(A-ALD and PK/PTA). Indeed, this approach
led to the highest yields ever reported of an
isoprenoid produced in S. cerevisiae and
impressively demonstrated how an “ideal”
combination of precursor supply pathways
based on stoichiometric analyses can be
designed to produce AcCoA derivatives at an
industrially competitive level.
D. Citrate-Oxaloacetate Shuttle
The major breakthroughs regarding production
of oleochemicals in S. cerevisiae have been
recently achieved by implementing the citrateoxaloacetate shuttle. If NADPH supply is not
concomitantly engineered, this pathway, like
the ATP-independent pyruvate to AcCoA
routes, has the lowest expected yield for FA
produced via FAS (74% of the theoretical maximum; van Rossum et al. 2016b) due to the
imbalance of NADH production and NADPH
requirement of FAS. Nevertheless, many oleaginous yeasts, such as Yarrowia lipolytica, which
produce large amounts of lipids in the cytosol,
rely on this system to provide cytosolic AcCoA
(Vorapreeda et al. 2012). The term “shuttle”
refers to the fact that AcCoA is formed from
pyruvate inside mitochondria via the native
PDH complex. Subsequently, the acetate moiety
is transferred to oxaloacetate by citrate
synthase (CS). The resulting citrate either can
enter the TCA cycle inside the mitochondria or
be exported to the cytosol to serve as a shuttle
for acetyl moieties. A cytosolic ATP citrate
lyase (ACL), an enzyme that is not present in
S. cerevisiae, is required to form AcCoA and
oxaloacetate under the expense of one ATP
molecule. The cycle is closed by transporting
oxaloacetate back into the mitochondria
through a citrate-oxaloacetate antiporter. In a
first attempt, the overexpression of a murine
ACL in S. cerevisiae led to a slightly increased
titer of total FA (Tang et al. 2013). The effect
was mostly pronounced during the stationary
phase, probably reflecting the redirection of the
metabolism toward respiration of the accumulated ethanol.
In the same study, isocitrate dehydrogenase genes IDH1
and IDH2 were deleted to promote the accumulation of
citrate; somewhat surprisingly, this intervention did
not affect the total content but only the saturation
profile of FA, suggesting that ACL activity and/or
other factors were rate limiting. Later studies, performed in strains engineered for n-butanol (Lian et al.
2014), hexadecanol (Feng et al. 2015), or mevalonate
(Rodriguez et al. 2016) production, showed that the
choice of the heterologous enzyme has a certain impact
on the pathway efficiency.
In most recent studies that reported the
highest titers of FA or their derivatives (Zhou
et al. 2016b; Yu et al. 2018), the citrateoxaloacetate shuttle was further optimized and
combined with strategies that increase the
NADPH supply. The overexpression of ACL
alone in a strain that was already engineered
to block FA degradation led to a moderate
improvement (up to 50%) of free FA titers,
which is consistent with previous studies
(Tang et al. 2013; Lian et al. 2014). A further
improvement was achieved by introducing a
transhydrogenase-like reaction sequence to
increase NADPH supply, exemplifying the
importance of redox cofactors as a driving
force for FA production. In this scheme, a cytosolic malate dehydrogenase (MDH) reduces
oxaloacetate to malate (oxidizing NADH), and
a cytosolic malic enzyme (MAE) subsequently
transfers hydrogen to NADP, whereby malate is
converted to pyruvate, which then re-enters
mitochondria and undergoes a new cycle
(Fig. 14.1). Additional overexpression of the
endogenous citrate transporter Ctp1 appeared
to have no beneficial effect on the FA production, suggesting that the export of citrate into
the cytosol may not have been a limiting factor
in this approach. In a follow-up study (Yu et al.
2018), several strategies to enhance the citrateoxaloacetate shuttle were tested. Overexpression of the endogenous (mitochondrial) PDH
components to enhance the mitochondrial synthesis of AcCoA did not lead to an increase in
FA titers in this study. However, the overexpression of the mitochondrial pyruvate carrier
subunits Mpc1 and Mpc3 and introduction of a
heterologous CS in addition to the native one
appeared to increase the flux through the
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
345
led to the highest yields ever reported of an
isoprenoid produced in S. cerevisiae and
impressively demonstrated how an “ideal”
combination of precursor supply pathways
based on stoichiometric analyses can be
designed to produce AcCoA derivatives at an
industrially competitive level.
D. Citrate-Oxaloacetate Shuttle
The major breakthroughs regarding production
of oleochemicals in S. cerevisiae have been
recently achieved by implementing the citrateoxaloacetate shuttle. If NADPH supply is not
concomitantly engineered, this pathway, like
the ATP-independent pyruvate to AcCoA
routes, has the lowest expected yield for FA
produced via FAS (74% of the theoretical maximum; van Rossum et al. 2016b) due to the
imbalance of NADH production and NADPH
requirement of FAS. Nevertheless, many oleaginous yeasts, such as Yarrowia lipolytica, which
produce large amounts of lipids in the cytosol,
rely on this system to provide cytosolic AcCoA
(Vorapreeda et al. 2012). The term “shuttle”
refers to the fact that AcCoA is formed from
pyruvate inside mitochondria via the native
PDH complex. Subsequently, the acetate moiety
is transferred to oxaloacetate by citrate
synthase (CS). The resulting citrate either can
enter the TCA cycle inside the mitochondria or
be exported to the cytosol to serve as a shuttle
for acetyl moieties. A cytosolic ATP citrate
lyase (ACL), an enzyme that is not present in
S. cerevisiae, is required to form AcCoA and
oxaloacetate under the expense of one ATP
molecule. The cycle is closed by transporting
oxaloacetate back into the mitochondria
through a citrate-oxaloacetate antiporter. In a
first attempt, the overexpression of a murine
ACL in S. cerevisiae led to a slightly increased
titer of total FA (Tang et al. 2013). The effect
was mostly pronounced during the stationary
phase, probably reflecting the redirection of the
metabolism toward respiration of the accumulated ethanol.
In the same study, isocitrate dehydrogenase genes IDH1
and IDH2 were deleted to promote the accumulation of
citrate; somewhat surprisingly, this intervention did
not affect the total content but only the saturation
profile of FA, suggesting that ACL activity and/or
other factors were rate limiting. Later studies, performed in strains engineered for n-butanol (Lian et al.
2014), hexadecanol (Feng et al. 2015), or mevalonate
(Rodriguez et al. 2016) production, showed that the
choice of the heterologous enzyme has a certain impact
on the pathway efficiency.
In most recent studies that reported the
highest titers of FA or their derivatives (Zhou
et al. 2016b; Yu et al. 2018), the citrateoxaloacetate shuttle was further optimized and
combined with strategies that increase the
NADPH supply. The overexpression of ACL
alone in a strain that was already engineered
to block FA degradation led to a moderate
improvement (up to 50%) of free FA titers,
which is consistent with previous studies
(Tang et al. 2013; Lian et al. 2014). A further
improvement was achieved by introducing a
transhydrogenase-like reaction sequence to
increase NADPH supply, exemplifying the
importance of redox cofactors as a driving
force for FA production. In this scheme, a cytosolic malate dehydrogenase (MDH) reduces
oxaloacetate to malate (oxidizing NADH), and
a cytosolic malic enzyme (MAE) subsequently
transfers hydrogen to NADP, whereby malate is
converted to pyruvate, which then re-enters
mitochondria and undergoes a new cycle
(Fig. 14.1). Additional overexpression of the
endogenous citrate transporter Ctp1 appeared
to have no beneficial effect on the FA production, suggesting that the export of citrate into
the cytosol may not have been a limiting factor
in this approach. In a follow-up study (Yu et al.
2018), several strategies to enhance the citrateoxaloacetate shuttle were tested. Overexpression of the endogenous (mitochondrial) PDH
components to enhance the mitochondrial synthesis of AcCoA did not lead to an increase in
FA titers in this study. However, the overexpression of the mitochondrial pyruvate carrier
subunits Mpc1 and Mpc3 and introduction of a
heterologous CS in addition to the native one
appeared to increase the flux through the
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
345