Yarrowia Lipolytica As A Model For Bio-Oil Production PDF

Progress in Lipid Research 48 (2009) 375387
Contents lists available at ScienceDirect
Progress in Lipid Research

journal homepage: www.elsevier.com/locate/plipres
Review
Yarrowia lipolytica as a model for bio-oil production

Athanasios Beopoulos a, Julien Cescut b, Ramdane Haddouche a, Jean-Louis Uribelarrea b,
Carole Molina-Jouve b, Jean-Marc Nicaud a,*
a
b
Microbiology and Molecular Genetic Laboratory, CNRS UMR2585, INRA UMR1238, AgroParisTech, INRA centre de Versailles-Grignon BP 01, F-78850 Thiverval-Grignon, France
Laboratoire dIngnierie des Systmes Biologiques et des Procds, CNRS UMR5504, INRA UMR792, INSA, 135 Avenue de Rangueil, F-31077 Toulouse, France
a r t i c l e
i n f o
Article history:
Received 24 July 2009
Received in revised form 18 August 2009
Accepted 20 August 2009
Keywords:
Lipid
Yeast
Yarrowia lipolytica
Triacylglycerol
Lipid particle
b-oxidation
Fermentation
a b s t r a c t
The yeast Yarrowia lipolytica has developed very efcient mechanisms for breaking down and using
hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large
amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable
tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism,
storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various
enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of
biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We
also discuss culture conditions, including the mode of culture control and the culture medium, as these
conditions can be modied to enhance the accumulation of lipids with a specic composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented
demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the
metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation
or accumulation in the form of intracellular lipid bodies.
2009 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lipid synthesis and accumulation factors in oleaginous microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Oleaginous yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Lipid accumulation pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
The de novo lipid synthesis pathway and non-polar lipid synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Biochemistry and regulation of lipid accumulation potential in oleaginous yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors affecting lipid accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Modes of culture for ensuring high levels of lipid accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Batch mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Continuous mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Fed-batch mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mastering lipid production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
The ex novo lipid accumulation pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
The b-oxidation degradation pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Growth conditions and genetic modifications favoring lipid production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
376
376
376
377
377
379
380
381
381
382
382
383
383
383
384
Abbreviations: AA, arachidonic acid; ACAT, acyl-CoA:cholesterol acyltransferase; ACC, acyl-CoA carboxylase; ACL, ATP citrate lyase; AMP, adenosine monophosphate; ATP,
adenosine triphosphate; DAG, diacylglycerol; DHAP, dihydroxyacetone phosphate; FFA, free fatty acids; GLA, c-linolenic acid; G-3-P, glycerol-3-phosphate; HS, hydrophobic
substrates; IMP, inosine 50 -monophosphate; LB, lipid body; MAG, monoacylglycerol; ME, malic enzyme; NADPH, nicotinamide adenine dinucleotide phosphate; PUFA,
polyunsaturated fatty acids; SCO, single-cell oil; SE, steryl esters; TAG, triacylglycerols (triglycerides).
* Corresponding author.
E-mail address: Jean-marc.nicaud@grignon.inra.fr (J.-M. Nicaud).
0163-7827/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.plipres.2009.08.005
376
A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387
6.
7.
Potential applications. . . . . . . . . . . . . . . . . . . .
Summary, conclusions and future directions .
Acknowledgements . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
The yeast Yarrowia lipolytica is often found in environments rich
in hydrophobic substrates, such as alkanes or lipids, and has developed sophisticated mechanisms for the efcient use of hydrophobic substrates (HS) as the sole carbon source [1,2]. One of the
most striking features of this yeast is the presence in its genome
of several multigene families involved in these metabolic pathways. The complexity and multiplicity of these genes enable Y.
lipolytica to use and valorize a wide range of hydrophobic substrates (HS). Using these mechanisms, this yeast can accumulate
lipids to levels exceeding 50% of cell dry weight [3]. Y. lipolytica
may therefore be considered an oleaginous yeast. Lipid accumulation is probably enhanced by the many protrusions on the cell surface, facilitating HS uptake from the medium [4]. The internalized
aliphatic chains are then broken down to meet needs for growth, or
accumulate in an unchanged or modied form. These lipids form
the storage lipid fraction, which consists mostly of triacylglycerols
(triglycerides) (TAG) and steryl esters (SE). In addition to direct
substrate assimilation from the medium, de novo TAG biosynthesis
is another energy storage process providing fatty acids for membrane phospholipid formation. SE formation and mobilization provide the sterols required for membrane proliferation. Storage
molecules accumulate in a specialized compartment of the cell
known as the lipid body (LB). Yeast lipid bodies consist of a lipid
core encased in a phospholipid monolayer, within which many
proteins with diverse biochemical activities are embedded [57].
Several of these proteins metabolize lipids and the LB therefore
probably plays a key role not only in lipid storage, but also in lipid
biosynthesis, metabolism, degradation and substrate trafcking
[6]. LB formation and function are tightly linked to the synthesis
of TAG and SE. A recently identied lipid-binding protein in Y.
lipolytica LB [8,9] has been implicated in lipid trafcking between
the cytoplasm and LB, suggesting that free (non-esteried) fatty
acids (FFA) probably accumulate in lipid bodies too [4,8,10,11].
A few models have been developed for the study of lipid metabolism. These models include Saccharomyces cerevisiae, which has
long been used as a genetic model in studies that have greatly improved our understanding of lipid metabolism [12]. The enzymes
involved in TAG biosynthesis, storage and degradation are very
similar between species, and particularly between yeasts, but S.
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384
385
386
386
cerevisiae is not an oleaginous yeast and accumulates only moderate amounts of lipids (less than 15% of its biomass). Furthermore,
unlike S. cerevisiae, which produces similar amounts of TAG and
SE, Y. lipolytica stores mostly TAGs (>90%). This yeast is also unusual in accumulating signicant quantities of FFA within the cell.
The unique features of Y. lipolytica, together with the availability of efcient genetic tools for this species, have stimulated interest in the use of this yeast as a model for bio-oil production, with
great potential for biotechnological applications. Several technologies, including various fermentation congurations, have been already used for single-cell oil (SCO) production by strains of Y.
lipolytica grown on various agro-industrial by-products or waste
[2,11]. The potential applications of these processes include the
production of reserve lipids with particular structures (e.g. oils enriched in essential polyunsaturated fatty acids) and the production
of nonspecic oils for use as renewable starting materials for the
synthesis of bio-fuels. This review aims to provide insight into
the routes of biosynthesis and degradation leading to the formation of oils and an overview of recent advances in the physiology
and genetics of Y. lipolytica relating to the assimilation of HS.
2. Lipid synthesis and accumulation factors in oleaginous

microorganisms
2.1. Oleaginous yeasts
Few microorganisms are known to accumulate lipids to a significant level. Those species able to do so to a level corresponding to
more than 20% of their biomass are described as oleaginous. Fewer
than 30 of the 600 species of microorganisms investigated in one
study were found to be oleaginous [1315]. The best known oleaginous yeasts include genus of Candida, Cryptococcus, Rhodotorula,
Rhizopus, Trichosporon and Yarrowia. On average, these yeasts accumulate lipids to a level corresponding to 40% of their biomass.
However, in conditions of nutrient limitation, they may accumulate lipids to levels exceeding 70 % of their biomass (Table 1). Nevertheless, lipid content and prole differ between species. For
instance, Cryptococcus curvatus and Cryptococcus albidus accumulate lipids to equivalent levels (58% and 65%, respectively), but
their fatty acid proles differ signicantly. C. curvatus accumulates
Table 1
Lipid contents and fatty acid proles of selected oleaginous yeasts [16]. Lipid contents are expressed, in terms of mass, as a fraction of dry cell mass (% glip g1
X , weight/dry
weight).
a
b
Species
% Lipid (glip gX1)
Major fatty acid residues (relative% w/w)

C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
Cryptococcus curvatus
Cryptococcus albidus
Candida sp. 107
Lipomyces starkeyi
Rhodotorula glutinis
Rhodotorula graminis
Rhizopus arrhizus
Trichosporon pullulans
Yarrowia lipolytica
58
65
42
63
72
36
57
65
36
25
12
44
34
37
30
18
15
11
Ta
1
5
6
1
2
0
0
6
10
3
8
5
3
12
6
2
1
57
73
31
51
47
36
22
57
28
7
12
9
3
8
15
10
24
51
0b
0
1
0
0
4
12
1
1
T means trace.
0 means none detected.
377
large amounts of palmitic acid, whereas oleic acid is the principal

fatty acid accumulating in C. albidus. By contrast, in Rhodotorula
species, lipid content diverges signicantly (Rhodotorula glutinis
and R. graminis accumulate lipids at levels corresponding to 72%
and 36% of their biomass, respectively), but fatty acid composition
remains similar. Y. lipolytica accumulates lipids to lower levels than
some other oleaginous species, but it is the only yeast known to be
able to accumulate such a high proportion of linoleic acid (more
than 50% of the fatty acid residues present Table 1).
2.2. Lipid accumulation pathways
Lipids may accumulate via two different pathways: (1) de novo
synthesis, involving the production, in dened conditions, of fatty
acid precursors, such as acetyl and malonyl-CoA and their integration into the storage lipid biosynthetic pathway (the Kennedy pathway, see below) and (2) the ex novo accumulation pathway,
involving the uptake of fatty acids, oils and triacylglycerols (TAG)
from the culture medium and their accumulation in an unchanged
or modied form within the cell. This pathway requires hydrolysis
of the hydrophobic substrate (HS), transport of the released fatty
acids within the cell, their re-assembly in the TAG and the steryl
ester (SE) fractions and their accumulation within the LB. The main
enzymes involved in these pathways are summarized in Table 2.
2.3. The de novo lipid synthesis pathway and non-polar lipid synthesis
Non-polar lipid synthesis in yeasts requires a constant supply of
coenzyme A (CoA)- activated FA for acylation of the glycerol backbone to synthesize TAG or the esterication of sterols to produce
steryl esters (SE). The rst two carbon atoms for de novo FA synthesis are provided by cytosolic acetyl-CoA, through citrate cleavage
by ATP-citrate lyase (ACL) in the TCA cycle. The fatty acid chain
then grows through the addition of units of malonyl-CoA (endoplasmic reticulum) or acetyl-CoA (mitochondria). Mitochondrial
acetyl-CoA for elongation is supplied by the breakdown of the lipids accumulated via the b-oxidation pathway. Malonyl-CoA is generated by acyl-CoA carboxylase (ACC) and is the principal source of
carbon atoms for de novo FA synthesis. For each step in the elongation of the growing FA acyl chain, two molecules of NADPH are required. This NADPH is generated principally by malic enzyme (ME).
These three enzymes (ACL, ACC and ME) are believed to play a crucial role in determining the potential for lipid accumulation and in
regulating this process (see below).
TAG synthesis generally follows the Kennedy pathway [17].
During the rst step of TAG assembly, glycerol-3-phosphate
(G-3-P) is acylated by G-3-P acyltranferase (SCT1) to generate
lysophosphatidic acid (LPA), which is further acylated by lysophosphatidic acid acyltransferase (SLC1) to generate phosphatidic acid
(PA). Upon dephosphorylation by phosphatidic acid phosphohydrolase (PAP), diacylglycerol (DAG) is released from PA. A gene
encoding PAP, YALI0D27016g, has just been identied in the
genome of Y. lipolytica (Table 1). This gene is 39% identical to the
PAH1 gene of S. cerevisiae. Pah1p belongs to the PAP1 enzyme
family, the members of which require Mg2+ [18] as a cofactor for
catalytic activity. The activity of this enzyme in S. cerevisiae is
regulated by lipids, nucleotides and phosphorylation [19,20].
In the nal step of TAG synthesis, DAG is acylated in the sn-3
position, via an acyl-CoA-dependent or acyl-CoA-independent
reaction (Fig. 1). The acyl-CoA-dependent reaction is catalyzed by
three enzymes: Dga1p, Are1p and Are2p. DGA1 encodes an acylCoA:diacylglycerol acyltransferase (ACAT), but is unrelated to the
DGAT1 subfamily encoding enzymes homologous to the acylCoA:cholesterol acyltransferases identied in plants and mammals
[21]. Instead, it belongs to the same family as the mammalian
DGAT2 gene. In Y. lipolytica, Dga1p seems to be the major enzyme
Table 2
Genes involved in fatty acid metabolism in Y. lipolytica and S. cerevisiae.a
Gene
SC name
EC number
YL ortholog
YL name
Function
GUT1
GPD1
GPD2
GUT2
PAP
SCT1
GPT2
SLC1
DGA1
LRO1
TGL3
TGL4
TGL5
ARE1
ARE2sc
ARE2yl
TGL1
POX1
POX2
POX3
POX4
POX5
POX6
MFE1
POT1
ACL1
ACL2
MAE1
ACC1
YHL032c
YDL022w
YDL059w
YIL155c
YMR165c
YBL011w
YKR067w
YDL052c
YOR245c
YNR008w
YMR313c
YKR089c
YOR081c
YCR048w
YNR019w
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
YALI0F00484g
YALI0B02948g
Glycerol kinase
Glycerol-3-phosphate dehydrogenase (NAD(+))
Glycerol-3-phosphate dehydrogenase (NAD(+))
Glycerol-3-phosphate dehydrogenase
Phosphatidate phosphatase
Glycerol-3-phosphate acyl transferase
Glycerol-3-phosphate acyl transferase
1-acyl-sn-glycerol-3-phosphate acyltransferase
Diacylglycerol acyltransferase
Phospholipid:diacylglycerol acyltransferase
Triacylglycerol lipase
Acyl-CoA:sterol acyltransferase
Acyl-CoA:sterol acyltransferase
Acyl-CoA:sterol/Diacylglycerol acyltransferase
Cholesterol esterase
Acyl-coenzyme A oxidase
Multifunctional beta-oxidation protein
Peroxisomal oxoacyl thiolase
ATP-citrate lyase, subunit a
ATP-citrate lyase, subunit b
Malic enzyme
Acetyl-CoA carboxylase
YKL140w
YGL205w
YKR009c
YIL160c
NP
NP
YKL029c
YNR016C
2.7.1.30
1.1.1.18
1.1.1.18
1.1.99.5
3.1.3.4
2.3.1.15
2.3.1.15
2.3.1.51
2.3.1.20
2.3.1.158
3.1.1.3
3.1.1.3
3.1.1.3
2.3.1.26
2.3.1.26
2.3.1.26
3.1.1.13
6.2.1.3
EC 4.2.1.74
EC 2.3.1.16
EC 1.1.1.38
EC 6.4.1.2
YALI0B13970g
YALI0D27016g
YALI0C00209g
YALI0E18964g
YALI0E32769g
YALI0E16797g
YALI0D17534g
YALI0F10010g
YALI0F06578g
YALI0D07986g
YALI0E32035g
YALI0E32835g
YALI0F10857g
YALI0D24750g
YALI0E27654g
YALI0C23859g
YALI0E06567g
YALI0E15378g
YALI0E18568g
YALI0E34793g
YALI0D24431g
YALI0E18634g
YALI0C11407g
Bioinformatic data were obtained from the Saccharomyces Genome Database (http://www.yeastgenome.org/) and the Genolevures database (http://cbi.labri.fr/Genolevures/).
NP; not present in this microorganism.
a
Genes, corresponding S. cerevisiae gene name and EC number, Y. lipolytica ortholog (gene name), and corresponding function. EC number: enzyme commission number.
378
Fatty acid synthesis

Acetyl-CoA
Malonyl-CoA
Sterol
Glucose
DHAP
Elongation
cycle
GUT2
GPD1
Glycerol
G-3-P
Acyl-CoA
GUT1
SCT1
LPA
SLC1
PA
ARE1
ARE2
PL
SE
DAG
LRO1
Acyl-CoA
DGA1, ARE1, ARE2
TAG
TGL1
Sterol
Neutral lipid
synthesis
PAP
FFA
Acyl-CoA
TGL3, TGL4
Glycerol
Mobilization
Degradation
POX 1-6
-oxydation
MFE1
THIO1
Acetyl-CoA
Fig. 1. Overview of the various pathways involved in fatty acid synthesis and in the storage and degradation of non-polar lipids. Synthesis of non-polar lipids (SE and TAG)
required sterol, acyl-CoA and glycerol-3-phosphate (G-3-P). Synthesis of fatty acids (Acyl-CoA) is catalyzed by the fatty acid synthase from the basic blocks acetyl-CoA and
malonyl-CoA through elongation cycles. G-3-P can be produced either from glycerol by the glycerol kinase encoded by GUT1 or from glucose via conversion of
dihydroxyacetone DHAP by the glycerol-3-phosphate dehydrogenase encoded by GPD1 gene. G-3-P can be oxidized to DHAP by the glycerol-3-phosphate dehydrogenase
encoded by GUT2 gene. The synthesis of SE is catalyzed by SE synthases encoded by ARE1 and ARE2. For the synthesis of TAG, three acyls are added to the G-3-P backbone
through enzymatic steps: rst, an acyl is added at the sn-1 position of G-3-P by a G-3-P acyltransferase to produce LPA (SCT1 gene), then a second acyl is added at the sn-2
position by a 1-acyl G-3-P acyltransferase (SLC1 gene) to produce phosphatidic acid (PA), which is then dephosphorylated by a phosphatidate phosphatase (PAP) yielding
DAG. Finally, the third acyl can be added at sn-3 position either by the acetyl-CoA-dependent pathway (directly from Acyl-CoA) by acyl-CoA:diacylglycerol acyltransferase
(DGA1) and by acyl-CoA:diacylglycerol acyltransferase/acyl-CoA:cholesterol acyltransferase (ARE1, ARE2) or by the acetyl-CoA-independent pathway (from a glycerophospholipid, PL) by the phospholipid:diacylglycerol acyltransferase (LRO1). Homologs to S. cerevisiae TAG lipases TGL1, TGL3 and TGL4 involved in SE and TAG mobilization have
been identied (Table 2). The FFA can then be degraded in the b-oxidation pathway which involved acyl-CoA oxidase (POX), multifonctional beta-oxidation protein (MFE1)
and the thiolase (THOI1). In Y. lipolytica, six genes (POX1POX6) coding for acyl-CoA oxidases are involved in the second step of the b-oxidation.
involved in TAG synthesis, accounting for 45% of DAG acylation

(Beopoulos et al., manuscript in preparation). In vivo essays have
shown that this enzyme preferentially makes use of oleic and palmitic acid as precursors for CoA-mediated acylation. The contribution of Dga1p in Y. lipolytica seems to increase during the growth
cycle, being greatest in the stationary phase. The tagging of Dga1p
with the uorescent protein EYFP showed Dga1p to be located
mostly at the surface of LB (Beopoulos et al., manuscript in
preparation).
Two steryl ester synthases, encoded by the ARE1 and ARE2
genes, have been identied as orthologs of the genes found in S.
cerevisiae, and have been shown to contribute to DAG acylation
by acting as acyl transferases in an acyl-CoA-dependent mechanism [22,12]. YlAre1p has an amino-acid sequence about 30% identical to that of its two orthologs in S. cerevisiae, whereas YlAre2p
has a lower level of sequence identity to Ylare1p (17%) and is more
similar to the diacylglycerol O-acyl transferase (DGAT) from the
plant Perilla frutescens (22%), human DGAT1 (28%) and DGAT1 from
the plant A. thaliana (25%) [23]. All these enzymes belong to the
acyl-CoA:cholesterol acyltransferase (ACAT) family, but their genes
display higher levels of sequence identity to the members of the
mammalian DGAT1 gene family [24]. Despite the differences between their sequences and that of Dga1p, Are proteins display
DGAT activity and are the major enzymes required for TAG synthesis during the exponential growth phase of Y. lipolytica. The ob-
served DGAT activity may result from the evolution of ARE genes
from an ancestral DGAT1 gene duplication in yeast. In vivo studies
have suggested that Are1p prefers saturated acyl-CoA substrates,
whereas Are2p prefers unsaturated acyl-CoAs and, more specically, incorporates oleic acid into TAG (Beopoulos et al., manuscript
in preparation).
In yeasts, the acyl-CoA-independent reaction is carried out by
lro1p, a protein with a sequence 27% identical to that of the human
lecithin cholesterol acyl-transferase (LCAT) [25]. This enzyme has
both phospholipase and acyltransferase functions. Unlike its human ortholog, yeast Lro1p cannot synthesize sterols and functions
as a phospholipid: diacyl glycerol acyl transferase (PDAT) [26]. The
acylation of DAG is an esterication reaction involving the sn-2
group of glycerophospholipids, preferentially PtdCho and PtdEtn.
The deletion of LRO1 in Y. lipolytica results in a 35% decrease in
TAG levels in vivo, with Lro1p making a minor contribution during
the exponential phase and a much greater contribution during the
stationary phase (Beopoulos et al., manuscript in preparation).
SE formation involves the reaction of a fatty acid molecule with
the hydroxyl group of sterols [27]. The SE fraction accounts for 50%
of storage lipids in S. cerevisiae [28], whereas only small quantities
of SE are synthesized (25%) in Y. lipolytica [29]. In S. cerevisiae, the
acyl-CoA:sterol acyltransferases Are1p and Are2p are the only SE
synthases involved in sterol esterication, as a double mutant lacking both ARE genes cannot synthesize SE [30]. Similar results have
been obtained for Y. lipolytica (Beopoulos et al., manuscript in preparation). In vivo measurements of SE accumulation in Y. lipolytica
have suggested that the Are proteins act in synergy and that their
relative contribution is probably greater in the exponential growth
phase.
Fatty acid (Acyl-CoA) synthesis is catalyzed by the fatty acid
synthase complex, with the basic acetyl-CoA and malonyl-CoA
building blocks. The acyl-CoA may be stored as sterol ester (SE)
or triacylglycerol (TAG). SE synthesis is catalyzed by an SE synthase
homologous to the human acyl-CoA:cholesterol acyltransferase
(ACAT) and SE mobilization is catalyzed by SE hydrolases, which
release sterol and FFA. TAG synthesis requires acyl-CoA and glycerol-3-phosphate (G-3-P). G-3-P may be produced from glycerol
or from dihydroxyacetone (DHAP). GUT1 encodes a glycerol kinase
that converts glycerol to G-3-P in the cytosol. The G-3-P produced
is then oxidized to DHAP by the glycerol-3-phosphate dehydrogenase encoded by the GUT2 gene and this DHAP may enter glycolysis or gluconeogenesis. G-3-P may also be used as a skeleton for
triacylglycerol synthesis. Three acyl groups are added to the G-3P backbone to generate TAG, and this process involves four
enzyme-catalyzed steps: (1) an acyl group is added at the sn-1
position of G-3-P by a G-3-P acyltransferase to produce LPA; (2)
a second acyl group is added at the sn-2 position by a 1-acyl G3-P acyltransferase (AGAT) to produce PA; (3) the PA is then
dephosphorylated by a phosphatidate phosphatase (PAP), yielding
DAG and (4) the third acyl group is added at the sn-3 position via
the acyl-CoA-dependent pathway or the acyl-CoA-independent
pathway. In the acyl CoA-independent pathway, this third acyl
group is supplied by a glycerophospholipid, whereas, in the acylCoA-dependent pathway, it is supplied by acyl-CoA. TAG can be
mobilized by conversion to FFA and DAG upon hydrolysis by TAG
lipase. The FFA generated may then be degraded by the b-oxidation
pathway which involved POX, MFE and THIO genes. This pathway
involves four enzyme-catalyzed steps. In Y. lipolytica, six genes
(POX1 to POX6) encoding acyl-CoA oxidases involved in the second
step of b-oxidation have been identied [31,2].
2.4. Biochemistry and regulation of lipid accumulation potential in
oleaginous yeast
Oleaginous microorganisms begin to accumulate lipids when an
element in the medium becomes limiting and the carbon source
(such as glucose) is present in excess. Many elements can induce
lipid accumulation. Nitrogen limitation is generally used in lipid
accumulation studies in microorganisms. Nitrogen limitation is
the easiest condition to control and is generally the most efcient
type of limitation for inducing lipid accumulation. During the
growth phase, the carbon ux is distributed between the four macromolecular pools (carbohydrate, lipid, nucleic acid, protein).
Nitrogen is essential for the protein and nucleic acid synthesis required for cellular proliferation. This process is therefore slowed by
nitrogen limitation. However, in conditions of nitrogen limitation,
the catalytic growth rate slows down rapidly, whereas the rate of
carbon assimilation slows more gradually. This results in the preferential channeling of carbon ux toward lipid synthesis, leading
to an accumulation of triacylglycerols within discrete lipid bodies
in the cells. If non-oleaginous microorganisms are placed in the
same nutrient-limiting medium, further cell proliferation tends
to cease, with carbon ux into the cell maintained but, in this case,
the carbon is converted into various polysaccharides, including
glycogen and various glucans and mannans.
During the transition between the growth phase (growth with
the production of catalytic biomass) and the lipid accumulation
phase (decrease in growth rate due to nutrient limitation and the
diversion of excess carbon to lipid production), some pathways
are repressed (nucleic acid and protein synthesis), whereas others
379
are induced (fatty acid and triacylglycerol synthesis). This transition is induced by the establishment of nitrogen limitation (see below). In addition, during the accumulation phase, precursors
(acetyl-CoA, malonyl-CoA and glycerol) and energy (ATP, NADPH)
are required for lipid synthesis. We will describe here the role of
the key enzymes involved in regulating lipid accumulation
potential.
AMP deaminase is activated by the exhaustion of nitrogen in
the medium during the growth of an oleaginous microorganism.
AMP deaminase catalyzes the following reaction [32]:
AMP ! IMP NH4

The activation of AMP deaminase decreases mitochondrial AMP
concentration and increases cellular ammonium concentration.
The decrease in AMP concentration inhibits isocitrate dehydrogenase, blocking the citric acid cycle at the isocitrate level. Aconitase
mediates the accumulation of citrate in mitrochondria, with exit
from the mitochondria mediated by the citrate/malate cycle
[32,33].
This reaction provides large amounts of acetyl-CoA for fatty acid
synthesis. Acetyl-CoA is provided by the cleavage of citrate coming
from the mitochondria by ATP-citrate lyase (ACL) in the cytosol.
ACL cleaves the citrate to give oxaloacetate and acetyl-CoA.
Citrate HS-CoA ATP ! acetyl-CoA oxaloacetate ADP Pi

This enzyme is absent from non-oleaginous yeasts, such as S.
cerevisiae, but has been shown to be present in Y. lipolytica.
Whereas the human ACL consists of a single protein encoded by
a single gene, the ACL enzymes of both Y. lipolytica and Neurospora
crassa consist of two subunits, Aclap and Aclbp, encoded by ACL1
(YALI0E34793g) and ACL2 (YALI0D24431g), respectively (Table 2).
This enzyme requires an ammonium ion for activation and is
dependent on adenosine mono- and diphosphate [16,34]. However, ammonium ions are scarce in the absence of nitrogen, due
to the induction of AMP deaminase [32,33].
In addition to acetyl-CoA, fatty acid synthesis requires a continuous supply of malonyl-CoA and NADPH. Malonyl-CoA can also be
generated from acetyl-CoA, in a reaction catalyzed by acetyl-CoA
carboxylase (Acc1p) [35].
Acetyl-CoA HCO3 ATP ! malonyl-CoA ADP P i

In mammalian cells, this enzyme is activated in the presence of
tricarboxylic acid intermediates, such as citrate [36]. In yeast, however, Acc1p undergoes allosteric activation as a function of citrate
concentration [37]. In Y. lipolytica, this enzyme is encoded by the
ACC1 gene, and is known as YALI0C11407 g (Table 2).
NADPH is required for the function of the fatty acid synthase
(FAS). It is thought that NADPH concentration is controlled by
the activity of malic enzyme (ME). This enzyme catalyzes the following reaction:
Malate NADP ! pyruvate NADPH

The rst evidence of ME involvement in lipid accumulation was
provided by the inhibition of this enzyme by sesamol in Mucor circinelloides, resulting in a decrease in lipid accumulation from 25%
to 2% of cell biomass [38]. Ratledge et al. subsequently demonstrated a direct correlation between decreasing ME activity during
the lipid accumulation phase and the extent of lipid accumulation
[39]. ME overproduction in M. circinelloides, through the expression
of the gene under the control of the strong constitutive promoter of
the glyceraldehyde-3-phosphate dehydrogenase gene (gpd1), was
recently shown to increase lipid accumulation by a factor of 2.5
[40]. In Y. lipolytica, this enzyme is encoded by the MAE1 gene
380
(YALI0E18634 g Table 2). However, preliminary results for ME

overproduction in Y. lipolytica indicate that NADPH concentration
is not limiting for lipid accumulation in this yeast.
3. Factors affecting lipid accumulation

Lipid accumulation depends primarily on microorganism physiology, nutrient limitation and environmental conditions, such as
temperature and pH. It is also affected by the production of secondary metabolites, such as citrate and ethanol.
The transition from catalytic growth to lipid accumulation generally occurs when excess carbon in the medium is associated with
a nutrient limitation affecting biomass production. Y. lipolytica and
S. cerevisiae have followed different courses of physiological evolution. S. cerevisiae catabolizes glucose efciently, glycerol to a lesser
extent, and fatty acids poorly. S. cerevisiae does not use fatty acids
very efciently. Indeed, the doubling time of cell populations on
this substrate is about 4 h. It is widely accepted that this poor lipid
utilization efciency results from a limited capacity for b-oxidation
(breakdown of FA to generate acetyl-CoA). S. cerevisiae cannot use
oils (mixture of TAG and FA) due to its inability to secrete lipases.
For S. cerevisiae (Fig. 2A), the presence of excess carbon (more glucose taken up than required for biomass production) induces a
metabolic shift from oxidative to oxido-reductive metabolism with
ethanol production. In aerobic batch culture, S. cerevisiae produces
ethanol, with a glucose-to-ethanol conversion yield of
1
0:548 Cmoletoh Cmolglc , a maximum specic growth rate of 0.43 h1
and an average ethanol volumic productivity of 3.3 g l1 h1 [41].
Y. lipolytica grows as well on glucose (lmax = 0.26 h1, [41,42])
as on oleic acid (lmax = 0.33 h1 [43]). In conditions of nutrient limitation in the presence of excess carbon, Y. lipolytica produces large
amounts of TCA-cycle intermediates, such as citric (CA), isocitric
acid (ICA), 2-ketoglutaric acid and pyruvic acid (for review see
[2]). It also converts excess carbon into TAG. By contrast to S. cerevisiae, Y. lipolytica (Fig. 2B) is a strict aerobic yeast unable to produce ethanol. Depending on C/N ratio, different governing
metabolisms can be observed: pure growth, organic acid production or conversion of excess carbon into lipids (triacylglycerols
and sterol esters). By monitoring growth, carbon excess in the
S. cerevisiae
anabolism of Y. lipolytica can be either oriented towards organic

acids production or lipids production (TAG, SE). Y. lipolytica is an
oleaginous yeast able to accumulate lipids to levels exceeding
50% of cell dry weight.
Lipid accumulation, in terms of lipid prole, amount, productivity and conversion yield, is inuenced by various operating conditions, such as the nature of nutrient limitation, pH, aeration and
temperature conditions. Published values for lipid accumulation
by the yeasts Candida sp. 107, R. glutinis and Y. lipolytica are presented in Table 3 as a function of the nature of nutrient limitation:
nitrogen, magnesium, zinc, iron or phosphorus. However, nitrogen
limitation is most commonly used to induce lipid accumulation
and gives the best conversion yield with glucose, reaching
0:22 glip g1
glu [44,45].
We will deal here specically with nitrogen limitation, which
remains the most efcient form of nutrient limitation for the
induction of lipid accumulation. The culture strategy involves
two phases, as a function of yeast metabolism. The rst is a growth
phase [48,49]. The growth of the yeast is then slowed by nitrogen
limitation and the lipid accumulation phase begins. The global conversion yield of glucose into lipids in batch culture depends on the
Table 3
Lipid content and lipid yield as a function of the nature of nutrient limitation for
Candida sp. 107 (a) Rhodotorula glutinis (b) and Yarrowia lipolytica (c).
Limitation
Yeast Culture
mode
% Lipid
glip g1
X
Maximum
yield
glip g1
glu
References
Nitrogen
Nitrogen
Nitrogen
Nitrogen
Nitrogen
Carbon
Phosphorus
Phosphorus and
nitrogen
Magnesium
Zinc
Iron
a
c
c
c
b
a
a
a
Batch
Continuous
Fed-batch
Batch
Fed-batch
Batch
Batch
Batch
37%
28%
38.6%
11%
72%
14%
31%
35%
0.22
0.11
0.22
0.017
0.255
0.07
0.15
0.052
[37]
[46]
[42]
[47]
[41]
[37]
[37]
[37]
a
b
b
Batch
Batch
Batch
32.3%
34%
45%
0.10
0.22
0.12
[37]
[44]
[44]
Y. lipolytica
Glucose
Glucose
Fatty Acid
transport
and
-oxydation
No
limitation
Alkane
Fatty acid
Fatty
acid
Oil
Glycerol
glycerol
Ethanol
Ethanol
Metabolite
Lipid
Respiration
capacity
Biomass
Metabolite
Biomass
Fig. 2. Schematic comparison of S. cerevisiae and Y. lipolytica metabolisms. S. cerevisiae catabolizes preferentially sugars (glucose, saccharose) rather than fatty acids. S.
cerevisiae cannot catabolize oils, because it does not secrete lipase. In the presence of excess sugar, S. cerevisiae produces ethanol. Y. lipolytica catabolizes alkanes, glucose, fatty
acids and glycerol. This yeast secretes lipases and is therefore able to grow on oils. In the presence of excess carbon, Y. lipolytica either produces metabolites or stores lipids.
-1
0.45
0.4
0.35
0.3
0.25
0.2
100
381
temperature. For example, R. glutinis produces 0:44 gAGI g1

AG unsaturated lipids at 15 C and 0:27 gAGI g1
AG at 30 C [44]. This modulation of the lipid prole probably results from an increase in
desaturase stability at low temperatures, with no such increase
in stability observed for the other enzymes. However, low temperatures do not favor lipid production, because they also lead to large
decreases in cellular activity and metabolism. There are few alternatives to genetic modication for the modulation of fatty acid
prole. One of these alternatives is the use of growth inhibitors,
such as cerulenin [58], or natural antimicrobial compounds, such
as Teucrium polium extracts [46].
0.5
[Cmollip.Cmolglc ]
conversion of glucose to lipids yield
150
200
250
300
-1
initial ratio C/N [Cmolglc.molN ]
350
400
Fig. 3. Changes in conversion yield for the conversion of glucose into lipids plotted
against C/N ratio for batches of R. glutinis [44].
duration of the growth phase and the transition to the accumulation phase. The duration of the growth phase depends on the C/N
ratio. The total substrate-to-lipid conversion yield therefore depends on the initial C/N ratio of the batch culture. Glucose-to-lipid
1
conversion yield increases from 0.25 to 0.40 Cmollip Cmolglu as C/
1
N ratio increases from 150 to 350 Cmolglu molN for the oleaginous yeast R. glutinis (Fig. 3) [44]. However, a C/N ratio exceeding
350 gC g1
N (data not shown) creates a severe nitrogen deciency,
leading to a rapid decrease in cell viability before the cells are able
to enter the lipid accumulation stage.
The immediate precursor of cellular lipid accumulation in oleaginous microorganisms is citric acid [15,5052]. In Y. lipolytica,
if the initial C/N molar ratio is high [80120 Cmol Nmol1], cell
growth may be followed by signicant citric acid production, with
low levels of lipid accumulation within cells. Papanikolaou et al.
assumed that ATP-citrate lyase was inactive in the presence of excess glucose, resulting in low levels of lipid accumulation [53].
However, if a cosubstrate was used, optimal C/N ratios of 35 Cmol
Nmol1 for lipid production from glucose and 180 Cmol Nmol1 for
lipid production from glycerol and glucose were obtained for batch
cultures [54,55]. In continuous culture conditions, Aggelis and
Komaitis used a medium with a C/N ratio of 66 Cmol Nmol1 [46].
In general, the maximum C/N ratio suitable for lipid accumulaY
1
CmolX Cmolsub , where q is
tion can be estimated from the ratio X=S
q
1
the nitrogen content of biomass [Nmol Cmol ], YX/S is the ratio of
theoretical biomass production to substrate, expressed as Cmol of
biomass/Cmol of substrate in conditions of carbon limitation in the
absence of by-product production [56].
If carbon is not limiting (present in large excess), the uptake of
carbon is limited only by the substrate transport system of the cell.
In this case, limiting concentrations of nitrogen in the medium lead
to the induction of lipid accumulation. The critical nitrogen concentration for lipid induction in Y. lipolytica has been found to be
about 103 mol l1[42]. It is important for nitrogen concentration
to exceed this threshold value to prevent the production of secondary metabolites (citric acid) that will otherwise affect lipid
accumulation.
Conversely, if the extracellular carbon supply is exhausted,
stored lipids may be mobilized. Thus, lipid accumulation is always
dependent on the inux of carbon substrates. This complex regulation makes it difcult to achieve high rates of lipid accumulation in
batch culture. In such conditions, lipid accumulation is always followed by citric acid production [47]. These ndings highlight the
need to manage both carbon and nitrogen ows to optimize lipid
accumulation and reduce citric acid production (see below).
Lipid prole can be modied by adjusting the temperature [57].
Fatty acid composition is dependent on culture temperature, because the degree of saturation generally decreases with decreasing
4. Modes of culture for ensuring high levels of lipid

accumulation
Bioprocesses for lipid production may be designed on the basis
of our knowledge of Y. lipolytica metabolism, taking into account
the ability of this yeast to produce large amounts of intermediates
and to accumulate large amounts of lipids and to break them down
by b-oxidation, even on a glucose substrate [42]. Four metabolic
states can be dened as a function of differences in C/N ux ratio
for a constant nitrogen ux (Fig. 4). The rst corresponds to a carbon ux lower than that required for growth. In this case, if the
cells have storage lipids, they make up the carbon decit by mobilizing these storage lipids (Fig. 4, state a). The second state corresponds to the maximum growth rate obtained with optimal
carbon inux from the medium, at a dened nitrogen ux rate. This
state results in maximal biomass production (Fig. 4, state b). The
third state corresponds to an inux of excess carbon from the medium, which has a high C/N ratio, resulting in a decrease in biomass
production and high levels of lipid production (Fig. 4, state c). The
fourth state results from a further increase in C/N ratio (Fig. 4, state
d), leading to the repression of lipid accumulation in favor of secondary metabolite production. If the desired end product of the
process is lipid, then the process must be designed so as to ensure
the maximal conversion of the carbon taken up into lipids, by minimizing by-product (citric acid) production and maximizing lipid
synthesis, holding the cells in state c.
Three different modes of culture are commonly used: batch,
fed-batch and continuous mode. Most of the processes described
in previous publications relate to batch mode [45,47,59].
4.1. Batch mode
In batch cultures, minerals and carbon substrates are initially
mixed in the bioreactor, with a high initial C/N ratio to boost lipid
accumulation. As nitrogen is actively consumed right from the
start of culture, the rC/rN ratio (residual carbon to residual nitrogen
ratio) continually increases, tending to innity. In this mode,
growth remains exponential whilst nitrogen is not limiting
(Fig. 4, state b). Granger showed that, in R. glutinis grown at
30 C with glucose, nitrogen limitation led to a decrease in the rate
of substrate consumption by a factor of four and an increase in lipid production rate by a factor of two to three [44]. Microbial
metabolism then shifts into phase c (Fig. 4). Nevertheless, citric
acid production is induced as a function of rC/rN ratio, resulting
in a shift of microbial metabolism into phase d (Fig. 4). Citric acid
production decreases the total conversion yield for the production
of lipids from carbon substrate. Thus, this conversion yield depends mostly on the ratio of biomass constituted during the
growth phase to lipids accumulated during the accumulation
phase in batch culture. Control of the ratio of carbon consumption
to nitrogen consumption is therefore essential to prevent citric
acid secretion, hence the importance of monitoring rC/rN ratio in
continuous and fed-batch cultures.
Carbon flow/growth carbon flow need
382
250%
200%
150%
100%
50%
0%
carbon
substrate
nitrogen
catalytic
biomass
lipids
citric
acid
Fig. 4. Yeast activity as a function of carbon ow rate for a xed nitrogen ow rate. State a: pure growth and mobilization of stored lipids; state b: pure growth; state c:
growth with lipid accumulation; state d: growth, lipid accumulation and citric acid production. The size of arrow is proportional to ow. The carbon substrate and nitrogen
arrows correspond to specic inux into cells. Catalytic biomass, lipids and citric acid arrows correspond to specic production rates (consumption for lipids, state a).
4.2. Continuous mode

Lipid production in continuous culture has been modeled by
Ykema et al. [56]. In a continuous culture, the C/N ratio in the culture medium and rC/rN are constant for a given dilution rate. For
low dilution rates, with intermediate C/N ratios promoting lipid
accumulation 40 gC g1
N , the lipid and biomass concentrations obtained are higher than those obtained for higher dilutions. Indeed,
at similar rC/rN ratios, low specic growth rates promote lipid accumulation. The optimization of the process therefore involves determining the optimal dilution rate with an optimal intermediate C/N
ratio (see Fig. 5).
as the substrate goes through three phases: a pure growth phase

(i), a transition phase (ii) and a lipid accumulation phase (iii).
During the growth phase, yeast metabolism results in the balanced distribution of carbon between the four main macromolecular pools (carbohydrate, lipid, nucleic acid, protein), with catalytic
biomass production (biomass without the accumulation of a macromolecular compound). This phase corresponds to state b in Fig. 4.
4.3. Fed-batch mode

The production of lipid by yeast may ensure that lipids are efciently and reproducibly produced [60]. If lipid production is to be
controlled, regulation of the environmental variables is require to
maximize the stability of the metabolic state. This stability can
be achieved through the precise control of nutrient ow rate.
Greater accuracy in the control of nutrient ow rate is associated
with greater control of metabolic state and more optimal production, which is the case for protein production processes [61] (for review see [62]).
In fed-batch culture, nitrogen and carbon ows are monitored
to control the specic growth rate and the rC/rN ratio. As shown
in Fig. 6, a fed-batch culture of Y. lipolytica at 28 C with glucose
Fig. 5. Volumetric productivity of Y. lipolytica biomass P(X) and lipid P(lip) plotted
against dilution rate on industrial glycerol. Cells were grown in single-stage
continuous cultures, with a xed C/N ratio at pH 6 0.05; T = 28 C and aeration
rate = 1.8 VVM. [55].
383
glucose, biomass, lipid, citric acid concentration

[arbitrary unit]
transition
lipid accumulation
nitrogen concentration [arbitrary unit]
growth
305
20
300
18
295
16
14
290
12
285
10
280
275
270
265
260
255
-2
10
20
30
40
50
60
70
80
time [h]
glucose [g]
NH4 [g]
lipids [g]
biomass [g]
citric acid [g]
Fig. 6. Modeling and prediction for a fed-batch culture of Y. lipolytica. The rst part of the growth (from 0 h to 27 h) corresponds to a pure growth phase, with a C/N ux ratio
equal to catalytic biomass production requirements. The second phase (transition from 27 to 40 h) corresponds to the establishment of nutrient limitation (nitrogen),
followed by the establishment of lipid accumulation. The last step (from 40 to 70 h) corresponds to the lipid accumulation phase, during which nutrient limitation is
1
controlled by optimizing the C/N ratio [around 20 Cmol molN to favor lipid accumulation.
The transition phase corresponds to the establishment of nitrogen

limitation, with excess carbon leading to the accumulation of lipids. This phase corresponds to the transition between state b and
state c. Lipid accumulation phase is the most extensive, corresponding to the establishment lipid production under constant
nitrogen limitation conditions, with a C/N ratio of about
20 Cmol Nmol1, preventing citric acid production.
5. Mastering lipid production
Lipid accumulation in oleaginous species is upregulated in culture media containing fatty acids or oils. Y. lipolytica has developed
sophisticated mechanisms for taking up and assimilating hydrophobic substrates. However, this process is counterbalanced by
beta oxidation, which mobilizes the accumulated lipids. Nevertheless, with a suitable culture medium and appropriate genetic modications, high levels of lipid accumulation can be achieved,
exceeding 60% of cell dry weight in some cases, with modication
of the prole of accumulated fatty acids.
5.1. The ex novo lipid accumulation pathway
Y. lipolytica has evolved elaborate strategies and adaptation
mechanisms for the efcient use of hydrophobic substrates, such
as n-alkanes, fatty acids and triacylglycerols. It has adapted to
the use of these substrates by evolving genes encoding surfactants
for their solubilization (liposan), by modifying its cell surface to
facilitate adhesion of hydrophobic droplets (cell surface protrusions), thereby maximizing cell-substrate contact and by developing complex transport mechanisms for the incorporation of these
compounds into the cell (for reviews see [2], [63], [64]). This adaptation of Y. lipolytica may have involved genome amplication and
successive evolution of the genes required for the utilization of diverse hydrophobic substrates (with respect to the chain lengths of
alkanes and fatty acids). Indeed, Y. lipolytica has at least 13 families
of genes involved in hydrophobic substrate utilization, reecting
the vast expansion of its genome with respect to those of other
ascomycetous yeasts [65,66]. For example, the lipase gene family
of this yeast has 16 members and the acyl-CoA oxidase family
has six members.
A ne example of the complex mechanisms developed by Y.

lipolytica is in the hydrolysis and incorporation of oil substrates.
The yeast secretes an extracellular lipase called lip2p, encoded by
the LIP2 gene. This gene encodes a precursor pre-pro-mature protein with a Lys-Arg (KR) cleavage site [67]. It simultaneously produces other intracellular lipases, such as Lip7p and Lip8p, which
may be released into the medium, depending on the substrate.
These lipases have different chain-length specicities, with Lip2,
Lip7 and Lip8 displaying maximal activity with oleate (C18), caproate (C6) and caprate (C10), respectively [68,69]. The released fatty
acids must then be transported into the cell. Kholwein et al. carried
out the rst study of FA transport in Y. lipolytica. They showed that
an energy-free transporter was required below a threshold of
10 lM, whereas at higher concentrations, lauric or oleic acid diffused freely [70]. This model also suggested that Y. lipolytica had
two different chain length-selective transporters. Papanikolaou
and Aggelis analyzed fatty acid uptake in Y. lipolytica during
growth on lipid-containing media with different substrate compositions [55]. Various mixtures of stearin and hydrolyzed rapeseed
oil were used for growth, and fatty acid uptake and proles were
analyzed. Regardless of the external fatty acid composition, all
fatty acids displayed similar incorporation constants for uptake
(0.023 h1), with the exception of C16:0 and C18:0, which had lower constants (0.01 and 0.013 h1, respectively) (Fig. 4). However, if
Y. lipolytica is grown on saturated fatty acids alone, the incorporation constants for C16:0 and C18:0 were signicantly higher
(0.018 h1) [71] (see Fig. 7).
5.2. The b-oxidation degradation pathway
In Y. lipolytica, FA are broken down in peroxisomes, via the fourstep b-oxidation pathway. Six different acyl-CoA oxidases (Aox1-6,
encoded by the POX1 to POX6 genes) catalyze the rst and rate-limiting step of b-oxidation. These acyl-CoA oxidases (Aox) have different activities and substrate specicities, as shown by gene
disruption in Y. lipolytica [31] and by expression and purication
in E. coli. Aox2p is very active and highly specic for long-chain
fatty acids, whereas Aox3p preferentially acts on short-chain fatty
acids. Thevenieau et al. [63] recently showed that Aox1p and
Aox6p are involved in breaking down dicarboxylic acids (DCA).
Incorporation constant (h-1)
384
0.030
C12:0
0.025
C18:2
C14:0
C18:3
C18:1
0.020
0.015
C16:0
C18:0
0.010
0.005
0.000
Fatty acid
Fig. 7. Fatty acid uptake in Y. lipolytica. Incorporation constant (k, h1) of fatty acids
during the growth of Y. lipolytica on lipid-containing medium (mixture of
hydrolyzed rapeseed oil and stearin). (Adapted from [71]).
Gene disruption analysis have also shown that the mechanism

of peroxisome entry depends on POX genotype and that these enzymes have no peroxisome targeting sequence (PTS). By contrast,
the acyl-CoA oxidase (Aox) complex of Y. lipolytica has been shown
to be assembled in the cytosol before its import into peroxisomes
as a heteropentameric, cofactor-containing complex, with Aox2p
and Aox3p playing a key role in this process [72]. Aox proteins
have also been shown to play a major role in peroxisomal division.
During peroxisome maturation, a membrane-bound pool of Aox
interacts with a membrane-associated peroxin, Pex16p. Pex16p
downregulates membrane ssion, thus preventing the excessive
proliferation of immature peroxisomal vesicles. The Dpox4 and
Dpox5 mutants form giant peroxisomes [73].
The second and third steps of b-oxidation are catalyzed by the
multifunctional enzyme encoded by the MFE gene, which displays
hydratase and dehydrogenase activities (Fig. 1). The fourth step is
catalyzed by the 3-ketoacyl-CoA-thiolase encoded by the POT1
gene. A decane-inducible peroxisomal acetoacetyl-CoA thiolase
(encoded by PAT1) has recently been identied and, together with
the thiolase encoded by POT1, this enzyme is thought to catalyze
the last step of b-oxidation. Changes in b-oxidation ux or the
complete abolition of this process might increase lipid accumulation (see below).
5.3. Growth conditions and genetic modications favoring lipid
production
The ability of Y. lipolytica to utilize and degrade HS has led to its
use in bioremediation processes and in fermentation techniques
for the production of intermediate metabolites, enzymes and
added-value oils. The capacity of this yeast to break down HS has
made it possible to decrease chemical oxygen demand (COD) signicantly in oil mill wastewater containing fats, sugars, phosphate,
phenols and metals [74]. Similar methods have been used for selective medium degradation [75] and fat separating processes in
wastewater purication. The production of intermediate metabolites was highlighted by Papanikolaou et al. [53], who used Y.
lipolytica to produce citric acid from raw glycerol, the major byproduct of biodiesel production units. A high C/N ratio, combined
with a buffered pH, resulted in rates of citric acid production of
35 g.l1, and low rates of lipid accumulation, probably due to the
downregulation of ATP-citrate lyase in the experimental conditions used. Similar results were obtained by Stottmeister et al.
[76], who used sunower oil or alkanes as a substrate for the production of up to 250 g l1 citric acid, in fed-batch cultures. Marty
et al. developed a mineral medium fullling the nutritional

requirements of Y. lipolytica for the production of the extracellular
lipase Lip2 which is known to have a high enantiomeric resolution
capacity and catalyzes re-esterication reactions [77], yielding
over 60,000 U l1 Lip2.
The ability of Y. lipolytica to utilize and accumulate HS has led
to the use of this yeast for the production of specic added-value
oils from raw materials. Lipid composition and accumulation
capacity in Y. lipolytica depend strongly on growth and culture
conditions, providing a broad range of choice for substrate/nal
product combinations. For example, replacing glucose by oleic
acid as the carbon source increases lipid accumulation capacity
and the size of lipid particles and also modies the composition
of lipids and proteins [78]. Depending on the composition of the
substrate used, this yeast selectively removes or assimilates FA
from the substrate to produce fats with a predetermined composition [43]. Papanikolaou et al. [79] used these properties to produce cocoa butter-like substances from mixtures of inexpensive
substrates, such as saturated fat and raw glycerol. In a similar procedure, Schrader et al. obtained high yields of c-lactone aroma
from castor oil [80].
Several procedures for increasing lipid production in Y. lipolytica
have been proposed. Fine adjustments of culture conditions can be
used to upregulate lipid metabolism: Bati et al. reported a major
effect of dissolved oxygen, nitrogen/carbon ratio, pH and amount
of oil substrate on lipid accumulation, resulting in yeasts containing 3770% lipid [81]. Aggelis et al. obtained yeasts that accumulated lipids to 43% of dry biomass, with a volumetric productivity
of 0.12 g lipid l1 h1, using industrial fats or glycerol both of
which are cheap industrial carbon side-products as the substrate
[46]. In addition to the regulation of nutrient levels and culture
conditions, genetic engineering approaches have been developed,
based on the potential of yeasts as cell factories for the production
of large amounts of oil with a particular lipid composition and
added-value metabolites. Redirection of carbon ux toward TAG
assembly mediated by deletion of the glycerol-3-phospate dehydrogenase gene (GUT2), an enzyme situated at the crossroad of lipid metabolism and DHAP production for glycolysis, increases lipid
accumulation to levels three times those observed in wild-type
strains [22]. The additional deletion of the POX1 to POX6 genes
encoding the acyl-CoA oxidases, completely abolishes b-oxidation,
preventing lipid mobilization. The resulting mutant strain had lipid
levels four times those of the wild-type strain. In addition, the POX
mutant genotypes affected lipid prole, as each POX gene has a different substrate specicity. POX2 deletion blocks the use of longchain fatty acids, whereas POX3 deletion decreases the uptake of
short-chain fatty acids [82]. In mutants producing only Aox4p, or
Aox1p and Aox6p, only a few, small intracellular LB are formed,
resulting in a so-called slim yeast phenotype. Slim yeasts cannot
accumulate the substrate, probably due to the feedback regulation
of oxidation, affecting HS transporters and downregulating the diffusion process. By contrast, strains producing either Aox2p alone or
both Aox2p and Aox4p form fewer, but larger, LB than the wild
type. The overexpression of Aox2p in the Dpox25 quadruple mutant restores lipid accumulation, by increasing non-polar lipid storage in the LB, resulting in an obese yeast phenotype. These
ndings suggest a regulatory mechanism preventing or enabling lipid storage as a function of the ability of the yeast to assimilate the
substrate from the medium. The LB phenotype also depends on the
acyltransferase prole of mutant strains, with strains lacking ARE2
sterol-acyltransferase being able to form large LB.
6. Potential applications
A potential application of Y. lipolytica or of oleaginous microorganisms in general is the production of single-cell oils (SCO).
Single-cell oils may be dened as edible oils obtained from microorganisms and of a similar type and composition to the oils and
fats obtained from plants or animals. SCO are now accepted as
biotechnological products fullling key roles in the supply of major
polyunsaturated fatty acids (PUFA), which are known to be essential for human nutrition and development. The commercial niche
targeted by SCO is that of dietary supplements enriched in docosahexaenoic acid (DHA), arachidonic acid (AA) and c-linolenic acid
(GLA) [3]. Picataggio et al. (pending US patent application Ser. No.
10/840,579) recently demonstrated the feasibility of engineering
Y. lipolytica for the production of x-3 and x-6 fatty acids (e.g.
18:3, x-3, ALA, a-linoleic acid; C18:3, x-6, GLA, c-linoleic acid;
18:4, x-3, STA, stearidonic acid; 20:3, x-3, ETrA, eicosatrienoic acid
and x-6, DGLA, dihomo-c-linoleic acid; 20:4, x-6, ARA, arachidonic
acid; 20:5, x-3, EPA, eicosapentaenoic acid and 22:6, x-3, DHA,
docosahexaenoic acid), by introducing and expressing heterologous
genes encoding the proteins of the x-3/x-6 biosynthetic pathway
in the oleaginous host. Damude et al. expressed in Y. lipolytica a
bifunctional D12/x3 desaturase from Fusarium moniliformis. This
strain produced a-linolenic acid (ALA, 18:3 D9,12,15) to levels corresponding to 28% of its cellular dry mass [83]. Similarly, GLA production was obtained by overproducing the D6 and D12 desaturases
from Mortierella alpina in Y. lipolytica under the control of a strong
constitutive promoter [84]. Furthermore, Dupont de Nemours have
identied and isolated genes encoding acytransferases suitable for
the transfer of these newly synthesized PUFA into TAG. These modied strains have been patented (US Patents 7267976, 7238482,
7256033) for the marketing of dietary supplements based on PUFA
to protect against cardiovascular disease.
One potential application of a Y. lipolytica strain with an altered
POX genotype grown on a medium with a specic composition
would be the production of dicarboxylic acids (DCA) or diacids
[2]. Y. lipolytica strains are also regarded as very promising agents
for the treatment of mineral oil pollution and plant oil waste. Oil
mill wastewater (OMW), in particular, is a major source of water
pollution as it contains fats, sugars, phosphate, phenols and metals.
Scioli and Volaro [74] reported an 80% decrease in the COD of
OMW after treatment for 24 h treatment with Y. lipolytica, whereas
Oswal [85] found this yeast to be capable of decreasing COD by 90%
in palm oil mill wastewater.
Efforts are currently being made to generate strains with extreme oil accumulation capacities and specic fatty acid compositions. These studies are based on the regulation of substrate
transport mechanisms, the overproduction or elimination of desaturases involved in TAG synthesis and the strict control of genes involved in fatty acid metabolism. The preliminary results obtained
are very promising and potential applications have been identied
in the areas of SCO and biodiesel production.
Re-engineering microbial metabolism to favor oil production for
fuel use seems to be the way forward to a third generation of biofuels: oils will be produced from more appropriate materials (such
as cellulose, glycerol, or even oil waste) in better ways. The capacity of Y. lipolytica to produce long-chain molecules and to adjust its
metabolism for the production of specic oils could lead to the production of more energy-efcient fuels.
7. Summary, conclusions and future directions
Studies of lipid accumulation are currently focusing on maximizing oil production in unicellular organisms, such as yeast or algae, and in plants, for the generation of bio-fuels, including
biodiesel. Like fossil hydrocarbons, TAG oils are highly concentrated stores of saturated hydrocarbons that can be oxidized to
generate energy. The metabolism of Y. lipolytica, oriented toward
the accumulation of lipids, and the unique capacity of this yeast
to use HS efciently, make this microorganism a prime candidate
385
for use in the production of bio-oils. The use of this yeast as a model organism for investigating the mechanisms underlying these
metabolic pathways has already provided insight into substrate
transport processes, the function and use of the diverse organelles
in metabolic processes and the identication and regulation of
genes involved in these processes.
However, although research on yeast has generated a number of
important results concerning TAG and SE synthesis, many questions remain unanswered. One of these questions concerns the
redundancy of the enzymes involved in lipid metabolism. The multiple functions of these proteins, which may act as acyltransferases
or hydrolases, generating similar products from similar substrates,
raises questions about the existence of multiple copies of enzymes
to serve as a back-up for important metabolic pathways. These enzymes may be subject to multiple regulation procedures, including
classical regulation at the transcriptional and translational levels,
post-translational modications of enzymes, and additional coordination at the organelle level. In addition, certain enzymes may
be involved in proteinprotein interactions or subject to differential regulation by auxiliary proteins, through processes that remain
to be claried [86].
For example, LB-associated proteins characteristically have PAT
domains in their primary sequences. The PAT domain is dened by
a conserved amino-acid sequence present in perilipin, adipophilin
(also named adipocyte differentiation-related protein, ADRP), and
the tail-interacting protein of 47 kDa (TIP47) [87]. The PAT proteins
(perilipin, adipophilin/adipose differentiation-related protein,
TIP47, and other related proteins) have structural and regulatory
functions and are thought to target lipid bodies through different
mechanisms [88]. Some PAT proteins are found exclusively on lipid
droplets (e.g. adipophilin), whereas others are found both in the
cytosol and on lipid droplets. The structural properties underlying
the binding of PAT proteins to lipid droplets provide a large number
of possibilities for regulating this distribution. Indeed, the localization of several proteins, such as TIP47, to lipid droplets is highly
regulated and can be induced by adding fatty acids to the medium
to trigger droplet formation [89]. However, the mechanism
underlying this regulation remains largely unclear.
It has been suggested that lipase access to the core of the lipid
droplet is regulated in part by PAT proteins, and the phosphorylation of perilipin may indeed be crucial for the regulation of lipase
access to substrates [90]. It has been estimated that PAT proteins
cover 1520% of the droplet surface, possibly resulting in steric
hindrance, restricting lipase access. However, the hydrolysis and
mobilization of sterol esters is less well understood. In S. cerevisiae,
sterol esters and TAG are hydrolyzed by three consecutive steps
(Yehl, Yeh2, and Tgl1 for SE, Tgl3, Tgl4, and Tgl5 for TAG) catalyzed
by enzymes present on the surface of lipid bodies [91,92]. Little is
known about the regulation of the activities of these enzymes, but
PAT proteins may be involved in the limiting step of hydrolysis. In
addition to the small number of lipid droplet-specic proteins
identied, such as PAT proteins, many cellular proteins with
known functions have been identied in lipid body fractions in
proteomic studies. Some of these proteins are involved in dynamic
cellular processes, suggesting a possible similar role in lipid droplet
biology [78,89,93]. However, both the localization of most of these
proteins to droplets and their functional roles remain to be validated. Another unanswered question relating to lipid metabolism
concerns LB biogenesis. Insight into this process might also provide
clues to the role of auxiliary proteins in the early steps of LP formation at the molecular level.
In conclusion, too few detailed studies of the functional and
structural properties of enzymes involved in lipid metabolism have
been carried out. In one such study, a polymorphism in DGAT was
recently identied as responsible for a quantitative trait locus
controlling high levels of oil production in maize. A single
386
amino-acid substitution boosted oil content by up to 41% and oleic

acid content to 107% [94]. An understanding of the molecular processes governing oil storage in lipid bodies might lead to novel approaches to the engineering of unicellular microorganisms.
Improvements in our understanding of the regulatory and structural properties of the enzymes involved in lipid metabolism and
the use of genetic techniques for the engineering of these organisms would make it possible to maximize cellular lipid storage
and, therefore, oil production per cell. A combination of modications to the culture conditions and the regulated expression of key
enzymes (native or indigenous) could be used to produce specic
lipid compounds with added value and of potential interest in
the oleochemical and petrochemical eld.
The study of de novo lipid accumulation as a result of the conversion of carbohydrate substrates, such as glucose, provides insight into the regulation of the entire lipid synthesis pathway.
The accumulation of lipids produced from these substrates is triggered by nutrient limitation. Nitrogen limitation is the principal
type of limitation studied and the C/N ratio is the key factor governing lipid accumulation. Most ex novo lipid accumulation studies
have been carried out in batch mode. However, the fed-batch mode
seems to be the best culture system, because carbon and nitrogen
ows can be perfectly controlled, making it possible to dissociate
growth and lipid accumulation and the absence of by-product
secretion. Novel approaches combining lipidomic, metabolomic
and genetic approaches and making use of fed-batch cultures will
undoubtedly provide a wealth of information about the regulation
of lipid metabolism.
Acknowledgements
A. Beopoulos was supported by the Lipicaero French national
research program (ANR-PNRB) No. 0701C0089. J. Cescut was supported by CNRS and Airbus. R. Haddouche was supported by the
European research program (LIPOYEAST). This work was nancially supported in part by Aerospace Valley. We thank Julie Sappa
of Alex Edelman and Associates for her excellent help in correcting
the English version of the manuscript.
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Progress in Lipid Research 48 (2009) 375387

Contents lists available at ScienceDirect

Progress in Lipid Research

Yarrowia lipolytica as a model for bio-oil production

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

2. Lipid synthesis and accumulation factors in oleaginous

% Lipid (glip gX1)

Major fatty acid residues (relative% w/w)

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

large amounts of palmitic acid, whereas oleic acid is the principal

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

Fatty acid synthesis

involved in TAG synthesis, accounting for 45% of DAG acylation

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

AMP ! IMP NH4

Citrate HS-CoA ATP ! acetyl-CoA oxaloacetate ADP Pi

Acetyl-CoA HCO3 ATP ! malonyl-CoA ADP P i

Malate NADP ! pyruvate NADPH

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

(YALI0E18634 g Table 2). However, preliminary results for ME

3. Factors affecting lipid accumulation

anabolism of Y. lipolytica can be either oriented towards organic

temperature. For example, R. glutinis produces 0:44 gAGI g1

conversion of glucose to lipids yield

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

4. Modes of culture for ensuring high levels of lipid

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

Carbon flow/growth carbon flow need

4.2. Continuous mode

as the substrate goes through three phases: a pure growth phase

4.3. Fed-batch mode

glucose, biomass, lipid, citric acid concentration

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

citric acid [g]

The transition phase corresponds to the establishment of nitrogen

A ne example of the complex mechanisms developed by Y.

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

Incorporation constant (h-1)

Gene disruption analysis have also shown that the mechanism

et al. developed a mineral medium fullling the nutritional

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

amino-acid substitution boosted oil content by up to 41% and oleic

A. Beopoulos et al. / Progress in Lipid Research 48 (2009) 375387

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