Growth characteristics of Saccharomyces rouxii isolated from chocolate syrup.

We investigated the growth parameters of Saccharomyces rouxii isolated from spoiled chocolate syrup. The optimum pH range for S. rouxii was 3.5 to 5.5, whereas the minimum and maximum pH values that permitted growth were 1.5 and 10.5, respectively. For cells grown in 0 and 60% sucrose the optimum water activity (aw) values were 0.97 and 0.96, respectively. The optimum temperature for S. rouxii increased with a decreasing aw regardless of whether glucose or sucrose was used as the humectant. The optimum temperatures for S. rouxii were 28 degrees C at an aw of greater than 0.995 and 35 degrees C at an aw of 0.96 to 0.90 in 2 X potato dextrose broth with sucrose. Increasing the sorbate concentration (from 0.03 to 0.10%) caused the growth of S. rouxii to become more inhibited between aws of greater than 0.995 and 0.82. S. rouxii did not grow when the sorbate level was 0.12% (wt/vol). At lower sorbate levels, the effect of sorbate on the growth of S. rouxii depended on the aw level. Lowering the aw enhanced the resistance of S. rouxii to increasing concentrations of potassium sorbate. Permeability and polyol production are discussed with respect to sorbate tolerance of S. rouxii at different aw levels.     

Effect of threonine, cystathionine, and the branched-chain amino acids on the metabolism of Zygosaccharomyces rouxii*

Zygosaccharomyces rouxii is an important yeast in the formation of flavor in soy sauce. In this study, we investigated the separate effects of exogenous threonine, cystathionine, and the branched-chain amino acids on the metabolism of Z. rouxii. The addition of these amino acids had significant effects on both Z. rouxii growth and glycerol and higher alcohol production. It also seemed that Z. rouxii displayed the Crabtree effect, which was independent of the added amino acids. Furthermore, we investigated the regulation of the metabolism of alpha-ketobutyrate, which is a key-intermediate in Z. rouxii amino acid metabolism. Threonine and cystathionine were introduced separately to stimulate the formation rate of alpha-ketobutyrate and the branched-chain amino acids to inhibit its conversion rate. Enzyme activities showed that these amino acids had a significant effect on the formation and conversion rate of alpha-ketobutyrate but that the alpha-ketobutyrate pool size in Z. rouxii was in balance all the time. The latter was confirmed by the absence of alpha-ketobutyrate accumulation.     

Regulation of aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii compared to Saccharomyces cerevisiae

To elucidate the growth inhibitory effect of threonine, the regulation of the aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii, an important yeast for the flavor development in soy sauce, was investigated. It was shown that threonine inhibited the growth of Z. rouxii by blocking the methionine synthesis. It seemed that threonine blocked this synthesis by inhibiting the conversion of aspartate. In addition, it was shown that the growth of Z. rouxii, unlike that of Saccharomyces cerevisiae, was not inhibited by the herbicide sulfometuron methyl (SMM). From enzyme assays, it was concluded that the acetohydroxy acid synthase in Z. rouxii, unlike that in S. cerevisiae, was not sensitive to SMM. Furthermore, the enzyme assays demonstrated that the activity of threonine deaminase in Z. rouxii, like in S. cerevisiae, was strongly inhibited by isoleucine and stimulated by valine. From this work, it is clear that the aspartate-derived amino-acid metabolism in Z. rouxii only partly resembles that in S. cerevisiae.     

Tools for the genetic manipulation of Zygosaccharomyces rouxii

A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP-kanMX-loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase.     

Production of higher alcohols during indonesian tapé ketan fermentation

A study was made of the higher alcohols (fusel oils) produced during the Indonesian tapé ketan fermentation using Amylomyces rouxii as the principal mold, alone or in combination with yeasts belonging to genera commonly found in the tapé ketan fermentation (Endomycopsis, Candida, and Hansenula). Total fusel oils increased with length of fermentation. Fusel oils detected in the product distillate included isobutanol and isoamyl and active amyl alcohols. No n-propanol was detected. Isobutanol and isoamyl alcohols were formed in the largest amounts. A. rouxii alone produced nearly the same quantity of fusel oils (total production, 275 mg/liter at 192 h) as it did in combination with Endomycopsis burtonii (total production, 292 mg/liter at 192 h).A. rouxii and Endomycopsis fibuliger produced fusel oils totaling 72 mg/liter at 32 h and 558 mg/liter at 192 h. A. rouxii in combination with Candida yeasts produced somewhat more fusel oils, ranging from 590 to 618 mg/liter at 192 h. A. rouxii in combination with Hansenula yeasts produced the least fusel oils, totaling 143 to 248 mg/liter at 192 h. During the first 36 h, production of fusel oils was higher at 30 and 35 degrees C than at 25 degrees C. At 48 h fusel oil production was slightly higher at 30 degrees C than at 35 degrees C. Beyond 48 h, production of fusel oils was higher at 25 degrees C. A. rouxii in combination with Hansenula anomala and Hansenula subpelliculosa produced considerable ethyl acetate, ranging from 145 to 199 mg/liter at 36 h and 354 to 369 mg/liter at 192 h.     

Expression of theSaccharomyces cerevisiae MPR1 gene encodingN-acetyltransferase inZygosaccharomyces rouxii confers resistance tol-azetidine-2-carboxylate

The osmotolerant yeast Zygosaccharomyces rouxii is sensitive to the toxic L-proline analogue, L-azetidine-2-carboxylate (AZC). The possibility of use of the Saccharomyces cerevisiae MPR1 gene (ScMPR1) encoding the AZC-detoxifying enzyme as a dominant selection marker in Z. rouxii was examined. The heterologous expression of ScMPR1 in two Z. rouxii strains resulted in AZC-resistant colonies, but that of ScMPR1 as a dominant marker gene in vectors was affected by a high frequency of spontaneously resistant colonies. The same was found for an AZC-sensitive S. cerevisiae strain in which the ScMPR1 was expressed. In both yeasts, ScMPR1 can be used only as an auxiliary marker gene.     

Substrate specificity and preference of Delta6-desaturase of Mucor rouxii

The Delta(6)-fatty acid desaturase is a key enzyme in the synthesis of an important fatty acid, gamma-linolenic acid. We have characterized, by heterologous expression in Saccharomyces cerevisiae, substrate specificity and preference of Delta(6)-desaturase of Mucor rouxii. Fatty acid supplementation was carried out based on the predicted enzyme topology, fatty acid phenotype and the corresponding metabolic pathway in M. rouxii. The enzyme has a broad substrate specificity as based on C15-C18. The result also supported classification of the M. rouxii Delta(6)-desaturase into a front-end desaturase. Interestingly, a relatively rare activity based on odd acyl chains and not described previously in other eukaryotic Delta(6)-desaturases was also observed.     

Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii

The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with both NaCl- and KCl-fortified growth media, with more tolerance being exhibited for the potassium salt. Washed and buffered cells from unfortified medium were challenged with a variety of compounds (and also with physical treatments) that potentially would elicit membrane perturbations. The efficacy of these brief treatments was judged primarily by monitoring subsequent viability. Change in the degree of expression of beta-fructofuranosidase (EC 3.2.1.26), which is cryptic in young cells of S. rouxii, was a second criterion. There was a linear correlation between cell death and enzyme expression for treatments with polyenes, detergents, some organic solvents which did not denature the enzyme, and various freeze-thaw regimens in graded amounts of glycerol. The species is relatively insensitive to polyene antimycotics, the order of decreasing effect being filipin, nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmotic shock than is Saccharomyces cerevisiae, but in neither species is beta-fructofuranosidase released to the medium. The sensitivity of S. rouxii to ionic detergents, but not to nonionic detergents, was rationalized as being due to cell wall discrimination against larger micelles for the nonionic examples. This was confirmed by showing that protoplasts were sensitive to both classes. In cultures older than 5 days the normal agreement between colony-forming units and methylene blue exclusion (another test of viability) no longer held. Delayed fermentation of sucrose by S. rouxii, which is a diagnostic feature of the species, is explained by death of some cells, expression of their beta-fructofuranosidase, and utilization of the monosaccharides by the surviving cells.     

Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I-VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type alpha and type beta) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

Effects of the amino-carbonyl reaction of ribose and glycine on the formation of the 2(or 5)-ethyl-5(or 2)-methyl-4-hydroxy-3(2H)-furanone aroma component specific to miso by halo-tolerant yeast

The formation of HEMF[2(or 5)-ethyl-5(or 2)-methyl-4-hydroxy-3(2H)-furanone], the aroma component specific to miso and soy sauce, was promoted by cultivating the halo-tolerant yeast, Zygosaccharomyces rouxii, in a medium including the amino-carbonyl reaction products based on ribose and glycine. The glucose concentration in the medium influenced the HEMF formation by Z. rouxii.     

THIAMINE AND NICOTINIC ACID: ANAEROBIC GROWTH FACTORS FOR MUCOR ROUXII.

Bartnicki-Garcia, S. (Rutgers, the State University, New Brunswick, N. J.), and Walter J. Nickerson. Thiamine and nicotinic acid: Anaerobic growth factors for Mucor rouxii. J. Bacteriol. 82:142-148. 1961.-Mucor rouxii requires preformed thiamine and nicotinic acid for anaerobic growth. Such requirements are not manifested during aerobic incubation. Aerobically, the fungus was shown to be able to synthesize both vitamins.The yeastlike form and the filamentous form of anaerobically grown M. rouxii exhibit the same vitamin requirements.Thiamine can be substituted by its thiazole moiety. Under certain conditions, nicotinic acid was partly substituted by tryptophan, kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid.Anaerobically. the fungus (thiamine requiring) was about ten times more susceptible to pyrithiamine antagonism than the same organism grown aerobically (thiamine independent).     

Biosorption of heavy metals using whole mold mycelia and parts thereof.

Biosorption of heavy metals was carried out using whole mycelia and selected components of Aspergillus niger, Rhizopus oryzae and Mucor rouxii. Binding of copper, cadmium, nickel and zinc was considerably improved by treating the cell wall fraction with 4 M NaOH at 121 degrees C. Chitosan contributed most to the biosorptive capacity. 0.96 mmol copper was bound by 1 g of the treated mycelium of M. rouxii DSM 1191.     

Activity of different ''killer'' yeasts on strains of yeast species undesirable in the food industry

Killer strains of the genera Saccharomyces, Hansenula and Kluyveromyces were tested for killing activity against yeasts that cause trouble in the food industry (in the genera Zygosaccharomyces, Kloeckera, Saccharomycodes and Schizosaccharomyces). Saccharomyces strains killed only Zygosaccharomyces rouxii strains, while non-Saccharomyces strains showed a wider anti-yeast spectrum. The Kluyveromyces phaffii killer strain was of particular interest because of its killer action against Kloeckera apiculata, Saccharomycodes ludwigii and Zygosaccharomyces rouxii.     

Microbial water relations. Effects of solute concentration on the respiratory activity of sugar-tolerant and non-tolerant yeasts.

The respiratory activity of the sugar-tolerant (osmophilic) yeast, Saccharomyces rouxii, and the non-tolerant species, Sacchromyces cerevisiae, were compared after growth in a complex basal medium, the medium supplemented with polyethylene glycol (mol. wt 200) to give a water activity of 0-95, and the medium supplemented with glucose (24 and 36%, w/v). The properties compared were Qo2 (glucose), NADH oxidase activity of isolated mitochondrial fractions, and cytochrome content. When grown in the basal medium S. cerevisiae was somewhat more active than S. rouxii by all criteria. Growth in the media supplemented were high glucose concentrations produced catabolite repression of respiration in S. cerevisiae but not in S. rouxii. The implications of this difference for polyol biosynthesis and the water relations of the sugar-tolerant species are discussed.     

Phosphofrucktokinase and glucose catabolism of Mucor and Penicillium species.

The primary catabolic pathways in the fungi Penicillium notatum and P. duponti, and Mucor rouxii and M. miehei were examined by measuring the relative rate of 14CO2 production from different carbon atoms of specifically labelled glucose. It was found that these organisms dissimilate glucose predominantly via the Embden--Meyerhof pathway in conjunction with the tricarboxylic acid cycle and to a lesser extent by the pentose phosphate pathway. Phosphofructokinase (EC 2.7.1.11) activity could not be detected initially in Penicillium species because of the interference from mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and NADH oxidase (EC 1.6.99.3). A combination of differential centrifuging and a heat treatment of Penicillium cell-free extracts in the presence of fructose-6-phosphate removed the interfering enzymes. The kinetic characteristics of phosphofructokinase from P. notatum and M. rouxii are described. The enzyme presents highly cooperative kinetics for fructose-6-phosphate. The kinetics for ATP show no cooperativity and inhibition by excess ATP is observed. The addition of AMP activated the P. notatum enzyme, relieving ATP inhibition; slight inhibition by AMP was observed with the M. rouxii enzyme. In contrast M. rouxii pyruvate kinase (EC 2.7.1.40) is activated 50-fold by fructose-1,6-diphosphate whereas pyruvate kinase from P. notatum and P. duponti were unaffected by fructose-1,6-diphosphate.     

Expression of plasma membrane proton-ATPase gene in salt-tolerant yeast Zygosaccharomyces rouxii is induced by sodium chloride

Abstract The amounts of mRNA and protein of plasma membrane proton-ATPase were measured in the salt-tolerant yeast Zygosaccharomyces rouxii by Northern and Western blot analyses. Although their amounts were independent of growth phase, their synthesis were induced when yeast cells were grown in the presence of NaCl or were subjected to NaCl shock. This finding was consistent with our previous result that plasma membrane proton-ATPase activity was elevated in Z. rouxii cells grown in medium containing high concentrations of NaCl.