Construction of Lactose-Assimilating and High-Ethanol-Producing

APPLIED

AND

Vol. 51, No. 2

ENVIRONMENTAL MICROBIOLOGY, Feb. 1986, p. 362-367

0099-2240/86/020362-06$02.00/0 Copyright © 1986, American Society for Microbiology

Construction of Lactose-Assimilating and High-Ethanol-Producing Yeasts by Protoplast Fusion F. FARAHNAK, T. SEKI,t DEWEY D. Y. RYU,* AND D. OGRYDZIAK University of California, Davis, California 95616

Received 26 April 1985/Accepted 22 September 1985 The availability of a yeast strain which is capable of fermenting lactose and at the same time is tolerant to high concentrations of ethanol would be useful for the production of ethanol from lactose. Kluyveromyces fragilis is capable of fermenting lactose, but it is not as tolerant as Saccharomyces cerevisiae to high concentrations of ethanol. In this study, we have used the protoplast fusion technique to construct hybrids between auxotrophic strains of S. cerevisiae having high ethanol tolerance and an auxotrophic strain of lactose-fermenting K. fragilis isolated by ethyl methanesulfonate mutagenesis. The fusants obtained were prototrophic and capable of assimilating lactose and producing ethanol in excess of 13% (vol/vol). The complementation frequency of fusion was about 0.7%. Formation of fusants was confirmed by the increased amount of chromosomal DNA per cell. Fusants contained 8 x 10-8 to 16 x 10-8 ,ug of DNA per cell as compared with about 4 x 10-8 ,ug of DNA per cell for the parental strains, suggesting that multiple fusions had taken place.

Yeast nitrogen base (YNB; Difco) medium was used as the synthetic minimal medium, and glucose was used as the carbon source. The sporulation media for K. fragilis contained 2% malt agar (Difco). This yeast sporulated after 24 to 48 h of incubation at 30°C on this medium. Spore isolation. Cells were harvested from the sporulation plate with 5 ml of sterile distilled water, centrifuged, washed again with sterile distilled water, and suspended in 5 ml of Zymolyase solution containing 10 mM 2-mercaptoethanol, 50 mM phosphate buffer (pH 7.5), and 0.5 mg of Zymolyase60,000 (Seikagake Kogyo Co., Tokyo, Japan) per ml. The suspension was incubated for 2 to 4 h at 30°C, during which time the cells were examined under the microscope for cell wall hydrolysis by monitoring the lysis of spheroplasts when they were subjected to a hypotonic environment such as distilled water. After most of the vegetative cells were eliminated, microscopic counts of the treated cell and spore mixture in a hemacytometer showed that about 90% of the population was spores. Mutagenesis. Ethyl methanesulfonate (EMS) was used as the mutagenizing agent. Spores were washed with sterile water three times, suspended in a solution containing 0.1 M potassium phosphate buffer (pH 8.0) and 7% EMS, and were incubated at 30°C for 1 h with occasional shaking. After this period, spores were washed with distilled water and treated with 5% sodium thiosulfate for 10 to 15 min. Enrichment method. The enrichment method used was the nystatin procedure described below by Snow (26). Ascospores were allowed to germinate in YPD for 18 h to fix the mutation. After this period, the cells were harvested and washed, and about 108 cells were transferred to 10 ml of YNB medium without amino acids in the presence of the antibiotic nystatin. Nystatin solution was prepared by making a 1:10 dilution of 1 mg of nystatin per ml in 95% alcohol with sterile distilled water and was used without additional sterilization. One milliliter of this 100-,ug/ml solution was added to the 10-ml culture. After incubation at 30°C for 1 h, the culture was washed three times, and 0.1 ml was spread onto YPD plates and incubated for at least 72 h at 30°C. After the colonies appeared, they were replica plated onto YNB medium

Lactose is one of the several least expensive carbon sources for ethanol production. Although attempts have been made to use lactose for alcohol production since 1940 (21), the major problem has been that Saccharomyces cerevisiae is unable to ferment lactose. The relatively small number of other microorganisms which are capable of fermenting lactose usually give a low yield of ethanol. Kluyveromyces fragilis has been known to be capable of fermenting lactose (15). However, it has been shown that only a fraction of the available lactose was converted to alcohol, possibly due to an alcohol inhibition (18). Whey, in addition to being an inexpensive feed-stock for ethanol production, is a serious pollutant in some cases. In 1974, for example, 32.5 x 109 lbs (ca. 14.7 x 109 kg) of whey was produced in the United States alone (18), which represented 1.6 x 109 lbs (ca. 7.3 x 108 kg) of lactose. If it could be converted into alcohol or other useful products, it would be a valuable carbon resource to the industry. In view of the importance and the need for development of a genetically constructed yeast strain which is capable of growing on lactose and producing ethanol in high concentration, we have undertaken this study. Protoplast fusion was a method of choice since it has been widely used for genetic improvement of industrial yeasts (9, 10, 17, 25, 27-30). This technique has been used extensively for intraspecific and interspecific transfer of nuclear genes of fungi (1, 2, 6-8). MATERIALS AND METHODS Microorganisms and media. The yeast strains used in this study were K. fragilis 55-55 (Department of Food Science and Technology, University of California, Davis) and S. cerevisiae STX 23-5B, ade4 trpl (Yeast Genetic Stock Center, University of California, Berkeley). Both yeast strains were grown and maintained on YPD medium, which contained 1% yeast extract (Difco Laboratories, Detroit, Mich.), 2% Bacto-Peptone, 2% glucose, and 2% Bacto-Agar. * Corresponding author. t On leave from Osaka University, Osaka, Japan.

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without amino acids to screen for auxotrophic strains. Colonies which grew on YPD medium but were unable to grow on YNB medium were further transferred to different pools of amino acids to elucidate their auxotrophic requirements. Protoplast preparation. S. cerevisiae and K. fragilis (Met-) were grown aerobically at 30°C to early stationary phase in 250-ml flasks containing 50 ml of YPD medium. Portions (5 ml) of each culture were collected and centrifuged at 500 x g for 5 min. Cells were then washed three times with sterile distilled water and were suspended in the protoplasting solution containing 0.6 M KCl, 10 mM 2mercaptoethanol, 50 mM phosphate buffer (pH 7.5), and 10 g of Zymolyase-60,000 per ml. The suspension was incubated at 30°C with occasional shaking, and it was checked periodically under the microscope for the formation of protoplasts. Practically all of the cells were converted into protoplasts within 1 h. These protoplasts were lysed when exposed to the hypotonic environment, as was confirmed by the microscopic examination. Protoplasts were collected by centrifugation at 1,000 x g for 10 min and washed at least three times with protoplasting buffer (0.1 M phosphate buffer [pH 7.5], 0.8 M sorbitol). Protoplast fusion. The protoplasts from both strains of yeasts were mixed and suspended carefully in polyethylene glycol solution (35% polyethylene glycol [molecular weight, 3,350; Sigma Chemical Co., St. Louis, Mo.], 10 mM CaCl; 0.8 M sorbitol) and the suspension was incubated for 20 min at room temperature. The fused cells were washed again with protoplasting buffer, and 0.1 ml of this suspension was mixed with 10 ml of regeneration medium (3% agar, 0.7% YNB medium without amino acids, 0.8 M sorbitol) and poured onto plates containing a thin bottom layer of agar with the same medium composition. Plates were sealed and incubated for a period of 3 to 7 days at 30°C. DNA extraction. Nucleic acid extraction was performed by using the Schneider procedure (23) with slight modifications (11). Cells were collected by centrifugation, washed, and suspended in 0.5 N perchloric acid at 0°C for 30 min. The suspension was then centrifuged, and cold-insoluble materials were washed twice with 0.5 N perchloric acid. Nucleic acids were then hydrolyzed by heating at 80°C for 20 min in 0.5 N perchloric acid. The hydrolysate was cooled to 0°C and centrifuged, and the supernatant was retained. Cells were reextracted at 80°C for another 20 min, and the two supernatant fractions were collected. DNA estimation. The DNA concentration was estimated by using diphenylamine (4). Two solutions were used. Solution A contained 1.5 g of diphenylalanine in 100 ml of glacial acetic acid and 1.5 ml of concentrated H2SO4. This solution was stored in a cool place and protected from light. Solution B contained 1.6% acetaldehyde. Just before the experiment, 20 ml of solution A was mixed with 0.1 ml of solution B to make solution C. A 1-ml portion of DNA extract (10 to 50 g/ml) was mixed with 2.5 ml of solution C, and the mixture was sealed and incubated at 30°C for 16 to 20 h). After the color development, the optical density of the solution was read at 600 nm against a DNA standard. Calf thymus DNA was used as the standard (0.4 mg of DNA per ml in 5 mM

NaOH). Test for hybridized DNA. Chromosomal DNA from the parent strains of S. cerevisiae and K. fragilis was isolated by the method of Rodriguez (22). The hybridization procedure described by Maniatis et al. (16) was used with minor modifications. DNA samples from parent strains and the fusant were

363

digested by HindlIl, electrophoresed in a 1% agarose gel, and transferred to nitrocellulose membrane. The plasmid YEP13, which contains the S. cerevisiae LEU2 gene, was used as the DNA probe. The confirmatory test for LEU2 gene present in parent strains and the fusant DNA were performed by using Bio-DNA probes and biotinylated alkaline phosphatase polymer colorimetric visualization. We used the DNA detection system of Bethesda Research Laboratories, Gaithersburg, Md. (catalog no. 8239 SA), a sensitive, nonradioactive method for detection of nucleic acids in Southern blot hybridizations. DNA probe was labeled with biotin by nick translation in the presence of dTTP analog biotin 11-dUTP (13). The biotin-tagged nucleotide was then incorporated into DNA by DNA polymerase I in the presence of the deoxynucleotide triphosphates, dATP, dGTP, and dCTP. The biotin-labeled DNA was then separated from those nucleotides that were not incorporated by exclusion chromatography by using gel filtration on a Sephadex G-50 column equilibrated with lx SSC (0.15 M sodium chloride, 0.015 M sodium citrate; pH 7.0) containing 0.1% (wt/vol) sodium dodecyl sulfate. Fractions (150 ,ul) were collected in 1.5-ml polypropylene microcentrifuge tubes. From each fraction, 1 to 2 pul was spotted onto a nitrocellulose filter and dried in a vacuum oven at 80°C for about 0.5 h. The biotinylated DNA was then detected by incubating the filter in avidin with biotinylated alkaline phosphatase polymer in a nitroblue tetrazolium-bromochloro-indolyl phosphate reaction mixture. The color development was allowed to proceed in the dark for 2 to 4 h (14). Isozyme analysis. For enzyme extraction, yeast cells were grown in 500-ml flasks containing 300 ml of YPD medium to the stationary phase under aerobic condition and harvested by centrifugation. The harvested cells were washed in extraction buffer (0.05 M Tris base, 0.007 M citric *acid monohydrate, 0.1% ascorbic acid, 1 mM mercaptoethanol, 0.1% cysteine hydrochloride, and 1% polyethylene glycol [molecular weight, 3,500]) (20). The washed cells were suspended in 10 ml of extraction buffer, and approximately 1 g of insoluble polyvinylpolyprollidone (Sigma) was added. The mixture was homogenized for 20 s in a Polytron homogenizer (Brinkman Instruments, Inc., Westbury, N.Y.); the mixture was kept in an ice bath during this procedure. The homogenate was centrifuged, and the supernatant was collected. For electrophoresis, 12% starch gels (Connaught Labs, Canada) were used. Two different gel buffer systems were used. Buffer system A (Tris citrate-lithium borate) had the following composition: 9 parts of Tris citrate buffer at pH 8.3 (0.05 M Tris base, 0.008 M citric acid monohydrate) and 1 part of lithium borate buffer at pH 8.3 (0.03 M lithium hydroxide, 0.2 M boric acid). The lithium borate buffer (pH 8.3) was used as the electrode buffer for analyses of esterase (EST). Buffer system B (histidine-Tris citrate) had the following composition: 0.006 M DL-histidine hydrochloric acid monohydrate (pH 7.0) as the gel buffer and 0.15 M Tris base-0.04 M citric acid monohydrate as the electrode buffer. Buffer system B was used for the analyses of alcohol dehydrogenase (ADH) and glutamate dehydrogenase (GDH). The method for the starch gel preparation and electrophoresis procedure used was the same as that used by Bringhurst et al. (3). For the gel staining, fresh stains were prepared just before the electrophoresis was completed each time. The gels were nicked at one corner to permit proper orientation and sliced horizontally twice to generate three pieces. Gel slices were incubated in the appropriate dye solutions in the dark at 37°C

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FARAHNAK ET AL.

A BC

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FIG. 1. Effect of various concentrations of ethylmethane sulfonate on the viability of spores (0) and vegetative cells (e).

until the bands were visible. The staining solutions had the following compositions: for EST, 0.04 g of Fast blue RR salt, 40 ml of H20, 50 ml of NaH2PO4 solution (27.8 g/liter), and 2 ml of 0.1% ov-naphthyl acetate; for ADH, 50 mg of NAD, 5 mg of phenazine methosulfate, 15 mg of dimethyl diazol diphenyltetrazolium bromide, 100 ml of 0.1 M Tris hydrochloride (pH 7.5), and 3 ml of ethanol; for GDH, 0.4 g of glutamic acid monosodium salt, 10 mg of NAD, 5 mg of phenazine methosulfate, 15 mg of dimethyl diazol diphenyltetrazolium bromide, 10 ml of 1 M Tris hydrochloride (pH 8.0), and the volume was adjusted to 100 ml with H20. Fermentation conditions. Fermentation medium contained 1% yeast extract, 2% peptone, and various amounts of lactose, maintained by feeding. The lactose was fed into the culture periodically to keep its concentration under 0.2 M to prevent substrate inhibition. The changes in the concehtration of lactose in the fermentor were monitored by lactose assay by a UV method (12). Fermentation was performed in 300-ml jacketed fermentation jars (Wheaton Industries, MilIville, N.J.). A constant-temperature bath was used to keep the fermentation temperature at 30°C. Each jar had an effluent line which was connected to a flask with a U-shaped side arm loop filled with water so that CO2 could be released and anaerobic conditions maintained. The fermentation medium was kept agitated by a magnetic stirring bar. TABLE 1. Average DNA content per cell for fusant and parental strains of yeasts Yeasts

FUS1 ....................................... FUS2 ....................................... FUS3 ....................................... FUS4 ....................................... FUS5 ....................................... FUS6 ....................................... FUS7 ....................................... FUS8 ....................................... FUS9 .......................................

FUS10 ..lO.................................

S. cere'isiae .................................. K.fragilis ....................................

(108 ,ug per cell) 8.0 8.9 10.8 10.8 22.5 7.8 7.8 8.6 9.3 13.7 3.9 4.7

Total DNA

FIG. 2. Southern blot test for hybridized DNA. Lane A, DNA from the fusant; lane 13, DNA from K.fragilis; lane C, DNA from S. cerevisiae.

Ethanol determination. Ethanol production was analyzed by using a gas chromatograph (model 5720A; Hewlett Packard Co., Palo Alto, Calif.) with a column having the packing material, 20 M polyethylene glycol on a 80/100 mesh celite 545 support. The injector and flame ionization detector temperatures were set at 200°C. The column temperature was 110°C. The column dimension was 1/8 in. (ca. 3.2 mm) in diameter and 6 ft (ca. 1.8 m) long. Nitrogen was used as the carrier gas.

RESULTS Sensitivity to EMS. The effect of EMS on the viability of vegetative cells and spores was studied before the sporulated culture was mutagenized. When the sporulating culture was treated with EMS, vegetative cells were far more susceptible than were the ascospores. At an EMS concentration of 7%, practically all the vegetative cells were killed, while about 40% of the spores were viable (Fig. 1). Therefore, a concentration of 7% EMS was tsed to expose the spores to the mutagen and to ensure that a large fraction of spores were still viable and could germinate and give rise to a vegetative population. Isolation of mutants. Colonies appeared 3 to 5 days after

VOL. 51, 1986

CONSTRUCTION OF YEASTS BY PROTOPLAST FUSION

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FIG. 3. Isozyme analyses. Isozyme pattelrns

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GDH, ADH, and EST from S. cerevisiae (larnes C, F. and 1), K. friagilis (lanes A, D, and G), and the fusant ( lanes B. E, and H), respectively.

the mutagenized spores were plated on Y'PD medium. Two classes of auxotrophs were isolated. The aluxotrophs isolated were found to be either methionine- or histidine-requiring mutant strains, occurring with a frequen cy of 13 and 4%, respectively (mean results of three de tcrminations with about 100 colonies). These mutants were used in the subsequent protoplast fusions to provide genet ilc markers for the selection of fusant products. Protoplast fusion prodlicts. The colonikes which grew on YNB medium (without amino acid) weire isolated as the fused hybrids between S. cerevisiae (Ad le Trp-), and Ki

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these hybridized fusants, since the palrent strains were auxotrophs and incapable of growing on this medium. The complementation frequency was found to be Q1.7%o. This

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value is defined as the ratio of the number of hybridized protoplasts capable of growing on the minimal medium to the number of colonies which grow on complete medium. Evidence that the recovered prototrophs were fusion products and not revertants was obtained from examination of the average DNA content of the fusant cells. It is reasonable to assume that fused hybrids should contain more DNA per cell. The results of DNA extraction and estimation of DNA content are shown in Table 1. The fusants had more DNA per cell than did the parental strains. Furthermore, the results indicate that multiple fusions had also taken place, suggesting that some of the fusants are polyploid. Hybridized DNA. The hybridization study revealed that

the plasmid harboring the 4EU2 gene could hybridize with HindIII DNA fragment in the S. cerevisiae and K. fragilis genomes. This indicated that the LEU2 gene hybridized with its homologous segment in the S. cereivisiae genome as well as a segment of DNA in K. faagilis chromosomal DNA. This plasmid showed three hybridization loci with the fusant genome, suggesting that the fusant was carrying the genome from both parent strains .(Fig. 2). The results from the isozyme analyses showed that the isozyme patterns from S. cerevisiae and K. fra gilis were significantly different in their electrophoretic mobility on starch gel (Fig. 3). For ADH pattern, S. cerelvisiae gave two bands while K. ffragilis gave only one. The fusant strain showed a combined isozyme pattern of both parental strains, that is, two bands which correspond to the ADH from S. ceres'isiae and one band which corresponds to that from K. fragilis (Fig. 3). The results of isozyme analyses for GDH and EST are also presented. The fusants showed four bands for GDH and three bands for EST, indicating that the hybrid strain carries the genes which correspond to these isozymes from both parent strains. Ethanol fermentation. To study the fermentation performances of fusants, it was first necessary to examine the substrate inhibition effect when lactose was used as the sole carbon source. The maximum growth rate was attained when the lactose concentration was about 0.06 M, and the growth rate decreased gradually with increasing lactose

concentration (Fig. 4). During the fermentation the lactose

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APPL. ENVIRON. MICROBIOL.

FARAHNAK ET AL.

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FIG. 6. Experimental protocol for inducing auxotrophic markers in a diploid yeast by mutagenizing the spores.

concentration was kept under 0.3 M. To achieve this in a batch fermentation, lactose was fed to the culture periodically to prevent substrate inhibition. The profiles of cell growth and ethanol production for the fusant no. 4 and its parent K. fragilis strain are presented in Fig. 5. In three separate fermentations, fusant no. 4 produced 13.3, 13.6, and 13.2% (vol/vol) ethanol while the parental strain produced only 10.5, 10.2 and 10.3% (vol/vol) ethanol. Statistical analysis showed the difference between fermentation performances of the fusant and the parent strains is significant with a confidence level higher than 90%. The fusants produced about 30% more ethanol than did the parent strain of K. fragilis. The fusants also yielded about 25% greater cell concentration as compared with the parent strain. These data suggest that fusants are more tolerant to ethanol than is the parent strain. On the other hand, the maximum specific rate of ethanol production was estimated to be about the same for both the fusant strain and the parent strain. This value was found to be about 0.6 g/g of cell per h. DISCUSSION In this study we used the protoplast fusion technique to improve a yeast strain for its ethanol tolerance and lactose assimilation. Although the specific rates of ethanol produc-

tion by fusant and parent strains were practically the same, the final ethanol yield of the fusant was significantly higher. The S. cerevisiae strain used in this study was a laboratory haploid strain which produced only about 13.5% ethanol by volume when fermented on glucose medium. The fusant is at least as tolerant to ethanol as is the parent strain of S. cerevisiae. This result suggests there is a good possibility of getting a hybridized fusant with higher ethanol tolerance when other strains of S. cerevisiae with higher ethanol tolerance become available. However, the major problem with using more ethanol-tolerant strains of S. cerevisiae is their higher ploidy, which causes difficulties in obtaining auxotrophic mutants necessary for creating fusants. The spontaneous reversion frequency of the mutant was about i0'. This low frequency strongly suggests that the prototrophs obtained may have been the result of complementation rather than the reversion to prototrophy. The fact that the fusants contain DNA fragments containing the LEU2 gene from both parents proves that these strains are fusants and not mutated revertants of one of the parental strains. K. fragilis 55-55 is a prototrophic homothallic yeast. We needed to find mutants with auxotrophic markers so that the fusants could be selected on the basis of complementation of those auxotrophic markers. Since it is difficult to obtain mutants from a diploid strain, spores rather than the vegetative cells were mutagenized (Fig. 6). It was reported that the structural gene for the enzyme ,B-galactosidase from K. lactis was transferred into S. cerevisiae (5). This strain, however, was still unable to grow on lactose because of problems involved with the transport of lactose across the cell membrane. It is also known that in Kluyveromyces lactis, the genes responsible for lactose utilization are not clustered, and there is no operon (24). However, there is no genetic information available as to whether the lactose utilization system in K. fragilis follows the same pattern. In view of these problems and difficulties involved with the recombinant DNA technique, the protoplast fusion technique may have some advantages over the gene manipulation method in obtaining a hybridized fusant. The physiological effects of osmotic pressure on lactose metabolism reported by Panchel et al. (19) is another example of the complexities involved in lactose metabolism and ethanol tolerance of yeast. Our results of isozyme analyses for ADH, GDH, and EST further strengthen our conclusion that the fusant obtained were genetically hybridized cells. The isozyme technique has been successfully and widely used in plant genetics and breeding for isolation and identification of truly hybridized fusant strains (28). The physiological and biochemical basis for ethanol tolerance are not as yet very well understood. Although the fusant obtained has been stable for about 6 months, judging from its fermentation activity, the long-term stability of the fusant will have to be evaluated. A great deal of work remains to be done along these lines. ACKNOWLEDGMENT We thank S. Arulsekar, D. E. Parfitt, and A. M. Dandekar at the University of California, Davis, for valuable assistance with isozyme analysis and stimulating discussions. LITERATURE CITED 1. Anne, J., H. Eyssen, and P. Desomer. 1976. Somatic hybridization of Penicillium roquefortii with P. chrysogenum after protoplast fusion. Nature (London) 262:719-721. 2. Anne, J., and J. F. Peberdy. 1975. Condition for induced fusion

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of fungal protoplast in polyethyleneglycol. Arch. Microbiol. 105:201-205. 3. Bringhurst, R. S., S. Arulsekar, J. F. Hancock, Jr., and V. Voth. 1981. Electrophoretic characterization of strawberry cultivars. J. Am. Soc. Hortic. Sci. 106:684-687. 4. Burton, K. 1955. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62:315-322. 5. Dickson, R. C. 1980. Expression of a foreign eukaryotic gene is Saccharomyces cervisiae: 3-galactosidase from Kluyveromyces lactis. Gene 10:347-356. 6. Ferenczy, L. 1976. Some characteristics of intra- and interspecific protoplast fusion products of Aspergillus nidulans and A. fumigatus, p. 171-182. In D. Dudits, G. L. Farkas, and P. Maliga (ed.), Cell genetics in higher plants. Akademiai Kiado, Budapest. 7. Ferenczy, L., F. Kevei, and M. Szyegedi. 1975. High frequency fusion of fungal protoplasts. Experientia 31:1028-1030. 8. Ferenczy, L., F. Kevei, and J. Zsolt. 1974. Fusion of fungal protoplasts. Nature (London) 248:793-794. 9. Ferenczy, L., and A. Maraz. 1977. Transfer of mitochondria by protoplast fusion in Saccharomyces cerevisiae. Nature (London) 268:524-525. 10. Gunge, N., and A. Tamaru. 1978. A genetic analysis of product of protoplast fusion in Saccharomyces cerevisiae. Jpn. J. Genet. 53:41-49. 11. Hutchinson, W. C., and H. N. Munro. 1961. Determination of nucleic acids in biological materials. Analyst 86:768-776. 12. Kurz, G., and K. Wallenfels. 1974. Lactose and other ,-Dgalactosides, p. 1180-1184. In H. V. Bergmeyer (ed.), Methods of enzymatic analysis, vol. 3, 2nd ed. Verlag Chemie International, Inc., Deerfield Beach, Fla. 13. Langer, P., A. Waldrop, and D. Ward. 1981. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78:6633-6637. 14. Leary, J., D. Brigati, and D. Wards. 1983. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc. Natl. Acad. Sci. USA 80:4045-4049. 15. Mahoney, R. R., T. A. Nickerson, and J. R. Whitaker. 1975. Selection of strain, growth condition, and extraction procedure for optimum production of lactase from Kluyveromycesfragilis. J. Dairy Sci. 58:1620.

367

16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 17. Maraz, A., M. Kiss, and L. Ferenczy. 1978. Protoplast fusion in Saccharomyces cerevisiae strains of identical and opposite mating type. FEMS Microbiol. Lett. 3:319-322. 18. O'Leary, V. S., R. Green, B. C. Sullivan, and V. H. Halsinger. 1977. Alcohol production by selected yeast strains in lactosehydrolysed acid whey. Biotech. Bioeng. 19:1019-1035. 19. Panchal, C. J., and G. G. Stewart. 1980. The effect of osmotic pressure on the production and excretion of ethanol and glycerol by a brewing yeast strain. J. Inst. Brew. 86:207-210. 20. Parfitt, D., and S. Arulsekar. 1985. Identification of plum x peach hybrids by isoenzyme analysis. J. Am. Soc. Hortic. Sci. 20:246-248. 21. Ragosa, M., H. H. Browne, and E. 0. Whittier. 1947. Ethyl alcohol from whey. J. Dairy Sci. 30:263-265. 22. Rodriguez, R. L., and R. C. Tait. 1983. Recombinant DNA techniques. Addison-Wesley Publishing, Inc., Reading, Mass. 23. Schneider, W. C. 1945. Extraction and estimation of deoxypentose nucleic acid and of pentose nucleic acid. J. Biol. Chem. 161 :293-331. 24. Sheetz, R. M., and R. C. Dickson. 1980. Mutations affecting synthesis of P-galactocidase activity in the yeast Kluyveromyces lactis. Genetics 95:877-890. 25. Sipiczki, M., and L. Ferenczy. 1977. Protoplast fusion of Schizosaccharomyces pombe auxotrophic mutants of identical mating type. Mol. Gen. Genet. 151:77-81. 26. Snow, R. 1966. An enrichment method for auxotrophic yeast mutants using the antibiotic nystatin. Nature (London)

211:20-21.

27. Stahl, U. 1978. Zygote formation and recombination between like mating types in yeast Saccharomycopsis lipolytica by protoplast fusion. Mol. Gen. Genet. 160:111-113. 28. Tanksley, S. D., and T. J. Orton (ed.). 1983. Isozymes in plant genetics and breeding. Elsevier Science Publisher BV, The

Netherlands.

29. Van Solingen, P., and J. B. Van Der Plaat. 1977. Fusion of yeast spheroplasts. J. Bacteriol. 130:946-947. 30. Whittaker, P. A., and S. M. Leach. 1978. Interspecific hybrid production between the yeasts Kluyveromyces lactis and Kluyveromyces fragilis protoplast fusion. FEMS Microbiol. Lett. 4:31-34.