To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30؇C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22؇C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol. The strains tested recovered their ability to yield maximal sporulation and tetrad formation after recovering their own mitochondria.

This review examines the desired characteristics and possibilities of genetic manipulation of industrial yeasts. Brewing, wine, and distillers' yeasts are characterized by good utilization of sugar, alcohol tolerance and production, flavor and aroma, flocculation, fermentation rate, cropping ratio, ability to utilize trisaccharides, dextrines and starch, rapid onset of fermentation, nonfoaming fermentation, and low H 2 S production. The characteristics required for a good bakers' yeast include high potential glycolytic activity, ability to adapt rapidly to changing substrates, high invertase activity, high potential maltose fermentation rate and quality in terms of dough fermentation, storage ability, and osmotic resistance to salts and sugars. In all of these cases, the major desirable factors are under genetic control and hence are potentially subject to improvement by genetic manipulation. However, this control is thought to be governed by several, in most cases, unidentified genes. So, although most genetic techniques developed for laboratory yeasts are potentially applicable to the study and improvement of the properties of industrial yeast strains, traits such as homothallism, aberration in mating behavior, polyploidy, aneuploidy, poor sporulation and poor spore viability, etc., make conventional hybridization and isolation of single spore clones more difficult.

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Saccharomyces cerevisiae baker's yeast mutants which produce 3 to 17 times as much lysine as the wild type, depending on the nitrogen source, have been selected. The baker's yeast strain was grown in a pH-regulated chemostat in minimal medium with proline as the nitrogen source, supplemented with increasing concentrations of the toxic analog of the lysine S-2-aminoethyl-L-cysteine (AEC). The lysine-overproducing mutants, which were isolated as AEC-resistant mutants, were also resistant to high external concentrations of lysine and to ␣-aminoadipate and seemed to be affected in the lysine biosynthetic pathway but not in the biosynthetic pathways of other amino acids. Lysine overproduction by one of the mutants seemed to be due to, at least, the loss of repression of the homocitrate synthase encoded by the LYS20 gene. The mutant grew slower than the wild type, and its dough-raising capacity was reduced in in vitro assays, probably due to the toxic effects of lysine accumulation or of an intermediate produced in the pathway. This mutant can be added as a food supplement to enrich the nutritive qualities of bakery products, and its resistance to ␣-aminoadipate, AEC, and lysine can be used as a dominant marker.

Three transformant (Mel ؉) Saccharomyces cerevisiae baker's yeast strains, CT-Mel, VS-Mel, and DADI-Mel, have been characterized. The strains, which originally lacked ␣-galactosidase activity (Mel ؊), had been transformed with a DNA fragment which possessed an ILV2-SMR1 allele of the ILV2 gene and a MEL1 gene. The three transformed strains showed growth rates similar to those of the untransformed controls in both minimal and semi-industrial (molasses) media. The ␣-galactosidase specific activity of strain CT-Mel was twice that of VS-Mel and DADI-Mel. The yield, Y X/S (milligrams of protein per milligram of substrate), in minimal medium with raffinose as the carbon source was 2.5 times higher in the transformed strains than in the controls and was 1.5 times higher in CT-Mel than in VS-Mel and DADI-Mel. When molasses was used, Y X/S (milligrams of protein per milliliter of culture) increased 8% when the transformed strains CT-Mel and DADI-Mel were used instead of the controls. Whereas no viable spores were recovered from either DADI-Mel or VS-Mel tetrads, genetic analysis carried out with CT-Mel indicated that the MEL1 gene has been integrated in two of three homologous loci. Analysis of the DNA content by flow cytometry indicated that strain CT-Mel was 3n, whereas VS-Mel was 2n and DADI-Mel was 1.5n. Electrophoretic karyotype and Southern blot analyses of the transformed strains showed that the MEL1 gene has been integrated in the same chromosomic band, probably chromosome XIII, in the three strains. Tetrads of CT-Mel strain segregated 4؉:0؊, 3؉:1؊ and 2؉:2؊, for both positive MEL1 Southern blots and ␣-galactosidase activity, indicating, as expected, integration of the MEL1 gene in two of three homologous chromosomes.