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YEAST
VOL.12: 199-205 (1996)
Selection of Yeast Cells with a Higher Plasmid Copy
Number in a Saccharomyces cerevisiae Autoselection
System
CONCETTA COMPAGNO, DANILO PORRO*, STEFANIA RADICE, ENZO MARTEGANI AND
BIANCA MARIA RANZI
Dipartimento di Fisiologia e Biochimica Generali, Sez. Biochimica Comparata, Universita di Milano,
via Celoria 26, 20133 Milano, Italy
Received 11 May 1995; accepted 7 August 1995
Autoselection systems allow the selection of a genetically engineered population independently of the growth
medium composition. The structure of a Saccharomyces cerevisiae population transformed with an autoselection
plasmid, in which a carbon-source-dependentmodulation of the plasmid copy number occurs, was analysed.
By means of flow cytometric procedures we tested the cell viability, dynamics of growth and heterologous protein
production at single cell level. Such analyses allow the identification and the tracking of a specific cellular
sub-population with a higher plasmid copy number which arises after the carbon source shift. The effects of the
cellular plasmid distribution on the dynamics of growth are also discussed.
KEY WORDS - S.
cerevisiae; autoselection plasmid; flow cytometry
INTRODUCTION
The first key factor required for successful production based on genetically engineered host cells is
the stability of the plasmid.
Autoselection systems represent a special class
of host-expression vectors which offer many
advantages for the improvement of processes related to the production of heterologous proteins.
In such systems, selection of the transformed
population occurs independently of the growth
medium composition, allowing the use of relatively
inexpensive complex media which are preferred
in industry for commercial-scale fermentations.
Different autoselection systems have been developed for Saccharomyces cerevisiae host cells
(Loison et al., 1986; Piper and Curran, 1990;
Unternahrer et al., 1991; Rech et al., 1992; Ayub
et al., 1992; Ludwig et al., 1993). The efficiency of
these systems is related to the opportunity to use
rich media which yield increased recombinant
protein productions (Loison et al., 1989; Napp and
Da Silva, 1993). Recently an autoselection system
has also been described for Kluyveromyces lactis
(Fleer, 1992).
*Corresponding author.
CCC 0749-503X/96/030199-07
0 1996 by John Wiley & Sons Ltd
Since autoselection systems are relatively new,
little information is available on the plasmid distribution in the cellular population. More knowledge on the state(s) of the population and on the
cellular distribution of the plasmid could be used
to improve the production processes.
Flow cytometry allows the study of the behaviour of a cell population following the analysis of
bioparameters at the single-cell level. Each cellular
population is heterogeneous with respect to cellular states such as mass, age and biochemical composition. The distributions of these properties in
the whole population provide information that
usually is lost when average properties of the
population are considered. The analysis of population growth dynamics (Alberghina and Porro,
1993), of the propagation of recombinant plasmids
(Wittrup et al., 1990) and of the metabolic state of
a cellular population (Porro et al., 1994) are some
examples of the application of this technique to the
studies of growing yeast populations.
We have previously reported the development of
an autoselection system based on the use of the
gene FBAZ (i.e. encoding the FDP aldolase
enzyme) to stabilize expression vectors in S.
cerevisiae host cells bearing a disruption of the
200
chromosomal FBAl gene. By using the inducible
UAS,,,
promoter to regulate the expression of
the FBAI gene, we have also obtained a system
in which the plasmid copy number is strongly
modulated by the carbon source used for growth
(Compagno et al., 1993).
In this work we report the studies performed to
gain insight into the population structure and the
cellular plasmid distribution in yeast populations
transformed with the autoselection plasmid
described above. By means of flow cytometric
procedures, we were able to study dynamics of
selection of a sub-population with a higher plasmid copy number during transient state(s) of
growth.
MATERIALS AND METHODS
Strain and growth conditions
The S. cerevisiae haploid strain WA6[pIA10]
( M A T a, ade2-1, canl-100, Jbal::URA3, leu2-3,
trpl-1, his3-11,15), in which the plasmid pIAlO
(carrying the wild-type gene for aldolase) complements the disruption of the corresponding chromosomal gene, was used in this study (Compagno
et al., 1993). Yeast cells were grown at 30°C in
minimal medium containing 0.67% (wh) Yeast
Nitrogen Base without amino acids (Difco Laboratory, Detroit, MI, U.S.A.) supplemented with the
required amino acids (50 pg ml- I). The carbon
source was 2% (w/v) glucose or 2% (wh) galactose.
In all cases, the cultures were grown in flasks in a
shaking incubator. For solid media, 2% (w/v) agar
was added. The cell number concentration was
determined, after sonication and appropriate dilution with Isoton (Coulter Electronics, England),
with a Coulter Counter ZBI equipped with a 70 pm
orifice.
Plasmid construction and shufling
Standard DNA manipulations were performed
according to Sambrook et al. (1989). Plasmid
pIlOG was constructed from pIAlO (Compagno
et al., 1993). The Escherichia coli LacZ gene was
obtained by the BamHI-StuI digestion of the plasmid pEl (Compagno et al., 1991) and was inserted
into the BamHI site of the plasmid pIA10, obtaining the plasmid pI1OG. The plasmid pIlOG was
used to transform WA6[p13A] cells and plasmid
shuffling was induced, selecting Trp+lLeu- cells in
which the plasmid p13A was substituted by the
PI 1OG plasmid.
C . COMPAGNO ET
AL.
Determination of the plasmid copy number
Total yeast DNA was prepared by the method
described by Nasmyth and Reed (1980). Total
DNA was digested with HindIII, run on agarose
gel and stained with ethidium bromide. The most
prominent bands seen were those due to plasmid
restriction fragments and those due to the restriction fragments of ribosomal DNA. The gels were
photographed and plasmid copy number was determined by comparing the band density of plasmid DNA with that of ribosomal DNA taken as
internal control (100 copies per cell; Broach, 1983)
using a scanning densitometer. The plasmid copy
number determination related to t=O in Table 2
was determined by a classical Southern approach
(Compagno et al., 1993).
Enzyme assays
Aldolase and P-galactosidase assays were performed as previously described (Compagno et al.,
1993).
ImmunoJiuorescent staining
The P-galactosidase immunofluorescent staining
was based on the procedure described by Eitzman
and Srienc (1991) with some modifications. Each
sample containing approximately 3 x lo7 cells was
collected, centrifuged and resuspended in 5 ml of
a fixation solution (3.7% formaldehyde; 1.3 Msorbitol in saline phosphate buffer (PBS: 3.3 mMNaH,PO,,
6.7 ~ M - N ~ , H P O ,0.2 mM-EDTA,
130 mM-NaC1, pH 7.5)) and incubated for 90 min
at room temperature (25°C). After recovery, the
cells were washed twice in 1-3M-sorbitol in PBS
and resuspended in a digestion solution containing
8 mg ml of Zymolyase 20T (ICN, Biomedicals),
27 mM-P-mercaptoethanol in 1.3 M-sorbitol-PBS.
The mixture obtained was vortexed gently and
incubated at 37°C for 30 min. The cells were then
centrifuged (2000 rpm, 10 min), washed once with
cold PBS and resuspended in 2.5 ml of cold PBS. A
100 p1 aliquot of each digested cell suspension
was resuspended in a microcentrifuge tube with
100 pl of a warm (37°C) PBS solution containing
10 mg ml - of bovine serum albumin (Sigma) and
the mouse anti-P-gal antibody (diluted 1:lOOO from
the stock solution; Sigma, Immuno Chemicals).
The cell suspension was incubated at 37°C for 1 h,
recovered by centrifugation (2000 rpm, 10 min)
and washed with 200pl of PBS. A second wash
was performed for 20 min at 37°C in PBS. After
recovery, cells were resuspended in a solution
~
20 1
AUTOSELECTION OF HIGH PLASMID COPY NUMBER
containing 2 pg/ml affinity-purified goat antimouse F(ab’)2 fragment conjugated with fluorescein isothiocyanate (FITC; Boehringer Mannheim,
Biochemicals) in PBS and incubated for 75 min at
37°C. After centrifugation, cells were washed twice
in 200yl of PBS, resuspended in PBS, sonicated
and analysed by flow cytometry.
Ethidium bromide, Concanavalin A (ConA)-FITC
staining andJlow cytometric analyses
Staining of dead cells with ethidium bromide,
staining of cell walls with conjugated ConA-FITC
and flow cytometric analyses were performed as
previously described (Martegani et al., 1993; Porro
and Srienc, 1995, respectively).
E
J
-I
w
10’
v
0
10
20
30
40
50
TIME (hours)
Figure 1. Growth curve of WA6[pIA10] transformed cells
during carbon source shift from galactose- to glucosecontaining medium. Cells precultured on galactose medium
were inoculated on glucose medium ( t = O ) . After 24 h of
growth, cells were reinoculated at a lower cell density in fresh
glucose medium. An unusually long lag phase can be observed.
RESULTS AND DISCUSSION
Analysis of cellular viability and growth properties
of transformed yeast cells during a carbon
source s h f t
The effect of the expression level of the selective
marker on the plasmid copy number has been
documented (Erhart and Hollemberg, 1983; Piper
and Curran, 1990). We have previously shown that
a carbon-source-dependent modulation of the
plasmid copy number can be also obtained by
cloning of the FBAl gene, the autoselective
marker, under the control of the UASGAL,.IO
promoter (i.e. plasmid pIA10) in cells bearing a
disruption of the chromosomal FBAl gene (i.e.
strain WA6; Compagno et al., 1993). WA6[pIA10]
S. cerevisiae transformed cells growing on galactose media exhibited a high level of aldolase (35 U mg - ’) in spite of a low plasmid copy number
(about five copies per cell), due to the effect of
UASGA, induction on FBAl gene expression. On
the other hand, in glucose media the repression of
the FBAl gene expression resulted in a low aldolase level (0.2 1 U mg - I ) and a high plasmid copy
number (about 80 copieskell). These observations
led us to wonder how yeast cells succeed in amplifying the plasmid copy number. At least two
different mechanisms could occur. In one case, the
plasmid copy number in each cell could increase
over time; on the other hand, the increase of
plasmid copy number should be related to the
selection of transformed cells bearing a higher
plasmid copy number. In order to discriminate
between the two hypotheses, we studied the
viability of the population and the dynamics of
growth during a typical transient state of growth
(Figure 1). The viability of the population during
shift from galactose- to glucose-containing media
was assessed by flow cytometry. The method to
estimate yeast cell viability relies on the uptake
from dead/damaged cells of ethidium bromide, a
dye which is normally excluded from viable cells
(De la Fuente et al., 1992; Martegani et al., 1993).
Analysis of staining patterns over time after the
carbon source shift (Figure 2) showed a deep
alteration of the cellular permeability during the
lag phase (B=27 h). In parallel with a restored
growth of the population, a cellular subpopulation with a low associated fluorescence
gradually rose (C=42 h, D=48 h). Such analysis
indicates that during the transient state of
growth, a relevant fraction of the transformed
WA6[pIA10] yeast cells died. Such a phenomenon
could be related to the long lag phase observed (see
Figure l), while the new growth following the lag
phase should be related to the accumulation of
viable cells.
In parallel, cell viability was estimated by the
classical approach of the plate-forming units obtained by plating on glucose samples taken from
the culture at different times during the lag phase.
In agreement with the flow cytometry analysis, we
observed that at the beginning of the lag phase,
most of the population was unable to grow on
glucose (i.e. only 0.5% of the population is viable),
while the number of colonies increased in the
course of this phase (at 31 h the fraction of viable
cells was 2% and at 46 h it was 9%). It is interesting
to note that the total amount of plasmid in all the
individual colonies analysed was high (60-80
copieskell, data not shown).
202
C. COMPAGNO ET AL.
d
L
-1
ii
FSC
d
K
W
8
-I
CONFI-FITC
W
FSC
LIN( FL2 1
Figure 2. Analysis of cell viability of WA6[pIAlO] transformed cells during carbon source shift from galactose- to
glucose-containing medium. Histograms A-D refer to the
samples withdrawn at 24 h (A), 27 h (B), 42 h (C) and 48 h
(D) of the experiment shown in Figure 1, treated with ethidium bromide and analysed by flow cytometry. Lin(FL2) is
the fluorescence signal (linear scale) related to ethidium
bromide.
Recently a novel flow cytometric procedure has
been developed to obtain information on the
growth properties of individual S. cerevisiue cells
in asynchronous culture. The method is based on
labelling the cell walls of growing cells with the
lectin ConA conjugated to a fluorescent marker
such as FITC. Cells growing in balanced growth
conditions are collected, stained with ConA-FITC
and resuspended in fresh medium. Because formation of new cell wall material in budded cells is
restricted to the bud tip, exposure of the stained
cells to growth conditions results in the production
of newborn daughter cells with a gradual decrease
in surface staining, which can thus easily be identified from the overall growing population (Porro
and Srienc, 1995). Transformed WA6[pIA 101 yeast
cells, precultured on galactose medium, were inoculated on glucose medium and, 24 h after the
nutritional shift, were stained with ConA-FITC
and resuspended in fresh glucose medium. At this
time all the cell walls were completely stained
(Figure 3A). Figure 3B shows the staining behaviour 7 h later (corresponding to t=31 h of growth
on glucose in Figure 1). On the right the evolution
of partially stained newborn daughter cells is
Figure 3. Dynamics of the cell-wall-tag for cells grown on
glucose. WA6[pIAIO] cells, cultured as described in Figure 1,
were stained with ConA-FITC after 24 h of growth on glucose
(A) and resuspended in fresh glucose medium. (B) 7 h after
resuspension partially stained cells (on the right) evolve as
newborn daughter cells; this point corresponds to 31 h of
growth on glucose. (C) 18 h after resuspension (42 h of growth
on glucose). (D) 24 h after resuspension (48 h of growth). FSC
is flow cytometric determination of cell volume (linear scale);
ConA-FITC is fluorescence signals related to Concanavalin
A conjugated to fluorescein (logarithmic scale). The arrows
indicate the direction of axes.
clearly evident. If it is assumed that the area of the
measured light-scattering signals at low angles
(FSC) reasonably reflects the size of cells, one can
directly estimate from these data some growth
properties of the growing transformed yeast
population. For example, it is interesting to
note that the average size of the overall population increased during the first 7 h (compare
FSC signals in Figure 3B and A). Even if during
this time it is not possible to note an appreciable
increase of the cell number concentration, all the
above considerations taken together indicate that
the lag time observed is only apparent. In fact,
from the area of the fluorescence signals associated
with the newborn cells, it is possible to calculate that this population evolves with a doubling
time of about 5 h. Figures 3C and D are related to
the staining pattern observed 18 h (t=42 h of
growth on glucose in Figure 1) and 24 h (t=48 h
in Figure 1) later. The figure clearly shows the
selection of a new population and its increase over
time.
203
AUTOSELECTION OF HIGH PLASMID COPY NUMBER
-
Table 2. Time-course of the specific 0-galactosidase
I
activity and of the plasmid copy number during the
E
2
growth of the WA6[pIAlOG] population on glucose
media.
W
m
I
3
~
Z
Time (h)*
-1
-I
~~
P-gal activity
(U I*g -
'>
COPY
number
W
0
0
10
20
30
40
50
TIME (hours)
Figure 4. Growth curve of WA6[pIlOG] transformed cells
during carbon source shift from galactose- to glucosecontaining medium. Cells precultured on galactose medium
were inoculated on glucose medium (t=O). After 24 h of
growth, cells were reinoculated at a lower cell density in fresh
glucose medium.
0
24
32
48
1
5
15
17
5
20
nd
80
*The values reported at the t=O refer to cells cultured on
galactose medium. At this time, cells were inoculated on glucose
medium, as described in Figure 4.
Table 1. Physiological parameters of WA6[pIA10] and
WA6[pIlOG] strains after 24 h of growth on glucose
medium.
WA6[pIA101
Cell number mi - '
Aldolase (U mg ')
Plasmid copy number
~
P-gal (U I*g- '1
5 x 106
0.05
10
-
WA6[pI1OG]
107
0.2
20
5
Analysis of cellular plusmid distribution during the
carbon source shvt
In order to correlate more tightly the dynamics
of growth to the increased plasmid content in the
population growing on glucose, we analysed the
distribution of the plasmid copy number at singlecell level during the nutritional shift. For this
purpose, the E. coli LacZ gene was inserted in the
plAlO plasmid under the control of the constitutive FBAI promoter. The resulting plasmid pI1OG
was used to obtain the WA6[pIlOG] strain (as
described in Materials and Methods). In this way
the LacZ gene expression should be proportional
to the plasmid copy number. WAG[pIlOG] transformed cells were cultured under the same conditions as shown in Figure 1 (Figure 4). On one
hand, a higher cell density than that observed for
the WA6[pIA10] cells was achieved after 24 h of
growth on glucose, while on the other hand a
shorter lag phase was observed when cells were
resuspended in fresh glucose medium. Table 1
summarizes some cellular parameters of the two
transformed yeast populations. The aldolase levels
L.OG( FL
Figure 5. Time-course analysis of fluorescence signals related
to P-galactosidase levels in WA6[pIlOG] cells, cultured as
described in Figure 4. (A) Cells grown on galactose; (B) cells
24 h after the nutritional shift; (C) cells after 48 h of growth on
glucose. Log(FL1) is the logarithm of fluorescence signals.
from WA6[pIlOG] cells after 24 h in glucose were
higher than from WA6[pIA10] cells. In addition,
the pIlOG plasmid content after 24 h of growth on
glucose was higher than the level of the pIAlO
plasmid. Table 2 compares the behaviour of the
specific P-galactosidase activity with the behaviour
of the plasmid copy number during 48 h of growth
on glucose. Analysis of the data reported supports
the assumption that the P-galactosidase content is
proportional to the plasmid copy number. Histogram A in Figure 5 reports the fluorescence
signals related to the P-galactosidase levels in
WA6[pI 10G] transformed cells growing on galactose. The plasmid copy number in these cells is
204
about five, as determined by Southern hybridization of the total D N A content (time t=O in Table
2). Histogram B reports the fluorescence signals
related to the P-galactosidase level in WA6[pI 10G]
transformed cells growing for 24 h on glucose. A
quantitative analysis of this bimodal histogram
indicated that for about 60% of the transformed
yeast population, the level of heterologous enzyme
is similar to that of the galactose culture (histogram A), while for the remaining yeast population,
a higher content is clearly visible. Finally, histogram C is related to the p-galactosidase content
from transformed yeast cells growing for 48 h on
glucose. Analysis of the staining pattern shown in
Figure 5 clearly indicates that the level of the
heterologous enzyme rose over time as a consequence of the transient state of growth (i.e. shift
from galactose- to glucose-containing media).
Therefore, such increase of the heterologous activity (i.e. of the plasmid copy number) seems to be
related to the selection of transformed cells bearing
a higher plasmid copy number. The unequal segregation of plasmid molecules a t cellular division
(Futcher, 1988; Morrissey and Cashmore, 1992)
could be the mechanism responsible for the selection of cells with a higher plasmid content and thus
with an aldolase level enabling growth on glucose.
The higher plasmid copy number observed in the
case of plasmid pIlOG in comparison to plasmid
pIAlO (see Table l), could allow a more rapid
selection of the sub-population, as indicated by the
shortened lag phase after 24 h of growth o n glucose (see Figures 1 and 4). A t this time, at least
30% of the population (Figure 5B) is already able
to grow because of the higher plasmid content.
In conclusion, in this work we have shown that
the singular feature of a carbon-source-dependent
modulation of the plasmid copy number coupled
to flow cytometric investigations allows the correlation of the distribution of a cellular component,
the plasmid, to dynamics of growth and selection
of a specific sub-population. Finally, the potential
of this kind of autoselection system should be
further tested with regard to both the long-term
stability of the amplified copy number and the
yield of heterologous protein. Studies along these
lines are in progress in our laboratory.
ACKNOWLEDGEMENTS
This project was supported by CNR, Progetto
Finalizzato Biotecnologie e Biostrumentazioni,
subproject no. 3 (B.M.R.).
C . COMPAGNO ET AL.
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