chain in the end product is determined by the
acyl chain of the FA precursor and by the specificity of the downstream enzymes.
A. Alkenes
Free FA can be converted to terminal alkenes by
a one-step decarboxylation pathway, catalyzed
by an H 2 O 2 -dependent cytochrome P450
enzyme, OleT (Chen et al. 2015; Zhou et al.
2018); a medium-chain FA-preferring nonheme
iron oxidase, UndA (Zhu et al. 2017a; Zhou
et al. 2018); or a membrane-bound desaturaselike enzyme, UndB (Zhou et al. 2018), together
with a reduction system. The major challenge
when applying these iron-dependent enzymes
is the inefficient electron transfer, resulting in
low titers in yeast due to inefficient cofactor or
reducing systems (Zhou et al. 2018).
B. Dicarboxylic Acids
Utilizing the v-oxidation pathway in S. cerevisiae enables the production of o-hydroxy- and
a, o-dicarboxylic acids (o meaning “last” C
atom when counted from the carboxyl group)
from free FA. Such a, o-dicarboxylic acids can
serve as raw material for commodities and
polymers (Han et al. 2017). Free FA can be
terminally hydroxylated by a cytochrome P450
enzyme, followed by an oxidation to a carboxyl
group by alcohol dehydrogenases and aldehyde
dehydrogenases. Feeding of medium-chain FA
to an S. cerevisiae strain expressing the cytochrome P450 enzyme CYP94C1, together with a
cytochrome reductase, ATR1 from A. thaliana,
allowed the production of o-hydroxy- and a, odicarboxylic acids with chain lengths ranging
from C10 to C16 (Han et al. 2017).
C. Fatty Aldehydes, Alkanes, and Alcohols
For the production of alkanes and alcohols, a
fatty aldehyde intermediate is used as a precursor. There are several possible routes for the
synthesis of fatty aldehydes. For instance, FA
can be reduced through action of a carboxylic
acid reductase (CAR) from Mycobacterium
marinum (Zhou et al. 2016b; Tang et al. 2017;
Henritzi et al. 2018), or it can be oxidatively
decarboxylated by an a-dioxygenase (a-DOX)
from Oryza sativa (rice) (Jin et al. 2016; Foo
et al. 2017). To be active, CAR requires a phosphopantetheinylation by a phosphopantetheinyl transferase (Akhtar et al. 2013). A third
possibility is the reduction of CoA-bound FA
to the corresponding aldehyde by fatty acylCoA reductases (AldFARs). Compared to
CAR, it was shown that AldFAR-type enzymes
are rather inefficient in yeast (Buijs et al. 2015;
Zhou et al. 2016b). Aldehydes can be converted
to odd-chain fatty alkanes (C nÀ1 ) by cyanobacterial aldehyde deformylating oxygenases
(ADO) or to fatty alcohols by endogenous alcohol dehydrogenases (ADHs) and aldehyde
reductases (ALR) (Buijs et al. 2015; Zhou et al.
2016b; Kang et al. 2017; Zhu et al. 2017a). Zhou
et al. (2016b) showed that the expression of a
CAR together with its activating enzyme, phosphopantetheine transferase NpgA from A.
nidulans, and an ADO, led to the synthesis of
long-chain alkanes (Zhou et al. 2016b). To supply sufficient electrons, a reducing system was
expressed additionally (Buijs et al. 2015). As
previous studies showed that CAR also shows
high activity toward medium-chain FA, Zhou
et al. (2016a) produced medium-chain alkanes
by screening different ADO orthologs and engineering their substrate binding sites (Zhu et al.
2017a). However, it is reported that ADO was
not able to compete with the alcohol-forming
enzymes (ADHs/ALRs) due to its low catalytic
efficiency (Buijs et al. 2015; Zhou et al. 2016b;
Foo et al. 2017; Kang et al. 2017; Zhu et al.
2017a). Another two-step pathway to produce
alkanes was demonstrated by Foo et al. (2017).
Expression of an a-DOX from rice led to the
production of long odd-chain fatty aldehyde
intermediates, which can subsequently be
deformylated to even-chain alkanes (C nÀ2 ) by
ADO (Foo et al. 2017). The advantage of a
dioxygenase is that it uses dioxygen instead of
NADPH for the production of aldehydes (Foo
et al. 2017). Odd-chain fatty aldehydes produced by a-DOX can also be oxidized to oddchain fatty alcohols through endogenous yeast
ADHs/ALRs (Jin et al. 2016). Zhou et al. (2016b)
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
357
acyl chain of the FA precursor and by the specificity of the downstream enzymes.
A. Alkenes
Free FA can be converted to terminal alkenes by
a one-step decarboxylation pathway, catalyzed
by an H 2 O 2 -dependent cytochrome P450
enzyme, OleT (Chen et al. 2015; Zhou et al.
2018); a medium-chain FA-preferring nonheme
iron oxidase, UndA (Zhu et al. 2017a; Zhou
et al. 2018); or a membrane-bound desaturaselike enzyme, UndB (Zhou et al. 2018), together
with a reduction system. The major challenge
when applying these iron-dependent enzymes
is the inefficient electron transfer, resulting in
low titers in yeast due to inefficient cofactor or
reducing systems (Zhou et al. 2018).
B. Dicarboxylic Acids
Utilizing the v-oxidation pathway in S. cerevisiae enables the production of o-hydroxy- and
a, o-dicarboxylic acids (o meaning “last” C
atom when counted from the carboxyl group)
from free FA. Such a, o-dicarboxylic acids can
serve as raw material for commodities and
polymers (Han et al. 2017). Free FA can be
terminally hydroxylated by a cytochrome P450
enzyme, followed by an oxidation to a carboxyl
group by alcohol dehydrogenases and aldehyde
dehydrogenases. Feeding of medium-chain FA
to an S. cerevisiae strain expressing the cytochrome P450 enzyme CYP94C1, together with a
cytochrome reductase, ATR1 from A. thaliana,
allowed the production of o-hydroxy- and a, odicarboxylic acids with chain lengths ranging
from C10 to C16 (Han et al. 2017).
C. Fatty Aldehydes, Alkanes, and Alcohols
For the production of alkanes and alcohols, a
fatty aldehyde intermediate is used as a precursor. There are several possible routes for the
synthesis of fatty aldehydes. For instance, FA
can be reduced through action of a carboxylic
acid reductase (CAR) from Mycobacterium
marinum (Zhou et al. 2016b; Tang et al. 2017;
Henritzi et al. 2018), or it can be oxidatively
decarboxylated by an a-dioxygenase (a-DOX)
from Oryza sativa (rice) (Jin et al. 2016; Foo
et al. 2017). To be active, CAR requires a phosphopantetheinylation by a phosphopantetheinyl transferase (Akhtar et al. 2013). A third
possibility is the reduction of CoA-bound FA
to the corresponding aldehyde by fatty acylCoA reductases (AldFARs). Compared to
CAR, it was shown that AldFAR-type enzymes
are rather inefficient in yeast (Buijs et al. 2015;
Zhou et al. 2016b). Aldehydes can be converted
to odd-chain fatty alkanes (C nÀ1 ) by cyanobacterial aldehyde deformylating oxygenases
(ADO) or to fatty alcohols by endogenous alcohol dehydrogenases (ADHs) and aldehyde
reductases (ALR) (Buijs et al. 2015; Zhou et al.
2016b; Kang et al. 2017; Zhu et al. 2017a). Zhou
et al. (2016b) showed that the expression of a
CAR together with its activating enzyme, phosphopantetheine transferase NpgA from A.
nidulans, and an ADO, led to the synthesis of
long-chain alkanes (Zhou et al. 2016b). To supply sufficient electrons, a reducing system was
expressed additionally (Buijs et al. 2015). As
previous studies showed that CAR also shows
high activity toward medium-chain FA, Zhou
et al. (2016a) produced medium-chain alkanes
by screening different ADO orthologs and engineering their substrate binding sites (Zhu et al.
2017a). However, it is reported that ADO was
not able to compete with the alcohol-forming
enzymes (ADHs/ALRs) due to its low catalytic
efficiency (Buijs et al. 2015; Zhou et al. 2016b;
Foo et al. 2017; Kang et al. 2017; Zhu et al.
2017a). Another two-step pathway to produce
alkanes was demonstrated by Foo et al. (2017).
Expression of an a-DOX from rice led to the
production of long odd-chain fatty aldehyde
intermediates, which can subsequently be
deformylated to even-chain alkanes (C nÀ2 ) by
ADO (Foo et al. 2017). The advantage of a
dioxygenase is that it uses dioxygen instead of
NADPH for the production of aldehydes (Foo
et al. 2017). Odd-chain fatty aldehydes produced by a-DOX can also be oxidized to oddchain fatty alcohols through endogenous yeast
ADHs/ALRs (Jin et al. 2016). Zhou et al. (2016b)
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
357