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Intergeneric hybridization in yeasts through protoplast fusion
FEMS MicrobiologyLetters 3 (1978) 309-312 © Copyright Federation of European MicrobiologicalSocieties Published by Elsevier/North-HollandBiomedicalPress
309
I N T E R G E N E R I C H Y B R I D I Z A T I O N IN YEASTS T H R O U G H P R O T O P L A S T F U S I O N A. PROVOST, C. BOURGUIGNON, P. FOURNIER, A.M. RIBET and H. HESLOT Department of Genetics, Institut National Agronomique, 16, Rue Claude Bernard, 75231 Paris Cedex 05, France Received 21 March 1978
1. Introduction Many yeast species have no clearly defined mating types, do not undergo meiosis, do not sporulate and are frequently polyploid, hence there are major difficulties in the improvement of these species for industrial purposes. Heterokaryons, as well as heterozygous diploids have been obtained through induced intra- and interspecific protoplast fusion in fungi [1-6] and yeasts [7-9]. Recombinants have also been selected in bacteria [10, 11 ], Streptomyces [ 12] and in the industrial asexual yeast Candida tropicalis [13]. In this paper, we show that recombinants can be obtained when protoplast fusion is induced between yeast strains belonging to different genera. These recombinants may show new useful characteristics and may be adaptable to a wider range of culture media. The induced fusion might also offer a method for transferring cytoplasmic organelles, such as mitochondria. This has already been done between strains ofS. cerevisiae [9].
2. Methods Strain C of Candida tropicalis (auxotrophic for cytosine and adenine) from our collection and strain CBS 2527 of Saccharomycopsis fibuligera were used. We found that the latter strain required methionine and pyridoxine. The intergeneric hybrids could thus be selected for complementation on a minimal medium. Protoplasts were obtained and fusion induced with polyethylene glycol (PEG), as described earlier for intraspeeifie fusion in Candida tropicalis [13]. The
same minimal (MM) and complete (YEA) media were also used. Parental protoplasts were subjected separately to the same PEG treatment and regeneration process. A control of the reversion rate on MM was thus obtained. No colonies were found on the control plates, indicating that prototrophic colonies observed in the experiment were true fusion products. Carbon assimilation tests were performed on plates of agar medium according to Lodder [14]. For the enzymatic assays, the strains were grown in a liquid medium containing soluble starch (10 g/l) and yeast extract (5 g/I). Cells were harvested by ce~rifugation; growth medium and cells were both tested. The centrifuged cells were disrupted in a Braun cell homogenizer with glass beads in 0.06 M phosphate buffer pH 7.0. The suspension was then centrifuged for 45 rain at 70 000 g and the supernatant was kept for assay. Protein content was determined as in Lowry et al. [15]. Glucoamylase activity was measured from the amount of glucose liberated, as determined by the method of Gascon et al. [ 1 6 ] . For biomass evaluation, the strains wexe grown in a medium prepared as follows: (NH-4)2SO4 (15 g), KH2PO4 (4.5 g) and soluble starch (10 g) were dissolved in 500 ml tap water and pH was adjusted at 4.5. Then MgC12.6H20 (0.85 g), NaC1 (0.5 g), CaCla (0.5 g) and FeCla (0.005 g) were added, together with vitamins (thiamin 5 mg, biotin 60/~g, pyridoxine 3 mg) and methionine (100 mg). Finally 11 ml of lactate buffer (160 mg sodium lactate in 15 ml lactic acid) were added before making up to 1 1 with tap water.
310 TABLE 1 Assimilation of various carbon compounds and morphology of parental and hybrid strains Symbols +, % - , indicate normal growth, weak growth and no growth respectively. Parental strains are shown as S (Saccharomycopsis fibuligeria) and C (Candida tropicalis). The results concern 13 hybrids out of 18; the 5 others were only tested for morphology: two represented a yeast-like (Y) form and three a pseudomycelial (P) form. Strains
S
C
1
L-Sorbose Erythritol Trehalose D-Xylose Inositol D-Mannitol Hexadecane Morphology
+ + P
+
+
+ +
+ +
+ + Y
+ + Y
2
3
4
+ +
+_ -
+ +_
+ + + P
+ + + P
+ + P
5
6
7
+
-
+
8
9
10
+ +
+ +
+ +
+ +
+ +
+ + -
+ + P
+ P
+ + Y
+ + Y
+ + Y
+ + P
3. Results and Discussion
11
12
13
+ + +_ + P
+ + + + Y
+_ + + -+ + + Y
groups: strains 4 and 11 were very similar to S.
fibuligera and strains 1, 5 , 7 , 13 to C tropicalis (9 out In t w o i n d e p e n d e n t experiments, a t o t a l o f 18 fusion p r o d u c t s were obtained. Their f r e q u e n c y , expressed as the ratio b e t w e e n t h e n u m b e r o f colonies growing on MM and the n u m b e r o f colonies growing on Y E A , is a b o u t 10 -s. This is s o m e w h a t lower that in o t h e r interspeeific fusions [ 4 - 6 ] and m a y be due to the great t a x o n o m i c difference b e t w e e n the t w o genera.
o f 10 characters identical to the parent species). The seven o t h e r strains e x h i b i t e d a range o f i n t e r m e d i a t e types. These data lead to the conclusion that the fusion p r o d u c t s were true hybrids. This is strengthened b y the following observations. Hybrids strains were f o u n d to have uninucleate cells and seemed at first t o be stable. H o w e v e r , t w o o f the p s e u d o m y c e l i a l hybrids, growing slowly on MM, were in fact leaky a u x o t r o p h s , stimulated b y m e t h i o nine. A f t e r transfer o f these t w o hybrids on various media, p r o t o t r o p h i c clones w i t h rapid g r o w t h and yeast-like f o r m appeared. These n e w clones did n o t differ f r o m the initial ones w i t h respect t o the assimilation o f carbon c o m p o u n d s . (The association o f these quite different genetic i n f o r m a t i o n s resulted in a higher protein c o n t e n t o f the cells g r o w n on soluble starch (Table 3).) O t h e r cases o f instability were observed: after subculturing the (so-called) i n t e r m e d i a t e hybrids, s o m e
On solid m e d i u m , S. fibuligera d e v e l o p e d a pseudomycelial f o r m and yeast-like ceils were v e r y rare, whilst on the same m e d i u m , C. tropicalis had a b u d d i n g yeast behaviour. T h e generation t i m e o f S. flbuligera was d o u b l e t h a t o f C. tropicalis in liquid MM. T h e assimilation o f seven c o m p o u n d s , for w h i c h the parental strains differed, was tested. G r o w t h habit was e x a m i n e d and presence o f amylo-1:6-glucosidase was also investigated. As s h o w n in Tables 1 and 2, the p r e s u m e d hybrids could be divided into three
TABLE 2 Amylo-1:6-glucosidase activity in cells and in growth medium of parental and hybrid strains Strains as in Table 1. The ability of C. tropicalis to grow on soluble starch is due to the presence of another amylolytic enzyme which was not investigated. Strains
S
C
1
2
3
4
5
6
7
8
9
10
11
12
13
Cells (crude extract) Growth medium
+
0
+
+
0
+
0
+
+
+
+
+
+
+
0
+
0
0
0
0
+
0
0
0
+
+
+
+
+
0
3.48 27.1 0.94
Dry weight a Protein content b Total protein a
5.56 32.1 1.78
C 5.56 34.6 1.92
1 3.89 23.4 0.91
2 3.83 31.2 1.19
3 3.95 28.2 1.11
4 6.23 29.8 1.86
5 2.2 29.6 0.65
6 5.49 32.8 1.8
7 5.05 33.8 1.71
8 5.36 34.0 1.82
9
5.54 33.2 1.84
10
4.41 32.7 1.44
11
5.71 30.0 1.71
12
5.5 41.3 2.27
13
a in g/1. b Mean o f two experiments, in percent of dry weight. The amount of proteins in cells was estimated from the nitrogen content determined by the Kjeldahl procedure [17].
S
Strains
Dry weight and protein content after culture on soluble starch Cultures were performed in fembach flasks containing 200 ml of medium. Cells were harvested after 72 h culture (i.e. in stationary phase, 4 - 8 • 108 ceUs/ml).
TABLE 3
t-*
312 changes were also observed in the assimilation spectrum, with increasing similarities to one or the other o f the parental strains. We therefore suggest that the genome o f the hybrids might consist o f the genome o f one parental species associated with a few chromosomes o f the other. These chromosomes could be lost under certain conditions.
Acknowledgements We thank Prof. P. Schaeffer for advice at several steps o f this work. Excellent technical assistance o f Mrs. Germain is gratefully acknowledged.
References [1] Ferenczy, L., Kevei, F. and Szegedi, M. (1975) Experientia 31, 1028-1030. [2] Anne, J. and Peberdy, J.F. (1976) J. Gen. Microbiol. 92, 413-417. [3] Dales, R.B.G. and Croft, J.H. 91977) FEMS Microbiol. Lett. 1,201-204.
[4] Ferenczy, L., Szegedi, M. and Kevei, F. (1977) Experientia 33,184-186. [5] Anne, J., Eyssen, H. and de Somer, P. (1976) Nature 262,719-721. [6] Kevei, F. and Peberdy, J.F. (1977) J. Gen. Microbiol. 102, 255-262. [7] Sipiczki, M. and Ferenczy, L. (1977) Mol. Gen. Genet. 151, 77-81. [8] Van Solingen, P. and Van der Plaat, J.B. (1977) J. Bacteriol. 130,946-947. [9] Ferenczy, L. and Maroz, A. (1977) Nature 268,524525. [10] Schaeffer, P., Cami, B. and Hotchkiss, R.D. (1976) Proc. Natl. Acad. Sci. USA 73, 2151-2155. [ 11 ] Fodor, K. and Alfoldi, L. (1976) Proc. Natl. Acad. Sci. USA 73, 2147-2150. [12] Hopwood, D.A., Wright, H.M., Bibb, M.J. and Cohen, S.N. (1977) Nature 268, 171-174. [13] Fournier, P., Provost, A., Bourguignon, C. and Heslot, H. (1977) Arch Microbiol. 115,143-149. [14] Lodder, J. (1970) In: The Yeast (Lodder, J., Ed.), pp. 75-83 North-Holland, Amsterdam. [15] Lowry, O.H., Rosebrough, N.J. Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275. [ 16 ] Gascon, S. and Lampen, J.O. ( 1968) J. Biol. Chem. 243, 1567-1572. [17] Herbert, D., Phipps, P.J. and Strange, R.E. (1971) In: Methods in Microbiology, 5B (Norris, J.R. and Ribbons, D.W., Eds.) pp. 218-221. Academic Press, New York.
309
I N T E R G E N E R I C H Y B R I D I Z A T I O N IN YEASTS T H R O U G H P R O T O P L A S T F U S I O N A. PROVOST, C. BOURGUIGNON, P. FOURNIER, A.M. RIBET and H. HESLOT Department of Genetics, Institut National Agronomique, 16, Rue Claude Bernard, 75231 Paris Cedex 05, France Received 21 March 1978
1. Introduction Many yeast species have no clearly defined mating types, do not undergo meiosis, do not sporulate and are frequently polyploid, hence there are major difficulties in the improvement of these species for industrial purposes. Heterokaryons, as well as heterozygous diploids have been obtained through induced intra- and interspecific protoplast fusion in fungi [1-6] and yeasts [7-9]. Recombinants have also been selected in bacteria [10, 11 ], Streptomyces [ 12] and in the industrial asexual yeast Candida tropicalis [13]. In this paper, we show that recombinants can be obtained when protoplast fusion is induced between yeast strains belonging to different genera. These recombinants may show new useful characteristics and may be adaptable to a wider range of culture media. The induced fusion might also offer a method for transferring cytoplasmic organelles, such as mitochondria. This has already been done between strains ofS. cerevisiae [9].
2. Methods Strain C of Candida tropicalis (auxotrophic for cytosine and adenine) from our collection and strain CBS 2527 of Saccharomycopsis fibuligera were used. We found that the latter strain required methionine and pyridoxine. The intergeneric hybrids could thus be selected for complementation on a minimal medium. Protoplasts were obtained and fusion induced with polyethylene glycol (PEG), as described earlier for intraspeeifie fusion in Candida tropicalis [13]. The
same minimal (MM) and complete (YEA) media were also used. Parental protoplasts were subjected separately to the same PEG treatment and regeneration process. A control of the reversion rate on MM was thus obtained. No colonies were found on the control plates, indicating that prototrophic colonies observed in the experiment were true fusion products. Carbon assimilation tests were performed on plates of agar medium according to Lodder [14]. For the enzymatic assays, the strains were grown in a liquid medium containing soluble starch (10 g/l) and yeast extract (5 g/I). Cells were harvested by ce~rifugation; growth medium and cells were both tested. The centrifuged cells were disrupted in a Braun cell homogenizer with glass beads in 0.06 M phosphate buffer pH 7.0. The suspension was then centrifuged for 45 rain at 70 000 g and the supernatant was kept for assay. Protein content was determined as in Lowry et al. [15]. Glucoamylase activity was measured from the amount of glucose liberated, as determined by the method of Gascon et al. [ 1 6 ] . For biomass evaluation, the strains wexe grown in a medium prepared as follows: (NH-4)2SO4 (15 g), KH2PO4 (4.5 g) and soluble starch (10 g) were dissolved in 500 ml tap water and pH was adjusted at 4.5. Then MgC12.6H20 (0.85 g), NaC1 (0.5 g), CaCla (0.5 g) and FeCla (0.005 g) were added, together with vitamins (thiamin 5 mg, biotin 60/~g, pyridoxine 3 mg) and methionine (100 mg). Finally 11 ml of lactate buffer (160 mg sodium lactate in 15 ml lactic acid) were added before making up to 1 1 with tap water.
310 TABLE 1 Assimilation of various carbon compounds and morphology of parental and hybrid strains Symbols +, % - , indicate normal growth, weak growth and no growth respectively. Parental strains are shown as S (Saccharomycopsis fibuligeria) and C (Candida tropicalis). The results concern 13 hybrids out of 18; the 5 others were only tested for morphology: two represented a yeast-like (Y) form and three a pseudomycelial (P) form. Strains
S
C
1
L-Sorbose Erythritol Trehalose D-Xylose Inositol D-Mannitol Hexadecane Morphology
+ + P
+
+
+ +
+ +
+ + Y
+ + Y
2
3
4
+ +
+_ -
+ +_
+ + + P
+ + + P
+ + P
5
6
7
+
-
+
8
9
10
+ +
+ +
+ +
+ +
+ +
+ + -
+ + P
+ P
+ + Y
+ + Y
+ + Y
+ + P
3. Results and Discussion
11
12
13
+ + +_ + P
+ + + + Y
+_ + + -+ + + Y
groups: strains 4 and 11 were very similar to S.
fibuligera and strains 1, 5 , 7 , 13 to C tropicalis (9 out In t w o i n d e p e n d e n t experiments, a t o t a l o f 18 fusion p r o d u c t s were obtained. Their f r e q u e n c y , expressed as the ratio b e t w e e n t h e n u m b e r o f colonies growing on MM and the n u m b e r o f colonies growing on Y E A , is a b o u t 10 -s. This is s o m e w h a t lower that in o t h e r interspeeific fusions [ 4 - 6 ] and m a y be due to the great t a x o n o m i c difference b e t w e e n the t w o genera.
o f 10 characters identical to the parent species). The seven o t h e r strains e x h i b i t e d a range o f i n t e r m e d i a t e types. These data lead to the conclusion that the fusion p r o d u c t s were true hybrids. This is strengthened b y the following observations. Hybrids strains were f o u n d to have uninucleate cells and seemed at first t o be stable. H o w e v e r , t w o o f the p s e u d o m y c e l i a l hybrids, growing slowly on MM, were in fact leaky a u x o t r o p h s , stimulated b y m e t h i o nine. A f t e r transfer o f these t w o hybrids on various media, p r o t o t r o p h i c clones w i t h rapid g r o w t h and yeast-like f o r m appeared. These n e w clones did n o t differ f r o m the initial ones w i t h respect t o the assimilation o f carbon c o m p o u n d s . (The association o f these quite different genetic i n f o r m a t i o n s resulted in a higher protein c o n t e n t o f the cells g r o w n on soluble starch (Table 3).) O t h e r cases o f instability were observed: after subculturing the (so-called) i n t e r m e d i a t e hybrids, s o m e
On solid m e d i u m , S. fibuligera d e v e l o p e d a pseudomycelial f o r m and yeast-like ceils were v e r y rare, whilst on the same m e d i u m , C. tropicalis had a b u d d i n g yeast behaviour. T h e generation t i m e o f S. flbuligera was d o u b l e t h a t o f C. tropicalis in liquid MM. T h e assimilation o f seven c o m p o u n d s , for w h i c h the parental strains differed, was tested. G r o w t h habit was e x a m i n e d and presence o f amylo-1:6-glucosidase was also investigated. As s h o w n in Tables 1 and 2, the p r e s u m e d hybrids could be divided into three
TABLE 2 Amylo-1:6-glucosidase activity in cells and in growth medium of parental and hybrid strains Strains as in Table 1. The ability of C. tropicalis to grow on soluble starch is due to the presence of another amylolytic enzyme which was not investigated. Strains
S
C
1
2
3
4
5
6
7
8
9
10
11
12
13
Cells (crude extract) Growth medium
+
0
+
+
0
+
0
+
+
+
+
+
+
+
0
+
0
0
0
0
+
0
0
0
+
+
+
+
+
0
3.48 27.1 0.94
Dry weight a Protein content b Total protein a
5.56 32.1 1.78
C 5.56 34.6 1.92
1 3.89 23.4 0.91
2 3.83 31.2 1.19
3 3.95 28.2 1.11
4 6.23 29.8 1.86
5 2.2 29.6 0.65
6 5.49 32.8 1.8
7 5.05 33.8 1.71
8 5.36 34.0 1.82
9
5.54 33.2 1.84
10
4.41 32.7 1.44
11
5.71 30.0 1.71
12
5.5 41.3 2.27
13
a in g/1. b Mean o f two experiments, in percent of dry weight. The amount of proteins in cells was estimated from the nitrogen content determined by the Kjeldahl procedure [17].
S
Strains
Dry weight and protein content after culture on soluble starch Cultures were performed in fembach flasks containing 200 ml of medium. Cells were harvested after 72 h culture (i.e. in stationary phase, 4 - 8 • 108 ceUs/ml).
TABLE 3
t-*
312 changes were also observed in the assimilation spectrum, with increasing similarities to one or the other o f the parental strains. We therefore suggest that the genome o f the hybrids might consist o f the genome o f one parental species associated with a few chromosomes o f the other. These chromosomes could be lost under certain conditions.
Acknowledgements We thank Prof. P. Schaeffer for advice at several steps o f this work. Excellent technical assistance o f Mrs. Germain is gratefully acknowledged.
References [1] Ferenczy, L., Kevei, F. and Szegedi, M. (1975) Experientia 31, 1028-1030. [2] Anne, J. and Peberdy, J.F. (1976) J. Gen. Microbiol. 92, 413-417. [3] Dales, R.B.G. and Croft, J.H. 91977) FEMS Microbiol. Lett. 1,201-204.
[4] Ferenczy, L., Szegedi, M. and Kevei, F. (1977) Experientia 33,184-186. [5] Anne, J., Eyssen, H. and de Somer, P. (1976) Nature 262,719-721. [6] Kevei, F. and Peberdy, J.F. (1977) J. Gen. Microbiol. 102, 255-262. [7] Sipiczki, M. and Ferenczy, L. (1977) Mol. Gen. Genet. 151, 77-81. [8] Van Solingen, P. and Van der Plaat, J.B. (1977) J. Bacteriol. 130,946-947. [9] Ferenczy, L. and Maroz, A. (1977) Nature 268,524525. [10] Schaeffer, P., Cami, B. and Hotchkiss, R.D. (1976) Proc. Natl. Acad. Sci. USA 73, 2151-2155. [ 11 ] Fodor, K. and Alfoldi, L. (1976) Proc. Natl. Acad. Sci. USA 73, 2147-2150. [12] Hopwood, D.A., Wright, H.M., Bibb, M.J. and Cohen, S.N. (1977) Nature 268, 171-174. [13] Fournier, P., Provost, A., Bourguignon, C. and Heslot, H. (1977) Arch Microbiol. 115,143-149. [14] Lodder, J. (1970) In: The Yeast (Lodder, J., Ed.), pp. 75-83 North-Holland, Amsterdam. [15] Lowry, O.H., Rosebrough, N.J. Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275. [ 16 ] Gascon, S. and Lampen, J.O. ( 1968) J. Biol. Chem. 243, 1567-1572. [17] Herbert, D., Phipps, P.J. and Strange, R.E. (1971) In: Methods in Microbiology, 5B (Norris, J.R. and Ribbons, D.W., Eds.) pp. 218-221. Academic Press, New York.