Appl Microbiol Biotechnol (2011) 90:1193–1206
DOI 10.1007/s00253-011-3212-8
MINI-REVIEW
An overview of lipid metabolism in yeasts and its impact
on biotechnological processes
Athanasios Beopoulos & Jean-Marc Nicaud &
Claude Gaillardin
Received: 25 January 2011 / Accepted: 25 January 2011 / Published online: 31 March 2011
# Springer-Verlag 2011
Abstract High energy prices, depletion of crude oil
supplies, and price imbalance created by the increasing
demand of plant oils or animal fat for biodiesel and specific
lipid derivatives such as lubricants, adhesives, and plastics
have given rise to heated debates on land-use practices and
to environmental concerns about oil production strategies.
However, commercialization of microbial oils with similar
composition and energy value to plant and animal oils
could have many advantages, such as being non-
competitive with food, having shorter process cycle and
being independent of season and climate factors. This
review focuses on the ongoing research on different
oleaginous yeasts producing high added value lipids and
on the prospects of such microbial oils to be used in
different biotechnological processes and applications. It
covers the basic biochemical mechanisms of lipid synthesis
and accumulation in these organisms, along with the latest
insights on the metabolic processes involved. The key
elements of lipid accumulation, the mechanisms suspected
to confer the oleaginous character of the cell, and the
potential metabolic routes enhancing lipid production are
also extensively discussed.
Keywords Yeast . Lipid metabolism . Bio-oils .
Biotechnology . Genetic engineering
A. Beopoulos : J.-M. Nicaud : C. Gaillardin (*)
AgroParisTech, UMR1319, Micalis,
Centre de Biotechnologie Agro-Industrielle,
78850 Thiverval-Grignon, France
e-mail: claude.gaillardin@grignon.inra.fr
A. Beopoulos : J.-M. Nicaud : C. Gaillardin
INRA, UMR1319, Micalis,
Centre de Biotechnologie Agro-Industrielle,
78850 Thiverval-Grignon, France
Introduction
The role of lipids, their precursors, and their derivatives in
eukaryotic cells is being increasingly recognized. The
emerging field of lipidomics, driven by the progress in
genetic engineering and amplified by the technological
advances in lipid analysis, is attracting more and more
interest for the study of lipid metabolism in various
organisms. Among them, the yeast Saccharomyces cerevi-
siae offers a number of advantages for lipidomics due to the
high accessibility of its molecular and classical genetics, the
ease of cultivation, and its short generation time. As a
consequence, the increasing range of available yeast
mutants offers great opportunities to perturb or to modulate
lipid metabolism to study the effects produced at the
molecular and/or cellular level (Nielsen 2009).
Besides S. cerevisiae, which has long been used for
fermentation of food and beverages, several other species
are nowadays widely considered for industrial production
of chemicals, pharmaceuticals, and proteins. Interestingly,
while the majority of yeasts are potentially well adapted to
these procedures, there is only a small class among them
which has the capacity to store lipids to over 20% of the
biomass. These yeasts, called oleaginous, are generally
non-pathogenic and are able to synthesize and accumulate
lipids with a composition similar to vegetable oils and fats.
As environmental protection issues and depletion of crude
oil supplies are becoming the overriding challenges faced
by oil industries, it comes not as a surprise to see
biotechnological companies increasingly investing on the
development of first- and second-generation biofuels from
plants and crop residues, respectively. Biodiesel, by means
of transesterified fatty acids (fatty acid methyl esters—
FAME) is biodegradable, non-toxic, and essentially free of
sulfur and aromatic components. However, more than 95%
1194
of the world’s biodiesel is derived from edible vegetable
oils, increasing the worldwide demand for vegetable oil
production (Gui et al. 2008). The most commonly used
vegetable oils for biodiesel production are palm, soybean,
rapeseed, and tung oil. It is estimated that by the end of this
year world biodiesel production will reach 11.1 million
tons/year with its production capacity rising to 32.6 million
tons/year, following an annual growth rate of 40% since
2002 (Thurmond 2008). As a consequence, from 2005 to
the present day, prices of a number of vegetable oils have
largely increased due to biofuel demand.
Today, however, it seems possible to consider micro-
organisms as factories for the production of molecules with
equivalent or improved properties compared to those
derived from fossil oils. The production line of microbial
oils from plant biomass may not compete with food
production if waste products such as lignocelluloses are
used as substrates. It could eventually present other
advantages compared to plants, such as shorter process
cycle, independence of season and climate, and easier
scale-up. Furthermore, the emergence of new downstream
processing techniques results in an optimistic forecast for
the commercialization of yeast–lipid products in the future.
An innovative approach towards this end uses white
biotechnology and metabolic engineering of microorgan-
isms for both the production of biofuels and non-fuel-oil-
derived products such as surfactants, solvents, paints,
waxes, plastics, and lubricants to name a few. It is
important to state, however, that only high valued oils have
any chance of being produced by such biotechnological
means as it is currently unrealistic to expect microorgan-
isms to produce main commodity oils and fats as cheaply as
do plant and animal sources.
Among microorganisms, yeasts, being unicellular, de-
void of endotoxins, amenable to genetic improvement and
suitable for large-scale fermentation, are particularly attrac-
tive for the development of biotechnological approaches.
More precisely, oleaginous yeasts seem to be the best
candidates for this purpose.
Oleaginous yeasts
Fewer than 30 of the about 600 yeast species are known to
be oleaginous (Rattray 1988; Ratledge 1989, 1994).
Oleaginous yeasts are typically found, but not exclusively,
in genera such as Candida, Cryptococcus, Rhodotorula,
Rhizopus, Trichosporon, Lipomyces, and Yarrowia (Ratlege
and Tan 1990). On average, these yeasts accumulate lipids
to levels corresponding to 40% of their biomass. However,
under conditions of nutrient limitation (see below), they
may accumulate lipids to levels exceeding 70% of their
biomass. They consist of 80–90% triacylglycerols (TAG)
Appl Microbiol Biotechnol (2011) 90:1193–1206
and a small fraction of steryl esters (SE), collectively called
neutral lipids. These storage lipids accumulate in a
specialized compartment of the cell known as the lipid
body (LB). The latter consists of a lipid core encased in a
phospholipid monolayer within which many proteins with
diverse functions are embedded (Zweytick et al. 2000;
Brown 2001; Fujimoto et al. 2008). The long prevailing
idea that LBs are simply storage compartments has been
however challenged in higher eukaryotic cells as several
LB proteins were shown to play key roles in lipid storage,
lipid biosynthesis, metabolism, degradation, and substrate
trafficking (Brown 2001; Beckman 2006). In S. cerevisiae,
LB are reported to contain mainly if not exclusively
enzymes involved in neutral lipid catabolism, whereas
biosynthetic enzymes are deemed to be present exclusively
in the ER (Czabany et al. 2007). In the oleaginous yeast
Yarrowia lipolytica, analysis of LB proteins evidenced
anabolic and catabolic enzymes, as well as proteins
controlling vesicle trafficking, a situation clearly reminis-
cent of that described in adipocyte cells (Athenstaedt and
Daum 2006). Moreover, it has recently been recognized
that free fatty acids (FFA) accumulate in LBs as well, and a
lipid-binding protein from the oleaginous yeast Y. lipolytica
LB has been involved in lipid trafficking between the
cytoplasm and LB (Mlickova et al. 2004b; Athenstaedt et
al. 2006; Beckman 2006; Ferreyra et al. 2006).
On another hand, lipid content and profile of LBs differ
between species. Table 1 illustrates the wide range of fatty
acids (FA) found in yeasts together with the extent of lipid
accumulation of the species undergoing fermentation on
glucose containing medium. For instance, Cryptococcus
curvatus and Cryptococcus albidus may accumulate lipids
to equivalent levels (58% and 65%, respectively), but their
fatty acid profiles vary: C. curvatus accumulates significant
amounts of palmitic acid (C16:0), whereas oleic acid
(C18:1, n−9) is preferentially accumulated by C. albidus.
In contrast, total lipid content varies significantly between
different Rhodotorula species, but fatty acid composition
remains similar. It can be acknowledged, however, that the
main fatty acids produced by oleaginous yeasts are myristic
(C14:0), palmitic (C16:0), palmitoleic (C16:1, n−7), stearic
(C18:0), oleic (C18:1, n−9), linoleic (C18:2, n−6), and
linolenic acid (C18:3). It has been suggested that such yeast
oils could be used as oil feedstocks for biodiesel production
that require C16–C18 fatty acids (Steen et al. 2010).
In all cases, process development will require further
metabolic engineering of the strains to increase as much as
possible lipid yields and production rates on cheap raw
materials as carbon sources. In order to prepare the stage
for these developments, we will first provide a description
of yeast lipid metabolism, assessing the key elements of
lipid accumulation and the mechanisms suspected to confer
the oleaginous character to these cells. We will frequently
Appl Microbiol Biotechnol (2011) 90:1193–1206 1195

Table 1 Lipid accumulation and fatty acid profiles of selected oleaginous yeasts grown on glucose containing medium (data adapted from
Ratledge and Wynn 2002 and complemented by our own experiences on Y. lipolytica)

Species Lipid accumulation (% D.W.) Major fatty acid residues (% w/w)

C16:0 C16:1 C18:0 C18:1 C18:2 C18:3

Cryptococcus curvatus 58 25 Trace 10 57 7 0

Cryptococcus albidus 65 12 1 3 73 12 0
Candida sp 107 42 44 5 8 31 9 1
Lipomyces starkeyi 63 34 6 5 51 3 0
Rhodotorula glutinis 72 37 1 3 47 8 0
Rhodotorula graminis 36 30 2 12 36 15 4
Rhizopus arrhizus 57 18 0 6 22 10 12
Trichosporon pullulans 65 15 0 2 57 24 1
Yarrowia lipolytica 36 11 6 1 28 51 1

make reference to the non-oleaginous yeast S. cerevisiae
which remains the best studied model for lipid metabolism,
and to Y. lipolytica, an oleaginous yeast which is genetically
tractable contrary to most other oleaginous species. How-
ever, other yeast models and species will be addressed too
whenever data are available.
Overview of yeast lipid metabolism
An intriguing question that comes to mind when looking at
the different lipid accumulation rates among yeasts,
whether oleaginous or not, concerns the origin of these
differences. Why do some microorganisms accumulate
lipids and others do not, and why do so vast fluctuations
in lipid content exist between oleaginous yeasts?
At first, we should differentiate the processes of lipid
synthesis and lipid accumulation. De novo lipid synthesis
provides for the synthesis of fatty acid precursors, such as
acetyl- and malonyl-CoA and for their subsequent elonga-
tion to an intermediate level (C14–C16), depending on the
enzymatic arsenal of each organism. These precursors then
feed into the lipid storage pathway, where the fatty acyl-
CoAs, assemble to form triacylglycerols (TAG) and steryl
esters (SE). The neutral lipids are then stored inside the LB
to serve as energy reserves. In the presence of external
sources of fatty acids, oils, or occasionally alkanes, yeasts
proceed to the incorporation of these substrates and their
subsequent modification for storage and accumulation. This
pathway, also known as the ex novo pathway, may require
the hydrolysis of the hydrophobic substrate (HS) and is
followed by the incorporation/transport of the released fatty
acids in the form of CoA-thioesters which feed into the
storage lipid pathway to generate neutral lipids. An early
review on fatty acid biosynthetic pathways in oleaginous
yeasts can be found in Ratledge and Wynn (2002), while
recent data on the biochemistry and regulation of fatty acid
synthesis and elongation in S. cerevisiae have been
reviewed by Tehlivets et al. (2007).
Fatty acid synthesis in yeasts
De novo fatty acid synthesis in yeast is carried out by the
fatty acid synthetase (FAS) enzymatic complex in the
cytosol and requires the constant supply of acetyl-CoA
and malonyl-CoA. The former is the initial biosynthetic
unit and the latter the elongation unit, providing two
carbons at each step of the growing fatty acid chain. The
origin of acetyl-CoA, however, differs in oleaginous and
non-oleaginous species. Non-oleaginous yeasts produce
acetyl-CoA from glycolysis of fermentable sugars by
breaking down pyruvate in the mitochondrion and by the
cytosolic pyruvate–acetaldehyde–acetate pathway. In ole-
aginous microorganisms, an additional source of acetyl-
CoA exists: its precursor is the excess of citrate exported
from the Krebs cycle out of the mitochondrion, via the
malate/citrate shuttle. Citrate is then cleaved within the
cytosol by ATP citrate lyase (ACL), an enzyme present in
all oleaginous microorganisms studied so far (Ratledge
2004):
Citrate þ CoA þ ATP ! acetyl ⁃⁃ CoA þ oxaloacetate
þ ADP þ Pi
It is thus quite likely that ACL is one of the enzymes
contributing to the oleaginous character of the cell, even if
some non-oleaginous yeasts exhibit ACL activity. Further-
more, this enzyme appears to be regulated by the presence
of exogenous fatty acids. In a study of various oleaginous
yeast species (Holdsworth et al. 1988), there was a
considerable decrease in ACL activity, in all species
examined, as soon as yeasts utilized endogenous or
1196
exogenous lipids. ACL is made up of two subunits,
encoded by the ACL1 and ACL2 genes and requires for its
activation an ammonium ion, which may be scarce in the
absence of nitrogen, or under high C/N conditions,
common approaches used in order to induce lipid accumu-
lation (see below) (Botham and Ratledge 1979; Ratledge
and Wynn 2002). This probably accounts for the difficulty
to maintain de novo FA synthesis when yeasts are fed
exogenous lipids, even if the C/N ratio is kept high.
Concerning malonyl-CoA, it is generated by the acetyl-
CoA carboxylase (ACC) enzyme by the condensation of an
acetyl-CoA unit with a bicarbonate anion:
acetyl ⁃⁃ CoA þ HCO3  þ ATP ! malonyl ⁃⁃ CoA
þ ADP þ Pi
The ACC enzyme appears to be crucial for de novo FA
synthesis as mutants invalidated for ACC are either
incapable of synthesizing their own FA (FA auxotrophs)
or may harbor low levels of residual ACC activity
(Tehlivets et al. 2007).
The FAS biosynthetic complex in yeasts is composed of
two subunits, Fas1 (beta subunit) and Fas2 (alpha subunit),
which are organized as a hexameric α6β6 complex.
Comprehensive reviews on fatty acid synthetases have
recently been published by Schweizer and Hofmann (2004)
and Tehlivets et al. (2007). Transcription of the FAS1, FAS2,
and ACC1 are all decreased in the presence of fatty acids. If
FAS2 is overexpressed in cells, the expression of FAS1 and
ACC1 is also increased, indicating coordinate regulation of
these three genes. In addition to fatty acid repression, these
three genes are coordinately regulated by an inositol/
choline response element, along with a number of genes
involved in phospholipid biosynthesis (Chirala 1992;
Hasslacher et al. 1993). It is thus likely that one of these
three enzymes acts as a limiting factor in lipid accumula-
tion. In keeping with this idea, a 40% increase in the total
fatty acid content of the non-oleaginous yeast Hansenula
polymorpha was achieved by overexpressing the ACC1
from the oleaginous fungus Mucor rouxii (Ruenwai et al.
2009).
For each step of the carbon chain elongation reaction,
FAS requires two molecules of NADPH. Cellular NADPH
can be either produced by the malic enzyme (malate
dehydrogenase carboxylase) by the reaction:
malate þ NADPþ ! pyruvate þ NADPH
or in the cytoplasm by the pentose phosphate pathway
through glucose-6-dehydrogenase, 6-phosphogluconate de-
hydrogenase, and NADPH isocitrate dehydrogenase. How-
ever, malic enzyme (ME) has been found in most
oleaginous microorganisms and has been suggested to form
a complex with ACL and FAS so as to facilitate FA
Appl Microbiol Biotechnol (2011) 90:1193–1206
elongation (Ratledge 2004). This was suggested by studies
on Mucor circinelloides and Aspergillus nidulans, where
inhibition or mutation of ME resulted in a loss of lipid
accumulation (Wynn et al. 1997). Reciprocally, the over-
expression of the NADP(+) ME of M. circinelloides
resulted in a 2.5-fold increase in lipid accumulation (Zhang
et al. 2007). This, however, would suggest that both a
mitochondrial and a cytoplasmic form of ME should exist
in yeasts, as in Schizosaccharomyces pombe and most
eukaryotic cells, but not in S. cerevisiae where a single gene
encoding a mitochondrial product has been identified
(Boles et al. 1998). A cytoplasmic form has indeed been
reported in Lipomyces starkei, but it was shown to strongly
prefer NAD+ over NADP+ (Tang et al. 2010). Furthermore,
a single mitochondrial form is predicted to exist in Y.
lipolytica (YALI0E18634g), albeit its overexpression did
not affect lipid accumulation (our unpublished data). Thus,
the existence of such a ME–FAS–ACL complex does not
seem to characterize all oleaginous yeasts and ME activity
or NADPH concentration and origin may not be the
limiting factors for lipid accumulation in oleaginous yeasts.
It should be stressed at this point that, besides the
classical (or FAS I) cytoplasmic pathway of FA synthesis
mediated by the Fas1/Fas2 complex, a mitochondrial
pathway has been evidenced in S. cerevisae and in most
eukaryotes, similar to the bacterial FAS II pathway (for a
recent review, the reader is referred to Hiltunen et al. 2009).
It relies on an acyl-carrier protein (ACP) and produces
octanoyl-ACP (a precursor of lipoic acid) and possibly
longer FA. Its inactivation in S. cerevisiae results in
mitochondrial defects (impairment of mRNA maturation,
cytochrome losses, and respiratory deficiency), but its role
in other yeasts has hitherto not been investigated.
Fatty acid modification reactions
The FAS end product depends on yeast species, but varies
between 14 and 16 carbon saturated fatty acids. Further
elongation or desaturation takes place in the ER by the
respective elongases and/or desaturases of the species.
The cyclic series of elongation reactions, reminiscent of
de novo lipid synthesis, require malonyl-CoA, provided by
the ACC. All elongation enzymes identified in yeasts
(ELO1, ELO2, ELO3, TSC13, PHS1, and YBR159w) are
localized in the endoplasmic reticulum (ER), where they are
presumably organized in a complex (Kohlwein et al. 2001;
Denic and Weissman 2007; Tehlivets et al. 2007; Kihara et
al. 2008). The major very long-chained fatty acid (VLCFA)
species in yeast is C26, predominantly present in an amide
linkage of the ceramide backbone of sphingolipids.
Desaturases are hydrophobic, membrane-bound proteins
endowed with at least three separate functions (cytochrome
b5 reductase, cytochrome b5 oxidase, dehydrogenase) to
Appl Microbiol Biotechnol (2011) 90:1193–1206
yield the final activity. Their substrate consists of either
acyl-CoA or acyl residues attached on the head of a
phospholipid, indicating that a number of specialized
acyltransferases and phosphatidylcholine metabolic
enzymes likely participate in these reactions (Dahlqvist et
al. 2000). The most commonly found desaturases in yeasts
are the Δ9 and Δ12 desaturase and, in some species such as
Rhodotorula glacialis and Debaryomyces hansenii, the Δ15
(both species) and Δ6 (R. glacialis) desaturases. The Δ9
desaturase catalyses the insertion of the first double bond
into a saturated fatty acid (C16:0 and C18:0 produced by
FA neosynthesis) and is the only desaturase that acts on a
saturated acyl-CoA rather than a phospholipid. Mutants
deficient in Δ9 activity are unable to grow in the absence of
exogenous unsaturated fatty acids (Goodrich-Tanrikulu et
al. 1994). The Δ12 desaturase catalyzes the insertion of the
second double bond (between carbons 12 and 13) into an
oleic acid substrate to produce linoleic acid (C18:2, n−6).
Further on, the Δ15 desaturase, if present, converts linoleic
acid to γ-linolenic acid (C18:3, n−3). The Δ6 desaturase of
R. glacialis produces C18:3 n−6 from linoleic acid, or
C18:4 n−3 from γ-linolenic acid (Amaretti et al. 2010).
Incorporation of HS substrates—the ex novo accumulation
pathway
The elongation and desaturation reactions also target HS
substrates incorporated by the ex novo pathway. This
pathway is more efficient in yeasts specialized in the
degradation and hydrolysis of hydrophobic substrates (HS),
notably the ascomycetous families of Endomycetaceae and
Saccharomycetoideae. The most common species include
Pichia jadinii, Candida rugosa, Y. lipolytica, and Torulop-
sis colliculosa. The mechanisms elaborated by these species
include a surface-mediated transport, carried out by
surfactants such as lipases and emulsifiers, resulting in the
reduction of the HS droplet size, and a direct interfacial
transport which entails the formation of protrusions allow-
ing HS droplet binding to the cell surface (Fickers et al.
2005a; Beopoulos et al. 2009b). Yeasts like Y. lipolytica
which are specialized in HS utilization also underwent
amplification and successive evolution of the genes
required for the utilization of HS of various chain length
(Dujon et al. 2004). In addition, several membrane proteins
such as acyl-CoA synthetases have been proposed as
possible fatty acid transporters or receptors, and have been
shown to enhance fatty acid uptake into cells (DiRusso and
Black 1999). This is the function of the FAT1/2 genes. FAT1
encodes an acyl-CoA synthetase required for both the
import of long-chain fatty acids and the activation of very
long-chain fatty acids (C20–C26). Disruption of FAT1
results in a decrease in the uptake of fatty acids (Faergeman
et al. 1997; Zou et al. 2002). FAT2, being a peroxisomal
1197
AMP-binding protein with a great similarity to acyl-CoA
synthetases, is also believed to participate in the import of
FA. In S. cerevisiae, it is localized in the peripheral
membrane and matrix (Biobel and Erdmann 1996). How-
ever, it is not required for growth under conditions in which
the peroxisomal β-oxidation of fatty acids provides the
carbon source (Hiltunen et al. 2003). Recently another
protein, Ypk1p, the yeast orthologue of the human serum
and glucocorticoid-induced kinase Sgk1, was found to be
required for the efficient uptake of fatty acids in S.
cerevisiae, and cells lacking Ypk1p fail to grow under fatty
acid auxotrophic conditions. Ypk1p is required for the
internalization step of endocytosis, suggesting that mem-
brane internalization is required for the uptake of fatty acids
(Jacquier and Schneiter 2010). Once inside the cytoplasm,
the FA is handled by several transporter systems such as the
fatty acid binding proteins (FABP), the ABC transporters,
or the PXA and PEX11 systems (Theodoulou et al. 2006).
Different yeast species may have elaborated different
systems, as for instance FABP are absent for S. cerevisiae
(Hiltunen et al. 2003), but very efficient in Y. lipolytica
(Dell'Angelica et al. 1992). The incorporated FA, in its
coenzyme A form, can then be directed either towards the
neutral lipid storage pathway in an unchanged or modified
form, or towards the β-oxidation pathway to be broken
down for energy production.
Alkane assimilation requires additional modifications in
order to serve for the synthesis of cellular compounds or for
FA biogenesis. Alkanes are classically considered to enter
directly in the cell and to then follow the monoterminal
oxidation pathway in the ER and the peroxisomes to be
converted into the corresponding fatty acids. However, a
recent analysis of Y. lipolytica mutants defective in ABC
transporter genes suggested that such proteins are involved
in the import or export of alkanes (Thevenieau 2006). The
oxidation reaction of the incorporated alkane is initiated by
a terminal hydroxylation carried out by a cytochrome P450-
dependent alkane monooxygenase system (AMOS, coded
by the ALK genes), for which a 12 gene family has been
described in Y. lipolytica (Iida et al. 2000; Dujon et al.
2004). The fatty alcohol produced is then transformed into
the corresponding aldehyde. This step is performed either
by a NAD+/NADP+-dependent fatty alcohol dehydrogenase
(encoded by the ADH genes) or by a fatty alcohol oxidase
(encoded by the FAO genes). Finally, the aldehyde is
converted into the corresponding FFA by a NAD+/NADP+-
dependent fatty aldehyde dehydrogenase (ALD genes).
Nevertheless, at this point, a dicarboxylic acid (DCA) can
be produced instead of a FFA by the intermediary of ω-
oxidation carried out by a cytochrome P450-dependent
fatty acid ω-hydroxylase (diterminal oxidation or ω-
oxidation) (Thevenieau et al. 2007). The fate of hydropho-
bic substrates is depicted in Fig. 1.
1198 Appl Microbiol Biotechnol (2011) 90:1193–1206

n-alkanes FFA TAG

protrusion LIP2
ABC1
P450
(AMOS)
FAT1/2
FFA-OH ER
FAO/ADH

cytosol
ALK1-12 aldehyde ALD
DCA FFA

ACL1/2 ER FAT1/2
TCA citrate acetyl-CoA
elongation/desaturation
ACC1/2 FAS1/2 acyl-CoA
NADP+ Sterol
MAE1?
acetyl-CoA malonyl-CoA
NADPH
ARE1

DGA1/2
SE lipid body
SCT1 SLC1 PAP1 LRO1
G3P LPA PA DAG TAG
TGL1
GUT2 GPD1
storage lipid synthesis Sterol
DHAP TGL2/3
mitochondrion
FAA1/4
acyl-CoA LC-FFA MC-FFA

PXA1/2
glucose fructose 1,6-bisP
FAA2
acyl-CoA FFA
POX1-6
MFE1
Carnitine shuttle POT1
or
Glyoxylate cycle acetyl-CoA
(pero-mito-cyto)
peroxisome

Fig. 1 Overview of lipid metabolism in yeasts with the corresponding
gene names. Model created from the available data by Y. lipolytica.
Abbreviations used for metabolic intermediates: DAG diacylglycerol,
DCA dicarboxylic acid, DHAP dihydroxyacetone phosphate, FFA free
fatty acids, FFA-OH hydroxylated fatty acid, G3P glycerol-3-
phosphate, LC-FFA long-chain fatty acids, LPA lysophosphatidic acid,
MC-FFA medium-chain fatty acid, PA phosphatidic acid, TAG
triacylglycerol, SE steryl ester, TCA tricarboxylic acid cycle. Gene
names refer to following encoded enzymatic activities (see text for
details): ACC acetyl-CoA carboxylase; ACL: ATP citrate lyase; ADH:
alcohol dehydrogenase; ALK: cytochrome P450 oxidase; ARE:
acyl-CoA:cholesterol acyltransferase; DGA: acyl-CoA:DAG acyl-
transferase; FAA: fatty acyl-CoA synthetase; FAD: fatty aldehyde
dehydrogenase; FAO: fatty alcohol oxidase; FAS: fatty acid synthetase;
FAT: fatty acid import and activation; GUT: G-3-P dehydrogenase/
glycerol kinase; LIP: lipase; LRO: phospholipid:diacylglycerol acyl-
transferase; MAE: malic enzyme; MFE: multifunctional enzyme; PAP:
phosphatidate phosphatase; POT: thiolase; POX: acyl-CoA oxidase;
PXA: peroxisomal acyl-CoA transporter; TGL: triacylglycerol lipase;
SCT: glycerol-3-phosphate acyltransferase; SLC: LPA acyltransferase

Neutral lipid formation TAG biosynthesis and homeostasis in S. cerevisiae, the

The fatty acids produced by the de novo lipid synthesis or
incorporated from the medium are then esterified either in
the glycerol backbone or in a sterol molecule to form TAGs
and steryl esters (SE), respectively. The whole process is
known as the storage lipid pathway, and its end products
form the neutral lipid fraction of the cell, packed inside the
LB. The TAGs are very efficient molecules for energy
storage as their prevalent hydrophobic properties allow
tight packing inside the LB without affecting the polar
hydrophilic environment of the cell. For recent reviews on
reader is referred to Czabany et al. (2007), Rajakumari et al.
(2008), Kohlwein (2010), and Gaspar et al. (2011).
At a branchpoint in neutral lipid synthesis, diacylgly-
cerol (DAG) is formed: it is the direct precursor of TAG
and phospholipids such as phosphatidylcholine and phos-
phatidylethanolamine. This molecule can be either pro-
duced by the glycerol-3-phosphate pathway or by the
monoacylglycerol (MAG) pathway. In this last one, DAG
is synthesized by direct acylation of MAG by an acyl-CoA:
diacylglycerol acyltransferase (Heier et al. 2010). Both
routes utilize fatty acyl-CoAs for the acylation of glycerol,
Appl Microbiol Biotechnol (2011) 90:1193–1206
but the MAG pathway is not yet found in all yeast cells
(Coleman and Lee 2004). In the glycerol-3-phosphate (G-3-
P) pathway, the sequential acylation of glycerol starts with
the formation of G-3-P. This molecule is either derived
from glycerol via glycerol kinase (GUT1) or synthesized
from dihydroxyacetone phosphate (DHAP) by G-3-P
dehydrogenase (GPD1). This last reaction is reversible
and is catalyzed by an isoform of the G-3-P dehydrogenase
(GUT2). Then, glycerol-3-phosphate acyltransferase
(SCT1) generates lysophosphatidic acid (LPA), which,
however, can also be formed by reduction of DHAP by
an NADPH-dependent acyl-DHAP reductase (AYR1). The
acylation of LPA is catalyzed by the LPA acyltransferase
(SLC1) to yield phosphatidic acid (PA). After dephosphor-
ylation of PA by phosphatidate phosphatase (PAP1), the
DAG is formed. Interestingly, Slc1p and Sct1p have been
reported to have a dual localization in the ER and the LB in
both S. cerevisiae and Y. lipolytica (Athenstaedt and Daum
2006).
Finally, the acylation of DAG is catalyzed either by an
acyl-CoA-dependent or independent reaction. The latter is
carried out by a phospholipid:diacylglycerol acyltransferase
(PDAT), commonly known as Lro1p in yeasts, using the
sn-2 group of phospholipids as an acyl donor (Dahlqvist et
al. 2000). However, although mammalian orthologues
of PDAT proteins (lecithin:cholesterol acyltransferase
family—LCAT) are able to synthesize sterols as well, in
yeasts such activity has not been observed. In S. cerevisiae,
Lro1p is localized in the ER, whereas in Y. lipolytica it
localizes to both the ER and the surface of LB (Athenstaedt
et al. 2006), playing a significant role in DAG acylation
during logarithmic growth in both organisms (Sandager et
al. 2002). This, apart from a direct involvement of LBs in
neutral lipid synthesis, may also indicate a more efficient
TAG synthetic pathway in oleaginous yeasts. Unfortunate-
ly, localization data from other yeasts are currently not
available. Concerning the second, acyl-CoA-dependent
pathway, two main protein families with diacyl-glycerol
transferase (DGAT) activity have been identified in higher
eukaryotes and called DGAT1 and DGAT2. The DGAT
proteins are admittedly integral proteins of the ER
membrane (Yen et al. 2008). However, recent data from
the oleaginous yeast Rhotorula glutinis indicate that this
yeast has a soluble homologue of DGA1, reassigned to a
third, soluble acyltransferase family, called DGAT3. The
enzyme is found to form a complex with acyl-ACP
synthetase, LPA acyltransferase, and PA phosphatase,
which may be advantageous for the cell in order to produce
TAG near the site of FA neosynthesis (Gangar et al. 2001).
This finding suggests that a cytosolic pathway of TAG
synthesis may exist in this oleaginous yeast. In S.
cerevisiae, one DGAT enzyme has been identified, Dga1p,
belonging to the DGAT2 diacylglycerol:acyltransferase
1199
family and is the major player for TAG biosynthesis
(Sandager et al. 2002). In both Y. lipolytica and S.
cerevisiae, Dga1p are integral ER proteins and are more
active during stationary phase, although they are expressed
during all growth phases; in Y. lipolytica, Dga1p is one of
the LB proteins identified by Athenstaedt et al. (2006).
Dga1p accounts for 87% of DAG esterification activity in
S. cerevisiae (Sandager et al. 2002), but only for about 45%
in Y. lipolytica (Beopoulos et al., AEM submitted). The
reason for these differences is that Y. lipolytica possesses a
second gene coding for a DGAT acyltransferase, this time
belonging in the DGAT1 family of DAG acyltransferases.
The protein, named Dga2p, accounts for about 30% of TAG
synthesis and appears to be more active during exponential
growth phase. Members of the DGAT1 family are highly
similar to acyl-CoA:cholesterol acyltransferases (ACAT),
catalyzing sterol esterification in eukaryotes. In S. cerevi-
siae, there are two ACAT proteins, Are1p and Are2p,
which are responsible for the whole process of sterol
esterification but also able to exhibit minor DGAT activity.
In Y. lipolytica, however, only one gene belongs to the
ACAT family, YlARE1, and its product is incapable of
carrying out DAG esterification. For what concerns the Y.
lipolytica Dga2p, although it exhibits considerable similar-
ity with YlAre1p, it is not involved in sterol esterification at
all. As a likely consequence of these species differences,
the neutral lipid composition of the two types of cells differ
markedly: in S. cerevisiae, LBs contain equivalent propor-
tions of SE and TAG, while in Y. lipolytica SE represent
only 5%, the rest being TAGs. Another interesting property
of Y. lipolytica is its ability to accumulate FFA as well.
Nevertheless, the possibility that these molecules are
accommodated in the LB is still under investigation, but
preliminary experimental data strongly suggest that this
may be the case (Beopoulos et al. 2008).
Lipid catabolism
Neutral lipids stored in the LB can be mobilized to fulfill
upon demand the cell requirements for sterols and fatty
acids. The hydrolysis of TAG by yeasts triacyglycerol
lipases provides DAG and FFA for membrane lipid
biosynthesis and the completion of the cell energy needs.
The number and function of intracellular lipases differs
between species: S. cerevisiae and Candida parapsilosis
possess three LB bound lipases (Tgl3p, Tgl4p, and Tgl5p),
each one with different affinity towards TAG, DAG, and
MAG (Kurat et al. 2006). Y. lipolytica, on the other hand,
may possess only two LB bound triacyglycerol lipases
(Tgl3p and Tgl4p), but, being more specialized in HS
degradation, has 16 identified lipase coding genes (encoded
by the LIP genes). Lip2p is secreted in the presence of
exogenous TAG, while Lip7p and Lip8p are membrane
1200
bound with different substrate specificities (Pignede et al.
2000; Fickers et al. 2005b). In addition, four more genes
encoding lipases of the esterase protein family are predicted
from genomic data in Y. lipolytica, adding up to 22 lipase
proteins. This outnumbers by far the number of lipases
found in other ascomycetous yeasts characterized until now.
These enzymes exhibit different substrate specificities and
expression patterns, strongly suggesting that they are
regulated depending on TAG content of cells and media
(Fickers et al. 2005b; Thevenieau et al. 2009).
For SE mobilization, not much data are available in
yeasts. In S. cerevisiae, three members of the sterol
hydrolase family (encoded by the YEH1, YLR020/YEH2,
and TGL1 genes, respectively) have been identified as
paralogues to the mammalian acid lipase family (Koffel et
al. 2005; Mullner et al. 2005). They are localized in the
plasma and LB membrane as well. The triple knockout
mutant has completely lost SE hydrolytic activity but is not
affected in TAG mobilization, demonstrating tight substrate
specificity of these enzymes (Daum et al. 2007). In Y.
lipolytica, a steryl ester hydrolase, homologous to S.
cerevisiae’s Tglp, has been identified in LB (Athenstaedt
et al. 2006).
Fatty acids, once hydrolyzed from their glycerol or sterol
backbone, are either directed towards phospholipid synthe-
sis or to the peroxisome where degradation through β-
oxidation takes place. The transport mechanism, however,
remains unclear. Available data from S. cerevisiae suggest
that long-chain fatty acids are activated in the cytoplasm by
the fatty acyl-CoA synthetase Faa1p and Faa4p and
transported to the peroxisomes by a complex of two ABC
transporters, PXA2 (PAT1) and PXA1 (PAT2) (reviewed by
Hiltunen et al. 2003), while medium-chain FA directly enter
peroxisomes to be activated by the peroxisomal acyl-CoA
synthetase Faa2p (Black and DiRusso 2007). Disruption of
PXA1 or PXA2 results in impaired fatty acid oxidation and
in decreased cell growth on medium containing long-chain
fatty acid as the sole carbon source (Hettema et al. 1996;
Swartzman et al. 1996). The substrate of the complex is
suggested to be the membrane-associated fatty acyl-CoAs
and the Pxa proteins flip the polar CoA group across the
membrane (Hettema et al. 1996). Cells lacking the Pxa1p/
Pxa2p transporter are capable of growth on medium-chain
fatty acids as laurate (C12:0), suggesting that these can
enter the peroxisome before their CoA activation: misloc-
alization of the fatty acid activation protein Faa2p to the
cytosol causes activation of medium-chain fatty acids
outside from the peroxisome, and their oxidation becomes
entirely dependent on the presence of Pxa1p/Pxa2p
(Hettema et al. 1996; Hettema and Tabak 2000).
Once inside the peroxisome, the degradation reactions of
acyl-CoAs are reminiscent of those of fatty acid synthesis
carried out by FAS. This time, however, it entails a four-
Appl Microbiol Biotechnol (2011) 90:1193–1206
reaction sequence resulting in a two-carbon shortening
(instead of lengthening) of the fatty acid. The cycle can
then be repeated until complete breakdown of the FA to
acetyl-CoA. However, intermediate fatty acids may leak
from this biochemical route, depending on the physico-
chemical properties of the specific acyl-CoA and on acetyl-
CoA concentrations. This “malfunction” could be exploited
for aroma production (cyclization of FA) or for polyhy-
droxyalkanoate synthesis after expressing the relevant
bacterial enzymes in yeast cells (PHA, see below).
The acyl-CoA oxidase gene family carries out the first
step of β-oxidation, i.e., the formation of a double bond at
the ω-2 position of the acyl-CoA. Gene representation of
this family varies between species. For instance, there are
three genes in Candida tropicalis, two in Candida maltose,
and only one in S. cerevisiae. In Y. lipolytica, six different
acyl-CoA oxidases (encoded by the POX1 to POX6 genes)
with different chain length specificities have been identi-
fied, broadening the adaptation capacity of this species for
HS utilization (Wang et al. 1998, 1999; Luo et al. 2002).
Two of these enzymes, encoded by POX1 and POX6, are
mostly implicated in dicarboxylic acid (DCA) degradation
(Thevenieau et al. 2009). Furthermore, the POX gene
products have been proposed to play a major role in
peroxisomal division in interaction with the membrane-
associated peroxin Pex16p (Guo et al. 2003). Modification
of the POX genotype in Y. lipolytica can lead to specific FA
production and can also result in “slim” or “obese”
phenotypes (see “Enhancement of lipid synthesis and bio-
oil production—the biotechnological approach” section
below).
In all yeasts studied so far, the multifunctional enzyme
encoded by the MFE gene catalyzes both the second and
third step of β-oxidation. This enzyme possesses two
functional domains exhibiting 2-enoyl-CoA hydratase and
3-hydroxyacyl-CoA dehydrogenase activities, respectively.
The unsaturated acyl-CoA is therefore successively
converted to a 3-hydroxyacyl-CoA and then to a 3-
ketoacyl-CoA, which is finally cleaved by the peroxisomal
ketoacyl-CoA thiolase (encoded by the POT1 gene),
generating an n−2 acyl-CoA and acetyl-CoA during the
fourth and final step of β-oxidation. However, recent
studies in Y. lipolytica identified a second decane-
inducible peroxisomal acetoacyl-CoA thiolase (encoded
by the PAT1 gene), which is thought to participate in the
fourth step of β-oxidation (Yamagami et al. 2001). The
acetyl-CoA formed by β-oxidation is then redirected to the
mitochondria towards the glyoxylate and the TCA (Krebs)
cycles, forming a sustainable energy recycling procedure.
Since acetyl-CoA is unable to cross a lipid bilayer, specific
shuttles involving acyl-carnitine intermediates and specific
translocases have to be used. The routes and export
pathways differ between S. cerevisiae and for instance
Appl Microbiol Biotechnol (2011) 90:1193–1206
Candida albicans, as S. cerevisiae is atypical among yeasts
in being unable to synthesize carnitine (for a review, see
Strijbis and Distel 2010). It is likely that the situation in
most oleaginous yeasts will be similar to that described in
C. albicans which has a complete carnitine biosynthetic
pathway (Strijbis et al. 2009).
Even though β-oxidation occurs in both peroxisomes
and mitochondria in mammals and most eukaryotes, in
yeasts only Sporobolomyces roseus has been demonstrated
to contain all the mitochondrial enzymes required for
mitochondrial β-oxidation. However, some yeast species,
including Y. lipolytica, possess mitochondrial acyl-CoA
dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl CoA
thiolase coding sequences, suggesting that at least some β-
oxidation could be carried out in the mitochondria (Shen
and Burger 2009; Shen et al. 2009). The exact role of this
hypothetical mitochondrial pathway remains to be assessed.
The possibility to exploit these findings for enhancing
lipid accumulation and customizing lipid products will be
addressed in the next sections, after a short introduction
on how lipid accumulation is initiated in oleaginous
microorganisms.
Setting on lipid accumulation
It is now well established that cultivation conditions,
nitrogen availability, temperature, pH, metal traces, and
mineral concentrations all influence lipid accumulation in
oleaginous yeasts (Ratledge and Wynn 2002). These begin
to accumulate lipids when an essential element for cell
proliferation becomes limiting in the medium and the
carbon source is present in excess. Once cell proliferation
becomes limiting, carbon is directly channeled into lipid
synthesis with the resulting buildup of neutral lipids. Then,
in the presence of an excess of carbon source, depletion of
any essential nutriment can be used for setting on lipid
accumulation, like nitrogen, magnesium, zinc, iron, or
phosphorus. However, nitrogen limitation is generally used
(often referred to as a high C/N ratio). This is the easiest
condition to control and it results in the most efficient
accumulation induction factors. The reason of this efficien-
cy lies in an additional regulation affecting the TCA cycle:
once nitrogen becomes limiting, the cell in order to produce
ammonium breaks down AMP through AMP desaminase
(AMP→IMP+NH4+). AMP is a molecule that happens to
be the co-substrate of isocitrate dehydrogenase in the Krebs
(TCA) cycle. The latter is responsible for the conversion of
citrate to isocitrate and the resulting excess of non-
metabolized citrate is transferred in the cytosol via the
citrate/malate shuttle to be converted to acetyl-CoA by
ACL. Nitrogen limitation induces therefore the excessive
production of the main precursor of fatty acid synthesis.
1201
When non-oleaginous microorganisms are placed in such
nutriment-limiting conditions, cell proliferation obviously
ceases, but excess carbon is now directed towards trehalose
and glycogen synthesis (Francois and Parrou 2001;
Ratledge and Wynn 2002).
Yeasts under nutriment limitation undergo three phases
of growth: the first is cell proliferation (exponential growth
phase), then growth slows down when nutriments become
limiting and the cells start to accumulate lipids, and finally
cells enter stationary phase where lipid accumulation
continues, but β-oxidation also starts in an effort to
remobilize the carbon stored, until cells can no longer
produce essential metabolites and switch off most metabol-
ic activity. The duration of each phase depends on the C/N
ratio if we take the example of nitrogen limitation. In order
to fine tune the C/N ratio, there are different culture options
such as batch, fed-batch, and continuous fermentation
modes. Briefly, in batch mode all minerals and carbon
substrates are initially mixed in the reactor, with a high
initial C/N ratio to boost lipid accumulation. No further
monitoring of the C/N ratio is possible in this mode of
culture. Granger and colleagues (Granger 1992) showed
that accumulation in R. glutinis in batch mode with
different C/N ratio could increase lipid production by a
factor of three. In the continuous mode, the C/N ratio stays
constant throughout culture and regulation of substrate
concentration leads to the fine tuning of growth rate: lower
growth rates promote more extensive lipid accumulation
(Ykema et al. 1986). Finally, fed-batch mode allows a
precise control of nutrient and substrate flow rates. This
ensures efficient and reproducible lipid production. This
last fermentation mode is the most accurate and has been
long used for protein production processes; it is indeed the
most reliable approach to use in a biotechnological
application (Beopoulos et al. 2009a).
Enhancement of lipid synthesis and bio-oil
production—the biotechnological approach
Organism selection, culture monitoring, and nutriment
limitation may all be used to enhance lipid production or
to alter lipid composition depending on the carbon source
utilized (Athenstaedt et al. 2006), but for commercializing
oil derivatives from yeasts it just may not be good enough.
Many genetic tools are nowadays available, ranging from
conventional in vivo genetics to genetic engineering using
single or multicopy integration of native or heterologous
genes.
The first attempts at ameliorating lipid accumulation
required modification of the expression of key enzymes
involved in lipid metabolism, so as to redirect carbon
supply towards lipid synthesis. We have already addressed
1202
the case of malic enzyme (ME) in various yeast species in
order to regenerate the NADPH+ cofactor supply for FA
synthesis. The same stands for the overexpression of ACL,
increasing the acyl-CoA support towards de novo lipid
synthesis. All these attempts gave impressive results in non-
oleaginous microorganisms (Ratledge and Wynn 2002), but
failed at touching the limiting steps in Y. lipolytica as stated
above. Another key enzyme that is currently under
investigation is acetyl-CoA carboxylase (ACC), which
catalyzes the first and rate-limiting step in de novo lipid
synthesis. ACC1/2 overexpression increases lipid accumu-
lation in non-oleaginous yeasts; however, no data are
available for oleaginous species to verify if the enzymatic
activity of ACC is indeed a limiting step.
Concerning the neutral lipid synthesis pathway, deletion
of the glycerol-3-phosphate dehydrogenase gene (GUT2) in
the oleaginous yeast Y. lipolytica undergoing ex novo lipid
accumulation in oleic substrate prevented conversion of
G3P to DHAP, which resulted in a 3-fold increase in lipid
accumulation compared to wild-type strain (Beopoulos et
al. 2008). The additional overexpression of the GPD1 gene,
catalyzing this time the conversion of DHAP to G3P,
resulted in a 4-fold increase in lipid accumulation (Dulermo
T., submitted). These modifications combined with dele-
tions of the six POX genes encoding acyl-CoA oxidases,
abolishing β-oxidation, gave rise to an obese yeast capable
of accumulating more than 80% of its mass as lipids.
The aforementioned acyltransferase families carry out
the final acylation step of the TAG formation. The differ-
ences in substrate specificity and regulation of these
enzymes may be linked to some extent to the differences
in lipid accumulation observed between oleaginous yeasts.
The assumption made is that the TAG to FFA ratio
regulates total lipid accumulation in cells: boosting up
TAG production by overexpressing these genes could yield
over-accumulating strains. Overexpression of the DGA2
gene under the control of POX2 constitutive promoter in Y.
lipolytica cultivated in oleic acid containing medium
resulted in a 78% increase of the TAG to FFA ratio with
a simultaneous increase of 63% in overall lipid accumula-
tion, compared to wild-type strain (Beopoulos et al., AEM
submitted). Selective deletion or expression of these
enzymes may lead to TAGs with specific acyl-chain length:
Dupont de Nemours currently tries to exploit these paths for
biotechnological applications and has recently deposited a
patent (U.S. Patent 7465565) claiming heterologous ex-
pression in Y. lipolytica of DGAT from various oleaginous
yeasts to alter the quality and increase the quantity of the oil
produced.
Modulation of the degradation pathways (ω- and β-
oxidation) is also of interest for potential biotechnological
applications. Acyl-CoA oxidase (POX genes) Y. lipolytica
mutants display altered lipid profiles as a result of the Aox
Appl Microbiol Biotechnol (2011) 90:1193–1206
substrate specificity. POX2 deletion blocks the use of long-
chain fatty acids, whereas deletion of POX3 decreases the
degradation of short-chain fatty acids. Mutant strains
deleted for POX2, POX3, and POX5 result in a “slim”
phenotype probably due to a feedback regulation of
oxidation. On the other hand, strains overexpressing
POX2 result in an obese phenotype (Mlickova et al.
2004a). This kind of manipulation is not possible for non-
oleaginous species like S. cerevisiae that encode a single
acyl-CoA oxidase.
Another approach, already commercialized by Safisis
(Lesaffre, France), makes use of the yeasts β-oxidation
pathway in order to produce aromatic compounds like the
peach aromatic additive γ-decalactone from ricinoleic acid
(C18:1-OH). The same is achieved by feeding hydroxy
fatty acids to a Y. lipolytica pox3-deleted strain in order to
abolish the degradation capacity of the C10 fatty acid
lactone precursor (Wache et al. 2003).
A different application taking advantage of oxidation
pathways in yeast is the production of dicarboxylic acids
(DCA), commonly produced by chemical synthesis for
nylon, resins, and adhesives or for biodiesel production. As
stated before, yeasts produce DCA as a result of ω-
oxidation. The main yeast species modified so far for DCA
production using genetic engineering are Candida cloacae,
Candida tropicalis, and Y. lipolytica. The first industrial
process was developed by Nippon Mining Company
(Japan) and afterwards exploited by Cathay Biotechnology
of Shanghai (China) and Cognis (Germany) using mutant
strains of C. tropicalis. In this strain, β-oxidation is
abolished in order to prevent degradation of the DCA,
and the cytochrome P450 monoxygenase and reductase
systems, involved in the hydroxylation step of ω-oxidation,
are overexpressed (Picataggio et al. 1992). Furthermore,
three fatty alcohol oxidases from C. tropicalis (FAO1-3
genes) are overexpressed, resulting in an increased produc-
tivity of DCA (Eschenfeldt et al. 2003). Currently, DCA
production is attempted in Y. lipolytica, by deleting POX
genes, overexpressing NADPH-dependent P450 reductase
and ALK (P450 cytochrome oxidase) genetic context in
order to improve substrate specificity and specific chain
length DCA (Iida et al. 2000; Thevenieau 2006).
Polyunsaturated fatty acid (PUFA) production is also of
great biotechnological interest as these composites are on
high demand for dietary supplements and fish farm nutri-
ments. Numerous nutritional recommendations have been
made for the inclusion of PUFAs in the diet for the
prevention of coronary heart problems and also for the
improvement of retinal and brain functions. Suitable
production hosts that have been considered are filamentous
fungi like M. circinelloides for γ-linolenic acid (18:3, n−6),
Mortierella alpina for arachidonic acid (20:4, n−6), and
Crypthecodinium cohnii and Schizochytrium for docosa-
Appl Microbiol Biotechnol (2011) 90:1193–1206
hexaenoic acid (22:6, n−3) production. All these organisms
are used, or have been used, commercially to produce these
SCOs, in stirred tank fermenters of up to 220 m3 capacity
(Ratledge 2001; Meng et al. 2009). Currently, non-
microbial sources of these materials are favored, includ-
ing many endangered species of fish. But new alter-
natives might be proposed, as shown for instance by
Dupont de Nemours who genetically engineered strains
of Y. lipolytica (patent WO/2006/052871) in order to
commercialize docosahexaenoic acid (DHA) as an alter-
native source to fish oil. Although Y. lipolytica accumu-
lates lipids to lower levels than other oleaginous species, it
is the only yeast known to accumulate a high proportion of
linoleic acid (more than 50% of the total FA—Table 1),
which is the intermediate substrate of DHA synthesis. The
DHA-producing strain of Y. lipolytica expresses Δ4, Δ5,
Δ6, and Δ7 desaturases of the oleaginous fungus M.
alpina, the C18–20, C20–22 elongases of the heterotro-
phic marine protist Thraustochytrium aureum and is
genetically modified for its TAG acyltransferases. Recent-
ly, a Dupont de Nemours Y. lipolytica strain producing up
to 34% (w/w) of eicosapentaenoic acid (EPA) was
characterized as safe, matching the safety profile of the
EPA commercial fish oil (Belcher et al. 2011). Other
studies target the production of ω-3 and ω-6 PUFA as γ-
linolenic acid (Damude et al. 2006).
Polyhydroxyalkanoates (PHA) are polyesters (bioplas-
tics) with interesting thermoplastic and elastomeric proper-
ties. The 3-hydroxyacyl-CoA intermediate of β-oxidation
(see above) is the substrate of PHA synthetase, a protein
found in a number of bacteria, but absent from yeasts. The
synthesis of medium chain length (MCL) PHA has been
achieved through peroxisomal targeting of the PHA
synthetase from Pseudomonas aeruginosa in S. cerevisiae
and Pichia pastoris (Poirier et al. 2001, 2002). The size of
the PHA monomers synthesized by these yeasts ranges
from five to 14 carbons and is generated by the degradation
of odd- or even-chain fatty acids via the peroxisomal β-
oxidation cycle. Recently, Haddouche and colleagues
expressed the PHA synthetase from P. aeruginosa in
different POX genetic backgrounds of Y. lipolytica, result-
ing in high yields of PHA production, depending on the
POX genotype (Haddouche et al. 2010). Further improve-
ments of PHA production by yeasts will require the
modification of the MFE enzyme in order to block β-
oxidation after the production of 3-hydroxyacyl-CoA, the
immediate substrate of PHA synthetase.
Finally, carotenoid production has been considered in
yeasts. Carotenoids represent a large group of structurally
diverse pigments, with an acyl chain ranging from C30 to
C50 carbons, synthesized by microorganisms and plants.
They are commercially used as food colorants, nutraceut-
icals, and for cosmetic and pharmaceutical purposes.
1203
Mammals, however, cannot synthesize carotenoids and
must obtain them through their diet. In general, the natural
biological systems that produce carotenoids are industrially
intractable and produce the compounds at such low levels
that production on a commercial scale is not practicable.
Therefore, most carotenoids used in industry are produced
by chemical synthesis, generating the need for more
environmental friendly biological processes for their pro-
duction. Some efforts have previously been made to
genetically engineer bacteria or fungi to produce high
levels of carotenoids (Visser et al. 2003). Biosynthesis of
these pigments in non-carotenogenic hosts such as yeasts
requires the heterologous expression of a geranylgeranyl
diphosphate synthetase encoding gene (crtE) to comple-
ment their own isoprenoid pathway. This enzyme catalyses
the head-to-tail condensation of an isoprenoid unit to the
isoprenoid precursor farnesyl diphosphate (C15), yielding
geranylgeranyl diphosphate (C20, GGDP). An additional
recombinant enzyme, phytoene synthetase (CrtB), is then
required for the head-to-head condensation of two GGDP
molecules to the colorless C40 carotenoid phytoene
(Sabirova et al. 2010). Expression of these enzymes in the
host Escherichia coli allowed the recombinant biosynthesis
of diverse cyclic and acyclic carotenoids as well (Schmidt-
Dannert 2000). In the need of improved systems, allowing
higher levels of production and facilitated purification,
efforts have been devoted to carotenoid production in
oleaginous yeasts such as Y. lipolytica (Ailey et al. 2007).
An interesting review presenting novel ideas for biocon-
version of fats and lipids by expressing bacterial genes in Y.
lipolytica was recently published (Sabirova et al. 2010).
Future works and perspectives
Negative environmental impact of fossil fuels and the need
of renewable energy have prompted research towards
biofuel production. The high cost and price imbalance
created by using plant oils or animal fat as petroleum
substitutes aroused interest in developing oleaginous micro-
organisms to obtain large amounts of oils for biodiesel
utilization. However, producing competitive biodiesel is
possible only if the oil is produced from cheap raw
materials as substrates. In the future, biofuel production
may combine use of plant leftovers to oil production by
yeasts. It would entail the screening for oleaginous yeast
strains tolerant to lignocellulose degradation products
(Chen et al. 2009) and evolutionary engineering of yeasts.
Such an approach has already been developed in S.
cerevisiae to improve xylose fermentation for ethanol
production (Garcia Sanchez et al. 2010).
Further optimization of yeast potentials for oil produc-
tion by means of substrate selection and genetic or
1204
metabolic engineering is crucial for the development of
future biotechnological applications. It remains essential
that the metabolism of more yeasts be studied as new
sequence data and more genetic tools become available.
This will lead to the understanding and the exploitation of
the mechanisms conferring massive lipid accumulation and
specific oil production in certain species. New methods
combining lipidomic, metabolomic, and genetic approaches
will undoubtedly provide much information about the
regulation of lipid metabolism. Combining these informa-
tions with subcellular localization data and feeding them
into models of the lipid pathways and their regulation may
provide novel ideas (Boer et al. 2007). In addition, making
use of the byproducts generated in the course of fatty acid
production to manufacture proteins, nutrients, steroids, and
aromatic components may reduce the production cost of the
lipid moieties and constitute a side biotechnological
application on its own.
In conclusion, even though few detailed studies in yeast
species other than S. cerevisiae and Y. lipolytica have been
carried out, increasing efforts are devoted towards the
manipulation and regulation of yeast lipid metabolism for
bio-oil production, building up an emergent biotechnolog-
ical field with many promising applications.
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