Kozo Ouchi

A mutated ARO4 gene for feedback-resistant DAHP synthase which causes both o-fluoro-dl-phenylalamine resistance and β-phenethyl-alcohol overproduction in Saccharomyces cerevisiae

Summaryo-Fluoro-dl-phenylalanine (OFP)-resistant mutants which overproduce β-phenethyl-alcohol were isolated from a laboratory strain of Saccharomyces cerevisiae. Cells of one of the mutants accumulated tyrosine and phenylalanine 1.5–3 fold more than did wild-type cells. Its 3-deoxy-d-arabino-hepturosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15), encoded by ARO4, was free from feedback inhibition by tyrosine. Genetic analysis revealed that the mutation was controlled by a single dominant gene, ARO4-OFP, encoding feedback-resistant DAHP synthase by tyrosine, and that this gene caused both the OFP resistance and β-phenethyl-alcohol overproduction. This was supported by molecular genetic studies using cloned ARO4 both from the wild-type and its mutant strain.

Factors affecting the ethanol productivity of yeast in molasses

The ethanol production by a laboratory yeast strain, X2180-1B, was less than half that by an alcohol yeast, YOY655, in a molasses medium containing 30% sugars, although X2180-1B produced approximately the same amount of ethanol as YOY655 in a nutrition medium with the same sugar content. The weak productivity of X2180-1B in the molasses was ascribed to the limitation of sucrose hydrolysis in the molasses. The invertase activity of X2180-1B was 0.019 (mmol sucrose/min/mg protein) in the nutrition medium, but substantially zero in the molasses, while that of YOY655 was 1.75 in the nutrition medium and 1.15 even under the inhibitory conditions in molasses. External addition of invertase greatly enhanced the ethanol productivity of only X2180-1B. The inhibitory factors of invertase in molasses were heat-stable and dialyzable substances.

Effects of yeast invertase on ethanol production in molasses

Abstract Ethanol fermentation by an alcohol yeast, YOY655, was slower in molasses than in a nutrition rich medium with the same sugar content. Osmolality was much higher in the molasses, and the slower fermentation in the molasses was ascribed to depressed fermentation under the high osmotic pressure. Yeast invertase was an important factor in regulation the osmolality and the fermentation rate in the molasses.

Isolation and genetic study of p-fluoro-dl-phenylalanine-resistant mutants overproducing β-phenethyl-alcohol in Saccharomyces cerevisiae

Summaryp-Fluoro-dl-phenylalanine (PFP)-resistant mutants which produce a large amount of β-phenethyl-alcohol, a rose-like flavor component, were isolated from the isogenic strains X2180-1A and X2180-1B of Saccharomyces cerevisiae. Cells of these mutants accumulated phenylalanine and tryptophan more than 3-fold times that of wild-type cells, while they accumulated less than half the tyrosine. The activity of prephenate dehydrogenase (PDG) (EC 1.3.1.12) was markedly decreased while that of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) was increased. Genetic analysis revealed that the mutation occurred at the TYR1 locus, encoding PDG, and that the mutated TYR1 gene, tyr1-pfp, caused both PFP resistance and β-phenethyl-alcohol overproduction. This was supported by molecular genetic studies with cloned tyr1-pfp DNA.

Molecular Breeding of a Sake Yeast with a Mutated ARO4 Gene Which Causes Both Resistance to o-Fluoro-DL-Phenylalanine and Increased Production of β-Phenethyl Alcohol

Abstract A sake yeast, Saccharomyces cerevisiae, was transformed with a YCp vector containing a mutated ARO4 gene (ARO4-OFP) derived from S. cerevisiae which encodes the 3-deoxy- d -arabino-heptulosonate-7-phosphate (DAHP) synthase released from feedback inhibition by tyrosine. The transformant showed feedback-insensitive DAHP synthase activity and simultaneously acquired both o-fluoro- dl -phenylalanine (OFP)-resistance and increased production of β-phenethyl alcohol and tyrosine. The greater production of the alcohol and its acetate, rose-like flavor components, improved the aroma and flavor of sake prepared experimentally with the transformant. The ARO4-OFP gene was also demonstrated to be available as a dominant selection marker in yeast vectors for the transformation of industrial strains of S. cerevisiae.

Reconstruction of ethanol fermentation in permeabilized cells of the yeast Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae cells from a stationary culture were permeabilized with 1% toluene, 4% ethanol and 0.075% Triton X-100. Not only sugars but also ATP, NAD + , magnesium and inorganic phosphate must be simultaneously added to initiate the ethanol fermentation. The optimal pH for the fermentation was between 6.9 to 7.0. Sucrose was a better substrate than glucose. Ethanol fermentation was greatly stimulated by the addition of 1 mM arsenate. Under this condition, permeabilized cells continued to produce ethanol for more than one hour at the rate of 0.141 mmol ethanol/min/mg protein. Methanol inhibited the fermentation with intact cells but did not inhibit the one using permeabilized cells. In contrast, propanol inhibited fermentations both with intact and permeabilized cells.

Cloning and characterization of the CSF1 gene of Saccharomyces cerevisiae, which is required for nutrient uptake at low temperature.

We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial bakers yeast that have practically no fermentation capacity at 5 degrees C and return to their normal capacity at 25 to 40 degrees C. CSF1 was cloned by functional complementation of the Csf phenotype. CSF1 contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids. The nucleotide sequence was identical to that of the YLR087C gene in the Saccharomyces genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein. Gene disruption shows that CSF1 is required for growth and fermentation only at low temperatures. Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10 degrees C. The disruptant cells had the same glucose uptake rates as the parental cells at 30 degrees C, but three- to fivefold-lower rates than the parental cells at 10 degrees C. These findings suggest that CSF1 associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.

Feedback-insensitive mutation of 3-deoxy-d-arabino-hepturosonate-7-phosphate synthase caused by a single nucleotide substitution of ARO4 structural gene in Saccharomyces cerevisiae

The Saccharomyces cerevisiae ARO4 gene product 3-deoxy- d -arabino-hepturosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15), the first common enzyme in aromatic amino acid biosynthesis, is subject to feedback inhibition by tyrosine. A mutated ARO4 gene for feedback-insensitive DAHP synthase which causes both o-fluoro- dl -phenylalanine-resistance and β-phenethyl-alcohol-overproduction was cloned. The DNA sequence of the mutant gene revealed a single nucleotide difference in the structural region from its wild-type gene. The mutation results in a glutamine-to-lysine amino acid substitution in the DAHP synthase.

A DNA construct useful for specific chromosome loss in Saccharomyces cerevisiae

Abstract A GAL1-R/RS-RS cassette DNA was constructed to induce the loss of a specific chromosome in Saccharomyces cerevisiae. The cassette is composed of two specific recombination sites (RS) derived from the pSR1 plasmid of Zygosaccharomyces rouxii, and the site-specific recombinase gene, R, placed downstream of the GAL1 promoter. To delete a certain chromosome, the cassette was inserted into a site on that chromosome in a diploid cell by homologous recombination between a DNA fragment cloned on the cassette and the relevant site on the chromosome. When the transformant was cultivated in galactose medium, elimination of the target chromosome occurred by R-promoted site-specific recombination between two unequal RS sites. Using this method, we demonstrated the loss of chromosomes III, V, VII and XV in heterozygous diploids. Aneuploids appeared at a frequency of 45–85% in the colonies examined after the induction of chromosome loss and were easily distinguished since they gave rise to smaller colonies than did the diploids. We confirmed that the aneuploids often duplicated the residual monosomic chromosome to restore the chromosome balance during mitotic growth. Such diploids formed colonies that were as large as the parent. This method is useful for conversion of heterozygous chromosomes into homozygous (or uniparental) ones in hybrid strains, and is also useful for chromosome loss mapping.

Effect of chromosome loss on the leavening ability of Saccharomyces cerevisiae in dough

SummaryTo investigate the leavening ability of yeast in dough, chromosome loss was induced by benomyl treatment in YOY1037, a diploid between a baking strain and a laboratory strain, and its effect on the leavening ability was studied. When benomyl-treated cells were spread on plates with a dye indicator for ploidy, about 20% of the visible colonies were stained dark blue or dark purple; the rest stained pale blue, similar to the diploid YOY1037. Strains showing the MATα phenotype, and non-galactose fermenting strains, apparently having lost particular chromosomes, were observed only in those with darkcoloured colonies. Strains with dark-coloured colonies showed a wider range of leavening ability than did those with pale-coloured colonies.