Current Genetics

Curr Genet (1992)21:339-344

9 Springer-Verlag 1992

Complementation of the Saccharomyces cerevisiae srbl-1 mutation: an autoselection system for stable plasmid maintenance Sandra B. Rech 1,, Lubomira I. Stateva l, 2 and Stephen G. Oliver 1 1 Manchester BiotechnologyCentre, University of Manchester Institute of Science and Technology(UMIST), PO Box 88, Manchester M60 1QD, UK 2 Institute of Molecular Biology,BulgarianAcademyof Sciences, BG-1113 Sofia, Bulgaria Received October 8/November 15, 1991

Summary. The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain. When trfinsformed into a fragile srbl-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used. Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol). However, in the absence of the osmoticum, nearly 100 % of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth. Key words: Yeast - Saccharomyces cerevisiae - Lysis mutants - Plasmid stability

Introduction The high level expression of heterologous genes in Saccharomyces cerevisiae is most easily achieved by cloning such genes into high-copy-number, autonomously replicating, plasmid vectors. However, these plasmids are usually very unstable and are quickly lost from the cells. Different selection systems have been devised to solve this problem. The most commonly used is based on the complementation of an auxotrophic mutation in the host by a plasmid-borne wild-type gene (Gerbaud et al. 1979). This type of selection requires the growth of the transformants in defined minimal media, which are usually unsuitable for industrial fermentations. Recently, an autoselection system based on the use of double ura3, f u r l mutants (lacking functional orotidine-5'-phosphate decarboxylase and uracil phosphoribosyl transferase) was developed (Loison et al. 1986). Strains bearing these two mutations can be grown in complex media, including * Present address: Faculdade de Farmacia, Universidade Federal do Rio Grande do Sul, Porto Alegre 90.000, Brazil Offprint requests to: S. G. Oliver

those in common industrial use, when transformed with plasmids carrying the URA3 gene. For industrial strains, which are prototrophic and often polyploid, drug (Webster and Dickson 1983; Kaster et al. 1984; Zhu et al. 1985; Kunze et al. 1989) or metal (Fogel and Welch 1982; Fogel et al. 1983; Karin et al. 1984; Henderson et al. 1985) resistance have been used as positively selectable genetic markers for both transformation and the stable maintenance of plasmids. An alternative approach for such hosts is based on the transformation of sensitive yeast strains with plasmids containing a eDNA copy of the yeast MldsRNA, bearing genes determining both the killer toxin and its immunity factor (Bussey and Meaden 1985). Stable maintenance of such plasmids has been reported provided the medium contains appropriate drugs, active agents like Cu 2 +, or if the medium pH is kept relatively constant (in the case of the killer toxin immunity selection). These special requirements, which are often difficult or expensive to apply in large scale fermentations, are a major drawback of these systems. A further complication is the high level of constitutive or inducible resistance to the selective agent which is found in many host strains. Recently, a gene was cloned (Stateva etal. 1991) which complements the nuclear recessive mutation srbl1, shown in earlier studies (Kozhina et al. 1979) to determine the ability of the S. cervisiae fragile mutant VY1160 to spontaneously lyse upon osmotic shock (Venkov et al. 1974). S. cerevisiae srbl-1 mutants grow normally only in the presence of osmotic stabilisers in the medium. When transformed with the cloned SRB1 gene, the mutants lose both their growth dependence on the presence of the osmoticum and their ability for lysis upon osmotic shock. In this paper, we report on the stability of autonomously replicating plasmids which contain the SRB1 gene and their potential exploitation in industrial processes.

Material and methods Strains andplasmids. The followingS. cerevisiae strains were used: DL-=MAT~, leu2-3, 2-112, his3-11,15, ura3-251, 372, 328,

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sodl::LEU2, cir + (kindly provided by Dr. A.P.G.M. van Loon) SLU15 = MAT~, leu2, ura3, srbl-1, tsl (from our laboratory collection of fragile srbl-1 mutants). E. coli HB101 was used for transformation and amplification of plasmid constructs. The plasmid YEpSS1 (see Fig. 1) was constructed as follows: YEp352 (Hill et al. 1986) was digested with BamHI and ligated to the 2.0 kb SOD1containing fragment derived from pYEUMn (Schrank et al. 1988). The resultant construct, YEpS1, was digested with HindlII and Sail and ligated to the 4.5 kb HindIII-SalI fragment containing the SRB1 gene (Stateva et al. 199t). Media. Yeast: The batch culture experiments were performed in YEPD (Sherman et al. 1970) with and without 10% w/v sorbitol as an osmotic stabiliser. The synthetic minimal medium used for the chemostat culture experiment was prepared as described by Brown et al. 1984. Bacteria: E. coli strains were grown in LB medium (Sambrook et al. 1989) supplemented with the necessary antibiotics. Culture conditions. For the batch culture experiments, a single transformed clone was inoculated in 20 ml YEPD and grown at 30 ~ under agitation until mid-exponential phase, when a sample was withdrawn and used for plasmid stability determination and inoculation of a fresh culture. The continuous culture experiments were performed in an LH2000 fermenter (top-stirred fermenter with nominal volume of 2 1from LH Engineering Ltd., Stoke Poges, UK) maintained under the following conditions: 1.5 1 working volume; 1.5 1 min -1 air supply rate; temperature 30_+0.1~ pH 5_+0.1; stirrer speed, 500 rpm. Plasmid stability determination. Aliquots were withdrawn from the cultures at appropriate time intervals and plated for single colonies onto YEPD agar plates. One thousand colonies were screened at each time point for plasmid loss by replicating them onto appropriate single omission selective media. DNA preparation. Yeast DNA was prepared from sheroplasts (Davis et al. 1980). Bacterial plasmid DNA was isolated on a large scale by the alkaline lysis method of Birnboim and Doly (1979) as described by Sambrook et al. (1989). Small scale DNA preparation (minipreps) was performed by the method of He et al. (1990). All DNA fragments used for subcloning or radioactive labelling were gel purified by the freeze-squeeze method of Tautz and Renz (1983).

phase, when the cells were growing at their ~max, according to the equation given by Pirt (1975).

Analysis of SOD activity. Superoxide dismutase (SOD) activity was monitored in native polyacrylamide gels by the method of Beauchamp and Fridovich (1971), as described in Schrank et al. (1988). Protein concentration of the extract was determined by the method of Bradford (1976). Usually 30-50 ~tl of the protein extracts were loaded to give an approximately equal amount of total protein from the different samples.

Results Instability o f SRBl-containing plasmids, transformed in wild-type strains I n the p r e s e n t p l a s m i d - s t a b i l i t y i n v e s t i g a t i o n , p l a s m i d s c o n t a i n i n g the S O D 1 gene were used. This is a gene w h i c h codes for y e a s t M n s u p e r o x i d e d i s m u t a s e , a r e p r e s e n t a tive o f the u n i v e r s a l f a m i l y o f s u p e r o x i d e d i s m u t a s e s ( F r i d o v i c h 1974, 1975). S u p e r o x i d e d i s m u t a s e s are o f c o m m e r c i a l interest a n d have been suggested for use in the t r e a t m e n t o f a n u m b e r o f diseases, i n c l u d i n g c a r d i a c i s c h a e m i a ( M c C o r d 1985). Therefore, c o n d i t i o n s for stable m a i n t e n a n c e o f S O D l - c o n t a i n i n g p l a s m i d s m i g h t p r o v e i m p o r t a n t for i n d u s t r i a l a p p l i c a t i o n a n d so p r o v i d e a m o d e l for the p r o d u c t i o n o f p h a r m a c e u t i c a l l y i m p o r t a n t p r o t e i n s in yeast. E a r l i e r e x p e r i m e n t s , c a r r i e d o u t with different autonomously replicating plasmids containing SOD1, s h o w e d t h a t all o f t h e m were very u n s t a b l e in S. cerevisiae D L - . B o t h b a t c h a n d c o n t i n u o u s culture e x p e r i m e n t s i n d i c a t e d t h a t the p l a s m i d s were u s u a l l y lost f r o m the cell p o p u l a t i o n in a b o u t 50 g e n e r a t i o n s ( d a t a n o t shown). I n a n a t t e m p t to e v a l u a t e the p o t e n t i a l o f using the c l o n e d S R B 1 gene ( S t a t e v a et al. 1991) as a basis o f a self-selection system for stable p l a s m i d m a i n t e n a n c e , we con-

Enzymatic reactions. Restriction enzymes, calf intestinal alkaline phosphatase (CIP), T 4 DNA ligase, and DNA polymerase I were from Pharmacia, Milton Keynes, UK. All reactions were performed according to the instructions of the manufacturers. DNA probes were radiolabelled with a2p adCTP (Amersham, Aylesbury, UK) by nick translation (Rigby et al. 1977). Unincorporated nucleotides were separated by the spun-column procedure (Sambrook et al. 1989).

AMP

Southern analysis. The standard protocol for Southern analysis was used (Sambrook et al. 1989). The 2.0 kb BamHI fragment from pYEUMn (Schrank et al. 1988) was used as a probe for the SOD1 gene, and the 4.0 kb BamHI fragment (Stateva et al. 1991) from YEpI3::SRB1 for the SRB1 gene.

.RV

URA3

-lindIII

YEpSS1 12181 bp

Transformation. E. coli strains were transformed by the C a 2 + method as described by Sambrook et al. (1989). Wild-type S. cerevisiae strains were transformed by the spheroplast method of Hinnen et al. (1978) as modified by Burgers et al. (1987). Fragile srbl-1 strains of S. cerevisiae were transformed by Philipova's (1985) modification of the method of Ito et al. (1983) as detailed in our previous publication (Stateva et al. 1991).

SOD1

Sal I

Determination of maximum specific growth rate by wash-out kinetics. The maximum specific growth rate (g~ax) of yeast strains was determined by wash-out kinetics from the chemostat culture (Esener et al. 1981; Walmsley et al. 1983). A biphasic exponential fall in cell density was observed and I.tmax was calculated from the second

2~

SRB1

Fig. 1. Map of YEpSS1. Details about the constructions of the plasmid are given in Materials and methods. (N), SRB1 gene; ([]), SOD1; ( ~ , URA3; (--), 2l-t sequences; ( - - ) , pBR322 sequences

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structed a series of 2 Ix-based autonomously replicating plasmids designated YEpS (Fig. 1 and see Materials and methods). The two main representatives of this series are the plasmids YEpS1 and YEpSS1. In addition to the URA3 marker, the first one contains the gene SOD1 coding for Mn superoxide dismutase, the second one also has the gene SRB1, which complements the lysis mutation srbl-1. The wild-type strain D L - was transformed with the plasmids YEpS1 and YEpSS1 and their rate of loss determined in batch culture experiments. The results (Fig. 2) indicate that both plasmids were lost rapidly from the cell population; the plasmid YEpSSI being more unstable than YEpS1, perhaps as a result of its larger size.

Y* P l a s m i d - c o n t a i n i n g cells

100 90 80 70 6O 50 40 30 20

Batch culture plasmid stability of YEpSSI transformed into a fragile srbl-1 mutant

i0 0

The experiment described above demonstrated that plasmid YEpSS1 was very unstable in a wild-type host. In an alternative experiment, we transformed the same plasmid into the fragile mutant SLU15. The resultant transformants are no longer dependent for growth upon the presence of an osmotic stabiliser in the nutritional medium, as shown by complementation studies using the SRB1 clone (Stateva etal. 1991). The stability of YEpSS1 transformed into the srbl-1 mutant SLU15 was determined in batch culture experiments using YEPD medium withand without 10% w/v sorbitol. The results (Fig. 3) show a striking difference in the maintenance of the plasmid YEpSS1 under the two kinds of growth conditions used. In the presence of the osmotic stabiliser (10% w/v sorbitol), the plasmid was completely lost from the srbl-1 population after approximately 45 generations; this result is similar to that observed with the wild-type strain, D L - (Fig. 2). However, when the same transformant was grown in the absence of the osmotic stabiliser, the trailsforming plasmid YEpSS1 proved very stable, being retained in almost 100% of the cells over a period of 80 generations.

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lOO

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Fig. 2. Batch culture kinetics of the accumulation of plasmid-free cells from the D L - strain. Cells were kept in the exponential phase by serial transfers to fresh medium (see Materials and methods). YEpSI loss is indicated by (-m-); YEpSS1 loss is indicated by ( + + )

% P l a s m i d - c o n t a i n i n g cells

1oo 90 80 70 60 50

40 30 20

Continuous culture experiments with YEpSS1 transformed in SLU15 To further evaluate the utility of fragile srbl-1 hosts for the stable maintenance of SRBl-containing plasmids, we grew SLU15 transformed with YEpSS1 under continuous culture conditions in synthetic minimal medium with and without 10% w/v sorbitol. Aliquots of the culture were taken at intervals and used for plasmid stability determination and DNA isolation for Southern analysis with the SOD1 and SRB1 genes as hybridisation probes. Figure 4 shows the results of the plasmid stability determination. In good agreement with the batch culture, the chemostat culture experiment also showed that, when an osmotic stabiliser is included in the nutritional medium, the YEpSS 1 plasmid is very unstable. After approximately 45 generations of continuous exponential growth,

10 0 10

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Generations

Fig. 3. Batch culture kinetics of the accumulation of plasmid-free cells from SLU15, grown in the presence ( + + ) and in the absence ( s ) of 10% w/v sorbitol. Cells were kept in the exponential phase by serial transfers to fresh medium (see Materials and methods)

100% of the cells had lost the transforming plasmid. In contrast, the same plasmid was very stable when the srbl1 transformant was grown in the absence of an osmotic stabiliser. Nearly 100% of the cells retained the plasmid after 80 generations of growth.

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The stable maintenance of YEpSS1 in the srbl-1 transformant grown continuously in the absence of sorbitol was also verified by Southern analysis of DNA isolated from the cells after different periods of time. The results (Fig. 5) show that, during the whole period tested, the cells retained the plasmid in an unaltered form as indicated by the hybridisation signals obtained with both SOD! and SRB1 probes.

Determination of maximum specific growth rate (#max) The maximum specific growth rate (~tmax)is an important parameter in the characterisation of microorganisms for

% Plasmid-containing cells

use in industrial fermentation processes (Pirt 1975). The ~tmaxof the two host strains studied, and of their different transformed derivatives, was determined by the wash-out kinetics method of Esener et al. (1981), as described by Walmsley et al. (1983). The results (Table 1) show that the fragile mutant SLU15 is characterised by a significantly longer doubling time compared to the wild-type strain D L - . However, the generation time of the two strains is affected to a different extent after transformation. While the introduction of the plasmid YEpS 1 increases considerably the doubling time of the wild-type strain D L - , there is virtually no change in the Td of the fragile mutant, compared to the plasmid-free strain SLU15, when the former is transformed with the larger YEpSS1 plasmid.

Determination of SOD activity by PAGE

lO0 90 80 70 60 50 40 30 20 10 0

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Fig. 4. Continuous culture kinetics of the accumulation of plasmidfree cells from the SLU15 transformant, grown with ( + ) and without (-- -- ) 10% w/v sorbitol

Since the system described above is potentially designed for stable maintenance of plasmids containing heterologous genes, we carried out SOD1 activity tests to demonstrate that the transformed fragile cells not only retained the plasmids, but expressed the cloned gene. For this purpose, we prepared protein extracts from cells of SLU15 transformed with YEpSS1 and grown for approximately 40 generations under continuous culture conditions. For comparison, the untransformed SLU] 5 strain was also used. Figure 6 shows that both strains produce Cu/Zn SOD, as indicated by the presence of the lower molecular weight band in lanes 1 and 2. However, the expression of the chromosomal SOD1 gene encoding MnSOD is virtually undetectable in the untransformed strain (lane 1), reflecting the fact that mitochondriat enzymes are the minor species in yeast. In contrast, there is a strong band in the transformant (lane 2) with the same mobility as the Mn SOD marker run in parallel in lane 3. The fact that, even in the transformant, there is less Mn SOD than Cn/Zn SOD activity may indicate that there is insufficient capacity to transport the Mn SOD precursor to the mitochondrion where it is activated.

Fig. 5. Southern analysis of YEpSS1 transformed into SLUI5 and grown continuously in the absence of 10% sorbitol. Lane 1, SLU15 untransformed; lane 2-6, SLU15-YEpSSI transformant after a different number of generations: lane 2-15; lane 3-30; lane 4-45; lane 5-60; lane 6 - 7 5

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Fig. 6. Determination of SOD activity by PAGE. Lane 1, SLU15 parental; lane 2, SLU15 transformed with EYpSS1 and grown for 45 generations in the absence of osmoticum; lane 3, Mn SOD marker

Table 1. Continuous culture growth parameters of S. cerevisiae DL- and SLUI5 transformed with different plasmids Strain/plasmid

~max(h-l)

Td(h)_+SEM"

DLDL-/YEpS1 DL /YEpSS1 SLU15 SLU15/YEpSSI

0.201 0.i68 N.D. b 0.145 0.147

3.45 +_0.10 4.12 ___0.17 4.78 _+0.21 4.71 _+0.19

a The results were obtained by a number of independent determinations, until the standard error of the mean (SEM) was less than 5% of P'm.~ b Because of high instability of the transforming plasmid, this transformant was not amenable to the wash-out experiment

Discussion

Any type o f autoselection system where: (1) the transforming plasmids are stably maintained, (2) the production of heterologous proteins is independent of the growth medium composition and/or does not require any selective agent, and (3) non-productive cells do not accumulate in the culture, is of particular interest in biotechnology. The results presented above provide evidence for a system which possesses these characteristics. It is based on: (1) the use of existent srbl-1 fragile mutants of the yeast S. cerevisiae (Venkov et al. 1974, Kozhina et al. 1979) which grow normally only on addition o f an osmotic stabiliser to the nutritional medium but spontaneously lyse upon osmotic shock, and (2) a D N A fragment which contains the S R B 1 gene (Stateva et al. 1991) and can, therefore, complement the s r b l - I mutation.

In our autoselection system, a fragile srbl-1 mutant is transformed with an autonomously replicating plasmid which contains the cloned wild-type gene S R B 1 . This renders the growth of the transformant independent of the addition of an osmotic stabiliser to the nutritional medium. Therefore, quite advantageously, any normal nutritional medium becomes selective for transformants which harbour plasmids containing the S R B I gene. Such plasmids are stably maintained in both batch- and continuous-culture conditions for at least 80 generations. These same plasmids, however, when transformed into a wild-type strain or into a fragile mutant grown in the presence of 10% sorbitol, are rapidly lost from the cell population. The system not only makes any nutritional medium selective, it also provides conditions for the survival o f only the plasmid-containing cells, since the fragile cells will eventually lyse upon loss of the S R B 1 containing transforming plasmid. Therefore, with the proposed system, non-productive cells do not accumulate in the culture. U p o n subsequent osmotic lysis, biomass of the plasmid-free cells is made available to the growing plasmid-containing cells. The system, however, has some disadvantages. First, the mutation srbl-1 has been shown to be ochre (Stateva and Tafrov, unpublished), so a more stable mutation is desirable. The cloned gene S R B 1 offers the opportunity to overcome this problem by creating stable mutations in vitro and using them to replace the wild-type S R B 1 sequence. We have taken advantage of this approach and created a disruption mutant. Experiments are under way to evaluate its potential as a host in our autoselection system. Second, the srbl-1 mutants grow very slowly and are not suitable for industrial purposes. A possible way to overcome this problem might be through the use of poly-

344 ploid derivatives (Stateva et al. 1988). Such strains possess the following advantages: unlike the haploid srbl-1 mutants, the srbl-1 hybrids of higher ploidy (2n, 3n, 4n) are prototrophic for growth and have been successfully grown in industrial medium based on molasses. U n d e r these conditions they grow nearly as fast as industrial strains of S. cerevisiae. In addition, these strains will further improve the stable maintenance of the transforming plasmids due to their higher ploidy (Mead et al. 1986; Spalding and Tuite 1989). The selection system described in this paper has two main features. First, it makes any ordinary nutritional medium selective, as long as it is compatible with yeast cell growth. Second, the combination of the srbl-1 host and the SRBI gene-containing plasmids eliminates the problem of accumulation of non-productive cells in the culture. A further advantage of our system over ones in which an essential gene is deleted or disrupted (e.g., the ede9 complementation system, Unternahrer et al. 1991) is that untransformed host cells m a y be simply maintained on osmotically buffered media. There is no need to carry the wild-type gene on a maintenance plasmid which must be displaced by the vector carrying the gene encoding the desired protein product. F o r these reasons, we consider the proposed system advantageous to the other systems described so far in the literature, and believe it m a y prove applicable for industrial purposes.

Acknowledgements. SBR was supported by a scholarship from CNPq, Brazil.

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