Appl Microbiol Biotechnol (2003) 61:6976

DOI 10.1007/s00253-002-1196-0

ORIGINAL PAPER

B. S. Ferreira C. R. C. Calado F. van Keulen

L. P. Fonseca J. M. S. Cabral M. M. R. da Fonseca

Towards a cost effective strategy for cutinase production by a

recombinant Saccharomyces cerevisiae: strain physiological aspects
Received: 27 July 2002 / Revised: 4 November 2002 / Accepted: 8 November 2002 / Published online: 24 January 2003

Springer-Verlag 2003

Abstract Although the physiology and metabolism of the

growth of yeast strains has been extensively studied,
many questions remain unanswered where the induced
production of a recombinant protein is concerned. This
work addresses the production of a Fusarium solani pisi
cutinase by a recombinant Saccharomyces cerevisiae
strain induced through the use of a galactose promoter.
The strain is able to metabolise the inducer, galactose,
which is a much more expensive carbon source than
glucose. Both the transport of galactose into the cell
required for the induction of cutinase production and
galactose metabolism are highly repressed by glucose.
Different fermentation strategies were tested and the
culture behaviour was interpreted in view of the strain
metabolism and physiology. A fed-batch fermentation
with a mixed feed of glucose and galactose was carried
out, during which simultaneous consumption of both
hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in
terms of hexoses, incurred with this fermentation strategy
were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy,
namely a fed-batch fermentation with a feed of galactose.

Introduction
Cutinases are hydrolytic enzymes that degrade cutin, a
cuticular polymer of higher plants with numerous and
very diverse potential commercial applications (Carvalho
et al. 1999). In order to obtain an efficient and low cost
production system for cutinase from Fusarium solani pisi,
this enzyme was overproduced in the recombinant
B. S. Ferreira ()) C. R. C. Calado F. van Keulen L. P. Fonseca
J. M. S. Cabral M. M. R. da Fonseca
Centro de Engenharia Biolgica e Qumica,
Instituto Superior Tcnico,
Av. Rovisco Pais, 1049-001 Lisbon, Portugal
e-mail: bsf@ ist.utl.pt
Tel.: +351-21-8417233
Fax: +351-21-8480072

Saccharomyces cerevisiae strain SU50. For heterologous

expression of cutinase cDNA, the mature fragment of the
gene was cloned into a yeast expression vector. The
cutinase gene is fused to the invertase signal sequence to
achieve efficient cutinase secretion. Expression is controlled by the strong inducible Gal7 promoter (van
Gemeren et al. 1995).
The productivity of fermentations employing recombinant micro-organisms depends on multiple factors,
namely interacting relationships between microbial physiology, copy number and stability of the plasmid, and
gene expression. S. cerevisiae offers several advantages
as a host for the production of heterologous proteins: (1)
availability of a large amount of information concerning
its molecular biology and physiology; (2) ease of genetic
manipulation; (3) ability of performing post-translational
modifications; (4) long history of utilisation in traditional
industrial processes, being generally regarded as safe.
Furthermore, S. cerevisiae holds advantages with regard
to product secretion, such as a well characterised
secretory pathway. Also, it normally secretes only a very
small number (5%) of its own proteins to the medium,
which is advantageous for product recovery (Smith et al.
1985; Bitter et al. 1987; Das and Shultz 1987; Calado et
al. 2001). Product loss due to degradation of cutinase by
proteases secreted by S. cerevisiae is negligible (Verrips
et al. 2000). Nevertheless, as a host strain, S. cerevisiae
still poses several problems, namely:
1. Glucose repression: common industrial carbon sources
are composed of a mixture of sugars that are used
sequentially. A glucose derepressed strain would use
all the sugars of the complex medium simultaneously,
shortening the production time and avoiding environmental problems. In addition, glucose is known to
repress the GAL-mediated expression system.
2. Crabtree effect: alcoholic fermentation may occur
even under fully aerobic conditions, negatively affecting biomass yields and also the cutinase yield as
heterologous cutinase produced during fermentation is
correlated to biomass produced (Verrips et al. 2000).

70

In addition, high ethanol concentrations decrease the

amount of recombinant protein produced (Park et al.
1995; Kapat et al. 2000) and proteases that degrade the
recombinant protein may be expressed during growth
on ethanol (Gimenez et al. 2000). The strain used in
this work is known to exhibit a strong Crabtree effect
(Giuseppin et al. 1993). Alcoholic fermentation is
currently minimised during aerobic yeast growth by
operating in the fed-batch mode under conditions
where the carbon source supply limits the growth rate
and the full aeration capacity of the vessel is utilised.
The strain used in this work is able to metabolise the
inducer, galactose, which is much more expensive than
glucose. Thus, an optimised fermentation strategy should
enhance cutinase productivity while minimising galactose
consumption.
The experiments performed were aimed at providing
fundamental knowledge to allow a better understanding of
the physiological aspects involved in recombinant cutinase production by yeast. The first fermentation consisted
of a batch phase on glucose, followed by a fed-batch
period during which galactose was added as carbon
source. To better understand the effects of culturing the
strain on galactose, a batch fermentation was performed
with galactose as carbon source. Next, a third fermentation was carried out, including a batch and a fed-batch
period, using a mixture of glucose and galactose as carbon
source in both periods, to study glucose repression and the
possibility of combining the higher biomass productivity
obtained on glucose with the inductive action of galactose. Complex media were used, aiming at mimicking the
conditions of the industrial process.

Materials and methods

Bioreactor
A 5 l bioreactor (Biostat MD; Braun, Melsungen, Germany),
equipped with a jacketed glass vessel, height/diameter ratio of 2,
and three equally spaced Rushton turbines, was used. The air flow
rate, dissolved oxygen, pH, temperature, stirrer speed and volume
of acid and base added were computer logged. Dissolved oxygen
was monitored with a polarographic electrode (Ingold, Urdorf,
Switzerland). Zero and 100% calibration of the electrode were
performed at 30C and 500 rpm by sparging nitrogen and air,
respectively, until stable signals were obtained. Anti-foam (silicon
anti-foaming agent; Merck) was added manually.
Fermentation A: fed-batch fermentation with galactose
feeding
Initially, the bioreactor contained 2 l culture medium: 20 g/l yeast
extract (Difco), 10 g/l peptone (BDH, Poole, Dorset, UK), 20 g/l
d(+)-glucose (Merck) and 10% (v/v) inoculum. A solution of 200 g/
l d(+)-galactose (Sigma, Munich, Germany), 35 g/l yeast extract
(Difco) and 45 g/l peptone (BDH, UK) started being fed with a
peristaltic pump (Watson Marlow 505Di, Falmouth Cornwall, UK)
immediately after the depletion of the ethanol produced during the
batch period. The feed rate was constant and set to 116 ml/h until
1.68 l medium (density =1,110 kg/m3) had been added. The amount
of medium added was monitored gravimetrically (PB1502 balance;
Mettler-Toledo, Greifensee, Switzerland). The fermentation was
carried out at 30C and the pH was maintained at 6.00.5 with 2 M
NaOH and 2 M HCl. Dissolved oxygen was kept above 60% and
30% saturation during the batch and the fed-batch periods,
respectively, at a constant air flow rate of 2.0 lN/min, by variation
of the stirring speed. A condenser was coupled to the fermenter air
outlet and cooled with water at 4C to minimise losses by
evaporation.
Fermentation B: batch fermentation on galactose
The bioreactor contained 4.0 l culture medium: 10 g/l yeast extract
(Difco, Detroit Mich.), 10 g/l peptone (BDH), 20 g/l d(+)-galactose
(Sigma) and 10% (v/v) inoculum. Temperature and pH were
controlled as before. Dissolved oxygen was kept above 30%
saturation at a constant air flow rate of 3.9 lN/min by varying the
stirring speed.

Strain
The S. cerevisiae strain SU50 (MATa, ciro, leu2-3,112, his4-519,
can1) containing the expression vector pUR7320 was used. The
strain was constructed and kindly provided by the Unilever
Research Laboratory, Vlaardingen, The Netherlands. The plasmids
contained ribosomal DNA sequences for chromosomal integration
and a LEU2d gene for selection on leucine-lacking plates (van
Gemeren et al. 1995). The stock cultures were maintained in a 50%
(v/v) mixture of selective medium (Leu agar) and glycerol (Merck,
Darmstadt, Germany) at 80C.
Inoculum preparation
The composition of the selective medium for inoculum preparation
was: 6.7 g/l yeast nitrogen base without free amino acids (Difco,
Detroit, Mich.), 20 g/l d(+)-glucose (Merck), supplemented with
20 mg/l l-histidine (Merck). The inoculum was grown overnight in
shake flasks at 30C and 200 rpm in an orbital shaker (Agitorb
160E, Aralab, Oeiras, Portugal) until a dry cell weight between 1.1
and 1.8 g/l was obtained (Calado et al. 2002a).

Fermentation C: fed-batch fermentation with glucose and

galactose feeding
To start with, the bioreactor contained 2 l culture medium: 20 g/l
yeast extract (Difco), 10 g/l peptone (BDH), 15 g/l d(+)-glucose
(Merck), 5 g/l d(+)-galactose (Sigma) and 10% (v/v) inoculum.
Feeding with a solution of 10 g/l yeast extract (Difco), 20 g/l
peptone (BDH), 45 g/l d(+)-glucose (Merck) and 10 g/l d(+)galactose (Sigma) through a peristaltic pump (Watson Marlow 202
U1) was switched on at 19.3 h. The feed rate was exponential until
645 ml medium had been added. Thereafter, between 26 h and 32 h,
the flow rate was constant and equal to 233 ml/h. Temperature and
pH were controlled as before and the total amount of medium fed,
1.85 l, was monitored gravimetrically as in fermentation A.
Dissolved oxygen was set at 30% saturation at a constant air flow
rate of 1.9 lN/min.
Analytical procedures
Determination of cell dry weight
Cell dry weight (cdw) was obtained by filtering a known volume of
culture through 0.20 mm pore size filters (Whatman, Clifton, N.J.)
predried in an infrared dryer (Mettler LP16) at 105C until constant

71
weight. The wet cell mass obtained was washed with distilled water
and dried on the filter in the infrared drier to constant weight.
Analysis of sugars and metabolites
Samples taken from the fermentation medium were centrifuged for
10 min at 3,000 g (Sigma 201; Braun). The supernatants were
analysed for glucose, galactose, acetate and ethanol with enzymatic
kits (d-glucose kit 716251, lactose/d-galactose kit 176303, acetic
acid kit 148261 and ethanol kit 176290, all from Boehringer
Mannheim, Germany).
Determination of extracellular protein concentration
The protein concentration was determined by the method of
Bradford (1976).
Cutinase activity assay
The cutinase estereolytic activity was determined spectrophotometrically, following the hydrolysis of p-nitrophenylbutyrate at
400 nm (Calado et al. 2002a). One unit of activity was defined as
the amount of enzyme required to convert 1 mol p-nitrophenylbutyrate to p-nitrophenol per minute.
Exhaust gas analysis
On-line exhaust gas analysis was carried out with a quadrupole
mass spectrometer (Spectra International, Edgeware, Middlesex,
UK) after appropriate calibration with gaseous mixtures (Ferreira et
al. 1998). The exhaust gas was dried through a Dimroth condenser
cooled with water at 4C, before entering the mass spectrometer.
The data for the calculation of oxygen and carbon dioxide
concentrations were logged by computer.

Results
Fermentation A: fed-batch fermentation with galactose
feeding
Production of cutinase by S. cerevisiae was first studied
by adopting a typical fed-batch fermentation strategy: a
first period of biomass growth on glucose, followed by a
fed-batch period on galactose for cutinase production
(Fig. 1).
A typical profile of a batch fermentation of S.
cerevisiae on glucose was obtained in the initial batch
period: a first stage in which glucose is consumed with
simultaneous production of biomass and ethanol, followed by a second stage, after glucose depletion, when
the culture grows by metabolising the ethanol produced
during the earlier stage. The presence of ethanol at time
zero is due to the ethanol introduced with the inoculum.
Oxygen limitation never occurred throughout the fermentation, given its relatively low volume and good mixing.
Thus, ethanol production was a result of the Crabtree
effect. Indeed, the specific glucose uptake rate, qS, was
1.75 g g1 h1, higher than the reported threshold qS
(0.54 g g1 h1; Kppeli 1986; Cortassa and Aon 1998),
above which the respiratory pathway is saturated and
ethanol is produced as a result of the onset of the

Fig. 1 Fermentation A, a fed-batch fermentation of Saccharomyces

cerevisiae on galactose, after a batch period on glucose for biomass
growth. Dry cell weight, and glucose, galactose, ethanol, acetate
and protein concentrations over the time course are shown. F
Substrate feed rate, RQ respiratory quotient, OUR oxygen uptake
rate, CPR carbon dioxide production rate

fermentative pathway. When the metabolism is not

exclusively oxidative, the respiratory quotient (RQ) is
greater than 1, confirmed by measurements at the
beginning of fermentation (Fig. 1). Ethanol and acetate
concentrations followed a similar profile during the batch
glucose consumption period. Next, both ethanol and
acetate concentrations decreased until almost full depletion of acetate and, later, ethanol. The theoretical RQ
during the aerobic oxidation of ethanol is 0.67, in good
agreement with the RQ of 0.66 obtained experimentally.
Upon ethanol exhaustion, the RQ increased to a fairly
constant value close to 1.
At 17.5 h of fermentation, galactose began to be fed at
a constant rate (116 ml/h). From this time on, acetate
accumulation was observed until complete exhaustion of
ethanol, at which time acetate started to be consumed
until its exhaustion. During the first 10 h of feeding, most
of the galactose simply accumulated. Nevertheless, both
biomass growth and protein production still occurred
(Fig. 1). Acetate accumulated both at the start of
induction and later at the start of galactose uptake.

72

galactose concentration, although oxygen limitation did

not occur. Acetate was present at a fairly constant
concentration throughout the fermentation, despite the
very low flux through glycolysis.
Fermentation C: fed-batch fermentation with glucose
and galactose feeding

Fig. 2 Fermentation B, a batch fermentation of S. cerevisiae strain

SU50 on galactose. OUR, CPR, dry cell weight, concentrations of
glucose, galactose, ethanol, acetate, and protein, and cutinase
activity during the time course are shown. Due to the noise of
oxygen measurements, the OUR values presented were filtered with
a Butterworth filter

Growth on galactose showed two different stages: an

exponential phase, followed by almost linear growth.
Fermentation B: batch fermentation on galactose
A batch fermentation on galactose was performed to
clarify some observations during fermentation A, namely
the limited specific galactose uptake rate and the apparent
importance of acetate to interpret the time course of the
fermentation (Fig. 2). Initially, ethanol carried over from
the inoculum was consumed. Acetate concentration was
apparently increasing at the time of the first sample. The
carbon dioxide production rate (CPR) shows a peak in the
first 5.5 h that was not accompanied by any detectable
peak in oxygen uptake rate (OUR), suggesting that the
culture was fermenting glucose present in the inoculum.
A large peak is observed in both OUR and CPR profiles
during the period in which the culture was growing on
ethanol. The abrupt drop of OUR and CPR at about 16 h
coincides with the exhaustion of ethanol. Due to a
problem in the mass spectrometer calibration, the OUR
was consistently higher than the CPR, and thus no
quantitative information can be drawn from the exhaust
gas analysis. The results clearly show that growth was
linear and was accompanied by a linear decrease in the

The galactose uptake rate is much lower than the glucose

uptake rate, leading to increased times for growth and
production (i.e. low volumetric productivities) when
galactose is used as the sole carbon source. It would be
advantageous if the strain could grow on glucose and,
simultaneously, produce cutinase due to the presence of
galactose. The transport of galactose into the cell,
necessary for galactose induction to occur, is repressed
by the presence of glucose (zcan and Johnston 1999).
Additionally, wild strains of S. cerevisiae continuously
growing on galactose stop metabolising galactose upon
addition of a glucose pulse, but restart metabolising
galactose when the glucose concentration drops (Sierkstra
et al. 1992). To investigate the influence of glucose on
galactose uptake and protein production, a fed-batch with
a mixed feed of glucose and galactose was performed
(Fig. 3). A batch growth was carried out for biomass
build-up prior to the feeding period, on a medium
containing glucose and galactose so that when the fedbatch phase was started the cells were already able to
metabolise galactose.
The behaviour of the culture during the first 12 h
closely followed that obtained when growing the strain
batchwise on glucose. Glucose was readily consumed,
with concomitant production of ethanol and acetate. Upon
glucose exhaustion, no immediate galactose consumption
was observed, the carbon source being ethanol. The
synthesis of enzymes necessary for galactose utilisation
explains this delay in galactose uptake. Acetate concentration increased until about 15 h of fermentation, 3 h
after glucose depletion, and only then did galactose
consumption start. At this stage, galactose and ethanol
were consumed simultaneously. A slight increase in the
protein concentration was also observed upon glucose
exhaustion, i.e. when the culture became unrepressed and
was able to take up galactose.
At the beginning of the exponential feeding period, the
concentration of galactose decreases until about 20 h and
a slight accumulation of glucose is detected. Galactose
starts accumulating thereafter, while practically all the
glucose fed is consumed with consequent ethanol formation in addition to cell mass. The galactose concentration
increases sharply until a fairly constant concentration is
reached between 4 and 4.5 g/l, despite the increasing
galactose input. This concentration level is also maintained when the feeding rate is kept constant. During the
feeding period, both ethanol and acetate build up. The
CPR increases sharply while feeding is exponential,
indicating that the carbon source is being metabolised
mostly through the fermentative pathway. During the

73

although some galactose remained in the broth, both

growth and protein production ceased.

Discussion

Fig.3 Fermentation C, a fed-batch fermentation of S. cerevisiae

strain SU50 on glucose and galactose. Feeding rate profile, OUR,
CPR, dry cell weight, concentrations of glucose, galactose, ethanol,
acetate, and protein, and cutinase activity during the time course are
shown. Dotted vertical lines Times at which the feeding regime was
changed

period of constant feeding, the CPR decreases as a result

of a lower specific carbon uptake rate due to a constant
volumetric uptake at increasing biomass concentrations.
This means that the carbon flux to the individual cell
decreases and thus its fermented fraction also decreases.
During this period, an increase in the protein concentration is observed.
From 32 h to about 52 h, both ethanol and galactose
concentration decrease and the RQ remains close to 1,
similar to the situation towards the end of the batch phase.
At the end of this period, the galactose uptake rate is very
low, although the galactose concentration is still ca. 1 g/l.
Between 52 h and 63.5 h, the remaining ethanol is totally
consumed, resulting in a significant increase in biomass
concentration from 12.7 g/l to 19.2 g/l. The protein
concentration, however, remained constant. During this
period, the RQ decreases (to an average value of 0.65), as
observed previously when the culture was growing on
ethanol. Once ethanol was exhausted, acetate was
consumed, until its depletion at 67 h. Subsequently,

The production of cutinase from F. solani pisi by a

recombinant strain of S. cerevisiae was addressed as a
contribution to the future design of a fermentation
strategy to enhance the productivity of the fermentation
process, while minimising the cost of carbon sources and
inducer.
During fermentation A, galactose was fed to the
culture after a batch phase on glucose for biomass growth.
Surprisingly, most of the galactose accumulated during
the first 10 h of the feeding period. Nevertheless, both
biomass growth and protein production still occurred,
though at low rates. After induction, the cell starts to
assemble the machinery necessary for cutinase production, thus the biosynthetic pathways will be very active
and will consume NADPH (Mathews and van Holde
1990). Upon exhaustion of NADPH produced by, e.g. the
pentose-phosphate pathway, the NADPH required will
come from the production of acetate, a key source of
NADPH supply in yeast (Stephanopoulos et al. 1998).
Thus, the peak of acetate production observed between
17.5 h and 22.5 h is probably related to the production of
NADPH required for protein biosynthesis. The peak of
acetate between 27.5 h and 35 h, the period during which
galactose uptake started and attained its maximum rate,
probably corresponds to induction of genes encoding the
Leloir pathway enzymes. This would also explain the
apparent increase in acetate concentration at the time of
the first sample in fermentation B. Acetate was present at
a fairly constant concentration throughout fermentation B,
despite the very low flux through glycolysis, suggesting
that the cells are producing NADPH required for biosynthesis via production of acetate. The coincidence between
acetate production and initiation of cutinase production or
galactose consumption was also observed during fermentation C.
How galactose utilisation in the presence of glucose is
carried out is a key, and still controversial, issue in the
understanding of the regulation of hexose metabolism in
S. cerevisae (Horak and Wolf 1997, 2001; Meijer et al.
1998; Rohde et al. 2000). It is well known that glucose is
able to exert catabolite repression, and it is widely
accepted that galactose cannot be transported inside the
cell or metabolised in the presence of glucose. In
fermentation A, during the period in which galactose
accumulated, cutinase was produced, indicating that
galactose induction did occur, i.e. some galactose was
being transported into the cell, although another carbon
source, probably from the yeast extract (which contains
17.5% of carbohydrates that might be able to repress and
inhibit galactose consumption), was used. Galactose
transport was perhaps achieved through a constitutive
low-affinity transport system present in cells grown on
glucose (Lagunas 1993). This suggests that it might be

74

Fig. 5 Specific cutinase activity with respect to biomass in

fermentation A: taking into account total biomass (l) or biomass
produced only after induction (m)

Fig. 4 Glucose concentration (l) and glucose () and galactose

( ) specific uptake rates during fermentation C. The oscillations
observed on the graph are mainly a result of the cubic splines used
to interpolate the experimental concentrations

possible to achieve galactose induction while growing the

strain on glucose. This possibility was studied during
fermentation C. The glucose- and galactose-specific
uptake rates during the feeding period are shown in
Fig. 4. While the initial galactose was still being
metabolised, the feeding period was started with an
instantaneous glucose concentration increase and concomitant drop of galactose uptake rate. When the glucose
concentration decreased to values lower than 0.20 g/l,
galactose consumption resumed, although glucose continued to be metabolised. These results agree with the
findings of Meijer et al. (1998), according to which
glucose repression in S. cerevisiae seems to be related to
the glucose concentration rather than the glucose flux.
This suggests the feasibility of using cheaper feeding
solutions containing both glucose and galactose, which
will convey sufficient galactose for efficient induction,
the remaining carbon being supplied by the much cheaper
sugar, glucose.
No cutinase was produced prior to induction with
galactose, but it was produced as soon as galactose was
added, except in the batch phase of fermentation C due to
the repressive effect of glucose. Extracellular cutinase
activity and extracellular protein were linearly correlated.
The slope of the linear regression gives the specific
activity of cutinase: 22613, 1799 and 41413 U/mg
protein for fermentations A, B and C, respectively. As
reported in the literature (Verrips et al. 2000; Calado et al.
2002a, 2002b), cutinase and biomass production were
linearly correlated. The slope of these lines gives
the specific cellular activity, (7.900.39) 103,
(2.620.16) 104 and (1.400.60) 104 U/g cdw for fermentations A, B and C, respectively. This can be
converted into a cutinase to biomass yield with the

specific activity of purified cutinase, 630 U/mg protein

(Calado 2001), resulting in 13, 42 and 22 mg cutinase/g
cdw for fermentations A, B and C, respectively. The
highest cutinase cellular specific activity obtained in
fermentation B, where the growth rate was the lowest, is
consistent with the results of Verrips et al. (2000).
Brown and co-workers (2000) discussed the hypothesis
that, upon induction, existing cells are unlikely to form
cutinase to any significant extent because they may
continue in a repressed state and that, on the other hand,
daughter cells are likely to be less repressed, and
galactose induction will occur in these cells. Thus, the
specific cellular activity was calculated according to two
distinct approaches: (1) on the basis of the total biomass
(Eq. 1) and (2) taking into account only the cells produced
after induction (Eq. 2):
SA At =Xt S

SA At
Ai =Xt
Xi

where SA is the specific cellular activity, A is the

extracellular cutinase activity, X is cdw, and the subscripts t and i refer to the sampled time and the induction
time, respectively. The specific cellular cutinase activity
calculated with the total biomass increases during the first
hours after induction (Fig. 5), i.e. either a lag phase exists
corresponding to the time required for the cells to
assemble the machinery for cutinase formation, or only
the daughter cells are capable of producing cutinase.
When the specific cellular activity was calculated taking
only the cells formed after induction into account, it
immediately attained the levels observed throughout the
fermentation, suggesting that only the daughter cells are
capable of producing cutinase. Understanding which
situation best reflects reality will require further study.
The highest specific cellular activity and the lowest
growth rate observed during fermentation B indicate that
cells have directed most of their resources to cutinase
production. This, together with the hypothesis that two
different populations exist (induced and non-induced), is
crucial to the development and optimisation of a fed-

75
Table 1 Comparison of the performance of the fermentation
strategies tested. The values refer to pure cutinase, calculated by
dividing the cutinase activity by the specific activity of purified
cutinase, 630 U/mg protein (Calado et al. 2001). Results for
fermentation C were calculated at 50 h fermentation time
Fermentation
Cutinase on total hexoses
(mg cutinase/g hexoses)
Cutinase volumetric productivity
(mg cutinase l1 h1)
Cutinase concentration
(mg cutinase/l)
Hexose cost (/g cutinase)

B
6.36

11.0
546
18.8

22.6
4.45
416
6.05

8.25
4.68
338

tional fermentation strategy, i.e. a fed-batch fermentation

with galactose feeding. These results could aid development of a highly efficient cutinase production process
based on the appropriate design of the feeding regime
(rate and glucose/galactose ratio) and time of induction.
Acknowledgements This work was supported by Funda
o para a
Cincia e a Tecnologia, PRAXIS XXI programme (grant GGP
XXI/BD/2936/96 awarded to B.S. Ferreira and grant GGP XXI/BD/
18276/98 awarded to C.R.C. Calado). The authors wish to thank Dr.
Maarten Egmond, Dr. Maurice Mannessi and Dr. Arthur Fellinger
and Unilever Research Laboratory for providing the transformed S.
cerevisiae strain.

4.41

References
batch strategy, specifically with regard to the optimum
time for induction.
Table 1 compares the cutinase/sugar yield, the volumetric productivity, the cutinase concentration and the
cutinase production cost in terms of hexoses for the three
fermentations. The prices of glucose and galactose were
obtained by averaging values of pharmacopoeia grade
reagents from various manufacturers, sold in packs of 1 kg
(12.6/kg for glucose and 137/kg for galactose). These
costs will obviously be lower for bulk quantities but their
ratio will remain roughly the same, making the comparison meaningful. Fermentation B gave the highest cutinase yield on total hexoses, but since it was carried out on
galactose only, it was not the most cost effective in terms
of hexoses. Since both growth and galactose uptake rates
were low, this fermentation was the longest, giving a
relatively low cutinase volumetric productivity. Fermentation A exhibited the highest productivity, which can be
ascribed to both the short fermentation duration and the
high cutinase production. The short duration of fermentation A has the additional benefit of lower energy
expenditure. However, the cutinase yield on total hexose
was the lowest and cutinase was only produced when
galactose was used as the only hexose, resulting in
fermentation A being the least cost effective option in
terms of hexose consumption. Fermentation C was
evaluated at 50 h fermentation time, i.e. when the
extracellular cutinase activity levelled off. This fermentation was the most cost effective in terms of hexose
consumption. However, cutinase concentration in fermentation C was the lowest (Table 1), implying more
costly downstream processing. However, the specific
activity of cutinase was the highest, possibly leading to
lower costs in the final purification steps, often the most
significant in terms of overall production costs.
To define an efficient production strategy, both the
composition of the cultivation medium, especially the
glucose/galactose ratio, and the medium feeding rate,
have to be optimised. Fermentation C, carried out with a
mixed feed of glucose and galactose, was shown to be the
most cost effective with regard to hexose consumption
(Table 1). Sugar costs were only 23% of those of
fermentation A, which was carried out with a conven-

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