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JTRANSIENT RESPONSES OF YEASTS TO GLUCOSE EXCESS 3< ? / it
\^ lï%>
fP
JProefschrift
ter verkrijging van de graad van doctor aan de Technische Universiteit Delft op gezag van de Rector Magnificus, prof. drs. P.A. Schenck, in het openbaar te verdedigen ten overstaan van een commissie aangewezen door het College van Dekanen op dinsdag 13 juni 1989 te 16.00 uur
door
Hendrik van Urk
scheikundig ingenieur
geboren te 's Gravenhage
TR diss
1738
Prof. dr. J. G. Kuenen.
Dr. J. P. van Dijken heeft als toegevoegd promotor in hoge mate bijgedragen aan het totstandkomen van
het proefschrift.
Transient responses of yeasts to glucose excess / Hendrik van Urk.
-Haarlem: Thesis. Published as thesis Delft, 1989. - With ref. - With summary
in Dutch. ISBN 90-5170-017-2. SISO 670 UDC 663.12 NUGI 821. Subject heading:
Hoewel dit dankwoord is geplaatst aan het begin van dit proefschrift is dit zo ongeveer het laatste gedeelte dat uit de tekstverwerker komt rollen. Toch is er een reden te noemen waarom dit dankwoord niet achterin maar voorin dit proefschrift is afgedrukt. Het voor U liggende proefschrift zou namelijk nooit verschenen zijn als er niet nog vele andere mensen bijgedragen zouden hebben aan het tot stand komen van dit boekje. Ik vind het daarom belangrijk dat U, voor U dit proefschrift leest, weet wie er allemaal aan dit werk hebben bijgedragen. Al deze mensen wil ik hierbij dan ook bedanken. Met name wil de volgende personen bedanken voor alles wat zij in de afgelopen jaren concreet en minder concreet voor mij betekend hebben.
De eerste persoon die hier genoemd moet worden is ongetwijfeld mijn begeleider, de toegevoegd promotor, Hans van Dijken. Hans jij bent een begeleider van ongeëvenaarde klasse. Je stond altijd en op de gekste tijden klaar, je opmerkingen waren kritisch en altijd opbouwend. Bovendien versta je de kunst om op de juiste momenten iemand, en dus ook mij, een hart onder de riem te steken zelfs onder de voor jou steeds groter wordende werkdruk de laatste jaren.
Ook Lex Scheffers heeft een belangrijke bijdrage geleverd aan het tot stand komen van dit proefschrift. Vooral ben ik jou dankbaar voor de manier waarop je de manuscripten met me hebt doorgenomen. Door jouw kritische vermogen en geduld zijn de stukken in kwaliteit vooruit gegaan.
Gijs jij had een iets minder frequent contact met mij dan mijn directe begeleiders Hans en Lex. Echter juist ook daardoor waren jouw opmerkingen tijdens de regelmatig weerkerende werkbesprekingen altijd fris en hebben vaak geleid tot nieuwe resultaten. Bedankt daarvoor.
Deze drie personen hebben wel in een bijzondere mate meegeholpen aan het tot stand komen van dit proefschrift. Echter, ik zou haast zeggen, zij zijn nog "maar" een topje van de ijsberg. Er zijn namelijk nog vele anderen die concreet werk gedaan hebben voor de hoofdstukken van dit proefschrift. Natuurlijk Peter Bruinenberg, die mijn begeleider was tijdens mijn afstudeeronderzoek. Verder wil ik de stagiairs bedanken die heel wat uitstekend werk hebben afgeleverd op een bijzonder prettige en gezellige manier: Paul Mak, Leo Voll, Jort Gerritsma en Jos Vorsselmans. Erik Postma wil ik ook hartelijk danken voor zijn bijdrage aan dit proefschrift. Wilma Batenburg, Guido Breedveld, Cornel Verduyn, Marten Veenhuis en Dick Schipper bedankt voor het helpen bij of uitvoeren van metingen.
Niet alleen de hulp en welwillendheid van wetenschappers is nodig voor het uitvoeren van een promotie-onderzoek. Daarom wil ik ook de volgende personen bedanken: Max Diederik de ideale koffieclubvoorzitter, bedankt voor je prettige en gezellige samenwerking, ook Nico Raaphorst stond altijd klaar om een handje te helpen en om over opera te praten. Jos Lispet is iemand die ik zeker wil bedanken, wat er ook voor problemen waren, jij had altijd een oplossing en bovendien een, al of niet door de beugel kunnende, mop daarbij. Verder bedank ik Gert v.d. Tholen en alle andere medewerkers van de instrumentenmakerij, Ton Verbeek de glasblazer, Bart Kerkdijk die altijd klaar stond met hulp en advies, Sjaak Lispet, Henk Dullaart en alle andere medewerkers van de diverse werkplaatsen.
Dan kom ik nu aan het rijtje mensen toe die niet in de vorm van experimenten e. d. hebben meegewerkt aan dit proefschrift maar die toch heel wat betekend hebben voor de sfeer waarin ik m'n werk mocht doen. Maudy Smith, Hans en Elly Bonnet en Francien de Jongh e.v.a hartelijk bedankt. In een bijzondere mate ben ik dankbaar aan mijn vader en ook aan mevrouw Muntingh die achter mij stonden tijdens het promotie-onderzoek..
Henk van Urk
Delft, 3 april 1989.
CONTENTS
CHAPTER
I Introduction
All illustrations and tables in this chapter have been published by others and have been reprinted by permission of the different publishers.
II Metabolic responses of Saccharomvces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose
limitation to glucose excess. 31
This paper has been published in the journal Yeast 4 (1988) 283-291, and has been reprinted by permission. (c) 1988 John Wiley & Sons Ltd., Chichester.
III Respiratory capacities of mitochondria of Saccharomvces cerevisiae CBS 8066 and Candida utilis CBS 621 grown under
glucose limitation. 47
This paper is in press Antonie van Leeuwenhoek 56 (1989) 000-000 and has been reprinted by permission.
© Kluwer Academie Publishers, Dordrecht- Printed in The Netherlands.
IV Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharo
mvces cerevisiae CBS 8066 and Candida utilis CBS 621. 57
This paper is in press in the journal Biochimica et Biophvsica Acta and has been reprinted by permission.
(c) Elsevier Science Publishers b. v., Amsterdam.
V A transition-state analysis of metabolic fluxes in
Crabtree-positive and Crabtree-negative yeasts. 77
PAGE 7
PAGE 9
and Environmental Microbiology.
VI Glucose transport in positive and
Crabtree-negative yeasts. 95
This paper has been submitted for publication in the Journal of General Microbiology.
VII The mechanism of the short-term Crabtree effect: 111 An overall view.
Summary. 123
Samenvatting. 125
CHAPTER I
INTRODUCTION
1.1 Physiological effects in yeasts
Though yeasts have been used for "biotechnological" purposes since ancient history (Chen & Chiger, 1985), detailed scientific knowledge on these organisms is rather recent. Pasteur, who lived from 1822-1890, was one of the first yeast researchers who studied the physiological behaviour of yeasts under several cultivation conditions. He observed, for instance, the effect that some yeasts tend to show higher fermentation rates when grown under anaerobic conditions in comparison with aerobic conditions. This effect, which is also found in other organisms, later has been called the "Pasteur effect".
Subsequently other physiological phenomena were discovered and described as general effects:
- The Custers effect (Custers, 1940; Scheffers, 1966; Wijsman et al., 1984). This effect is the opposite of the Pasteur effect and is found especially in Brettanomyces species (Custers, 1940). The Custers effect has been described as the inhibition of fermentation by the absence of oxygen.
- The Kluyver effect (Kluyver & Custers, 1940; Sims & Barnett, 1978): Some yeasts, which are able to ferment glucose anaerobically, may assi-milate different sugars aerobically but they do not ferment these sugars under anaerobic conditions. This phenomenon is called the Kluyver effect.
- Yet another effect, first found in tumour cells (Warburg, 1926; Crabtree, 1929), is the so-called Crabtree effect (De Deken, 1966; Fiechter et al., 1981). This effect concerns the aerobic fermentation of glucose, despite the presence of sufficiënt oxygen.
In contrast to the rather phenomenological description of these effects in the beginning of this century, description in mechanistic terms is possible in some cases due to profound biochemical knowledge nowadays. For instance, detailed knowledge on the control of the redox balances, or NAD(P)(H) balances (van Dijken & Scheffers, 1986; Bruinenberg, 1986), is a key to the understanding of many of these problems.
(Scheffers, 1961, 1966; Wijsman et al., 1984). Wijsman et al. (1984) found that the yeast Brettanomvces intermedius immediately stopped fermenting and growing upon transition from aerobic to anaerobic conditions. However, when acetoin, a hydrogen acceptor, was added to these anaerobic cultures the ability to grow and ferment was immediately recovered. Wijsman et al. (1984) explained the Custers effect in B. intermedius in terms of the inability of this yeast to form glycerol. Because B. intermedius tends to form acetate under all circumstances, the NADH produced can not be reoxidized in this yeast by formation of glycerol. Addition of acetoin, however, results in a recovery of the NAD(H) balance.
The Kluyver effect, in many cases, still is not understood (Sims & Barnett, 1978; Barnett & Sims, 1982). Sims & Barnett (1978) proposed that the transport of the different sugars, in yeasts that display the Kluyver effect, is an oxygen-dependent step. The mechanism of this oxygen-dependent sugar transport still is unclear. However, the Kluyver effect cannot be covered with only one general explanation as can be concluded from work by Bruinenberg (1986) (see also van Dijken & Scheffers, 1986). He found that xylose is assimilated under aerobic conditions by the yeast Candida utilis. However, xylose could not be fermented under strictly anaerobic conditions. Bruinenberg (1986) showed that this, also, is a consequence of redox problems. The first step of xylose metabolism is its NADPH-dependent reduction to xylitol (see Fig. 1 ) . The NADP produced in this way is again reduced to NADPH by the enzyme glucose-6-phosphate dehydrogenase. However, the oxidation of xylitol is an NAD -dependent step in C. utilis (Fig. 1 ) . Therefore, under anaerobic conditions there would be a net production of NADH because no transhydrogenase is present in this organism (Bruinenberg, 1986) (transhydrogenase catalyses the reaction NADH + NADP+<- NAD+ + NADPH).
Yeasts such as Pichia stipitis. which also possess an NADH-dependent xylose reductase are able to ferment xylose anaerobically (Bruinenberg, 1986). The Kluyver effect with respect to xylose seems to be a result of the regulation of the NAD(P)(H) balance.
The Pasteur effect, which is the inhibition of fermentation in the presence of oxygen, only occurs to a significant degree when the respiration rate under aerobic conditions is high compared to the aerobic fermentation rate (Lagunas, 1986). Under anaerobic conditions the reducing equivalents produced have to be reoxidized via fermentation. Under aerobic conditions reoxidation occurs also via respiration. This explains the anaerobic
CHAPTER I
stimulation of fermentation, as under anaerobic conditions the competition between fermentation and respiration for NADH does not occur.
Also the Crabtree effect is explained in terms of the need of reoxidation of NADH via either the respiratory or fermentative pathway (Fiechter et al., 1981; Kappeli, 1986). Some yeasts perform aerobic alcohol ie fermentation when grown on glucose: The Crabtree effect (De Deken, 1966). This
fermentation, which is energetically unfavourable, occurs, according to Kappeli (1986), because the respiratory capacity of e.g. Saccharomvces species is not sufficiënt for competing effectively with fermentation for NADH.
The aim of this thesis was to further investigate the mechanism of the Crabtree effect, which is detrimental to the economy of bakers' yeast production. Special attention is paid to the role of the respiratory capacity in this phenomenon.
3C02 p 3 p e n l o s e - P « . 3 glucose - $ - p *>— 6NAOPH 6NADP sTl ^ ^ exylilol 6 N » D _ _ l | 6 N A O H | 6 xylulose 6 penlose-P 3 l r i o s e - P 3he«ose-P 3hexos»-P |9 elhgnol|
Figure 1. From Bruinenberg et al. (1983b). Schematic representation of
xylose metabolism in case of alcoholic fermentation. 1: xylose reductase; 2: xylitol dehydrogenase; 5: glucose-6-phosphate dehydrogenase.
1.2 Background and scope of this thesis
In the industrial production of bakers' yeast it is essential to obtain a high cell yield on the sugar-containing substrate, molasses. In modern bakers' yeast production a fed-batch fermentation process is used (Reed, 1982; Barford, 1987). The growth rate during production is kept below 0.2
h by adjusting the sugar supply rate. This growth rate is below the maximal growth rate, /i , of this organism. The reason why the growth rate is kept at a submaximal value is that at high growth rates alcohol ie fermentation sets in (Rieger et al., 1983; Postma et al., 1989). This, of course, is undesirable because ethanol production means a loss of substrate and a decrease of the cell yield. The occurrence of alcohol ie fermentation in yeasts at high growth rates is called the Crabtree effect (De Deken, 1966; Fiechter et al., 1981).
In practice, alcohol ie fermentation cannot be prevented entirely by producing the yeast at a low growth rate. This is due to the fact that
3
mixing in the huge industrial fermenters (> 200 m ) is not ideal. Imperfect mixing leads to sugar concentration gradients. In some regions in the reactor, therefore, the sugar concentration will be high. It is known that even in cells grown at a lower growth rate high sugar concentrations trigger alcohol production (Woehrer & Roehr, 1981; Petrik et al., 1983; Verduyn et al., 1984b): During the production process the yeast cells will be exposed to a low sugar concentration but, when the cells suddenly enter a region where the sugar concentration is high, fermentation will set in within a time span of 1 min. This transient effect is called the short-term Crabtree effect (Petrik et al., 1983) as opposed to the occurrence of alcohol ie fermentation under steady-state conditions at high growth rates which is called the long-term Crabtree effect (De Deken, 1966; Fiechter et al., 1981).
The imperfect mixing during bakers' yeast production also results in oxygen gradients. At some places in the reactor the oxygen transfer rate may limit the respiration rate and, therefore, also lead to alcoholic fermentation (Sweere et al., 1988a, b ) . It is therefore important to distinguish between the oxygen and the sugar (Crabtree) effect. This thesis deals with a study on the mechanism underlying the undesirable aerobic fermentative response of bakers' yeast as a consequence of sudden changes in sugar concentration, the so-called short-term Crabtree effect. Although the Crabtree effect is mostly associated with the physiological responses of yeasts, it is a more widespread phenomenon. For instance, the effect has also been found in mammalian tumour cells and in bacteria. In the following paragraph a genera! discussion of the Crabtree effect is presented.
CHAPTER I
2. The mechanism of the Crabtree effect
As mentioned above it is important to discriminate between two types of effects:
- The long-term Crabtree effect is the aerobic fermentation by cells during steady-state conditions. Full physiological adaptation to this fermentative state has therefore been achieved.
- The short-term Crabtree effect is the sudden fermentative response under fully aerobic conditions upon addition of excess sugar to cells that did not ferment before this addition. In this case no adaptation to this fermentative state has occurred.
Where possible the two effects will be discussed separately.
2.1 The Crabtree effect in tumour cells
When reviewing the literature on the Crabtree effect, it is inevitable to include information on this effect in tumour cells because it first has been described in these cells (Warburg, 1926; Crabtree, 1929).
In the literature published on the Crabtree effect in tumour cells it is often rather difficult to distinguish long-term and short-term effects. In most of the studies, especially the older reports, manometric techniques (Warburg, 1926) have been used. In these studies the effect of glucose addition on the endogenous respiration of tumour slices was investigated. Therefore, they deal with transient responses (short-term effect). However, as pointed out below, frequently a long-term effect is evident as wel!.
The history of the Crabtree effect starts with the research on tumour cells (Minami, 1923; Warburg, 1926). The hypothesis of Warburg (1956, 1967) on the cause of cancer was that the respiratory system of tumor cells has been damaged. This hypothesis has been rejected by most of the cancer researchers (c. f. Racker & Spector, 1981). It could be established that the respiratory activity of tumour tissues is not significantly different from that observed in normal tissues (Aisenberg, 1961; Weinhouse, 1982) although Warburg disputed this (1956). However, an important observation made by Warburg (1926) still stands, namely that all tumours show a high glycolytic activity under aerobic conditions as compared to non-malignant tissues. (It must be emphasized here that especially in the early publications on this subject the term glycolytic activity is used as a synonym for lactate production). Since the observation of aerobic lactate formation by tumour
cells upon addition of glucose was first made by Warburg (1926), this effect is called the Warburg effect (Racker, 1972).
In 1929 a paper by Crabtree was published on carbohydrate metabolism of tumours. He observed that the respiratory activity, Q„ (see Table 1 ) , of
u2
tumour cells was lower upon addition of glucose in comparison to conditions where xylose was added. Xylose is not metabolised by the tumour cells and, therefore, no glycolytic activity is present.
Table 1. Recalculated data from Crabtree (1929). Comparison of tumour
metabolism in presence of glucose or xylose. The respiratory activity Qn ? -1 -1
(jumol CL consumed x h x (mg dry tissue) ) . Lactate production under aerobic (Q-||ctate) o r anaero')ic ^ïactate^ conc'itions (/lm0'' x n x (mg dry tissue) ) . Tissue Crocker sarcoma * * *» » j > j Jensen's rat sarcoma »» Average Glucose \ 0.35 0.44 0.57 0.41 0.66 0.46 0.48 0.41 0.47 added 0.2 %
o°
2 xlactate 0.50 0.60 0.65 0.70 0.74 0.39 0.50 0.37 0.56 Xylose \ 0.45 0.48 0.60 0.50 0.66 0.54 0.57 0.47 0.53 added 0.2 % 0o
2 ^lactate 0 0 0 0 0 0 0 0 0 QN2 ^lactate 0.05 0.05 0.07 0.03 0.04 0.05 0.04 0.05 0.05Although the suppressing effect of glucose was not very substantial (about 11 %, Table 1 ) , it was almost invariably found in the separate experiments. Crabtree established the rul e that "glycolytic activity exerts a significant checkinq effect on the capacitv for respiration of tumour tissue". Medes & Weinhouse (1958) and Bloch-Frankenthal & Weinhouse (1957) observed that this effect is only present at high glucose concentrations (>1 itiM). At low glucose concentrations no inhibition of respiration was found.
CHAPTER I
The effect of the glycolytic activity on the respiratory activity has been called the Crabtree effect since the publication of Crabtree (1929). It should be remarked that this effect, though clearly related to it, is not the same as the Warburg effect: The Crabtree effect is the suppression of respiration whereas the Warburg effect is the production of lactate under aerobic conditions. Both effects may be present at the same time.
A rather complete review on tumour metabolism in relation to the Crabtree effect was presented by Aisenberg (1961). Only few publications have appeared since then on this subject (Hepp et al., 1966; Tsuiki et al., 1968; Gosalvez et al., 1975; Sussman et al., 1980; Racker & Spector, 1981). Most of these publications deal with the inhibition of respiration in tumour cells upon addition of glucose. Interestingly, when oxygen consumption is measured using modern equipment, namely oxygen electrodes, during the first 30 s upon addition of glucose to tumour cells no inhibition but a stimulation of the respiratory activity can be observed (Sussman et al., 1980) (Fig. 2 ) .
4 m M Glucose
t— 2min-H
Figure 2. From Sussman et al. (1980). The changes in respiration rate following the addition of 4 mM glucose to ascites tumour cells. The numbers indicate the rate of oxygen consumption (/«nol 0?/min) by 22 mg wet weight of
cells.
After the first 30 s, however, the respiratory activity decreases to values below that present before glucose addition. Though respiration increases as an immediate response to glucose addition, also lactate production sets in immediately (Sussman et al., 1980).
The inhibition of respiration in tumour cells by glucose has been explained in terms of competition between glycolysis and respiration for inorganic phosphate, P ^ and ADP (Gatt & Racker, 1959; Wu & Racker, 1959a, b; Aisenberg, 1961). The fact that the inhibition of respiration is eliminated by addition of the uncoupler dinitrophenol, DNP, is in favour of this hypothesis (Warburg, 1926; Aisenberg, 1961; Tsuiki et al., 1968; Racker & Spector, 1981). However, the other phenomenon, namely lactate production, is not eliminated but it is stimulated upon addition of DNP (Racker & Spector, 1981). Sussman et al. (1980) refined the above hypothesis stating that the ratio ATP/(ADP + P.) determines the behaviour of tumor cells. This refinement was based on the pattern of the intracellular concentrations of these compounds after glucose addition. The stimulation of respiration during the first 30 s and the inhibition after that period (Fig. 2) could be explained from the course of the ATP/(ADP + P,) ratio. The higher this ratio the more substrate level phosphorylation will occur and lead to lactate formation.
However, in my opinion, the hypothesis of Racker and Sussman is more a description than an explanation of the Crabtree or Warburg effect. In other words, the question to be asked is: What causes the glycolysis to effectively compete for P. and ADP with respiratory-coupled phosphorylation? It seems reasonable to assume that the activities of glycolytic enzymes (Table 2 ) , citric-acid cycle- and respiratory enzymes play a role. Indeed, White (1958, Table 2) found that in tumour tissues some of the glycolytic enzymes are present at elevated levels, especially the enzyme lactate dehydrogenase, like in a number of other diseases. It may, therefore, be concluded that high activities of glycolytic enzymes and especially of lactate dehydrogenase are responsible for the high glycolytic activities and thus for the lactate production.
Wenner et al. (1952) found also low activities of the citric-acid cycle enzymes aconitase and 2-oxoglutarate dehydrogenase. Hepp et al. (1966) reported the presence of a high deacylase and a low thiokinase activity (Fig. 2) in tumour cells leading to acetate formation. So, apart from an elevated lactic dehydrogenase activity in cancer cells (Table 2 ) , leading to lactate formation, in some cases also acetate may be produced when the oxidation of acetyl-CoA cannot compete with the deacylase activity (Fig. 3 ) .
CHAPTER I
Table 2. From Aisenberg (1961) (with data from White et al. (1958). Elevated
serum enzyme activity in human disease. The number of patients with elevated enzyme activities from the group of patients that is tested is given in this table.
Aldolase Phosphohexose
isomerase
Lactic dehydrogenase Serum glutamic oxal
-acetic transaminase Isocitric dehydrogenase Cancer 36 of 104a 42 of 88 57 of 75 16 of 51 Myocardial infarction 36 of 69a 28 of 69 68 of 69 51 of 69 11 of 69 Angina pectoris 23 of 6 1a 27 of 61 59 of 61 40 of 61 Infectious hepatitis 5 of 14 13 Of 14 7 of 14 7 of 14 Expressed as numbers of p a t i e n t s . GLUCOSE A FYRUVATE■
11
LACTATE ACETATE + CoA h H2° (Deacylase) A T P (Thiokinase) Ir - > ACETYL-CoA 4 If FATTY ACID » C 02 + H20Figure 3. From Hepp et al (1966). Schematic view of acetyl-CoA metabolism in
tumours.
From the above it will be clear that the metabolic behaviour of tumour cells is governed by both long- and short-term effects. The tumour cells do have repressed levels of oxidative enzymes (Wenner et al., 1952; Hepp et al., 1966) which is, of course, a long-term effect. However, because the respiratory activity is only inhibited upon addition of glucose to tumour
cells (Crabtree, 1929 (Table 1 ) ; Bloch-Frankenthal & Weinhouse, 1957; Medes & Weinhouse, 1958) also a short-term effect does exist.
2.2.1 The long-term Crabtree effect in bacteria
The Crabtree effect in bacteria has not been studied as extensively as in tumour cells and yeasts. Only in one case the Crabtree effect in bacteria has been mentioned as such, namely by Doelle (1981) who studied the behaviour of Escherichia coli in glucose-limited chemostats under aerobic conditions. He found fermentation products at high dilution rates, as is also the case with certain yeasts (Fiechter et al., 1981). Also Ishikawa & Shoda (1983) found a low cell yield with E. coli cultures at high dilution rates which may result from acetate production. Doelle states that the phosphorylation site I is repressed at high growth rates and in contrast the activity of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase increases. The repression of site I wi11 result in an inhibition of respiration while, on the other hand, a stimulation of glyceraldehyde 3-phosphate dehydrogenase may lead to a stimulation of glycolytic activity. However, these authors did not explicitly establish that cultures growing at high dilution rates were truly glucose-limited. For instance, tracé element limitation may result in a change in metabolic pattern. This has been shown by Rieger et al. (1983) and Kappeli et al. (1985b): With yeast chemostat cultures, any limitation in the tracé element composition of the medium may lead to formation of fermentation products.
2.2.2 The short-term Crabtree effect in bacteria
A study by Neijssel & Tempest (1976) reveals some important information on the behaviour of bacteria under transient-state conditions. They studied the response of aerobically grown Klebsiella aeroaenes upon addition of excess glucose to glucose-limited, non-fermenting, cultures. An immediate increase of respiratory activity upon the addition of glucose was observed which was accompanied by the formation of acetate. However, the growth rate did not increase instantaneously. Similar effects were found with E. coli and Bacillus subtilis (Neijssel, personal communication). Neijssel & Tempest (1976) concluded that uncoupling of catabolism and anabolism occurred upon disturbance of the steady state, because after a pul se glucose is consumed at a high rate whereas the anabolic capacity is low. In other words, a
CHAPTER I
limited anabolic reactivity leads to formation of overflow products. Similar arguments have been put forward by Teixeira de Mattos (1983) who pulsed anaerobically grown cultures of K. aerogenes with glucose. He found an immediate production of lactate, via the energy-inefficient methylglyoxal bypass, and no increase of growth rate. On the other hand, Koch (1971) reported that E. coli does have an anabolic overcapacity. Similar results have been reported by Steekstra et al. (1988) who pulsed glucose-grown K. aerogenes with fructose. They found relatively low fructose consumption rates but an immediate increase of growth rate; moreover, no lactate production could be detected.
2.3.1 The long-term Crabtree effect in yeasts
The Crabtree effect in yeasts was first described by De Deken (1966) (Table 2 and 3 ) . He stated: "The Crabtree effect must be considered as the repression of an energy-producing svstem. respiration. bv another energy-producinq svstem, fermentation". This could be concluded from a clear correlation between fermentation rate and respiration rate of Saccharomyces cerevisiae grown with different sugars (Table 3). On the other hand, the results obtained with other yeast species grown on glucose (Table 4) do not invariably confirm this clear inverse correlation between fermentation rate and respiration rate. A possible explanation for these diverging results may be found in differences in growth rates and glucose consumption rates between the species tested. These parameters, unfortunately, were not tested by De Deken (1966).
Since the report by De Deken, many papers have appeared on the repressive effects of glucose on respiratory enzymes and other mitochondrial enzyme systems (Polakis & Bartley, 1965; Jayaraman et al., 1966; Beek & von Meyenburg, 1968; Neal et al., 1971; Knöpfel, 1972; Haarasilta & Oura, 1975; Petrik et al., 1983). In general it was concluded that the fermentation is a consequence of a repression of respiratory enzymes. However, when better cultivation techniques became available von Meyenburg (1969) could show that the long-term Crabtree effect in yeasts is only present at high dilution rates (D> 0.25 h" ) (Fig. 4) in glucose-limited cultures of S. cerevisiae.
Table 3. Recalculated data from De Deken (1966). Rates of fermentation
expressed as C02 produced via fermentation (qC Q fe r m e ntation^ anc* rates °^
-1 7 -1
respiration (qn ) , both expressed as /imol x h x 10 cells ; and doubling u2
times (t.) of Saccharomvces cerevisiae (normal strain) on various hexoses.
Carbon source Glucose Fructose Mannose Galactose \ 1.2 1.5 2.7 5.0 ^COpfermentation 19.5 17.3 11.5 3.8 td (h) 0.88 0.88 1.05 1.20
DILUTI0N RATE D = SPECIFIC GROWTH RATE fj
Figure 4. From von Meyenburg (1969). Aerobic growth of Saccharomvces
cerevisiae H 1022 in glucose-limited chemostats. Culture dry weight X (g/l), specific oxygen uptake Q0 and carbon dioxide production G>0 (mmol x g x
h ) and respiratory quotiënt RQ (QC Q / QQ ) as a function of the dilution
CHAPTER I
Table 4. Recalculated data from De Deken (1966). Presence of the Crabtree
effect in various yeasts grown on glucose. The CO„ production caused by fermentation ( q ™ fe r m e ntation^ ^as '3een correctec' f °r t n e c^ ? evo^vec^ a s a consequence of respiration. The respiration rate (qQ ) and the CO„
- 1 7 -1 production rate are expressed as /imol x h x 10 cells .
Organism Saccharomyces cerevisiae S. chevalieri S. fragilis S. italicus S. oviformis S. pasteurianus S. turbidans S. carlsbergensis Schizosaccharomyces pombe Candida utilis C. tropicalis C. monosa Trichosporon fermentans Hansenuia anomala Debaryomyces globosus Pichia fermentans Schwanniomyces occidentalis Brettanomyces lambicus Torulopsis dattila T. sphaerica T. glabrata T. colliculosa T. sake Nematospora coryli Nadsonia fulvescens \ 1.2 0.3 6.1 0.0 0.0 0.5 0.9 0.0 0.0 7.5 6.9 5.3 3.9 6.0 3.1 6.1 2.3 0.3 0.0 6.4 2.7 3.3 5.3 ^OLfermentation 19.5 22.7 0.5 23.6 15.3 14.7 17.1 17.1 10.2 0.0 0.0 0.0 0.0 0.0 5.6 0.3 0.0 2.3 13.0 0.9 9.8 0.0 7.3 Crabtree effect + + -+ + + + + + -+ -+ + -+ . + -+ +
NOTE. The absence of a respiration value means that it was too low to be measured in the presence of the high rate of aerobic fermentation.
At low dilution rates, and thus low rates of glycolysis, no repression of the synthesis of respiratory enzymes was found, and no ethanol was produced. Apparently, when the rate of glycolysis is low, respiration can effectively compete with fermentation. Thus, under these conditions a Pasteur effect does exist (Lagunas, 1986) which is defined as the suppression of alcohol ie fermentation by oxygen. At high dilution rates, however, the Crabtree effect overrules this Pasteur effect. The antagonism between these two effects was noted by De Deken who called the Crabtree effect the "contre-effet Pasteur".
15 L S 10 | <D ü c o o "> E o m "0 0.1 0.2 0.3 Dilution rate (rr1)
Respirative glucose Respiro-fermentative metabolism glucose metabolism
Figure 5. From Kappeli (1986). The Op uptake rate ( — ) and biomass concentration ( ) of glucose-limited chemostat cultures of Saccharomvces uvarum H 2055 as a function of the dilution rate.
In later publications by the group of Fiechter some of the conclusions drawn by Beek & von Meyenburg were corrected (Rieger et al., 1983; Petrik et al., 1983). It was shown by this group that the repression of respiration at high dilution rates as observed by Beek & von Meyenburg (1968) and von
2+
Meyenburg (1969) was caused by Mn -limited conditions at these high dilution rates (Rieger et al., 1983). In the presence of sufficiënt amounts
2+
CHAPTER I
capacity (qQ m a x) of about 7-8 mmol. g ^ . h "1. This qQ m a x is reached at the
dilution rate where fermentation sets in (Fig. 5 ) .
Barford et al. (1981) and Postma et al. (1989), working with other S. cerevisiae strains, observed that the oxygen consumption increased to a value of 12 mmol 02 . g"1. h"1. These authors also reported that the
respiratory activity increased disproportionally at dilution rates above 0.3 h , at which a small amount of acetate (< 0.6 mM) but no ethanol was yet present in the culture (Fig. 6 ) . Above D= 0.38 h"1 the ethanol production
set in.
Figure 6. From Postma et al. (1989). Specific rate of oxygen uptake O , carbon dioxide production • , and cell yield D , as a function of the dilution rate in glucose-limited cultures of S. cerevisiae CBS 8066. (Acetate is produced at D > 0.3 h"1 and ethanol is produced at D > 0.38
I T1) .
The occurrence of acetate at dilution rates of above 0.3 h"1 (Postma et al.,
1989) in cultures of S. cerevisiae CBS 8066 is probably caused by the presence of high pyruvate decarboxylase activities and relatively low acetyl-CoA synthetase acitivities (Fig. 7 ) . Acetate may have an uncoupling effect which, therefore, explains the relatively high oxygen consumption rates at 0.3 < D < 0.38 h"1 (Fig. 7 ) . Postma et al. (1989) concluded that
fermentation is required at high dilution rates because the respiratory-coupled phosphorylation wi11 not be efficiënt enough to supply the ATP needed for growth. Moreover, the activity of the enzymes acetaldehyde dehydrogenase and acetyl-CoA synthetase seemed repressed at high dilution rates (Postma et al., 1989).
glucose
1 . t , t
' \
pyruvate »- acetaldehyde »~ ethanol
acetate » -«
A
acetyl-CoA♦
TCA cycleI
C 02t
Figure 7. From Postma et al. (1989). Alternative routes of pyruvate
catabolism in yeasts. The enzymes are indicated by numbers. 1: pyruvate dehydrogenase complex; 2: pyruvate decarboxylase; 3: acetaldehyde dehydrogenase; 4: acetyl-CoA synthetase; 5: alcohol dehydrogenase.
2.3.2 The short-term Crabtree effect in veasts
Literature on the short-term Crabtree effect in yeasts is not abundant (Petrik et al., 1983; see Fig. 8) this in contrast to the number of papers on the long-term Crabtree effect (Polakis & Bartley, 1965; Fiechter et al., 1981; Kappeli, 1986; Kappeli & Sonnleitner, 1986; Postma et al., 1989). As has been mentioned in paragraph 2.3.1, much emphasis is layed on a limited respiratory capacity of Saccharomvces species. The hypothesis, put forward by Kappeli & Sonnleitner (1986, Fig. 5 ) , implies that Saccharomvces species possess only a limited maximal respiratory capacity, qQ m a x. As long
CHAPTER I
pyruvate concentration will be low, which results in an efficiënt oxidation of pyruvate via the oxidative pathway, thus preventing alcohol ie fermen-tation (at D < 0.16 h for the strain presented in Fig. 5 ) . When the glucose consumption rate is too high (at D > 0.16 h" , Fig. 5 ) , and respiration cannot keep pace with the pyruvate production, the pyruvate decarboxylation to
Pulse
30 60 Time (min)
Figure 8. From Petrik et al. (1983). Short-term response of S.uvarum after a glucose pulse to cells growing under glucose limitation. ■ , Biomass; ▲ , glucose; • , ethanol; A , acetate; O , Qn ; D , Qr n ; T , RQ. The dilution
02 ^C02
j v>2 V,U2
rate of the chemostat was 0.1 h . A t zero time 25 g glucose/l was added.
acetaldehyde, subsequently reduced to ethanol, can play a role of importance. The competition between the enzymes pyruvate dehydrogenase and pyruvate decarboxylase is important in this respect. The affinity for pyruvate of the latter enzyme is low (Holzer, 1961) and, therefore, plays an important role in the Crabtree effect (Schmitt & Zimmermann, 1982; Petrik et al., 1983). Petrik et al. (1983), in line with the hypothesis of Kappeli, stated that the fermentative response as shown in Fig. 7 must be a
consequence of a high glucose consumption rate and the subsequent limitation by the maximal respiratory capacity of his strain.
On the other hand, no serious attention has been paid by the group of Kappeli and Fiechter to a possible anabolic limitation in S. cerevisiae (or Saccharomvces strains). From the work by Neijssel and Tempest (see above), it is evident that a limitation in the anabolic processes upon addition of glucose to glucose-limited cultures may have important consequences. A bottleneck in anabolism may result in pyruvate accumulation, uncoupling of anabolic and catabolic processes and thus in fermentation in case the enzymes pyruvate decarboxylase and alcohol dehydrogenase are present.
The effect of repression of respiratory enzymes by glucose, although it may play a role in the long-term Crabtree effect (Polakis & Bartley, 1965), wil! not be important in the short-term Crabtree effect. Instead, the activity and regulation of glucose uptake systems (Romano, 1982; Bisson & Fraenkel, 1983; Spencer-Martins & van Uden, 1985a, b; de Bruijne et al., 1988; Postma et al., 1988) and glycolytic enzymes (Maitra & Lobo, 1971 a, b; Hirai et al, 1975; Banuelos & Gancedo, 1978; Ciriacy & Breitenbach, 1979; Navon et al., 1979; Lagunas & Gancedo, 1983; Reibstein et al., 1986) may have an important impact on the behaviour of yeasts upon addition of glucose to glucose-limited cells. A relatively high glucose consumption rate and a high glycolytic rate as compared to respiratory activity may result in a fermentative response.
2.4 Definition
It is obvious from this historical review that the original definition of the Crabtree effect (Crabtree, 1929) namely that "glycolytic activity exerts a significant checking effect on the capacity for respiration of tumour tissue" is not appropriate for the description of the behaviour of yeasts when cultured in glucose-limited chemostats. Terminology is rather confusing, because the fermentative behaviour of S. cerevisiae at high dilution rates when cultured under glucose limitation is still called the Crabtree effect although there is no inhibition of respiration. The term "Warburg effect" as introduced by Racker (1972) principally is better because it is a more phenomenological description of the aerobic fermentation without mentioning a mechanistic explanation of the effect. However, to prevent confusion when referring to literature on the Crabtree effect in yeast, we will maintain the use of this term and especially the
CHAPTER I
term: short-term Crabtree effect (Petrik et al.. 1983) which is the effect that some veasts immediatelv start to ferment under aerobic conditions, when excess glucose is added to non-fermenting. glucose-limited chemostat cultures.
2.5 Conclusions
From the review presented above several important parameters that are relevant when studying the Crabtree effect can be distilled. Six parameters can be distinguished: I: Glucose uptake rate.
II: Rate of glycolysis. III: TCA-cycle capacity.
IV: Respiratory capacity. V: Biosynthetic capacity. VI: Fermentative capacity.
The impact of each parameter on the occurrence of the Crabtree effect will be discussed separately:
I: Glucose uptake rate.
The first step of the metabolism of glucose is its uptake by the cell. As discussed above, at low glucose concentrations tumour cells do not show a Crabtree effect (Bloch-Frankenthal & Weinhouse, 1957; Medes & Weinhouse, 1958). The same effect can be observed with yeast cells (Woehrer & Roehr, 1981; Rieger et al., 1983; Verduyn et al., 1984b). Only when high glucose concentrations are present the Crabtree effect can be observed. On the other hand, other organisms do not show this effect (non-malignant tissues and e.g. Candida strains). Therefore, the regulation of the sugar uptake and its relation to the glucose concentration may play an important role in the occurrence of the Crabtree effect.
II: Rate of glycolysis.
As the glycolytic flux seems to be an important parameter in the occurrence of the Crabtree effect (Warburg, 1926; Gatt & Racker, 1959; Wu & Racker, 1959 a,b; Doelle, 1981; Petrik et al., 1983) a more detailed study could give more information on the role of glycolysis in this effect. High glycolytic rates result in pyruvate accumulation in a cell and may therefore lead to formation of overflow products. For instance, determination of the activities and regulation of several glycolytic enzymes may give useful information (White, 1958; Banuelos & Gancedo, 1978; Reibstein et al., 1986). The presence of high amounts of glycolytic enzymes may, for instance,
explain the ability of glycolysis to effectively compete for ADP and P. as pointed out by the group of Racker and others.
III: TCA-cycle capacity.
As discussed in paragraph 2.1, a limited capacity of the TCA cycle may lead to accumulation of pyruvate in a cell and may, therefore, lead to fermentation. This is very much related to the other relevant parameter namely
IV: The respiratory capacity.
When an organism possesses a limited respiratory capacity, overflow at the level of pyruvate may occur in the cell (Kappeli, 1986; Warburg, 1926). According to the hypothesis of Kappeli (1986) no fermentation will occur unless the maximal respiratory capacity of the organism is reached. However, in this hypothesis the importance af an anabolic limitation as pointed out by Neijssel & Tempest (1976) is neglected.
V: The biosynthetic capacity.
When an organism is suddenly exposed to high substrate concentrations, the biosynthetic capacity probably must be adapted to this new situation (Neijssel & Tempest, 1976). When there is no biosynthetic overcapacity present, pyruvate accumulation and an uncoupling of anabolic processes and respiration and fermentation will occur because the consumption of pyruvate and TCA-cycle intermediates is rate-limiting.
VI: Fermentative capacity.
A rather trivial, but important, parameter is the presence of lactate- or ethanol-producing enzymes. Moreover, the regulation of these enzymes is important because certain circumstances, e.g. glucose-limitation in chemostats at low dilution rates (Rieger et al., 1983; Postma et al., 1989), seem to favour oxidation of pyruvate. The kinetics of the enzymes pyruvate dehydrogenase and pyruvate decarboxylase are important in this respect (Holzer, 1961).
In this thesis it is attempted to find the order of importance of these six parameters in the occurrence of the short-term Crabtree effect.
3. The structure of this thesis
The experimental work has been approached by doing a comparative biochemical and physiological study on the behaviour of different yeast species under identical conditions. By doing so it was hoped that conclusions could be drawn about the most relevant parameters causing an organism to show a
CHAPTER I
fermentative or non-fermentative response upon addition of glucose to glucose-limited cultures. No other studies have dealt with this problem in a similar fashion.
In the chapters 2, 3 and 4 the yeasts Saccharomvces cerevisiae CBS 8066 and Candida utilis CBS 621 are compared on the basis of several relevant parameters. S. cerevisiae immediately starts to ferment upon addition of excess glucose to glucose-limited cultures, whereas C. utilis behaves as expected for a Crabtree-negative yeast.
Based on work presented in the literature (see paragraph 2 of this chapter) and the experience at our institute at the time this study was started (1983) the following studies were performed. In chapter 2 the response of S. cerevisiae and C. utilis upon glucose addition to aerobic, glucose-limited cultures, is analysed. Parameters such as glucose consumption rate, respiratory activity, growth rate, metabolite production rate and also accumulation of reserve carbohydrates are presented. Particular attention is paid to the relation between catabolism and anabolism under transient glucose stress conditions in yeasts.
In view of the hypothesis of Kappeli (1986) and his group (see above) more information on the respiratory potential of S. cerevisiae and C. utilis was essential. Therefore, the respiratory capacities of mitochondria from these yeasts were determined in vitro with organelles isolated from the two yeasts, grown under identical, glucose-limited conditions. The results of these experiments are discussed in chapter 3.
Because the rol e of pyruvate and pyruvate-metabolizing enzymes is important, localization, activities and kinetics of these enzymes were determined. A relation between activities of these enzymes and intracellular concentrations of pyruvate and phosphate and the short-term response of S. cerevisiae and C. utilis is discussed in chapter 4.
On the basis of the findings presented in chapters 2, 3 and 4, it was decided to extend the comparative study to more yeasts. By doing so it would be possible to make genera!isations of the conclusions presented, based on the work done with S. cerevisiae and C. utilis. Three other Crabtree-positive species were chosen: Torulopsis glabrata CBS 138, Schizosaccharomvces pombe CBS 356 and Brettanomvces intermedius CBS 1943. Also three other Crabtree-negative species were used: Hansenuia nonfermentans CBS 5764, Kluvveromvces marxianus CBS 6556 and Pichia stipitis CBS 5773. Also a leaky pyruvate decarboxylase mutant of S. cerevisiae was used (natnely strain gdc 2-122 (Schmitt & Zimmermann, 1982)) to evaluate the
importance of the presence of this enzyme. Similar experiments as presented in chapter 2 and 4 were carried out with these different yeast species. The results are presented in chapter 5.
In view of the observation that three of the Crabtree-positive yeasts reveal high glucose consumption rates and that three of the Crabtree-negative yeasts reveal low glucose consumption rates upon addition of glucose to glucose-limited cultures (chapter 5 ) , it was decided to study the regulation of glucose uptake in yeasts in order to find a possible relation between the occurrence of the short-term Crabtree effect and this sugar uptake. The results of this study are presented in chapter 6.
In the last chapter, chapter 7, it is attempted to formulate a hypothesis on the mechanism of the short-term Crabtree effect in yeasts, based on the results presented in this thesis and those in the literature as reviewed in this chapter.
CHAPTER II
Metabolic responses of Saccharomvces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose
limitation to glucose excess.
H. van Urk, P. R. Mak, W. A. Scheffers and J. P. van Dijken.
SUMMARY
When chemostat cultures of Saccharomvces cerevisiae CBS 8066 and Candida utilis CBS 621, grown under glucose limitation, were pulsed with excess glucose, both organisms initially exhibited similar rates of glucose and oxygen consumption. However, striking differences were apparent between the two yeasts with respect to the production of cell mass in the culture and metabolite excretion. Upon transition from glucose limitation to glucose excess, S. cerevisiae produced much ethanol but the growth rate remained close to that under glucose limitation. C. utilis. on the other hand, produced little ethanol and immediately started to accumulate cell mass at a high rate. This high production rate of cell mass was probably due to synthesis of reserve material and not caused by a high rate of protein synthesis.
Upon a glucose pul se both yeasts excreted pyruvate. In contrast to C. util is. S. cerevisiae also excreted various tricarboxylic acid cycle intermediates, both under steady-state conditions and after exposure to glucose excess. These results and those of theoretical calculations on ATP flows support the hypothesis that the ethanol production as a consequence of pyruvate accumulation in S. cerevisiae. occurring upon transition from glucose limitation to glucose excess, is caused by a limited capacity of assimilatory pathways.
INTR0DUCTI0N
Since the early studies by Warburg (1926) and Crabtree (1929) on the effect of glycolysis on respiration in tumour cells, many papers have appeared on the interrelation between respiration and fermentation in eukaryotic cells.
(Polakis & Bartley, 1965; De Deken, 1966; Barford et al. , 1981; Fiechter et al., 1981). The Crabtree effect has been defined by Fiechter et al. (1981) as: "The repression of respiratory activity by glucose under aerobic conditions and subsequent deregulation of glycolysis with formation of ethanol". Indeed, many investigators have observed repression of the synthesis of enzymes of the citric acid cycle and the respiratory chain (Polakis & Bartley, 1965; Beek & von Meyenburg, 1968; Knöpfel, 1972; Haarasilta & Oura, 1975; Petrik et al., 1983) and repression of the synthesis of mitochondria (Neal et al., 1971; Jayaraman et al., 1966) under conditions of glucose excess. However, the tendency of Crabtree-positive yeasts, such as Saccharomvces species, for alcoholic fermentation of glucose under strict aerobic conditions does not necessarily involve a long-term adaptation via repression of the synthesis of respiratory enzymes. It is well known from the manufacturing of bakers' yeast that under glucose limitation alcoholic fermentation does not occur in aerobically grown S. cerevisiae. If, however, glucose is suddenly added in excess, alcoholic fermentation sets in immediately. This so-called short-term Crabtree effect (Fiechter et al., 1981; Petrik et al., 1983) does not involve repression or inactivation of the existing respiratory potential, or at least not initially.
The immediate onset of alcoholic fermentation upon transition of S. cerevisiae from glucose limitation to glucose excess has been ascribed to a limited respiratory capacity of this yeast: Alcoholic fermentation becomes apparent when the rate of sugar uptake exceeds the capacity of the respiratory pathways (Petrik et al., 1983; Rieger et al., 1983). The results described in this paper indicate, however, that the occurrence of alcoholic fermentation in S. cerevisiae upon relief from glucose limitation does not necessarily result from a bottleneck in the respiratory metabolism but may be caused by a restricted assimilatory capacity.
MATERIALS AND METHODS
Micro-orqanisms and qrowth conditions
Saccharomvces cerevisiae CBS 8066 and Candida utilis CBS 621 were maintained on malt agar slopes. The yeasts were grown under glucose limitation at 30°C
CHAPTER II
in laboratory fermenters with a working volume of 1 1. The dissolved-oxygen tension was kept above 20 % of air saturation; pH was controlled
automatically at 5.0 by addition of 2 M KOH. The mineral medium was prepared according to Bruinenberg et al. (1983a) (see also Chapter IV). The reservoir glucose concentration was 12.5 g/l. The dilution rate was either 0.1 or 0.2 h"1.
Glucose-pulse experiments
After non-oscillating steady-state cultures had been obtained, the medium flow was stopped and glucose was added to the culture to an initial concentration of approximately 50 mM. Samples were taken aseptically and immediately (within 20 s) centrifuged for 2 min at 13,000 r.p.m. in an MSE microcentrifuge; it was determined that all cells were sedimented within 15 s. Supernatants were frozen until analysed for metabolites or protein.
Determination of dry weight
For dry-weight measurements nitrocellulose filters (Gelman, pore width 0.45 firn) were used. After removal of the medium by suction, the filter with the pellet was washed with demineralized water. The filter was then dried in a Sharp R-7400 magnetron oven for. 15 min. Cell mass production rate in the cultures was determined via linear regression of the logarithm of dry weight values at 2, 10, 20 and 30 min (phase I) and 30, 40, 50 and 60 min (phase II).
Determination of C and N content
For the determination of C and N contents of biomass, cells were washed twice with distilled water and dried at 70°C. Analyses were performed using a Perkin Elmer Elemental Analyser 240 B.
Analvsis of oxvaen consumption and CO? production
Oxygen and C02 were analysed in the dried off-gas (Permapure dryer, Inacom
Instruments) from the fermenter by using a Servomex OA-184 oxygen analyser and a Beekman C02 analyser. The gas flow rate (as measured in the exhaust
line) was determined with a water-filled precision gas flow meter (Schlumberger, Holland).
The C02 production and oxygen consumption in the fermenters was
calculated according to equations 1, 2 and 3:
\ 0Z - % a s , o u tx pC02,out " < W i nx pC°2,in> / Vm ™ %2 - % s , i nx P°2,in " %as,outx P°2,out> / Vm ^
Qgas.in = ^ " PC02,out " P°2,out> x V , o u t ' °-79 (3)
In which QC Q and QQ represent the C02 production and oxygen consumption
rates (mol/h); Q represents the gas flow rate (l/h); V is the mol ar volume at atmospheric pressure and room temperature (1); p(L and pC0„ stand for the volume fraction of 0- and CCL.
Analvsis of metabolites
Spectrophotometric assays were performed at 30 °C with a Hitachi 100-60 spectrophotometer. Acetate was assayed enzymatically with the Boehringer test kit no. 148261. Citrate and oxaloacetate were determined in an assay mixture containing 163 mM glycylglycine buffer, pH 7.8; 0.15 mM NADH; 0.2 mM ZnCl2; and sample.The extinction was read at 340 nm. The reaction for
oxaloacetate determination was then started by adding 6 U/ml of malate dehydrogenase and the second extinction was read. Oxaloacetate was never present at detectable levels. Citrate could then be determined by measuring the increase in extinction following the addition of 0.24 U/ml citrate lyase.
Ethanol was assayed according to the colorimetric method of Verduyn et al. (1984a). Glucose was determined with the GOD-PAP method of Boehringer. Glycerol was assayed enzymatically with the Boehringer test kit no. 148270.
The assay mixture for malate determination consisted of 250 mM glycylglycine-glutamate buffer, pH 9; 2.4 mM NAD+;1.6 U/ml glutamate
oxaloacetate transaminase; and sample. The malate concentration was calculated from the increase in extinction at 340 nm as a result of the addition of 6 U/ml malate dehydrogenase.
2-0xoglutarate was assayed with the following mixture: 240 mM tri ethanolamine.HCl buffer, pH 7.6; 1 mM ADP; 0.15 mM NADH; 32 mM
CHAPTER II
ammoniumacetate; and sample. The concentration of 2-oxoglutarate was calculated from the increase in extinction at 340 nm following the addition of 12 U/ml glutamate dehydrogenase.
The assay mixture for the determination of pyruvate consisted of 124 mM triethanolamine.HCl buffer, pH 7.6; 0.30 mM NADH; 1.5 mM EDTA; and sample.The concentration of pyruvate was calculated from the increase in extinction at 340 nm following the addition of 2 U/ml lactate dehydrogenase. Succinate was determined with an assay mixture consisting of 108 mM glycylglycine buffer, pH 8.4; 0.015 mM MgS0«.7 H20; 0.15 mM NADH; 0.04 mM
CoA; 0.054 mM ITP (inosin triphosphate); 0.076 mM phosphoenolpyruvate; 2 U/ml lactate dehydrogenase; 1.67 U/ml pyruvate kinase; and sample. The concentration of succinate was calculated from the increase in extinction at 340 nm following the addition of 0.5 U/ml succinyl-CoA synthetase.
Calculation of ATP production
ATP production needed for growth can be calculated from the equation:
" + V T P'ATP * q. A HATP,m
Y
qATP, needed = (4)
ATP
in which q.jp neeclecj [mmol ATP x (g cells)" x h" ] represents the amount of
ATP that is required for growth at a given specific rate ji (h~ ) . The values for specific rate of ATP consumption in maintenance processes (qATp m) and
Y.jp were taken from von Meyenburg (1969) as 1.572 mmol ATP x (g cells)" x h" and 0.012 g cells/mmol ATP, respectively.
In non-fermenting aerobic steady-state cultures the rate of ATP production, qAjp> is linearly proportional to the specific rate of oxygen consumption:
qA T P= k x q ^ (5)
It is assumed that the ATP consumption rate from equation (4) and the ATP production rate from equation (5) are equal. It is relevant to note that k in this equation does not only depend on the P/0 ratio but also on ATP produced via substrate-level phosphorylation in glycolysis and TCA cycle. On the basis of the above-mentioned values for Y . ™ and q.jp , and the
measured oxygen consumption rates ,qQ , under steady-state conditions ( 3.0
+ 0.5 and 5.6 ± 0.5 mmol x (g c e l l s ) " ^ h"1 for D= 0.1 and 0.2,
respectively), k was calculated to be 3 + 0.4 for both S. cerevisiae and C. utilis.
In the calculation of ATP production during the glucose pulse experiments equation (5) should be corrected because of additional substrate-level phosphorylation caused by ethanol and acetate formation, and ATP consumption due to glycerol production. Substrate-level phosphorylation yields 1 mol of ATP for 1 mol of ethanol or acetate produced, whereas 1 mol of ATP is consumed for every mol of glycerol formed. Moreover, the corrections should take into account that production of 1 mol of acetate causes consumption of 1 mol of CL and that in production of 1 mol of glycerol 0- consumption is diminished by 0.5 mol. The rate of ATP production from the fraction of glucose that is completely oxidized to CO- is:
qATP,complete oxidation= k x ^q02" qacetate+ 0'5 x qglycerol* (6)
The rate of ATP production via oxidative phosphorylation gained or lost due to acetate and glycerol formation can be given as:
qATP,acetate/glycerol= P / 0 x (2 x qacetate" ''glycerol (7)
Excluding ATP production via substrate-level phosphorylation due to ethanol and acetate production and ATP consumption due to glycerol production we define:
qATP,respiration qATP,complete oxidation+ qATP,acetate/glycerol (8)
The total rate of ATP production, including ATP produced via substrate-level phosphorylation during ethanol and acetate production and ATP consumption during glycerol production, can be calculated with equation:
qATP,total" qATP,respiration + qethanol + qacetate " qglycerol * '
In our calculations a P/0 ratio of 1.1 was used according to von Meyenburg (1969).
CHAPTER II
Protein determination
Protein concentrations in culture supernatants were determined by the Lowry method. Bovine serum albumin (Sigma, fatty acid-free) served as a Standard. The protein content of whole cells was determined as follows: Cells were harvested and washed once with demineralized water. Cell suspensions (about 6 mg/ml) were then boiled in 1 M KOH for 10 min. After cooling, CuS0-.5 H20
was added to a final concentration of 25 mM. After 5 min this mixture was centrifuged in an MSE microcentrifuge and the absorbance at 555 nm was determined with a Hitachi 100-60 spectrophotometer.
Glvcogen determination
Glycogen content of cells was determined by method 4 from Quain (1981). The glucose released at the end of this procedure was determined as mentioned above.
Trehalose determination
Trehalose was determined according to Stewart (1975) with the following modifications: After the extraction procedure, to 200 /J1 of the combined supernatants containing the extracted trehalose, 17.5 /il 4 % (v/v) hydroxyl
amine was added, and with 1 M sodium acetate buffer (pH 5.9) the final volume was brought to 1 ml, leading to a pH of approximately 5.7. Then 0.2 U of trehalase (Sigma T8778) was added and the mixture was incubated for 2 h at 37 °C. It was established by using a Standard trehalose solution that all the trehalose added was indeed hydrolysed to glucose by this procedure. The glucose released at the end of this procedure was determined as mentioned above.
Biochemicals and enzvmes
These were obtained from Boehringer Mannheim unless stated otherwise.
RESULTS
Anabolic reactivitv
In order to elucidate the mechanism of the rapid alcohol ie fermentation known to occur in S. cerevisiae after relief of glucose limitation, a comparative study was made of the transient behaviour of S. cerevisiae and C. utilis following the addition of a glucose pulse to glucose-1imited chemostat cultures. The results presented in Figure 1 and Table 1 illustrate the marked differences in anabolic reactivity between the two yeasts under these conditions.
When S. cerevisiae was pulsed with glucose, the rate of biomass production ( M Q U ) during the first 30 min, designated as phase I, was equal to the dilution rate at which the organism was pregrown (Table 1 ) . C. utilis. on the other hand, immediately accumulated biomass at a higher rate. In order to investigate if this "growth rate", as determined by dry weight measurements, was related to production of protein (/*ppn-r)> the protein and N-contents were measured (Table 2 ) . In contrast to S. cerevisiae. C. utilis showed a sharp decrease of protein and N-content during phase I. This indicates that C. utilis accumulates endogenous reserves in this phase. Indeed, during phase I the glycogen content increased considerably (Table 2 ) . Trehalose remained virtually absent (not shown). The growth rate in phase I, based on protein production, ^pR0-r, is related to the biomass production rate based on the measurement of dry weights, /i™., according to the equation:
"PROT = "DW + 2 X (1n P0.5" ln PS S} (10)
in which Ps s and PQ 5 are the protein content of the cells (g/g) during steady state and 0.5 h after addition of glucose. If the errors in the protein determinations are taken into account (Table 2 ) , it can be estimated that also in C. utilis protein synthesis continued at approximately the same rate at which it was precultured.
During the second phase of the experiment (30-60 min after the glucose pulse, Table 1 and 2 ) , the biomass production rate of both organisms was approximately the same. Moreover, the protein content of the cells remained constant.
Table 1. Fluxes, q [mmol x g cells x h ] , during the first and second phase (see text) of glucose pulse experiments after pre-cultivation under glucose limitation at D=0.1 h" or D=0.2 h" . Standard deviation values are given for two to four separate experiments . /inu is the production rate of biomass, determined by dry weight measurements.
"DW (h_1) ^glucose ^ethanol ^acetate ''glycerol \ qco2 S. cerevisiae D=0.1 h"1 Phase I 0.09 + 0.03 5.4 ± 0.5 5.6 + 0.8 1.3 + 0.5 0.5 6.6 ± 0.3 12.0 + 0.9 Phase II 0.31 + 0.03 9.0 ± 1.0 7.7 ± 0.7 1.3 + 0.6 0.5 7.5 ± 0.3 15.7 + 1.4 D=0.2 h'1 Phase I 0.19 + 0.02 6.7 ± 0.7 6.7 ± 1.3 1.3 + 0.4 0.4 7.7 + 0.3 12.0 + 0.3 Phase II 0.32 ± 0.02 9.5 ± 1.0 8.6 ± 1.3 0.8 + 0.5 0.1 8.1 + 0.3 15.5 + 0.5 C. utilis D=0.1 h"1 Phase I 0.59 + 0.02 5.2 + 0.8 0.1 + 0.1 0.2 + 0.2 0 7.3 + 0.3 6.7 + 0.3 Phase II 0.37 + 0.04 5.2 + 0.3 0.2 + 0.1 0.3 + 0.3 0 8.4 ± 0.3 7.9 ± 0.3 D=0.2 h"1 Phase I 0.53 + 0.03 5.5 + 1.2 0.1 ± 0.05 0 0 7.0 ± 0.3 6.3 + 0.3 Phase II 0.33 + 0.02 5.6 + 0.8 0.6 + 0.3 0.1 ± 0.1 0 8.4 ± 0.3 7.3 ± 0.3
