et al. (2014b) overexpressed a plant MalCoA
synthetase, which ligates malonic acid and
CoA to generate MalCoA directly. This
improved the productivity of strains engineered for polyketide production and increased
the total FA amount. Since the native Acc1 is
known to be inactivated by phosphorylation by
the kinase Snf1 upon glucose depletion, different strategies were developed to circumvent
this regulatory mechanism, including relocalization of the mitochondrial ACC from S. cerevisiae (Hfa1) into the cytosol (d’Espaux et al.
2017) and site-directed mutagenesis to prevent
phosphorylation (Choi and Da Silva 2014; Shi
et al. 2014b). All of them led to remarkable
improvements in yields of MalCoA-derived
products.
The specific activity of the phosphorylation-resistant
mutant Acc1
S1157A was higher compared to wild-type
Acc1 even when measured in extract from glucosegrown cells (Choi and Da Silva 2014), suggesting that
the protein might be partly phosphorylated also on this
carbon source (not only on ethanol). In another study,
a second (in silico predicted) phosphorylation site
(S659) was mutated to alanine in addition to S1157,
which led to an increase in specific activity and total
FA yields compared to a control harboring the S1157A
mutation alone (Shi et al. 2014b). Considering that Snf1
is activated by AMP (Sanz 2003), we hypothesize that
phosphorylation resistance may become even more
relevant on glucose-grown cells when the PDH bypass,
which produces AMP in the ACS reaction (Fig. 14.1), is
overexpressed.
Another important consideration for strain
design is the sensitivity of FA chain lengths to
the AcCoA/MalCoA ratio. Elevated concentration of MalCoA is known to favor the elongation of the FA carbon chains by FAS (Sumper
et al. 1969). Indeed, the expression of
Acc1
S1157A (Choi and Da Silva 2014; Hofbauer
et al. 2014; Besada-Lombana et al. 2017) and
Acc1
S1157A, S659A (Zhou et al. 2016b) led to a
higher proportion of longer FA chains. This
demonstrates that, especially if tight chain
length control is desired, the relative abundance of the precursor molecules must be subtly balanced. By affecting the lipid composition
of the membranes, the ACC hyperactivity not
only changes the product profiles but also
appears to negatively influence cell growth,
which was attributed to an imbalanced synthesis of long-chain FA or depletion of intermediates (Shi et al. 2014b). On the other hand, the
expression of Acc1
S1157A was shown to improve
the resistance of yeast to medium-chain FA (see
Sect. IV.B). All these observations demonstrate
that balancing the ACC activity is a non-trivial
task and several factors such as the AcCoA
abundance, product chain length, and toxicity
must be taken into account. The development
of product-specific biosensors (see Sect. IV.C)
will accelerate the screening of strains producing the desired chain length in a highthroughput manner, which will greatly facilitate
the balancing of AcCoA and MalCoA supply.
To summarize, the endogenous supply of
cytosolic precursors (AcCoA and MalCoA),
redox cofactors (NADH or NADPH), and
energy equivalents (ATP) via the PDH bypass
is not sufficient for production of FA and derivatives in S. cerevisiae beyond the proof-ofconcept level. Although there are no systematic
studies comparing the FA yields in dependence
on different precursor supply routes, stoichiometric analyses (van Rossum et al. 2016b) and
experimental data outlined above show that
there is no “one best” precursor supply pathway valid for all AcCoA-derived products. Since
the choice of the FA elongation system (FAS vs.
reverse b-oxidation; see Sect. III) dictates the
redox cofactor requirement, the appropriate
precursor supply pathway must be chosen
accordingly. Moreover, to minimize the
amount of carbon that needs to be diverted
for energy supply, the native PDH bypass
must be fully replaced by a combinatorial precursor supply configuration, as predicted by
stoichiometric analyses for different model
compounds (van Rossum et al. 2016b) and
experimentally shown for the isoprenoid farnesene (Meadows et al. 2016). Considering that
very promising improvements in FA productivity, reaching total yields up to 30% of the theoretical maximum, have been recently achieved
(Yu et al. 2018), it is conceivable that similar
combinatorial approaches can lead to further
advances in the production of oleochemicals.
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
347
synthetase, which ligates malonic acid and
CoA to generate MalCoA directly. This
improved the productivity of strains engineered for polyketide production and increased
the total FA amount. Since the native Acc1 is
known to be inactivated by phosphorylation by
the kinase Snf1 upon glucose depletion, different strategies were developed to circumvent
this regulatory mechanism, including relocalization of the mitochondrial ACC from S. cerevisiae (Hfa1) into the cytosol (d’Espaux et al.
2017) and site-directed mutagenesis to prevent
phosphorylation (Choi and Da Silva 2014; Shi
et al. 2014b). All of them led to remarkable
improvements in yields of MalCoA-derived
products.
The specific activity of the phosphorylation-resistant
mutant Acc1
S1157A was higher compared to wild-type
Acc1 even when measured in extract from glucosegrown cells (Choi and Da Silva 2014), suggesting that
the protein might be partly phosphorylated also on this
carbon source (not only on ethanol). In another study,
a second (in silico predicted) phosphorylation site
(S659) was mutated to alanine in addition to S1157,
which led to an increase in specific activity and total
FA yields compared to a control harboring the S1157A
mutation alone (Shi et al. 2014b). Considering that Snf1
is activated by AMP (Sanz 2003), we hypothesize that
phosphorylation resistance may become even more
relevant on glucose-grown cells when the PDH bypass,
which produces AMP in the ACS reaction (Fig. 14.1), is
overexpressed.
Another important consideration for strain
design is the sensitivity of FA chain lengths to
the AcCoA/MalCoA ratio. Elevated concentration of MalCoA is known to favor the elongation of the FA carbon chains by FAS (Sumper
et al. 1969). Indeed, the expression of
Acc1
S1157A (Choi and Da Silva 2014; Hofbauer
et al. 2014; Besada-Lombana et al. 2017) and
Acc1
S1157A, S659A (Zhou et al. 2016b) led to a
higher proportion of longer FA chains. This
demonstrates that, especially if tight chain
length control is desired, the relative abundance of the precursor molecules must be subtly balanced. By affecting the lipid composition
of the membranes, the ACC hyperactivity not
only changes the product profiles but also
appears to negatively influence cell growth,
which was attributed to an imbalanced synthesis of long-chain FA or depletion of intermediates (Shi et al. 2014b). On the other hand, the
expression of Acc1
S1157A was shown to improve
the resistance of yeast to medium-chain FA (see
Sect. IV.B). All these observations demonstrate
that balancing the ACC activity is a non-trivial
task and several factors such as the AcCoA
abundance, product chain length, and toxicity
must be taken into account. The development
of product-specific biosensors (see Sect. IV.C)
will accelerate the screening of strains producing the desired chain length in a highthroughput manner, which will greatly facilitate
the balancing of AcCoA and MalCoA supply.
To summarize, the endogenous supply of
cytosolic precursors (AcCoA and MalCoA),
redox cofactors (NADH or NADPH), and
energy equivalents (ATP) via the PDH bypass
is not sufficient for production of FA and derivatives in S. cerevisiae beyond the proof-ofconcept level. Although there are no systematic
studies comparing the FA yields in dependence
on different precursor supply routes, stoichiometric analyses (van Rossum et al. 2016b) and
experimental data outlined above show that
there is no “one best” precursor supply pathway valid for all AcCoA-derived products. Since
the choice of the FA elongation system (FAS vs.
reverse b-oxidation; see Sect. III) dictates the
redox cofactor requirement, the appropriate
precursor supply pathway must be chosen
accordingly. Moreover, to minimize the
amount of carbon that needs to be diverted
for energy supply, the native PDH bypass
must be fully replaced by a combinatorial precursor supply configuration, as predicted by
stoichiometric analyses for different model
compounds (van Rossum et al. 2016b) and
experimentally shown for the isoprenoid farnesene (Meadows et al. 2016). Considering that
very promising improvements in FA productivity, reaching total yields up to 30% of the theoretical maximum, have been recently achieved
(Yu et al. 2018), it is conceivable that similar
combinatorial approaches can lead to further
advances in the production of oleochemicals.
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
347