explored ADHs/ALRs of yeasts and could show
that Adh5 plays an important role in the production of long-chain alcohols, as overexpressing ADH5 together with CAR increased the
amount of long-chain alcohols (Zhou et al.
2016b). So far, the expression of fatty alcohol
pathways resulted in the production of a pool of
long- or medium-chain fatty alcohols with different chain lengths. Recently, our group was
able to selectively produce the diesel-like alcohol 1-octanol in S. cerevisiae by combining a C8
FA-producing FAS with a two-step reduction
pathway composed of CAR together with the
phosphopantetheinyl transferase Sfp from
Bacillus subtilis and the aldehyde reductase
Ahr from E. coli (Henritzi et al. 2018). It has
been shown that Ahr accepts a broad range of
aliphatic aldehydes with chain lengths from C4
to C16 (Akhtar et al. 2013).
Another way to produce fatty alcohols is a
one-step reduction of the fatty acyl-CoA, catalyzed by fatty acyl-CoA reductases (AlcFARs)
(Runguphan and Keasling 2014). Zhou et al.
(2016b) expressed the CAR/Adh5 pathway
together with a fatty acyl-CoA reductase to produce fatty alcohols simultaneously from free FA
and acyl-CoAs (Zhou et al. 2016b). Other labs
have also reported the functional expression of
several heterologous AlcFARs (Feng et al. 2015;
d’Espaux et al. 2017). The advantage of onestep reactions, like the direct conversion from
fatty acyl-CoAs to alcohols by AlcFARs or the
FA decarboxylation pathway for alkene production, is the reduced intermediate metabolite
loss and the circumvention of toxic intermediates (Chen et al. 2015). Besides medium-chain
and long-chain FA-derived chemicals, very
long-chain FA-derived products also play an
important role as ingredients for lubricants,
detergents, polymers, photographic filmprocessing agents, coatings, cosmetics, and
pharmaceuticals (Yu et al. 2017). Yu et al.
(2017) were able to selectively produce docosanol (C22) from very long-chain FA by rewiring
the native FA elongation system and overexpressing a heterologous mycobacteria FAS I
system, which provides high levels of C22 FA
as direct precursor, together with a specific
AlcFAR (Yu et al. 2017).
D. Wax Esters and Fatty Acid Ethyl Esters
Further compounds of interest include wax
esters, which are typically esters of long-chain
FA and long-chain alcohols and are used in
personal care products, lubricants, or coatings
(Wenning et al. 2017). They can be synthesized
by wax ester synthases (WS) from alcohols and
fatty acyl-CoA thioesters (Shi et al. 2012; Runguphan and Keasling 2014). Esters of ethanol
and FA with chain lengths ranging from C14 to
C20 (FAEE) represent suitable diesel fuels (Shi
et al. 2012). Shi et al. (2012) could functionally
express different WSs from different species
and characterize their substrate preference.
Unfortunately, some of the WSs from bacteria
are bifunctional enzymes, which function as
WS and acyl-CoA:diacylglycerol acyltransferase
(DGAT), resulting in TAG formation, leading to
a depletion of the acyl-CoA precursor pool (Shi
et al. 2012). Eriksen et al. (2015) investigated
the heterologous expression of a FAS I from
Brevibacterium ammoniagenes coupled with a
WS/DGAT to produce FAEEs. This strategy has
the advantage of providing additional FA for
growth supplementation and to supply the FA
needed for FAEE synthesis (Eriksen et al. 2015).
The microbial synthesis of wax esters by the
esterification of a (very) long-chain fatty acylCoA with a primary, very long-chain fatty alcohol was reported by Wenning et al. (2017). The
group combined the expression of a heterologous FAR with the expression of a plant WS to
synthesize different jojoba-like wax esters. The
used FAR reduced long-chain fatty acyl-CoAs,
which resulted from de novo FA biosynthesis
and elongation, to a long-chain alcohol. The
plant-derived WS enabled the esterification of
this alcohol with a long-chain fatty acyl-CoA in
S. cerevisiae (Wenning et al. 2017).
Overall, there are still many obstacles that
have to be overcome when using these heterologous enzymes, such as the challenging expression in the heterologous yeast host, low
catalytic efficiency, inefficient electron transfer,
or loss of intermediates by competing pathways. These issues have to be tackled in order
to increase yields and titers of FA-derived compounds. One promising strategy could be a
358
L. Baumann et al.
that Adh5 plays an important role in the production of long-chain alcohols, as overexpressing ADH5 together with CAR increased the
amount of long-chain alcohols (Zhou et al.
2016b). So far, the expression of fatty alcohol
pathways resulted in the production of a pool of
long- or medium-chain fatty alcohols with different chain lengths. Recently, our group was
able to selectively produce the diesel-like alcohol 1-octanol in S. cerevisiae by combining a C8
FA-producing FAS with a two-step reduction
pathway composed of CAR together with the
phosphopantetheinyl transferase Sfp from
Bacillus subtilis and the aldehyde reductase
Ahr from E. coli (Henritzi et al. 2018). It has
been shown that Ahr accepts a broad range of
aliphatic aldehydes with chain lengths from C4
to C16 (Akhtar et al. 2013).
Another way to produce fatty alcohols is a
one-step reduction of the fatty acyl-CoA, catalyzed by fatty acyl-CoA reductases (AlcFARs)
(Runguphan and Keasling 2014). Zhou et al.
(2016b) expressed the CAR/Adh5 pathway
together with a fatty acyl-CoA reductase to produce fatty alcohols simultaneously from free FA
and acyl-CoAs (Zhou et al. 2016b). Other labs
have also reported the functional expression of
several heterologous AlcFARs (Feng et al. 2015;
d’Espaux et al. 2017). The advantage of onestep reactions, like the direct conversion from
fatty acyl-CoAs to alcohols by AlcFARs or the
FA decarboxylation pathway for alkene production, is the reduced intermediate metabolite
loss and the circumvention of toxic intermediates (Chen et al. 2015). Besides medium-chain
and long-chain FA-derived chemicals, very
long-chain FA-derived products also play an
important role as ingredients for lubricants,
detergents, polymers, photographic filmprocessing agents, coatings, cosmetics, and
pharmaceuticals (Yu et al. 2017). Yu et al.
(2017) were able to selectively produce docosanol (C22) from very long-chain FA by rewiring
the native FA elongation system and overexpressing a heterologous mycobacteria FAS I
system, which provides high levels of C22 FA
as direct precursor, together with a specific
AlcFAR (Yu et al. 2017).
D. Wax Esters and Fatty Acid Ethyl Esters
Further compounds of interest include wax
esters, which are typically esters of long-chain
FA and long-chain alcohols and are used in
personal care products, lubricants, or coatings
(Wenning et al. 2017). They can be synthesized
by wax ester synthases (WS) from alcohols and
fatty acyl-CoA thioesters (Shi et al. 2012; Runguphan and Keasling 2014). Esters of ethanol
and FA with chain lengths ranging from C14 to
C20 (FAEE) represent suitable diesel fuels (Shi
et al. 2012). Shi et al. (2012) could functionally
express different WSs from different species
and characterize their substrate preference.
Unfortunately, some of the WSs from bacteria
are bifunctional enzymes, which function as
WS and acyl-CoA:diacylglycerol acyltransferase
(DGAT), resulting in TAG formation, leading to
a depletion of the acyl-CoA precursor pool (Shi
et al. 2012). Eriksen et al. (2015) investigated
the heterologous expression of a FAS I from
Brevibacterium ammoniagenes coupled with a
WS/DGAT to produce FAEEs. This strategy has
the advantage of providing additional FA for
growth supplementation and to supply the FA
needed for FAEE synthesis (Eriksen et al. 2015).
The microbial synthesis of wax esters by the
esterification of a (very) long-chain fatty acylCoA with a primary, very long-chain fatty alcohol was reported by Wenning et al. (2017). The
group combined the expression of a heterologous FAR with the expression of a plant WS to
synthesize different jojoba-like wax esters. The
used FAR reduced long-chain fatty acyl-CoAs,
which resulted from de novo FA biosynthesis
and elongation, to a long-chain alcohol. The
plant-derived WS enabled the esterification of
this alcohol with a long-chain fatty acyl-CoA in
S. cerevisiae (Wenning et al. 2017).
Overall, there are still many obstacles that
have to be overcome when using these heterologous enzymes, such as the challenging expression in the heterologous yeast host, low
catalytic efficiency, inefficient electron transfer,
or loss of intermediates by competing pathways. These issues have to be tackled in order
to increase yields and titers of FA-derived compounds. One promising strategy could be a
358
L. Baumann et al.