III. Strategies for Chain Length
Control
The majority of microbially synthesized FAs
are 14–20 carbon atoms in length (referred to
as long-chain FA). Mainly saturated and monounsaturated FA with 16 or 18 carbon atoms are
synthesized in S. cerevisiae (Cottrell et al. 1986).
Engineering microbes like E. coli, S. cerevisiae,
or oleaginous yeasts (e.g., Yarrowia lipolytica)
toward FA and FA derivatives with alternating
chain length is desired due to their compelling
properties as biofuels; for the cosmetic, healthcare, and pharmaceutical industries; or as platform compounds (Lennen and Pfleger 2013).
In microorganisms however, biosynthesis
of short- ( C4), medium- (C6–C12), or very
long- (!C22) chain FA is rare and low in quantity. For this reason strategies for chain length
control through a focus on the main anabolic
(FA biosynthesis) and catabolic (b-oxidation)
pathways have been developed.
A. Manipulation of the Natural Fatty Acid
Biosynthesis and Elongation Machinery
Fatty acid biosynthesis follows a conserved
chemistry among different types of FAS
enzymes which differ in their general organization. Type I FAS systems (mainly eukaryotic)
carry all catalytic domains in one multifunctional complex (Schweizer and Hofmann
2004). However, fungal type I FAS greatly differ
from animal type I FAS in their architecture
(Leibundgut et al. 2008).
In type I systems, FA biosynthesis is
initiated by the transfer of the starter unit
AcCoA to the acyl-carrier protein (ACP) by
the acetyltransferase (AT) domain of FAS. Subsequently, the ACP domain delivers the acetyl
residue to the ketosynthase (KS) domain. Similarly, the extension unit MalCoA is loaded onto
the free ACP by the malonyl-palmitoyl transferase (MPT) domain. Acetyl and malonyl moieties are condensed by the KS domain to a bketoacyl intermediate, which is then further
processed. The b-ketoacyl intermediate is
reduced by the ketoacyl reductase (KR) domain
to form 3-hydroxyacyl-ACP, which is dehydrated by the dehydratase (DH) domain and
eventually reduced by the enoyl reductase
(ER) domain to a saturated acyl chain containing two additional carbon atoms. The reductive
steps at the KR and ER domains are strictly
NADPH-dependent. After completion of one
cycle, the acyl chain is either condensed with
another malonyl moiety for further elongation
or released by the MPT domain as a CoA-bound
thioester in fungal FAS (Lomakin et al. 2007).
After release of acyl-CoA esters from the FAS
complex, thioesterases (TE) cleave the thioester
bond between the acyl chain and CoA to release
free FA.
As a strategy for enrichment of short- and
medium-chain FA, expression of short- and
medium-chain-specific TE for early termination of FA biosynthesis has been established
in multiple studies (Leber and Da Silva 2014;
Fernandez-Moya et al. 2015; Xu et al. 2016; Zhu
et al. 2017b). Combined with fungal FAS
enzymes, soluble TE only have limited or no
access to FAS-bound acyl-CoA or acyl-ACP
esters, and protein engineers have therefore
developed strategies to locate TE within the
FAS complex. For instance, some type I FAS
(e.g., from Rhodosporidium toruloides and
Aplanochytrium kerguelense) harbor two
redundant ACP domains, which are located
inside of the FAS scaffold. It is therefore possible to replace one of them by a short-chain TE
to enable a direct access to acyl intermediates
inside the reaction chamber. The implementation of this strategy proved to be more efficient
than the expression of free TE and increased
short- and medium-chain FA production by 3to 15-fold (Zhu et al. 2017b). In Y. lipolytica
FAS, replacement of the MPT domain by a
short- and medium-chain-specific TE resulted
in an increase of up to 29% of C12–C14 FA of
the total FA content (Xu et al. 2016).
As an alternative approach, the yeast FAS
has been replaced by non-fungal enzymes.
Human FAS (hFAS) carries its own TE domain,
which releases the acyl moieties directly from
the ACP (Leibundgut et al. 2008) and has a
more flexible structure than yeast FAS
(Brignole et al. 2009). Expression of hFAS, in
which its own TE domain was deleted, in com348
L. Baumann et al.
Control
The majority of microbially synthesized FAs
are 14–20 carbon atoms in length (referred to
as long-chain FA). Mainly saturated and monounsaturated FA with 16 or 18 carbon atoms are
synthesized in S. cerevisiae (Cottrell et al. 1986).
Engineering microbes like E. coli, S. cerevisiae,
or oleaginous yeasts (e.g., Yarrowia lipolytica)
toward FA and FA derivatives with alternating
chain length is desired due to their compelling
properties as biofuels; for the cosmetic, healthcare, and pharmaceutical industries; or as platform compounds (Lennen and Pfleger 2013).
In microorganisms however, biosynthesis
of short- ( C4), medium- (C6–C12), or very
long- (!C22) chain FA is rare and low in quantity. For this reason strategies for chain length
control through a focus on the main anabolic
(FA biosynthesis) and catabolic (b-oxidation)
pathways have been developed.
A. Manipulation of the Natural Fatty Acid
Biosynthesis and Elongation Machinery
Fatty acid biosynthesis follows a conserved
chemistry among different types of FAS
enzymes which differ in their general organization. Type I FAS systems (mainly eukaryotic)
carry all catalytic domains in one multifunctional complex (Schweizer and Hofmann
2004). However, fungal type I FAS greatly differ
from animal type I FAS in their architecture
(Leibundgut et al. 2008).
In type I systems, FA biosynthesis is
initiated by the transfer of the starter unit
AcCoA to the acyl-carrier protein (ACP) by
the acetyltransferase (AT) domain of FAS. Subsequently, the ACP domain delivers the acetyl
residue to the ketosynthase (KS) domain. Similarly, the extension unit MalCoA is loaded onto
the free ACP by the malonyl-palmitoyl transferase (MPT) domain. Acetyl and malonyl moieties are condensed by the KS domain to a bketoacyl intermediate, which is then further
processed. The b-ketoacyl intermediate is
reduced by the ketoacyl reductase (KR) domain
to form 3-hydroxyacyl-ACP, which is dehydrated by the dehydratase (DH) domain and
eventually reduced by the enoyl reductase
(ER) domain to a saturated acyl chain containing two additional carbon atoms. The reductive
steps at the KR and ER domains are strictly
NADPH-dependent. After completion of one
cycle, the acyl chain is either condensed with
another malonyl moiety for further elongation
or released by the MPT domain as a CoA-bound
thioester in fungal FAS (Lomakin et al. 2007).
After release of acyl-CoA esters from the FAS
complex, thioesterases (TE) cleave the thioester
bond between the acyl chain and CoA to release
free FA.
As a strategy for enrichment of short- and
medium-chain FA, expression of short- and
medium-chain-specific TE for early termination of FA biosynthesis has been established
in multiple studies (Leber and Da Silva 2014;
Fernandez-Moya et al. 2015; Xu et al. 2016; Zhu
et al. 2017b). Combined with fungal FAS
enzymes, soluble TE only have limited or no
access to FAS-bound acyl-CoA or acyl-ACP
esters, and protein engineers have therefore
developed strategies to locate TE within the
FAS complex. For instance, some type I FAS
(e.g., from Rhodosporidium toruloides and
Aplanochytrium kerguelense) harbor two
redundant ACP domains, which are located
inside of the FAS scaffold. It is therefore possible to replace one of them by a short-chain TE
to enable a direct access to acyl intermediates
inside the reaction chamber. The implementation of this strategy proved to be more efficient
than the expression of free TE and increased
short- and medium-chain FA production by 3to 15-fold (Zhu et al. 2017b). In Y. lipolytica
FAS, replacement of the MPT domain by a
short- and medium-chain-specific TE resulted
in an increase of up to 29% of C12–C14 FA of
the total FA content (Xu et al. 2016).
As an alternative approach, the yeast FAS
has been replaced by non-fungal enzymes.
Human FAS (hFAS) carries its own TE domain,
which releases the acyl moieties directly from
the ACP (Leibundgut et al. 2008) and has a
more flexible structure than yeast FAS
(Brignole et al. 2009). Expression of hFAS, in
which its own TE domain was deleted, in com348
L. Baumann et al.