1983 Boulton Ratledge Use of Transition Studies in Continuous Cultures of Lipomyces Stavkeyi to...

8/12/2019 1983 Boulton Ratledge Use of Transition Studies in Continuous Cultures of Lipomyces Stavkeyi to Invest Physiology

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ournal of General Microbiology (1983), 129 2871-2876. Printed in Great Britain 287 1

Use of Transition Studies in Continuous Cultures of Lipomyces stavkeyi, anoleaginous yeast, to Investigate the Physiology of Lipid AccumulationBy CH RI STO PHE R A. BOULTO N* A N D C O L I N R A T L E D G E

Department of Biochemistry, University of Hull, Hull HU6 7 R X , U . K .Received 23 February I983 ; evised 5 May 1983)

The physiological changes that occurred in chemostat cultures of an oleaginous yeast, Lipomycesstarkeyi CBS 1809 undergoing transitions from steady-state carbon-limiting to steady-statenitrogen-limiting conditions have been investigated. A sequence of events has been identifiedinitiated by the presentation of excess glucose to the culture and culminating in theaccumulation of substantial quantities of intracellular lipid. The intracellular concentrations ofadenine nucleotides and citrate have been determined and their possible regulatory significancein the control of catabolic and anabolic pathways is discussed.

I N T R O D U C T I O NAn oleaginous micro-organism may be defined as one that has the potential to accumulate

substantial lipid (typically 25 to 70 of the dried biomass) when cultivated under optimalconditions (Ratledge, 1982). These conditions are satisfied when the medium is limiting in anutrient, which is usually nitrogenous, and when there is an excess of the principal carbonsource, which is usually glucose.As a result of previous work carried out examining oleaginous yeasts, an hypothesis has beenadvanced to explain the phenomenon (Botham Ratledge, 1979). Briefly, this states that underconditions of nitrogen-limitation the excess glucose continues to be transported into the cell andis then dissimilated by the glycolytic and pentose phosphate pathways, eventually leading to theproduction of intramitochondrial acetyl-CoA. As a result of glucose catabolism, there is anincrease in the intracellular adenylate energy charge with a concomitant decrease in theintracellular concentration of AMP.

Botham Ratledge (1979) provided evidence that under these conditions the intramitochon-drial NAD+-dependent isocitrate dehydrogenase of oleaginous yeasts would be inactivated,thereby placing a stricture on the oxidative role of the tricarboxylic acid cycle. Subsequentstudies indicated that under conditions of nitrogen-limitation, citrate synthase would be fullyactive (Boulton Ratledge, 1980), resulting in the accumulation of citrate within mitochondriavia equilibration with aconitase. Citrate may then be transported into the cytosol in exchangefor malate (Evans et al . , 1983) and there be cleaved into acetyl-CoA and oxaloacetate byATP :citrate lyase, thus providing precursor C, units for lipogenesis. This latter enzyme hasbeen well-documented from mammalian sources (Srere, 1975), but in yeasts appears to beconfined to those species that are potentially oleaginous (Boulton Ratledge, 1981a). Inaddition, preliminary studies indicated that ATP :citrate lyase may catalyse the rate-determining step in fatty acid biosynthesis in oleaginous yeasts as well as fulfilling a regulatoryrole in controlling rates of lipogenesis in these organisms (Boulton Ratledge, 19816).

Our laboratory has been one of the few to investigate growth of oleaginous yeasts incontinuous culture (Gill et al., 1977; Hall Ratledge, 1977; Ratledge Hall, 1979). Thesestudies indicated that lipid concentrations could be achieved similar to those observed in batchcultures providing the medium had a high C :N ratio and a dilution rate of 0.02 to 0.06 h-l wasused. However, the behaviour of oleaginous yeasts in chemostat culture has so far only beeninvestigated after the establishment of steady-state conditions of carbon- or nitrogen-limitation.0022-1287/83/0001-1107 $02.000 983 S G M


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2872 C . A . B O U LT O N A N D C . R A T L E D G EThis report describes the results of experiments in which various metabolic parameters were

monitored when chemostat cultures of the typical oleaginous yeast, Lipornyces starkeyiCBS 1809,underwent transitions from conditions of carbon- to nitrogen-limitation. In so doingit was hoped that the sequence of events leading to accumulation of lipid could be identifiedfollowing introduction of excess glucose into the growth vessel. In addition, it was consideredthat a study of the physiological changes occurring during the transitions would providecorroborative evidence for the validity of the hypothesis already outlined, since this was mainlybased on results of experiments performed with isolated enzymes.

M E T H O D SYeast and medium. The yeast used in this study was Lipomyces starkeyi CBS 1809. In the majority of experimentsa glucose/salts medium was used as described by Botham Ratledge (1979). Carbon-limiting medium contained

10g glucose 1-I and 1-5g NH,C1 I- . Transitions were initiated by replacing the inflowing medium with oneidentical in composition but containing 100g glucose 1-l. In certain experiments, a defined medium was used ofthe following composition (g 1-*): KH, P04, 7.0; Na,HPO,, 2.0; MgS0 ,.7H20, 1.5; CaC1,.2H20, 0-1FeCl, .6H20,0408; ZnSO,. 7H20,0.001 ; hiamine, 0 005; calcium pantothenate, 0 005; 4-aminobenzoic acid,0.005; nicotinic acid, 0 005; riboflavin, 0 005 and pyridoxin.HC1, 0.005; with .biotin at 5 pg 1-l.

Cultural conditions. Transition experiments were performed in a conventional 8 litre chemostat with anoperating volume of 5.21. Yeasts were grown at 3 0 C and a dilution rate of 0.06 h-' and the culture wasmaintained at pH 5 5 by the automatic addition of NaOH. Air was delivered to the vessel at 5 1 min-' and theculture was stirred at 600 r.p.m. Foaming was controlled by the timed addition of polyglycol P2000 antifoam(Bevaloid Ltd, Beverley, U.K.) to a final concentration of approximately 0.1% (v/v).Sampling procedures. For citrate and adenine nucleotide analyses a rapid sampling device was fitted to thechemostat as described by Knowles (1977). This method gave a quenching time of less than 1 (Knowles, 1977).Monitoring ofgrowth. Washed yeast samples were dried to constant weight in tared vials by treatment in avacuum oven at 80 C.Determination o glucose. The concentration of glucose in culture filtrates was determined using a commerciallyavailable kit (GOD-Perid, Boehringer-Mannheim GmbH, Mannheim, W. Germany).Determination o fN H: . Concentrations of NH,+ n culture filtrates were determined by the method of ChaneyMarbach (1962).Determination o lipid concentration. Total lipid content of yeast was determined by a method based on that ofFolch et a / . (1957) as described previously (Boulton Ratledge, 1981a .Determination o intracellular adenine nucleotide concentrations. Yeast was removed from the chemostat using arapid sampling device and quenched in 1.4M-H2S0,. Adenine nucleotides were determined in the quenchedsamples using the modified luciferin-luciferase procedure of Chapman et a/. (1971) as described by BothamRatledge (1979).Determination of intracellular c itrate concentration. Yeast (1 8 ml) was removed from the chemostat using a rapidsampling device and quenched with 4 ml 1.4 M-H~SO,.After neutralizing the sample with 1 M-NaOH,precipitated protein and cell debris were removed by centrifugation and the supernatant retained for analysis. Atthe time of sampling, samples of spent medium were retained to determine extracellular citrate concentrations.

Citrate was determined by the coupled citrate lyase-malate dehydrogenase spectrophotometric procedure ofWilliamson Corkey (1969).Determination o A T P :citrate lyase activity. The specific activity of ATP :citrate lyase EC 4. I . 3.8) wasdetermined in crude French pressure cell extracts as described previously (Boulton Ratledge, 1981a .

R E S U L T S AND D I S C U S S I O NIn recent studies on the biochemistry of microbial lipid accumulation the oleaginous yeast,Lipomyces starkeyi CBS 1809, has been used (Boulton Ratledge, 1981a, b). Boulton

Ratledge (1981a ) confirmed that this yeast could accumulate substantial lipid when grown incontinuous culture, and this organism was employed in the transition experiments describedhere.

Initial experiments (Fig. 1a) showed that under steady state carbon-limited conditions thebiomass, of approximately 5 g l- , contained 4 (wlw) lipid. When the transition was initiated,the biomass increased as soon as excess glucose was introduced into the growth vessel and thiswas accompanied by a rapid decrease in the NH,+ concentration in the medium. However,


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Physiology of lipid accumulation 2873

Time after transition (h)

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0 8 16 24 32Time after transition (h)

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0 8 16 24 32Time after transition ( h )Fig. 1 a)Biomassa),ipid content 0 ) nd residual concentrations of NH,+ m) and glucoseu)during a transition from carbon- to nitrogen-limitation were compared with b) variation in theintracellular concentrations of A M P a),ADP (v), ATP a)nd energy charge v ) , a nd c )intracellular (+) and extracellular 0) itrate concentration.

although the culture became nitrogen-limited from the third to fourth hour, as gauged by thedisappearance of this nutrient from the medium, no appreciable lipid accumulation occurreduntil approximately 24 h after initiation of the transition. This was a reproducible occurrence(see Fig. 2) and similar results were obtained with the oleaginous yeast, Candida 107 (Boulton,1982). These results indicated that there was not a simple causal relationship between the onsetof nitrogen-limitation and the accumulation of lipid. It remained, therefore, to investigate theintervening physiological changes that must have occurred.Fluctuations in intracellular adenine nucleotide concentrations during transitionsfrom carbon- tonitrogen-limitation

Gill et al. (1977) demonstrated that the specific rate of lipid synthesis [expressed as g lipidsynthesized (g lipid-free yeast)-' h-I ] in chemostat cultures of Candida 107 was of a similar orderunder both carbon- and nitrogen-limiting conditions. This has been confirmed for otheroleaginous yeasts (Boulton Ratledge, 1981a ; C. T. Evans, unpublished work). The suggestionhas been made, therefore, that nitrogen-limitation does not induce higher lipogenic rates, butthat other biosynthetic pathways dependent upon a supply of nitrogen decline such thatlipogenesis eventually becomes the dominant feature of the metabolism of the yeast.

Botham Ratledge (1979) considered that the primary mechanism responsible forcontrolling the flux of metabolism between lipogenic and other pathways, in oleaginous yeasts,was probably the variation in intracellular concentrations of adenine nucleotides. The validityof this concept was tested by following changes in adenine nucleotide content during a transitionfrom carbon- to nitrogen-limitation as compared with the time course for lipid accumulation.


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28 74 C A . B O U L T O N AND C R A T L E D G EThe results (Fig. 1b) showed that there was an 11 fold decrease in the content of AMP during

the course of the transition accompanied by smaller increases in the levels of ADP and ATP.The computed energy charge [(ATP +ADP)/(ATP + ADP + AMP)] increased during thetransition from 0.13 to 0-57. Thus, the observed increase in energy charge was in most par t due tothe dramatic fall in intracellular AMP content. Furthermore, the rate of decrease in AMPcontent was most rapid in the initial part of the transition, a basal level being achievedapproximately 7 h after the disappearance of nitrogen from the medium. This result supports thesuggestion that the decrease in AMP content and concomitant increase in energy chargeoccurred as a direct consequence of nitrogen depletion, which itself was the primary signal todivert the bulk of the yeast's metabolism into a largely anabolic mode in order to accommodatethe excess carbon source.

The adenine nucleotide contents and computed energy charge values were of a similar order tothose determined for batch cultures of Candida 107 (Botham Ratledge, 1979). However, theywere clearly not typical of the majority of biological systems so far examined. Thus, Atkinson(1977) reported that the energy charge values of all cells under all, or nearly all, conditions liewithin the range 0.87 to 0-94. However, due attention was paid to the experimental proceduresemployed in this study such that reacidification of the cell debris after the first treatment withH 2SO4 did not yield further adenine nucleotides. Furthermore, the observed low energy chargevalues could not be explained by adenine nucleotide excretion and the sampling method wassufficiently rapid to prevent enzymic degradation of ATP and ADP. In addition, Botham (1978)demonstrated that the use of other quenching agents did not increase the observed yields ofadenine nucleotides from yeast samples.Fluctuations in the intra- and extracellu lar citrate concen tration during transitionsfrom carbon tonitrogen-limitation

The mechanism by which variations in adenine nucleotide concentrations could lead tocitrate accumulation, as postulated by Botham Ratledge (1979), has already been discussed.Citrate may serve a dual role in oleaginous micro-organisms; firstly, in the supply of precursoracetyl units for lipogenesis via the intermediacy of ATP :citrate lyase (Boulton Ratledge,1981a ) and secondly, as a possible activator of acetyl-CoA carboxylase, an effect reported fornumerous lipogenic systems (Volpe Vagelos, 1976).

When Lipomyces starkeyi CBS 1809 was cultivated under conditions of carbon-limitation, theextracellular citrate concentration was 12 p~ and, during a transition this gradually increased to38 PM when steady-state nitrogen-limiting conditions were achieved. The initial intracellularcitrate content of 3.8 nmol (mg dry weight)-' rapidly fell when the transition was initiated;however, when nitrogen became exhausted from the medium, the value rapidly increased toreach a plateau of 6.5 nmol (mg dry weight)-l (Fig. 1c). Knowles (1977) assumed that theintracellular water content of micro-organisms lies within the range 2 to 4 pl (mg dry weight)-'.From this it follows that the citrate content of carbon-limited yeast was 0.95 to 1.9 mM and thisincreased to 1-6 to 3.2 mM during the transition.

Our results may be interpreted in the following fashion. When the transition was initiated, theculture was limited by neither carbon nor nitrogen. Presentation of additional glucose would bereflected in an overall increase in the rate of metabolism of the yeast. Since at this stage therewould be no stricture placed upon the operation of the tricarboxylic acid cycle, as AMP levelswere relatively high, an increase in oxidative rates would account for the observed decrease inintracellular citrate content. As the transition progressed, nitrogen became exhausted from themedium between 3 and 4 h, and this was accompanied by a rapid fall in the intracellular AMPcontent. This latter metabolite reached its lowest value between 8 and 11 h and this wasimmediately followed 8 to 13 h) by a rapid increase in intracellular citrate levels. This providespowerful corroborative evidence for the proposal of Botham Ratledge (1979) that the declinein AMP concentration is responsible for channelling carbon flow into citrate synthesis and,subsequently, for the accumulation of lipid.

In previous communications we provided evidence suggesting that ATP :citrate lyasecatalyses the rate-limiting step in lipogenesis inL starkeyi (Boulton Ratledge, 1981a , b ) . This


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Physiology of lipid accumulation 2875

161-

121---MW* 81 -EmC

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is contrary to the generally accepted premise that this position is occupied by acetyl-CoAcarboxylase in the majority of biological systemsso far studied (Volpe Vagelos, 1976; BlochVance, 1977). The results presented here support our contention in that the citrate content of theyeast increased when the organism entered a lipogenic phase. This would indicate that either therate of citrate efflux from mitochondria or the rate of citrate cleavage was rate-limiting underthese conditions, depending in which cellular compartment the citrate accumulated. When thetransition was initiated, the extracellular citrate concentration immediately increased. Thiscitrate must have been derived from a cytosolic citrate pool since citrate could not have left themitochondrion without first passing through the cytosol. This argues therefore that ATP :citratelyase must operate at a lower rate (i.e. is the rate-limiting step) than the rate of citrate efflux fromthe mitochondrion.

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Variations in the specijic activity of A T P : itrate lyase during transitions from carbon- tonitrogen-limitationEvidence for the involvement of ATP:citrate lyase in many mammalian tissues has been

provided by the observation that its activity positively correlates with altered lipogenic ratesinduced by nutritional or hormonal factors (Kornacker Lowenstein, 1963, 1964, 1965a, b ) .This is an induction-repression phenomenon (Yen Mack, 1980). We showed that ATP : itratelyase occurs solely in oleaginous micro-organisms; however, there was no correlation betweenobserved enzyme activity and lipid content (Boulton Ratledge, 1981a ) .

This result was confirmed in that, when the activity of ATP :citrate lyase was monitored in aseparate transition experiment, no significant variation was observed (Fig. 2). Therefore, clearlythe regulatory role we have ascribed to ATP :citrate lyase must be effected at the enzyme ratherthan the gene level. Possible strictures placed upon the activity of the enzyme in vivo would notbe detected in the artificial conditions of the in vitro enzyme assay mixture.

Although the evidence presented so far supports the hypothesis of Botham Ratledge (1979),an explanation is still required as to why constant AMP and citrate concentrations were attainedat 11 and 13 h, respectively, after the initiation of the transition, although appreciable lipidaccumulation did not commence until approximately 26 h (Fig. 1). The situation is furthercomplicated in that examination of the data presented in Fig. 1 a ) shows that the bulk of theincrease in biomass occurred after nitrogen disappeared from the medium. Clearly this could nothave been due to growth, nor was it explained by utilization of the excess glucose to accumulatelipid. We interpret this increase in biomass as representing biosynthesis of a short-term storageproduct, which has been tentatively identified as largely trehalose (C. T. Evans, unpublisheddata). Thus, under steady-state carbon-limiting conditions, the metabolism of the yeast is gearedto scavenge all available carbon. When an excess of carbon is made available to the culture, it is

C

8.?3:z

I I I I I0 8 16 24 32 J

Time after transition (h)Fig. 2. Variation in the specific activity of ATP:citrate lyase a) ompared with the biomass A),lipid content 0)nd pattern of utilization of NHZ 1)uring a transition from carbon- to nitrogen-limitation.


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2876 C . A . B O U L T O N A N D C . R A T L E D G Eimmediately assimilated but, because it cannot be immediately metabolized presumablybecause key enzymes such as phosphofructokinase are not fully active, it is initially stored in areadily mobilized form. When carbon-excess conditions persist, the metabolism of the yeastadapts so that the carbon stored as carbohydrate, which may be regarded as an intermediate, isutilized to synthesize lipid, which may therefore be regarded as a long-term carbon store. Itfollows that there must be a mechanism which acts as a signal to mobilize carbohydrate andutilize the released carbon to synthesize lipid. This signal has not as yet been identified but ispossibly also related to changes in energy charge. Examination of the data presented in Fig. 1shows that when lipid was being synthesized most rapidly 24to 28 h), this was accompanied bya second cycle of rapid increase in energy-charge. Possibly, variations in adenine nucleotideconcentrations may control the flux of metabolism between gluconeogenesis and lipogenesis.However, it is equally possible that the observed variations in adenine nucleotide levels arethemselves a consequence of the intraconversion of carbon between carbohydrate and lipid andas such of no regulatory significance.

R E F E R E N C E SATKINSON, . E. (1977). Cellular Energy Metabolismand its Regulation. New York: Academic Press.BLOCH,K . VAN CE, . (1977). Control mech anisms inthe synthesis of saturated fatty acids. Annual Reviewof Biochemistry 46, 263-298.BOTHAM, . A . (1978). The mechanism of lipid accumu-lation in a yeas t, Candida 107. Ph .D thesis, Universi tyof Hull, U . K .BOTHAM,. A. RATLED GE, . (1979). A biochemicalexplanation for lipid accumulation in Candida 107and other oleaginous micro-organisms. Journal ofGeneral Microbiology 114 361-375.BOULTON,C. A. (1982). The biochemistry of lipidaccumulation in oleaginous yeasts. Ph.D thesis, Uni-versity of Hull, U.K.BOULTON, . A. RATLEDGE,. (1980). Regulatorystudies on citrate synthase in Candida 107, anoleaginous yeast. Journal of General Microbiology121, 4 4 1 4 4 7 .BOULTON, . A. RATLEDGE,. (1981a ) . Correlationof lipid accumulation in yeasts with possession ofA T P :citrate lyase. Journal of General Microbiology

BOULTON, . A. RATLE DGE,. (1981 b ) . ATP :c i tra telyase he regulatory enzym e for lipid biosynthes isin Lipomyces starkeyi? Journal of General Microbi-ology 127, 423426 .C H A N E Y , . L. MARBACH, . P. (1962). Modifiedreagents for the determination of urea and amm onia.Clinical Chemistry 8, 130-1 32.CHAPMAN , . G., FALL,C. ATKINSON, . E. (1971).Adenylate energy charge in Escherichia coli duringgrowth and starvation. Journal of Bacteriology 108,EVANS, . T . , SCRAGG, . H . RATLEDGE,. (1983).A comparative study of citrate efflux from mito-chondria of oleaginous and non-oleaginous yeasts.

European Journal of Biochemistry 130, 195-204.FOLCH, ., LEES,M . SLOANE-STANLEY,. H . (1957).A sim ple method for the isolation and purification oftotal lipids from anim al tissues. Journal of BiologicalChemistry 226, 497-509.GILL,C.O. HALL,M . J RATLEDGE,. (1 977). Lipidaccumulation in an oleaginous yeast Candida 107growing on glucose in a single-stage continuous

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culture. Applied and Environmental Microbiology 33,HALL,M. J. RATLEDGE,. (1977). Lipid accumu-lation in an oleaginous yeast Candida 107) growingon glucose under various conditions in a one- andtwo-stage continuous culture. Applied and Environ-mental Microbiology 33, 577-584.KNOWLE S, . J (1977). Microb ial metabo lic regulationby adenine nucleotide pools. Symposia of the Society

o r General Microbiology 27, 241 -283.KORNACKER, . S. LOWENSTEIN, M . (1963). Therelation between the rates of citrate cleavage andfatty acid synthesis in rat liver preparations. Bio-chemical Journal 89, 27P.KORNACKER, . S. LOWENSTEIN, M . (1964).Citrate cleavage and acetate activation in livers ofnormal and diabetic rats. Biochimica et biophysicaacta 84 4 9 0 4 9 2 .K O R N A C K E R ,. S . LOWENSTEIN, M. (1965a).Citr ate and the conversion of carbohydrate into fa t .Th e activities of citrate cleavage enzyme a nd acetatethiokinase in livers of starved and re-fed rats.Biochemical Journal 94, 209-2 5.K O R N A C K E R , . S . LOWENSTEIN,. M. (19653).Citr ate and the conversion of carbohydrate into fat.Activities of citrate cleavage enzyme of no rmal a nddiabetic rats. Biochemical Journal 95 832-837.

RAT LED GE, . (1982). Microbial fats and oils: anassessment of their comm ercial potential. Progres s inIndustrial Microbiology 16, 119-206.RATLEDGE,. HALL,M. J (1979). Accumulation oflipid by Rhodotorula glutinis in continuous culture.Biotechnology Letters 1, 115- 120.S R ER E,P. A. (1975). Enzymology of formation andbreakdown of citrate. Advances in Enzymology 43VOLPE, J VAGELOS, . R. (1976). Biosynthesis offatty acids, Physiological Reviews 56, 339-41 7.WILLIAMSON,. R . C OR KEY,. E. (1969). Assays ofintermediates of the citric acid cycle and relatedcompou nds by fluorometric enzyme metho ds. Meth-ods in Enzymology 12, 435-5 13.YEN,C. S . MACK,D. 0. 1980). Immun oquantitationof hepatic ATP :citrate lyase in th e starved, sucrose-refed, and fat-refed rat. Nutrition Repor ts Internation-al 22, 245-252.

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