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J. Biol. Chem., Vol. 278, Issue 35, 32596-32601, August 29, 2003
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-Oxidation in Saccharomyces cerevisiae*
From the Laboratoire de Biotechnologie Végétale, Institut d'Écologie, Université de Lausanne, CH-1015 Lausanne, Switzerland
Received for publication, May 28, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-oxidation was analyzed in
Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis
in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate
synthase, of the -oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis
of polyhydroxyalkanoate was dependent on the -oxidation enzymes acyl-CoA
oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase
multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs,
and on the peroxin PEX5, which is involved in the import of proteins
into the peroxisome. In wild type cells grown in media containing fatty acids,
the polyhydroxyalkanoate monomer composition was largely influenced by the
nature of the external fatty acid, such that even-chain monomers are generated
from oleic acid and odd-chain monomers are generated from heptadecenoic acid.
In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate,
3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant
deficient in the peroxisomal 3-ketothiolase (fox3
0)
growing either on oleic acid or heptadecenoic acid as well as in wild type and
fox30 mutants grown on glucose or raffinose,
indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis
were generated from the degradation of intracellular short- and medium-chain
fatty acids by the -oxidation cycle. Inhibition of fatty acid
biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from
intracellular fatty acids but still enabled the use of extracellular fatty
acids for polymer production. Mutants affected in the synthesis of lipoic acid
showed normal polyhydroxyalkanoate synthesis capacity. Together, these results
uncovered the existence of a substantial futile cycle whereby short- and
medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway
are directed toward the peroxisomal -oxidation pathway. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Pseudomonads, such as Pseudomonas oleovorans and Pseudomonas
aeruginosa, typically synthesize PHA containing 3-hydroxyacids ranging
from 6 to 14 carbons in length
(2,
3). P. oleovorans can
synthesize PHA when cells are grown on alkanoic acids as the carbon source
(2,
3). The nature of the PHA
produced is related to the substrate used for growth and is typically composed
of monomers that are 2n (n 
0) carbons shorter than the
substrate. For example, growth of P. oleovorans on dodecanoate
generates PHA containing 3-hydroxydodecanoic acid, 3-hydroxydecanoic acid,
3-hydroxyoctanoic acid, and 3-hydroxyhexanoic acid
(5). In these cells, PHA is
synthesized by the PHA synthase from the polymerization of 3-hydroxyacyl-CoA
intermediates generated by the -oxidation of alkanoic acids. PHAs have
also been produced in a number of recombinant eukaryotes, including insect
cells (6), yeast
(7–9),
and plants
(10–13).
Beyond its value as a biopolymer for commercial applications, PHA can be
used as a valuable indicator of the flux of carbon through various metabolic
pathways providing substrates for its synthesis. To date, this has been mostly
exploited in transgenic Arabidopsis thaliana expressing the PHA
synthase from P. aeruginosa in the peroxisome
(14–18).
In these plants, PHA containing saturated and unsaturated 3-hydroxyalkanoic
acids ranging from 6 to 16 carbons is synthesized during germination using
intermediates of the -oxidation of fatty acid derived from the
degradation of triacylglycerides
(14). Manipulation of the
plant fatty acid composition, either through the use of mutants in fatty acid
desaturases or by feeding external fatty acids, demonstrated that the PHA
monomer composition adequately reflects both the nature and quantity of fatty
acid directed toward peroxisomal -oxidation
(15). From these initial
studies, peroxisomal PHA was subsequently used to reveal the existence of a
futile cycle of fatty acids in transgenic plants expressing a medium-chain
fatty acyl-acyl carrier protein thioesterase in leaves or developing seeds
(15,
16) as well as in developing
A. thaliana embryos deficient in the synthesis of triacylglycerides
(16). The PHA monomer
composition has also been used to analyze the pathway of degradation of
unsaturated fatty acids in plants
(17).
Recently, the synthesis of PHA from intermediates of -oxidation has
been demonstrated in Saccharomyces cerevisiae expressing the PHA
synthase from P. aeruginosa in the peroxisome
(7). Growth of these
recombinant S. cerevisiae in media containing oleic acid or
heptadecenoic acid resulted in the accumulation of PHA containing either
even-chain monomers from 6 to 14 carbons in length, or odd-chain monomers from
5 to 13 carbons, respectively
(7). In an effort aimed at
understanding the factors influencing the flux of fatty acids toward
-oxidation, we have examined PHA synthesis in mutants of S.
cerevisiae affected in various enzymes involved in fatty acid metabolism.
In this study, we show that the synthesis of PHA in the fox30
mutant deficient in the peroxisomal 3-ketothiolase reveals the presence of a
substantial futile cycle of intermediates of the cytoplasmic fatty acid
biosynthetic pathway toward peroxisomal -oxidation in yeast growing in
media containing various carbon sources.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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according to Sambrook
et al. (19). Wild
type S. cerevisiae strain BY4742 (mat
his31 leu20 lys20
ura30) as well as the mutants
fox10 (YGL205w::kanMX4),
fox20 (YKR009c::kanMX4),
fox30 (YIL160c::kanMX4), and
pex50 (YDR244W::kanMX4), all
derived from the strain BY4742, were obtained from EUROSCARF
(www.uni-frankfurt.de/fb15/mikro/euroscarf/index.html).
S. cerevisiae harboring the PHA synthase gene from P.
aeruginosa was maintained in leucine-deficient media (0.67% yeast
nitrogen base without amino acids (Difco, Detroit, MI), 0.5% ammonium sulfate,
2% glucose, and 0.69 grams/liter leucine drop-out supplement (Clontech) with
or without 200 µg/ml gentamycin). For experiments analyzing PHA synthesis
in cells growing on media containing fatty acids, a stationary phase culture
was harvested by centrifugation, and cells were washed once in water and
resuspended at a 1:10 dilution in fresh media containing 0.1% (w/v) glucose,
2% Pluronic F-127 (w/v) (Sigma), and 0.1% (v/v) fatty acids. Cells were grown
for an additional 3–4 days before harvesting for PHA analysis. A similar
protocol was used for cells grown on media containing carbohydrates, except
that the cells were diluted 1:40 in leucine-deficient media supplemented with
either 2% (w/v) glucose or 3% (w/v) raffinose. In some experiments, cerulenin
(Sigma) was added at a final concentration of 25–50 µM,
and fatty acids (oleic acid, palmitic acid, and tridecanoic acid) were added
at concentrations between 0.005–0.01%. DNA Constructs—The PHAC1 synthase from P. aeruginosa was modified for peroxisomal targeting by the addition, at the carboxyl end of the protein, of the last 34 amino acids of the Brassica napus isocitrate lyase, as described previously (14). The plasmid Yiplac128-PHA containing the modified PHAC1 gene is under the control of the S. cerevisiae catalase A (CTA1) promoter and terminator region, as described previously (7). To construct the plasmid CTA-PHA, the cassette containing the CTA1 promoter-PHAC1 synthase-CTA1 terminator was excised from Yiplac128-PHA and cloned into the replicative shuttle plasmid Yiplac111 (20). In the plasmid GPD-PHA, the PHAC1 gene is under the control of the constitutive glycerol-3-phosphate dehydrogenase promoter (21). This plasmid was constructed by first excising the PHA synthase along with the CTA1 terminator from the CTA-PHA plasmid with EcoRI and blunt-ended with Klenow DNA polymerase. The fragment was then subcloned into the vector p415-GPD (21) at the SmaI site. Plasmids were transferred into the various S. cerevisiae strains by the lithium acetate procedure (22), and transformants were recovered on media without leucine.
Analysis of Fatty Acids and PHA—Analysis of fatty acids and PHA was performed essentially as described previously (7, 14).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Oxidation Mutants of
S. cerevisiae—Synthesis of PHA in cells expressing a peroxisomal
PHA synthase from the plasmid CTA-PHA and grown on medium containing oleic
acid was examined in wild type BY4742 cells as well as in three mutants having
a deletion in the genes encoding the enzymes of the core peroxisomal
-oxidation cycle. These mutants were deficient either in the acyl-CoA
oxidase (FOX1), the multifunctional protein (FOX2) possessing the 2-enoyl-CoA
hydratase II and R-3-hydroxyacyl-CoA dehydrogenase activities, or the
3-ketothiolase (FOX3). Also used was a mutant deficient in the peroxin PEX5,
which is involved in the import, into the peroxisomes, of proteins having a
type 1 peroxisomal targeting signal, including FOX1, FOX2, and the modified
PHA synthase. Wild type cells grown for 3 days on oleic acid produce
0.06% PHA (weight/dry weight) containing even-chain monomers from 6 to 14
carbons that are generated by the degradation of oleic acid via the
peroxisomal -oxidation cycle, namely the monomers H14:1, H14:0, H12:0,
H10:0, H8:0 and H6:0 (3-hydroxy acid monomers are identified with the prefix
H, followed by the number of carbons and the number of unsaturated bonds)
(Fig. 1A). The
presence of both 3-hydroxytetradecenoic acid (H14:1) and
3-hydroxytetradecanoic acid (H14:0) agrees with the generation of the
corresponding acyl-CoA by the -oxidation of fatty acids having a
cis-unsaturated bond at an odd-numbered carbon
(23). No PHA was detected in
the fox10, fox20, and
pex50 mutants expressing the PHA synthase (data not
shown). These results are in agreement with the fact that the
3-hydroxyacyl-CoAs used by the PHA synthase are generated by the peroxisomal
-oxidation pathway. Expression of the PHA synthase in the
fox30 mutant resulted in the accumulation of
0.006% (weight/dry weight) of a PHA that contained only the monomers
H8:0, H10:0, and H12:0 (Fig.
1B). These results were unexpected, because the only
3-hydroxyacyl-CoA that could be generated in the
fox30 mutant from the degradation of the external
oleic acid was 3-hydroxyoctadecenoyl-CoA, which is itself not a substrate for
the PHA synthase because this enzyme in yeast accepts only 3-hydroxyacyl-CoAs
between 5 and 14 carbons in length
(7).
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PHA Synthesized in the fox30 Mutant Is Derived from
Intracellular Short- and Medium-chain Fatty Acids—The presence of
PHA in the fox30 mutant raised the question as to the
source of the carbon that can generate peroxisomal 3-hydroxyacyl-CoAs from 8
to 12 carbons in length. Analysis of the synthetic media used to grow cells
combined with experiments wherein external fatty acids were added to the
growth media revealed that the level of contaminating fatty acids present in
the growth media was at least one order of magnitude below the minimum level
of fatty acids required to influence PHA synthesized
fox30 cells (data not shown). Thus, contaminating
fatty acid cannot explain the PHA synthesized in fox30 cells.
The presence of an alternative 3-ketothiolase that could functionally
complement the FOX3 enzyme and generate shorter-chain 3-hydroxyacyl-CoAs from
oleic acid was examined. Wild type and fox30 cells
expressing the peroxisomal PHA synthase were grown in media containing 0.1%
oleic acid and 0.1% lauric acid. Wild type cells grown on both oleic and
lauric acid produced 5-fold more PHA compared with cells grown only on oleic
acid (Fig. 1C). The
monomer composition of the PHA synthesized from the co-feeding was shifted
toward shorter saturated monomers, with the H14:1 and H14 monomers becoming
undetectable. This shift in monomer composition is explained by the fact that
degradation of lauric acid will generate only even-chain saturated monomers
equal to and lower than 12 carbons. In sharp contrast, the PHA synthesized in
the fox30 cells grown on oleic and lauric acid was composed
exclusively of the monomer 3-hydroxydodecanoic acid
(Fig. 1D). Thus,
although the external lauric acid was able to access the peroxisomal
-oxidation enzymes in fox30 cells, only
3-hydroxydodecanoyl-CoA could be generated from it, indicating the absence of
an alternative to the FOX3 peroxisomal 3-ketothiolase for the generation of
shorter 3-hydroxyacyl-CoA.
Wild type cells expressing the PHA synthase and grown on media containing
10cis-heptadecenoic acid generated a PHA composed of the
saturated odd-chain monomers H5:0, H7:0, H9:0, and H11:0, as well as the
unsaturated monomer H13:1 (Fig.
2A). This monomer composition is in agreement with the
pathway of degradation of fatty acids having a cis-unsaturated bond
at an even-numbered carbon
(24). Although H8:0, H10:0,
and H12:0 monomers were also detectable in the PHA produced from these cells,
their amount represent less than 0.5 mol %. Thus, in wild type cells fed with
10cis-heptadecenoic acid, the PHA monomers are primarily
derived from the degradation of the external fatty acid. In contrast, the
growth of fox30 cells on media containing
10cis-heptadecenoic acid resulted in a PHA containing only the
monomers H8:0, H10:0, and H12:0, a composition unchanged relative to the
growth of fox30 on oleic acid
(Fig. 2B). These
results indicated that the PHA synthesized in fox30
cells grown on 10cis-heptadecenoic acid cannot be derived from
the degradation of the external fatty acids but rather must come from
metabolically synthesized substrates. Furthermore, the absence, in the
fox30 mutant, of a 3-ketothiolase that can function
in the -oxidation cycle means that any fatty acid that reaches the
-oxidation cycle in the fox30 mutant can only be
converted to a 3-hydroxyacyl-CoA of equal chain length. Together, these
results support the hypothesis that PHA synthesized in
fox30 cells fed with either oleic acid or
10cis-heptadecenoic acid are derived from intracellular fatty
acids or fatty acyl esters of 8, 10, and 12 carbons in length that reach the
peroxisomal -oxidation cycle.
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Synthesis of PHA in fox30 Cells Grown on
Carbohydrates—In contrast to wild type, the fox30
mutant cannot utilize external fatty acids as a carbon source to grow because
the -oxidation pathway is blocked. Thus, fox30 cells
grown in media containing 0.1% glucose and 0.1% oleic acid are essentially
starved for carbon once the glucose is consumed. To assess whether the
synthesis of PHA in fox30 cells was dependent on
these particular growth conditions, the synthesis of PHA was monitored in
cells grown on carbohydrates as the principal carbon source. Because the CTA1
promoter used in the CTA-PHA vector is activated by fatty acids but repressed
by glucose (7,
25,
26), a new vector named
GPD-PHA was constructed by putting the PHA synthase under the control of the
constitutive glycerol-3-phosphate dehydrogenase promoter. Western analysis of
wild type cells transformed with the plasmid GPD-PHA revealed the expression
of the PHA synthase to approximately the same level in cells grown in media
containing either glucose, raffinose, or oleic acid (data not shown).
Synthesis of PHA in wild type cells transformed with GPD-PHA and grown in
media containing 10cis-heptadecenoic acid was essentially the
same as that reported for the plasmid CTA-PHA, with the PHA being composed
mainly of the odd-chain monomers H5:0, H7:0, H9:0, H11:0, and H13:1
(Fig. 2C). In
contrast, fox30 cells transformed with GPD-PHA and
grown in media containing 10cis-heptadecenoic acid produced a
PHA containing only the H8, H10, and H12 monomers
(Fig. 2D).
Wild type and fox30 cells transformed with the
plasmid GPD-PHA and grown for 4 days in media containing 2% glucose synthesize
0.01% (weight/dry weight) PHA (Fig. 3,
A and B). The monomer composition for these two
strains was similar and included the monomers H6:0, H8:0, H10:0, and H12:0. In
contrast to glucose, raffinose does not repress the genes involved in
-oxidation (27,
28). Growth of cells for 4
days in media containing raffinose resulted in an 4-fold increase in PHA
synthesized in fox30 and wild type cells compared
with growth in media containing glucose, although the monomer composition
remained similar for all treatments (Fig.
3, C and D). No PHA was detected in
fox10, fox20, or
pex50 cells transformed with the GPD-PHA plasmid and
grown on raffinose. Together, these results demonstrate that, in cells growing
on carbohydrates as the main carbon source, synthesis of PHA from
intracellular fatty acids of 6, 8, 10, and 12 carbons occurs and requires a
functional peroxisome and -oxidation cycle.
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The Contribution of Pathways of Lipoic Acid and Fatty Acid Biosynthesis
to Peroxisomal PHA—The synthesis of PHA in
fox30 cells grown in media containing raffinose was
monitored over 5 days (Fig. 4).
The quantity of PHA per unit dry weight of cells remained relatively constant
during the initial lag phase, decreased during the exponential growth phase,
and increased during the stationary phase. These data indicate that PHA
synthesis in fox30 still occurs when cells have
ceased to divide and are in nutrient-limiting conditions. Although some PHA
could also be synthesized during the exponential phase, the rate of PHA
accumulation is lower than the rate of increase of the cell mass.
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The impact of inhibiting fatty acid biosynthesis on PHA production was
tested in the fox30 strain. Cerulenin, an inhibitor
of the fatty acid synthase (FAS)
(29), was added at 48 h in the
exponential growth phase of a fox30 culture growing
on raffinose. The addition of cerulenin limited growth of cells as observed by
the lower density of the culture (Fig.
4). Furthermore, the addition of cerulenin quickly abolished the
increase in PHA that was observed after 48 h in cultures growing without
cerulenin.
The effect of cerulenin on the monomer composition of synthesized PHA was
also examined in fox30 cells that were grown in media
containing 3% raffinose, 0.01% oleic acid, 0.005% palmitic acid, and 0.01%
tridecanoic acid as well as with and without cerulenin. Oleic and palmitic
acids were added as a source of fatty acids necessary for the synthesis of
membrane lipids and the maintenance of cell growth after the addition of
cerulenin. As shown in Fig.
5A, growth of fox30 cells in
this complex medium containing a low amount of tridecanoic acid and without
cerulenin lead to the synthesis of PHA containing the H13 monomer as well the
H6, H8, H10, and H12 monomers. The addition of cerulenin resulted in a 9-fold
increase in the proportion of the H13 monomer as well as a 4-fold reduction in
the amount of PHA relative to cultures grown without cerulenin
(Fig. 5B). These
results are in agreement with the hypothesis that even-chain PHA monomers are
derived from intermediates of the cerulenin-sensitive FAS, whereas the
odd-chain H13 monomer is derived from the degradation of the external
tridecanoic acid via the cerulenin-insensitive -oxidation cycle.
Furthermore, the overall decrease in PHA produced in cells grown in media with
cerulenin and 0.01% tridecanoate would indicate that, in the absence of
inhibitor, the contribution of the internal fatty acids derived from FAS to
PHA synthesis was larger than the contribution of the external fatty acid.
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Lipoic acid is an eight-carbon fatty acid modified with sulfur insertion
and synthesized via the FASII system present in the mitochondria. The
contribution of the lipoic acid pathway to the generation of intracellular
substrates for peroxisomal PHA synthesis was examined in the oar1 and
cem1 mutants deficient in the mitochondrial FASII enzymes essential
for lipoic acid synthesis (30,
31). PHA synthesis in wild
type and the oar1 and cem1 mutants transformed with the
GPD-PHA construct and grown in media containing raffinose was similar in both
quantity and monomer composition (data not shown), indicating that the
mitochondrial FASII does not contribute to the flux of short- and medium-chain
fatty acids toward the peroxisome -oxidation pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-oxidation pathway
(7). This is supported by the
observation that deletion mutants in the acyl-CoA oxidase and the
multifunctional protein, the first two enzymes of the -oxidation pathway
that are required for synthesis of R-3-hydroxyacyl-CoA, do not
produce PHA. The requirement of a functional peroxisome is also indicated by
the absence of PHA in the pex50 mutant deficient in
the import, into the peroxisome, of proteins having a type 1 peroxisome
targeting signal, including proteins such as FOX1, FOX2, and the PHA synthase.
In cells grown in media containing an external fatty acid as the main source
of carbon, PHA monomer composition is directly influenced by the nature of the
external fatty acids. Thus, feeding with oleic acid yields a PHA containing
even-chain monomers exclusively (Fig.
1A), whereas feeding with
10cis-heptadecenoic acid yields a PHA containing primarily
odd-chain monomers (Fig. 2, A and
C). These results indicate that, under these conditions,
the main 3-hydroxyacyl-CoAs going into PHA are primarily derived from the
-oxidation of external fatty acids.
The synthesis of PHA containing the H8:0, H10:0, and H12:0 monomers in the
fox30 mutant grown on oleic acid
(Fig. 1B) was
surprising, because the only 3-hydroxyacyl-CoA that could be generated from
oleic acid entering a -oxidation cycle that is truncated at the
3-ketothiolase step would be 3-hydroxyoctadecenoyl-CoA, which is itself not a
substrate for the PHA synthase. In contrast to wild type cells, PHA
synthesized in fox30 cells grown in media containing
10cis-heptadecenoic acid does not contain any odd-chain
monomers but rather the monomers H8:0, H10:0, and H12:0, indicating that the
substrates for PHA synthesis in the fox30 mutants are not
derived from the external fatty acids (Fig.
2, B and D). However, in
fox30 cells grown in the presence of both oleic acid
and lauric acid, PHA containing the H12:0 monomer exclusively is generated, a
result expected if the main substrate entering the truncated -oxidation
cycle is derived from the external lauric acid
(Fig. 1D). This
demonstrates that, similar to wild type cells, fox30 cells are
still competent to acquire fatty acids from the external media, target them to
the peroxisome, and initiate their degradation through -oxidation.
Together, these results indicated that the 8-, 10-, and 12-carbon monomers
found into PHA synthesized in fox30 cells grown on oleic and
10cis-heptadecenoic acids are derived from intracellular fatty
acids (or fatty acyl-esters) of 8, 10, and 12 carbons.
The monomer composition of the PHA synthesized in fox30
cells grown on media containing either fatty acids, glucose, or raffinose
changed little with the exception of the presence of the additional H6:0
monomer only in cells grown on carbohydrates in addition to the H8:0, H10:0,
and H12:0 monomer (Figs. 2 and
3). Furthermore, the monomer
composition and quantity of PHA synthesized in wild type and
fox30 cells grown on carbohydrates were remarkably
similar. The absence of both the H14:1 and H14:0 in PHA from wild type cells
grown on carbohydrates is important, because H14:1 is an indicator of the
degradation of palmitoleic and oleic acids, whereas H14 is an indicator of the
degradation of palmitic and stearic acids. These four fatty acids account for
>98% of the fatty acid found in cells at either the exponential or
stationary growth phase, whereas fatty acids of 
14 carbons account for
<2% (data not shown, and Ref.
32). This indicates that, in
wild type cells as well as fox30 grown on carbohydrates, the
fatty acids that enter the peroxisomal PHA are not derived from the turnover
of all fatty acids found in membranes but rather are enriched in short- and
medium-chain fatty acids. The observation that the addition of cerulenin, a
powerful inhibitor of FAS, inhibits the accumulation of PHA in cells grown on
raffinose as well as increases the proportion of odd-chain monomers at the
expense of even-chain monomers in cells grown in media containing tridecanoic
acid indicates that the premature release of short- and medium-chain fatty
acids from the FAS complex contributes to the overall carbon flux toward
peroxisomal -oxidation. Furthermore, the demonstration that a mutation
in the pathway of mitochondrial lipoic acid biosynthesis did not affect PHA
synthesis from glucose indicates that it is the cytoplasmic FAS complex that
mainly contributes to this carbon flux.
The amount of PHA synthesized in fox30 cells was
influenced by the carbon source used for growth with the maximal amount
obtained with raffinose (0.055% weight/dry weight), whereas glucose and oleic
acid gave lower amounts (0.012 and 0.006% weight/dry weight, respectively).
The increase of PHA in raffinose compared with glucose is likely to be due, at
least in part, to the fact that in contrast to glucose, raffinose does not
repress transcription of the genes of the -oxidation cycle
(27,
28). Although the
-oxidation genes should be fully activated in media containing oleic
acid, the amount of PHA synthesized was lowest. One explanation for this could
be that the 3-hydroxyoctadecenoyl-CoA produced in
fox30 cells grown on oleic acid may inhibit the flux
of intracellular short- and medium-chain fatty acids through the truncated
-oxidation cycle and thus decrease carbon flux to PHA.
It is striking that the amount of PHA synthesized in wild type cells grown
on raffinose (0.05% weight/dry weight; Fig.
3C), wherein the PHA is largely derived from
intracellular short- and medium-chain fatty acids, is similar to or slightly
lower than the amount of PHA synthesized in wild type cells grown either on
oleic acid (0.06% weight/dry weight; Fig.
1A) or 10cis-heptadecenoic acid
(0.14–0.18% weight/dry weight; Fig.
2, A and C), wherein the bulk of
-oxidation intermediates going into PHA are derived from the external
fatty acids and where carbon flux through -oxidation cycle is expected
to be maximal because cells depend on it for growth. Although PHA synthesized
in yeast peroxisomes may not be directly proportional to the carbon flux to
the -oxidation cycle under all growth conditions, these results
nevertheless indicate that the flux of short- and medium-chain fatty acids
derived from FAS toward peroxisomal -oxidation is substantial and
clearly larger than the turnover of long-chain fatty acids.
The molecular mechanism responsible for this futile cycling of short- and
medium-chain fatty acids in yeast is unclear at present. Fatty acids lower
than 14 carbons are present only in trace amounts in yeast lipids, with the
bulk of fatty acids being C16:0, C16:1, C18:0, and C18:1
(32). In yeast, fatty acids
are released from the FAS complex as acyl-CoAs by the malonyl/palmitoyl
transferase domain of the -subunit of FAS, which plays the dual role by
loading malonyl substrate moieties in chain elongation and translocating the
palmitoyl moieties to a CoA acceptor in chain termination
(33). Experiments with
purified FAS from yeast indicated that the primary products released from the
FAS complex in vitro are long-chain acyl-CoAs
(34,
35). However, it has also been
demonstrated that the presence of the acyl-CoA binding protein ACBP1 in an
in vitro reaction mixture results in an enhanced release of
medium-chain acyl-CoAs from the FAS complex, indicating that the presence of
other proteins or co-factors can greatly influence the distribution of fatty
acyl-CoAs released from FAS
(35). The presence of a futile
cycle of short- and medium-chain fatty acids in yeast may thus indicate that
the release of short- and medium-chain acyl-CoAs from FAS in vivo is
considerably larger than would be suspected from either in vitro
experiments or fatty acid composition of lipids. Further experiments are
required to determine whether this is specific to the FAS of yeast or whether
it also applies to the FAS of plants and animals.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 41-21-692-4222; Fax:
41-21-692-4195; E-mail:
yves.poirier{at}ie-bpv.unil.ch.
1 The abbreviations used are: PHA, polyhydroxyalkanoate; CTA, catalase; FAS,
fatty acid synthase; GPD, glycerol-3-phosphate dehydrogenase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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