FORMATION OF HIGHER ALCOHOLS FROM AMINO ACIDS DERIVED FROM YEAST PROTEINS

When yeast, which had previously been grown in an amino acid rich medium and labelled with ¹⁴C-leucine, was transferred to a strongly nitrogen-limiting medium containing glucose and ammonium sulphate, the 3-methylbutanol obtained after completed fermentation was radioactive, indicating that about 13% of the leucine of yeast was transformed to this alcohol, and that at least 10% of 3-methylbutanol formed was derived from the protein of yeast. This in turn suggests that protein turnover in yeast partly occurs in such a way that carbon skeletons of amino acids are rejected and only the amino group is re-utilized.     

The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic,Mg2+-activated acetaldehyde dehydrogenase

The deduced translation product of an open reading frame on the left arm of chromosome XVI of Saccharomyces cerevisiae, with the systematic name of YPL061w, is 500 amino acids in length and shares significant homology with aldehyde dehydrogenases. Amino acids 2 to 16 of the protein encoded by YPL061w were found to be identical to the N-terminal 15 amino acids of the purified cytosolic, Mg²⁺-activated acetaldehyde dehydrogenase (ACDH) of S. cerevisiae. This enzyme is thought to be involved in the production of acetate from which cytosolic acetyl-CoA is then synthesized. Deletion of YPL061w was detrimental to the growth of haploid strains of yeast; an analysis of one deletion mutant revealed a maximum specific growth rate (in complex medium containing glucose) of one-third of that displayed by the wild-type strain. Mutants deleted in YPL061w were also unable to use ethanol as a carbon source. As expected, the cytosolic, Mg²⁺-activated ACDH activity had been lost from the mutants, although the mitochondrial, K⁺-activated ACDH was readily detected. YPL061w has been registered with the name of ALD6 in the Saccharomyces Genome Database and the nucleotide sequence submitted to GenBank as part of accession number U39205. © 1997 John Wiley & Sons, Ltd.     

Simultaneous utilization of ammonia,free amino acids and peptides during fermentative growth of Saccharomyces cerevisiae

The efficiency of nitrogen use by yeast is one of the key determinants of the successful completion of alcoholic fermentations. In this work the growth of Saccharomyces cerevisiae S288c in a synthetic medium containing ammonia and free amino acids, supplemented with yeast hydrolysate, was studied. Experiments with ¹⁵NH₄Cl and ¹⁵N‐labelled yeast hydrolysate were carried out to gain insight into which of these three classes of assimilable nitrogen sources yeast cells prefer. Co‐consumption of all three sources was observed; approximately 40% of the total nitrogen in the yeast protein fraction originated from yeast hydrolysate, while free amino acids and ammonia contributed 40 and 20%, respectively. The results indicate that several amino acids are more readily obtained from peptides, most likely when the uptake of their free forms is competitively inhibited and/or repressed. During the second half of each fermentation, a decrease in the incorporation of yeast hydrolysate‐derived nitrogen was observed. These results highlight the nutritional role of peptides in various yeast fermentations. Copyright © 2016 The Institute of Brewing & Distilling     

FORMATION OF HIGHER ALCOHOLS FROM 14C-LABELLED VALINE AND LEUCINE

The formation of isobutanol and 3-methylbutanol during alcoholic fermentation by brewer's yeast has been studied by adding uniformly ¹⁴C-labelled valine and leucine to the complex amino acid mixture which is used as nitrogen source. In the presence of ¹⁴C-leucine, only 3-methylbutanol becomes radioactive whereas, in the presence of ¹⁴C-valine, both isobutanol and 3-methylbutanol acquire label. The specific radioactivity of these compounds is, in all cases, inferior to that of the parent amino acids showing that a part of these higher alcohols is always formed by the synthetic functions of the yeast. The results suggest that the regulating effect of the valine and leucine level of the medium on the synthesis of these amino acids in yeast cells is rather limited. They furthermore indicate that intact assimilation of amino acids is not the only mode of their utilization, even in media with excessive levels of amino acids. An attempt is made to explain the dependence of the anabolic and catabolic modes of higher alcohol formation upon the nitrogenous nutrient level of the medium. The presence of radioactive isoleucine as impurity in the ¹⁴C-leucine used has allowed an approximative calculation of the formation of 2-methylbutanol from isoleucine which is very similar to the formation of isobutanol from valine.     

AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

Studies on Wheat Steep Liquor Produced During Starch Production

Chemical analysis of wheat steeping liquor, produced from soaking of wheat grains revealed the presence of 1% total soluble solids with 7,15% proteins. A method was devised to obtain large amounts of precipitates with protein content up to 9,84%. Glutamic acid and lysine constitute the largest proportions of the amino acids. Nine essential amino acids were found. The raw wheat steeping liquor and the supernatant resulting after separation of the protein were used as a growth medium for the production of fodder yeast Candida tropicalis.     

Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast,Pichia pastoris and characterization of the secreted product

Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q³⁰²) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0·47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA. Some approaches to minimizing the proteolysis of scu-PA-Q³⁰² were attempted. Addition of Triton X-100, l-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q³⁰² and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation. Co-expression of scu-PA-Q³⁰² and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu-PA-Q³⁰² was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose. The purified protein had a molecular weight of 47 kDa. It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain. N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast. Scu-PA-Q³⁰² closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.     

Biochemical composition of two red seaweed species grown on the Brazilian coast

BACKGROUND: Algae species have been used as an important source of food because they are highly nutritive considering their vitamin, protein, mineral, fiber, essential fatty acid and carbohydrate contents. However, a large number of seaweeds have been poorly studied, especially Brazilian species. Two red macroalgae species from the Brazilian coast (Plocamium brasiliense and Ochtodes secundiramea) were assessed with respect to their total lipid, fatty acid, total nitrogen, protein, amino acid and total carbohydrate contents. RESULTS: The total lipid contents (dry weight) were 36.3 and 35.4 g kg^?1; fatty acid contents were 9.3 and 12.1 g kg^?1; total nitrogen contents were 37.4 and 24.9 g kg^?1; protein contents were 157.2 and 101.0 g kg^?1; amino acid contents were 127.5 and 91.4 g kg^?1; and total carbohydrate contents were 520.3 and 450.7 g kg^?1 for P. brasiliense and O. secundiramea, respectively. CONCLUSION: Considering these compositions, both algae species were determined to have sources of protein, essential amino acids and carbohydrates similar to the edible seaweeds Laminaria japonica and Palmaria palmata. Copyright © 2011 Society of Chemical Industry     

Production of Candida utilis biomass as aquaculture feed

Fish offal-peat compost was hydrolysed and the resultant liquid extracts used as substrate in the cultivation of the yeast Candida utilis. The yeast was able to grow in this culture medium, attaining a growth yield of approximately 260 g biomass kg^?1 total carbohydrate consumed. The biomass produced had a good amino acid composition, with a protein content of 520 g kg^?1, and a relatively low lipid content. Dry yeast was used as a feed component in the diet of cultivated rainbow trout (Oncorhynchus mykiss) in which it substituted well for other, traditional sources of protein.     

Production of Polysaccharide by Rahnella aquatilis with Whey Feedstock

The rates were monitored on biomass increase, polysaccharide production and viscosity development of whey broth and a control synthetic broth during fermentation by Rahnella aquatilis and organic acids, lactose, peptides and free amino acids were measured. Growth curves were similar and characterized by maximum specific growth rates of 0.61 h^?1 for whey and 0.63 h^?1 for synthetic medium. The yields of polysaccharide were 0.59 g/g_lactose for the synthetic medium and 0.56 for whey. Small peptides (<4,000 Da) and most free amino acids in both fermentation media were consumed within 24h.     

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca²⁺-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.     

XV. Yeast sequencing reports. Nucleotide sequence of the GDS1 gene of Saccharomyces cerevisiae

We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae. The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database. GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms. Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product. The sequence has been deposited in the GenBank data library under Accession Number U18262.     

Composition and nutritional evaluation of Aspergillus oryzae biomass grown on palm oil processing effluents

Aspergillus oryzae biomass grown on effluents produced during the extraction of palm oil was found to have a crude protein content of 39.6 g 100 g^?1 and a ‘true protein’ content of 32.1 g 100 g^?1. Amino acid analysis showed that the essential amino acid content was 58.2 g 16 g^?1 N and the essential amino acid index was calculated to be 87.4. The sulphur-containing amino acids were present at a combined level of 2.8 g 16 g^?1. In rat feeding trials the biomass had a biological value of 0.68 ± 0.03, a net protein utilisation of 0.65 ± 0.05 and a true digestibility of 0.96 ± 0.05 based on its crude protein content. No unusual fatty acids were detected in the biomass.     

Yeast mutant affected for viability upon nutrient starvation: Characterization and cloning of the RVS161 gene

In yeast, nutrient starvation leads to entry into stationary phase. Mutants that do not respond properly to starvation conditons have been isolated in Saccharomyces cerevisiae. Among them the rvs161 mutant (RVS for R educed V iability upon S tarvation) is sensitive to carbon, nitrogen and sulphur starvation. When these nutrients are depleted in the medium, mutant cells show cellular viability loss with morphological changes. The mutation rvs161-1 is very pleiotropic, and besides the defects in stationary phase entry, the mutant strain presents other alterations: sensitivity to high salt concentrations, hypersensitivity to amino acid analogs, no growth on lactate or acetate medium. The addition of salts or amino acid analogs leads to the same morphological defects observed in starved cells, suggesting that the gene could be implicated mainly in the control of cellular viability. The gene RVS161 was cloned; it codes for a 30,252 daltons protein. No homology was detected with the proteins contained in the databases. Moreover, Southern analysis revealed the presence of other sequences homologous to the RVS161 gene in the yeast genome.     

The Branched-Chain Amino Acid Permease Gene of Saccharomyces cerevisiae,BAP2, Encodes the High-Affinity Leucine Permease (S1)

The amino acid leucine has been shown previously to be transported into a yeast cell by at least three permeases: the general amino acid permease, a high-affinity permease (S1) and a low-affinity permease (S2). We isolated the gene BAP2 as a multicopy suppressor of the YPD⁻ phenotype of aat1leu2 yeast. BAP2 has been identified previously as encoding an amino acid permease which transports branched-chain amino acids. In order to align the genetic and biochemical studies of leucine uptake we completed a detailed kinetic analysis of yeast strains in which the BAP2 gene was disrupted and compared this to the kinetics of uptake of the parental strain. We demonstrate that BAP2 encodes the high-affinity leucine permease previously called S1. © 1997 John Wiley & Sons, Ltd.     

Sequence analysis of a 14·2 kb fragment of Saccharomyces cerevisiae chromosome XIV that includes the ypt53, tRNALeu and gsr m2 genes and four new open reading frames

As part of the EU yeast genome program, a fragment of 14 262 bp from the left arm of Saccharomyces cerevisiae chromosome XIV has been sequenced. This fragment corresponds to cosmid 14-14b and is located roughly 130 kb from the centromere. It contains four new open reading frames which encode potential proteins of more than 99 amino acids, as well as the ypt53, tRNA^Leu and gsr m2 genes. The putative protein N2212 is similar to the ribosomal protein S7 from humans. N2215 contains several predicted transmembrane elements. N2231 contains regions which are rich in acidic, as well as basic, residues which could form α-helical structures. Similar regions are found in a variety of proteins including glutamic acid rich protein, trichohyalin, caldesmon, Tb-29 and several cytoskeleton-interacting proteins. The sequence has been entered in the EMBL data library under Accession Number X85811.     

SOME CONSIDERATIONS OF THE FLOCCULATION CHARACTERISTICS OF ALE AND LAGER YEAST STRAINS*

While some ale yeast strains are able to flocculate when cultured in a defined medium of glucose, ammonium salts, vitamins and ions, others require the presence of a nitrogen-containing inducer in the growth medium. On the other hand, all flocculent lager strains examined to date are able to flocculate after being cultured in a defined medium and do not appear to require the addition of inducer material to the growth medium. The inducer material present in wort has been identified as peptide. By the use of ion exchange chromatography the peptide fraction that induces flocculation has been found to contain a high level of acidic amino acid residues with a very similar structure to that reported for the α-factor involved in sexual agglutination of haploid α and a cells of Sacch. cerevisiae. Studies on the adsorption of Ca⁺⁺ ion by the cell wall failed to reveal any significant differences in total uptake between flocculent and non-flocculent cultures. It would appear that Ca⁺⁺ ions are bound less tightly by non-flocculent cells than by flocculent cells. The contribution of calcium to flocculation is not the absolute amount of this ion adsorbed by the yeast cell wall but rather the stereo-specific manner by which it is bound, i.e., its position relative to the three-dimensional structure of the yeast cell wall.     

Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Screening of drug‐resistant mutants of sake yeast strains has been a major method for creation of superior strains. We attempted to create a valproic acid (VPA)‐resistant mutant strain from sake yeast Kyokai No. 7 (K7). VPA is a branched‐chain fatty acid and is an inositol synthesis inhibitor in mammals and yeast. We succeeded in isolating a mutant of strain K7 that can survive long‐term in a VPA‐containing medium. This strain, K7‐VPA^LS, is significantly more resistant to not only VPA‐induced cell death but also ethanol in comparison with the parent strain. Further experiments showed that the new strain is likely to have a deficiency in inositol and/or phosphatidylinositol synthesis. The major characteristics of sake brewed by strain K7‐VPA^LS (compared with K7) were lower amino acidity, higher isoamyl acetate content without an increase in the isoamyl alcohol level and changes in constituent organic acids, particularly higher malate and succinate but lower acetate concentrations. In addition, taste sensor analysis revealed that K7‐VPA^LS‐brewed sake has milder sourness and higher saltiness or richness than K7‐brewed sake does. High isoamyl acetate production may be related to a deficiency in phosphatidylinositol because this compound directly inhibits alcohol acetyltransferase, an enzyme responsible for isoamyl acetate synthesis. Strain K7‐VPA^LS grew more rapidly than the parental strain did in a medium containing acetate as a sole carbon source, indicating that K7‐VPA^LS effectively assimilates acetate and converts it to malate and succinate through the glyoxylate cycle. Thus, strain K7‐VPA^LS shows improved characteristics for brewing of high‐quality sake. Copyright © 2017 The Institute of Brewing & Distilling     

Nutritional evaluation of the fungus Phycomyces blakesleeanus as a protein source

The quality of the protein of the carotenogenic fungus Phycomyces blakesleeanus Burgeff, strain NRRL 1555 ( - ), was evaluated with rats and observed to have a protein efficiency ratio of 1.4. The limiting amino acids were tryptophan, sulphur-containing amino acids, lysine and isoleucine. Upon incorporation into diets containing 100 g kg⁻¹ protein the proportions of the above mentioned amino acids of the optimal recommended dietary allowances for growing rats were 33.0%, 57.4%, 74.5% and 87.2% respectively. Furthermore, the fungal protein was found to contain an excess of histidine about eight fold more than the recommended dietary allowance for growing rats. Some safety aspects were examined and, although the fungus was consumed in high dietary concentrations, 242 g kg⁻¹ of the diet, no pronounced toxicological effects were observed.