bination with the short-chain-specific TE
CpFatB1 (Cuphea palustris) or TEII (Rattus
norvegicus) as free proteins enabled the production of C6–C10 FA (Leber and Da Silva
2014). Substituting the TE domain of hFAS by
TEII in the same polypeptide chain substantially increased the production of short- and
medium-chain FA.
In contrast to type I FAS, the catalytic
domains of the bacterial type II FAS system
are expressed as single enzymes. This allows a
simpler manipulation of the subunits, and acyl
intermediates are freely accessible for TE.
Expression of the E. coli system (acpS, acpP,
fabB, fabD, fabG, fabH, fabI, fabZ) in combination with the TE fatB from Ricinus communis in
a FAS-deficient S. cerevisiae strain significantly
increased the total FA titer and shifted the FA
profile toward C14 FA production (FernandezMoya et al. 2015).
A minimally invasive strategy to rewrite the
chain length control relies on rational engineering of yeast FAS by site-directed mutagenesis. For instance, an early release of shorter
acyl chains (C6–C10 or C14) was achieved by
introducing bulky residues like tryptophan into
the KS domain to shorten the acyl-ACP binding
channel in S. cerevisiae (Gajewski et al. 2017;
Zhu et al. 2017b) or Y. lipolytica (Rigouin et al.
2017) FAS. Furthermore, it was shown that
decreasing the affinity of the MPT domain for
malonyl moieties by introducing one point
mutation disfavors the chain elongation and
promotes the release of shorter-chain (C6–
C10) FA (Gajewski et al. 2017). One additional
mutation was introduced into the AT domain
for a more frequent priming with AcCoA, as
this was expected to shift the product profile
toward shorter chains; however, this modification proved only effective in combination with
mutations in the MPT and/or KS domain. Single or multiple mutations in the three domains
caused different ratios of C6–C10 FA, with
some of the mutants exhibiting a considerable
specificity, e.g., for C8 FA (Gajewski et al. 2017).
Thus, rational engineering of FAS holds a great
promise for narrowing down the product profile to the desired chain length.
Very long-chain FAs (C22–C26) are present
in S. cerevisiae at low abundance (Welch and
Burlingame 1973) but are essential compounds,
e.g., for sphingolipids (Oh et al. 1997). After
their release from the FAS complex, FAs are
elongated at the ER membrane from (longchain) acyl-CoA precursors. Elongases with
different product specificities (Elo1, Elo2, and
Elo3) carry out the elongation of acyl-CoA
intermediates with MalCoA. Elo1 elongates
medium- and long-chain compounds (C14–
C16) to C18 FA, Elo2 elongates compounds up
to 22 C atoms, and Elo3 elongates compounds
up to 26 C atoms (Toke and Martin 1996; Oh
et al. 1997). Reminiscent of cytosolic FA biosynthesis, two reductions and one dehydration
step are necessary to complete very long-chain
FA synthesis. To selectively increase the pool of
C22, overexpression of ELO2 and deletion of
ELO3 are necessary (Yu et al. 2017), whereas
overexpression of only ELO3 is sufficient for
enrichment of C26 FA (Wenning et al. 2017).
Alternatively, FAS I from mycobacteria has
been shown to naturally generate FA with 22–
26 carbon atoms (Kaneda et al. 1995). Expression of FAS I from Mycobacterium vaccae in an
Elo2-/Elo3-deficient strain increased the C22
FA pool by fourfold (Yu et al. 2017).
B. Reversal of b-Oxidation as an Orthogonal
Pathway for Fatty Acid Biosynthesis
The b-oxidation cycle naturally is an FA degradation pathway (for review see Hiltunen et al.
2003). In each turn of the cycle, FAs are
truncated by removing two carbon atoms
from the FA chain, thereby generating AcCoA.
Reversing all reactions of the b-oxidation
can consequently be used as an alternative synthetic pathway for FA production. The single
reactions of the b-oxidation are equilibriumbalanced, and the functionality of the enzymes
in the reverse direction has been demonstrated
(Dellomonaco et al. 2011; Clomburg et al. 2012).
The reverse b-oxidation pathway starts with the
condensation of two AcCoA to acetoacetyl-CoA
by a thiolase, followed by the reduction of the
b-ketogroup to 3-hydroxyacyl-CoA by a reductase/dehydrogenase, dehydration to transenoyl-CoA by a hydratase/dehydratase, and a
final reduction by another reductase/dehydro14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
349
CpFatB1 (Cuphea palustris) or TEII (Rattus
norvegicus) as free proteins enabled the production of C6–C10 FA (Leber and Da Silva
2014). Substituting the TE domain of hFAS by
TEII in the same polypeptide chain substantially increased the production of short- and
medium-chain FA.
In contrast to type I FAS, the catalytic
domains of the bacterial type II FAS system
are expressed as single enzymes. This allows a
simpler manipulation of the subunits, and acyl
intermediates are freely accessible for TE.
Expression of the E. coli system (acpS, acpP,
fabB, fabD, fabG, fabH, fabI, fabZ) in combination with the TE fatB from Ricinus communis in
a FAS-deficient S. cerevisiae strain significantly
increased the total FA titer and shifted the FA
profile toward C14 FA production (FernandezMoya et al. 2015).
A minimally invasive strategy to rewrite the
chain length control relies on rational engineering of yeast FAS by site-directed mutagenesis. For instance, an early release of shorter
acyl chains (C6–C10 or C14) was achieved by
introducing bulky residues like tryptophan into
the KS domain to shorten the acyl-ACP binding
channel in S. cerevisiae (Gajewski et al. 2017;
Zhu et al. 2017b) or Y. lipolytica (Rigouin et al.
2017) FAS. Furthermore, it was shown that
decreasing the affinity of the MPT domain for
malonyl moieties by introducing one point
mutation disfavors the chain elongation and
promotes the release of shorter-chain (C6–
C10) FA (Gajewski et al. 2017). One additional
mutation was introduced into the AT domain
for a more frequent priming with AcCoA, as
this was expected to shift the product profile
toward shorter chains; however, this modification proved only effective in combination with
mutations in the MPT and/or KS domain. Single or multiple mutations in the three domains
caused different ratios of C6–C10 FA, with
some of the mutants exhibiting a considerable
specificity, e.g., for C8 FA (Gajewski et al. 2017).
Thus, rational engineering of FAS holds a great
promise for narrowing down the product profile to the desired chain length.
Very long-chain FAs (C22–C26) are present
in S. cerevisiae at low abundance (Welch and
Burlingame 1973) but are essential compounds,
e.g., for sphingolipids (Oh et al. 1997). After
their release from the FAS complex, FAs are
elongated at the ER membrane from (longchain) acyl-CoA precursors. Elongases with
different product specificities (Elo1, Elo2, and
Elo3) carry out the elongation of acyl-CoA
intermediates with MalCoA. Elo1 elongates
medium- and long-chain compounds (C14–
C16) to C18 FA, Elo2 elongates compounds up
to 22 C atoms, and Elo3 elongates compounds
up to 26 C atoms (Toke and Martin 1996; Oh
et al. 1997). Reminiscent of cytosolic FA biosynthesis, two reductions and one dehydration
step are necessary to complete very long-chain
FA synthesis. To selectively increase the pool of
C22, overexpression of ELO2 and deletion of
ELO3 are necessary (Yu et al. 2017), whereas
overexpression of only ELO3 is sufficient for
enrichment of C26 FA (Wenning et al. 2017).
Alternatively, FAS I from mycobacteria has
been shown to naturally generate FA with 22–
26 carbon atoms (Kaneda et al. 1995). Expression of FAS I from Mycobacterium vaccae in an
Elo2-/Elo3-deficient strain increased the C22
FA pool by fourfold (Yu et al. 2017).
B. Reversal of b-Oxidation as an Orthogonal
Pathway for Fatty Acid Biosynthesis
The b-oxidation cycle naturally is an FA degradation pathway (for review see Hiltunen et al.
2003). In each turn of the cycle, FAs are
truncated by removing two carbon atoms
from the FA chain, thereby generating AcCoA.
Reversing all reactions of the b-oxidation
can consequently be used as an alternative synthetic pathway for FA production. The single
reactions of the b-oxidation are equilibriumbalanced, and the functionality of the enzymes
in the reverse direction has been demonstrated
(Dellomonaco et al. 2011; Clomburg et al. 2012).
The reverse b-oxidation pathway starts with the
condensation of two AcCoA to acetoacetyl-CoA
by a thiolase, followed by the reduction of the
b-ketogroup to 3-hydroxyacyl-CoA by a reductase/dehydrogenase, dehydration to transenoyl-CoA by a hydratase/dehydratase, and a
final reduction by another reductase/dehydro14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
349