J. Basic Microbiol. 32 (1992) 6. 369-372
(Institute of Biology, Romanian Academy of Sciences, Bucharest: 'Institute for Physics and Nuclear Engineering. Bucharest MG-4 and 'Chemical and Pharmaceutical Research Institute. Bucharest. Romania)
Electrically induced protoplast fusion for ergosterol-producing yeast strain improvement DORINAAVRAM,ILEANAPETCU', MIHAIRADU', FLORICA DAN^ and RODICASTAN (Recv,iLwl 1.5
Jniiitnrj5
1992/Accepted 26 M q . 1992)
Electrofusion was employed for hybrid construction in ergosterol-producing yeast strains. Some fusion products proved to be hybrid with respect to ergosterol content and to remain stable over several generations.
Yeasts belonging to Saccharomyces are the main source of ergosterol (EL-REFAIand EL-REFAI1968), the precursor of ergocalciferol (vitamin D2). It is well known that an increase in ploidy level can subsequently lead to an enrichment of some cellular metabolites. KOSIKOVet al. (1977) obtained polyploids improved in ergosterol content by sexual hybridization. Generally, industrial yeasts lack or have very inefficient sexual processes and as a result, the use of sexual hybridization for their improvement is limited (SPENCERand SPENCER 1983). A more suitable approach towards hybrid formation in industrial yeasts is protoplast fusion. Chemically induced protoplast fusion has been widely used for intraspecific, interspecific and intergeneric fusion in order to breed industrial yeast strains (SPENCERet al. 1990), but polyethylen glycol (PEG), the main fusogenic agent, has a well known toxic effect on the cell. Electrically induced protoplast fusion in yeasts has been initiated by ZIMMERMAN (198 l), but it has been studied especially from basic standpoint (ZIMMERMAN 1986, NODAet al. 1990). There are only a few trials to use electrofusion for industrial yeast improvement (AARNIOand SUIKHO1991). In this paper we describe the construction of ergosterol improved hybrids by electrofusion. The conditions for electrofusion are also discussed as well as the stability of the fusion products.
Materials and methods Microorganisms: Saccharoii7yces carlshergensis ATCC9880 and Succhrrronzjws species were used. Succharon~yceswas isolated from nature and it was industrially used for ergosterol production. Mutants resistant to NiCl, ( 5 mM), CuSO, (7 mM) CdSO, (0.8 mM) were chosen for fusion experiments in order to facilitate the selection of the fusion products. Culture media: The growth medium was YPDMedium (1 % yeast extract, 1 YOpcptone, 2% glucose). The fermentation medium contained molasses as carbon source with 5% reducing substances and 0.5% residual mycelium from penicillin production as nitrogen source. Protoplast isolation was performed on exponentially growing cells, essentially by the common method involving two succesive steps: 1. a pretreatment with beta-mercapto-cthanol (10-300 mM) and 2. a treatment with snail gut juice (0.4-20 mgiml).
D. AVRAMet id.
3 70
Eleetrofusion was carried OUI by ZIMMERMAN'S method ( 198 1). Our specific experimental arrangement and method have been described elsewhere (PETCUet d . 1990) and is also presented in the Table 1. The electrofusion yield was expressed as the number of protoplasts involved in fusion divided by the number of protoplasts initially aligned betwzeen the electrodes. Reversion was performed in YPD medium osmotically stabilized by 0.6 M KCI. The revertants were then replicaplated on YPD medium supplemented with 5 mhf NiCI2, 7 mM CuSO, and 0 . 8 m CdSO,, ~ on which the fusion products were selected. Ergosterol content was assayed by the method of
BR.AlVIK
and ORADES (1967).
DNA content per cell was determined in stationary growing cells by the diphenylamine method (GILES and MYFKS1965). using herring sperm D N A as standard.
Results and discussion
The success of the electrofusion process depends on several experimental parameters, some related to protoplast isolation and others to the electric field conditions (NADOet 01. 1990). We assumed that a good yield of electrofusants could be obtained only in a homogenous population of protoplasts with a complete removal of the cell wall and undamaged plasma membrane. To fulfil these requirements we used several variations for protoplast isolation in which the amounts of beta-mercapto-ethanol, snail gut juice and time treatment varried. For each set of isolation conditions we checked the electrofusion yields in different electrofusion media. Table 1 present two extreme isolation conditions in which we obtained protoplasts with very different electrofusion yields, although their viability seemed to be unaffected. In this Table 1 Electrofusion yields for Strcc,/iuloriz!,c~~,s sp. Protoplast Isolation Conditions
I . -pretreatment with 300 mhz beta-mercaptoethanol 30 min, 30 C. 1 x 10' ccllsml -treatment with 20 mg ml snail gut juice, 2. h, 30 C 1 x 10' c e k m l 2. -pretreatment with 14.3 mhl beta-mercaptoethanol. 30 min 30 C 1 x 10' cells'rnl -treatment with 0.4 m g m l snail gut juice, 2 1 h 30 ' C t x 1O'cells'mI
Electrofusion Media
Electrical Parameters
Fusion Yield
Dielectro- Fusion Electrical Pulse -~ phoretic Dura- Number field Intensity tion of Pulses
Yo
A. 0.7 M Manitol B. 0.7 M Manitol + 0.5 mg. ml pronasc
2MHz 200 V'cm 2MH7 200 V.cm
3KV'cm
20ps 2
8
XKVcm
20ps 2
20
A. 0.7 M
2 MHz 200 V t m
4.3 KV/cm
14ps 2
16
2 MHz 200 V/cm
5.6 KV/cm
16ps 2
85
2 MHz 200 V k m
8 KV/cm
2ops 2
61
Manitol B. 0.7 31 Manitol + 0.5 mg;ml pronase C. 0.7 hi Manitol + 0.1 mMCa + 0.5 mM Mg' ' +
371
Electrofusion of ergosterol-producing yeasts
respect, we consider that the low electrofusion yield obtained in the first variant (Table 1, I-A and I-B) was determined by the damage of the plasma membrane caused by the high concentrations of beta-mercapto-ethanol and snail-gut juice. The electrofusion yields were two and four fold higher, respectively, when the isolation conditions were milder (Table 1, 2-A, and 2-B). As can be seen, for a proper electrofusion process the presence of pronase or Ca' and Mg' was absolutely necessary (Table 1, 1-B, 2-B and 2-C). Protoplast isolation in S. carlsbergensis was tested in the same conditions as in Sacc/iaronzyces sp. and similar results were registered. The conditions in which we obtained the highest electrofusion yields (Table 1, 2-B) were used for hybrid construction. From sixteen fusion products selected on the basis of the resistance to heavy metals, thirteen (81.25%) had higher levels of ergosterol than their parents and most of them were polyploid (Table 3). The highest ergosterol yields were obtained in experiment C, when the parents were two different mutants of Saccharoniyces. This fact was predictable because Sacclzaroniyces had a higher ergosterol content than S. carlsbergmsis. Fusion products FPAA16 and FPAA2 exceeded Saccliaroniyces by 119% and 105%, respectively. +
+
Table 2 Ergosterol content and ploidy level of the fusion products and of the parental strains Strain
Total ergosterol mg/gDCW')
24.28-Dehi- Pure erProducdroergoste- gosterol tivity rol mg/gDCW') mg/gDCW') YO
DNA content
44.6 27.1
29 15.4
32.19 31.5
2n 2n
Experiment A : Snccliaromyces CuRNiSX S. carlshergeiisis CuSNiR FPAB72 47.2 27 20.2 14 FPAB77 44.66 33.3 11.3 13 FPAB2 91.5 63.1 28.4 30
33 34 66
2n 2n 4n
Experiment B: S. cnrlsbergeiisis NiRCdSX S. cnrlsbergerisis NiSCdR FPBBl2 43 26.1 16.9 13 FPBBl3 44 21.4 22.6 13 FPBB 16 31.8 15.8 16 10 FPBB2O 35.4 17.5 17.9 14 FPBB23 43.2 21.3 21.9 17
60 67 60 62 66
4n 4n 4n 4n 4n
Experiment C: Snceharomjws sp. CuRNiSX Sncclmrornjws sp. CuSNiR FPAAl 99.5 67.9 21.6 20 FPAA2 93.6 61.6 32 25 FPAA 11 76.1 52.8 23.3 18 FPAA I 2 68.5 47.9 20.6 16 FPAAl3 37.9 22.3 15.6 14 FPAA14 95.9 65.2 30.7 23 F P A A 16 77.8 43.6 34.2 28 FPAA8 38.4 26.9 11.5 12 FPAA3 54.6 35.6 19 28
68 60 65 67 36 64 51 32 33
4n 4n 4n 4n 2n 4n 3n-4n 2n 2n
Sncckaronij~essp. S. carlshergensis
') DCW. cell dry weight
15.6 11.7
13 9.9
Ploidy Level
fg/cell
372
D. AVRAM et trl.
At the same time. one of the fusion products obtained between Sclcchnronij~cesand S. c~nr/.dwgrrisic(Experiment A - Table 3) exceeded the most productive parent (Sacc ~ h ~ ~ o Jt by ~ ~ 82%. j ~ c ~ . ~ As could be expected. the ergosterol content of the fusion products obtained between two different mutants of S. c~crrlshrrgemis(Experiment B. Table 2 ) was lower than that of the fusion products obtained in the other two experiments. However, it could be noticed that two fusants ( FPBBl3 and FPBB23) surpassed S. cwtsbergetlrsis by approxirnatively 90 9 4 . The stability of the fusion products after cultivating them 10 times in fermentation medium without selective pressure was good. Eleven fusion products from thirteen tested (84.6%) maintained their ergosterol content at a stably high level. Moreover, the stability could be considered encouraging because the fusion products have kept their ergosterol content unchanged after twelve months since they were obtained.
References AARI'IO.T. H . and SUIHKO. M. L.. 1991. Flectrofusion of an industrial baker's yeast strain with a sour dough yeast. Appl. Biochem. Biotechnol.. 27. 65 -74. B u i v t n . 0. W. cind ORADES. J. L.. 1967. Spectrophotometric semimicrodetermination of ergosterol in
yeast. Agric. Food Chem.. 55. 361 -370. EI.-REFAI. A. H. and EL-REFAI. I . A.. 1968. Sterol production of yeast strains. Z. allg. Mikrobiol., 8, 355 - 360. Gir.r-s. K . W. and MYERS.A.. 1965. An improled diphenilaniine method for estimation of deoxyribonucleic acid. Nature. 206. 93-97. KOSIKOV.K. W.. LIAPUNOVA.T. S.. RAEVSRIA.0 . G.. SEMIKHATOVA. N. M.. KOCHKINA,I. B. and MEISSEI..M. N.. 1917. Synthesis of ergosterol of hybrids and strains of Sac,c,hciroii!r,e~~.~ of different ploidy. Mikrobiologia. 46. 86-91. NODA.K.. TOGA\VA. Y . and YAMADA, Y.. 1990. Quantification of physical and cyto-physiological conditions for clcctrofusion of Saccharo/i(j,cescer-rrisiw. Agric. Biol.. Chem., 54, 2023- 2028. PETCU.1.. RADU. M.. AVRAM.D.. V ~ s s u T. . and BREZEANU. A,. 1990. Electrofusion ofyeast proloplasts. Rev. Roum. Riol.. 35. 115-120. SPENCER. J . F. T. and SPENCER. D. M.. 1983. Genetic improvement of industrial yeasts. Ann. Rev. Microbiol.. 37. 121 - 142. SPF.NCER. D. M.. RI.YNOI 11s.N. and SPENCI.K. J. F. T.. 1990. Protoplast fusion - Application to industrial yeasts. I n : Yeast Technology (J. F. T. SPENCER & D. M. SPENCER. eds.). p. 348-353., Springer Verlag Berlin. ZIMMERMANN. U . and S C H E m I c r t . P.. 1981. High frequency fusion of the plant protoplasts by electric fields. Planta. 151. 26-32. . U.. 1986. Electrical breakdou n. electropermeabili7ation and electrofusion. Rev. Physiol. Biochcm. Pharmacol.. 105. 175-257. Mailing address: Dr. DORINA AVRAM.Institute of Biology. Spl. Indepcndcntei 296. 17148 Bucharest, Romania
(Institute of Biology, Romanian Academy of Sciences, Bucharest: 'Institute for Physics and Nuclear Engineering. Bucharest MG-4 and 'Chemical and Pharmaceutical Research Institute. Bucharest. Romania)
Electrically induced protoplast fusion for ergosterol-producing yeast strain improvement DORINAAVRAM,ILEANAPETCU', MIHAIRADU', FLORICA DAN^ and RODICASTAN (Recv,iLwl 1.5
Jniiitnrj5
1992/Accepted 26 M q . 1992)
Electrofusion was employed for hybrid construction in ergosterol-producing yeast strains. Some fusion products proved to be hybrid with respect to ergosterol content and to remain stable over several generations.
Yeasts belonging to Saccharomyces are the main source of ergosterol (EL-REFAIand EL-REFAI1968), the precursor of ergocalciferol (vitamin D2). It is well known that an increase in ploidy level can subsequently lead to an enrichment of some cellular metabolites. KOSIKOVet al. (1977) obtained polyploids improved in ergosterol content by sexual hybridization. Generally, industrial yeasts lack or have very inefficient sexual processes and as a result, the use of sexual hybridization for their improvement is limited (SPENCERand SPENCER 1983). A more suitable approach towards hybrid formation in industrial yeasts is protoplast fusion. Chemically induced protoplast fusion has been widely used for intraspecific, interspecific and intergeneric fusion in order to breed industrial yeast strains (SPENCERet al. 1990), but polyethylen glycol (PEG), the main fusogenic agent, has a well known toxic effect on the cell. Electrically induced protoplast fusion in yeasts has been initiated by ZIMMERMAN (198 l), but it has been studied especially from basic standpoint (ZIMMERMAN 1986, NODAet al. 1990). There are only a few trials to use electrofusion for industrial yeast improvement (AARNIOand SUIKHO1991). In this paper we describe the construction of ergosterol improved hybrids by electrofusion. The conditions for electrofusion are also discussed as well as the stability of the fusion products.
Materials and methods Microorganisms: Saccharoii7yces carlshergensis ATCC9880 and Succhrrronzjws species were used. Succharon~yceswas isolated from nature and it was industrially used for ergosterol production. Mutants resistant to NiCl, ( 5 mM), CuSO, (7 mM) CdSO, (0.8 mM) were chosen for fusion experiments in order to facilitate the selection of the fusion products. Culture media: The growth medium was YPDMedium (1 % yeast extract, 1 YOpcptone, 2% glucose). The fermentation medium contained molasses as carbon source with 5% reducing substances and 0.5% residual mycelium from penicillin production as nitrogen source. Protoplast isolation was performed on exponentially growing cells, essentially by the common method involving two succesive steps: 1. a pretreatment with beta-mercapto-cthanol (10-300 mM) and 2. a treatment with snail gut juice (0.4-20 mgiml).
D. AVRAMet id.
3 70
Eleetrofusion was carried OUI by ZIMMERMAN'S method ( 198 1). Our specific experimental arrangement and method have been described elsewhere (PETCUet d . 1990) and is also presented in the Table 1. The electrofusion yield was expressed as the number of protoplasts involved in fusion divided by the number of protoplasts initially aligned betwzeen the electrodes. Reversion was performed in YPD medium osmotically stabilized by 0.6 M KCI. The revertants were then replicaplated on YPD medium supplemented with 5 mhf NiCI2, 7 mM CuSO, and 0 . 8 m CdSO,, ~ on which the fusion products were selected. Ergosterol content was assayed by the method of
BR.AlVIK
and ORADES (1967).
DNA content per cell was determined in stationary growing cells by the diphenylamine method (GILES and MYFKS1965). using herring sperm D N A as standard.
Results and discussion
The success of the electrofusion process depends on several experimental parameters, some related to protoplast isolation and others to the electric field conditions (NADOet 01. 1990). We assumed that a good yield of electrofusants could be obtained only in a homogenous population of protoplasts with a complete removal of the cell wall and undamaged plasma membrane. To fulfil these requirements we used several variations for protoplast isolation in which the amounts of beta-mercapto-ethanol, snail gut juice and time treatment varried. For each set of isolation conditions we checked the electrofusion yields in different electrofusion media. Table 1 present two extreme isolation conditions in which we obtained protoplasts with very different electrofusion yields, although their viability seemed to be unaffected. In this Table 1 Electrofusion yields for Strcc,/iuloriz!,c~~,s sp. Protoplast Isolation Conditions
I . -pretreatment with 300 mhz beta-mercaptoethanol 30 min, 30 C. 1 x 10' ccllsml -treatment with 20 mg ml snail gut juice, 2. h, 30 C 1 x 10' c e k m l 2. -pretreatment with 14.3 mhl beta-mercaptoethanol. 30 min 30 C 1 x 10' cells'rnl -treatment with 0.4 m g m l snail gut juice, 2 1 h 30 ' C t x 1O'cells'mI
Electrofusion Media
Electrical Parameters
Fusion Yield
Dielectro- Fusion Electrical Pulse -~ phoretic Dura- Number field Intensity tion of Pulses
Yo
A. 0.7 M Manitol B. 0.7 M Manitol + 0.5 mg. ml pronasc
2MHz 200 V'cm 2MH7 200 V.cm
3KV'cm
20ps 2
8
XKVcm
20ps 2
20
A. 0.7 M
2 MHz 200 V t m
4.3 KV/cm
14ps 2
16
2 MHz 200 V/cm
5.6 KV/cm
16ps 2
85
2 MHz 200 V k m
8 KV/cm
2ops 2
61
Manitol B. 0.7 31 Manitol + 0.5 mg;ml pronase C. 0.7 hi Manitol + 0.1 mMCa + 0.5 mM Mg' ' +
371
Electrofusion of ergosterol-producing yeasts
respect, we consider that the low electrofusion yield obtained in the first variant (Table 1, I-A and I-B) was determined by the damage of the plasma membrane caused by the high concentrations of beta-mercapto-ethanol and snail-gut juice. The electrofusion yields were two and four fold higher, respectively, when the isolation conditions were milder (Table 1, 2-A, and 2-B). As can be seen, for a proper electrofusion process the presence of pronase or Ca' and Mg' was absolutely necessary (Table 1, 1-B, 2-B and 2-C). Protoplast isolation in S. carlsbergensis was tested in the same conditions as in Sacc/iaronzyces sp. and similar results were registered. The conditions in which we obtained the highest electrofusion yields (Table 1, 2-B) were used for hybrid construction. From sixteen fusion products selected on the basis of the resistance to heavy metals, thirteen (81.25%) had higher levels of ergosterol than their parents and most of them were polyploid (Table 3). The highest ergosterol yields were obtained in experiment C, when the parents were two different mutants of Saccharoniyces. This fact was predictable because Sacclzaroniyces had a higher ergosterol content than S. carlsbergmsis. Fusion products FPAA16 and FPAA2 exceeded Saccliaroniyces by 119% and 105%, respectively. +
+
Table 2 Ergosterol content and ploidy level of the fusion products and of the parental strains Strain
Total ergosterol mg/gDCW')
24.28-Dehi- Pure erProducdroergoste- gosterol tivity rol mg/gDCW') mg/gDCW') YO
DNA content
44.6 27.1
29 15.4
32.19 31.5
2n 2n
Experiment A : Snccliaromyces CuRNiSX S. carlshergeiisis CuSNiR FPAB72 47.2 27 20.2 14 FPAB77 44.66 33.3 11.3 13 FPAB2 91.5 63.1 28.4 30
33 34 66
2n 2n 4n
Experiment B: S. cnrlsbergeiisis NiRCdSX S. cnrlsbergerisis NiSCdR FPBBl2 43 26.1 16.9 13 FPBBl3 44 21.4 22.6 13 FPBB 16 31.8 15.8 16 10 FPBB2O 35.4 17.5 17.9 14 FPBB23 43.2 21.3 21.9 17
60 67 60 62 66
4n 4n 4n 4n 4n
Experiment C: Snceharomjws sp. CuRNiSX Sncclmrornjws sp. CuSNiR FPAAl 99.5 67.9 21.6 20 FPAA2 93.6 61.6 32 25 FPAA 11 76.1 52.8 23.3 18 FPAA I 2 68.5 47.9 20.6 16 FPAAl3 37.9 22.3 15.6 14 FPAA14 95.9 65.2 30.7 23 F P A A 16 77.8 43.6 34.2 28 FPAA8 38.4 26.9 11.5 12 FPAA3 54.6 35.6 19 28
68 60 65 67 36 64 51 32 33
4n 4n 4n 4n 2n 4n 3n-4n 2n 2n
Sncckaronij~essp. S. carlshergensis
') DCW. cell dry weight
15.6 11.7
13 9.9
Ploidy Level
fg/cell
372
D. AVRAM et trl.
At the same time. one of the fusion products obtained between Sclcchnronij~cesand S. c~nr/.dwgrrisic(Experiment A - Table 3) exceeded the most productive parent (Sacc ~ h ~ ~ o Jt by ~ ~ 82%. j ~ c ~ . ~ As could be expected. the ergosterol content of the fusion products obtained between two different mutants of S. c~crrlshrrgemis(Experiment B. Table 2 ) was lower than that of the fusion products obtained in the other two experiments. However, it could be noticed that two fusants ( FPBBl3 and FPBB23) surpassed S. cwtsbergetlrsis by approxirnatively 90 9 4 . The stability of the fusion products after cultivating them 10 times in fermentation medium without selective pressure was good. Eleven fusion products from thirteen tested (84.6%) maintained their ergosterol content at a stably high level. Moreover, the stability could be considered encouraging because the fusion products have kept their ergosterol content unchanged after twelve months since they were obtained.
References AARI'IO.T. H . and SUIHKO. M. L.. 1991. Flectrofusion of an industrial baker's yeast strain with a sour dough yeast. Appl. Biochem. Biotechnol.. 27. 65 -74. B u i v t n . 0. W. cind ORADES. J. L.. 1967. Spectrophotometric semimicrodetermination of ergosterol in
yeast. Agric. Food Chem.. 55. 361 -370. EI.-REFAI. A. H. and EL-REFAI. I . A.. 1968. Sterol production of yeast strains. Z. allg. Mikrobiol., 8, 355 - 360. Gir.r-s. K . W. and MYERS.A.. 1965. An improled diphenilaniine method for estimation of deoxyribonucleic acid. Nature. 206. 93-97. KOSIKOV.K. W.. LIAPUNOVA.T. S.. RAEVSRIA.0 . G.. SEMIKHATOVA. N. M.. KOCHKINA,I. B. and MEISSEI..M. N.. 1917. Synthesis of ergosterol of hybrids and strains of Sac,c,hciroii!r,e~~.~ of different ploidy. Mikrobiologia. 46. 86-91. NODA.K.. TOGA\VA. Y . and YAMADA, Y.. 1990. Quantification of physical and cyto-physiological conditions for clcctrofusion of Saccharo/i(j,cescer-rrisiw. Agric. Biol.. Chem., 54, 2023- 2028. PETCU.1.. RADU. M.. AVRAM.D.. V ~ s s u T. . and BREZEANU. A,. 1990. Electrofusion ofyeast proloplasts. Rev. Roum. Riol.. 35. 115-120. SPENCER. J . F. T. and SPENCER. D. M.. 1983. Genetic improvement of industrial yeasts. Ann. Rev. Microbiol.. 37. 121 - 142. SPF.NCER. D. M.. RI.YNOI 11s.N. and SPENCI.K. J. F. T.. 1990. Protoplast fusion - Application to industrial yeasts. I n : Yeast Technology (J. F. T. SPENCER & D. M. SPENCER. eds.). p. 348-353., Springer Verlag Berlin. ZIMMERMANN. U . and S C H E m I c r t . P.. 1981. High frequency fusion of the plant protoplasts by electric fields. Planta. 151. 26-32. . U.. 1986. Electrical breakdou n. electropermeabili7ation and electrofusion. Rev. Physiol. Biochcm. Pharmacol.. 105. 175-257. Mailing address: Dr. DORINA AVRAM.Institute of Biology. Spl. Indepcndcntei 296. 17148 Bucharest, Romania
