respiratory efficiency and metabolize partitioning as ... · pdf fileas regulatory phenomena...

Respiratorypartitioningin yeasts

efficiency and metabolizeas regulatory phenomena

M. A. ALEXANDER and T. W. JEFFRIES

Institute for Microbial and Biochemical Technology, Forest Products Laboratory, †Madison, WI

Studies of metabolic regulation in yeasts have a long history. Yeasts have served as models for theregulation of fermentaive metabolism. Yeasts differ in their partitioning of metabolism betweenrespiration and fermentation, that is, between their use of oxygen and organic compounds as terminalelectron acceptors. The respirative route is assumed to yield more ATP. Many yeasts carry outmetabolism solely by the respirative route. Other show a predominantly fermentaive metabolism,even when oxygen is freely available. Fermentaion under aeobic conditions, that is, when respirationshould be possible, was believed to result from repression of respiration by fermentation. Thisphenomenon is known as the Crabtree effect. However, recent work has shown that aerobicfermentation results from an inherently-limited respiratory capacity of some yeasts, rather than from aspecific repression of respiration, Nevertheless, considerable enzymatic evidence exists that suggeststhat a Crabtree effect does indeed operate in some yeasts, specifically Saccharomyces cerevisiae.Because multiple electron transport systems are known to exist in yeasts, repression of the normalATP-producing system can be accompanied by the induction of an alternate pathway. No decrease inthe overall rate of oxygen utilization would then be apparent. Repression would, however, affect theyield of ATP from oxidative metabolism. This effect should be detectable using a suitable analysis ofgrowth energetics. To this end, a model has been developed and applied to a variety of yeasts in orderto examine them for changes in respiratory efficiency indicative of a Crabtree effect. A Crabtree effectconsistent with previous enzmatic findings was detected in S cerevisiae and S. uvarum, but not inSchizosaccharomyces prombe. New regulatory classifications based on model findings are proposedand methods for independently verifying these findings are outlined.

Keywords: Crabtree effect; alternate respiration; P:O; model; respiratory capacity

Regulation of sugar metabolism ingrowing yeastsEarly work: the Pasteur and Crabtree effects

During the era of classical biochemistry, the metabo-lism of sugars by yeasts was investigated and de-

† Maintained in cooperation with the University of Wisconsin-Madison.The use of trade, firm, or corporation names in this publication is forthe information and convenience of the reader. Such use does notconstitute an official endorsement or approval by the U. S, Depart-ment of Agriculture of any product or service to the exclusion ofothers which may be suitable.Dr. Alexander’s present address is Bioprocess Engineering (1450-89-1), The Upjohn Company, Kalamazoo, MIAddress reprint requests to Dr. Jeffries at the Institute for Micro-bial and Biochemical Technology, Forest ProductsMadison, WI 53705, USAReceived 24 January 1989; revised 15 February 1989

1990 Butterwotth Publishers

scribed in terms of regulatory effects of sugar andoxygen. One of these effects is named after LouisPasteur, who, more than a century ago, observed thatfermentation by Brewers’ yeast (Saccharomyces cere-visiae) decreased in the presence of oxygen. 1 A secondeffect, essentially antithetical to the Pasteur effect, isnamed after Herbert Crabtree, who noted in 1929 that“the glycolytic activity of tumor cells exerts achecking effect on their respiration.”2

Although the observations of Pasteur were madewith yeasts, while those of Crabtree were made withmammalian tumor cells, the metabolism of glucose bygrowing yeast is more consistent with a Crabtree effectthan a Pasteur effect. In 1948, Swanson and Cliftonfirst demonstrated that glucose utilization by growingS. cerevisiae in aerated cultures proceeded almostcompletely via fermentation.3 Fermentative assimi-lation dominated over aerobic oxidation as long as

Enzyme Microb. Technol., 1990, vol. 12, January 2

Regulatory phenomena in yeasts: M. A. Alexander and T. W. Jeffries

there was an appreciable amount of glucose present.This phenomenon of “aerobic fermentation” by grow-ing yeasts is contrary to the Pasteur effect (inhibitionof fermentation by respiration) but consistent with theCrabtree effect (inhibition of respiration by fermen-tation).

A mechanism for the Pasteur effect in terms ofallosteric effects of ATP and citrate on phosphofructo-kinase (PFK) has been advanced by SolS.4 The citricacid cycle is feedback controlled at the level of isoci-trate dehydrogenase, the activity of which is adjustedto the ATP requirements of the cell by its allostericdependence on AMP. Increasing the rate of oxygendelivery will produce an increase in the energy charge,resulting in a decrease in citric acid cycle activity andan increase in citrate levels. Increased levels of citrateallosterically inhibit PFK, restricting glycolytic activ-ity. 4 Note that the operation of the Pasteur effectrequires that the ATP requirements of the cell (i.e.growth) do not increase with the introduction ofoxygen. If they increased by a sufficient amount, nochange in the energy charge would occur and the citricacid cycle activity and citrate levels would be unaf-fected. Thus, the Pasteur effect is expected tobe most pronounced in nongrowing cultures. In addi-tion, PFK is stimulated by the prescnce of ammonium.which is present under growing conditions.4 Indeed,most observations of the Pasteur efffect have typicallybeen made with nongrowing Cells.5 Experimentalassessment of the Pasteur effect in growing cultureshas shown it to be less pronounced6or totally ab-s e n t .5,7

It is the Crabtree effect and not the Pasteur effectthat appears to be the relevant regulatory phenomenonfor growing yeast. Growing yeast are, moreover, ofgreater commercial interest than resting yeast. DeDeken 8 examined glucose metabolism during aerobicgrowth of 25 yeasts to determine if excess CO2 forma-tion occurred relative to oxygen utilization. ExcessCO2 (i.e. aerobic fermentation) indicated operation ofa Crabtree effect, which was defined as a repression ofone energy source (respiration) by another (fermenta-tion), and was found in about half of the yeasts tested.8

Thus, the occurrence of aerobic fermentation wasconsidered equivalent to the existence of repression ofrespiration by glycolysis.

There are actually two definitions of the Crabtreeeffect used implicitly by De Deken and subsequentworkers. One is purely phenomenological: “ethanolproduction under fully aerobic conditions;” the otheris mechanistic: “the repression of respiration by gly -colysis.” These two definitions can become blurred inpractice, leading to circular reasoning. For example, ayeast is first identified as Crabtree positive by itsdisplay of aerobic fermentation. Later, it is assumedthat respiratory repression occurs in this yeast simplybecause it is Crabtree positive,

For the present, we will use the occurrence ofethanol production under fully aerobic conditions toclassify yeasts as “acrobic-fermenting” (AF) yeasts.By fully aerobic conditions it is meant that the dis-

solved oxygen tension (DOT) is above the criticaloxygen concentration everywhere in the fermentationvessel. The critical oxygen concentration is that con-centration at which the oxygen uptake system of thecell is saturated. When the DOT exceeds the criticalconcentration, uptake is independent of DOT. Thoseyeasts that do not show ethanol production under fullyaerobic conditions will be termed “aerobic-respiring”(AR) yeasts. Later, a more satisfactory classificationwill be proposed. The term “Crabtree effect” will bereserved exclusively for the repression of respiration.For a yeast to show repression of respiration (i.e.a Crabtree effect), it is necessary (but not sufficient)that this yeast show aerobic fermentation. Thus, allCrabtree-positive yeasts are also AF yeasts, but thereverse is not necessarily true. One of the objectives ofthis paper will be to identify AF yeasts that also showa Crabtree effect. We do not consider strictly respira-tive yeasts, that is yeasts that do not exhibit fermenta-tion under any circumstances.

Relation of the Crabtree effect with thepetite mutation and anaerobic growth

In a companion paper, De Deken identified a cor-relation between aerobic fermentation (which waspresumed to reflect a Crabtree effect) and the occur-rence of the petite mutation. 9 The petite mutation wasfirst described in 1949 and involves a loss of capacityfor respiration.10,11 By growing S. cerevisiae in thepresence of acriflavin, yeasts are obtained that lackseveral components of the respiratory chain12 and socarry out little respiration. Upon plating onto acrifla-vin-free plates, the acriflavin-treated cells give rise todwarf colonies (petites) which retain respiratory defi-ciency over the course of vegetative growth. 10-l2 Thepetite mutation involves an extrachromosomal fac-tor13,14 and is specifically induced15 by acridines, suchas euflavin or acriflavin, that inhibit the synthesis ofrespiratory enzymes.12

De Deken treated with euflavin the same 25 yeastsstudied in his other paper. With some of the yeasts.variants were obtained that were unable to grow onglycerol and so were considered respiratorily defi-cient. These yeasts were considered petite - positive.With two exceptions, petite - positive yeasts were alsoAF yeasts.9

Bulder 16 noted that the failure of some yeasts togive rise to petite mutants did not result from thefailure of the respiratory inhibitor to produce themutation. but rather from the lethality of the petitemutation. Plating acriflavin-treated cells onto acrifla-vin-free plates gave rise to microcolonies that werepresumably composed of petite mutants that had failedto grow beyond a few generations. Apparently, theloss of respiratory competence was lethal in theseyeasts. A correlation between the ability of the parentstrain to grow under anaerobic conditions and theoccurrence of the petite mutation was noted, implyingthat the petite mutation is lethal for those yeasts


Review

unable to grow in the absence of oxygen (i.e. thatrequire respiration for growth).

Comparisons of the ability of yeasts to grow anaero-bically are complicated by the requirement for certaingrowth factors in the absence of oxygen. Cochin, apupil of Pasteur, first demonstrated that malt extractdoes not support batch growth of S. cerevisiae afterrepeated serial transfers under strictly anaerobic con-ditions, whereas a medium containing yeast extractdoes. 17 Andreasen and Stier18,19 identified ergosteroland unsaturated fatty acids as the factors required forextended anaerobic growth. Certain steps in the bio-synthesis of these compounds are mediated by cyto-chrome P-450 and involve molecular oxygen.20 Thus,these compounds cannot be formed under strictanaerobic conditions.

Unlike S. cerevisiae, the failure of some yeasts togrow without oxygen is not due to nutritional factors.Even when placed in a glucose medium containing anextract of aerobically-grown Candida utilis, this ARyeast fails to grow more than two or three generationsin the absence of oxygen. 17 The same thing is observedfor a number of other AR yeasts: Candida shehatae,Pichia stipitis, Candida tenuis,, Pichia segobiensis,and Pachysolen tannophilus, pointing to a correlationbetween the AR classification and the absence ofanaerobic growth.17 These yeasts ferment anaero-bically, 17’21-24 so their failure to grow probably doesnot result from a lack of ATP.

Inability to grow in the absence of oxygen is furtherevidenced by the failure of P. tannophilus to incorpo-rate radiolabeled glucose or xylose into trichloroaceticacid-insoluble cellular material, even though both sug-ars are utilized for ethanol production under suchconditions .24 The absolute requirement for oxygentypical of AR yeasts is well demonstrated by theobservation that shifting a continuous culture of C.shehatae from oxygen-limited growth to anaerobicconditions results in the immediate cessation ofgrowth, although fermentation continues unabated.25

Thus far, a correlation between the AF classi-fication (which presumably reflects respiratory re-pression), the petite mutation, and the ability to growanoxically has been described. This correlation can beexplained in terms of the varying importance of respi-ration in the metabolism of different yeasts. For AFyeasts, such as S. cerevisiae, respiration seems to beoptional; they can grow in the absence of oxygen,produce viable mutants that lack respiratory ability,and presumably repress their respiration in favor offermentation when in the presence of large amounts ofa fermentable sugar. Aerobic respiring yeasts, such asC. utilis, appear to have a fundamental requirement forrespiration: they do not grow in the absence of oxy-gen, do not produce viable respiration-deficient mu-tants, and do not repress their respiration.

This simple picture is disrupted by the observationthat the AF yeast Schizosaccharomyces pombe doesnot grow under anaerobic conditions,26 nor does it giverise to petite mutants. 27 De Deken had classified thisyeast as petite - positive, but did not characterize the

mutants further. In a later, more thorough study,Heslot and co-workers clearly demonstrated that S.pombe is petite- negative. 27 Thus, S. pombe resemblesthe AR yeasts in its requirement of oxygen for growth.The picture can be restored by asserting that it isrespiratory repression (i.e. the Crabtree effect), andnot aerobic fermentation, that is correlated with thepetite mutation and anaerobic growth. This conceptimplies that repression of respiration does not occur inS. pombe, but does occur in S. cerevisiae. Testing thisidea would require detection in S. cerevisiae, but notS. pombe, of some characteristic that is more closelyassociated with respiratory repression than is aerobicfermentation.

Enzymatic and further physiologicalevidence for respiratory repression in yeast

The observation of aerobic fermentation per se doesnot necessarily require that repression of respirationactually occurs. The existence of repression is sup-ported by numerous observations of decreased levelsof respiratory enzymes in fermentative or “glucose-repressed” cells, versus those in depressed cells.6

Most of these studies have been hampered by experi-mental difficulties arising from reliance on batch cul-ture techniques.17 For example, cells from batchgrowth on high glucose concentrations are considered“glucose-repressed, ” while those grown on low con-centrations are “derepressed.” Batch growth of S.cerevisiae on glucose is diauxic: ethanol production byaerobic fermentation occurs until glucose is ex-hausted, after which the accumulated ethanol is me-tabolized. 28 Thus, a comparison of cells harvested atthe same time after growth on high versus low initialconcentrations of glucose is really a comparison ofglucose-fermenting versus ethanol-oxidizing cells.17

Apparent differences between the cells may simplyreflect the different substrates and not a specific effectof repression.

It was not until continuous culture techniques wereused that a satisfactory analysis of the nature of theCrabtree effect could be made. When S. cerevisiae isgrown on glucose in continuous culture, two distinctmetabolic regimes are seen.29 At low dilution rates,metabolism is completely respirative: cell yields areclose to 0.5 g g-1 and no ethanol is formed. When thedilution rate is raised beyond a critical value (DCRIT),respirofermentative metabolism is observed. Such me-tabolism is characterized by lower growth yields andethanol formation. With continuous culture, cellsshowing both respirative (derepressed) and respirofer-mentative (repressed) metabolism can be obtained onthe same substrate.

In his pioneering study of glucose metabolism by S.cerevisiae H1022 in continuous culture, von Meyen-berg29 noted that the specific rate of oxygen utilization(Qo2) increased linearly with dilution rate, reaching amaximum value at DCRIT. Qo2 declined with increasingdilution rate for D > DCRIT and ethanol production

4 Enzyme Microb. Technol., 1990, vol. 12, January


increased. 29 This decline in Qo2 associated with theonset of ethanol production was-taken as evidence forrepression of respiration. A similar decrease in Qo2

was subsequently observed for another strain of S.cerevisiae also.30 Beck and von Meyenberg28 mea-sured the titers of malate dehydrogenase and NADH-linked glutamate dehydrogenase as a function of dilu-tion rate. They found a sharp decrease in the levels ofboth enzymes that was associated with the transitionfrom respirative to respirofermentative metabolism atDCRIT. Similar observations have been made for TCAcycle and respiratory chain enzymes.6 In aggregate,these observations provide substantial evidence forrepression of respiration in aerobic fermentation (atleast for S. cerevisiae).

Is repression of respiration a relevantfactor in yeast metabolism?

Aerobic fermentation is the result ofrespiratory limit at ion, not repression

A decade after von Meyenberg’s study, Barford andHall3l reported the results of continuous culture exper-iments with S. cerevisiae UNSW 703100, in whichthey found no evidence for respiratory repression.Like von Meyenberg, they observed increasing Qo2 upto DCRIT. For dilution rates above DCRIT, however,Qo2 remained constant at the value attained at DCRIT

. Identical behavior was obtained with glucoseor galactose as carbon source.

The cultures used by Barford and Hall had beenadapted to respirofermentative metabolism. For glu-cose, this was accomplished by prolonged culture at adilution rate of 0.45 h -1

, which is equal to the maxi-mum specific growth rate (µ MAX) of the unadaptedyeast on glucose. Adaptation was accompanied by anincrease in µ MAX from 0.45 to 0.60 h-1. For othersugars and ethanol, adaptation could be achieved byserial batch cultivation on the desired substrate, andwas usually accompanied by an increase in µ MAX of0.14–0. 15 h-1. The adapted cultures displayed eitherconstant Qo2 in batch culture, or constant Qo2 for D >DCRIT, in continuous culture, leading Barford and Hallto suggest that repression did not occur in yeastsadapted to a fully respirative condition.

In a subsequent paper,32 Barford and co-workerstested this idea by evaluating the eftcets of adaptionon the Qo2 profile in continuous culture using S .cerevisiae H1022 (the same strain used by von Mey-enberg). Above DCRIT, Qo 2 remained constant at

for this yeast also. In a third paper.33 Barfordand Hall advanced the idea that the respiratory capac-ity of S. cerevisiae may become saturated and exhibita maximum specific oxygen uptake rate after suitableadaptation. In this case, aerobic fermentation occurs,not because respiration is repressed, but because eventhe fully-developed respiratory capacity of S. cere-visiae is too small to utilize oxidatively all the sugartaken up.

Glucose metabolism by S. cerevisae H1022 wasevaluated yet again by Rieger and co-workers in1983. 34 They found invariant Qo2 values for D > DCRIT,

just as did Barford and co-workers. Furthermore, theyobtained evidence that a nutritional limitation in themedium used by von Meyenberg may have producedhis findings of decreased Qo2.

34 Constant Qo2: values forD > DCRIT have also been observed with Saccharomy-ces uvarum35 and S. pombe. 36 These results and theanalysis of the results of shifts in dilution rate35,37 andco-metabolism of glucose-ethanol mixtures34,38 haveestablished that aerobic fermentation is a consequenceof the limited respiratory capacity of AF yeasts.39-41

Having a mechanistic explanation for aerobic fermen-tation, we can now refer to AF yeasts as respiration-limited and to AR yeasts as respiration-unlimited.

With the adoption of the new concept of respiratorylimitation, the old concept of respiratory repressionhas been largely discarded as an important factor insugar metabolism by yeasts. Repression certainly oc-curs. When a continuous culture of S. uvarum isshifted from a dilution rate lower than DCRIT to onehigher, decreases in titers of malate dehydrogenaseand cytochromes a, b, and c occur over 16–24 h, yetQo2 does not change.35,37 The lack of correlation be-tween Qo2, and the titers of respiratory enzymes sug-gests that allthough repression occurs, it is not relevantto the observed metabolic behavior. Käppeli37 hassuggested that changes in titers of respiratory enzymesreflect adaptation to the new respirofermentativephysiological state caused by saturation of the limitedrespiratory capacity of Saccharomyces -type yeasts.Conversely, Barford and Hall31-33 suggest that re-pression of respiration results from a failure to achievea complete adaptation to rcspirofermentative metab-olism.

The key observation that has dispelled respiratoryrepression as a relevant regulatory system is the lackof change in Qo2 associated with the onset of respi-rofermentative metabolism. For example, if repressionwere a relevant factor in S. uvarum, it should give riseto a decline in Qo2, corresponding to the decline inenzyme titers, yet none is observed. This argument isvalid provided that oxygen is utilized solely by thenormal, cytochrome~containing respiratory system. Ifan alternate respiratory system were present alongwith the normal one, this system could increase itsactivity in response to repression of the normal one.The result would be a decrease in the rate of normalrcspiration without a corresponding decrease in Qo2.Such a change should have detectable physiologicaland metabolic effects. This idea will be exploredfollowing a review of respiratory systems in yeasts.

Respiratory systems in yeasts

Normal respiration

The primary respiriation in yeasts is the cytochrome-containing electron transport chain, which consists of


Review

rotenone.45,46 In contrast, provided site I is active,

plants. 48 It occurs in protozoa and even in some loweranimals. 48 This common cyanide-insensitive repira-

four complexes located in the inner mitochondrialmembrane (Figure 1). Electron transfer between thesecomplexes is mediated by two mobile carriers: theubiquinone/ubiquinol redox couple (also called coen-zyme Q) and cytochrome c. The first complex trans-fers electrons from NADH to ubiquinone and is knownas the NADH–CoQ reductase complex. This transferis coupled to phosphorylation via proton transport atsite I and is inhibited by rotenone or piericidin. 42

The second complex is succinate dehydrogenase,which carries out the oxidation of succinate to fuma-rate, transferring the electrons to ubiquinone. It is notcoupled to phosphorylation. The third complex, CoQ–cytochrome c reductase, oxidizes ubiquinol back toubiquinone, passing the electrons to cytochrome c.Electron transfer is coupled to phosphorylation viaproton transport at site 11. The complex containscytochromes b and c1 in a 2:1 ratio.42 Electron trans-fer from cytochrome b to c1 and the reverse transfer toubiquinol is blocked by antimycin.43 The last complextransfers electrons from cytochrome c to oxygen andis called cytochrome c oxidase. Electron transfer iscoupled to phosphorylation via proton transport at siteIII and is blocked by cyanide or carbon monoxide. Thecytochrome c oxidase complex contains cytochromesa + a3 and a copper-containing protein. 42

In yeasts, fungi, and plants, but not in animals,

NADH dehydrogenases exist which are located on theouter surface of the inner mitochondrial membrane .44

These dehydrogenases can accept electrons fromNADH or NADPH45 located outside the mitochon-drion (Figure 1). Because NAD(H) does not permeatethe inner mitochondrial membrane, special mecha-nisms that shuttle NAD(H) across this membrane arenecessary in animal cells.42 Yeasts can oxidize cy-toplasmic NAD(P)H directly and so do not requirethem. Electrons accepted by these external dehy-drogenases are passed to ubiquinone, and so bypasssite I-driven phosphorylation. Thus, oxidation of ex-ogenous NAD(P)H by intact mitochondria proceedswith an apparent P : O of 2 and is insensitive to

oxidation of malate + pyruvate, which produces intra-mitochondrial NADH, proceeds with an apparent P: Oof 3 and is sensitive to rotenone. 45,46

Alternative forms of respiration

The existence of respiration that is resistant to cyanidehas been known in yeasts for a long time.47 Cyanide-insensitive respiration is widespread in other fungi and

tion is specifically inhibited by hydroxamates.49 An-other cyanide-insensitive respiration that is hydrox-amate-insensitive has been detected in the yeasts S.pombe, 50-52 S. cerevisiae, 53 Kluyveromyces lactis, 54

Hanseula saturnus, 55 and Endomycopsis capsularis .56

It is susceptible to high concentrations of azide,however. Thus, following Edwards,57 we will refer tothe first type of alternate respiration as hydroxamate-sensitive (HAS) and to the second as azide-sensitive(AZS).

HAS respiration branches off from the normalrespiratory chain at ubiquinone (Figure 1). 43,58,59 Elec-tron transfer between ubiquinone and oxygen via theHAS pathway does not generate a proton gradient andso does not drive ATP production .60-64 Because itbypasses sites II and III, HAS respiration is insensi-tive to either cyanide or antimycin. Electrons flowingthrough the HAS pathway do not produce ATP aftersite I proton translocation at ubiquinone and so pro-duce, at most, one ATP per electron pair (see Figure1).

AZS respiration is believed to be mitochondrial andnonelectrogenic. Succinate is oxidized by normal res-piration, but not by AZS respiration, whereas exoge-nous NAD(P)H is oxidized by both, 56 implying thatelectrons from NAD(P)H, but not succinate, haveaccess to AZS respiration. Apparently, the AZS path-way dots not communicate with the normal respira-tory chain at ubiquinone. The position of the AZSpathway relative to the others is not known at presentand so this pathway does not appear in Figure 1.

Occurrence of alternate respirations

The appearance of HAS respiratiton in the stationaryphase of batch culture is frequently observed. 62 Condi-



tions similar to stationary phase have been artificiallyproduced with Saccharomycopsis lipolytica by shak-ing dense suspensions of exponential-phase cells inmedium lacking nitrogen for 90 min, during which timean HAS respiration develops that roughly equals res-piration in the absence of cyanide .59 The developmentof alternate respiration in stationary phase may reflecta change in the purpose of respiration from energyproduction to redox-balance maintenance as growthdeclines.

Alternate respiratory systems often appear whenthe normal pathway is blocked or limited in some way.This has been accomplished by use of respiratoryinhibitors such as antimycin for both HAS and AZSrespiration. 54,64-66 Drugs such as chloramphenicol thatimpair mitochondrial protein synthesis have also beenused to obtain HAS respiration. 58,65-67

Sustained growth in the absence of an elementrequired for the synthesis of a key component of thenormal respiratory chain (e.g. copper) can serve toartificially “repress” normal respiration, permittingthe development of alternate respiration. Copper is anessential constituent of the cytochrome c oxidasecomplex. Growth of C. utilis in continuous culture ona copper-limited medium gives rise to a variant strainpossessing HAS in lieu of normal respiration.60 Thisstrain lacks cytochromes a + a3, which are com-ponents of the cytochrome c oxidase complex. Addi-tion of copper under nongrowing conditions does notrestore the activity of cytochromes a + a3, indicatingthat the cytochrome c complex is absent and notmerely inactive in the variant strain. Growth in cop-per-containing medium regenerates normal respirationand cytochromes a + a3, without loss of HAS respira-tion. Apparently, HAS becomes derepressed in re-sponse to the artificially-imposed repression of cyto-chrome c oxidase produced by copper deficiency.

Development of alternate respiration usually occursin response to limitation of growth or to repression/inhibition of normal respiration. For yeasts having alimited capacity for oxygen utilization, Qo2 cannotexceed its maximum value of Q(),CRr~. For such yeasts,a repression of normal respiration accompanied by thedevelopment of alternate respiration would result inthe replacement of normal with alternate respiration atconstant Qo2 This replacement would lead to a drop inthe ATP yield resulting from oxygen utilization andshould be detectable by examination of growth ener-getic. This idea is explored in the next section.

Effect of respiratory efficiency (P : O)on growth yieldsBy growing C. utilis in continuous culture undervarious limitations, mitochondria with different P : Oratios (ATP generated per oxygen atom) can be iso-lated. 68 Mitochondria isolated from cells grown underglycerol, ammonium or magnesium limitation possesssite I and have a P : O of three.69Those from cellsgrown under iron70 or sulfur71 limitation lack site I andhave a P : O of two. 69 AS described above, cultivation

with copper limitation gives rise to variant cells pos-sessing HAS, but lacking normal respiration. Site I isactive in the mitochondria from these cells, givingthem a P: O of one. 60 When cell yields from oxygen(Yo2) were calculated for glycerol-limited (P: O = 3),iron--limited (P: O = 2), and copper-limited variantcells (P: O = 1), respective values of 47.6, 30, and 15.8g cells (mol oxygen)-1 were obtained. 60,72 These valuesfall in a 3:1.9:1 ratio, almost exactly as expected fromthe corresponding P: O ratios. This correspondenceimplies that the growth yield from ATP (YATP) isunaffected by changing mitochondrial efficiency, al-lowing mitochondrial P: O to be inferred from ananalysis of growth energetic.

When the petite -negative yeast Endomyces mag-nusi is grown in the presence of antimycin, HASrespiration is observed.64 In the absence of antimycin,normal respiration is observed. The growth rate ofcells showing normal respiration is 2.6-fold greaterthan those showing HAS respiration, yet the rate ofoxygen uptake is 20% less.64 Thus, the Yo2 for growthemploying normal respiration is about three timesgreater than that employing HAS respiration.

A similar result is obtained with Sporobolomycesruberrimus. 73 Cultures grown in the presence of anti-mycin produce only about 70% of the biomass of thosegrown in the absence of antimycin, and they exhibitHAS respiration. Assessment of the respiration ratesof cells from both cultures shows that the antimycin-grown cells utilize oxygen at nearly twice the rate ofthe control. Once again, the Yo2 of growth mediated bynormal respiration is about three times greater thanthat mediated by HAS respiration.

The finding of a threefold greater Yo2, for growthusing normal versus HAS respiration suggests that theP: O of the former is 3, whereas that of the latter is 1.This inference assumes that YATP is unaffected by thechanges in respiratory P: O produced when respira-tory systems are interchanged. Provided site I ispresent, electron transport via the normal respiratorychain does indeed proceed with a P: O of 3, whereasthat proceeding by the HAS route proceeds with aP: O of 1 (Figure 1). The values inferred from growthenergetic correspond well with the correct values.Thus, growth energetic can be used to infer changesin the relative amounts of different respirations.

Analysis of growth energetic can giveevidence for respiratory repression

An analysis of growth energetic can indicate changesin respiratory efficiency, from which changes in therelative amounts of normal versus alternate respirationcan be inferred. Specifically, by analyzing the ener-getic associated with respirofermentative metabolismby respiration-limited yeasts, we can infer whethernormal respiration remains unchanged or is beingreplaced by an alternate form. In the latter case, arepression of (normal) respiration (i.e. a Crabtreeeffect) would be identified.


Review

In this paper, we develop a model that can beapplied to a variety of yeasts, both respiration-limitedand respiration-unlimited. The model is based on that

39 which explicitly uses theof Sonnleitner and Käppeh,concept of limited respiratory capacity to account foraerobic fermentation. Respirative and fermentativemetabolism are considered as a pair of parallel reac-tions. The former is favored over the latter, reflectingthe lower Km for pyruvate of pyruvate dehydrogenase

74 Respirative metabo-versus pyruvate decarboxylase.lism is limited to a maximum rate by the inherentlylimited respiratory capacity of S. cerevisise. Thus,metabolism proceeds solely by the respirative routeuntil its maximum rate is reached at DCRIT. This rate isequal to the specific rate of sugar utilization (Qs) atDCRIT (QsCRIT). For dilution rates greater than DCRIT, Qsexceeds QsCRIT and the excess is utilized via fermenta-tive metabolism.

This model is extended to respiration-unlimitedyeasts in this paper. A simple analytical format is usedthat is amenable to graphical analysis, eliminating theneed for numerical solution by computer. More impor-tantly, the key parameters of Sonnleitner and Käp-peli’s model are interpreted in terms of a respiratoryefficiency parameter (β) that can be thought of as theweighted average of the P: O values of all activerespiratory pathways. Thus, by employing a simplegraphical analysis of metabolic data from continuousculture, changes in respiratory efficiency can be in-ferred, even when Qo2 data are not available. Thisanalysis is then used to provide a new operationaldefinition for the Crabtree effect that is closely relatedto (normal) respiratory repression.

The ModelIn the following section, expressions are developedrelating the specific rates of sugar utilization, oxygenutilization, and ethanol production to the specificgrowth rate. The specific growth rate (µ) was chosenas the independent variable because it is equal todilution rate (D) in continuous culture at steady stateand so is under experimental control. When discussingsteady-state continuous cultures, the parameters µand D will be used interchangeably.

Conceptual ideas on which the modelis based: metabolic stoichiometry

We start with the concept formulated by Sonnleitner39 Metabolism is considered as a pair ofand Käppeli.

parallel processes: respirative and fermentative me-tabolism:

YsR and YsF refer to the yield (g g -1) of biomass fromsugar for respirative and fermentative metabolism,respectively. YE refers to the effective yield of ethanol

from sugar in g g-1, which is not necessarily equal tothe theoretical yield coefficient (Ψ E) of 0.51 g g-1.Unlike Sonnleitner and Käppeli, an explicit stoichio-metric parameter for oxygen is not employed in equa-tion (1). Rather, the quantity of oxygen used tometabolize 1 g of sugar via respirative metabolism isdetermined by energetic, which will be discussedlater. The rates of respirative metabolism (equation 1)and fermentative metabolism (equation 2) are denotedby QR and QF. Their units are expressed as specificrates: g sugar (g dry wt)-1 h -1.

We can write an expression for the overall specificgrowth rate by summing the contributions from equa-tions (1) and (2):

We define µR and µF as the specific rates of biomassproduction (i.e. growth) resulting from respirative andfermentative metabolism. The overall specific rate ofsugar utilization is obtained by a similar sum:

Metabolic energetic

We use an energetic analysis similar to that used byBarford and Hall,33 except that it is expressed indifferential form. Growth is assumed to be energylimited. That is, a differential increase in µ requires aproportional differential increase in the specific rate ofATP production (QATP):

The proportionality coefficient (YATP) is the yield ofbiomass from ATP in g mol-1. YATP is assumed con-stant in the model because YATP and β cannot bedetermined independently for respirative metabolismand the purpose of the model is to detect changes in β.We have already seen that the assumption of constantYATP allowed correct inferences to be made concern-ing relative growth yields with C. utilis, E. nzagnusi,and S. ruberrimus having variable respiratory effi-ciencies.

ATP production occurs as a result of respirationand glycolysis. Each gram of sugar consumed isassumed to undergo glycolysis with the concomitantproduction of 1/90 mole of ATP. That is, all carbonassimilated into biomass is assumed to be derived fromglycolytic pyruvate. Each mole of oxygen consumed isassumed to give, on average, 2 β moles of ATP. Asmentioned earlier, β is a parameter reflecting respira-tory efficiency and represents the effective P: O forrespiration as a whole. It will be treated as a functionof µ. Thus, QATP is given by

Equation (6) is clearly an oversimplification. Allsugar utilized does not undergo glycolysis; a fractionbypasses glycolysis to make anabolic precursors andNADPH via the pentose shunt. The effect of thissimplification will be to overestimate glycolytic ATP



production, thus underestimating respiratory ATPproduction and respiratory efficiency (i.e. β). Theeffect on β will be small, however.

Differentiation of equation (6) and substitution intoequation (5) yields a differential relation betweensubstrate uptake and growth rate in terms of energeticparameters:

An expression for β in terms of QO2, QS, and µ can beobtained by integrating equation (7) subject to thefollowing condition:

following rearrangement:

Equation (9) is valid for both respirative and respi-rofermentative metabolism. It is useful for analyzingmetabolic rates in terms of changing respiratory effi-ciency.

Metabolic kinetics

Sugar uptake is assumed to follow saturation kinetics:

Here QSMAXis the maximum value of QS and KS is the

half saturation constant (in g 1 -1). Both of theseparameters are assumed to be constant under allCultural conditions.

Development of the model

The concepts presented in the previous section will beused to develop expressions relating QS, Qo2 and QE.to µ for both respirative and respirofermentative me-tabolism of both respiration-limited and respiration-unlimited yeasts.

1. Respirative metabolism of both respiration-limitedand respiration-unlimited yeasts. U rider fully aerobicconditions, purely respirative metabolism occurs at allvalues of D with respiration-unlimited yeasts, but onlyfor D < DCRIT with respiration-limited yeasts. Forpurely respirative metabolism, µF and QF are zero andequations (3) and (4) become

Differentiation of equation (12) and substitution intoequation (7) yields, upon rearrangement:

When YsR is a constant, QS will be linearly related topby equation (12), in which case we can write:

For constant β , equation (14) gives equal to aconstant, implying a linear relation between QO2, and µ.

From this it follows that if both QS and Qo2 are linearfunctions of µ , then β is constant. For this case(constant β and YsR) equation (13) can be integratedusing the following initial condition:

Here Qo,,, is simply the intercept of the linear relationbetween QO2 and µ that is seen when both β and YsR

MYO2 can be interpreted as the marginalare constants.yield of biomass from oxygen in respirative metabo-lism, that is, the additional quantity of biomass (ingrams) produced by the input of an additional quantityof oxygen (in moles). The relation of MYO2 to β is givenby

In practice, MYO2 is obtained from the reciprocal of theslope of a plot of experimental QO2 values versus wasshown in Figure 2. YsR is obtained-as the reciprocal ofthe slope of a plot of Qs versus µ for µ < µ CRIT. GivenMYO2 and YsR, β is obtained from equation (16). QSCRIT

and Q(),,) are identified as shown in Figure 2.

2. Respirofermentative metabolism by respiration-limited yeasts. Respirative metabolism gives way torespirofermentative metabolism at µ CRIT for respira-tion-limited yeasts. This occurs because these yeastshave a limited respiratory capacity characterized by amaximum oxygen utilization rate equal to QO2CRIT.39Thus, the maximum values of QR and µR are reached atµCRIT and so are equal to QSCRIT and µCRIT. With thisequations (3) and (4) yield, upon rearrangement:

An alternate expression for QS can be obtained forµ > µ CRIT by substituting QO2CRIT for QO2 in equation (7):

Figure 2 Graphical determination of model parameters for arespiration-limited yeast. MYo2, YsR, and YsF are determined fromthe slopes of plots of Qo2 and Qs versus µ as shown in the figure.Respirative metabolism of respiration-unlimited yeasts is ana-lyzed in the same way as that of respiration-limited yeasts.


Review

When the product of β and Qo2CRIT is constant,the second term on the right-hand side of equation (19)vanishes and equation (19) can be integrated subject tothe initial condition:

Comparison of equation (20) with equation (18) showsthat YsF is equal to YATP/90), provided the product of βand Qo,c,l, does not change with µ. The assumption ofinvariant f3Q0,,RI, implies that ATP production byrespiration is unaffected by fermentative metabolism,that is, no repression of respiration by fermentationoccurs. When this is true, YsF can be interpreted as theactual cell yield resulting from fermentative metabo-lism. In anaerobic metabolism, respiration efficiencycannot affect the cell yield and YsFis necessarily equalto YATP/90. This result forms the basis for directexperimental assessment of YATP values. Only foranaerobic conditions can the ATP produced by sugarcatabolism be known with any certainty. Thus, YATP

values can be obtained only for anaerobic growth, andare typically close to 10 g mol -1 for a wide range ofsubstrates and microbes. 75,76 We will use this valuethroughout this paper.

If normal respiration were repressed, the productBQo,,.R,T would decrease and YsF would not be equal toYATP/90. In particular, YsF would not have the samevalue as the anaerobic cell yield; it would be lowerbecause fermentation-derived ATP must be used tomake up for the loss in respiration-derived ATPcaused by repression.

From equation (18) it is evident that

An expression for QE can be obtained from equa-tions (2) and (21):

YE must be less than its theoretical value Ψ E of 0.51g g-1 for respiration-limited yeasts because some ofthe sugar metabolized fermentatively is assimilatedinto biomass and so is unavailable for fermenta-tion. As a first approximation, each gram of bio-mass formed represents one gram of sugar lostto fermentation, in which case we can write:

Equation (23) is reasonably accurate for small YSF.

3. Respirofermentative metabolism of respiration-unlimited yeasts. Respiration-unlimited yeasts have norespiratory limitations and so display respirative me-tabolism for all dilution rates under fully aerobicconditions. Respirofermentative metabolism is ob-served only when the supply of oxygen is limitedexternally. This class of yeasts is also different fromrespiration-limited yeasts in that they do not exhibitgrowth as a consequence of fermentative metabolism.That is, µF is always zero and ~R = µ under allconditions. With this, equation (4) gives:

Under semiaerobic conditions, growth in contin-uous culture is oxygen-limited and high concentrationsof sugar appear in the chemostat.25 Thus, S > KS

in equation (10) and QS is equal to its maximum valueQSMAX for all µ. Substituting QSMAX for QS in equa-tion (24) and solving for QF gives:

Since fermentative growth does not occur withrespiration-unlimited yeasts, YSF is zero and YE. isequal to Ψ E by equation (23). Thus, with equations (2)and (25) we have

Recall that for respirative metabolism, QS = IJYsR,from which we obtain QSMAX = µ MAX/YSR. Substitutionof this result into equation (26) gives:

Since YSR, values are typically close to 0.56,1725 and Ψ E

is equal to 0.51 g g-1, the first term in equation (27) isapproximately equal to unity and we have

Substitution of QS = Qs,{~Y into equation (7) gives theenergetic that applies to oxygen-limited growth:

The subscript OL refers to oxygen-limited conditions.A proportional relation between growth and oxygenuptake is often observed for low-to-moderate oxygen-limited growth rates,25,77 in which case equation (29)can be integrated to give:

Here OLYO 2 (g mol-1) is the yield of biomass fromoxygen for oxygen-limited growth.

A graphical interpretation of respirofermentativemetabolism of respiration-unlimited yeasts in terms ofequations (28) and (30) is shown in Figure 3. Values forQsM.X and ,OLYO2 are found as shown in the figuure.

Figure 3 Graphical determination of model parameters forsemiaerobic metabolism of a respiration-unlimited yeast



Application of the modelParameter evaluation

Figure 4 shows plots of QS and Qo2 data versus µ fromcontinuous culture of S. cereuisiae H1022 on glu-cose.34,39 The experimentally observed values of µ CRIT

and µ MAx are 0.3 h-1 and 0.45 h - 1, respectively.Clearly, the graphical interpretation shown in Figure 2was valid for this yeast. A value for Qs~,X of 3.5 g g-1

h -1 was given by Sonnleitner and Kappeli39 and ap-pears in Table 1. Other parameter values were ob-tained graphically as shown in Figure 2 and appear inTable f. This procedure was applied to data from a

variety of respiration-limited yeasts in Figures 5–8.The results appear in Table 1.

The data for S. pombe ATCC 26189 were in-complete: respirative metabolism was not studied(Figure 7). Thus, a value for Q020 could not be obtainedand it was assumed to be zero. Reliable values for Qo2

were not obtained at the higher dilution rates becauseof the very low cell densities present.8 In experimentswith mixed sugars, in which cell densities were higher,Qo, was constant and independent of D. When mea-sured in cultures having sufficiently high cell densities,Qo, values were largely independent of carbon sourceand were consistent with an average value of 3.7 ± 0.3


Review

mmol g-1 h -1, which was used for Q(~,, ,,1 in Table 1.Metabolism of S. cerevisiae UNSW 703100 on glucose(Figure 8) was not everywhere consistent with Figure2. For µ > µ CRIT and µ ~ 0.15 h-1 the data wereconsistent and could be analyzed to give the parame-ters in Table 1.

Figure 9 shows data for C. shehatae on xyloseunder fully aerobic conditions. 25 Both relations were

clearly linear and were analyzed as shown in Figure 2for µ < µ CRIT. The parameters obtained appear inTable 2. Data for Candida utilis 17and Candida purap-silosis 30 under fully aerobic conditions were analyzedin the same way with the results shown in Table 2.

Figure 10 shows data for continuous culture of C.shehatae on xylose under oxygen limitation .25 Thedata were consistent with Figure 3 and the parameters



obtained are shown in Table 2. A OLYo, of 35 g mol - 1

for oxygen-limited batch culture of Candida sp XF217has been reported77 from which β OL of 1.75 wasobtained (see footnote 2, Table 2).

interpretation of metabolism in terms of βFigures 4 through 10 also show β calculated as afunction of µ using equation (9). Examination of thevariation of β with µ for respiration-limited yeastsreveals two general trends. In the Saccharomycesyeasts (Figures 4, 6, and 8), β declines by about oneunit as µ increases from zero to µ MAX , whereas βremains approximately constant for S. pombe (Figures5 and 7).

For S. cerevisiae UNSW 703100 (Figure 8), thedrop in β occurs for µ < µ CRIT. This drop in β occursbecause of the increase in the slope of the relationbetween Q02 and µ for µ >0.15 h-1. The change in βwas calculated assuming a constant YATP of 10 g mol-1.Alternatively, a change in YATP at constant β couldalso account for the increase in slope. These two waysof interpreting Figure 8 are not equivalent. This can beseen by comparing the overall and marginal values of βand YATP as shown in Table 3. The marginal value of βrefers to the (small) increase in ATP production result-ing from a (small) increase in oxygen uptake. Simi-larly, the marginal YATP refers to an increase in µresulting from an increase in QATP. The marginalvalues of β suggest that the decline in β overall withincreasing µ is consistent with two parallel respiratorysystems, one having a β of about 2 and the otherhaving a much lower value. Similarly, the marginalYATP suggests that variable overall YATP results fromthe operation of three parallel growth processes, twohaving YATP values of about 10 and one having a muchlower value.

The metabolic patterns displayed by S. cerevisiaeUNSW 703100 in Figure 8 are consistent with eithermultiple parallel growth processes having differentYATP values or multiple respiratory systems havingdifferent β (i.e. P: O) values. The latter hypothesis isthe more plausible, because multiple respiratory path-ways having the necessary characteristics are known

to exist. In contrast, there are no known mechanismsconsistent with multiple growth processes with differ-ent YATP values. In order for YATP to vary, the amountof ATP required for protein, nucleic acid, lipid, orcarbohydrate synthesis would have to change. Allbiochemical evidence suggests that the ATP require-ments are fixed characteristics of the pathways. Thus,we will assume that the variable respiratory systemhypothesis is correct.

For S. cerevisiae H1022 and S. uvarum (Figures 4and 6), a drop in β occurs for µ greater than µ cRIT.Recall from the model development that when theproduct of β and Qo2 is not constant for µ > µ CRIT, YsF

is not equal to the anaerobic cell yield. For example,the cell yield observed for anaerobic glucose culture ofS. cerevisiae HI022 is 0.10 g g-1.80 The glycerol-corrected yield (YsF) is 0.11 g g-1. This correction isrequired because glycerol-forming metabolism, whichis necessary for maintenance of the redox balance, 17,81

does not contribute to ATP production. Thus, theobserved anaerobic cell yield is consistent with thepredicted value of YATP/90 for a YATP of 10 g mol-1.YsF for this same yeast in aerobic culture is 0.052(Table 1). This low value is a consequence of the lossof respiratory efficiency manifest by the decrease in βshown in Figure 4. Because of the declining β, fermen-


Review

tation-derived ATP must be used to make up for theloss of respiration-derived ATP. As a result, less ATPis available for the formation of new biomass, and theapparent yield of biomass from fermentative metabo-lism (YsF) is lower. It must be stressed that the truefermentative yield is unaffected by declining respira-tory efficiency. It remains equal to its anaerobic valueof YATP/90. In general, the model parameter YsF willbe equal to the actual fermentative cell yield onlywhen respiratory deficiency is constant. When it isnot, YsF is only an apparent yield and has no simpleenergetic interpretation.

A declining value of /3Q0,C,,, will necessarily givea value of YsF lower than YATP/90, which for a YATP

of 10, is equal to 0.11. Changes in β reflect changes inthe relative rates of normal versus alternate respira-tion. For constant QO,C,,,, a decrease in β implies adecrease in the rate (i.e. repression) of normal respira-tion (NQO,). A decrease in Qo,,,,, at constant β likewiseimplies a- decrease in ‘Qo,. Thus, YsF values lowerthan 0.11 may be used as an indicator of repression ofnormal respiration. This idea is exploited in Table 4, inwhich YsF values for a number of yeasts appear. TheYsF values for S. cerevisiae (except UNSW 703100)and S. uvarum were significantly smaller than 0.11,indicating that repression occurs in these yeasts. Thevalues for S. pombe were reasonably close to 0.11,indicating that repression does not occur in theseyeasts. The assignment of S. pombe to the Crabtree-negative category is in accordance with its character-ization as petite-negative27 and incapable of anaerobicgrowth .26

Also shown are values for two Brettanomyces spe-cies. For B. lambicus, no definite DCRIT was observedand the cell yield was approximately constant for all

dilution rates.83 For B. intermedius, a definite DCRIT

was observed, allowing the calculation of a YsF of0.12 ± 0.04. Based on this value, this yeast is tenta-tively assigned to the Crabtree-negative category.

Instead of inferring repression of normal respirationfrom changes in β, ‘Qo, can be calculated as a functionof Qs, µ, and a reference β value (βΝ) using equation(8):

β N is calculated using equation (9) for a referencecondition in which Qo, is assumed to be composed of100% normal respiration. ‘Qo, calculated by equation(31) can be compared with enzymatic and other data toprovide evidence for the reality of repression of nor-mal respiration. Examples of the application of equa-tion (31) follow.

Figure (11) shows Qc), and titers of several respi-ratory enzymes for S. uvarum before and after ashift in dilution rate from 0.14 h-1 (< µ CRIT) to 0.21 h-1

(> µ CRIT).37 Also shown are biomass and ethanol

concentrations. Prior to the shift, Qo2 was assumedto be 100% normal respiration, allowing a value for β Nof 1.37 to be obtained from equation (9) (values for ~[)and Q020 of 1.5 and 0.9 were obtained for S. uvarumfrom Table 1). This value of ~N was then used inequation (31) to calculate the effects of the shift on‘Qo,. Both the cytochrome titers and ‘Qo, declined toabout half their values before the shift. Malate dehy-drogenase fell even further. This correspondence be-tween declining enzymatic titers and ‘Qo, stronglysuggests that (normal) respiratory repression occurredas a result of this shift, implying that a Crabtree effectoperates in S. uvarum.

Figure 12 shows Qo, data for continuous culture of



S. cereuisiae H1022 from three researchers, vonMeyenberg 29 and the groups of Barford32 and Rieger.34

The data of each are consistent with different inter-cepts (Qo,,, and so the intercept-corrected rate (Qo, –Q020) was plotted. The respirative data for the Barfordand Rieger groups fall on the same line; thus the slope(1/MYo2) and, hence, β (by equation 16) is the same forboth. Here we see the generality of β among the dataof different workers, which is indicative of its validityas a fundamental concept.

The data of von Meyenberg are clearly differentfrom the other two. Energetic analysis shows that therespiratory efficiency for D < DCRIT was low ( β = 1.3,Table 1) compared with the others ( β = 1.85). Thesuspected nutritional limitation in the medium used byvon Meyenberg appears to have produced a lessefficient respirative metabolism. β increased with dilu-tion rate for respirofermentative metabolism (D >D CRIT), however, rising to 2.1 at µ MAX (Table 1).Apparently, the QCJ, measured by von Meyenberg forlarge dilution rates was composed solely of normalrespiration. ‘Qo, was calculated with equation (31)(BN = 1.85) using the data of Rieger et al. and wascompared with the Qo, of von Meyenberg (Figure 12).The normal respiratory component (NQ02 ) of the over-all respiration observed by Rieger and co-workersdecreased with dilution rate in a remarkably similarway as the Qo, (apparently all normal respiration)measured by von Meyenberg. Thus, the use of anutritionally sufficient medium by Rieger et al. did noteliminate repression, but simply served to mask it.

Since the Qo, data of Barford et al. are essentiallyidentical to those of Rieger et a/., we conclude thatrepression of normal respiration (i.e. the Crabtree

effect) occurs with S. cerevisiae H1022, regardless ofadaptation, medium, or other “handling” effects.

Thus far, we have inferred that (normal) respiratoryrepression appears to be common in yeasts of thegenus Saccharomyces, but not in other respiration-limited genera such as Schizosaccharomyces or Bret-tanomyces. This repression is consistent with theobservations of von Meyenberg28 and the enzymaticfindings of Käppeli et al. 37 The major exception to thisgeneralization was S. cerevisiae UNSW 703100,whose value of YsF puts it into the Crabtree-negativecategory (see Table 4), in sharp opposition with theassignment of all the other S. cerevisiae strains asCrabtree-positive. Recall that this yeast was adaptedto respirofermentative growth, which results in anincrease in pMAX and Qo,, ,,,. The unadapted strainis very similar to S. cerevisiae HI022 in that ithas similar Qs,,~X, Qo,, ,,, and PMAX values (Table 1).Assuming that pcRrr for the unadapted strain is thesame as that for the adapted strain (and S. cerevisiaeH1022), the YsF value for the unadapted strain of S.cerevisiae UNSW 703100 is much smaller than 0.11,indicating that a Crabtree effect does occur with theunadapted yeast but not with the adapted one. Thehypothesis of Barford and Hall that respiratory re-pression can be removed by adaptation to a fullyrespiratory condition appears to be partially correct. Itis true for S. cerevisiae UNSW 703100, but not forH1022. Adaptation appears to involve selection for avariant strain with a depressed alternate respiration.The sudden drop in β and increase in Qo2 shown inFigure 8 and analyzed in Table 3 can be interpreted asan overflow of respiration into the alternate pathwayas the normal pathway approaches saturation.


Review

DiscussionComparison of the present model with themodel of Sonnleitner and KäppeliThe primary difference between this model and that ofSonnleitner and käppeli 39,40 is that energetics consid-erations have a prominent position in the former,whereas stoichiometric considerations dominate thelatter. In the stoichiometric approach, overall metabo-lism is considered as the sum of two independent,parallel reactions corresponding to respirative andfermentative metabolism. These reactions have fixedstoichiometric coefficients that are determined frommass balances on carbon, oxygen, and hydrogen usingcell elemental composition data and the correspondingexperimentally determined cell yields (i.e. YsR andYsF). YsR is obtained directly from aerobic growthdata. Similarly, YsF could be obtained from anaerobicgrowth data. However, the value so obtained did notgive a good fit of aerobic fermentation data when usedin the model. Thus, YsF was determined from theaerobic fermentation data using a goodness-of-fit cri-terion. The YsF value obtained in this way was onlyhalf the value consistent with anaerobic growth, lead-ing Sonnleitner and Käppeli to hypothesize that differ-ent nutritional requirements or ethanol inhibition maybe responsible for the difference. As described earlier,the energetics model interprets this difference as a sideeffect of respiratory repression. In reality, the fermen-tative cell yield is not different; it only appears to beso.

The energetics model has the advantage of explan-atory power. For example, the metabolic patternsshown in Figure 8 are inconsistent with a fixed stoichi-ometry. Analysis of these patterns using the energeticsmodel shows that they are very likely the result of theaction of multiple respiratory systems. On the otherhand, the stoichiometric model can be readily ex-tended to substrates other than sugars, for whichbiosynthetic oxygen demand must be considered,whereas the energetic model cannot.

Implications of differences in relationbetween β and µ among yeasts

Comparison of the parameters for respiration-unlimited (Table 2) and respiration-limited (Table 1)shows that β values for respirative (fully aerobic)metabolism of the former are at least 50% higher thanthose for respirative metabolism (</..LMAx) of the latter,suggesting that at least one proton translocation site isinactive in the mitochondria of respiration-limitedyeasts. Under normal growing conditions, all threesites are active in mitochondria from the respiration-unlimited yeasts C. utilis45,46,68,69,83 and E. magnu-

has been a matter of controversy for a long time.83 It isoften reported to be present in stationary phase83,85 orstarved cells. 86 However, cells harvested from expo-nential 83 or early stationary phase85,87 are generallyreported to lack site I. Site I can be induced by aerobic

incubation under starved conditions .87 The findinghere of a lower apparent P: O for Saccharomycesyeasts is consistent with the frequently observed ab-sence of site I activity in actively growing cells.

Beta for C. shehatae under oxygen limited condi-tions (i.e. @OI_) is smaller than β under fully aerobicconditions (Table 2). Also, /301. rises with increasingdilution rate (Figure 10). The data shown are forculture on xylose, whose utilization gives rise to aredox imbalance that results in NADH overproductionin the cytoplasm. 21 Up to 6.7 mmol of cytoplasmicNADH per gram xylose can be produced, dependingon the activity of NADH-linked xylose reductases,which recycle NADH back to NAD. 88 Since xyloseutilization for C. shehatae is constant at 0.55 g g -1 h-1

under semiaerobic conditions (Table 2), a constantflux of up to 3.6 mmol g-1 h-1 of cytoplasmic NADH isgenerated, which requires an oxygen utilization rate ofup to 1.8 mmol g-1 h - 1 for its oxidation. In actuality,not all of this NADH is re-oxidized: some is used forproduction of xylitol.

Recall that oxidation of cytoplasmic NADH pro-ceeds with a P: O of 2 rather than 3 (Figure1). Thus,at low dilution rates, when QCj, is small, a large fractionof Q(), will be used to oxidize cytoplasmic NADH andβ will be approximately 2. As dilution rate and Qo,increase, a decreasing fraction of the total respirationresults from oxidation of cytoplasmic NADH, and βwill gradually increase. Under fully aerobic condi-tions, rates of xylose and oxygen utilization risetogether with dilution rate (Figure 9). Thus, oxidationof cytoplasmic NADH is never a major fraction of thetotal respiration and P: O values are high.

Concluding remarks

It has been shown that the Crabtree effect can beidentified by apparent fermentative cell yield (YsF)values in aerobic continuous culture that are less than0.11 g g -1 as shown in Table 4. If Qo, data areavailable, one can identify a Crabtree effect directly bya drop in the (calculated) normal respiratory rate(~Qo, ) associated with the onset of respirofermenta-tlve metabolism at DCRIT.

Yeasts identified as Crabtree-positive using one ofthe above methods fall into the first of three distinctregulatory categories identifiable using the model.Crabtree-positive yeasts are necessarily respiration-limited. In addition, they are petite- positive and ableto grow anoxically in suitable media, indicating thatrespiration is totally optional for these yeasts. Theprototype Crabtree-positive yeast is S. cerevisiae.

Crabtree-negative, respiration-limited (CNRL)yeasts fall into the second category. They showaerobic fermentation but no detectable repression andare petite -negative. Respiration is more important inthese yeasts. Although they are capable of utilizingfermentative metabolism for growth, they will notgrow in the complete absence of oxygen. Examples ofCNRL yeasts are S. pombe and, possibly, B. inter-medius.

sii. 83,84 The existence of site I in saccharomyces yeasts



The third category are the respiration-unlimitedyeasts, which are necessarily Crabtree-negative, sothis term need not be applied to them. They areincapable of anaerobic growth and do not form stablepetite mutants. The growth of respiration-unlimitedyeasts has the greatest requirement for oxygen. Insemiaerobic culture showing low to moderate growthrates, the growth of these yeasts is proportional tooxygen utilization. 25,79 That is, the oxygen-limitedgrowth of respiration-limited yeasts is consistent witha constant ‘l-Y() , regardless of the extent of fermenta-tion, implying that fermentative metabolism is irrele-vant to growth (i.e. YsF is always zero).

The subtle difference in the ability of respiration-limited and respiration-unlimited yeasts to make use offermentative metabolism for growth is well illustratedby a comparison of the growth characteristics of S.pombe ATCC 26189 on xylulose with those of C.shehatae on xylose. Both of these yeasts are Crabtree-negative and incapable of anaerobic growth. The pri-mary difference between them is their respiratorycapacity.

Under excessive aeration, C. shehatae develops thefaster growth rate (0.28 vs 0.23 h-1, and utilizes sugarfar more efficiently (cell yield on sugar of 0.5 vs 0.2 gg -1). When oxygen is in short supply, the relevantgrowth yield is ‘LY(j,, the cell yield from oxygen foroxygen-limited growth. Consider the situation inwhich oxygen availability limits the extent of respira-tive growth to half its maximal value. Under theseconditions, C. shehatae shows a µ of 0.14 h-1and aOLYo2 of 35 g mol -1 (Figure 10). In contrast, S. pombewould show a µ of about 0.19 h-1(= µ MAX – µ CRIT/2)and a ‘LYO, of 103 g mol-1 (= 2p/Qol,,,, ). Thus, under50% oxygen limitation, S. pombe enjoys 35% fastergrowth at three times the cell yield.

When oxygen availability falls to zero, the advan-tage of S. pombe over C. shehatae vanishes, as neithercan grow, even with glucose as substrate. Crabtree-positive yeasts like S. cerevisiae can grow. however,and so possess a clear advantage over all others underconditions of extreme oxygen limitation or anaerobicconditions.

The ecological significance of respiratory limitationis apparent. A respiration-limited yeast, by virtue of itsrestricted respiration capacity and ability to growfermentatively, obtains an advantage over respiration-unlimited yeasts under conditions of carbon suffi-ciency and oxygen limitation at the cost of a disadvan-tage under conditions of oxygen sufficiency and car-bon limitation. Crabtree-positive yeasts gain the extraadvantage of anaerobic growth at the expense ofrespiratory repression.

Finally, we note that the model findings used todetect the Crabtree effect are directly testable usingrespiratory inhibitors. For example, the great increasein Qo, in respirative metabolism of adapted S. cere-visiae’ UNSW 703100 (Figure 8) is associated with adrop in β, which implies that an alternate respirationdevelops. If this is true, as much as half of the Q(J,observed for D > 0.25 should be cyanide resistant,

The CN-insensitive respiration detected (if any) couldbe tested for its sensitivity to salicyl hydroxamate(SHAM) in order to classify it as HAS or AZS. If it isthe former, SHAM could be added to cultures tosuppress HAS respiration and allow direct observationof normal respiration for comparison to ~Q[jz. Rote-none sensitivity of semiaerobic xylose cultures versusfully aerobic xylose cultures of respiration-unlimitedyeasts can be used to verify whether site I is active(Figure 1). Since mostly cytoplasmic NADH is oxi-dized by the former, we should expect no effect ofrotenone on these cultures, whereas some effect ofrotenone should be observable for the latter.

Alternative evidence for decreased efficiency ofrespiration at high dilution rates has recently beenprovided by Postma and co-workers. 91 They observedan inflection in the Qo, versus µ relation and a de-crease in the cell yield similar to that shown in Figure8. They attributed this change to a decrease in respira-tory efficiency. The change in inflection was ac-companied by a marked decrease in specific activity ofacetyl-CoA synthetase and the appearance of acetate.incorporating propionate into the medium caused asimilar decrease in cell yield. They hypothesized fromthese observations that organic acids uncouple theproton driving force in the mitochondria, therebydecreasing P: O.

Further research into the mechanisms determiningrespiratory efficiency and metabolize partitioning inboth Crabtree-positive and Crabtree-negative yeasts isstill required.

Nomenclature


Review

References


respiratory efficiency and metabolize partitioning as ... · pdf fileas regulatory phenomena...

Documents