress the defect of Δrer2 to wild-type levels. These results strongly suggest that Srt1p possesses the cis-prenyltransferase activity by itself.

Over-expression of SRT1 suppresses the Δrer2 mutant. (A) Growth phenotype of Δrer2 suppressed by SRT1. Wild-type (YPH500, closed diamonds), Δsrt1 (SMY30, shaded squares), Δrer2 carrying RER2 on a single-copy plasmid (SMY20-2, open triangles), Δrer2 carrying SRT1 on a multicopy plasmid (SMY20-3, open diamonds) and rer2-2 (SNH23-7D, open circles) were inoculated into MCD medium and cultured at 30 °C. OD600 was measured and plotted at each time point. (B) SRT1 suppresses the glycosylation defect of Δrer2. The glycosylation state of carboxypeptidase Y was analysed by immunoblotting of total cell lysates of the early logarithmic phase. Lane 1, Δrer2 expressing RER2 on a single-copy plasmid (SMY20-2) lane 2 Δrer2 expressing SRT1 on a single-copy plasmid (SMY20-4) lane 3, Δrer2 expressing SRT1 on a multicopy plasmid (SMY20-3) lane 4, Δrer2 (SMY20) harbouring vector only lane 5, Δsrt1 (SMY30). m, mature CPY ug, underglycosylated CPY.

Srt1p has cis-prenyltransferase activity

To biochemically prove the cis-prenyltransferase activity of Srt1p, we first tried to purify Srt1p using E. coli expression systems or directly from yeast lysates, but neither method was successful. Therefore, we used the membrane fraction of the Δrer2 cells expressing SRT1 under the authentic promoter on a multicopy plasmid (referred to as the SRT1-dependent cells) as the enzyme source of Srt1p. For a comparison, the membrane fractions were also prepared from the wild-type, rer2 and Δsrt1. Initially, the membrane fractions were prepared from cells in the early logarithmic phase (1.2∼1.7 × 10 7 cells/mL) and assayed for cis-prenyltanrsferase activity with FPP and [1– 14 C]-IPP as substrates. However, the activity only increased marginally in the SRT1-dependent cells as compared with the rer2 mutant (Fig. 2). In consideration of a possibility that some factors in the soluble fraction are necessary for the full activity of Srt1p, synthesis of dolichyl compounds was also tested using the total cell lysate. Even in this experiment, the activity of dolichol synthesis was still very low in the SRT1-dependent cells (data not shown). As described later, we found that the expression level of Srt1p changes with the progress of the growth phase and increases in the late-logarithmic and stationary phases. Accordingly, the membrane fractions were re-prepared from cells in the stationary phase (1.2 × 10 8 cells/mL) and assayed. Under this condition, the high activity of cis-prenyltransferase was detected in the SRT1-dependent cells (Fig. 2). These results indicate that Srt1p by itself possesses the cis-prenyltransferase activity and that it is up-regulated in the stationary phase. The deletion of SRT1 alone did not affect the cellular activity, indicating that the genomic copy of SRT1 has only a minor contribution to total cis-prenyltransferase activity under normal conditions.

Srt1p has cis-prenyltransferase activity. Membrane fractions were prepared from cells of the wild-type (YPH500), Δsrt1 (SMY30), Δrer2 harbouring RER2 on a single-copy plasmid (SMY20-2), Δrer2 harbouring SRT1 on a multicopy plasmid (SMY20-3), and rer2-2 (SNH23-7D) strains in the early logarithmic or stationary phase and assayed for cis-prenyltransferase activity in vitro.

The chain length of these reaction products was also assessed. The polyprenyl products of the in vitro cis-prenyltransferase assay were further treated with phosphatase and subjected to reversed-phase thin-layer chromatography followed by fluorography (Fig. 3). Interestingly, the main products synthesized by the SRT1-dependent cells were longer by two isoprene units than those of the Δsrt1 (the RER2-dependent) or wild-type cells. This result indicates that cis-prenyltransferase determines the chain length of dolichol and that Rer2p and Srt1p have different properties to terminate the isopentenyl condensation reaction.

Analysis of polyprenols synthesized by Rer2p and Srt1p in vitro. Products of the cis-prenyltransferase assay were treated with phosphatase and analysed by reversed-phase thin-layer charomatography with an LKC-18 plate and an acetone : water (39 : 1) solvent system. WT, wild-type (YPH500) RER2, Δsrt1 (SMY30) SRT1, Δrer2 harbouring SRT1 on a multicopy plasmid (SMY20-3).

Quantification of dolichol and Dol-P

In addition to the enzymatic activities in vitro, the cellular contents of dolichols and Dol-Ps were quantified for the mutant cells. Dolichol and Dol-P were extracted sequentially from cells in the middle-logarithmic phase (4∼5 × 10 7 cells/mL) as described in Experimental procedures. Dolichol and Dol-P fractions were analysed by reversed-phase HPLC, which gives a separation of individual compounds according to chain length. Figure 4 shows results from the analysis of the dolichol fraction. Total contents of dolichols and Dol-Ps are summarized in Fig. 5. Wild-type contained almost equal amounts of dolichols and Dol-Ps, and C75 and C80 were the main constituents in both fractions. This result is consistent with a previous report for the wild-type yeast ( Adair & Cafmeyer 1987 ). The dolichol content of the rer2 mutant was decreased to about 25% of that of wild-type. In the rer2 mutant, small peaks due to dolichols of longer chain-length became obvious, which were not observed in the wild-type cells. Srt1p may be somewhat up-regulated in rer2. The level of Dol-Ps in rer2 was too low to estimate by this method. In the SRT1-dependent cells, the HPLC pattern of the dolichol fraction changed markedly each peak was a doublet. There is a report that this HPLC condition provides a partial separation of α-unsaturated (polyprenol) and α-saturated (dolichol) components with the same length and polyprenol is eluted slightly faster than dolichol ( Ekstrom et al. 1984 ). We confirmed this by using the standard compounds C95-polyprenol and C95-dolichol. The retention times of these standards coincided with those of one set of peaks in the SRT1-dependent cells, as indicated in Fig. 4D. Therefore, we concluded that the split peak corresponded to polyprenol and dolichol with the same length and that the SRT1-dependent cells contained a significant amount of polyprenols, which was hardly detected in the wild-type cells. The total amount of dolichols and polyprenols in the SRT1-dependent cells was about twice that of the wild-type, although 30% of it was in the unusual α-unsaturated form. Consistent with the in vitro analysis, the main peaks of dolichols (and polyprenols) in the SRT1-dependent cells were C95–C105, which were apparently longer than those observed in the wild-type cells (C75-C80). The unsaturated form was observed for every chain-length, suggesting that there is no clear preference for length in the reduction reaction. Despite the fact that the SRT1-dependent cells contained a sufficient amount of dolichols, the level of Dol-Ps was extremely low (13% of wild-type). We could not detect phosphorylated polyprenols, but this may be simply due to the overall low level of phosphorylated forms. The distribution of Dol-Ps in terms of chain length was similar to that of free dolichols and polyprenols.

Reversed-phase HPLC analysis of dolichol fractions. Dolichol fractions extracted from cells of the wild-type (YPH500, A), Δsrt1 (SMY30, B), rer2-2 (SNH23-7D, C), and Δrer2 harbouring SRT1 on a multicopy plasmid (SMY20-3, D) were analysed with an ODS column and 2-propanol : methanol (3 : 2) as eluent. The elution positions of C95-polyprenol and C95-dolichol are indicated in (C) and (D).

Contents of dolichols and Dol-Ps in mutant strains. Dolichol and Dol-P fractions were extracted from cells of the wild-type (YPH500), Δsrt1 (SMY30), rer2-2 (SNH23-7D) and Δrer2 harbouring SRT1 on a multicopy plasmid (SMY20-3) and quantified as described in Experimental procedures. Average values (µg/g [wet weight] of cells) of two independent experiments are shown. n.d., not detectable.

Level of Rer2p and Srt1p changes along the growth stage

To detect the Srt1 protein, a 3HA-tag was introduced at the C-terminus of Srt1p. This Srt1-3HAp was completely functional, because the SRT1-3HA gene suppressed the temperature sensitivity of the rer2 mutant as well as the authentic SRT1 (data not shown). SRT1-3HA was expressed in Δsrt1 cells on a single-copy or multicopy plasmid, and their products were analysed by immunoblotting with the anti-HA monoclonal antibody (16B12). Srt1-3HAp was detected as a single band around 45 kDa in a dose-dependent manner (data not shown). In the course of these experiments, we noticed that the amount of Srt1-3HAp increased in the late-logarithmic phase. So, the level of Srt1p and Rer2p was carefully analysed through the growth phase using the Δrer2 Δsrt1 cells that were expressing the 3HA-taged SRT1 and RER2 genes under their own promoters (SMY241). The 3HA-RER2 gene was also functional, as reported previously ( Sato et al. 1999 ). Total lysates were prepared at each point and 60 µg of protein was subjected to immunoblotting (Fig. 6). The amount of Srt1-3HAp gradually increased as the growth phase progressed, and reached a maximum in the late-logarithmic and stationary phases. In contrast, the highest level of 3HA-Rer2p was detected in the early logarithmic phase. Thus, the expression of two cis-prenyltransfrerases is differently regulated during the yeast life cycle.

Expression levels of RER2 and SRT1 vary over growth phases. Total cell lysates (60 µg) were prepared from Δrer2 Δsrt1 cells expressing 3HA-RER2 and SRT1-3HA on single-copy plasmids (SMY241) at the time points indicated by arrows in (A) and analysed by immunoblotting using the anti-HA monoclonal antibody (B).

Srt1p is localized to lipid particles

A subcellular fractionation experiment using the Δsrt1 cells harbouring SRT1-3HA on a single-copy plasmid revealed that almost all the Srt1-3HAp was recovered in the membrane fraction, similarly to 3HA-Rer2p (data not shown). The subcellular localization of Srt1p was further assessed by indirect immunofluorescence microscopy. The Δsrt1 cells expressing SRT1-3HA on a single-copy plasmid were grown to stationary phase and subjected to immunofluorescence observation with the anti-HA antibody. Srt1-3HAp showed punctate and ring-like structures, as shown in Fig. 7A–C. These structures exhibited a round shape, which were unlikely to be a typical pattern of the Golgi. This staining pattern is rather similar to that of lipid particles which accumulate in the stationary phase in yeast. Together with the fact that Srt1p is increased in the stationary phase, these results led us to speculate that Srt1-3HAp is localized to lipid particles. To evaluate this possibility, we performed a double immunofluorescence of Srt1–3HAp and sterol Δ 24 -methytransferase Erg6p, a marker protein of lipid particles in yeast ( Leber et al. 1998 ). To detect Erg6p, 3myc-tagged ERG6 was constructed, which could complement the cycloheximide sensitivity of Δerg6 completely. The staining of Srt1-3HAp overlapped very well with that of Erg6-3mycp (Fig. 7D–F). When the cells were collected at the early logarithmic phase, the staining of Srt1–3HAp was too weak to detect the particular localization.

Subcellular localization of Srt1-3HAp. (A) Immunofluorescence of Srt1-3HAp. Δsrt1 (SMY30) cells harbouring SRT1-3HA on a single-copy plasmid were grown to the stationary phase and subjected to indirect immunofluorescence observation with the anti-HA monoclonal antibody. (B) DAPI staining of the same field. (C) Nomarski image of the same field. (D, E) Double staining of Srt1-3HAp and Erg6-3mycp. Δsrt1 (SMY30) cells harbouring SRT1-3HA and ERG6-3myc on single-copy plasmids were grown to the stationary phase and observed by indirect immunofluorescence microscopy with anti-HA monoclonal (D) and anti-myc polyclonal (E) antibodies. (F) Nomarski image of the same field.

The localization of Srt1p was also examined using a fusion protein between GFP and Srt1p. The expression of GFP-SRT1 by the authentic SRT1 promoter was able to suppress the rer2 mutant, however, the level of GFP-Srt1p was not enough to observe the signal of GFP (data not shown). Therefore, GFP-SRT1 was over-expressed under the strong TDH3 promoter in Δsrt1 and observed in living cells. GFP-Srt1p showed punctate structures that were similar to the immunofluorescence of Srt1–3HAp (Fig. 8C). Such a punctate pattern was observed throughout the growth phase when GFP-SRT1 was over-expressed under the TDH3 promoter. We further compared the subcellular localization of Srt1p with that of Rer2p. We previously reported that 3HA-Rer2p was localized to punctate structures which seemed to overlap or closely associate with the ER when it was observed by immunofluorescence ( Sato et al. 1999 ). In this study we constructed a GFP fusion with Rer2p as well and confirmed that it is functional (data not shown). When GFP-RER2 was over-expressed under the TDH3 promoter in Δrer2, a continuous ER pattern and some dots associated with the ER were observed in the early logarithmic phase (Fig. 8A). In contrast, GFP-Srt1p did not show such a continuous ER pattern (Fig. 8C). The hydrophobic dye Nile Red, which is known to stain lipid particles ( Greenspan et al. 1985 ), gave a pattern which was very similar to GFP-Srt1p (Fig. 8E), supporting our conclusion that Srt1p resides mostly in lipid particles. It should also be noted that lipid particles were sometimes clearly visible in Nomarski images (Fig. 8E,F), again overlapping with GFP-Srt1p.

Subcellular localization of GFP-Srt1 and GFP-Rer2 fusion proteins. rer2-2 (SNH23-7D) expressing GFP-Rer2p (A and B) and Δsrt1 (SMY13) expressing GFP-Srt1p (C and D) were observed for GFP fluorescence. For comparison, wild-type cells (SNY9) were stained with Nile Red to visualize lipid particles (E and F). The GFP fusions were expressed under the TDH3 promoter on a single-copy plasmid. (A and C) GFP fluorescence. (E) Nile Red staining. (B, D and F) Nomarski images of the same field.


Isoprenoid Diphosphate Concentration in Yeast saccharomyces cerevisiae - Biology

Head: Professor Grażyna Palamarczyk

The overall focus of our work concentrates on basic studies concerning glycosylation and regulation of the cell wall glycoproteins. Two interrelated lines of research concern the role of protein glycosylation for the antifungal drug resistance and pathogenesis (lead by G.Palamarczyk) and the metabolic engineering of the Trichoderma sp., with the aim to improve biotechnological properties of the strains (lead by J.Kruszewska).

Molecular mechanisms in protein glycosylation in the yeast: Saccharomyces cerevisiae and Candida albicans

Group leader: Prof. Grażyna Palamarczyk
Staff: Dr. Anna Janik, Ph.D. students: M.Sc. Monika Pasikowska, M.Sc. Mateusz Juchimiuk

This project concerns the basic studies on glycosylation and regulation of the cell wall glycoproteins and their impact on the cell wall integrity. This area, although important, is still only in part understood even in geneticaly tractable microorganism such as S. cerevisiae, and, despite its potential importance in medical mycology, is unexplored in the human pathogenic fungi such as C. albicans.

The vast majority of eukaryotes (fungi, plants, animals) synthesise asparagine (Asn) linked N-glycans by means of a lipid precursor dolichyl-phosphate linked oligosaccharide. The isoprenoid lipid, dolichyl phosphate, is also involved in the O-mannosylation pathway which is essential for the cell wall integrity maintenance.

We have demonstrated that the glycosylation defect resulting from the synthesis of dolichyl phosphate results in the alterations of the S. cerevisiae cell wall structure and composition (Orłowski et al.2007).

Subsequently we would like to characterize the key steps in dolichol biosynthesis and dolichol dependent glycosylation and establish how alterations in these reactions affect the cell wall and pathogeneity of C. albicans.

Such information will inform the field of host/ fungal pathogen interactions and may be exploitable by the antifungal drug industry.

To this end we have isolated the genes involved in dolichol, and dolichyl phosphate formation and demonstrated that impaired dolichol synthesis affects formation of the hyphal (virulent) forms of C. albicans (Orłowski 2008, PhD Thesis). Subsequently we plan to construct a set of glycosylation mutants and test their sensitivity to the antifungal drugs as well as virulency.

All together, this project will increase our fundamental understanding of the molecular and biochemical regulation of cell wall glycoprotein assembly in C. albicans with the aim of better understanding of fungal pathogenicity and selection of targets for antifungal therapy.

Research grants:
2004-2005 "Euro Cell Wall, Fungal cell wall as a target for antimicrobial therapy", (5FP UE)
2005-2008 "The cell wall assembly in S. cerevisiae and C. albicans as a target for the antifungal drugs",
(Ministry of Science and Higher Education)

Selected publications:

  1. Kuranda K., François J., Palamarczyk G. The isoprenoid pathway and transcriptional response to its inhibitors in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. (2009) - Epub ahead of print
  2. Kuranda K., Grabinska K., Berges T., Karst F., Leberre V., Sokol S., François J., Palamarczyk G. The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. (2009) 9(3): 381-90
  3. Orłowski J., Machula K., Janik A., Zdebska E., Palamarczyk G. Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions. Yeast (2007) 24(4): 239-52
  4. Kuranda K., Leberre V., Sokol S., Palamarczyk G., François J. Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. Mol Microbiol. (2006) 61(5): 1147-66
  5. Grabińska K., Sosińska G., Orłowski J., Swiezewska E., Berges T., Karst F., Palamarczyk G. Functional relationships between the Saccharomyces cerevisiae cis-prenyltransferases required for dolichol biosynthesis. Acta Biochim. Pol. (2005) 52(1): 221-32
  6. Kamińska J., Kwapisz M., Grabińska K., Orłowski J., Boguta M., Palamarczyk G., Zoładek T. Rsp5 ubiquitin ligase affects isoprenoid pathway and cell wall organization in S. cerevisiae. Acta Biochim Pol. (2005) 52(1): 207-20
  7. Grabińska K., Palamarczyk G. Dolichol biosynthesis in the yeast Saccharomyces cerevisiae: an insight into the regulatory role of farnesyl diphosphate synthase. FEMS Yeast Res. (2002) 2(3): 259-65
  8. Janik A., Sosnowska M., Kruszewska J., Krotkiewski H., Lehle L., Palamarczyk G. Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation. Biochim. Biophys. Acta. (2003) 1621(1): 22-30

Genetic engineering of fungi important in biotechnology and biocontrol

Group leader: Assoc. Prof. Joanna Kruszewska

Staff: Dr. Urszula Perlińska-Lenart Senior Assistant, Dr. Wioletta Górka-Nieć Senior Assistant, M.Sc. Patrycja Zembek Ph.D. student, M.Sc. Sebastian Graczyk Ph.D. student

We have studied the influence of changes in the activity of enzymes engaged in the glycosylation pathways on protein production and secretion.

Expression of the yeast DPM1 gene (coding for DPM-synthase) in T. reesei increased the intensity of protein glycosylation and secretion, and caused ultrastructural changes in the fungal cell wall. We have undertaken further biochemical and morphological characterization of the DPM1 expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increase in chitin content and changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble β-(1, 6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants.

It was observed in S. cerevisiae that disruption of PMT genes, coding for protein O-mannosyltransferases catalyzing the transfer of the first mannosyl residue to proteins during their O-glycosylation, resulted in cell wall alterations.

Disruption of the pmt1 gene in Trichoderma caused a significant decrease in the total activity of protein O-mannosyltransferases and led to osmotic sensitivity of the strain, indicating an essential role of the PMTI protein activity for cell wall synthesis. Disruption of the pmt1 gene decreased protein secretion but had no effect on glycosylation of secreted proteins, which suggests that PMTI protein O-mannosyltranferase does not take part in glycosylation of these proteins.

To study influence of higher activity of protein O-mannosyltransferases on protein production and secretion we integrated an additional copy of the pmt1 gene into the Trichoderma genome. This integration unexpectedly resulted in silencing of pmt1 expression. Strains carrying the additional copy of pmt1 gene exhibited lower total activity of protein O-mannosyltransferases, lower O- and N-glycosylation of secreted proteins and showed defects in their cell wall composition. At the same time, the strains grew slowly on solid medium and were hypersensitive to an antifungal reagent, Calcofluor white. These results indicate that protein O-manosyltransferases are required for proper cell wall formation, and their decreased activity influences not only O- but also N-glycosylation.

We also have cloned and analysed function of dpm2 and dpm3 genes coding for subunits of DPM synthase. It was found that apart from the catalytic subunit DPMI, also the DPMIII subunit was essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzyme activity. We also characterized S. cerevisiae strains expressing dpm1,2,3 or dpm1,3 genes and analyzed the consequences of dpm expression on protein O- and N-glycosylation in vivo and on the cell wall composition.

Research grants:

2009-2012 "Engineering of Trichoderma strains with enhanced biocontrol capacities", (Structural Funds, POIG,
Ośrodek Przetwarzania Informacji)

2006-2009 "Up-regulation of biocontrol capacities of Trichoderma", (Ministry of Science and Higher Education)

2005-2008 part of the project “The functional genomics of the model microorganisms in molecular studies of inherited
human diseases and in mechanism of pathogenesis”, (Ministry of Science and Higher Education)

Selected publications:

  1. Górka-Nieć W., Pniewski M., Kania A., Perlińska-Lenart U., Palamarczyk G., Kruszewska J.S. Disruption of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition. Acta Biochim.Pol. (2008) 55: 251-260
  2. Kruszewska J.S., Perlińska-Lenart U., Górka-Nieć W., Orłowski J., Zembek P. Palamarczyk G. Alterations in protein secretion caused by metabolic engineering of glycosylation pathways in fungi. Acta Biochim.Pol. (2008) 55: 447-456
  3. Górka-Nieć W., Bańkowska R, Palamarczyk G., Krotkiewski H., Kruszewska J.S. Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP mannose and cis-prenyltransferase activity. Biochim. Biophys. Acta (2007) 1770: 774–780
  4. Perlinska-Lenart U., Bankowska R., Palamarczyk G., Kruszewska J.S. Overexpression of the Saccharomyces cerevisiae RER2 gene in Trichoderma reesei affects dolichol dependent enzymes and protein glycosylation. Fungal Genet. Biol. (2006) 43(6): 422-9
  5. Perlińska-Lenart U., Orłowski J., Laudy A.E., Zdebska E., Palamarczyk G., Kruszewska J.S Glycoprotein hypersecretion alters cell wall in Trichoderma reesei strains expressing the Saccharomyces cerevisiae dolichylphosphate mannose synthase gene. Appl. Environ. Microbiol. (2006) 72: 7778-7784
  6. Perlińska-Lenart U., Kurzątkowski W., Janas P., Kopińska A, Palamarczyk G., Kruszewska J.S Protein production and secretion in an Aspergillus nidulans mutant impaired in glycosylation. Acta Biochim. Pol. (2005) 52: 195-205

Institute of Biochemistry and Biophysics Polish Academy of Sciences
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Enzyme Scaffolds

Protein scaffolds, where specific enzymes are recruited onto synthetic constructs, have been used to localize desired biosynthetic pathway enzymes to improve product formation via substrate channeling. Dueber et al. (2009) successfully demonstrated the use of synthetic scaffolds comprised of metazoan protein-protein interaction domains and ligands in Escherichia coli, increasing mevalonate production 77-fold. There were concerns about the efficiency of this approach for S. cerevisiae, since native proteins may interact with the scaffold domains (Siddiqui et al., 2012). However, the metazoan synthetic scaffold improved the production of resveratrol in S. cerevisiae by 5-fold relative to the non-scaffold system and by 2.7-fold relative to a protein fusion strategy (Zhang et al., 2006 Wang and Yu, 2012). Nonetheless, optimization of the system was still important depending on the stoichiometry of the SH3 and pDZ domains in the protein scaffold, the increase in resveratrol production varied from 1.2 to 5-fold over the control.

Another successful synthetic scaffold in S. cerevisiae is based on the cohesin and dockerin-enzyme interaction found in the surface cellulosomes of Clostridium cellulovorans and other microorganisms (Mechaly et al., 2001). Engineered cellusome-like complexes localizing cellulases have been successfully expressed on the surface of S. cerevisiae to improve ethanol production (Fan et al., 2012 Tsai et al., 2013 Tang et al., 2018). Recently, the “largest cellulolytic complex” was successfully constructed on the surface of the yeast Kluyveromyces marxianus (Anandharaj et al., 2020). However, utilization of the dockerin-cohesin interaction as an intracellular metabolic scaffold in yeast has been limited. Kim and Hahn (2014) took advantage of the high binding affinity between cohesion and dockerin (Kd 㰐 � M) (Stahl et al., 2012) to create a substrate channeling module in the cytosol of S. cerevisiae. Increasing numbers (2, 3, or 7) of cohesin domains were included on the scaffold and the C terminal dockerin fusion proteins of heterologous AlsS, AlsD, and endogenous Bdh1 proteins were overexpressed. 2,3-Butanediol production increased by 37% compared to the scaffold-free system in fed-batch fermentation with occasional glucose feeding although the increase was limited, this was the first study to utilize the interaction between cohesin and dockerin in the yeast cytosol and for metabolic engineering. In a follow-up study (Kim et al., 2016), this synthetic substrate channeling strategy was used to redirect carbon flux from pyruvate to 2,3-butanediol using a heterologous AlsS or to lactate using heterologous LdhA, rather than to ethanol. The native Pyk1 (for pyruvate synthesis) was fused to a cohesin domain and AlsS to a dockerin domain colocalizing the two enzymes, resulting in a 38% increase in 2,3-butanediol production and a 46% decrease in ethanol production compared to the native strain. However, this strategy was unsuccessful in redirecting the flux toward lactate formation the dockerin-fused LdhA had a Ϣ-fold lower specific activity relative to wildtype LdhA (11.6 and 27.9 U/nmol, respectively). As discussed in the previous section, the reduction of enzymatic activity presents a significant challenge to fusion-based colocalization strategies.

The dockerin-cohesin interaction was also utilized to colocalize the ethyl acetate biosynthesis enzymes onto the surface of lipid droplets (thus combining both scaffolding and organelle targeting strategies), resulting in 2-fold increase in the production rate (Lin et al., 2017). In S. cerevisiae, ethyl acetate is synthesized by the enzymes Ald6, Acs1, and Atf1, with only Atf1 targeted to the lipid droplets. To target Ald6 and Acs1, the authors screened and identified the protein oleosin as a promising candidate to direct enzymes of interest to the lipid droplets. Cohesin domains were fused to the oleosin proteins while the corresponding dockerin domains were fused to Ald6 and Acs1. After an extensive screening of promoters and scaffold optimization, the ethyl acetate specific titer increased 1.7-fold compared to the strain without scaffold.

An alternate scaffolding strategy is based on the interactions between affibodies and anti-idiotypic affibodies. The 58-residues affibodies (or Z-domains) are a class of non-immunoglobulin affinity proteins derived from Staphylococcus aureus protein A (Llom et al., 2010). They possess high specificity and binding affinity toward their target proteins (0.3 pM� μM) (Ståhl et al., 2017). In a recent study, Tippmann et al. (2017), utilized the ZTaq:anti-ZTaq (Kd = 0.7 μM) and ZIgA:anti-ZIgA (Kd = 0.9 μM) interactions to create a functional synthetic scaffold in S. cerevisiae. A scaffold linked anti-ZTaq and anti-ZIgA, and ZIgA and ZTaq were fused with farnesyl diphosphate synthase and farnesene synthase, respectively. By optimizing the amino acid linkers and the enzyme:scaffold ratio, a 135% increase in the yield of farnesene on glucose was achieved. This affibody scaffold was also functional in E. coli, in which its utilization resulted in a 7-fold increase in PHB production, demonstrating its versatility as a scaffold platform for metabolic engineering.

Scaffolds were also used to create an artificial Gal2-xylose isomerase complex to improve xylose utilization in S. cerevisiae (Thomik et al., 2017). In this study, WASP-homology 1 (WH1) from rat N-WASP was used as the scaffold construct and a xylose isomerase (XI) was fused with WH1 ligand (WH1L) at the N-terminus. To recruit the XI-WH1 scaffold complex to the Gal2 transmembrane protein, a pair of synthetic coiled-coil zippers with high binding affinity for one another was used: SYNZIP1 (SZ1) and SYNZIP2 (SZ2) (Reinke et al., 2010). In this configuration, Gal2 was fused with SZ2 at the N-terminus by either the flexible or helical linker, while the WH1 scaffold was used with SZ1 at the N-terminus by the flexible linker. The synthetic scaffold allowed the Gal2-xylose isomerase complex to form, enabled the yeast strain to uptake and process xylose, and resulted in higher ethanol/xylitol ratio, a proxy measurement for xylose consumption. Kang et al. (2019) used an alternate means to localize pathway enzymes into a multienzyme complex via RIDD and RIAD, short peptides with high binding affinity (KD = 1.2 nM), to improve lycopene production in S. cerevisiae. The two key enzymes Idi and CrtE, were tagged with RIAD and RIDD, respectively. The resulting complex yielded 2.3 g/L of lycopene after 144 h fermentation, 58% higher than the control strains.

In a recent study, Han et al. (2018) utilized Tya, a part of the Ty1 retrotransposon, to spatially recruit the key enzymes to improve farnesene and farnesol production in S. cerevisiae. Tya is a 49-kDa protein that self-assembles into a shell, similar to virus-like particles (VLPs) (Marchenko et al., 2003). Tya was fused to either the C-terminus or the N-terminus of three key isoprenoid enzymes: tHMG1 (a truncated form of HMG-CoA reductase from S. cerevisiae), IspA (from E. coli), and AFS1 (from Malus domestica) or DPP1 (from S. cerevisiae) to create synthetic metabolons to drive carbon flux toward farnesene or farnesol. Titer reached 930 ± 40 mg/L in the best-performing strain, a 3.1-fold increase relative to 300 ± 11 mg/L for free enzymes. The best-performing strain for farnesol production produced 882 ± 15 mg/L, 3.8-fold higher than the control strains expressing free enzymes (231 ± 14 mg/L).

With increased interest in scaffolds to channel substrates toward formation of desired products, additional protein interactions have been screened for their scaffolding potential. Curvature Thylakoid1A (CURT1A) protein, isolated from the thylakoid membrane of Arabidopsis thaliana, has been identified as a prospective membrane-bound scaffolding module (Behrendorff et al., 2019). CURT1A can form homo-oligomers in the membrane. By fusing fluorescence proteins to several variants of CURT1A, CURT1A fusion proteins were shown to scaffold onto the endoplasmic reticulum of S. cerevisiae, resulting in the fluorescence signal being localized to the membrane.


RESULTS AND DISCUSSION

Experimental design and methodology.

Following the complete sequence determination of the S. cerevisiae genome, Affymetrix DNA microarrays have emerged as a powerful tool for examining the simultaneous expression pattern of more than 6,000 yeast genes (10, 14, 21, 33, 52). Because of the scope of each data set obtained from a microarray hybridization, treatments used in this study were limited to a single concentration at a single time point. Rich medium (YPD) was used so as not to limit the growth rate, and one doubling time (90 min) was selected for the duration of exposure to agent at 0.5 times the MIC. Cells were exposed to agents and harvested during the mid-logarithmic-growth phase.

RNA was prepared following exposure of the parent strain, BY4743, to one of five previously characterized azoles (clotrimazole, fluconazole, itraconazole, ketoconazole, and voriconazole), a novel imidazole, PNU-144248E (shown in Fig. ​ Fig.1), 1 ), an allylamine (terbinafine), or a morpholine (amorolfine) as described in Materials and Methods. In addition, RNA was prepared from three strains each bearing a homozygous deletion of ERG2, ERG5, or ERG6 and from two untreated control cultures. Figure ​ Figure1 1 shows the genes involved in the biosynthesis of ergosterol from farnesyl pyrophosphate in S. cerevisiae.

Treatments used in the study and their relationships to ergosterol biosynthesis. Gene names are as listed in reference 9). Genes in deletion strains are boldfaced and boxed. Arrows point to the sites of action of the antifungal agents. The structure of PNU-144248E is shown under the azoles.

Identification of gene expression patterns.

Each Affymetrix yeast genome set represents 6,593 ORFs, including 172 control genes. Thus, the 11 data sets from the treatments comprising this study represent 72,523 data points. Hybridization intensity values are expressed in ADI units, as described in Materials and Methods. Following normalization to account for variations in chip intensity (described in Materials and Methods), a filter was applied requiring the experimental ADI value to be above 50 ADI units, thereby limiting the data to 29,428 points. Use of a second filter requiring the baseline ADI value to be greater than 50 ADI units further limited the data to 20,697 points. The ADI values used in the filters were chosen after examination of the background ADI value calculated by the GeneChip software and the ADI values for selected unexpressed genes (e.g., haploid-specific genes in diploid cells 寚ta not shown]) with both approaches, a value of up to � ADI units corresponded to no signal.

Genes for which ADI ratios changed beyond 1 standard deviation were considered to be responsive to the treatment. By this criterion, 1,154 genes responded with increased mRNA levels in at least one treatment 1,358 genes responded with decreased mRNA levels relative to the baseline. To distinguish gene responses to perturbations in ergosterol biosynthesis from other transcriptional changes, genes responding in at least 5 of the 11 experimental treatments were considered in the subsequent analysis. The intention of selecting by these criteria is to identify genes that have a convergent pattern of expression across many individual treatments, which may be indicative of a common response.

A total of 156 genes showed significant increases in transcript levels in five or more treatments, and 78 showed significantly decreased transcript levels in five or more treatments. These were annotated using the biological roles assigned by the Yeast Protein Database (YPD) (18). The number and characteristics of the responsive genes grouped according to biological role are shown in Table ​ Table1. 1 . The category with the largest number of responses (hits) is “unknown” group of 53 genes. Next most abundant are the 36 responsive mitochondrial genes, followed by 22 genes involved in biosynthesis of lipids, fatty acids, and sterols. This group includes nine genes in the ergosterol pathway. The category “other related genes” refers to genes that are related to ergosterol perturbation by other experimental results these genes are described in Tables ​ Tables2 2 and ​ and3 3 and Fig. ​ Fig.2 2 and ​ and3. 3 . The category “other genes” includes responsive genes each of which was the sole representative of a particular biological pathway, and whose relationship to ergosterol perturbation could not be discerned.

TABLE 1

Genes responding in five or more treatments

Biological role a No. of genes b No. of hits c Hit/gene ratioNo. of drug hits d Drug hit/ gene ratioNo. of KO hits e KO hit/ gene ratioNo. (%) of genes responding to saturated culture% of genes responding to: Direction of change f
Drug treatmentKO
Unknown f g
 Up261545.921244.77301.150 (0)8020Up
 Up/sat'd8526.50354.38172.138 (100)6733Up
𠀽own11676.09565.09111.000 (0)8416Down
𠀽own/sat'd8506.25415.1391.138 (100)8218Down
Mitochondrial321946.061454.53491.5323 (72)7525Up
4256.25205.0051.251 (25)8020Down
Lipid/fatty acid/sterol161016.31633.94382.381 (6)6238Up
6376.16284.6791.502 (33)7624Down
Protein synthesis and processing15906.00734.87171.130 (0)8119Up
9546.00424.67121.336 (67)7822Down
Stress15906.00593.93312.075 (33)6634Up
5316.20255.0061.204 (80)8119Down
Membrane associated8455.62394.8860.750 (0)8713Up
8506.25364.50141.753 (38)7228Down
Amino acid biosynthesis9586.44434.78151.677 (78)7426Up
Cell cycle control7385.43314.4371.000 (0)8218Up
Chromatin associated6355.83294.8361.000 (0)8317Up
Carbohydrate metabolism6355.83244.00111.835 (83)6931Up
Vesicle associated5295.80234.0061.200 (0)7921Down
Cell wall biosynthesis5336.60265.2071.403 (60)7921Up
Nucleotide metabolism5285.60193.8091.803 (60)6832Down
Other related genes4246.00194.7551.251 (25)7921Down
3175.67113.6762.001 (33)6535Up
Other genes12665.50584.8380.672 (17)8812Down

TABLE 2

Genes encoding products associated with cell envelope structure or𠂟unction

Function of gene product(s) Gene(s) a
Involved in protein glycosylation ALG5 2,4 , ALG3, ALG9, ALG6 3,3 , ALG8 1,4 , ALG10, transferase (PMT family PMT2 3,2 ) CWH41, GLS2 1,5 , MNS1
O-glycosylated proteinsAGA1, AGA2, BAR1, CTR1, CTS1 2,3 , CWP1, CWP2, DAN1, FET3, FLO1, FLO5, FLO9, FLO10, FUS1, GAS1, GIT1, HKR1, HSP150, KEX2, KNH1, KRE1 3,4 , KRE9 2,3 , MID2, MSB2, PEX15, PGM1, PGM2, PIR1, PIR3, PRB1 1,4 , SAG1, SEC20, SED1, SED4, SLG1, SRO4, SSR1, STA1, STA2, TIP1 2,5 , TIR1 3,3 , TIR2, YLR110C
Responsive membrane proteins b BAP2 2,3 , BAP3 0,6 , DIP5 3,4 , FET4 1,7 , HNM1 3,5 , HXT1 0,5 , HXT3 2,4 , PHO87 0,5 , RCS1 1,4 , SMF1 3,4 , YDR373W 1,4 , YGR138C 0,5 , YKL146W 1,4 , YNL065W 0,6 , YNL321W 2,3 , YOR161C 2,3 , YOR271C 2,4
GPI-anchored proteinsAGa1, AGA1, CWP1, CWP2, EGT2 1,4 , FLO1, FLO5, FLO9, GAS1, ICWP, KRE1 3,4 , PRY3, SED1, TIP1 2,5 , TIR1 3,3 , TIR2, YAP3, YCR089W, YDR055W 0,5 , YDR134C, YDR534C, YEL040W, YER150W 0,5 , YGR189C, YJR151C, YLR110C, YNL300W 3,7 , YOR009W, YOR214C
Involved in synthesis of β-1,6-glucanCWH53, FKS2, KNR4, HKR1, KRE1 3,4 , KRE5, KRE6, KRE9 2,3 , KRE11, SKN1
Osmotic-stress related
 Part of osmotic-stress response signal pathwayHOG1 2,3
 Transcription factor with a role in salt tolerance CIN5 1,6
 Induced by osmotic stressDDR48 3,4 , DDR2, GRE1, GRE2 3,4 , GRE3, HOT1, PTP2, PTP3, SIP18, SKN7, SKK2, SSK1, STE11, SLN1, SHO1, YPD1
Responsive proteins involved in secretion b VID24 0,7 , RET2 3,4 , SEC17 0,5 , COP1 2,5 , LST8 3,4 , NHX1 0,5 , YGL054C 0,6 , YHR138C 0,5

Genes involved in the biosynthesis of ergosterol and membrane components. Boldfaced genes were responsive in the study boldface italics indicate genes with decreased transcript levels. Superscripts indicate the number of treatments to which the gene responded. The first number in each superscript is the number of genetic perturbations (out of a total of three) which elicited a response, and the second number is the number of drug treatments (out of a total of eight) which elicited a response. Lists of genes were obtained from the YPD (18) and reference 9.

TABLE 3

Genes encoding mitochondrial proteins

Gene product(s) Gene(s) a
Electron transport complexes, inner mitochondrial membrane:
 Ubiquinol cytochrome c reductase complex IIICOB, CYT1 2,4 , COR1 2,3 , QCR2, QCR6 1,4 , QCR7, QCR8, QCR9, QCR10, RIP1 1,5
𠀼ytochrome c (anaerobic isoform) CYC7 1,5
𠀼ytochrome c oxidaseCOX1, COX2, COX3, COX4 3,4 , COX5A 2,4 , COX5B 1,8 , COX6, COX7, COX8 0,5 , COX9, COX12, COX13 3,4
𠀺TP synthaseATP1 3,5 , ATP2, ATP3, ATP4, ATP5, ATP6, ATP7, ATP8, ATP9, ATP14 0,6 , ATP15 3,4 , ATP16 0,5 , ATP17, ATP20, INH1 0,6 , TIM11
�P/ATP carrier proteinAAC1, AAC3 1,4 , PET9 3,3 , YPL134C 1,4 , YPR021C
Elements of the TCA cycleCIT1, ACO1 2,4 , IDH1, IDH2, KGD1 1,5 , KGD2, LPD1, LSC1, LSC2, SDH1 2,4 , SDH2, SDH3, SDH4, FUM1 2,4 , MDH1
Other responding mitochondrial proteinsARG5,6 0,5 , ARG7 1,6 , COT1 3,5 , GPD2 3,4 , IDP1 0,5 , ILV5 2,3 , ILV6 3,5 , MRP1 0,6 , PFK27 0,6 , SHM1 1,4 , SOM1 0,6 , SOD2 2,4 , TIM23 3,3 , YAH1 2,3 , YIL154C 0,6

Genes involved in the biosynthesis and utilization of heme. Boldface, italics, and superscripts are as described for Fig. ​ Fig.2. 2 . Lists of genes were obtained from the YPD (18).

Genes were also categorized by the relative number of responses due to chemical versus genetic perturbation. The percentage of the response that was due to chemical perturbation ranged from highs of 87 to 88%, in the case of membrane-associated proteins, to a low of 62% for the class of lipid-, fatty acid-, or sterol-related genes. Analysis using a k-means algorithm did not reveal transcriptional patterns associated with particular treatment classes (data not shown). Interestingly, the ergosterol genes were highly responsive to genetic disruption of ERG2, ERG5, and ERG6. The 10 responsive ergosterol genes had increased transcript levels in all three mutant strains (Fig. ​ (Fig.2) 2 ) with the exception of the particular gene disrupted in each strain.

As a means to assess the specificity of the ergosterol response identified in this study, the behavior of these responsive genes was examined following the transition from logarithmic growth to saturation. Table ​ Table1 1 shows the number of responsive genes in each category whose transcript levels change as the cells enter saturated growth and undergo the diauxic shift (10, 18 G. F. Bammert and J. M. Fostel, unpublished data). The proportion of responsive genes which are altered in response to saturated growth varies from 0% of genes in several pathways to highs of 78 and 83% of ergosterol-responsive genes related to amino acid and carbohydrate metabolism, respectively. This suggests that some groups of transcripts may be responding to alterations in the metabolic rate rather than to ergosterol perturbation directly.

Responsive genes in the ergosterol pathway.

Figure ​ Figure2 2 shows the genes involved in the biosynthesis of ergosterol from acyl coenzyme A (acyl-CoA), highlighting some of the responsive genes identified above. Most notably, nine genes in the ergosterol pathway responded with increased transcript levels to the conditions used in this study. The ergosterol pathway represented the highest proportion of responsive genes identified, in agreement with previous studies showing that this pathway is the target of azoles and is responsive to modulations of the ergosterol level.

The biosynthesis of ergosterol involves the coordination of many factors by which the cell regulates the synthesis of this essential component. ERG19 is reported to contribute to the regulation of flux through the mevalonate pathway (3) and is increased in response to perturbations here. ERG3 expression increases following treatment with antifungal agents (43) and in strains with mutations in ERG2, ERG5, and ERG6 (2). These data are supported by the observations reported here: ERG3 expression increased following drug treatment and in the three ERG deletion strains. Additionally, expression of NCP1, which encodes NADP-cytochrome P450 reductase and the electron donor for squalene epoxidase, lanosterol 14α-demethylase, and sterol C-22 desaturase (45), increases fivefold in a strain constitutively overexpressing ERG11 (48), consistent with coordinate increases in the transcript levels of these two genes in this study.

Measurements using promoter fusions in various genetic backgrounds found a number of genes affecting ERG9 expression (23). Two treatments overlapped those reported here: exposure to ketoconazole and expression in an ERG2 disruptant. In both cases ERG9 expression increased (23). While ERG9 did not meet the criterion of change in five or more treatments used here, levels of the ERG9 transcript did increase in four treatments: ERG2 and ERG6 knockouts and exposure to fluconazole or PNU-144248E. The difference in the ketoconazole treatments used in the two studies (18 h the study reported in reference 23 versus 90 min here) may account for the difference, as observed in Fig. ​ Fig.5 5 for ERG25.

Expression time course following exposure to ketoconazole. (A) Culture OD at the times sampled (solid bars, untreated culture heavily shaded bars, 4 μM ketoconazole open bars, 8 μM ketoconazole light shaded bars, 16 μM ketoconazole) and change in TEF1 level as culture goes into saturation (line, average of three measurements of untreated culture in panels B, C, and D error bars, standard errors). (B through D) Responses of ERG25, YER067W, and YNL300W transcripts (solid symbols) and an internal TEF1 control (open symbols). Culture was left untreated (circles) or treated with ketoconazole at 4 μM (triangles), 8 μM (squares), or 16 μM (diamonds).

Another gene in the pathway, ERG1, did not change significantly in any of the treatments used here, including terbinafine, although Erg1p enzyme activity does increase following terbinafine exposure (28). Erg1p activity responds to oxygen and sterol limitation (34) and appears to be regulated by protein localization or other factors (28). Taken together, these observations suggest that changes in Erg1p activity arise from posttranscriptional regulation.

In a study using promoter fusions as a readout of transcriptional changes, Dimster-Denk et al. (11) observed 19 genes with a change in expression greater than 1.5-fold following 21 h of exposure to fluconazole relative to expression levels in untreated cells. Of these 19 responsive genes, 7 were found to be responsive in this study (ERG2, ERG3, ERG4, ERG5, ERG6, ERG11, and ERG19) and ERG9 expression was changed in four treatments. The other 11 genes had transcript levels too low to be measured reliably, including 5 mating-response genes not expressed in the diploid strain used here (ERG8, ERG12, ARE2, COQ7 ֺlso referred to as CAT5], FAR3, FIG1, HEM14, MFA1, MFA2, MOD5, and STE2). Responses by ERG24 and ERG25 were seen on the microarray but were not reported by Dimster-Denk et al. Thus, the two methods are in agreement on the identification of responsive genes among genes detected by both. The change in transcript level was not always in the same direction in the two studies, however. Decreases in ERG3, ERG4, ERG5, ERG6, and ERG11 transcript levels at 21 h were reported by Dimster-Denk et al. (11), while these genes showed increased transcript levels following 90 min of exposure as measured by microarrays. It is more likely that this reflects differences between the biological responses of these genes at the different exposure times than a discrepancy between the methods used to measure expression.

Other responsive genes related to lipid, fatty acid, and sterol biosynthesis.

In addition to participating in cell membranes, esterified ergosterol is found in lipid particles, which may serve as storage reservoirs or as intermediates in intracellular transport (53, 55). ARE1 is one of the two genes responsible for the esterification of ergosterol, the final step in the pathway leading to ergosteryl, and was responsive in this study. Another responsive gene, CYB5 (encoding cytochrome b5), was identified by an ability to overcome ketoconazole hypersensitivity when overexpressed in an erg11 background (47) and may help protect the organism from azole exposure. ACH1 (encoding acetyl-CoA hydrolase) may be responding to an overproduction of sterol intermediates formed during inhibition of the pathway. FAS1 (encoding fatty-acyl-CoA synthase) deletion strains have reduced levels of ergosterol esters and sphingolipids, indicating a possible role in lipid biosynthesis and metabolism (9). LCB1 encodes serine C-palmitoyltransferase, the first enzyme involved with the biosynthesis of the long-chain base component of sphingolipids. An increase in the level of transcripts of this transferase following perturbation of the ergosterol pathway suggests an interaction between the ergosterol and sphingolipid biosynthetic pathways in yeast.

Transcript levels of several other genes involving lipid, fatty acid, and sterol metabolism decreased in this study. The SUR2 product hydroxylates the sphingoid C-4 of ceramide (15), and the decrease observed in the level of this transcript further suggests an interaction between the sphingolipid and sterol pathways. ELO1 encodes an enzyme responsible for the elongation of fatty acids. A decline in the level of this transcript under the conditions of this study may indicate a compensatory response in cellular fatty acid content to limited sterols following perturbation of the ergosterol pathway. OLE1, encoding δ-9 desaturase, is needed for the formation of unsaturated fatty acids and also shows a decreased transcript level here. OLE1 is repressed by the presence of saturated fatty acids (12) thus, the decline in OLE1 transcript levels may indicate an increase in saturated fatty acid levels, possibly another compensatory response to altered ergosterol. Interestingly, the fatty-acid-responsive repression of OLE1 is mediated through the FAA1 and FAA4 products (7), and the level of FAA4 transcripts decreased here. A connection of fatty acids to perturbation of the ergosterol pathway may suggest a restructuring of the cell membrane in response to reduced ergosterol levels.

Other responsive pathways.

While ergosterol is found throughout the cell membranes, it is most abundant in the plasma membrane and secretory vesicles and is important for mitochondrial respiration (9, 36, 55). Depletion of ergosterol with concomitant accumulation of sterol intermediates can result in alterations in membrane functions, synthesis and activity of membrane-bound enzymes, and mitochondrial activities, as well as in uncoordinated behavior of the yeast cell (36, 49). The changes in transcript pattern reported in Table ​ Table3 3 may reflect these stresses.

It can be predicted that perturbations of ergosterol levels within the cell may affect the functioning of mitochondrial enzymes. This pattern does indeed emerge with the increase in transcript levels of several components of the mitochondrial electron transport system, as shown in Table ​ Table3. 3 . Transcript levels of four members of the cytochrome c oxidase complex, COX4, COX5A, COX8, and COX13, were increased. Similarly, transcript levels increased for four members of the cytochrome c reductase complex, RIP1, CYT1, QCR6, and COR1, and five genes encoding subunits of ATP synthase, ATP1, ATP14, ATP15, ATP16, and INH1. Other genes involved in energy generation that showed increased transcript levels are listed in Table ​ Table3, 3 , as are a number of other responsive genes encoding mitochondrial proteins.

Levels of transcripts from four of the five members of the hypoxic gene family (ANB1, COX5b, CYC7, and HEM13) are reduced in response to treatments used in this study (Table ​ (Table4). 4 ). Two of these genes, CYC7 and COX5b, encode the hypoxic isoforms of cytochrome c and cytochrome c oxidase. Since their expression levels depend on the level of available oxygen (6, 25), the decrease in transcript levels of these anaerobiosis-induced genes may be occurring in response to increased levels of intracellular oxygen. Another line of evidence to support this hypothesis is the observation that transcripts from three genes involved with oxidative-stress response (GRE2, YDR453C, and SOD2) are increased (Table ​ (Table4). 4 ). In the course of normal catalysis, cytochrome c oxidase transfers two electrons to molecular oxygen, and incomplete reactions can result in the formation of reactive oxygen species. Machida et al. (32) have shown that exposure to farnesol results in the generation of reactive oxygen species through an indirect effect on mitochondrial electron transport. Farnesol can be generated from farnesyl pyrophosphate, a precursor to sterols which may accumulate if late stages of ergosterol biosynthesis are inhibited. All these observations are consistent with increased oxidative stress following perturbation of ergosterol biosynthesis.

TABLE 4

Genes responding to oxygen stress

Function or response Gene(s) a
Induced by hypoxiaAAC1, ANB1, COX5B 1,8 , CPR1, CYC7 1,5 , ERG11 3,3 , HEM13 0,7 , HMG2, OLE1 2,7 , ROX1, SUT1
Induced by anaerobiosisCYB2, ERG11 3,3 , TIP1 2,5 , TIR2, TIR1 3,3
Pentose phosphate pathway shuntZWF1 3,4
Major oxidant-scavenging enzymesCCP1, CTT1, TSA1, SOD1, SOD2 2,4 , TRR1, TRX1, TRX2, GLR1, YDR453C 2,4 , YCL035, AHP1, GRE2 3,4

Perturbation of mitochondrial electron transport could arise from a decrease in ergosterol levels in the inner membrane due to interference with ergosterol biosynthesis or from a direct interaction between the chemical agents used and the mitochondrial enzyme complexes. For example, metal-chelating drugs block reduction at the ubiquinol oxidation site of the cytochrome c complex (5), and azoles inhibit their target, lanosterol demethylase, through an interaction with the heme iron component of the complex (54). Since more than half of the responsive mitochondrial genes are found to respond both to genetic alterations and to drug treatment, it is likely that the effect is mediated through ergosterol biosynthesis and does not arise solely as a direct consequence of drug action.

Interestingly, one drug-specific pattern was observed. Transcript levels of HEM1, the first gene involved in the biosynthesis of heme, increased in the eight drug treatments but not in the three deletions. This suggests a compensatory response to drug treatment not represented in the mutant strains. Transcript levels of HEM13, the rate-limiting step of heme biosynthesis (57), were reduced in response to seven drug treatments and, again, in none of the deletion strains. HEM13 is repressed by heme and oxygen (1, 22), raising the possibility that levels of one or both are elevated following treatment. This is consistent with the hypothesis of increased intracellular oxygen suggested by the responses of anaerobiosis-induced and oxygen stress genes described above. Heme also plays a role in sensing intracellular oxygen levels (57).

Heme plays a central role in sterol synthesis and regulates the transcription of several genes involved with this process (37, 46). The accumulation of 5-aminolevulenic acid, the product of Hem1p, derepresses 3-hydroxy-3-methyl-glytaryl CoA reductase, leading to increased levels of 2,3-oxidosqualene (31). Heme is required for the enzymatic activities of Erg3p (C-5 sterol desaturase) and Erg5p (C-22,23 desaturase) (36). Erg11p also contains heme and shows heme-regulated expression (48). Other genes regulated by heme are listed in Fig. ​ Fig.3. 3 . In the present study, levels of transcripts from four heme-induced genes are increased and levels of transcripts from three heme-repressed genes are decreased, consistent with induction of heme-regulated expression, and suggesting elevated intracellular heme levels following treatment, particularly treatment with chemical agents.

Correlation of the expression pattern of a novel azole to other conditions.

Included in this analysis was the expression profile in response to treatment with a compound containing an azole moiety. PNU-144248E contains an imidazole ring, yet is structurally distinct from the other azoles tested. The rationale for including it in this study was to determine if exposure to this compound would result in a pattern of expression similar to that seen in response to treatment using azoles with known biochemical functions, thereby suggesting a similar mode of action. This information could then be used to assess the predictive ability of expression profiles observed in response to an agent with an unknown mode of action. Of the 156 genes with increased transcript levels following ergosterol perturbation, 144, or 92%, also had increased transcript levels in response to treatment with PNU-144248E. All of the ergosterol, lipid, fatty-acid, and sterol metabolism genes and 17 of the 19 genes involved with energy generation were included (the exceptions were QCR6 and COX13). Twenty-nine of the 34 unknowns were also included. From the set of 78 genes with decreased transcript levels following ergosterol perturbations, 40, or 51%, also had decreased transcript levels following treatment with PNU-144248E. These data suggest that PNU-144248E does indeed behave as an azole as measured by cellular responses at the level of gene transcription.

PDR5 transcript levels increased following treatment with PNU-144248E. This gene encodes a member of the multidrug resistance pump family homologous to genes which are upregulated in azole-resistant isolates of C. albicans (41). It might be anticipated that PDR5 transcript levels increase in all of the drug treatments however, they were unaltered in other treatments used in this study. It is possible that PDR5 requires a longer exposure time for full induction and that PNU-144248E was the only treatment of sufficient potency to elicit a response in the 90 min of exposure used here. PDR5 transcript levels in cells treated with mucidin did not increase until 2 h of exposure (35), and the response to ergosterol perturbation may follow similar kinetics.

Comparison of microarray measurements with PCR measures of transcript levels.

Microarrays represent a new technology for measuring transcript levels. Control experiments indicate that measures using this technique are quantitative however, these experiments were carried out for a limited subset of the genes on the array (52). It was of interest to compare microarray ADI measurements for responsive genes to measurements of the same RNA preparation using the Taqman quantitative PCR system. This was performed for two genes of interest, ERG25 and YER067 (Fig. ​ (Fig.4), 4 ), one showing an increase in response to treatment and the other a decrease.

Comparison of expression measurements by RT-PCR and microarrays. Each data point represents the measurement of a given gene by each method under a specific treatment, as indicated by the label. Squares, ERG25 circles, YER067W solid symbols, untreated control. The vertical axis is the change from the TEF1 level larger numbers signify more cycles needed and thus less starting material in the sample. The horizontal axis indicates the microarray intensity in ADI units, normalized as described in Materials and Methods ADI units are proportional to transcript levels.

Data from the experiments shown in Fig. ​ Fig.4 4 were normalized as described in Materials and Methods. Microarray ADI measures are proportional to transcript abundance, while PCR measures of Ct are inversely proportional, i.e., more-abundant transcripts require fewer cycles to achieve detectable levels, and hence the Ct is lower than for less-abundant material. For this reason the correlation seen in Fig. ​ Fig.4 4 suggests good agreement between measures of transcript levels by these two methods.

Changes in transcript levels over time.

A single doubling time was selected for the duration of treatment with chemical agents in this study. It is likely that different transcripts may be responsive at earlier or later times and that the responsive transcripts detected herein may peak at times other than 90 min. In order to assess the response kinetics for genes of interest, measures of transcripts from responsive genes were made following different times of exposure to several of the agents used in this study. ERG25 is a late-stage transcript in the ergosterol pathway and has previously been shown to be responsive to ergosterol levels (29). YER067W and YNL300W are uncharacterized ORFs which respond to ergosterol perturbation but do not change in many other treatments tested (data not shown). YER067W is a ploidy-regulated gene (13). YNL300W is predicted to be a glycosylphosphatidylinositol (GPI)-anchored protein (16) with weak homology to the potential cell wall stress sensor Mid2p (18, 24). Microarray measurements show that both ERG25 and YNL300W transcripts decrease under conditions where the growth rate is slowed, for example, entry into stationary phase, while YER067W transcript levels increase under these conditions (data not shown). This is opposite to the direction of response to ergosterol perturbation, where microarray data show that YER067W levels increase and ERG25 and YNL300W transcripts decrease.

Figure ​ Figure5 5 shows the response kinetics for these three genes during a 24-h exposure to ketoconazole. Panel A shows the growth of the cultures used in this experiment both by OD and by the concomitant change in the TEF1 level as the cultures become saturated between 5 and 24 h. Panels B, C, and D show the PCR measures of ERG25, YER067W, and YNL300W RNA, respectively, from cells exposed to ketoconazole at three different doses. All show the TEF1 measure and the experimental transcript. In all cases, TEF1 is relatively unchanged by treatment and serves to normalize slight differences in the total RNA in each reaction tube during logarithmic growth (0, 60, 90, 180, and 300 min). In contrast, exposure to differing concentrations of ketoconazole results in an almost dose-dependent response in the three transcripts measured, and the direction of change is opposite that seen during growth saturation.

One interesting exception is the ERG25 response. The initial microarray measure found a 1.8-fold increase following a 90-min treatment with ketoconazole, and this was confirmed by PCR (Fig. ​ (Fig.4). 4 ). In the time course experiment shown in Fig. ​ Fig.5, 5 , however, ERG25 levels were not observed to increase until the 3-h point, when they were 2.5 cycles or approximately 5.6-fold increased relative to levels in untreated cells. This most likely reflects a difference in sensitivity (a 1.8-fold change is within one PCR cycle) or small differences between the cultures used in the two experiments. This experiment illustrates the different strengths of the two methods used. Microarrays reveal the transcript profile at a particular time and state and can be used to identify all transcripts responding under the conditions of the study. Quantitative PCR probes can then be generated to follow the kinetics of expression of transcripts of interest, allowing easy measurement of their response to a variety of treatments.

Conclusions.

Genome-wide transcriptional changes in S. cerevisiae observed in response to treatment of the cells with chemical agents correlate with responses to genetic alterations in the same biosynthetic pathway. A number of responsive genes which encode products with functions impinging on ergosterol biosynthesis or products related to membrane structure and function were identified. This supports the interpretation of expression profiles to define the mode of action of a drug. In addition to changes in transcript level relating directly to ergosterol biosynthesis, expression changes suggestive of interruption of heme biosynthesis and increased intracellular oxygen tension were also observed, indicating additional effects perturbing the ergosterol pathway. The approach used to identify genes responsive to the treatments studied does not rely on prior understanding of the biological effects of the treatments. Thus, it is possible to contemplate applying this method to predict the mode of action of novel agents with antifungal activity. The novel azole PNU-144248E has provided a validation of this method, causing transcriptional changes with a high degree of correlation to those seen following treatment with other azoles with known biological effects. This study has outlined a method to identify genes of interest common to a particular cellular response that will be of utility in the future study of novel antifungal compounds.


Abstract

Photoaffinity labeling is a useful technique employed to identify protein–ligand and protein–protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with Km (6.6 μM) and Vmax (947 pmol min –1 mg –1 ) values comparable or better than a-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing a-factor peptides.


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Affiliations

Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China

Xiaomei Lv, Fan Wang, Pingping Zhou, Lidan Ye, Wenping Xie, Haoming Xu & Hongwei Yu

Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou, 310027, China

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Contributions

H.Y., X.L. and W.X. conceived the project and designed the experiments. X.L., F.W. and P.Z. performed the experiments. X.L., L.Y. and H.X. wrote and revised the manuscript. All authors discussed the results and commented on the manuscript.

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