Direct Visualization of Structurally Similar Polysaccharides in Single Yeast Cells in Vivo by Multivariate Analysis Assisted Raman Microspectroscopy.

The demand for functional food ingredients like ß-glucan has risen enormously in recent times owing to its use in many fields including the food and beverage, cosmetics, pharmaceuticals, and biotechnology industries. Among the many natural sources of glucans such as oats, barley, mushrooms, and seaweeds, yeast has a special advantage in the industrial production of glucans. However, characterizing glucans is not straightforward as there are many different structural variations such as α- or ß-glucans with various configurations which vary in their physical and chemical properties. Currently, microscopy, chemical or genetic approaches are followed to study glucan synthesis and accumulation in single yeast cells. However, they are time-consuming, lack molecular specificity, or are practically not feasible for real applications. Therefore, we developed a Raman microspectroscopy based method to identify, distinguish, and visualize structurally similar glucan polysaccharides. By employing multivariate curve resolution analysis, we successfully separated Raman spectra of α- and ß-glucans from mixtures with high specificity and visualized heterogeneous molecular distributions during the sporulation of yeasts at the single-cell level in a label-free manner. We believe such an approach when combined with a flow cell can achieve the sorting of yeast cells based on the accumulation of glucans for various applications. Further, such an approach can also be extended to various other biological systems to investigate structurally similar carbohydrate polymers in a fast and reliable manner.

Identification of novel coenzyme Q10 biosynthetic proteins Coq11 and Coq12 in Schizosaccharomyces pombe.

Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S. cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S. pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S. pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.

Benzoic acid inhibits Coenzyme Q biosynthesis in Schizosaccharomyces pombe.

Coenzyme Q (CoQ, ubiquinone) is an essential component of the electron transport system in aerobic organisms. Human type CoQ10, which has 10 units of isoprene in its quinone structure, is especially valuable as a food supplement. Therefore, studying the biosynthesis of CoQ10 is important not only for increasing metabolic knowledge, but also for improving biotechnological production. Herein, we show that Schizosaccharomyces pombe utilizes p-aminobenzoate (PABA) in addition to p-hydroxybenzoate (PHB) as a precursor for CoQ10 synthesis. We explored compounds that affect the synthesis of CoQ10 and found benzoic acid (Bz) at >5 µg/mL inhibited CoQ biosynthesis without accumulation of apparent CoQ intermediates. This inhibition was counteracted by incubation with a 10-fold lower amount of PABA or PHB. Overexpression of PHB-polyprenyl transferase encoded by ppt1 (coq2) also overcame the inhibition of CoQ biosynthesis by Bz. Inhibition by Bz was efficient in S. pombe and Schizosaccharomyces japonicus, but less so in Saccharomyces cerevisiae, Aureobasidium pullulans, and Escherichia coli. Bz also inhibited a S. pombe ppt1 (coq2) deletion strain expressing human COQ2, and this strain also utilized PABA as a precursor of CoQ10. Thus, Bz is likely to inhibit prenylation reactions involving PHB or PABA catalyzed by Coq2.

CoQ10 production in Schizosaccharomyces pombe is increased by reduction of glucose levels or deletion of pka1.

Coenzyme Q (CoQ) is an essential component of the electron transport system that produces ATP in nearly all living cells. CoQ10 is a popular commercial food supplement around the world, and demand for efficient production of this molecule has increased in recent years. In this study, we explored CoQ10 production in the fission yeast Schizosaccharomyces pombe. We found that CoQ10 level was higher in stationary phase than in log phase, and that it increased when the cells were grown in a low concentration of glucose, in maltose, or in glycerol/ethanol medium. Because glucose signaling is mediated by cAMP, we evaluated the involvement of this pathway in CoQ biosynthesis. Loss of Pka1, the catalytic subunit of cAMP-dependent protein kinase, increased production of CoQ10, whereas loss of the regulatory subunit Cgs1 decreased production. Manipulation of other components of the cAMP-signaling pathway affected CoQ10 production in a consistent manner. We also found that glycerol metabolism was controlled by the cAMP/PKA pathway. CoQ10 production by the S. pombe ∆pka1 reached 0.98 mg/g dry cell weight in medium containing a non-fermentable carbon source [2% glycerol (w/v) and 1% ethanol (w/v) supplemented with 0.5% casamino acids (w/v)], twofold higher than the production in wild-type cells under normal growth conditions. These findings demonstrate that carbon source, growth phase, and the cAMP-signaling pathway are important factors in CoQ10 production in S. pombe.

Studying anti-oxidative properties of inclusion complexes of α-lipoic acid with γ-cyclodextrin in single living fission yeast by confocal Raman microspectroscopy.

α-lipoic acid (ALA) is an essential cofactor for many enzyme complexes in aerobic metabolism, especially in mitochondria of eukaryotic cells where respiration takes place. It also has excellent anti-oxidative properties. The acid has two stereo-isomers, R- and S- lipoic acid (R-LA and S-LA), but only the R-LA has biological significance and is exclusively produced in our body. A mutant strain of fission yeast, Δdps1, cannot synthesize coenzyme Q10, which is essential during yeast respiration, leading to oxidative stress. Therefore, it shows growth delay in the minimal medium. We studied anti-oxidant properties of ALA in its free form and their inclusion complexes with γ-cyclodextrin using this mutant yeast model. Both free forms R- and S-LA as well as 1:1 inclusion complexes with γ-cyclodextrin recovered growth of Δdps1 depending on the concentration and form. However, it has no effect on the growth of wild type fission yeast strain at all. Raman microspectroscopy was employed to understand the anti-oxidant property at the molecular level. A sensitive Raman band at 1602cm-1 was monitored with and without addition of ALAs. It was found that 0.5mM and 1.0mM concentrations of ALAs had similar effect in both free and inclusion forms. At 2.5mM ALAs, free forms inhibited the growth while inclusion complexes helped in recovered. 5.0mM ALA showed inhibitory effect irrespective of form. Our results suggest that the Raman band at 1602cm-1 is a good measure of oxidative stress in fission yeast.

Schizosaccharomyces japonicus has low levels of CoQ10 synthesis, respiration deficiency, and efficient ethanol production.

Coenzyme Q (CoQ) is essential for mitochondrial respiration and as a cofactor for sulfide quinone reductase. Schizosaccharomyces pombe produces a human-type CoQ10. Here, we analyzed CoQ in other fission yeast species. S. cryophilus and S. octosporus produce CoQ9. S. japonicus produces low levels of CoQ10, although all necessary genes for CoQ synthesis have been identified in its genome. We expressed three genes (dps1, dlp1, and ppt1) for CoQ synthesis from S. japonicus in the corresponding S. pombe mutants, and confirmed that they were functional. S. japonicus had very low levels of oxygen consumption and was essentially respiration defective, probably due to mitochondrial dysfunction. S. japonicus grows well on minimal medium during anaerobic culture, indicating that it acquires sufficient energy by fermentation. S. japonicus produces comparable levels of ethanol under both normal and elevated temperature (42 °C) conditions, at which S. pombe is not able to grow.

Urea enhances cell lysis of Schizosaccharomyces pombe ura4 mutants.

Cell lysis is induced in Schizosaccharomyces pombe ∆ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ∆ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ∆ura4 cells.

Cloning and characterization of decaprenyl diphosphate synthase from three different fungi.

Coenzyme Q (CoQ) is composed of a benzoquinone moiety and an isoprenoid side chain of varying lengths. The length of the side chain is controlled by polyprenyl diphosphate synthase. In this study, dps1 genes encoding decaprenyl diphosphate synthase were cloned from three fungi: Bulleromyces albus, Saitoella complicata, and Rhodotorula minuta. The predicted Dps1 proteins contained seven conserved domains found in typical polyprenyl diphosphate synthases and were 528, 440, and 537 amino acids in length in B. albus, S. complicata, and R. minuta, respectively. Escherichia coli expressing the fungal dps1 genes produced CoQ10 in addition to endogenous CoQ8. Two of the three fungal dps1 genes (from S. complicata and R. minuta) were able to replace the function of ispB in an E. coli mutant strain. In vitro enzymatic activities were also detected in recombinant strains. The three dps1 genes were able to complement a Schizosaccharomyces pombe dps1, dlp1 double mutant. Recombinant S. pombe produced mainly CoQ10, indicating that the introduced genes were independently functional and did not require dlp1. The cloning of dps1 genes from various fungi has the potential to enhance production of CoQ10 in other organisms.

Label-free Chemical Imaging of Fungal Spore Walls by Raman Microscopy and Multivariate Curve Resolution Analysis.

Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as α-glucan, ß-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both α- and ß-glucans while spore wall is exclusively made of α-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future.

Vacuolar amino acid transporters upregulated by exogenous proline and involved in cellular localization of proline in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, the AVT genes (AVT1-7), which encode vacuolar amino acid transporters belonging to the amino acid vacuolar transport (AVT)-family, were significantly upregulated in response to exogenous proline. To reveal a novel role of the Avt proteins in proline homeostasis, we analyzed the effects of deletion or overexpression of the AVT genes on the subcellular distribution of amino acids after the addition of proline to the cells grown in minimal medium. Among seven AVT gene disruptants, avt1Δ and avt7Δ showed the lowest ratios of vacuolar proline. Consistently, overexpression of the AVT1 gene specifically enhanced the vacuolar localization of proline. Since double disruption of the AVT1 and AVT7 genes did not completely abrogate vacuolar accumulation of proline, it is presumed that Avt1 has a dominant role, and Avt7 and other Avt proteins have redundant functions, in the localization of proline into the vacuolar lumen. In contrast, deletion of the AVT3 gene increased vacuolar proline, although the highly expressed AVT3 gene interfered with the accumulation of proline in the vacuole. Based on these results, it appears that Avt3 is the major protein involved in the export of proline from the vacuole. We also observed vacuolar membrane localization of GFP-fused Avt1, Avt3, and Avt7 proteins. Taken together, our data suggest that the AVT genes induced by exogenous proline are involved in the bidirectional transport of proline across the vacuolar membrane.

Proline accumulation protects Saccharomyces cerevisiae cells in stationary phase from ethanol stress by reducing reactive oxygen species levels.

During fermentation processes, Saccharomyces cerevisiae cells are exposed to multiple stresses, including a high concentration of ethanol that represents toxicity through intracellular reactive oxygen species (ROS) generation. We previously reported that proline protected yeast cells from damage caused by various stresses, such as freezing and ethanol. As an anti-oxidant, proline is suggested to scavenge intracellular ROS. In this study, we examined the role of intracellular proline during ethanol treatment in S. cerevisiae strains that accumulate different concentrations of proline. When cultured in YPD medium, there was a significant accumulation of proline in the put1 mutant strain, which is deficient in proline oxidase, in the stationary phase. Expression of the mutant PRO1 gene, which encodes the γ-glutamyl kinase variant (Asp154Asn or Ile150Thr) with desensitization to feedback inhibition by proline in the put1 mutant strain, showed a prominent increase in proline content as compared with that of the wild-type strain. The oxidation level was clearly increased in wild-type cells after exposure to ethanol, indicating that the generation of ROS occurred. Interestingly, proline accumulation significantly reduces the ROS level and increases the survival rate of yeast cells in the stationary phase under ethanol stress conditions. However, there was not a clear correlation between proline content and survival rate in yeast cells. An appropriate level of intracellular proline in yeast might be important for its stress-protective effect. Hence, the engineering of proline metabolism could be promising for breeding stress-tolerant industrial yeast strains. Copyright © 2016 John Wiley & Sons, Ltd.

Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis.

Coenzyme Q10 (CoQ10) is essential for energy production and has become a popular supplement in recent years. In this study, CoQ10 productivity was improved in the fission yeast Schizosaccharomyces pombe. Ten CoQ biosynthetic genes were cloned and overexpressed in S. pombe. Strains expressing individual CoQ biosynthetic genes did not produce higher than a 10% increase in CoQ10 production. In addition, simultaneous expression of all ten coq genes did not result in yield improvements. Genes responsible for the biosynthesis of p-hydroxybenzoate and decaprenyl diphosphate, both of which are CoQ biosynthesis precursors, were also overexpressed. CoQ10 production was increased by overexpression of Eco_ubiC (encoding chorismate lyase), Eco_aroF(FBR) (encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase), or Sce_thmgr1 (encoding truncated HMG-CoA reductase). Furthermore, simultaneous expression of these precursor genes resulted in two fold increases in CoQ10 production.

Functional conservation of coenzyme Q biosynthetic genes among yeasts, plants, and humans.

Coenzyme Q (CoQ) is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3-9) that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana) to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.

Polypeptone induces dramatic cell lysis in ura4 deletion mutants of fission yeast.

Polypeptone is widely excluded from Schizosaccharomyces pombe growth medium. However, the reasons why polypeptone should be avoided have not been documented. Polypeptone dramatically induced cell lysis in the ura4 deletion mutant when cells approached the stationary growth phase, and this phenotype was suppressed by supplementation of uracil. To determine the specificity of this cell lysis phenotype, we created deletion mutants of other genes involved in de novo biosynthesis of uridine monophosphate (ura1, ura2, ura3, and ura5). Cell lysis was not observed in these gene deletion mutants. In addition, concomitant disruption of ura1, ura2, ura3, or ura5 in the ura4 deletion mutant suppressed cell lysis, indicating that cell lysis induced by polypeptone is specific to the ura4 deletion mutant. Furthermore, cell lysis was also suppressed when the gene involved in coenzyme Q biosynthesis was deleted. This is likely because Ura3 requires coenzyme Q for its activity. The ura4 deletion mutant was sensitive to zymolyase, which mainly degrades (1,3)-beta-D glucan, when grown in the presence of polypeptone, and cell lysis was suppressed by the osmotic stabiliser, sorbitol. Finally, the induction of cell lysis in the ura4 deletion mutant was due to the accumulation of orotidine-5-monophosphate. Cell wall integrity was dramatically impaired in the ura4 deletion mutant when grown in the presence of polypeptone. Because ura4 is widely used as a selection marker in S. pombe, caution needs to be taken when evaluating phenotypes of ura4 mutants.

Functional analysis of the C-terminal region of γ-glutamyl kinase of Saccharomyces cerevisiae.

γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ(1)-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.

A subunit of decaprenyl diphosphate synthase stabilizes octaprenyl diphosphate synthase in Escherichia coli by forming a high-molecular weight complex.

The length of the isoprenoid-side chain in ubiquinone, an essential component of the electron transport chain, is defined by poly-prenyl diphosphate synthase, which comprises either homomers (e.g., IspB in Escherichia coli) or heteromers (e.g., decaprenyl diphosphate synthase (Dps1) and D-less polyprenyl diphosphate synthase (Dlp1) in Schizosaccharomyces pombe and in humans). We found that expression of either dlp1 or dps1 recovered the thermo-sensitive growth of an E. coli ispB(R321A) mutant and restored IspB activity and production of Coenzyme Q-8. IspB interacted with Dlp1 (or Dps1), forming a high-molecular weight complex that stabilized IspB, leading to full functionality.

Proline as a stress protectant in the yeast Saccharomyces cerevisiae: effects of trehalose and PRO1 gene expression on stress tolerance.

Proline is considered to be a stress protectant in the yeast Saccharomyces cerevisiae, as is trehalose. In this study, we constructed yeast strains that accumulate proline but not trehalose and that increase proline levels in response to stress. Our results suggest that proline does not complement the function of trehalose, and that moderate expression of the PRO1 gene encoding gamma-glutamyl kinase is important to stress tolerance of yeast cells.

Self-cloning baker's yeasts that accumulate proline enhance freeze tolerance in doughs.

We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding gamma-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.

Gene expression profiles and intracellular contents of stress protectants in Saccharomyces cerevisiae under ethanol and sorbitol stresses.

In response to osmotic stress, proline is accumulated in many bacterial and plant cells. During various stresses, the yeast Saccharomyces cerevisiae induces glycerol or trehalose synthesis, but the fluctuations in gene expression and intracellular levels of proline in yeast are not yet well understood. We previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. In this study, we examined the relationships between the gene expression profiles and intracellular contents of glycerol, trehalose, and proline under stress conditions. When yeast cells were exposed to 1 M sorbitol stress, the expression of GPD1 encoding glycerol-3-phosphate dehydrogenase is induced, leading to glycerol accumulation. In contrast, in the presence of 9% ethanol, the rapid induction of TPS2 encoding trehalose-6-phosphate phosphatase resulted in trehalose accumulation. We found that intracellular proline levels did not increase immediately after addition of sorbitol or ethanol. However, the expressions of genes involved in proline synthesis and degradation did not change during exposure to these stresses. It appears that the elevated proline levels are due primarily to an increase in proline uptake from a nutrient medium caused by the induction of PUT4. These results suggest that S. cerevisiae cells do not accumulate proline in response to sorbitol or ethanol stress different from other organisms.