Cross-breeding of distillers' yeast by hybridization of spore derived clones

0 K e y w o r d s : S a c c h a r o m y c e s cerevisiae , sporulation, hybridization, selection , f e r m e n t a t i o n , e t h a n o l t o l e r a n c e 1 Department of Physiology, Carlsberg Laboratory , Gamle Cadsberg Vej 10, DK-2500 Copenhagen Valby 2 Danish Distillers Ltd. , Raffinaderivej 10, DK-2300 Copenhagen S Four distillers' yeasts, strains A, B, C and D yielded 1-, 2-, 3- and 4-spored asci upon sporulation. Isolated a and a mater strains, recovered from 4-spored asci by microdissection, were intercrossed to produce a total of 425 hybrids. The meiotic segregants and hybrids were tested for fermentation kinetics in a synthetic sucrose medium using a small scale batch system. A few hybrids exceeded the performance of the parent strains with respect to fermentation rate and final degree of fermentation. A population derived from random mass mating of spores was subjected to selection for ethanol tolerance by repeated fermentations. Among a total of 56 clones tested most gavethe same or a higher final ethanol concentration than the reference strain also when the fermentation rate was lower. 1. I N T R O D U C T I O N The distilling industry requires fast fermenting ethanol tolerant yeasts which possess the ability to produce high concentrations o f ethanol while remaining viable. A range o f genetic and physiological factors affect the fermentation performance o f yeasts. T h e response o f yeast cells to ethanol is complex and has been shown to be strain dependant and affected by m a n y genes (10, 15, 16, 17, 32). Hybridization and selection are considered important tools for the i m p r o v e m e n t o f yeast strains in the brewing ( l 1, 17, 33), baking (4, 12, 34, 35) and winemaking (10, 29, 32) industries. Mutagenesis has been used in the breeding o f ethanol tolerant wine yeasts (1) and selective - hybridization has produced hybrids with a better wine making potential (10, 32). Ethanol resistant variants have been recovered by selection in batch (8) as well as continuous cultures (3, 2 l) and protoplast fusion has been used to create strains with improved fermentation characteristics (22, 27). In addition to the genome, the e n v i r o n m e n t is important for ethanol tolerance and fermentation performance (6, 18). Osmotic pressure (25), the presence o f unsaturated fatty acids and sterols (2, 5, 19, 30, 31) and temperature (13, 14, 18, 23, 24) influence the effect o f ethanol on yeast cells. Inheritance o f the fermentation ability and tolerance to ethanol formed during the fermena adel hisl ctadel hisl Production strain DDSF 102") Production strain DDSF Y) Production strain DDSF 6") Production strain DDSF 72") Course manual on yeast genetics, Cold Spring Harbor Laboratory (28) Course manual on yeast genetics, Cold Spring Harbor Laboratory (28) A79xD64 Population derived from random mass mating of spores from AD236 "~Theproduction strains are from the Danish Distillers Ltd. Culture Collection. tation were in the present work studied in production strains, meiotic segregants and hybrids. Small scale batch fermentations using a synthetic sucrose medium were used in the evaluation of fermentation performance as this method is simple and reliable and thus useful when large numbers of yeast strains are to be tested. 2. MATERIALS AND M E T H O D S 2.1. Strains and media The principal strains of Saccharomyces cerevisiae used in this study are listed in Table I. Strain A is a production strain used both as a distillers' yeast and for the manufacture of bakers' yeast strains. The following media were employed (28): YPD contained 1% Difco Bacto Yeast Extract, 2% Difco Bacto Peptone, 2% Dextrose. Presporulation medium was 0.8% Difco Bacto Yeast Extract, 0.3% Difco Bacto Peptone, 10% glucose (pH 5.0). Sporulation medium was 1% potassium acetate, 0.1% Difco Bacto Yeast Extract, 0.05% glucose. The synthetic fermentation medium consisted of 30% sucrose, 0.67% Difco Yeast Nitrogen Base without amino acids, and was buffered with t% succinic acid and 0.6% sodium hydroxide. Plates were solidified with 2% Difco Bacto Agar. 2.2. Sporulation and spore recovery After two days of growth on a pre-sporulation plate, yeast cells were transferred to a sporulation plate and incubated 3-5 days at 30 ~ Sporulation was detected by staining with malachite green - safranine (26). Clones from single spores were obtained by microdissection (28). Isolation of random spores proceeded as described by GJERMANSENand SIGSGAARD(11), except for the ascus opening and the killing of the vegetative cells, where the procedure described by CHRISTENSEN(7) was used. 2.3. Determination of mating type After 2 days at 30 ~ on YPD plates spore colonies were transferred to sporulation medium and tested for sporulation as described in 2.2. Non-sporulating colonies were considered to be potential mater strains. Mating ability was determined by mixing, on YPD plates, with the tester strain (D273-11 a) and the a tester strain (D286-2a). After 2 days the cell mass was replica plated onto sporulation medium; subsequent sporulation after 2-3 days on sporulation medium was taken as an indication of mating. A colony was designed mating type a when sporulation occurred after mixing with the cttester and as mating type ct when sporulation took place after mixing with the a tester. For practical purposes, the colonies which would not sporulate after mixing with either of the tester strains were classified as non-maters. 2.4. Construction of hybrids The isolated strains of opposite mating type were crossed pairwise by mixing them on a YPD plate and incubating them at 30 ~ for 24 hours. Subsequently, the mixture was streaked on YPD plates for single colonies and the larger single colonies were isolated and checked for sporulation. 2.5. Test fermentatiGn and analysis of the fermented substrate Hybrids were propagated by inoculating cell material from a distinct colony into 10 ml YPD in test tubes followed by aerobic incubation at 30 ~ for 20 hours. Approximately 1.108 cells from this culture were transferred to 250 ml, YPD at 30 ~ and allowed to grow aerobically at 30 ~ for 20 hours. The cell density was then approximately 1.108 cells, ml~. The yeast cells were harvested by centrifugation and washed with sterile cold distilled water. Approximately 5.109 cells were suspended in 10 ml fermentation medium in 250 ml conical flasks stoppered with fermentation locks filled with concentrated sulphuric acid. The fermentation was allowed to take place with agitation for 44 hours at 30 ~ interrupted by weight determinations at 0, 2, 4, 8, 20, 22, 24, 27 and 44 hours. The decrease in weight measures the CO2 loss from the system, a loss which is proportional to the ethanol produced and the reduction in sucrose. A weight loss of 1.00 g corresponds to the formation of 1.05 g ethanol. The final ethanol concentration was determined either by the oxidative method (9) or alternatively by HPLC in a BIO-RAD Ion Exclusi (...truncated)