Journal - Physiology and Genetics of The Dimorphic Fungus Yarrowia Lipolytica

FEMS Microbiology Reviews 19 (1997) 219^237
Physiology and genetics of the dimorphic fungus

Yarrowia lipolytica
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Gerold Barth a *, Claude Gaillardin b
;
a
Institut fuër Mikrobiologie, Technische Universitaët Dresden, Mommsenstrasse 13, D-01062 Dresden, Germany
b
Laboratoire de Geèneètique Moleèculaire et Cellulaire, Institut National de la Recherche Agronomique-Centre National de la Recherche
Scienti¢que, Institut National Agronomique Paris-Grignon, F-78850 Thiverval-Grignon, France
Received 15 September 1996; accepted 5 December 1996
Abstract
The ascomycetous yeast Yarrowia lipolytica (formerly Candida, Endomycopsis, or Saccharomyces lipolytica) is one of the
more intensively studied `non-conventional' yeast species. This yeast is quite different from the well-studied yeasts
Saccharomyces cerevisiae and Schizosaccharomyces pombe with respect to its phylogenetic evolution, physiology, genetics, and
molecular biology. However, Y. lipolytica is not only of interest for fundamental research, but also for biotechnological
applications. It secretes several metabolites in large amounts (i.e. organic acids, extracellular proteins) and the tools are
available for overproduction and secretion of foreign proteins. This review presents a comprehensive overview on the available
data on physiology, cell biology, molecular biology and genetics of Y. lipolytica.
Keywords: Yarrowia lipolytica ; Physiology; Cell biology; Cell cycle; Genetics
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
2. Natural habitats, taxonomy, and phylogenetic relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
3. Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
3.1. Assimilation of carbon sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
3.1.1. Utilization of hydrocarbons as carbon source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
3.1.2. Fatty acids biosynthesis and degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
3.1.3. Assimilation of alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
3.1.4. Assimilation of acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
3.2. Secretion of metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
3.2.1. Organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
3.2.2. Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
3.2.3. Secretion of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
* Corresponding author. Tel.: +49 (351) 463 7617;

fax: +49 (351) 463 7715; e-mail: [email protected]
0168-6445 / 97 / $32.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 1 6 8 - 6 4 4 5 ( 9 7 ) 0 0 0 0 2 - 8
220 G. Barth, C. Gaillardin / FEMS Microbiology Reviews 19 (1997) 219^237
4. Cell biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
4.1. Peroxisome biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
4.2. Secretion and genes involved in of the secretory pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
4.3. Dimorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
5. Cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
5.1. Mating and mating type alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
5.2. Sporulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
6. Genetics and molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
6.1. Mutagenesis and mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
6.2. Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
6.3. Genetic linkage groups and chromosome maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
6.4. ARS and centromeres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

6.5. Repetitive elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
6.5.1. Ribosomal RNA and other RNA genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
6.5.2. Transposons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
6.6. Features of protein encoding genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
6.7. Plasmids and VLPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
6.8. Mitochondrial genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
7. Genetic tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
9. Note added in proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
1. Introduction citric acid) when grown on these substrates [2].

Large-scale industrial production of citric acid or
In the last two decades the group of so called of SCP using Y. lipolytica thus permitted the accu-
`non-conventional' yeasts received more and more mulation of extensive data on its behavior in very
attention in fundamental research and biotechnol- large fermentors.
ogy. The term `non-conventional' was originally Three reviews have been already published which
used to di¡erentiate this group of yeasts from the describe the historical development of this research
9
more commonly used `conventional' and already 9 and summarize main data on physiology, genetics,
well studied yeasts Saccharomyces cerevisiae and, molecular biology, and biotechnological application
with some restrictions, Schizosaccharomyces pombe. of this fungus [3^5].
Nowadays about ten species among these non-con- This review will give a comprehensive overview on
ventional yeasts have been investigated more inten- the physiology and genetics of this yeast. It will also
sively with respect to their physiology, genetics, mo- show recent areas of interest which include both fun-
lecular biology, and biotechnical application. A damental and applied topics.
summary of these data and the special tools devel-
oped has been published recently [1].
The ascomycetous yeast Yarrowia lipolytica (orig- 2. Natural habitats, taxonomy, and phylogenetic
inally classi¢ed as Candida lipolytica) is one of the relationship
more intensively studied `non-conventional' species.
With the emergence of single-cell protein (SCP) proj- Y. lipolytica strains are readily isolated from dairy
ects in the mid-sixties, a strong industrial interest products, but also from shoyu or from salads con-
arose from the fact that strains of this species were taining meat or shrimps. The inability of this yeast to
able to use n-para¤ns (which were cheap and abun- survive under anaerobic conditions permits their easy
dant in this period) as sole carbon source. It was also elimination from dairy products, contrary to Kluy-
observed that Y. lipolytica was able to produce high veromyces marxianus strains for example [6]. An ex-
amounts of organic acids (2-ketoglutaric acid and tensive description of assimilated substrates is done
G. Barth, C. Gaillardin / FEMS Microbiology Reviews 19 (1997) 219^237 221
by Kreger-van Rij [7] and Barnett et al. [8]. Glucose, quence comparison of genes encoding well conserved
alcohols, acetate, and hydrophobic substrates like functions (glycolytic genes, ribosomal RNA genes)
fatty acids or alkanes, but not sucrose are used as locate Y. lipolytica on an isolated branch, clearly
carbon sources. separated from S. pombe on one hand, and from
The species was originally classi¢ed as a Candida, the bulk of other ascomycetous yeasts on the other
since no sexual state had been described. The perfect [16,17]. A truly positive aspect of this divergence is
form of C. lipolytica was identi¢ed in the late sixties that the observation of structural conservation be-
by Wickerham at the Northern Regional Research tween Y. lipolytica and other yeast genes is much
Laboratory of the USDA at Peoria. A culture iso- more likely to re£ect functional constraints than
lated in 1945 from a jar of ¢ber tailings in a corn when it happens between closely related yeasts.
processing plant was found to form asci attached to

hyphal elements when put on suitable media. One to
four spores of various size and shape could be iso- 3. Physiology
lated from these asci, but spore viability was found
to be very low. Two mating types, called A and B, 3.1. Assimilation of carbon sources
were identi¢ed among the progeny. Nearly all other
wild type isolates from the species would mate to one 3.1.1. Utilization of hydrocarbons as carbon source
of these two types, albeit at very low frequency, sug- Y. lipolytica can use n-alkanes and 1-alkenes as
gesting that most natural isolates are haploid (or carbon sources [18^21]. Polymethylated and chlori-
near haploid). The perfect form was reclassi¢ed ¢rst nated alkanes are also assimilated by this fungus
as Endomycopsis lipolytica [9], then as Saccharomy- [22,23]. However, only few enzymes are characterized
copsis lipolytica [10], and ¢nally as Yarrowia lipoly- and no gene involved in the early steps of degrada-
tica. tion of these compounds has so far been cloned.
Y. lipolytica is not considered as pathogenic [11]. The ¢rst step in the assimilation is likely to be an
Several observations suggested that Y. lipolytica emulsi¢cation at the cell surface to form small drop-
may have diverged considerably from other ascomy- lets which can be internalized. A 27 kDa extracellu-
cetous yeasts: high GC content, unusual structure of lar emulsi¢er, called liposan, is induced in cells grow-
rDNA genes coupled with a lack of RNA polymer- ing on n-alkanes [24,25]. Liposan contains 88%
ase I consensus sequences found in other yeasts [12], carbohydrate and 12% protein. After entry into the
higher eukaryotic-like size of snRNA [13], and of 7S cell, n-alkanes are hydroxylated by a cytochrome P-
RNA [14]. Homologous genes tend to display a low 450 monooxygenase system. Cytochrome P-450 was
level of similarity (typically in the 50^60% range at detected in several strains of Y. lipolytica [26^28].
amino acid level) with their counterparts in S. cere- The synthesis of this enzyme is induced during
visiae, K. lactis or C. albicans, thus hindering in most growth on n-alkanes, but not on glucose, ethanol
cases attempts to clone homologous genes by means or acetate. Di¡erences in sensitivity to glucose re-
of Southern hybridization. Naumova et al. [15] pression during growth on hexadecane and decane
showed that Y. lipolytica genes did not hybridize indicate the presence of di¡erent regulated cyto-
detectably with DNA from species of the genera Sac- chrome P-450 genes in Y. lipolytica [29]. Cytochrome
charomycopsis, Endomyces or Endomycopsella, some P-450 is localized in the membrane of the endoplas-
of which were formerly classi¢ed in the same genus mic reticulum as shown by subcellular fractionation
as Y. lipolytica. Similarly, only few genes of Y. lip- of alkane-grown cells of Y. lipolytica [29]. 1-alkanol
olytica seem to be directly expressed in S. cerevisiae, formed after the ¢rst step is further oxidized by a
suggesting that RNA polymerase II promoters and/ membrane-bound fatty alcohol oxidase [30,31], not
or associated transcriptional factors have diverged by NAD(P)-dependent alcohol dehydrogenases,
considerably. Recent data on 7S RNA genes, on in- which are also present in Y. lipolytica [32].
tron structure, or on ARS function con¢rmed that Several alkane-non-utilizing mutants have been
this species was quite peculiar when compared to isolated and characterized [33^36]. Uptake of n-alka-
most other yeasts. Evolutionary trees based on se- nes is inducible and due to active transport. Muta-
tions in 16 loci caused a signi¢cant reduction in n- tion include production of wax esters from fatty al-
alkane uptake [34]. Mauersberger [35] characterized cohols [45].
three subtypes of alkA mutants, which were unable The POT1 gene encoding a catabolic 3-oxoacyl-
to utilize either all types of n-alkanes (alkAa), or CoA-thiolase, which catalyzes the thiolytic cleavage
only short ones in the C8 to C12 range (alkAb), or of 3-oxoacyl-CoA thioesthers during L-oxidation of
only long alkanes in the C16 range (alkAc). These fatty acids, was cloned and characterized by Ber-
data indicate that Y. lipolytica contains either several ninger et al. [46]. Disruption of POT1 inhibited the
length speci¢c alkane-uptake systems or speci¢c cy- utilization of oleate but not the elongation of exter-
tochrome P-450 monooxygenases. nally added tridecanoic acid to higher-chain-length
homologues.
3.1.2. Fatty acids biosynthesis and degradation

Fatty acid biosynthesis and degradation were ini- 3.1.3. Assimilation of alcohols
tially investigated by Kamiryo et al. [37] and a few Y. lipolytica does not produce ethanol, but uses
reports were issued on the use of Y. lipolytica for the ethanol as carbon source at concentrations up to
stereospeci¢c conversion of hydroxylated long chain 3%. Higher concentrations of ethanol are toxic. Sev-
fatty acids into Q-lactones [38].
eral NAD - and NADP -dependent alcohol dehy-
Kohlwein and Paltauf [39] detected at least two drogenases were observed in Y. lipolytica [32]. There
fatty acid carrier systems in Y. lipolytica, one being

probably exist two NAD -dependent alcohol dehy-
speci¢c for C12 or C14 fatty acids, the other for C16 drogenases which di¡er in substrate speci¢city. Syn-
and C18 saturated or unsaturated fatty acids. Octa- thesis of both enzymes seems not to be repressible by
noic acid and decanoic acid are not taken up by this glucose or inducible by ethanol [32]. Three NADP -

yeast. Contrary to S. cerevisiae, an intracellular 100 dependent alcohol dehydrogenases were detected
kDa fatty acid binding protein is readily induced in which exhibited di¡erent substrate speci¢cities. The
Y. lipolytica when grown on palmitate [40]. A low occurrence of these enzymes varies depending on
molecular mass fatty acid binding protein (15 kDa) growth phase and carbon source of the media [32].
has been isolated from the cytosol of this yeast re-
cently [41]. 3.1.4. Assimilation of acetate
An anabolic acyl-CoA synthetase I activity was Most strains of Y. lipolytica grow very e¤ciently
found in peroxisomes, mitochondria and cytoplasm on acetate as sole carbon source. Concentrations up
and was shown to be required for the incorporation to 0.4% sodium acetate are well tolerated, higher
of exogenous fatty acids into cellular lipids. Mutants concentrations reduce the growth rate and concen-
de¢cient in this activity have been isolated [37]. trations above 1.0% inhibit the growth.
The gene FAS1, encoding the pentafunctional b- Several mutants have been isolated and character-
subunit of the fatty acid synthetase was cloned and ized, which are blocked in the utilization of acetate
sequenced [42,43]. Its overall structure was very sim- [33,36,47^50]. Nothing is known about the uptake of
ilar to that of S. cerevisiae. acetate by this yeast. Mutants blocked in the activity
A catabolic acyl-CoA synthetase II was found ex- of acetyl-coenzyme A synthetase were characterized
clusively in peroxisomes, and was required for the L- by Kujau et al. [36]. Acetyl-coenzyme A is needed for
oxydation of fatty acids. A mutant initially reported the induction of the glyoxylate cycle, which is not
as a¡ecting this activity was later shown to a¡ect induced in acetyl coenzyme A de¢cient mutants
another step (Kamiryo, personal communication). [36,47].
Five genes encoding acyl-CoA oxidases have been The induction of the glyoxylate pathway is neces-
identi¢ed and cloned (Nicaud, personal communica- sary for the utilization of alkanes, fatty acids, alco-
tion). hols and acetate. Isocitrate lyase, the key enzyme of
Use of Y. lipolytica cells for the production of Q- this cycle, and its encoding gene is well studied [49^
decalactone from alkyl ricinoleate (a derivative of 51,36,47,48]. The structural gene has been cloned
castor oil) has been patented by BASF and sequenced [52].
(DE4126997, 1993), [44,38]. Other possible applica- Dominant mutations in the gene GPR1 (glyoxylate
pathway regulator) [36] make the cells sensitive to do and Gaillardin, 1972). Furthermore, a haploid
low concentrations of acetate or ethanol even in strain selected as a suppressor mutant of a mutation
the presence of glucose. in methionine biosynthesis secretes very large
amounts of this organic acid during growth on glu-
3.2. Secretion of metabolites cose (patented by WeiMbrodt et al., 1988).
Production of 2-hydroxyglutaric and 2-ketogluta-
3.2.1. Organic acids ric acids from glucose has been described by Oogaki
3.2.1.1. Citrate, isocitrate. Wild type strains of et al. [61]. A process for very high yield production
Y. lipolytica secrete a mixture of citric and isocitric of isopropylmalic acid using a leucine auxotrophic
acid when grown on n-para¤ns as carbon source: a strain has been patented [62].
total yield of 130% compared with the substrate and

a ratio of 60:40 (citric to isocitric) was reported for 3.2.2. Lysine
strain ATCC 20114 by Akyiama et al. [53]. When the Mutations a¡ecting all 11 biosynthetic steps of
cells were supplied with mono£uoroacetate, this lysine except one have been identi¢ed. A single locus,
compound was transformed into mono£uorocitrate LYS1 encoding homocitrate synthase controls the
which competitively inhibited aconitase: this in ¢rst step [63,64]. The existence of isoenzymes hither-
turn improved the ratio of citric to isocitric acid to to prevented characterization of such mutants in S.
85:15. In an e¡ort to obtain a strain with low aco- cerevisiae. A detailed analysis of the LYS1 gene iden-
nitase activity, a mutant unable to use citrate as car- ti¢ed homocitrate synthase as a major check point of
bon source was ¢rst isolated, and further mutagen- the lysine biosynthetic pathway [65]. The LYS1 gene
ized in order to obtain a £uoroacetate sensitive has recently been cloned and sequenced [66]. Muta-
strain: this last strain yielded citric and isocitric acids tions at the LYS11 locus, unlinked to LYS1, were
in a ratio of 97:3, with a yield of 145% (w/w). initially isolated as suppressors of LYC1 mutations
Treèton and Heslot [54] observed that on glucose or [65]. They abolish completely homocitrate synthase
glycerol medium, the ratio of citrate to isocitrate was activity. It is unknown presently if LYS11 de¢nes a
92:8 vs 67:33 on n-para¤n. However the intracellu- regulator of LYS1, or a second subunit of homoci-
lar ratio of the two acids was roughly the same trate synthase.
(90:10) on all media. These authors further showed No mutant a¡ecting homoaconitate hydratase has
that both acids were equally well secreted into the been identi¢ed so far. This activity could not be pu-
growth medium, but that only citric acid was recon- ri¢ed away from aconitate hydratase. LYS6 and
sumed on para¤ns, whereas neither was reused on LYS7 mutations are defective in the third step of
glucose. the pathway (homoaconitase), thermosensitive mu-
Use of mutants unable to use acetate or citrate as tants at the LYS6 locus identi¢ed it as homoaconi-
carbon sources has been evaluated by Wojatatowicz tase structural gene. Homoisocitrate dehydrogenase
et al. [55]. A direct selection of citrate overproducing was puri¢ed to homogeneity. Two tightly linked loci,
strains has been described by McKay et al. [56]. LYS9 and LYS10, actually de¢ne the same structural
Processes have been described for producing citric gene encoding a bifunctional protein involved in suc-
acid from glucose or n-para¤ns in liquid cultures cessive dehydrogenation and decarboxylation of ho-
(for a review see Mattey, [57]) or with immobilized moisocitrate [67]. LYS8 mutants are unable to con-
cells [58] but also from date coats [59], tapioca starch vert K-ketoadipic acid into K-aminoadipic acid
hydrolyzates [60] and glucose hydrol [55]. suggesting that a single enzyme catalyzes this step
3.2.1.2. K-ketoglutarate and other organic acids. in Y. lipolytica vs. probably two isoenzymes in S.
Y. lipolytica strains grown on n-alkanes in the pres- cerevisiae. LYS2 and LYS3 are two closely linked
ence of a limited thiamine supply (a vitamin not loci, which control adenylylation of K-aminoadipic
synthesized by this yeast but required for K-ketoglu- acid, a rate-limiting step in vivo and the only one
tarate dehydrogenase activity) secrete large amounts in the pathway which is insensitive to the general
of K-ketoglutarate. Use of a diploid strain for K-ke- control of amino acid biosynthesis [65]. The two
toglutarate production has been patented (Maldona- last steps of the pathway are controlled by LYS4
and LYS5, respectively. The latter gene, encoding unique site on the propeptide (both occurring co-
saccharopine dehydrogenase, has been cloned and translationally during translocation into the endo-
sequenced [68,69]. plasmic reticulum), processive hydrolysis of a tail
Y. lipolytica uses lysine both as a nitrogen and as a of 9 N-terminal dipeptides (X-Ala or X-Pro) by a
carbon source, via the N-6-acetyllysine-5 aminoval- Golgi dipeptidyl aminopeptidase, followed by the
erate pathway, like Hansenula saturnus [70,71]. The cleavage of the propeptide at a Lys-Arg junction
LYC1 gene encoding the ¢rst step of the pathway, ahead of the mature part. The endopeptidase is en-
N-6-lysine acetyl transferase (LAT), was cloned and coded by the XPR6 gene, a KEX2 homolog which
sequenced [72] and shown to be induced by lysine. has recently been cloned and sequenced [87]. The
Combining mutations desensitizing homocitrate propeptide has been shown to be required for fold-
synthase (LYS1.5), as well as mutations simultane- ing, transit and activation of the mature part [88,89],

ously preventing lysine catabolism and relieving the a ¢nding which has since been extended to various
lysine e¡ect on saccharopine dehydrogenase (lyc1.5), proenzymes in S. cerevisiae.
yielded strains accumulating 40 times more lysine Regulation of AEP synthesis is complex and re-
than the wild type strain [73]. No lysine secretion £ects the carbon, nitrogen, sulfur and pH status of
in the growth medium was observed, indicating the cell, among others. A study of the promoter has
that very e¤cient systems existed for uptake and/or been initiated; internal deletions and in vivo foot-
retention within the cell. prints demonstrate that there are two main UASs
Both a high and a low a¤nity transport system located 700 bp (UAS1) and 40 bp (UAS2) upstream
have been described for lysine uptake [74] and mu- from the TATA box [90]. Each UAS seems to con-
tants a¡ecting either or both systems were identi¢ed. tain targets for general transcription factors (RAP1-
Intracellular lysine is stored in the vacuole [75]. Mu- and possibly ABF1-like). These UAS are able to ac-
tants a¡ecting vacuolar storage of lysine were iden- tivate a minimal promoter consisting of LEU2
ti¢ed among strains selected for reduced content of TATA box and initiation site (Madzak et al., to be
polyphosphates in the vacuole [76,77], although no published). Unlinked mutations a¡ecting transcrip-
direct relationship could be established between ly- tional control of XPR2 have been selected and iden-
sine and polyphosphate pools. A direct selection of tify at least four unlinked loci (Lambert et al., sub-
mutations preventing lysine retention within the cell mitted). One of these identi¢es a zinc-¢nger
was carried out, starting with a LYS1.5, lyc1.5 transcriptional activator (YlRim1p) which synthesis
strain: mutants displayed a complex phenotype in- and activity are controlled by ambient pH (Lambert
cluding reversion of the lyc1.5 mutation, mating-type et al., submitted).
switch, and mating-type context dependent pheno- 3.2.3.2. Acid extracellular protease. On rich YPD
types [78]. medium at pH 4.0, an acid protease activity was
detected in the growth medium. Three protein spe-
3.2.3. Secretion of proteins cies were initially described, of 28, 32 and 36 kDa
3.2.3.1. Extracellular alkaline protease (AEP). [91]. More recent data suggest that there is only one
Y. lipolytica strains grown on rich (YPD type) acid protease, which may undergo partial proteolysis
medium at pH 6.8 secrete large amounts (1^2 g in some strains and/or under certain growth condi-
l31 ) of an alkaline extracellular protease (AEP) tions (Young, unpublished). Interestingly, induction
[79,80]. of the acid protease occurs under conditions very
AEP is encoded by the XPR2 gene, one of at least similar to that used for inducing AEP, except for
11 genes controlling AEP synthesis, secretion and/or the pH of the medium. The gene encoding acid pro-
activity [81,82]. The gene has been cloned and se- tease has been cloned and sequenced [169], and its
quenced from three di¡erent strains [83^85]. AEP is disruption abolishes acid protease activity. It encodes
a 32 kDa protease of the subtilisin family, which is a preproenzyme with an unusually large 5P upstream
intracellularly processed from a 55 kDa glycosylated region. Interestingly, there seem to exist some con-
precursor [84,86]. Processing includes cleavage of a servation of the promoter elements of both acid and
15-amino acid presequence and glycosylation of a alkaline protease. Comparing the determinants of
the regulation of these two genes might thus be quite bound lipases did not, and di¡ered in several proper-
revealing. ties from the extracellular enzyme. Sugiura et al.
3.2.3.3. Extracellular RNase. A secreted RNase [101] and Ota et al. [102] solubilized two cell-bound,
activity is detected in Y. lipolytica cultures grown monomeric lipases of 39 and 44 kDa, which di¡ered
under conditions leading to alkaline protease secre- in their pH optima. Gomi et al. [103] showed that
tion, the major 45 kDa protein being partially de- the lipase(s) exist in the cell wall as an activator-
graded by AEP into 43 kDa and 34 kDa proteins bound complex, which is rapidly dissociated upon
[92]. RNase is probably synthesized as a 73 kDa enzyme puri¢cation. The extracellular, activator de-
glycosylated precursor [92], but de¢nite con¢rmation pendent activity may thus re£ect release of (up to
of the maturation process awaits determination of 50% of) the cell-bound acivity, perhaps under con-
the sequence of the gene. ditions leading to extracellular protease(s) induction.

3.2.3.4. Extracellular phosphatases. A cell wall- Further details on Y. lipolytica lipase(s) may be
bound acid phosphatase activity is induced if Y. lip- found in a recent review [104].
olytica is grown on media depleted of inorganic Mutants unable to use tributyrin as a carbon
phosphate sources. A glycosylated protein of 90^ source were isolated after nystatin enrichment [105],
200 kDa has been puri¢ed, which is converted into and were shown to de¢ne three complementation
a 60 kDa species upon endoglycosidase H treatment. groups [106]. A gene encoding a secretory protein
Antibodies directed against it cross-react with S. ce- highly homologous to fungal lipases has been re-
revisiae major acid phosphatase [93]. A kinetic study cently cloned in an independent approach (A. Do-
of this enzyme has been published [94]. minguez, personal communication) : this should rap-
è ton et al. [95] tried to clone its structural gene
Tre idly permit assessing if there is more than one lipase
by functional complementation of a pho5,pho3 mu- activity in Y. lipolytica.
tant of S. cerevisiae. This approach failed to identify Y. lipolytica lipase may be of interest for synthesis
PHO2, the gene of
the gene searched for, but yielded of 2,4-dimethylglutaric acid monoesters, transesteri-
a secreted minor phosphatase. The PHO2 gene prod- ¢cation of meso-cyclopentane diols [107] or for use
uct has no homology to other acid phosphatases, it in the leather industry or in cheese manufacturing
corresponds to an acid phosphatase activity with a (German patent DD-272867).
narrow substrate spectrum, the synthesis of which is An extracellular thermostable esterase of low mo-
induced in cells starved for inorganic phosphate. lecular weight (10 kDa), speci¢c for the 1-position of
3.2.3.5. Extracellular lipase and esterase. Y. lip- triglycerides has recently been described [108].
olytica strains display a lipase activity which acts
preferentially on oleyl residues at positions 1 and 3
of the glyceride. Lipase activities were examined by 4. Cell biology
di¡erent investigators on di¡erent strains. It is un-
known whether the reported discrepancies re£ect dif- 4.1. Peroxisome biosynthesis
ferences in the methodologies used, or the existence
of di¡erent types of strains. Peters and Nelson Y. lipolytica grows well on oleic acid and this is
[96,97] described a single glucose repressible activity accompanied by an extensive proliferation of peroxi-
with a pH optimum around pH 6.2^6.5. Zviagintze- somes. Antibodies directed against the targeting sig-
va et al. [98] also reported on a single cell wall-anch- nal Ser-Lys-Leu COOH (anti-SKL) of peroxisomal
ored lipase, whereas Kalle et al. [99] described two proteins of several yeasts, react strongly with three
cell-bound activities : a constitutive, glucose insensi- peroxisomal proteins of Y. lipolytica [109]. Using
tive lipase, and a second activity induced by sorbitan these two features, studies on peroxisome biosynthe-
monooleate. Ota et al. [100] described both an ex- sis have recently begun with the identi¢cation of 17
tracellular activity in cultures supplemented with a oleic acid non-utilizer mutants. Three of them were
protein-like fraction derived from soybean, and shown to a¡ect peroxisomal assembly (pay mutants)
cell-bound lipases. The extracellular lipase required by a rapid immuno£uorescence assay, using anti-
oleic acid as a stabilizer/activator, whereas the cell- SKL antibodies [110]. The punctate labelling pattern
observed with anti-SKL in wild type cells was lost in Several genes controlling early steps of the path-
the mutants, no peroxisomal structures could be ob- way have been characterized. A homologue of
served, and two peroxisomal marker enzymes (L-hy- SEC61 (one of the subunits of the translocation
droxyacyl-CoA dehydrogenase and catalase) failed pore into the endoplasmic reticulum) was cloned by
to localize to the particulate fraction in mutant cells. PCR and shown to be highly conserved between S.
Genes complementing pay mutations (PAY2, PAY4, cerevisiae and Y. lipolytica (68% identity at amino
PAY5, PAY32) were isolated by complementation acid level). A homologue of SEC62 was isolated us-
using a genomic library made in a replicating vector. ing the cDNA expression library to complement a
The PAY2, PAY4, PAY5 and PAY32 genes have sec62 thermosensitive mutant of S. cerevisiae. Its pre-
been sequenced [110^113]. Pay2p is a 42 kDa peroxi- dicted product shows only 37% identity with its S.
somal integral membrane protein. It is essential for cerevisiae counterpart (D. Swennen, unpublished).

matrix protein import and enlargement of peroxi- Rather close homologues of a signal recognition
somes [111]. PAY4 encodes a 112 kDa hydrophilic particle (SRP), involved in the targeting of secretory
protein, presumably localized in the cytoplasm, polypeptides towards the endoplasmic reticulum,
which is expressed at low levels on glucose contain- have been observed in Y. lipolytica and in S. pombe
ing medium and is strongly induced upon shifting [14]. On the contrary, S. cerevisiae SRP seems to
to oleic acid. Pay4p exhibits an ATP binding have diverged considerably and was identi¢ed later
site and appears closely related to Pas5p of Pichia [116]. Y. lipolytica SRP contains a typical 7S RNA,
pastoris (59% identity) and less to Pas1p of S. cere- which can be immunoprecipitated from whole cell
visiae, both involved in peroxisome assembly in extracts by human anti SRP antibodies [117]. This
their respective hosts [110]. PAY5 encodes a RNA is encoded by two unlinked genes, SCR1 and
peroxisomal integrale membrane protein homolo- SCR2, whose products share 94% identity [118]. Ei-
gous to the mammalian peroxisome assembly factor ther gene can be deleted, but the double deletion is
PAF1 [113]. PAY32 encodes an intraperoxisomal lethal [119], indicating that essential secretory pro-
protein of the matrix protein translocation ma- teins strictly require the SRP dependent pathway
chinery [112]. for secretion. This also seems to be the case for
AEP, whose synthesis and secretion are severely de-
4.2. Secretion and genes involved in of the secretory pressed under non-permissive conditions in condi-
pathway tional mutants of SCR1 or SCR2 [120,117]. Such
mutants were created by site directed mutagenesis
An essential gene called RYL1 has been isolated of conserved nucleotides in a loop binding Srp19p,
by hybridization [114], and may represent a homo- one of the six polypeptides associated to mammalian
logue of SEC4, which encodes a small G protein 7S RNA. The genes encoding two of these polypep-
involved in the control of secretory vesicle fusion tides, Srp19 (SEC65) and Srp54 have been cloned
with the plasma membrane. A homologue of S. and sequenced recently (M. Sanchez et al., unpub-
cerevisiae SEC14 was identi¢ed both by PCR and lished; D. Ogrydziak, unpublished).
by functional complementation of a S. cerevisiae In an attempt to identify new genes whose product
sec14 mutant using a cDNA expression library of may (directly or not) interact with the SRP, muta-
Y. lipolytica mRNA made in a S. cerevisiae expres- tions displaying colethality with conditional muta-
sion vector [115]: unexpectedly, the deletion of this tions within SCR2 were selected [121]. One of these
gene in Y. lipolytica proved to be viable and did not mutations identi¢es a gene (SLS1) whose product is
a¡ect protein secretion, although recent electron mi- predicted to be a resident protein in the lumen of the
croscopy data suggest that (derivatives of) the Golgi endoplasmic reticulum. Interestingly, deletion of this
apparatus look abnormal (Rambourg et al., unpub- gene leads to a thermosensitive phenotype, which is
lished). This deletion however prevented the yeast- further pronounced in the presence of a 7S RNA
mycelium transition characteristic of this species, thermosensitive mutation, and results in a dramatic
suggesting the existence of some link between Golgi decrease of the amount of AEP precursor synthesis:
function(s) and cellular di¡erentiation. this suggests that Sls1p may facilitate SRP-depend-
ent translocation of secretory precursors from the activity and polyamine cell pools increased in hyphal
lumenal side of the ER membrane. cells grown on N-acetylglucosamine-containing me-
dium [126].
4.3. Dimorphism Mutations in the genes SEC14 [115], GPR1 (G.
Barth, unpublished), and deletion of XPR6 (Ogryd-
Wild type strains of Y. lipolytica exhibit various ziak, personal communication) have strong e¡ects on
colony shapes, ranging from smooth and glistening the yeast to hyphae transition. However, it is not
to heavily convoluted and mat. The colony morphol- known whether the proteins encoded by these genes
ogy is determined both by growth conditions (aera- are directly involved in the regulation of this transi-
tion, carbon and nitrogen sources, pH etc...) and by tion. Morphological mutants completely unable to
the genetic background of the strain. Wild type iso- form hyphae, can be easily selected after visual in-

lates may give rise to various colonial types, each spection of the colonies [129].
giving rise to the other types : the reason for this
instability, which is not seen in laboratory strains,
is unknown. 5. Cell cycle
Y. lipolytica is a natural dimorphic fungus, which
forms yeast cells, pseudohyphae and septate hyphae 5.1. Mating and mating type alleles
[122,8]. True mycelium consists of septate hyphae
3 to 5 Wm in width and up to several mm in length. Extensive overviews on the life cycle, including
Apical cells often exceed 100 Wm, whereas segments mating processes and sporulation, are given by We-
are 50 to 70 Wm long. There is a single nucleus per ber and Barth [130], Weber et al. [3], and Heslot [4].
segments. Septa show a minute, ascomycete-type All natural isolates of Y. lipolytica so far tested are
central pore, unusual for other ¢lamentous yeasts, heterothallic. The mating type is determined by the
with endoplasmic reticulum extending through it two alleles MATA and MATB [131]. The MATA
from one segment to the next [123]. allele has been cloned [132]. Similar to heterothallic
The proportion of the di¡erent cell forms depends strains of S. cerevisiae haploid and diploid cells are
on the strain used, probably accounting for the mor- vegetatively stable. Kurischko and Weber [133] re-
phological di¡erences observed at colony level. Cer- ported spontaneous haploidization of some diploid
tain conditions are known, however, which cause cells after formation of zygotes. Furthermore, it was
preferential formation of yeast cells, or induce myce- observed in several laboratories that diploid strains
lial development [124^126]. Mycelial development spontaneously sporulate during prolonged storage
was inhibited by a de¢ciency of magnesium sulfate on complete media. Therefore, it is recommended
and ferric chloride or by the addition of cysteine or to prepare fresh diploid strains for genetic analysis.
reduced glutathione. Rodriguez and Domiguez [125] Mating frequencies of natural isolates are always
could induce a complete and reproducible yeast-my- very low (1% viable zygotes/cell or less). Attempts to
celium transition in minimal medium containing N- increase the mating response by inbreeding [134^
acetylglucosamine as sole carbon source, but this 136,49] failed to raise mating frequency above 15%
seems to be strain dependent, too. Guevara-Olvera viable zygotes/cell. Mating frequency is dependent on
et al. [126] enhanced the yeast-mycelial transition by several parameters besides the genetic background of
heat shocking the cells during the inoculation in me- strains : medium, cell density, growth phase, and
dium containing N-acetylglucosamine. temperature [134,137].
Some studies were done to detect physiological
changes occurring during yeast-hyphae transition 5.2. Sporulation
[127,128,126]. Comparison of the composition of
yeast and hyphal cells have shown that hyphal walls Y. lipolytica does not require nitrogen limitation
exhibit a higher content of aminosugars and a re- for induction of sporulation in contrast to S. cerevi-
duced content of protein (Vega and Domiguez, siae and some other yeasts. Diploid strains sporulate
1986 [127]). Furthermore, ornithine decarboxylase on solid or in liquid complete medium when glucose
is exhausted. Highest sporulation frequencies were mutants or of certain classes of mutants, spontane-
obtained with many strains if 1.5% sodium citrate ous mutability and frequency of reversions in di¡er-
was used as a carbon source [49] at temperatures ent strains of Y. lipolytica have been published
of 20³C to 30³C. High sporulation frequencies may [131,140,134,141,135,142].
also be obtained in liquid or on solid YM and V8 Many mutants have been described which show
media [134]. nutritional requirements for most of the amino acids,
Some natural isolates of Y. lipolytica sporulate adenine, uracil, and the vitamins biotin, nicotinic
quite well after mating but only inbred strains form acid, pantothenic acid, ribo£avin, and thiamine
a high proportion of complete tetrads and have a [131,134,143,135,142,4]). There are several colour
satisfactory level of germinating spores. mutants described which turn the colonies green,
Initial studies with Wickerham's strains were brown or red ([143,144,142]) but no red adenine re-

plagued by low mating frequencies, poor sporulation quiring mutants have been isolated. Several mutants
ability and low ascospore viability : these defects have been described which are resistant to antifungal
could be partially alleviated by inbreeding pro- agents or di¡erent analogues of amino acids and
grammes, but no perfect set of strains could be ob- carbon sources [142,145,63,64]. Copper resistance
tained. The proportion of complete tetrads is high varies widely among natural isolates of Y. lipolytica .
and the spore germination of these strains plateaued Two tightly linked metallothionein genes, transcribed
at 80%, which however makes tetrad analysis possi- by a bidirectional promoter, have been cloned and
ble (summary [5]). A very e¤cient method for selec- sequenced (Dominguez et al., unpublished).
tion of spores for random spore analysis using nys- Most of the isolated mutants reverted at the ex-
tatin has been developed [137]. pected frequency of 10

37 to 10
38 or less. However,
Since genetic data are mainly gathered currently some of the mutants reverted at much higher fre-
on strains of the E129^E150 series, it might be ad- quencies [142,78,36]. The reason for this high revert-
visable to localize new genes on this map. In any ibility has not been clari¢ed in most cases. The clon-
case, the existence of e¤cient methodologies for ing and sequencing of one of such a highly revertible
gene transfer/replacement and for retrieval of chro- allele ( GPR1^112 ) resulted in the detection of a ret-
mosomal mutations once the gene has been cloned, rotransposon which is involved in the reversion proc-
makes it likely that exchange of markers between ess [146].
di¡erent inbred lines will be much easier than in The nystatin method for enrichment of mutants
the past. [147,148] has been successfully adapted to concen-
trate auxotrophic and citrate non-utilizing mutants
of Y. lipolytica [134,142]. Enrichment of mutants us-
6. Genetics and molecular biology ing inositol-death has been reported (DeZeeuw, per-
sonal communication).
A fair amount of data both on genetics and mo-
lecular biology of this species had been accumulated 6.2. Genome

since the mid eighties when a transformation system
became available [138,139]. A G+C content of 49.6^51.7% has been reported
for Y. lipolytica [149^151]. Determination of deoxy-
6.1. Mutagenesis and mutants

U
adenosine content of exponentially growing haploid
9
cells yielded an estimated genome size of 4 10 Da
Several commonly used chemical and physical mu- or about 11 Mb [134], which would be smaller than
tagens have been tested on wild type and inbred that of S. cerevisiae . It was later reported that di¡er-
strains of Y. lipolytica , and many di¡erent mutants ent haploid strains of Y. lipolytica exhibit wide var-
have been isolated and characterized. This is not the iations in their DNA content estimated by this meth-
place to describe all these mutants, but some main od [152], but no absolute values were given.
classes and general aspects will be discussed. Results Chromosome separation by pulse ¢eld electrophore-
of extensive studies on cell inactivation, frequency of sis yielded much larger genome estimates in the
range of 12.7 to 22.1 Mb for wild type or laboratory has been selected to assign 36 cloned genes by hy-
strains [15]. A pronounced chromosome length poly- bridization. Ribosomal RNA genes were found on
morphism was observed between di¡erent isolates, as four of the ¢ve chromosomes. The linkage groups
well as a variation of the number of chromosomal appear conserved among di¡erent independent lines.
bands detected (4 to 6), which may re£ect both gel
artifacts (chromosome comigration) and aneuploidy 6.4. ARS and centromeres
(evidence for a duplication of the URA3 marker on
separate chromosomes was observed in a strain dis- In Y. lipolytica an origin of replication alone is
playing 6 bands). Natural isolates of Y. lipolytica unable to maintain a plasmid extrachromosomally,
appeared to have widely divergent genetic structures, but it absolutely requires a centromeric sequence
which may account for the low spore viability ob- [129,156].

served in the initial inbreeding programs. ARS have been isolated up to now
Three di¡erent
CEN) and a
[129,157], each carrying a centromere (
6.3. Genetic linkage groups and chromosome maps nearby chromosomal origin of replication (ORI) :
ORI3018/CEN3, ORI1068/CEN1, ORI4002/CEN4 . 1
Based on tetrad dissection and random spore anal- ORI1068 and ORI3018 have been shown by two di-
ysis, a genetic map encompassing more than 60 mensional gel electrophoresis to be included in re-
markers was established for a certain inbred line gions of initiation of replication in the chromosome
[135,153]. Linkage was observed 8 to 10 times (Fournier and Vernis, unpublished), whereas all
more frequently between random markers than in three CEN sequences are able to induce chromosome
S. cerevisiae, suggesting that the average recombina- breakage when integrated at the LEU2 locus. CEN1
tion per kb was lower than in S. cerevisiae. Five and CEN3 were shown additionally to be closely
linkage groups were de¢ned at that time but linkage linked genetically to a chromosomal centromere,
studies have proceeded at slow pace since that time and to correspond to a strong pausing site of the
[154]. Linkage groups were also de¢ned for German polymerase. ORI and CEN functions are carried by
lines [155] and for industrial strains (De Zeeuw, per- ARS inserts,
two independent regions of the original
sonal communication), but since marker exchange and can be exchanged between di¡erent ARS : a giv-
between the di¡erent lines is di¤cult, no pooled set en CEN sequence can thus apparently confer ARS
of data is available so far. No centromere linked function to any chromosomal ORI, thus opening the
marker has been conclusively identi¢ed, but strains way to the cloning of many ORI. Several new ORIs
carrying centromeres tagged with a marker have were recently isolated from a DNA library made in a
been recently constructed (Fournier et al., 1993 CEN non-transforming vector (Chasles and Four-
[156]) and should help in this regard. nier, to be published). One of them ( ORIX009)
Inbred strains of Y. lipolytica tend to display 5 was shown to be repeated at several places in
chromosomes in the range of 2 to 5 Mb (P. Fournier, the genome, another one originated from rDNA
S. Casaregola, unpublished). It should be noted that spacer.
at this stage 5 is still an estimate for the chromosome ORI and CEN sequences of Y. lipolytica show no
number of the species : comigration may occur in this homology to S. cerevisiae or K. lactis corresponding
range of size. Some bands are fuzzy, which may be sequences (nor to S. pombe), and the existence of a
due to the presence of rDNA repeats, so that speci¢c clear functional consensus for these sequences in Y.
chromosome breakage will be required in order to lipolytica has still to be assessed.
get de¢nite results. As expected from the study of the
initial isolates from which the inbred lines were de-
rived, each line shows a completely di¡erent chromo-
1
Nomenclature of ORI and CEN. The CEN number refers to
the chromosome number in strain E150 map. Four digits are
some pattern. An average size of 15 to 18 Mb has
used to de¢ne ORIs : the ¢rst refers to the chromosome number
been estimated for inbred lines (Fournier and Nguy-
in strain E150 map, the three following are speci¢c of each ORI.
en, unpublished). A highly inbred strain (E150) giv- For ORIs which are repeated in the genome, the ¢rst digit is re-
ing consistently 5 well resolved chromosomal bands placed by an X.
6.5. Repetitive elements Ylt1 belongs to the Ty3/gypsy group of retrotrans-

posons and contains two overlapping large open
6.5.1. Ribosomal RNA and other RNA genes reading frames (YltA and YltB). The predicted
Genetic data and hybridization of separated chro- YltA gene product shows high similarity to retroviral
mosomes showed that most if not all wild type iso- Gag encoded proteins. YltB encodes protein domains
lates of Y. lipolytica contained several clusters of with some similarity to retroviral gene products en-
rDNA units located on 1^4 [12,158]. Up to ¢ve types coded by the pol gene. In the YltB gene, these pre-
of repeated units were identi¢ed in a single strain, dicted products occur in the order protease, reverse
di¡ering in length and structure of the non-tran- transcriptase, RNase H and integrase (Barth et al., in
scribed spacer DNA. All isolates so far tested preparation).
showed this polymorphic rDNA structure, but one

strain (ATCC 18944) was reported to contain a sin- 6.6. Features of protein encoding genes
gle type of the unit [159].
Genetic recombination within the clusters occurs Several motifs similar to consensus sequences for
during meiosis, leading to expansion or reduction of transcriptional factor binding in S. cerevisiae can be
individual clusters. Thus, crossing individual strains observed in Y. lipolytica promoters, including TATA
within a given Y. lipolytica inbred line results in the boxes, CT rich blocks, TUF/RPG or ABF1 binding
progeny in a strain-speci¢c reshu¥ing of rDNA sites, or UASGCN and UASLEU . The function of these
genes supplied by each parent [158]. Strain typing sequences has not been assessed in most cases, with
can thus be done by simple inspection of hyperdense one exception so far [90], and the corresponding
bands on digests of total chromosomal DNA. transcription factors have not been isolated. One
The total number of repeated units varies around gene encoding a transcription factor has recently be-
a mean value of 50^60 [159]. The contribution of come available: CRF1. Transcription starting points
the rDNA repeats to interchromosome exchange have been mapped in a few cases and often occur
seems to be low in inbred lines and in wild type within a CCAAA type of structure, 20^30 bp down-
isolates. stream from the TATA box.
The 5SRNA genes are not part of the rDNA clus- Positions 31 and 33 are strongly conserved up-
ters as in most other Saccharomycetoideae, but are stream of the initiator ATG: an A is observed in 12/
dispersed throughout the genome as in S. pombe 15 cases at position 31 and in 11/15 cases at position
[12,159] and higher eukaryotic cells. 33. An A is found at both positions in strongly ex-
Small nuclear RNAs U1, U2, U4, U5 and U6 were pressed genes like XPR2, PGK1, TPI1, PYK1, ICL1
analyzed by Roiha et al. [13]. Their sizes appeared and LEU2.
much closer to that of human snRNA than to those Introns have been detected in several Y. lipolytica
of S. cerevisiae. genes (5 intron containing genes among 37 known
sequences). Two genes show two introns (CDC42
6.5.2. Transposons and SEC14) separated by a short exon. The 5P end
A retrotransposon, called Ylt1, was detected in the of the intron (donor site) is GTGAGTPu in all cases.
genome of Y. lipolytica [146]. It is 9.4 kb long and The 3P internal consensus (branch site) is TACTAAC
can transpose in the genome. This retrotransposon is in all cases but one (cgCTAAC in the ¢rst intron of
bounded by a long terminal repeat (LTR), the zeta SEC14), and is separated by one or two nucleotides
element, which is 714 bp long, highly conserved, and only from the 3P end of the intron: CAG.
can exist also as a solo element. Ylt1 and solo zeta Most Y. lipolytica genes show a typical signal for
elements are £anked by a 4-bp directly repeated ge- transcription termination TAG...TA(T)GT...TTT,
nomic sequence. The copy numbers of Ylt1 and solo which is located upstream of the site of poly A ad-
zeta are dependent on the strain examined, but at dition [115].
least 35 copies of Ylt1 and more than 30 copies of Codon usage appears to be di¡erent from that of
the solo zeta element per haploid genome have been S. cerevisiae [160] and similar to that of Aspergillus
observed. (A. Dominguez, unpublished).
6.7. Plasmids and VLPs 7. Genetic tools
No DNA plasmid was detected in a systematic Nowadays many genetic tools are available which
survey of 24 wild type isolates (Treèton, unpublished), make this yeast to an excellent model organism for
but a linear dsRNA of 4.9 kb was observed in sev- addressing several fundamental questions. Powerful
eral strains [161,162]. This RNA is encapsidated methods have been developed and well characterized
within virus-like particles of 50 nm diameter. The strains and plasmids are available now for transfor-
capsid seems to be composed of two major polypep- mation, expression and secretion of foreign genes. A
tides of 83 and 77 kDa [162,163]. Based on hybrid- detailed description of these methods as well as a
ization data, there seems to exist at least two types of summary of the available strains, plasmids, cloned
dsRNAs in di¡erent strains, which show little ho- genes etc. is given in the review by Barth and Gail-

mology to one another [162]. An additional linear lardin [5].
dsRNA molecule of about 6 kb was detected in
some strains besides the smaller 4.9 kb long dsRNA
(Barth, unpublished) similar to the situation in killer 8. Conclusion
strains of S. cerevisiae. No homology was found
compared with genomic DNA of Y. lipolytica, nor The `non-conventional' yeast Y. lipolytica turns
with dsRNAs from S. cerevisiae. No killer phenotype out to be a superb model organism, both exquisitely
associated with these VLPs could be evidenced. Cur- original and generally meaningful. With the recent
ing after UV treatment did not lead to any pheno- development of powerful gene ampli¢cation systems
typic change. [168], the identi¢cation of a retrotransposon [146],
and the characterization of strongly regulated or
6.8. Mitochondrial genome constitutive promoters [52,90] the stage is now set
for concentrating on speci¢c biological problems in
The mitochondrial genome of Y. lipolytica has a this yeast. Investigation of mechanisms of gene ex-
buoyant density of 1.687 g cm33 and a GC content pression and protein secretion for expression of het-
of 24.9% [151]. It consists of a circular molecule of erologous genes are mainly done for biotechnological
14.5 mm and its restriction map was established by application. Fundamental studies are directed now
Wesolowski et al. [164] on strain W29. The arrange- on the structure and functioning of the genome
ment of genes for ATPase subunits, rRNA and which is in some aspects very di¡erent from what
4SRNA are conserved with respect to S. cerevisiae is seen in other yeasts: structure and maintenance
and K. lactis. Several mitochondrial genes, including of polymorphic and dispersed rRNA genes, absolute
those for ATPase subunit 6, 8 and 9, cytochrome requirement for a centromeric function for the main-
oxidase subunit 3, NADH oxidoreductase (ubiqui- tenance of extrachromosomal plasmids, and genetic
none) subunit 4 and tRNA genes, have been cloned structure of natural populations which consist of
[165,166]. The restriction map of mitDNA seems to widely divergent haploid lines. Other aspects of Y.
be well conserved among di¡erent isolates, as judged lipolytica biology have begun to be explored, like
from the mobility of the corresponding hyperdense determinants of the mating type, control of the di-
bands observed on digests of total genomic DNA morphic transition from yeast to hyphae, alkane and
stained with ethidium bromide. fatty acid metabolism, structure and function of ret-
A single mitochondrial mutation leading to oligo- rotransposons, glyoxylic pathway, peroxisome bio-
mycin resistance was described by Matsuoka et al. genesis, etc.
[167]. It has been used in protoplast fusion experi-
ments.
9. Note added in proof
Addition to Section 6.3. It has been shown that

chromosome band no. 3 was a doublet. Therefore,
the chromosome number is six and the genome size genic Candida species and relatives. J. Bacteriol. 173, 2250^
2255.
is 18^20 Mb instead of 15^18 Mb.
[17] Okuma, M., Hwang, C.W., Masuda, Y., Nishida, H., Sugiya-
ma, J., Ohta, A. and Takagi, M. (1993) Evolutionary position
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FEMS Microbiology Reviews 19 (1997) 219^237

Physiology and genetics of the dimorphic fungus

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Keywords: Yarrowia lipolytica ; Physiology; Cell biology; Cell cycle; Genetics

* Corresponding author. Tel.: +49 (351) 463 7617;

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1. Introduction citric acid) when grown on these substrates [2].

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of 20³C to 30³C. High sporulation frequencies may [131,140,134,141,135,142].

media [134]. nutritional requirements for most of the amino acids,

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tatin has been developed [137]. pected frequency of 10

makes it likely that exchange of markers between ess [146].

the past. [147,148] has been successfully adapted to concen-

trate auxotrophic and citrate non-utilizing mutants

of Y. lipolytica [134,142]. Enrichment of mutants us-

A fair amount of data both on genetics and mo-

lecular biology of this species had been accumulated 6.2. Genome

became available [138,139]. A G+C content of 49.6^51.7% has been reported

for Y. lipolytica [149^151]. Determination of deoxy-

6.1. Mutagenesis and mutants

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6.5. Repetitive elements Ylt1 belongs to the Ty3/gypsy group of retrotrans-

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Addition to Section 6.3. It has been shown that

ma, J., Ohta, A. and Takagi, M. (1993) Evolutionary position

cleotide sequence of small-subunit ribosomal RNA gene. Bio-

sci. Biotech. Biochem. 57, 1793^1794.

Chem. (Tokyo) 33, 929^938. 192, 892^893.

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technol. 43, 43^73. (1967) Comparative growth of Candida lipolytica on glucose

New York. acid composition of pristane-grown cells. Agric. Biol. Chem.

Characteristics and Identi¢cation. pp. 683^684. Cambridge biol. 1154^1155.

Leeuwenhoek 38, 357^360. course of yeast growth on di¡erent substrates. Mikrobiologija

bouraudia 17, 71^78. of cytochrome P-450 in yeast cells growing on hexadecane.

Yarrowia lipolytica homologous to signal recognition particle. of Microorganisms, Berlin 63,

[32] Barth, G. and Ku

(ADH) in yeast. II. NAD

krobiol. 19, 381^390. 607^613.

[35] Mauersberger, S. (1991) Mutants of alkane oxidation in the 157, 899^908.

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no. ICL1 of the yeast Yarrowia lipolytica. Yeast 3, 255^262.

coenzyme A synthetase. Yeast 8, 193^203. Microbiol. 31, 251^258.

4390^4394. Induction and citric acid productivity of £uoro-actetate-sensi-

Pichia ohmeri. Biotechnol. Lett. 14, 665^668. è te

Genet 226, 310^314. (1983) Extracellular production of D-(+)-2-hydroxyglutaric

14-hydroxy unsaturated fatty acids. J. Org. Chem. 56, 5237^ US4407853.

5239. [63] Gaillardin, C., Fournier, P. and Sylvestre, G. (1976) Mutants

nol. Plant Fats Oils 162^176. J. Bacteriol. 125, 48^57.

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593^600. tion machinery. J. Cell. Biol. 131, 1453^1469.