BACTERIOLOGICAL REVIEWs, Sept. 1975, p. 197-231 Vol. 39, No. 3 Copyright © 1975 American Society for Microbiology Printed in U.SA. Lipids of Yeasts JAMES B. M. RATTRAY,6 ANGELO SCHIBECI, AND DENIS K. KIDBY Departments of Chemisty and Microbiology, University of Guelph, Guelph, Ontario, Canada
INTRODUCTION ...... 198 CELLULAR LIPIDS ...... 198 Extraction ...... 198 Subcellular Distribution ...... 198 Cytoplasmic (plasma) membrane ...... 198 Intracytoplasmic elements ...... 199 Cell wall ...... 199 LIPID COMPONENTS ...... 199 Fatty Acids ...... 199 Chemical nature ...... 199 Mutant strains ...... 201 Biosynthesis ...... 201 Biological significance ...... 202 Taxonomy ...... 202 Hydrocarbons and Sterols ...... 202 Hydrocarbons ...... 202 Sterols (i) Chemical nature ...... 202 (ii) Mutant strains ...... 204 (iii) Biosynthesis ...... 204 (iv) Biological significance ...... 206 Glycerophospholipids ...... 207 Chemical nature ...... 207 Biosynthesis ...... 208 Cellular distribution ...... 208 Biological significance ...... 208 Sphingolipids ...... 209 Chemical nature ...... 209 Biological significance ...... 209 Glycolipids ...... 209 FACTORS INFLUENCING CELLULAR LIPID COMPOSITION ...... 209 Growth Cycle ...... 209 Sporulation ...... 210 Carbon Source ...... 210 Glucose ...... 210 Hydrocarbons ...... 211 Nutrients ...... 213 Nitrogen ...... 213 Phosphorus ...... 213 Growth factors ...... 213 (i) Inositol ...... 213 (ii) Pantothenic acid ...... 213 (iii) Vitamin B6 ...... 213 (iv) Biotin ...... 214 Miscellaneous Additives ...... 214 Sodium chloride ...... 214 Choline and ethanolamine ...... 214 Benzo(a)pyrene and dibenzanthracene ...... 214 Propanediol ...... 214 Oxygen ...... 214 Respiratory-deficient (Crabtree-positive) yeasts (i) Lipid composition ...... 214 (ii) Lipid supplementation of growth medium ...... 215 Respiratory-sufficient (Crabtree-negative) yeasts ...... 216 Temperature ...... 216 pH ...... 217 CONCLUDING COMMENTS ...... 217 LITERATURE CITED ...... 219 197 198 RATTRAY, SCHIBECI, AND KIDBY BACTERIOL. REV. INTRODUCTION form-methanol (2:1, vol/vol), the extract is con- The earlier literature on yeast lipids has centrated, and the lipids are solubilized in chlo- been reviewed by Hunter and Rose (127). More roform. Failure to purify the initial extract by recently much research has been undertaken in washing with water and re-extracting can re- this area of yeast biochemistry particularly in sult in falsely high lipid values (126). Thus the fields of membranology of mitochondria variations in reported total lipid values may be (174, 175), the possible commercial production related to the conditions ofextraction. The most of fat (246), and the control of opportunistic reproducible results have been obtained after organisms (104). The major purpose of this re- hydrolysis and recovery of the total fatty acid view has been to describe and evaluate the component which can then be used as an index present state of knowledge of yeast lipid bio- of total lipid (248). chemistry. In particular the importance of var- The total lipid content ofyeast cells is subject ious factors influencing the cellular composi- to a variety of influences. However, it is possi- tion and distribution has been discussed. ble to group the various yeasts, based on a low, medium, or high lipid content (Table 1). This CELLULAR LIPIDS loose classification cannot be accorded taxo- nomic significance as there are obvious over- Extraction laps; thus members ofthe genus Candida occur Yeast lipids have been defined in two opera- in all three groups. tional forms dependent upon the ease of extrac- tion. The "readily extracted" lipids are generally Subcellular Distribution best recovered (218, 232, 298) using a neutral Various membranous structures have been two-solvent extractant of chloroform-methanol recognized (184) in yeast and have been as- (35, 81); "bound" lipids require an acidified ex- sumed by Hunter and Rose (127) to contain the tractant to affect their release (5). A "bound" bulk of the cellular lipids. In general, unfrac- fatty acid fraction can be recovered by metha- tionated membranous systems possess a nolic potassium hydroxide treatment of cells lipid:protein ratio of approximately 1 (88, 163, previously extracted with chloroform-methanol 176). However, more detailed studies require (255). Acid (2) and base (3, 254) hydrolysis may the availability of procedures for the isolation be required to permit subsequent total extrac- and separation of these systems in a high de- tion ofthe various bound forms ofyeast sterols. gree of purity. Incomplete recovery ofpolar phospholipids may Cytoplasmic (plasma) membrane. Methods result unless there is sufficient water in the for the isolation and identification ofyeast cyto- extracting medium to produce a two-phase sys- plasmic membrane have been discussed by Schi- tem (224). Generally polyphosphoinositides are beci et al. (262, 263). Two general approaches best extracted with an acidified solvent system have been employed (Fig. 1). One method in- (251), although Steiner and Lester (282) have volves the conversion of cells to protoplasts by employed a basic system of ethanol-water- enzymic digestion of the cell wall and recovery diethyl ether-pyridine (15:15:15:1) in the isola- ofa total membrane fraction after osmotic lysis. tion of di- and triphosphoinositides from yeast. This procedure has been employed with Saccha- Lipid is most efficiently recovered from romyces cerevisiae (17, 37, 176, 262) and Can- freeze-dried or freeze-thawed yeast cells (275) dida utilis (88). Isolation of the plasma mem- and this procedure is commonly employed. Me- brane can then be achieved by isopycnic centrif- chanical disintegration of the cell (170) and ugation (262). Treatment of protoplasts with drying yeast at moderate temperatures (171) lipase and various phospholipases (211) pro- also enhance lipid extraction. Particular care duced no observable morphological changes. must be taken to avoid possible enzymic decom- Thus consideration that the enzymic action position of the phospholipid components during may be detrimental to the membrane integrity the operative steps (109, 316). Activation of has not yet been substantiated. The second pro- membrane-bound phospholipases may also cedure for plasma membrane preparation in- arise on treatment of cells with acetone or volves the mechanical disintegration of the cell aqueous ethanol, particularly at room tempera- and the recovery of the cell envelope (187, 291, ture (171). A convenient procedure for lipid ex- 292), i.e., wall plus plasma membrane. A traction has been proposed by Letters (171) in- plasma membrane-enriched fraction can then volving the heating of yeast cells with 80% be obtained by differential centrifugation ethanol at 80 C for 15 min to deactivate en- either before (187) or after removal of the cell zymes and split lipid-protein linkages. The wall fraction by enzymic digestion (291). How- yeast residue is then extracted with chloro- ever, the second procedure has been found (68) VOL. 39, 1975 LIPIDS OF YEASTS 199 TABLE 1. Total cell lipid of various strains ofyeasts' Lipid (% cell dry wt) Strain Reference Low (<5%) Candida albicans 214 C. lipolytica 201 C. utilis 61, 311 Lipomyces starkeyi 58 Rhodotorula glutinis 311 Saccharomyces fragilis 216 Medium (5 to 15%) Blastomyces dermatitidis 65 C. lipolytica 51, 147, 148, 218, 311 C. scottii 147 C. tropicalis 201, 311, 312 Debaromyces hansenii 195 Endomycopsis vernalis 343 Hanseniasporo valbyensis 112 Hansenula anomala 311 Histoplasma capsulatum 66, 213 L. starkeyi 58 Mucor rouxii 255 Pullularia pullulans 194 R. glutinis 147 R. graminis 311 S. carlsbergensis 225, 267 S. cerevisiae 44, 128, 140, 170, 176 High (>15%) B. dermatitidis 5 Candida 107 311 Cryptococcus terricolus 232 E. vernalis 343 H. capsulatum 5 H. duboisii 5 L. lipofer 189 L. starkeyi 58, 278, 295 R. glutinis 352 R. gracilis 78 R. graminis 110 S. cerevisiae 48 Trigonopsis variabilis 266 a Cell cultivation under different conditions; lipid extraction generally involved the treatment of freeze- dried cells with a neutral chloroform:methanol system. to produce a high degree of contamination due tamination by particles of lipid-rich plasma to entrapment of intracellular components. membrane should always be considered. A lipid The importance of lipids in the yeast plasma content of 3 to 10% of the cell wall dry weight membrane structure has been evaluated by has been reported (180). A significant quantity Kopp (161) in the light of ultrastructure stud- of the cell wall lipid occurs in a bound form ies. except in the notable cases of Candida albi- Intracytoplasmic elements. Ultracentrifu- cans and S. cerevisiae (Table 2). Kidby and gal methods, applied to yeast, have permitted Davies (155) have discussed the biological activ- the isolation of nuclei (252), vacuoles (121, 131, ity of the cell wall in terms of a proposed struc- 188, 317), "promitochondria" (57, 260), mitochon- ture, but the role ofthe lipid component has not dria (1, 15, 26, 49), and the inner and outer yet been resolved. mitochondrial membranes (1, 15, 26). Particles which are almost wholly lipid in composition, LIPID COMPONENTS containing 90 to 95% ofthe total weight as lipid, have been isolated from S. cerevisiae (52). Fatty Acids Cell wall. A cell wall preparation is gener- Chemical nature. Significant amounts (ap- ally obtained (67, 143, 256) after rupture of the proximately 5% of total lipid) of free fatty acid cell and removal ofthe intracytoplasmic compo- have been found in S. cerevisiae (44, 128), Lipo- nents and plasma membrane by centrifugation myces starkeyi (295), Candida tropicalis (102) and frequent washings. The possibility of con- and various other members of the genus Can- 200 RATTRAY, SCHIBECI, AND KIDBY BACTERIOL. REV.
dida (311). However, free fatty acid can also 80 sterylester + h arise by the enzymic hydrolysis of cellular phos- triglycerideac pholipids during the extraction procedure (316). stero +digtyceride
A typical lipid composition for yeast (Fig. 2) phospholipid would be anticipated to have a total fatty acid 60 content of 70 to 90%. In certain cases, however, the total fatty acid content of S. cerevisiae (140;1 unpublished findings from our laboratory), Can- e dida lipolytica (218), and Hansenula valbyensis &o (112) has been found to account for approxi- mately 40% of the total lipid extract. Several factors have been examined to account for these apparent discrepancies in fatty acid composi- tion. Investigations ofpossible nonlipid contam- c
METHOD A METHOD B .11 ~~~~~~~~~0 011 f S.cerevisiae Scarlsbergensis C.lipolytica C.tropicalis i ON*% ) rupture FIG. 2. General lipid composition determined for CELL S. cerevisiae (44, 128, 138), S. carlsbergensis (225, %.Woo at267), C. lipolytica (51, 311), and C. tropicalis (102, 311). Vertical bars represent range of observed val-
hgh. osmotic centrifuge ination of the extract (112), the presence ofhigh PIshock be,
TABLE 3. Sterol components and presumed enzyme lesion in selected mutant strains of S. cerevisiae
Mutant Sterol occurrence Metabolic lesion Refer-ence olerg (growth require- ? 144 ments for fatty acid + ergosterol) erg (growth requirement Conversion of 145 for ergosterol) squalene to lanosterol Ergosterol-deficient Lanosterol 4,14-Dimethylcholesta-8,24-dien-3,t3oI C14 demethyl- 314 ase 4,14-Dimethylergosta-8,24 (28)-dien-3,-ol 14-Methylergosta-8,24 (28)-dien-3,-ol pol 1 (nys 1) (polyene [ny- Zymosterol C24 methyl 24 statin]-resistant) transferase Cholesta-5,7,24-trien-3,3-ol Cholesta-5,7,22,24-tetraen-3,-ol pol 2 (polyene resistant) Ergost-8-en-3p-ol 8:A7 Isomer- 24 ase Ergosta-8,22-dien-3p-ol Ergosta-5,8,22-trien-3,-ol Fecosterol pol 3 (nys 3) Ergosta-7,22-dien-33-ol 5,6-Dehydro- 24, 229 genase Ergosta-8,22-dien-3p-ol Ergosta-7,22,24 (28)-trien-33-ol Ergosta-8,22,24 (28)-trien-3f-ol Episterol Fecosterol pol 5 Episterol 22,23-Dehydro- 24 genase Ergosta-5,7-dien-3,&ol Ergosta-5,7,24 (28)-trien-3,&ol Ergosta-8,14,24 (28)-trien-3p-ol 204 RATTRAY, SCHIBECI, AND KIDBY BACTERIOL. REV. like cells of M. rouxii (254). Acid-labile steryl presumed affected enzyme systems indicates derivatives, presumably including steryl glyco- close metabolic relatedness (85, 203). The na- sides, have been observed in S. cerevisiae (2, ture of the sterol component resulting from a 17). defect in a particular synthetic enzyme system (ii) Mutant strains. Various mutant strains is summarized in Table 3. ofS. cerevisiae have been employed (Table 3) in (iii) Biosynthesis. All the sterols named in the elucidation of the pathways ofsterol biosyn- Fig. 3 have been isolated from various S. cere- thesis. Particular use has been made of mu- visiae strains and have been considered as possi- tants resistant to polyene antibiotics. Details ble biosynthetic intermediates by Barton and on the particular genetic lesions are generally his co-workers (23-25). The efficiency with lacking but the information available on the which cell-free extracts have been able to con-
(C043)
HO
s PAFOEa- .-- 40
(CH3) (04 -3)
HOOWCX ZMSEO FECOEISTEROL
q'r ERGOSTA-5,7,22,24(28)- ERGOSTEROL ERGOSTA-5,7-DIEN-9-OL TETRAEN-V-OL FIG. 3. Possible pathways ofmetabolic conversion ofsterols in yeast; named compounds have been isolated from S. cerevisiae (25, 84, 333). VOL. 39, 1975 LIPIDS OF YEASTS 205 vert these postulated intermediates has been tion of cholesta-7,24-dien-3,&ol from zymosterol examined and the available details on some of would require the activity of A58:A7 isomerase the proposed enzyme systems have been re- (24). viewed by Weete (333). Information, however, Several pathways possibly exist for the en- on the sequence of action of these enzymes is zymic conversion ofthe two C24 methylene ster- generally lacking, but evidence for a multiplic- ols, fecosterol and episterol. The molecular ity of sterol biosynthetic pathways in yeast has events presumably involve a 5,6-dehydrogenase been presented (84). for the introduction of additional unsaturation The initial step of lanosterol conversion in- in ring B, a 22,23-dehydrogenase for the dehy- volves the removal of the two methyl groups at drogenation of the side chain and a methylene position 4 and the methyl group at position 14. reductase for the reduction of the methylene Inability to carry out the demethylation at posi- group at position 24. The absence of any of tion 14 has been found (314) to be accompanied these three systems would be anticipated to by the accumulation of 4,14-dimethyl sterol de- result in the accumulation of different sterols, rivatives. This finding suggests that the other whereas the presence of all three systems 4-methyl group is subject to earlier removal. would permit the formation of ergosterol. The Sterols carrying a methyl group at position 4 relationship between the availability of a partic- undergo little methylation at position 24 (204, ular reaction mechanism in vivo and sterol 228). S-adenosylmethionine:AA sterol methyl product is given in Table 4. A major biosyn- transferase (C24 methyl transferase), involved thetic pathway from episterol to ergosterol has in the methylenation process, apparently shows been deduced (83, 84) as involving the sequen- greatest substrate specificity for cholesta-7,24- tial introduction of unsaturation at position dien-3/3-ol (116) although zymosterol (204) is 22,23 and then at position 5,6 followed by the also used. This enzyme activity has been found reduction of the methylene group at position 24. to be primarily located in the promitochondria Additional knowledge on the characterisitics or mitochondria of S. cerevisiae (304) and to be ofthe enzyme systems involved in sterol biosyn- enhanced by the presence of a fermentable car- thesis in yeast should provide a better under- bon source and molecular oxygen (279). Forma- standing of the mechanisms controlling the
TABLE 4. Relationship between the occurrence ofanticipated enzyme systems and products ofreaction during sterol biosynthesis in yeast Reaction system Sterol product Present Absent 5,6-Dehydrogenase Ergost-7,24(28)dien-3,&ol (episterol) 22,23-Dehydrogenase Methylene reductase 5,6-Dehydrogenase 22,23-Dehydrogenase Ergosta-5,7,24(28)trien-3p3-ol Methylene reductase 22,23-Dehydrogenase 5,6-Dehydrogenase Ergosta-7,22,24(28)trien-3P-ol Methylene reductase Methylene reductase 5,6-Dehydrogenase Ergost-7-en-3,8-ol 22,23-Dehydrogenase 5,6-Dehydrogenase Methylene reductase Ergosta-5,7,22,24(28)tetraen-3,8-o1 22,23-Dehydrogenase 5,6-Dehydrogenase 22,23-Dehydrogenase Ergosta-5,7-dien-3,8-ol Methylene reductase 22,23-Dehydrogenase 5,6-Dehydrogenase Ergosta-7,22-dien-3,8-ol Methylene reductase 5,6-Dehydrogenase Ergosterol 22,23-Dehydrogenase Methylene reductase 206 RATrRAY, SCHIBECI, AND KIDBY BACTERIOL. REV. process. The activity of hydroxymethylglu- eral capacity to bind sterol in vitro (305). taryl-CoA reductase (EC 1.1.1.34) participating The major function ofsterol in yeast has been in the initial biosynthetic step, of mevalonate regarded by Proudlock et al. (242) to be one of formation, has been considered (150, 151) to be structural influence upon the dynamic state of under feedback control of ergosterol or some membranes. Comparison may therefore be acidic products of ergosterol metabolism. Con- made with the observed biological roles of cho- trol ofsterol biosynthesis may occur (204) at the lesterol in influencing phospholipid-protein in- reaction step catalyzed by S-adenosylmethio- teractions, membrane permeability and nine:A24 sterol methyl transferase which has membrane-bound enzyme activity in animal sys- been suggested (307) to be competitively in- tems (227). The specificity for C28 sterols in hibited and possibly repressed by ergosterol. yeast does not appear to be absolute, since cer- Studies (345) made on three strains ofS. cerevis- tain mutants of S. cerevisiae (94, 144, 145) or iae have revealed that the conversion of er- wild-type S. cerevisiae growing anaerobically gosta-5,7,22,24(28)-tetraen-3(3-ol to ergosterol is (97, 242) can have the growth requirement for subject to differing metabolic controls. The ex- ergosterol met by supplementation with var- istence of several biosynthetic routes for ergos- ious C27 or C2, sterols. The chemical characteris- terol suggests the possibility of several regula- tics associated with functional membrane ster- tory mechanisms. ols have been reviewed by Nes (212), but a Various factors influencing the synthesis of major requirement is that the molecule pos- sterol by yeast have been discussed by Hamil- sesses a highly planar nature (242). Absence of ton-Miller (106). In particular, the composition compounds with the required steroid conforma- of the growth medium (77, 128) and stage ofthe tion would be expected to produce a function- growth cycle (44, 76) have been noted to have an ally impaired membrane (212). effect on the amount of sterol produced. The Ergosterol has been implicated (258) as a com- availability of oxygen requires special atten- ponent of a particular membranous system con- tion as it governs both the type of sterol synthe- cerned with the initiation of cell division in C. sized (59, 60, 256) and also the quantity of albicans. The capacity for the sterol-induced sterol. The lower levels of ergosterol occurring recovery from the damaging effects of ultravi- under anaerobic conditions in S. cerevisiae (84, olet irradiation is considered (258) to be species 140, 309) and the yeast-like form of Mucor ge- specific. Decrease in the total sterol content of nevenis (99) have been reported to be accompa- yeast mitochondria has been associated with a nied by increased amounts of squalene. The depression in oligomycin sensitivity (299) and apparent accumulation of this hydrocarbon re- an increase in the temperature of phase transi- flects, in part at least, the observed sensitivity tion (54) of adenosine 5'-triphosphatase activ- to lack of oxygen of several reaction steps in- ity. Replacement of ergosterol by the less cluding oxidative cyclization of squalene (com- planar ergosta-8(9),22-dien-3,-ol in certain mu- pare reference 206), oxidative demethylation tant strains of S. cerevisae resulted in a low- (199, 206), oxidative desaturation of ring B ered transition temperature for mitochondrial (313), and methylation at position 24 (204). In S-adenosylmethionine-A24 sterol methyl trans- certain mutants ofS. cerevisiae the inability to ferase and cytochrome oxidase (308). This effect form ergosterol has been traced (21) to lesions was attibuted to a less efficient packing ofmem- in the biosynthesis of porphyrins rather than brane phospholipids. Although ergosterol has sterols and indicates the probable importance of been found (306) to be intimately associated an active respiratory chain in the conversion of with yeast cytochrome oxidase, it is considered squalene to ergosterol. nonessential for enzyme activity. Thus Thomp- (iv) Biological significance. Both free and son and Parks (306) have suggested that var- esterified sterol have been detected in various ious lipid combinations may provide suitable membranous systems of S. cerevisiae (249), al- environments for enzyme activity. though the suggestion has been made by Rose Interaction of membrane-bound sterol with and his co-workers (125, 128) that steryl esters the polyene macrolide antibiotics (104, 105, 106, may be preferentially concentrated in certain 164) has stimulated much interest both as a intracellular structures rather than the plasma possible means of controlling opportunistic membrane. Significant quantities of free and yeast (104) and as an experimental system for esterified sterol occur in the cell wall of C. studying the function of sterol in membranes albicans (32), B. dermatitidis (67), and H. cap- (106). Yeasts containing ergosterol appear to be sulatum (67). Although the cell wall of S. cere- especially sensitive to the action ofpolyene anti- visiae has been found (17, 217) to be deficient in biotics, whereas a low sterol content is associ- sterol, its mannan component possesses a gen- ated with resistance (20, 41). Variable yeast VOL. 39, 1975 LIPIDS OF YEASTS 207 sensitivity to polyenes has been correlated with various lysoderivatives, phosphatidyl glycerol differences in the sterol composition of several (PG), phosphatidyl glycerol phosphate (PGP), strains of Candida (12, 103, 105, 346) and S. diphosphatidyl glycerol (DPG), phosphatidic cerevisiae (20, 85, 203, 309, 344). Fryberg et al. acid (PA), phosphatidyl monomethylamino- (85) have concluded that polyene affinity is ethanol, phosphatidyl dimethylaminoethanol, greatest with A5' 7-sterols, e.g., ergosterol, and diphosphoinositol (DPI), and triphosphoinositol lowest with the less planar A8' 24-sterol, (TPI) (90, 132, 281). The relatively high values namely, fecosterol. Impaired access of polyene of 11% and 8% reported (132) for lysophosphati- antibiotic to sterol located in the cell surface dyl choline (LPC) and lysophosphatidyl etha- membrane has been considered (231) in explana- nolamine (LPE), respectively, in S. cerevisiae tion of resistance, but the chemical nature of may represent artifact formation during isola- the sterol component is believed (105) to be tion. Polyphosphoinositides are not frequently more important. A major consequence ofnysta- encountered in yeast, but the occurrence of PI- tin action is the leakage of the bulk of the DPI-TPI in molar ratios of 140:4:1 has been intracellular K+ from C. albicans (107) and S. observed in S. cerevisiae (169, 282). Comparable cerevisiae (319). These findings endorse the con- very low levels of DPI have been noted (239) cept of membrane sterol being associated with inKloeckera brevis. Specific examination for the permeability factors. presence of plasmalogens in S. cerevisiae (140) and Lipomyces lipofer (189) have revealed only Glycerophospholipids trace quantities. Most phospholipids are characterized by a Chemical nature. Most yeast species have a high level of unsaturated fatty acid which is phospholipid content of 3.0 to 7.0% of the cell replaced to a significant extent by 16:0 acid in dry weight (171). The various phospholipids and the PS-PI component of C. tropicalis (64), S. their abbreviated names are shown in Fig. 4. A cerevisiae (17, 176, 290), Schizosaccharomyces representative distribution (as the percentage pombe (339), B. dermatitidis (67), andH. capsu- oftotal phospholipid) of35 to 55% phosphophati- latum (67). Whereas the PS component of L. dyl choline (PC), 20 to 32% phosphatidyl etha- starkeyi has been determined (295) to be almost nolamine (PE), 9 to 22% phosphatidyl inositol entirely 1-palmitoyl, 2-oleoyl-sn-glycero-3-phos- (PI), 4 to 18% phosphatidyl serine (PS), and phoryl-L-serine, the PC and PE components are <10% minor components has been described by composed of several molecular species. The na- Letters (171). The minor phospholipids include ture of the molecular species of PC, however,
1112011 HOCH1 o CI2°O"-OH 0- FATTY ACYL COA _ -ELYClRO-3-PDOSPHO3IC ACID 0 9 _ 0 I_CH2OdCR1 ATP £9? CH120CR1 C Tr, t5? 0oCH20LR1A.".. TRI.ACYL- | R2COCH o -CUUCH GLYCUO 0 R2CCUHI IDOSITOL CH20H CH2-OOH CH20-CDF 1,2-DlACYL-_C-GLYCIOL P1ISPRATIDIC AID CDP-1,2-DIGLYCERIDE (PA) CDP-CHIOLINE CDP-EYTAUOLAXINZ L-SERI\\ LYCEROL
R0CH0 9 9 9J\ 0 CI20CR1 o %CI2CR1 Co2 o CH2OCR1 o CH2OCR1 CHI2OH R2COCH o0~ R2COCH o R2tOCH o NH3 R2LOCH o HCOII CH20p--OlCH2Ni(CH3)3 CII20p-0C02CII2NH3 CH2OA-0C112&I CH°-OCI2 0- 0- too 0 PNOSPIATIDYL CHOLINE PDlOSPATIDYL ETHAOLOINE PW)SPHATIDYL SEREIE PROSPRATIDYL GLYCEROL (PC) (pp.) (PS) (PC) 9 (CH3) 0 ~ 0 0 L C1120CR1C CH OC 0 o 1C12OCR1 CI2JI CR, R2CCCH o RzCOCI 0 R2?OCH 0 HCOII IICOCR3 R2tOCH o CH20P-OCH2CH2NH(CH3)2 CH2OP-OCH2CH2NtI2CH3 CH20-OCt12 CH2OCR4 CH2OA-OC6H6(OFI)5 0- PHOSPIETIDYL D(PETHYL- PNOSPHEATIDYL (OPETHYL- DIPHOSPTIDYL GLYCEROL PHOSPATIDYL INOSITOI A.MrNOTRUANOL (PDtE) A.4rOETHANOL (P41E) (npr) (PI) FIG. 4. Possible pathways of metabolic conversion ofglycerophospholipids in yeast. All species have been found in S. cerevisiae (see reference281). ADP, Adenosine 5'-diphosphate; CTP, cytidine 5'-triphosphate; and PP, pyrophosphate. 208 RATTRAY, SCHIBECI, AND KIDBY BACTERIOL. REV. can vary depending on the particular biosyn- ently involve two general processes. Direct de- thetic pathway involved (325). saturation of intact PC has been observed in Biosynthesis. The possible pathways of bio- Torulopsis utilis (300) and C. lipolytica (243). synthesis and metabolic interrelationships of The deacylation-reacylation process is believed the various phospholipids in the eukaryotic cell (296) to be involved in the metabolic turnover of have been discussed by Mangnall and Getz PE but not PS in L. starkeyi. The metabolic (183) and are summarized in Fig. 4. The forma- significance of the specific deacylation of PI tion of PA in S. pombe (339) and S. cerevisiae occurring at the cell surface accompanied by (281, 339) has been determined to involve the the secretion of glyceryl phosphoryl inositol (11) specific acylation of sn-glycero-3-phosphoric awaits further explanation. acid. Cytidine diphosphate (CDP)-diglyceride, Cellular distribution. The glycerophospho- which can arise from PA (129, 281), appears to lipid content (as percentage of total lipid) ofthe be the major precursor of most phospholipids. cell wall has been determined to range from Evidence has, however, been obtained for the zero in S. cerevisiae (17, 217) to 15% in Trigo- occurrence of a CDP-nitrogenous base reaction nopsis variabilis (266) and 39% in C. albicans with 1,2-diacyl-glycerol in the formation of PE (32). Although slight differences have been (281) and PC (53, 324, 325). An alternate mecha- noted (132, 249) in the intracellular distribution nism for the formation of PC in S. cerevisiae of the major phospholipids in S. cerevisiae, spe- involves the sequential methylation ofPE, phos- cific localization of certain minor components phatidyl monomethylaminoethanol, and phos- may exist. Thus DPG is primarily associated phatidyl dimethylaminoethanol, but the proc- with yeast mitochondria although this occur- ess is depressed in the presence ofcholine (325). rence is not absolute (53). Increasing quantities Synthesis ofPE can also involve the decarboxyl- ofDPG have been taken (132) as an index ofthe ation of PS (281). Reaction of CDP-diglyceride development state ofmitochondria. Fully devel- with serine and inositol has been implicated oped mitochondria have also been found (226) to (281) in the synthesis ofPS and PI, respectively, possess lower PI values than promitochondria. although an alternate but undefined mecha- Mitochondriogenesis, in general, is accompa- nism has been considered (339) to be of major nied by a high turnover of PE and PS (135). importance for PI formation in S. pombe. DPG Biological significance. The relative con- synthesis in S. cerevisiae has been found (53) to stancy of the total phospholipid component in involve reaction of CDP-diglyceride with PG, yeast is suggestive of some primary function. although other possible routes for polyglycero- Phospholipids have been considered (140) to be phospholipids have been discussed (276). An major structural components of yeast mem- exchange of the alcohol unit between the var- branes in which a preferential concentration of ious phosphatidyl derivatives would allow a unsaturated fatty acid is of importance. Mito- rapid interchange of phospholipid types. Stei- chondria depleted of unsaturated fatty acid, ner and Lester (281) have shown that there is and consequently depressed in oxidative phos- interconversion (via CDP-diglyceride) between phorylation capacity, have been noted (114) to endogenous PI and PS (and thence PE as well). have unchanged quantities of total phospho- A detailed study by Cobon et al. (53) has lipid but a lower content of PI. The precise shown that yeast microsomal fractions contain importance of specific phospholipids in yeast the two general pathways for PC synthesis membranous systems has not been established, (Fig. 4), as well as the systems for the formation but is presumably comparable to that in mem- of PA, PS, and PI. The synthesis of DPG is branes of high organisms (190). A cyclic turn- restricted to the mitochondria which also con- over of polyphosphoinositides involving tain enzyme systems for the formation of PE PI -* [DPI and TPI] -* PI, accompanied by an and PG. Mitochondria of yeast apparently dif- alteration in adenylate energy charge has been fer from those of animal cells in possessing a suggested (301) as possibly facilitating the diffu- limited ability to synthesize the acidic phospho- sion of ions across membranes in S. cerevisiae. lipids PA, PS, and PI. Phosphatidylinositol Hydrolysis of PGP to PG has been considered kinase activity, responsible for the conversion (63) to be important in the active transport of D- of PI to DPI, has been suggested by Wheeler et galactose and amino acids. Variations between al. (338) to be primarily associated with the phospholipid levels and enzyme activities in plasma membrane in S. cerevisiae. Ready solu- yeast are not believed (30) to be linked by any bilization of this enzyme, however, may occur causal relationship. Evidence is available, how- (302). ever, for the protective action of phospholipid The mechanisms for modification of the fatty against oligomycin action on yeast mitochon- acid composition of glycerophospholipids appar- drial adenosine 5'-triphosphatase (56, 273). Sub- VOL. 39, 1975 LIPIDS OF YEASTS 209 stantiation has not been obtained for a possible complex sphingolipid containing inositol, phos- correlation between the total cellular phospho- phorus, mannose, and high levels of 2-hydroxy- lipid component in B. dermatitidis (65) and H. hexacosanoic and hexacosanoic acids was deter- capsulatum (213) and virulent properties to- mined (217) to be present in the cell wall and ward mice. plasma membrane of baker's yeast. C18-phyto- sphingosine was determined (315) to be the ma- Sphingolipids jor and dehydrophytosphingosine and dihydro- Free phytosphingosine bases containing 18 C sphingosine were determined to be the minor atoms and 20 C atoms in the ratio of 9:1 have long-chain base components. The possible occur- been found (156) to account for 2 to 3% of the rence of other inositol sphingolipids, which are total lipid of Candida intermedia, whereas stable to mild alkaline methanolysis, has been fully and partially acetylated derivatives ofphy- considered (283). Four monoinositol phospho- tosphingosine and dihydrosphingosine have rylceramide components have been identified been reported (100, 286, 336) inHansenula cifer- (272) in S. cerevisiae. Preliminary studies (10) rii. More generally, however, sphingosines oc- have shown that phosphosphingolipids have a cur as integral units of several yeast lipids metabolic origin in PI. ranging in structure from the relatively simple Biological significance. The role ofsphingo- cerebrins to the complex glyco- and inositol lipids in yeast is unknown. It has been sug- phosphoryl sphingolipids. A variety of sphingo- gested (235) that the presence ofacetylated phy- sines, dihydrosphingosines, and phytosphingo- tosphingosine on the cell surface of H. ciferrii sines have been determined as components of may be responsible for the tendency ofthe yeast sphingolipids in Torulopsis utilis (277), C. to form pellicles in liquid media. utilis (327), and S. cerevisiae (315, 328). The mechanisms for possible metabolic interrela- Glycolipids tionships between the various sphingolipids In addition to the complex glycosphingolipids have been discussed by Gatt and Barenholz small quantities of other glycolipids have been (89). determined in S. cerevisiae (17, 28, 217, 315). At Chemical nature. Mixtures of ceramides least four different acyl glucose derivatives (cerebrins) consisting of various phytosphingo- have been noted (38). Glycolipids are character- sine and dihydrosphingosine derivatives have ized by a relative abundance of odd-chain and been identified in T. utilis (277). The material >C08 fatty acids (17, 217). The presence ofsteryl was characterized by the presence of C18 to C26 glycosides, sulfolipids and acyl glucoses has saturated and a-hydroxy acids with 2-hydroxy- been observed (315) in the cell envelope of S. hexacosanoic acid predominating. The various cerevisiae. Steryl glycosides appear to be re- cerebrins isolated from S. cerevisiae contained stricted to the plasma membrane whereas mon- n-hexacosanoic acid, 2-hydroxyhexacosanoic ogalactosyl diglycerides and sulfolipids are dis- acid and (+)erthyro-2,3-dihydroxyhexacosanoic tributed throughout the cell (17). acid accompanied by minor quantities of C2 to Acyl glucose, which has been noted to occur C27 saturated unbranched 2,3-dihydroxy acids intracellularly when glucose is present in the (123, 238, 335). growth medium, has been suggested as being Simple cerebroside-type lipids (O-glycosyl cer- involved in the storage or transport of glucose amide) are generally present in only trace quan- in yeast (38). tities but consist primarily of a sphingosine base, 2-hydroxyoctadecanoic acid, and D-galac- FACTORS INFLUENCING CELLULAR tose as isolated from S. cerevisiae (328) and C. LIPID COMPOSITION utilis (327). High levels of hexacosanoic and 2- hydroxyhexacosanoic acids characterized a gly- Growth Cycle cosphingolipid present in the cell envelope of A variety of conditions influence the growth baker's yeast (217). Some species of Torulopsis, of yeast and must be taken into account in any Candida, Cryptococcus, and Rhodotorula are meaningful assessment ofthe cell lipid composi- highly efficient in the production of glycolipids tion. In closed systems, such as batch cultures, which are subsequently excreted into the me- the composition of the medium is modified by dium (285). each successive generation of cells and must Several inositol phosphoryl sphingolipids are ultimately result in unbalanced growth. Steady present (272, 283) in S. cerevisiae including states, however, can be obtained when the mannosyl di-(inositol phosphoryl) ceramide yeast is grown in highly controlled open sys- which has been determined (284) to account for tems, e.g., the computer-controlled chemostat. approximately 20% of lipid-soluble inositol. A The considerable advantages associated with 210 RATTRAY, SCHIBECI, AND KIDBY BACTERIOL. REV. the use of synchronous cultures have recently of sporulation. A correlation has been noted been reviewed by Hartwell (111). (130) between increasing sterol level and the Initial stages of growth of S. cerevisiae in formation of membranous systems in develop- batch culture have been found (44) to involve a ing asci. Two main periods of particular lipid progressive decrease in total lipid, mainly at synthesis have been observed (119, 130) during the expense of the triglyceride component. sporulation. The initial phase is predominantly Thus the energy requirements of the develop- associated with the formation of phospholipid ing cell would appear to be met, in part at least, and triglyceride which coincides with mem- by fatty acid oxidation. Increase in lipid produc- brane development. The relative proportions of tion has been noted during the late exponential the various phospholipids remain unaltered phase of growth of S. cerevisiae (44) and C. (130). Synthesis of primarily neutral lipid oc- lipolytica (218, 311). Entrance to the stationary curs in the second phase and coincides with the phase, however, results in a rapid decrease in appearance of mature asci. Abundant lipid total lipid content due to depletion ofthe carbon granules are apparent in the germinating asco- source (61). Cellular senescence arising from spores but diminish during several cell division carbon source deprivation has been associated cycles in the transition from spore to vegetative (202) with the induction of lipid peroxidase ac- cell (280). tivity. Subsequent peroxidation might be antici- Certain hydrocarbon-utilizing ascosporogen- pated to produce a lowering of membrane poly- ous yeasts, e.g., Endomycopsis lipolytica, may ethenoid acid content and hence biological activ- sporulate as a result of contact between the ity. The unsaturated fatty acid component of cells and growth medium alkanes. This process microsomal and supernatant fractions varies may be a special characterization associated with the state of cell growth, ultimately reach- with hydrocarbon utilization (177). ing a value of 70% of the total acid in the late exponential phase (45). Increase in cell wall Carbon Source unsaturated fatty acid accompanies the growth In addition to various environmental factors, of C. tropicalis on n-alkanes but not glucose the nature of the carbon source has a pro- (253). A high content of polyunsaturated fatty nounced effect on the quantity and composition acid has been determined (311) to be associated of yeast lipid. with the increased quantity of triglyceride Glucose. Yeasts growing on glucose as car- present in C. lipolytica entering the stationary bon source show various responses which re- phase of growth. flect differences in metabolic behavior. A broad Decreased growth rates, as determined with categorization into Crabtree-positive (respira- chemostat cultures of C. utilis, have been ob- tory-deficient) and Crabtree-negative (res- served (61, 192) to result in increased quantities piratory-sufficient) yeasts has been suggested of polyunsaturated acids, particularly at the by De Deken (62). The Crabtree-positive yeasts, latter stages of growth. In S. cerevisiae, growth exemplified by many members of the genus rate, at least as influenced by temperature, Saccharomyces, metabolize glucose primarily has been found (128) to have no effect on fatty via glycolysis. On the other hand the Crabtree- acid composition. Increased synthesis ofPC and negative yeasts generally lack glycolytic en- steryl ester, however, results from lowered zymes (205) and oxidize glucose via aerobic me- growth rate. These general alterations in lipid tabolism in which the pentose phosphate path- patterns may reflect differences in the develop- way is of major importance (198). The two yeast ment of intracellular membranous systems. categories, therefore, show different capacities Further studies in this area would be profita- in the utilization of increasing concentrations ble. of glucose in the growth medium. Thus Crab- tree-positive yeasts exhibit diauxic growth in Sporulation which glucose fermentation and ethanol produc- Sporulation in the diploid yeast S. cerevisiae tion occur initially followed by a second phase is markedly dependent upon the state of cell involving oxidation of the accumulated ethanol growth and the composition ofthe medium (82). (323). Catabolite repression can occur in the The process is accompanied by an increase in Crabtree-positive yeast but is of little signifi- cellular lipid (50, 130). Unsaponifiable matter cance in Crabtree-negative yeasts growing on constitutes the major lipid components at the media containing less that 10%o glucose (73, initiation of sporulation, particularly with cells 271). in the exponential phase of growth (50). Sterol The quantities of total lipid, steryl ester, and can comprise up to 81% ofthe total lipid and has phospholipid have been found (138) to be de- been ascribed (50) a special role in the induction creased in S. cerevisiae, a Crabtree-positive VOL. 39, 1975 LIPIDS OF YEASTS 211 yeast, as the glucose concentration of the incu- while n-alk-l-enes are used less efficiently and bation medium was increased from 2 to 10 by fewer yeasts (160, 246). Variations in growth g/liter. These lipid changes were accompanied response have been noted (236) between two by a slight increase in unsaturation ofthe fatty strains of C. lipolytica utilizing specific hydro- acid component. The fatty acid pattern of the carbons. In general C10 to C18 n-alkanes show glucose-repressed cell would appear to be inter- little toxicity toward yeast due to low water mediate between those described by Jollow et solubility (91). The dispersed state, however, of al. (140) for aerobically and anaerobically the substrate hydrocarbon influences yeast grown S. cerevisiae. An active synthesis ofphos- growth, and is best achieved by use of a Vortex pholipid has been noted (135) during glucose fermenter. Hydrocarbon assimilation appears repression in S. cerevisiae. The process of de- to require intimate association between yeast repression was accompanied by a drastic reduc- cells and efficiently emulsified hydrocarbon tion in phospholipid biosynthesis. The quantity and air droplets (34, 36, 96, 330). The growth of total phospholipid, however, remained unaf- rate ofa Candida sp. has been shown (245) to be fected, although the PE component was par- markedly increased in the presence ofhigh con- tially replaced by PS. A decrease in the amount centrations of n-alkanes. Compared to glucose ofDPG has been noted (39) at very high glucose as carbon source, growth of Candida 107 on concentration (50 g/liter), but this effect has various n-alkanes gives a lower yield of cells been attributed rather to a simultaneous state (311). A lower specific growth rate has also been of anaerobiosis (178). observed (73) for C. tropicalis incubated with n- The Crabtree-negative yeasts, including hexadecane compared to glucose. In particular many members of the genus Candida, show a the utilization of hydrocarbons by yeast ap- tendency to accumulate lipid when the glucose pears to be more heavily dependent upon an concentration of the medium is increased (14, active respiratory system and the availability 138). High quantities of triglyceride, in excess of oxygen than does the metabolism of glucose of 80% of the total lipid, have been observed in (73). The main factor influencing the yield of glucose-grown L. starkeyi (296), and C. lipolyt- yeast incubated with hydrocarbon is probably ica, C. tropicalis, C. utilis, Candida 107, Han- the capacity to utilize the various initial prod- senula anomala, Rhodotorula glutinis, and ucts of metabolism (207). Rhodotorulagraminis (311). The major accumu- Utilization of n-alkanes by yeast has been lating triglyceride species in L. starkeyi, Can- determined (95) to be a two-stage process. Al- dida 107, and R. graminis has been determined kane metabolism is initially associated with (295, 296, 311) to be saturated-unsaturated-satu- growth and then follows a zero order reaction. rated. A slight increase in the quantity of total The precise mechanism of n-alkane uptake has phospholipid occurs in glucose-grown L. star- not been established, but alkane-grown cells keyi and involves increased proportions ofPC at show a greater capacity to assimilate hydrocar- the expense of PS. The major molecular species bon than glucose-grown cells (92). Metabolic of PC and PE have been found to be 1-C16:l, 2- conversion ofup to 25% ofthe assimilated hydro- C18:1 and 1-C18:1, 2-C18:2 and to be considerably carbon can occur (246). Fatty acid is the major different from those present in cells grown in product of metabolism although small quanti- the absence of glucose. The PS component has ties ofn-alkanols may also accumulate (33, 158, been identified as almost entirely 1-palmi- 160). Pathways of alkane degradation have toyl - 2 - oleoyl - sn - glycero - 3 - phosphoryl - L - been observed to occur both in the cytoplasm serine (295, 296). Crabtree-negative yeasts and the mitochondria (166) and may involve possess the general capacity to synthesize poly- several types of mechanism (160, 166). Alkane unsaturated fatty acids, especially at high con- oxidation is an adaptive process in C. lipolytica centrations ofglucose and oxygen (14, 138, 296). (141) but is apparently dependent on both adap- These acids are primarily associated with the tive and constitutive enzyme systems (87, 167, phospholipid component ofL. starkeyi (295). 168). Yeast possesses a limited capacity for the Hydrocarbons. Considerable attention has utilization of intracellular fatty acid and may been focused on the growth of yeast on by- excrete excess free acid into the medium (321). products of the petroleum industry. Although A competition has been observed (27) between the capacity ofutilize hydrocarbons is generally hydrocarbon and short-chain (C12) fatty acid in nonexistent in Saccharomyces and many other the growth of C. tropicalis. yeasts (185, 261), it is an efficient process in Quantitative rather than qualitative differ- certain genera, especially Candida and Toru- ences have been found in the cell lipid ofseveral lopsu (33, 160, 261). Growth preferentially oc- yeast strains grown on n-alkanes (233). Growth curs on n-alkanes with chain length C9 to C18 of various members of Candida on n-alkanes 212 RATTRAY, SCHIBECI, AND KIDBY BACTERIOL. REV. compared to glucose has been variously re- The composition of lipid obtained after ported to result in greater (201, 218, 245) or growth of C. tropicalis on individual n-alkanes lower (51, 201, 311) quantities of total lipid. is shown in Fig. 6. Apart from some presently These differences may reflect varying meta- inexplicable deviations, increasing alkane bolic capacities of different strains, variations chain length generally results in increased in environmental conditions or a combination amounts of triglyceride but decreased quanti- of these effects. In addition an accurate quanti- ties of (sterol + partial glyceride) and (steryl tation of the lipid must be made by ensuring ester + wax). The phospholipid content appears the complete chromatographic removal of any to be independent of substrate hydrocarbon ex- contaminating hydrocarbon (311). cept for the very high value observed with The nature of the fatty acid component of growth on n-pentadecane. The quantity of tri- cellular lipid reflects (Fig. 5) the composition of glyceride in Candida 107 grown on various n- the alkane substrate (122, 126, 201, 233, 245, alkanes has been shown (108, 311) to be consid- 321, 326) as well as the growth phase ofthe cells erably lower than the occurring in glucose- (201, 321). Shorter-chain ( C, TRIGLYCERIDE PHOSPHOLIPID STEROL STERYL ESTER t 10 - 4 GLYCERIDE + WAX in 1 I 0.0 So 10 6O Ol_, ...... z .- 0 z 2401 J 0 * SATURATED ED MONOETHENOID Q POLYETHENOID FIG. 5. Relationship between cellular fatty acid ALKANE SUBSTRATE (number of carbon atoms) composition obtained after growth ofC. tropicalis on FIG. 6. Lipid composition obtained after growth individual n-alkanes and glucose (201). Cells were of C. tropicalis on individual n-alkanes (311) or a harvested during the late exponential growth phase. mixture (M) of (C14 to C20)n-alkanes (102). VOL. 39, 1975 LIPIDS OF YEASTS 213 An increase in cellular phospholipid is Candida sp., grown on n-alkanes, has been achieved with yeast grown on n-alkanes rather observed (245) to occur in a nitrogen-deficient than on glucose (311). The composition of the medium. Similar general findings have been phospholipid derived from C. tropicalis grown obtained (343) on decreasing the level of the on C04 to C20 n-alkanes has been determined nitrogen source for Endomycopsis vernalis. (64) to be (as percentage of the total) 41% PC, Phosphorus. Phosphorus is an essential 37% PE, 8% PS, 5% LPC, 10%o fully acylated growth factor for yeast (293). Increasing the DPG, and 5% fully acylated PG. A higher level phosphate concentration of the medium has of unsaturated and short-chain fatty acids is been found (339) to have little effect on the associated with phospholipid than with triglyc- phospholipid composition ofS. pombe. Compari- eride (64, 312). Heptadeca-9,12-dienoic acid oc- son of phosphate-limited with carbon-limited curs primarily at position 2 of the phospholipid cultures of S. cerevisiae has shown (137) in- molecule (64). A consequence of the increased creased amounts of total lipid mainly as triglyc- phospholipid level in yeast grown on n-alkanes eride in the phosphate-limited culture. Only is the observed proliferation of the cell plasma small alterations were apparent in the composi- membrane and intracytoplasmic elements (179, tion of the polar lipids and fatty acid. In the 207, 222). It has not been established whether case of phosphate-limited C. utilis, however, the observed (179) thickening of the plasma the increased content of lipid had an altered membrane and the development of deep inva- fatty acid pattern and the major phospholipid ginations are a requirement for, or a conse- components were largely replaced by three uni- quence of, hydrocarbon being the sole carbon dentified phosphate-free polar lipids (137). source. Extension of the cell surface area could Growth factors. Different yeast species have result in more efficient assimilation of the sub- highly specific vitamin requirements (293). Defi- strate (330). In addition the hydrophobic nature ciency in certain of these growth factors can of the plasma membrane may govern the trans- lead to pronounced alterations in the cell lipid. port of hydrocarbon into the cell and could con- (i) Inositol. Deficiency in inositol results in stitute the basis for the varying growth re- increased quantities of lipid, primarily as tri- sponses of Candida and S. cerevisiae (27). The glyceride, in Saccharomyces carlsbergensis several advantages of examining membranous (139, 172, 225, 267). The phospholipid compo- systems derived from alkane-grown yeasts nent remains essentially unaltered except for a have been stressed (220, 270, 271). Variations in decrease in PI. The accumulated lipid has been the fatty acid composition of mitochondria ob- observed (172) to occur as globules in the cell tained from yeast grown on different n-alkanes cytoplasm and to be accompanied by an appar- have been found (270, 271) to influence mem- ent alteration in the nuclear membrane. Inosi- brane fluidity and enzyme activities. Further tol deficiency influences the composition but exploitation of alkane growth will be ofvalue in not the quantity of cell wall lipid in S. cerevis- studies on membrane structure-activity rela- iae (237). Johnston and Paltauf (139) have con- tionships. sidered the consequence of inositol deficiency to be due to a more active fatty acid synthetase Nutrients system. (ii) Pantothenic acid. Omission of panto- The general aspects of yeast nutrition have thenic acid from the culture medium results in a been discussed by Suomalainen and Oura (293). marked decrease in the total lipid content ofS. Certain nutritional factors have been recog- cerevisiae (86, 124) and H. valbyensis (112). The nized to have a particular influence on cell lipid reduction in the quantity of the phospholipid composition. component has been reported (86, 124) to be Nitrogen. All yeasts are capable of utilizing accompanied by various changes in the unsatu- ammonium salts, especially ammonium sul- rated fatty acid content. These lipid disturb- fate, as a source of nitrogen (293). Members of ances have been associated with impaired mito- the genus Saccharomyces, however, differ from chondrial development as determined from elec- C. utilis in being incapable of assimilating ni- tron microscopic examination (86) and de- trates (293). Comparison of the influence of creased quantities of mitochondrial DPG and NO3- and NH41 revealed (102) that the more PE (124). Pantothenic acid deficiency has been highly oxidized nitrogen source resulted in a suggested (86) to be very similar to an anaero- lower growth yield of C. tropicalis. The lipid bic state with respect to mitochondriogenesis. content was characterized by increased levels of (iii) Vitamin B6. Decreased total lipid and a triglyceride and a partial replacement of 18:2 marked reduction of 16:1 acid has been ob- acid by saturated fatty acid. The highest served (112) with H. valbyensis grown in the growth yield and production of fatty acid for a absence ofvitamin B6. Although the total phos- 214 RATTRAY, SCHIBECI, AND KIDBY BACTERIOL. REV. pholipid level was unaltered, PI and phyto- Oxygen sphingolipid were partially replaced by PC and Oxygen has a pronounced effect on the PE. An intimate relationship between vitamin growth, general metabolism, and lipid composi- B6 and the biosynthesis of unsaturated fatty tion of yeast. The more rigorously controlled acid and certain phospholipids has been consid- system of chemostat culture is best employed to ered (112). Studies (215) made on S. carlsber- achieve oxygen-deficient conditions which can gensis suggest that the effect of thiamine in be then studied independently of other growth producing lowered quantities ofcellular unsatu- factors. Knowledge of the history of the inocu- rated fatty acid is a consequence of induced lum is also critical to the proper interpretation vitamin B6 deficiency. of either microaerobic or anaerobic cultures. (iv) Biotin. Impaired growth and lowered Yeasts have been broadly classified into "respi- levels of C06 and C08 acids resulted from the ratory-sufficient" and "respiratory-deficient" growth of S. cerevisiae on a biotin-deficient strains when considering the predominant medium (289). Unsaturated fatty acids have mechanism for energy derivation and require- been considered to have a biotin-sparing effect ment for molecular oxygen (62). Many studies since their addition to the incubation medium on the development of a respiratory-deficient to permitted normal growth. a respiratory-sufficient state have been made on the facultative anaerobe S. cerevisiae and Miscellaneous Additives have been reviewed by Linnane and his co- workers (174, 175). Sodium chloride. Growth of C. albicans in Respiratory-deficient (Crabtree-positive) the presence of increasing concentrations (O to yeasts. (i) Lipid composition. The general dif- 10%) of NaCl has been noted (55) to inhibit cell ferences occurring in the lipid composition ofS. growth and to produce considerable elevation of cerevisiae as a consequence of growth under the lipid content (0.3 to 6.3% ofcell weight). The aerobic and anaerobic conditions are shown in associated decrease in unsaturated fatty acid Fig. 7. The lipid of anaerobically grown cells involved mainly 18:1 acid. has a lower total level, a highly variable glycer- Choline and ethanolamine. Supplementa- ide fraction, decreased phospholipid and sterol tion of the growth medium ofS. cerevisiae with components, and increased hydrocarbon con- choline has been shown (224) to increase the tent. The major changes in the phospholipid synthesis of phospholipid mainly as PC. The composition of anaerobic cells are depicted in pathway ofPC synthesis involving the methyla- tion ofPE (Fig. 4) appears to be repressed under these conditions (324). On the other hand only small increases in total phospholipid and PE occurred on medium supplemented with etha- nolamine (224). Benzo(a)pyrene and dibenzanthracene. Incubation of S. cerevisiae under aerobic or anaerobic conditions with small quantities (0.15 to 5 mg/liter) ofthe carcinogenic hydrocar- bon benzo(a)pyrene has been found (18, 210) to reduce the cell lipid content. A particularly drastic reduction of the phospholipid compo- nent to 10% of the normal value occurred. De- LE creased levels of all phospholipid classes in both II' whole cells and protoplast membranes was par- tially compensated by an increase in the mono- glyceride component. Similar, but less pro- nounced, effects were observed with the weakly carcinogenic agent dibenzanthracene(ac). Propanediol. Good growth of L. starkeyi on PHOSPHOLIPD STEROL propane-1,2-diol can be obtained after "train- GLYCERIDE HYDROCARBON diol of ing" (294). The presence of analogues FIG. 7. Lipid composition obtained after growth glycerophospholipids has been deduced, but no of S. cerevisiae under aerobic (44, 128, 140, 162), evidence was obtained for the formation of sim- anaerobic (140, 162) and lipid-supplemented anaero- ple diol fatty acid ester. Minor quantities of bic (140,162) conditions. Cells were harvested during these diesters have been noted in soil yeasts mid-exponential-early-stationary growth phase. (28). Vertical bars represent range ofobserved values. VOL. 39, 1975 LIPIDS OF YEASTS 215 Fig. 8. Lowered DPG and PE and incrreased PC accompanied by a decrease in the quantities of and PI levels occur and are particularlLy obvious cellular unsaturated fatty acid and a sterol to on entering the stationary phase of girowth (90, approximately 1/4 the values of aerobically 140). The observed (132, 171) high c ontent of growing cells (98). Supplementation of anaero- lysoderivatives in anaerobically gnown cells bically growing S. cerevisiae with ergosterol may be attributed to phospholipase activity. dispersed in Tween 80, which also serves as a Whereas 80 to 90% of the fatty acid coomponent source ofoleic acid, alters cellular lipid composi- associated with glyceride and phosp]holipid in tion as shown in Fig. 7. Although the trend is aerobically grown S. cerevisiae has been found toward increased glyceride content and appar- (140) to be 16:1 and 18:1 acids, thee lipid of ent replacement of hydrocarbon by sterol, the anaerobic cells is characterized by a high con- amount and composition of phospholipid re- tent (up to 50% of the total acid) of E3:0 to 14:0 mains unaltered. Both the glyceride and phos- acids and a low level of unsaturated fatty acid pholipid fractions are characterized by the in the phospholipid fraction. replacement of the short-chain fatty acid com- Differences in lipid composition ofaerobically ponent by high levels of 18:1 acid (140). Asso- and anaerobically grown S. cerevisuze are re- ciated with these lipid changes is the appear- flected in the state of mitochondrialI develop- ance of mitochondria-like structures (140, 329). ment. Specifically the decreased content of Promitochondria, in contrast to the whole cell, DPG in anaerobic cells can be takLen as an have been found (226) to possess a markedly indication of impaired mitochondrial synthesis decreased sterol component but a significantly (132, 171, 226). Promitochondria hiave been increased phospholipid level on lipid supple- noted (226) to be characterized by decreased mentation of the incubation medium. The PI DPG and PE levels and elevated PI content. In content was reduced but the values for DPG, addition a particularly low amount of ergos- PC, and PS approximated those observed with terol is present (226). The high levelIs of satu- developed mitochondrio (226). rated and short-chain fatty acids iassociated The lipid requirement of anaerobically grow- with the lipids of promitochondria (97, 226, 331, ing cells can be satisfied, but a source of fatty 332) have been found (332) to be rapidly re- acid by itself is not sufficient (8, 9, 59). Growth- placed by 16:1 and 18:1 acids after aerc)bic induc- promoting activity is most pronounced with tion of mitochondria over a period off 30 to 120 oleic and linoleic acids (9) and less efficient with min. A marked increase also occurs iin the level linolenic acid, octadec-9-ynoic acid, and 11,12- of mitochondrial phospholipid after aLeration of methylene stearic acid (9, 173). Increasing the anaerobically grown cells (320). Chiange from degree of fatty acid unsaturation may extend anaerobic to aerobic conditions restilts in in- the exponential phase of growth but produces creased membrane fluidity (31) and thie develop- no difference in growth rate or quantities of ment of mitochondrial function (178) total lipid and phospholipid (7). A high propor- (ii) Lipid supplementation of grrowth me- tion (54 to 65%) of the cell fatty acid has been dium. Oxygen deficiency is generadly recog- shown (7) to be derived directly from the acid nized (8, 9) as the reason for sterol aiid unsatu- supplement. Comparison of cells grown on oleic rated fatty acid auxotrophy in S. icerevisiae. or linoleic acid-supplemented media revealed The inhibition of growth obtained on;unsupple- that the cytoplasmic membrane resulting from mented media under anaerobic coIaditions is the more unsaturated acid was more suscepti- ble to osmotic lysis (7). 50 A definite requirement for sterol supplemen- tation appears to exist (8). Addition of 10 ppm .r__- U Aerobic (10 pg/ml) of ergosterol has been determined Anaerobic (59) to replace the normal oxygen requirement -5 B of various strains of S. cerevisiae. Generally, 0 25- any sterol possessing the hydroxyl group at I position 3 with axial orientation is effective in U) 0 permitting anaerobic growth (242). A further degree of specificity would, however, appear to H. I E~Th exist as yeast grown under anaerobic condi- e1nF 11 M n tions and cholesterol supplementation showed PS PI PE PC DP)G OTHER considerable replacement of the C27 sterol by FIG. 8. Phospholipid composition obitained after ergosterol after aeration (97). Studies (41) on growth of S. cerevisiae under aerobic (910, 128, 140) Schizosaccharomyces japonicss, which is not and anaerobic (90, 140) conditions. Vvertical bars auxotrophic for ergosterol, have revealed poly- represent range of observed values. ene sensitivity during aerobic growth and resist- 216 RATlRAY, SCHIBECI, AND KIDBY BACTERIAL. REV. ance under anaerobic growth. These findings yeasts generally show a tendency to raise the can be interpreted as being suggestive of a lipid content and degree of unsaturation as the change in the composition of the plasma mem- environmental temperature is dropped below brane sterol component. Differences between that for optimal growth. strains of S. cerevisiae with regard to their Decrease in the growth temperature from 25 oxygen requirement may reflect variations in to 10 C for the mesophile C. lipolytica has been sterol composition rather than the total sterol determined (147, 148) to result in increased content (59, 60). A close correlation has been lipid and a higher ratio of linoleic acid to oleic suggested (230) between loss ofrespiratory com- acid. The relative levels of linoleic and oleic petency and suppression of sterol synthesis in acids dropped to the values of the inoculum at yeast. Impaired sterol biosynthesis could be a the end of active growth at 25 C. A similar re- primary consequence of oxygen deficiency (29, adjustment was incomplete at 10 C (Fig. 9). 59). The general assumption has been made (84, Growth of C. utilis in either batch culture (80) 157, 309) that the oxygen-sensitive process is in or in the chemostat at fixed growth rate (40, the conversion of squalene to ergosterol. De- 193) results in higher proportion of16:1 and 18:3 pressed 3-hydroxy, 3-methyl-glutaryl CoA re- acids at 10 C compared to 30 C. The specific ductase has also been observed (29) in anaerobic alterations in 16:0, 18:1 and 18:2 acids are sub- cells but would be expected to result in a lower ject to limitation of glucose or nitrogen in the quantity of squalene as well as of sterol. Expo- medium (193). A pronounced increase of 18:2 sure of anaerobic cells to oxygen appears to acid has been determined (102) in C. tropicalis cause rapid enzyme induction for ergosterol bio- grown at 28 C compared to 38 C. Cyclic varia- synthesis (29). The rate offormation ofboth free tion of 18:1 and 18:3 acids has been observed and esterified sterol, however, proceeds at a (193) in a psychrophilic Candida sp., but is slower rate than in aerobically grown cells (181). Respiratory-sufficient (Crabtree-nega- 60 ok25'C, ,,A 00IOC\ Ad| 2 A has been tive) yeasts. general conclusion so O\/ 0 reached (73, 250) that the rates of synthesis of cellular components in strains of Candida are 40 0 T O controlled by the capacity of the respiratory pathways. Thus the availability of oxygen gov- 240 lA A 0.4 erns the ability ofC. utilis to synthesize polyun- 026 saturated fatty acid (14, 40). At high oxygen 0 C, tension the occurrence of linolenic acid was 10 2~~~B0 greatest (14), whereas at low oxygen the degree 60 of unsaturation decreased and greater concen- trations of C16 accumulated at the expense of 50 C18 acids (40). Growth of C. -lipolytica on hex- 6 0 adec-1-ene at low oxygen tension resulted in the 301 incorporation offatty acid derived directly from 40 20 oxidation of the hydrocarbon. Formation of C16 20 and C18 acids predominated at higher aeration 7 rates (159). It would, therefore, appear that low oxygen inhibits both the elongation and oxy- gen-dependent desaturase systems. The fatty acid patterns of total lipid and phospholipid of C. utilis grown under low oxygen have been found (14) to be similar. The mitochondrial con- tent ofoleic and linoleic acids inCandidaparap- silosis grown under microaerobic conditions 0 4 S 2 16 0 S 16 24 32 40 46 was unaltered, although the quantity of oleic TIME, HOURS in was acid the whole cell depressed compared FIG. 9. Changes in fatty acid composition of (A) to aerobic cultures (250). total lipids, (B) phosphatidyl choline, and (C) phos- Temperature phatidylethanolamine at25 C and 10 C. (Reproduced from reference 148 by kind permission of M. Kates The influence of temperature on the growth and the Canadian Journal of Biochemistry.) Sym- and metabolism of yeasts has been reviewed bols: growth curves (0), linoleic acid (0), and oleic (287). In common with most living organisms, acid (A). VOL. 39, 1975 LIPIDS OF YEASTS 217 temperature independent. No marked altera- has been found (70) to be most marked at pH tion in fatty acid composition occurs on decreas- 5.6. Examination ofC. lipolytica showed (71) an ing the growth temperature of S. cerevisiae increase in unsaturated fatty acid and polygly- (128), which, in any case, appears incapable of cerophospholipid and a decrease in PC and PE synthesizing polyunsaturated fatty acid (136). at lower pH values. The production of ergos- The desaturase system involved in unsaturated terol in C. tropicalis growing on n-alkanes in fatty acid formation has been found (196) to be pH dependent (303). Significantly lower quanti- stimulated in C. utilis, a Crabtree-negative ties of total lipid occur in L. lipoferus (347) and yeast, at lower temperatures. Decrease in the R. glutinis (348) at pH values not supporting hypothetical mean melting point of the cellular optimal growth. acids may also be achieved at lower tempera- tures through an increase in the content of CONCLUDING COMMENTS TABLE 5. General effects produced by culture factors on the lipid content and composition ofyeasts ~~~~~Total Phos- Ster- Factor Condition I Reference lipid pholi- ol Unsaturation pid Organism Species; strain a a a a Growth rate Decreased 128, 192 (+)b ( (+)(++), 0)b Development (i) Lag and early exponen- 44, 45 (-)b (+) 0 (+) stage tial phases (ii) Laterandpostexponen- 44, 45, 192, 218, 311 (+) (-) 0 (-) tial phases (iii) Sporulation 50, 119, 130 (+) (+) (+) (+) Carbon source (i) Increased glucose level Crabtree positive 39, 138 (-) (-), 0 (-) (-) Crabtree negative 14, 39 (+) 0, (+) (+) (+) (ii) Hydrocarbon level (compared to glucose level) Crabtree positive 185, 246, 261 C C C C Crabtree negative 201, 218, 245, 246 (+) (-) 51, 108, 201, 311 (-) (+) (+) Reflects sub- strate chain length Nitrogen Decreased level 245 (+) source Phosphorus Increased level 293 (+) 0 0 source Growth fac- (i) Inositol or nicotinic 112, 139, 172, 225, 267 (+) 0 tors acid deficiency (ii) Biotin, pantothenic 86, 112, 124, 289 (-) (-) acid or vitamin B6 de- ficiency P02 W High (aerobic) 14, 44, 128, 140, 162 (+) (+) (+) (+) (ii) Low (anaerobic) 40, 140, 162 (-) (-) (-) (-) pCO2 High 47 (+) (+) pH More acidic 47, 71, 347, 348 0, (-) 0 0 (+) Temperature Decreased from 25 to 40,128, 147, 148, 193 (+) (+) (-) (+) 10 C a Considerable variations may occur. b (+) Enhanced; (-) depressed; and (0) generally unaffected. c Little or no growth. 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