The 40°C fermentation took only 33 h as compared to 42 h at 30° C. These results indicate that K. marxianus is a promising host for the extracellular production ...
Appl Microbiol Biotechnol (1994) 42:516-521
© Springer-Verlag 1994
ORIGINAL PAPER M. Hensing • H. Vrouwenvelder • C. Hellinga R. Baartmans • H. van Dijken
Production of extracellular inulinase in high-cell-density fed-batch cultures of Ifluj/veromyces marxianus
Received: 25 March 1994 / Received revision: 3 June 1994 / Accepted: 15 June 1994
Abstract The production of extracellular inulinase (131,2-D-fructan fructanohydrolase, EC 3.2.1.7) was studied in fed-batch cultures of the yeast Kluyveromyces marxianus CBS 6556 at 30 and at 40° C. At both temperatures, the final biomass concentration exceeded 100 g.1 -I and more than 2 g enzyme. L -1 of culture supernatant was produced. The biomass yield on 02 at 40°C was substantially lower than at 30° C. Nevertheless, at 40°C a growth rate of 0.20 h -1 could be maintained for a longer period than at 30° C. The unexpected higher O2-transfer rate at 40°C is probably due to a lower viscosity of the culture broth. The 40° C fermentation took only 33 h as compared to 42 h at 30° C. These results indicate that K. marxianus is a promising host for the extracellular production of heterologous proteins under the control of the inulinase promoter.
Introduction Yeasts of the genus Kluyveromyces are used for the industrial production of /3-galactosidase (EC 3.2.1.23) and, more recently, also for the production of heterologous proteins (van den Berg et al. 1990; Fleer et al. 1991; Martinez et al. 1992; Bergkamp et al. 1992). Application of Kluyveromyces yeasts for the production of heterologous proteins offers the advantage of well-established fermentation processes. Furthermore these yeasts have the GRAS (generally regarded as safe) status, which makes them particularly suitable for production of pharmaceutical and food-grade proteins. M. Hensing • H. Vrouwenvelder • R. Baartmans H. van Dijken (N~) Department of Microbiology and Enzymology, Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. Fax: +31-15-133141 C. Hellinga Department of Bioprocess Engineering, Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands
K. marxianus CBS 6556 possesses several characteristics that make it an attractive host for the industrial production of proteins (Rouwenhorst 1990). It exhibits a high growth rate (P-max of 0.86 h-l) at 40° C on a defined mineral medium supplemented with sucrose as a carbon and energy source. This yeast is also able to grow rapidly on low-cost industrial substrates such as whey, molasses and spent sulphite liquor (Hensing et al. unpublished). The high temperature optimum for growth of this yeast is especially interesting since this facilitates cooling during large-scale fermentations for which heat transfer is known to be a limiting factor. K. marxianus CBS 6556 is able to grow on various polysaccharides such as pectin, xylan and inulin (Hensing et al. unpublished). Since all these compounds are hydrolysed extracellularly to monomers, K. marxianus possesses the natural ability to excrete enzymes. This is a desired property for a cost-effective down stream processing of low- and medium-value enzymes. Growth of K. marxianus on inulin and sucrose proceeds via the action of an extracellular inulinase (/3-1,2-D-fructan fructanohydrolase, EC 3.2.1.7). The enzyme is secreted into the culture fluid but also partially retained in the cell wall. Enzyme secreted in the culture fluid is a dimer whereas the enzyme retained in the cell wall is a tetramer (Rouwenhorst et al. 1990). Inulinase may have applications for the saccharification of fructans of plant origin (Jerusalem artichoke, chichory). However, production of extracellular inulinase was primarily used as a model system for the production of heterologous proteins in K. rnarxianus. The synthesis of inulinase in K. marxianus is repressed when growth is not sucrose-limited (Rouwenhorst et al. 1988; Parekh and Margaritis 1985; GrootWassink and Hewitt 1983). We therefore employed carbon-limited growth conditions for the production of the enzyme. Fed-batch cultivation rather than chemostat cultivation was chosen to investigate the possibility of an industrial production of extracellular enzymes with this yeast at high cell densities.
517
Materials and methods Micro-organism and maintenance K. marxianus var. marxianus CBS 6556 was obtained from the
Yeast Division of the Centraalbureau voor Schimmelcultures (CBS), Delft, The Netherlands, and maintained on YEPD agar slopes. YEPD contained, per litre of demineralized water: yeast extract (Difco Laboratories Detroit, USA), 10 g; Difco Bactopeptone, 20 g; glucose, 20 g. Inoculum production The inocula for the fed-batch fermentations were pregrown in sucrose-limited chemostat cultures at a dilution rate of 0.1 h -1, at the same temperature and pH as the subsequent fed-batch cultivation. Cultivation was performed in a laboratory fermentor (Applikon, Schiedam, The Netherlands) with a working volume of i 1 at an aeration rate of 21.rain 1 and a stirrer speed of 800 rpm. The pH was controlled automatically at 5.0 for fermentations at 30° C and at 4.5 for cultivation at 40° C by addition of 1 M KOH. The dissolved-O2 tension (DOT) was measured with a polarographic Oa electrode (Ingold, Urdorf, Switzerland) and was maintained above 50% air saturation. The medium used for chemostat cultivation contained per litre: (NH4)~SO4, 5 g; MgSO4.7H20, 0.5 g; KHzPO4, 3 g; ethylenediaminetetraacetic acid (EDTA), 15 mg; ZnSO4-7H:O, 4.5 mg; MnCla'7H20, i mg; COC12'6HaO, 0.3 mg; CuSO4' 5H20, 0.3 rag; Na2MoO4'2H20, 0.4mg; CaC12"2HaO, 4.5 mg; FeSO4"7HaO, 3 rag; H3BO3, i mg; KI, 0.1 mg; biotin, 0.1 rag; Ca-pantothenate, 1 rag; nicotinic acid, i rag; silicone antifoaming agent (BDH, Poole, Dorset, UK) 0.15 ml. The mineral medium was sterilised at 120° C for 20 rain. Sucrose (CSM, Amsterdam, The Netherlands) was sterilised separately at 110°C for 20 rain and added to a final concentration of 5 g.1-1.
50 mg; KI, 5 mg; biotin, 5 mg; Ca-pantothenate, 100 rag; nicotinic acid, 100 rag; the antifoaming agent Struktol J673 (Struktol Co., Stow, USA), 0.13-0.33 ml depending on the amount of biomass present in the fermentor. Sucrose was added to the medium stock to give a final concentration of 400 g.t 1for cultivations at 30°C and of 500 g.1 1 for cultivations at 40° C. The feed medium was not sterile but medium components were dissolved in sterilized demineralized water. Upon microscopic examination no contamination was observed during the fed-batch cultivation. The medium was pumped from 20-1 reservoir vessels into the reactor using a controllable Watson Marlow model 503 U pump (Smith and Nephew WatsonMarlow, Falmouth, Cornwall, UK) with flow rates between 0.2 and 4.8 l'h-1. The growth rate was kept at 0.2 h -1 until 02 limitation became apparent. The exponential feed was calculated and regulated by an ONSPEC computer programme (Heuristics, Sacramento, USA) using Eq. 1. F~ = ( ~
+ ms).(C×°'v°/, e ~'t
) \ Csi/
(1)
Where Fs is the flow rate of the medium feed (1.h-Z), t* is the specific growth rate (h-l), Y~xis the biomass yield on substrate (g cells.g substrate -1), ms is the maintenance coefficient (g substrate.g cells-l.h 1), Cxo is the initial biomass concentration (g'l-1), V0 is the initial culture volume (1), Csi is the substrate concentration in the feed (g substrate.1 1), and t is the time (h) after starting the feed. A maintenance coefficient of 0.024 g glucose.g lbiomass-h-1 was used (Roels 1983). Other parameters are described in the text. The feed rate was not corrected for the amount of ammonium hydroxide added or the total volume of the culture samples withdrawn from the fermentor. When the exponential feed could no longer be maintained as a result of Oa limitation a linear feed was employed as indicated in the text. Measurement of enzyme production
Pilot-scale fermentations Fed-batch cultivation was carried out in a fermentor with a working volume of 1001 (Applikon). Ammonium hydroxide (7.5 M) was used as a titrant. The DOT was measured with a polarographic 02 electrode (Ingold, Urdorf, Switzerland) and kept above 30% of air saturation with mixtures of air and pure 02, at a constant impeller speed of 800 rpm. The pressure in the fermentor was adjusted manually up to a maximum of 2.5 bar absolute pressure. The amount of medium added in the fed-batch phase was recorded by continuous monitoring of the weights of the reservoir vessels and the fermentor (both rested on an electronic balance).
Cells were haivested by centrifugation. Inulinase activities were determined according to Rouwenhorst et al. (1988) by incubating 50 txl of an appropriately diluted supernatant sample with 950 pA of 0.1 M sodium acetate buffer, pH 5.0, containing 65 mM sucrose. After 5 min at 50° C the reaction was stopped by the addition of 4 ml of 0.4 M TRIS-HC1, pH 7.8. The glucose released was determined with Boehringer D-Glucose test kit no. 716251. One unit (U) of inulinase is defined as the amount of enzyme catalysing the release of i ~mol glucose, rain-1 under the above-mentioned conditions. The amount of inulinase was calculated by using a specific activity of 1500 U.mg -1 protein for the purified supernatant enzyme (Rouwenhorst et al. 1990).
Batch phase
Determination of dry weight
The fermentor with approximately 30 1 of medium was sterilized at 121° C for 45 rain. After sterilization, sterile sucrose was added to a final concentration of 10 g.1-1. A medium twice the strength of that used for the continuous cultivation was employed. Aeration was automatically adjusted to keep the DOT above 50% air saturation at a constant stirrer speed of 800 rpm. After depletion of sugar, which was indicated by a rise in DOT and a decrease in COa production and 02 consumption, the fed-batch phase was started.
For dry-weight determination, culture samples containing 5-100 mg cells were filtered over pre-dried 0.45 ixm polysulfone filters (Gelman Sciences, USA). Cells were washed three times with demineralised water and dried in a microwave oven (Amanda, USA) at 700 W output for 15 min.
Fed-batch phase For the fed-batch phase the medium feed consisted of (amounts per litre): (NH4)2SO4, 10g; MgSO4"7H20, 5g; KH2PO4, 20g; EDTA, 750mg; ZnSO4'7H20, 225 mg; MnC12"7H20, 50mg; COC12'6H20, 15rag; CuSO4"5HzO, 15rag; Na2MoO4-2H20, 20mg; CaC12-2H20, 225mg; FeSO4'7H20, 150mg; H3BO3,
Metabolite analysis Organic acids, alcohols and sugars in the supernatant were determined by HPLC on an HPX-87H column (300 x 7.8 ram; Bio-rad) at 30°C as described by Verduyn et al. (1990). Biomass composition analysis The carbon, hydrogen, nitrogen and oxygen contents of the biomass were determined with a Carlo-Erba EA 1108 Elemental
518 analyser. Cells were washed three times with demineralised water and freeze-dried before analysis. Gas analysis Oz and CO2 in the exhaust gas were analysed with a paramagnetic Oz analyser (Servomex, model 1100, Crowborough, UK) and an infrared CO2 analyser (Beckman Instruments, model 870, USA) according to van Urk et al. (1988).
Results Physiology of K. marxianus in sucrose-limited chemostat cultures In industrial fermentations 02 transfer is a crucial parameter. In order to achieve high cell densities and to avoid by-product formation it is required to maintain aerobic conditions. This can only be achieved via carbon-limited growth at a rate well below p,. . . . T o establish the physiological characteristics of K. marxianus under these conditions its behaviour in carbon-limited chemostat cultures was studied. A dilution rate of 0.2 h -1 was chosen since in industrial practice, high-celldensity fed-batch cultivation involves growth at or below this rate. Two sets of conditions were studied: growth at 30°C and p H 5.0 and growth at 40°C and p H 4.5. From the results listed in Table 1 it is apparent that during growth at 40°C the 02 requirements for biomass synthesis are much higher than at 30 ° C. From these data it follows that growth of K. marxianus at 30 or 40 ° C can be described by Eq. 2 and 3, respectively. In these equations the amounts are given in mmoles. The amount of biomass formed includes an assumed ash content of approximately 4.5%. 609 C12H22Oll + 730 NH2- + 3628 02 --* 1000 C3.63H6.7502.18No.73(100 g biomass) + 3678 CO2 + 730 H ÷ + 4419 H 2 0
(2)
731 C12H22Oll + 620 NH2- + 4910 02 1000 C3.73H6.8102.2zNo.62(100 g biomass) + 5042 CO2 + 620 H + + 5566 H 2 0
(3)
The reactor, containing 34 1 of medium, was inoculated with 1 1 of a sucrose-limited chemostat culture to a final density of 0.48 g. 1-1. Cells were then grown batchwise for 6.5 h until a cell concentration of 4 g.1-1 (Fig. 1A) was reached and all of the sucrose was consumed. H e r e a f t e r the fed-batch part of the fermentation was initiated (Fig. 1B). The feed rate was increased exponentially to maintain a growth rate of 0.2 h-1. The pressure in the reactor was gradually increased to 2.5 bar and instead of air, a mixture of air and pure 02 was used. A constant growth rate was possible until the biomass reached 50 g.1-1 at a volume of 45 1 (Fig. 1A and B). Thereafter, a linear feed was applied to avoid 02 limitation. Due to a decrease in the O r t r a n s f e r rate it was necessary to lower the feed rate a few times (Fig. 1B). The linear feed resulted in a decrease in the growth rate from 0.2 h 1 down to 0.05 h 1 at the end of the fermentation (Fig. 1B). The final volume was 93 1 and the biomass concentration was 103 g dry weight. 1 1 (Fig. 1A). The biomass increase slowed down during the phase of linear feed. This is due to the higher proportion of substrate used for maintenance purposes. The amount of inulinase in the culture supernatant increased from 0.003 g.1-1 to 2.0 g.1-1 at the end of the fermentation (Fig. 1C). Throughout the process, sucrose limitation was maintained to control the growth rate, and to avoid byproduct formation. The residual sugar concentration and the concentration of usual fermentation products such as ethanol, glycerol and pyruvate were below 1 mM. However, acetate and succinate reached 4 and 3 mM, respectively, at the end of the fermentation (data not shown).
Fed-batch fermentation at 40°C
From these equations the biomass yield on 02 at 30 and 40°C can be calculated as 0.86 and 0.64g biomass'g -1 02, respectively. The amount of inulinase produced at both temperatures was similar (Table 1).
Table 1 Influence of temperature on the physiology of Kluyveromyces marxianus grown in sucrose-limited chemostat cultures at a dilution rate of 0.2 h-1 (U units)
Fed-batch fermentation at 30°C
Fermentations at 40°C were performed in the same way as at 30 ° C. After a batch period of 6.3 h the biomass reached 4.5 g'1-1 (Fig. 2a). Thereafter an exponentially increasing feed was applied to maintain a constant growth rate of 0.2 h -1. In order to avoid 02 limitation, the same strategy as used in the 30 ° C fermentations was employed. Although the biomass yield on 02
Parameters
30° C
40° C
Biomass yield on substrate (g biomass.g-1 substrate) Biomass yield on 02 (g biomass-g-1 02) Biomass composition Specific production rate of inulinase (U-mg -1 dry weight.h-I)
0.48
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Fig. l a - c Fed-batch cultivation of Kluyveromyces marxianus at 30° C. a Culture volume (O) and dry weight ([3). b Substrate feed ( - ) and growth rate (0). The exponential feed of 0.2 h -~ was started after 6.5 h at a rate of 0.1601 .h -~. After 20 h a linear feed of 2.5 1.h -~ was applied to maintain a dissolved 02 tension (DOT) of 30% air saturation. The feed rate was changed to 2.01"h ~ after 22.1h, to 1.71.h -~ after 28.7 h, to 1.21.h -~ after 37.5 h and to 0.9 after 42.6 h. Changes in the feed rate were made to maintain a DOT above 30% air saturation, c Supernatant inulinase (0)
a n d t h e s o l u b i l i t y o f 0 2 in w a t e r is l o w e r at 4 0 ° C t h a n at 30 ° C, a g r o w t h r a t e of 0.2 h -1 c o u l d b e m a i n t a i n e d m u c h l o n g e r at 40 ° C. This was p o s s i b l e until t h e b i o m a s s r e a c h e d 82 g.1 1 at a v o l u m e o f 59 1. T h e f e e d p r o f i l e o f t h e f e r m e n t a t i o n is g i v e n in Fig. 2b. A f t e r t h e b a t c h p h a s e t h e f e e d r a t e was e x p o n e n t i a l l y i n c r e a s e d f r o m 0.17 1-h-1 to 4.77 1. h ~and t h e n a l i n e a r f e e d was a p p l i e d . I n c o n t r a s t t o t h e s i t u a t i o n at 3 0 ° C t h e c o n s t a n t f e e d - r a t e o f 4.77 1.h -1 c o u l d b e m a i n t a i n e d witho u t a d e v i a t i o n of t h e D O T f r o m 30% air s a t u r a t i o n . T h e use o f t h e c o n s t a n t f e e d l e d to a d e c r e a s e o f t h e g r o w t h r a t e f r o m 0.2 h -1 to 0.067 h - 1 at t h e e n d o f t h e p r o c e s s (Fig. 2b).
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Fig. 2a-c Fed-batch cultivation of K. marxianus at 40° C. a Culture volume (O) and dry weight ([Z). b Substrate feed ( - ) and growth rate (0). The exponential feed of 0.2 h-1 was started after 6.3 h at a rate of 0.17 1-h-land increased to 4.77 1-h -1 after 23 h. At that time, the feed rate was not further increased, to maintain a DOT above 30% air saturation, e Supernatant inulinase (O)
F e r m e n t a t i o n at 40 ° C r e s u l t e d in 1 2 0 g d r y w e i g h t . 1 - 1 a n d an i n u l i n a s e c o n c e n t r a t i o n in t h e s u p e r n a t a n t of 2 . 5 8 g - 1 - 1 ( 3 . 8 7 x 1 0 6 u n i t s - 1 - 1 ) a f t e r 3 3 h (Fig. l c ) . A s o b s e r v e d d u r i n g f e r m e n t a t i o n at 30 ° C, t h e c o n c e n t r a t i o n o f sugars was well b e l o w 2 mM o v e r t h e w h o l e f e r m e n t a t i o n p e r i o d , e x c e p t for t h e b a t c h p h a s e (data not shown). In the batch phase a rapid consumpt i o n o f f r u c t o s e a n d g l u c o s e was o b s e r v e d . T h e occurr e n c e o f t h e s e sugars is d u e to e x t r a c e l l u l a r h y d r o l y s i s o f t h e s u c r o s e b y inulinase. B y - p r o d u c t f o r m a t i o n at 40 ° C (Fig. 3) was m o r e p r o n o u n c e d t h a n at 30 ° C. A c e t a t e a c c u m u l a t e d to a c o n c e n t r a t i o n o f 18 mM a f t e r 24 h of f e r m e n t a t i o n . D u e to t h e switch to a l i n e a r f e e d a f t e r 23.3 h, p a r t o f t h e a c e t a t e was c o n s u m e d . T h e r e a f t e r a c e t a t e i n c r e a s e d again, while s u c c i n a t e a n d g l y c e r o l c o n c e n t r a t i o n s d e c r e a s e d . D e s p i t e t h e s e r e l a t i v e l y high a c e t a t e c o n c e n t r a t i o n s , n o d e v i a t i o n in t h e e x p e c t e d g r o w t h p a t t e r n was o b s e r v e d .
520
20
by the cells. Bearing these complications in mind, it can nevertheless be concluded that this high-cell-density i fed-batch cultivation of K. marxianus results in a high --~5 productivity. Initially a temperature of 30°C was chosen for fer10' mentation studies because of the higher biomass yield "8 on 02 as compared to that observed at 40° C (Table 1). Surprisingly, an exponential growth rate of 0.2 h -1 5' could be maintained longer at 40°C than at 30°C (Figs. 1B and 2B). This may have been caused by a low0 er viscosity of the culture broth at 40° C, resulting in a 10 20 3'0 much higher O2-transfer rate. Thus, not only the easier Time[h] cooling of the reactor but also the higher 02 transfer Fig. 3 Formation of glycerol (IT), succinate (E3) and acetate (0) rate makes fermentation at 40°C the obvious choice. At 40°C the fermentation took 33 h as compared to during fed-batch cultivation of K. marxianus at 40° C 42 h at 30° C. At 40° C, however, the formation of organic acids, in particular acetic acid, was more pronounced. Since acetic acid readily becomes toxic at low ~ 100t culture pH (Baronofsky et al. 1984; Verduyn et al. £ 1990), this phenomenon requires further study. The excretion, at low levels, of metabolites is probably due to locally elevated sugar concentrations occur-~ 60 '~" 801 "6 ring before the highly concentrated substrate feed is completely mixed. K. marxianus CBS 6556 has a strong ,0 tendency to produce pyruvate and acetate when ex-5 posed to excess sugar (Hensing et al., unpublished re~6 20 sults). During large-scale fermentation this tendency 0m may be a problem, because of the presence of sugar 10 20 30 40 0 gradients in the reactor. Time[h] In Fig. 4 the advantage of high-cell-density fermenFig. 4 Inulinase production as a percentage of the total amount tation is illustrated: 50% of the inulinase was formed of enzymeobtained during cultivationat 30° C ([3) and 40° C (O). during the final 6 h of the 40° C fermentation or the last Note that the fermentation time at 40°C is shorter than at 30°C 13 h in the 30°C fermentation. The high volumetric production makes this type of process attractive from a commercial point of view. Also the high protein concentration that was obtained is of importance since waDiscussion ter extraction during downstream processing is expenSucrose-limited fed-batch cultivation of K. rnarxianus sive. Detailed descriptions of high-cell-density fermentaCBS 6556 resulted in biomass densities exceeding 100 g dry weight-1 -a, both at 30 and at 40°C. The inulinase tion of yeasts producing homologous or heterologous concentration in the culture supernatant exceeded proteins are scarce. Only limited information is pre3 x 106 U x1-1, corresponding to a protein concentra- sented in articles about the production of heterologous tion of more than 2 g'1-1 (Rouwenhorst et al. 1990). proteins by the methylotrophic yeasts Pichia pastoris Estimation of the overall productivity of the fermenta- and Hansenula polymorpha (Clare et al. 1991; Janowicz tions with respect to extracellular inulinase is, however, et al. 1991). H. polymorpha secreted up to 1.4 g-1 -a of complicated since the amount of inulinase in the cul- the heterologous Schwanniomyces occidentalis glucoature supernatant is not equivalent to the total amount mylase during a high-density fermentation in which of inulinase produced. Continuous-culture studies have 130 g biomass "1-1 w a s obtained. In another case 740 mg shown that only half of the inulinase synthesized is se- product" 1-1 and 60 g biomass, l-a was reached in a pericreted in the culture fluid by K. marxianus. The re- od of 63 h (Gellissen et al. 1992). With P. pastoris mainder is mostly retained in the cell wall and a small 12g'1-1 of intracellular tetanus toxin fragment C amount is located intracellularly (Rouwenhorst et al. (Clare et al. 1991) has been obtained in a fermentation 1988). Furthermore, the amount of supernatant inuli- process that took 6 days. Extracellular production of nase is not the product of the volume of the culture the Saccharomyces cerevisiae invertase (EC 3.2.1.26) broth and the enzyme concentration since a significant with P. pastoris reached levels up to 2.5 g-1-1 during a fraction of the broth volume is occupied by the cells fermentation of 256 h (Tschopp et al. 1987). The procthemselves. For example, if the specific volume of the ess developed here for the production of extracellular cells is assumed to be 2 ml.g-1 dry weight is taken, it inulinase with K. marxianus can reach the same protein follows that at least 20% of the final volume is occupied levels in a much shorter time.
~
521 U p to now, S. cerev&iae has been the most commonly used host for the production of heterologous proteins (Buckholz and Gleeson 1991). Nevertheless, this yeast has some negative properties such as its Crabtreepositive character and a tendency to hyperglycosylate secreted glycoproteins. Therefore many other yeasts are presently studied as alternative hosts for heterologous gene expression. The best-known expression systems are the methylotrophic yeasts P. pastor& and H. polymorpha ( R o m a n o s et al. 1992). In this paper we have shown that the yeast K. marxianus, from a physiological point of view, is also a promising host organism for heterologous-protein production: the yeast can easily be grown to high cell densities in a short period of time and produces large amounts of secreted inulinase.
Acknowledgements The authors are indebted to their enthusiastic colleagues for assistance during the night shifts. The critical discussions with Professor Planta, Ruud Geerse and Ronald Bergkamp from the Free University of Amsterdam and with John Verbakel, Holger Toschka, Marco Giuseppin and Robert Rouwenhorst from the Unilever Research Laboratory, Vlaardingen, are highly appreciated. We thank Lex Scheffers of our department for a critical reading of the manuscript. This study was financed by Unilever Research, Vlaardingen, and the Dutch Ministry of Economical Affairs. References Baronofsky JJ, Schreurs WJA, Kashket ER (1984) Uncoupling by acetic acid limits growth of and acetogenesis by Clostridium thermoaceticum. Appl Environ Microbiol 48:1134-1139 Berg JA van den, Laken KJ van de, Ooyen van AJJ, Renniers TCHM, Rietveld K, Schaap A, Brake AJ, Bishop RJ, Schultz K, Moyer D, Richman M, Shuster JR (1990) Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Bio/Technology 8:135-139 Bergkamp RJM, Kool IM, Geerse RH, Planta RJ (1992) Multicopy integration of the a-galactosidase from Cyamopsis tetragonoloba into the ribosomal DNA of KIuyveromyces lactis. Curr Genet 21:365-370 Buckholz RG, Gleeson MAG (1991) Yeast systems for the commercial production of heterologous proteins. Bio/Technology 9:1067-1072 Clare JJ, Rayment FB, Ballatine SP, Sreekrishna K, Romanos MA (1991) High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio/Technology 9:455-460
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