Co-production of S-adenosyl-l-methionine and glutathione from spent brewer’s yeast cells

Both S-adenosyl-l-methionine (AdoMet) and glutathione (GSH) are important small molecules with pharmaceutical importance. The co-production of AdoMet and GSH using abundant spent brewer’s yeast cells from the beer industry and with l-methionine supplement was successfully realized. Experimental data showed that improvement of GSH productivity was accompanied by AdoMet accumulation. AdoMet productivity of 40–45 mg g⁻¹ (DCW) was successfully achieved and an additional 13–18 mg g⁻¹ (DCW) GSH was synthesized in spent brewer’s yeast cells.     

Reinvestigation of the primary structure of brewer's yeast tRNA3Arg

The primary structure of brewer's yeast tRNA₃^Arg has been reinvestigated using postlabelling techniques. Several errors have been found at the 5′ and 3′ ends of the molecule. The sequences found previously are replaced by 5′ pG-C-U-C(or U)G and U-G-A-G-U-G(or C)C-C-A_OH.     

An investigation by ion-exchange chromatography of a chromium,amino acid and nicotinic acid mixture

The mixture of chromium, nicotinic acid and the amino acids glycine, glutamic acid and cysteine which stimulates the rate of CO₂ production in a yeast bioassay system was subjected to the separation scheme based on ion-exchange chromatography which has been used to separate the chromium- containing fractions in brewer's yeast, [S.J. Haylock, P.D. Buckley and L.F. Blackwell, J. Inorg. Biochem., 18, 195 (1983)]. Four chromium-containing fractions (C2 to C5) were obtained by salt gradients and two further fractions (G1 and G2) were obtained using a pH gradient. All were amino acid-containing complexes of chromium and all except C5 also contained nicotinic acid. However, none of the isolated chromium fractions showed any activity in a yeast bioassay. On the basis of previous work, the activity of the original mixture was attributed to the presence of an oxygen-coordinated trans chromium(III)-dinicotinate complex. Biologically- inactive chromium complexes such as Cr(glu)₂(H₂O)⁺₂ and Cr(gly)₂(H₂O)⁺₂ after elution by ammonium hydroxide from Dowex 50W-X12 cation- exchange columns, stimulated the rate of CO₂ production in the yeast bioassay. Elution with other bases, such as lithium hydroxide, potassium hydroxide and sodium hydroxide led to inactive fractions in all cases. A warning is therefore given that the use of ammonium hydroxide-elution of ion-exchange columns to isolate glucose tolerance factor fractions from biological samples (such as brewer's yeast) can lead to active fractions which do not relate to the native material.     

Clonal propagation of Trifolium Pratense,T. Resupinatum and T. Subterraneum by direct somatic embryogenesis on cultured immature embryos

Direct somatic embryogenesis on immature zygotic embryos in vitro has been confirmed for Trifolium pratense and extended to T. resupinatum and T. subterraneum. For all species direct embryo cloning can be achieved on an appropriate basal medium supplemented with 1gl^–1 yeast extract and 0.05 mgl^–1 BAP. Basal medium/sucrose formulation, level of yeast extract and level of BAP affected the nature of in vitro responses. In particular, for T. pratense and T. subterraneum lowering of the yeast extract level suppressed embryoid initiation, and raising of the BAP level stimulated formation of nodular morphogenic callus. For T. resupinatum alteration of the basal medium/sucrose formulation changed the tissue site of embryoid initiation from hypocotyl to cotyledons or both. Control of embryoid initiation is briefly discussed.Abbreviation BAP 6-benzylaminopurine     

Multiple α-Glucoside Transporter Genes in Brewer’s Yeast

Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain MAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure.     

The influence of linoleic acid and linolenic acid on adult moth emergence of Homona coffearia from meridic diets in vitro

The fatty acids linoleic and linolenic acids have been found to be critical dietary supplements for the successful adult emergence of Homona coffearia reared in meridic diets in vitro. When the levels of these fatty acids were sub-optimal, the moths were able to emerge only partially and a few that emerged fully were deformed with naked wings. In the presence of optimal amounts of these fatty acids, the Δ⁷ sterol, ergosterol, available in the brewer's yeast provided in the diets was found to be adequate for growth and adult emergence. The availability of linoleic acid, which appeared to be the more critical requirement, was sufficient for successful adult emergence, provided that the diet was supplemented with additional amounts of ergosterol. The above two fatty acids seemed to be the only critical dietary supplements offered by whole tea leaves incorporated in a previously described oligidic diet. The provision of a dietary source of a Δ⁷ sterol was found essential as this insect was unable to utilize cholesterol.     

Phosphorus-31 NMR of rat brain in vivo with bloodless perfluorocarbon perfused rat

Rat brain in vivo has been examined by ³¹p NMR under conditions of normal blood perfusion (hematocrit 38%) and under conditions in which a perfluorocarbon blood substitute, devoid of any phosphorus containing compounds, largely replaced the animal's normal blood supply (hematocrit 7%). These studies demonstate that 2,3-diphosphoglycerate does not — as has been suggested — contribute to, and thus does not interfere with, the ³¹p NMR analysis of rat brain in vivo. However, low intensity ³¹P resonances assigned to choline phosphate, glycerol 3-phosphorylethanolamine, and glycerol 3-phosphorylcholine are observed. “High energy phosphorus” metabolite levels show no marked change over two hours with perfluorocarbon blood substitution from those of the normal blood perfused animal. This supports use of perfluorocarbon media for tissue perfusion in vitro and for ¹⁹F NMR vascular imaging in vivo.     

Bakers' yeast protease a purification and enzymatic and molecular properties

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is inactivated by yeast protease A (EC 3.4.23.8). A complete purification procedure for protease A from bakers' yeast, which lacks the acidic activation step used by other workers, and the major properties of the enzyme are shown. The enzyme is homogeneous as judged by disc gel electrophoresis. Its molecular weight, calculated from both sodium dodecyl sulfate-disc gel electrophoresis and gel filtration experiments, is around 45,000. The protein does not possess quaternary structure. The isoelectric point is 4.1. Carbohydrate content is around 8%. Amino acids analysis and sulfur analysis reveal the presence of 1-SH group and two disulfide bridges. The free-SH group does not seem to be involved in catalysis. Amino terminal analysis shows that isoleucine is at the amino terminal position. The pH optima are 2.4 for the hydrolysis of azocasein and casein, and 3.3 for the hydrolysis of hemoglobin. The K_m value for hemoglobin is 1.7 × 10^?5m. The inhibition exerted by pepstatin on the proteolytic activity of protease A is pH dependent. Among various yest enzyme substrates only uridine nucleosidase is inactivated by protease A.     

Effect of high concentration of nutrients onBacillus thuringiensis cultures

Summary Bacterial insecticide production using a strain ofBacillus thuringiensis var. kurstaki was studied in batch culture considering the influence of increasing concentration of components of a glucose — yeast extract — mineral salts medium.It was found that spore counts were increased from 1.08×10¹² spores. 1^–1 to 7.36×10¹² spores. 1^–1 and toxin level from 1.05 mg.ml^–1 to 6.85 mg.ml^–1, when the concentration of glucose was increased from 8 to 56 (g 1^–1), with the corresponding increase in the rest of medium components. Higher concentration of nutrients inhibit either spore count or toxin production.Preliminary experiments of fed-batch cultures which allows the use of high amounts of nutrients were also carried out. In this case spore counts of 1.2×10¹³ spores.1^–1 were achieved.     

Production of solasodine by calli from different parts of Solanum eleganifolium Cav. plants

Callus tissues from different explants (hypocotyl, cotyledon, root, leaf and fruit) of Solanum eleagnifolium Cav. were cultured on a modified Murashige-Skoog medium, with 1 mg.1^–1 2,4-D as the sole growth regulator. The presence of the alkaloid solasodine was determined by spectrophotometric and TLC methods. Its concentration ranged from 1.00 to 2.15 mg.g^–1 DW. The calli from different explants showed a direct association between the solasodine production and their growth, although they have a different production rate. It was also observed that about the seventh week of culture the metabolite concentration decreased in all cases.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - TLC thin layer chromatography     

Carbon isotope effect on the enzymatic decarboxylation of pyruvic acid

The decarboxylation of pyruvic acid by the thiamine pyrophosphate dependent pyruvate decarboxylase from brewer's yeast is accompanied by a carboxyl carbon isotope effect $\text{k}{}^{12}\text{k}{}^{13}\text{= 1.0083±0.0003}$ at 25°, pH 6.8. The small size of the isotope effect indicates that decarboxylation is not rate-determining in the overall reaction. The rate constant for decarboxylation of the enzyme-bound pyruvate-thiamine pyrophosphate complex is greater by about a factor of five than the rate constant for dissociation of this complex to form free pyruvate and the enzyme-thiamine pyrophosphate complex.     

Biosorption of heavy metals from acid mine drainage onto biopolymers (chitin and α (1,3) β-D-glucan) from industrial biowaste exhausted brewer’s yeasts (Saccharomyces cerevisiae L.)

A biosorption process has been developed for the bioremediation of heavy metal-contaminated acid drainages from Merladet and Faith open-cast mines, located in western Spain. The process is based on the physico-chemical properties for the adsorption, ion exchange, and complexation of metal ions by biopolymers (chitin and α (1,3) β-D-glucan) from industrial biowaste exhausted brewer’s yeast (Saccharomyces cerevisiae L.). Firstly, the chemical composition (U, Mn, Al, Fe, Cu, Zn, and Ni) and the physico-chemical and ecological states of these acid mine drainages were characterised. Furthermore, the selectivity for Zn, Cu, Mn, Ni, and Al the first order kinetics and the performance of the metals biosorption process by exhausted brewer’s yeast were evaluated with polluted acid synthetic waters and mine drainages. The biosorption equilibria were reached in 10 ∼ 15 min following Langmuir type isotherms with higher affinity constants for metal-biosorbent binding for synthetic waters than for acid mine drainages. The efficiency of the process with real water samples was markedly lower for the case of Mn, and zero for Zn and Al. An antagonistic interference on the biosorption of a metal due to the presence of other metals is proposed. Finally, the ecotoxicity of the acid mine drainage was removed when it was incubated with brewer’s yeast trapped in polyurethane foam.     

Gypsy/Ty3-class retrotransposons integrated in the DNA of herring,tunicate, and echinoderms

Eight new examples of retrotransposons of the Gypsy/Ty3 class have been identified in marine species. A 525-nt pol gene-coding region was amplified using degenerate primers from highly conserved regions and has extended the range of recognition of Gypsy/Ty3 far beyond those previously known. The following matrix shows the percentage AA divergence of the translations of this segment of the pol gene coding region. Spr2 Strongylocentrotus purpuratus, sea urchin 39 Por2 Pisaster ochraceus, starfish 46 45 Cprl Clupea pallasi, herring 51 52 41 Cirl Ciona intestinalis, tunicate bar52 49 49 55 P. orchraceus, starfish 55 60 60 62 62 Spr3 S. purpuratus, sea urchin 55 61 60 63 61 24 Tgrl^* Tripneustes gratilla, sea urchin 56 61 60 63 58 26 27 Lvrl^* Lytechinus variegatus, sea urchin 57 62 60 64 62 27 10 29 Sprl^* S. purpuratus 58 61 62 65 61 15 27 30 31 Spr4 S. purpuratus 72 72 74 75 72 73 72 72 73 72 Por3 P. ochraceus The underlines separate three groups of retrotransposons that can be recognized on the basis of this amino acid sequence. The new upper group shows surprising amino acid sequence similarity among members from the DNA of herring, sea urchin, starfish, and a tunicate. For example, the herring element differs by only 41 % from the Ciona element and 46% from the sea urchin element. The group between the lines includes members close to previously known elements (marked by asterisks) and has so far been found only in sea urchins. The two upper groups differ from each other by 55–60% and yet members of both groups (e.g., Sprl and Spr2) are integrated into the DNA of one species-S. purpuratus. Below the lower underline is listed the only known representative of a very distant group, which occurs in starfish DNA. In spite of large divergence, amino acid sequence comparisons indicate that all of the elements shown in the array are members of the LTR-containing class of retrotransposons that includes Gypsy of Drosophila and Ty3 of yeast. Of all known mobile elements this class shows the closest sequence similarity to retroviruses and has the same arrangement of genes as simpler retroviruses.Correspondence to: R.J. Britten     

Genetic improvement of brewer’s yeast: current state, perspectives and limits

Brewer’s yeast strain optimisation may lead to a more efficient beer production process, better final quality or healthier beer. However, brewer’s yeast genetic improvement is very challenging, especially true when it comes to lager brewer’s yeast (Saccharomyces pastorianus) which contributes to 90% of the total beer market. This yeast is a genetic hybrid and allopolyploid. While early studies applying traditional genetic approaches encountered many problems, the development of rational metabolic engineering strategies successfully introduced many desired properties into brewer’s yeast. Recently, the first genome sequence of a lager brewer’s strain became available. This has opened the door for applying advanced omics technologies and facilitating inverse metabolic engineering strategies. The latter approach takes advantage of natural diversity and aims at identifying and transferring the crucial genetic information for an interesting phenotype. In this way, strains can be optimised by introducing “natural” mutations. However, even when it comes to self-cloned strains, severe concerns about genetically modified organisms used in the food and beverage industry are still a major hurdle for any commercialisation. Therefore, research efforts will aim at developing new sophisticated screening methods for the isolation of natural mutants with the desired properties which are based on the knowledge of genotype–phenotype linkage.     

The relationship of chromium to the glucose tolerance factor. II

After incubation with CrCl₃·6H₂O (or⁵¹CrCl₃·6H₂O) for 25 days, a sterile growth medium, whole yeast cells harvested after growth on a similar chromium-containing medium for the same period, and the spent growth medium remaining after removal of the yeast were each subjected to the separation procedure reported previously [S.J. Haylock, P.D. Buckley and L.F. Blackwell, J. Inorg. Biochem., in press]. The results obtained showed that most of the eleven chromium-containing fractions isolated previously were artifacts formed as a result of direct reaction between the chromium and components of the medium. An anionic complex (which was the major chromium-containing fraction isolated) was identified as a chromium-glucose complex, but one possessing no biological activity. The biologically active chromium-containing fractions (P-3 and P-4) that were only present after yeast had been grown in the medium were further purified, however, during the purification steps, the biological activity was cleanly separated from the chromium material for both P-3 and P-4. Fraction P-4 was subsequently shown to consist of approximately 90% tyramine, but pure tyramine was not active in the yeast bioassay. Although the structure of the glucose tolerance factor-active component in fraction P-3 could not be determined due to the presence of high concentrations of salt that could not be separated on gel filtration columns, the results show that the glucose tolerance factor from brewer's yeast can no longer be regarded as a chromium complex.     

Contractility of rat testicular seminiferous tubules in vitro: Prostaglandin F1α and indomethacin

The frequency of spontaneous in vitro contractions of seminiferous tubules of the rat appeared to be increased in a dose-dependent manner by prostaglandin F_1α. PGF_1α treatment increased the tonus of the smooth muscle cells in the wall of the tubules as indicated by a reduction in the diameter of the tubules. When the tubules were rinsed successively with fresh Tyrode's solution, the contractile frequency was diminished. Returning the original bathing medium to the tubules restored their contractile frequency, as did treatment of the rinsed tubules with PGF_1α (10⁻⁷ M). Pre-injecting the rats with indomethacin tended to reduce the contractile frequency of the extirpated tubules. Treating the tubules with a solution of indomethacin for 90 min. in vitro was more effective than pretreatment in vivo in reducing contractile frequency, but a combination of these two procedures produced the greatest inhibition. PGF_1α restored the contractile frequency of the indomethacin-treated tubules. Our results indicate that PGs modulate the in vitro contractility of the tubules.     

A rapid method for monitoring intracellular lipid production using1H NMR spectroscopy

Summary A method has been developed for rapid mondtoring of lipid production in the intact yeast cells ofRhodotorula gracilis using¹H NMR spectroscopy.     

Total synthesis of PGC2 methyl ester. Comparison with enzymatically produced material

The synthesis of PGC₂ methyl ester is described. Comparison of the synthetic material with the methyl ester prepared from natural PGC₂ showed the two to be identical, thus confirming the structure assignment. The physical and biological data of PGC₂ methyl ester are presented.Horton and Jones have recently shown that PGA₁ (Ic) and PGA₂ (Ia) are deactivated on incubation with cats blood (1,2). Jones has proposed that this deactivation involves the enzymic conversion of PGA (I) to an 11,12-double bond isomer (PGC, II) which is subsequently isomerized by base to the inactive PGB (III) (3). Structure assignment of the PGC's in the earlier study were based on chromatographic mobilities and uv and mass spectrometric properties. We report here a total synthesis of PGC₂ methyl ester (IIb). Comparison of the biological and physical properties of this material with those of diazomethane-esterified natural material confirms the earlier structure assignment for PGC₂ (IIa).     

Production of ethanol by adsorbed yeast cells

Summary Various ion exchange resins were tested for their ability to adsorb cells of Saccharomyces cerivisiae with the ultimate intention of developing a packed bed immobilized cell reactor for the continuous production of ethanol. The resins varied greatly in their ability to adsorb cells - the least effective resins retained less than 1 mg S. cerivisiae cells (dry weight)/g of resin (dry weight), and the most effective, 130–140 mg cells/g of resin. A column reactor packed with adsorbed yeast cells was operated continuously for over 200 hours using a 12% (w/v) glucose medium at dilution rates of 1.1 h^-1 and 1.44 h^-1 (based on void volume). High ethanol productivities of 53.1 and 62.0 g ethanol/l-h were obtained.     

Asp133 Residue in NhaA Na+/H+ Antiporter Is Required for Stability Cation Binding and Transport

Na⁺/H⁺ antiporters have a crucial role in pH and Na⁺ homeostasis in cells. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and revealed a previously unknown structural fold, which has since been identified in several secondary active transporters. This unique structural fold is very delicately electrostatically balanced. Asp133 and Lys 300 have been ascribed essential roles in this balance and, more generally, in the structure and function of the antiporter. In this work, we show the multiple roles of Asp133 in NhaA: (i) The residue's negative charge is critical for the stability of the NhaA structure. (ii) Its main chain is part of the active site. (iii) Its side chain functions as an alkaline-pH-dependent gate, changing the protein's conformation from an inward-facing conformation at acidic pH to an outward-open conformation at alkaline pH, opening the periplasm funnel. On the basis of the experimental data, we propose a tentative mechanism integrating the structural and functional roles of Asp133.