carbon metabolism compared to the FA biosynthesis route and offers a larger choice of potential precursor supply routes (see Sect. II,
Fig. 14.1).
IV. Chassis Engineering
To achieve high FA production levels in S. cerevisiae, several obstacles have to be addressed
within the chosen production pathway. Blocking the degradation of FA has been achieved
through the disruption of different enzymes
involved in b-oxidation. An issue that remains
to be tackled is the toxicity of some FA. We
will discuss different methods that have or
could be employed to engineer more robust
chassis. Furthermore, several biosensor systems have been developed, which can be used
in high-throughput screenings to further speed
up the search for best performing strains.
A. Elimination of By-product Formation and
Fatty Acid Degradation
To increase FA pools, it is important not only to
increase precursor pools (see Sect. II, Fig. 14.1)
but also to downregulate competing pathways
as well as to prevent degradation through specific gene deletions or overexpressions.
1. Prevention of b-Oxidation
The degradation, i.e., b-oxidation, of the
already synthesized free FA or their activated
forms, the acyl-CoAs, takes place in the peroxisomes (Hiltunen et al. 2003; van Roermund
et al. 2003). In S. cerevisiae, b-oxidation of
saturated FA is a cyclic mechanism mainly catalyzed by the three enzymes Pox1, Fox2, and
Pot1 (Hiltunen et al. 2003). Pox1 is an acyl-CoA
oxidase catalyzing the first step of the degradation of an acyl-CoA molecule. The dehydrogenated intermediate is then modified by the
multifunctional enzyme Fox2, which acts as an
enoyl-CoA hydratase as well as a 3-hydroxyacyl-CoA dehydrogenase, followed by thiolytic
cleavage through Pot1, a 3-ketoacyl-CoA thiolase. These cyclic steps are repeated until the FA
molecule is completely degraded (Hiltunen
et al. 2003).
Prior to peroxisomal b-oxidation, free FAs
are activated to their acyl-CoA form. In S. cerevisiae, this reaction can be catalyzed by five
enzymes, Faa1, Faa2, Faa3, Faa4, and Fat1
(Black and DiRusso 2007). Long-chain FAs are
primarily activated by Faa1 and Faa4 in the
cytosol (Scharnewski et al. 2008) and transported into the peroxisomes by the heterodimeric ATP-binding cassette transporter Pxa1/
Pxa2 (Hettema et al. 1996). Fat1 is a multifunctional enzyme, which imports long-chain FA
into the peroxisomes and also has an acylCoA synthetase activity for very long-chain FA
(Zou et al. 2002). Medium-chain FAs, on the
other hand, are assumed to enter peroxisomes
by passive diffusion or spontaneous flipping
and are then activated by the peroxisomal
Faa2 (Knoll et al. 1994; Hettema et al. 1996;
Hettema and Tabak 2000). The role of Faa3 is
not entirely clear yet. It showed some activity
on very long-chain FA; however, its in vitro
activity on long-chain FA was much lower
than that of Faa1 and Faa2 (Johnson et al.
1994; Knoll et al. 1994).
The effects of disrupted FA activation, boxidation, or both, on FA titers have been
studied extensively. In S. cerevisiae strains
engineered for increased FA production, a positive effect on free FA titers was detected when
deleting FAA1 and FAA4 separately, as well as
together; however, only minimal increases were
observed when deleting POX1 (Li et al. 2014;
Runguphan and Keasling 2014). Another group
found that the two triple deletion mutants
Dfaa2Dpxa1Dpox1 and Dfaa1Dfaa4Dfat1 produced more free FA than the original strain
(Leber et al. 2015). The combination of all six
knockouts in one strain showed the highest
titers with 1.3 g L
À1 free FA, corresponding to
18% of the maximum theoretical yield (Leber
et al. 2015).
Apart from these rather general b-oxidation disruptions, there have also been
approaches for a chain length-specific disruption of b-oxidation, involving FAA2, PEX11,
and ANT1 (Leber et al. 2016). Pex11 is thought
to transport short- and medium-chain FA into
the peroxisomes, where they are activated by
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
351
Fig. 14.1).
IV. Chassis Engineering
To achieve high FA production levels in S. cerevisiae, several obstacles have to be addressed
within the chosen production pathway. Blocking the degradation of FA has been achieved
through the disruption of different enzymes
involved in b-oxidation. An issue that remains
to be tackled is the toxicity of some FA. We
will discuss different methods that have or
could be employed to engineer more robust
chassis. Furthermore, several biosensor systems have been developed, which can be used
in high-throughput screenings to further speed
up the search for best performing strains.
A. Elimination of By-product Formation and
Fatty Acid Degradation
To increase FA pools, it is important not only to
increase precursor pools (see Sect. II, Fig. 14.1)
but also to downregulate competing pathways
as well as to prevent degradation through specific gene deletions or overexpressions.
1. Prevention of b-Oxidation
The degradation, i.e., b-oxidation, of the
already synthesized free FA or their activated
forms, the acyl-CoAs, takes place in the peroxisomes (Hiltunen et al. 2003; van Roermund
et al. 2003). In S. cerevisiae, b-oxidation of
saturated FA is a cyclic mechanism mainly catalyzed by the three enzymes Pox1, Fox2, and
Pot1 (Hiltunen et al. 2003). Pox1 is an acyl-CoA
oxidase catalyzing the first step of the degradation of an acyl-CoA molecule. The dehydrogenated intermediate is then modified by the
multifunctional enzyme Fox2, which acts as an
enoyl-CoA hydratase as well as a 3-hydroxyacyl-CoA dehydrogenase, followed by thiolytic
cleavage through Pot1, a 3-ketoacyl-CoA thiolase. These cyclic steps are repeated until the FA
molecule is completely degraded (Hiltunen
et al. 2003).
Prior to peroxisomal b-oxidation, free FAs
are activated to their acyl-CoA form. In S. cerevisiae, this reaction can be catalyzed by five
enzymes, Faa1, Faa2, Faa3, Faa4, and Fat1
(Black and DiRusso 2007). Long-chain FAs are
primarily activated by Faa1 and Faa4 in the
cytosol (Scharnewski et al. 2008) and transported into the peroxisomes by the heterodimeric ATP-binding cassette transporter Pxa1/
Pxa2 (Hettema et al. 1996). Fat1 is a multifunctional enzyme, which imports long-chain FA
into the peroxisomes and also has an acylCoA synthetase activity for very long-chain FA
(Zou et al. 2002). Medium-chain FAs, on the
other hand, are assumed to enter peroxisomes
by passive diffusion or spontaneous flipping
and are then activated by the peroxisomal
Faa2 (Knoll et al. 1994; Hettema et al. 1996;
Hettema and Tabak 2000). The role of Faa3 is
not entirely clear yet. It showed some activity
on very long-chain FA; however, its in vitro
activity on long-chain FA was much lower
than that of Faa1 and Faa2 (Johnson et al.
1994; Knoll et al. 1994).
The effects of disrupted FA activation, boxidation, or both, on FA titers have been
studied extensively. In S. cerevisiae strains
engineered for increased FA production, a positive effect on free FA titers was detected when
deleting FAA1 and FAA4 separately, as well as
together; however, only minimal increases were
observed when deleting POX1 (Li et al. 2014;
Runguphan and Keasling 2014). Another group
found that the two triple deletion mutants
Dfaa2Dpxa1Dpox1 and Dfaa1Dfaa4Dfat1 produced more free FA than the original strain
(Leber et al. 2015). The combination of all six
knockouts in one strain showed the highest
titers with 1.3 g L
À1 free FA, corresponding to
18% of the maximum theoretical yield (Leber
et al. 2015).
Apart from these rather general b-oxidation disruptions, there have also been
approaches for a chain length-specific disruption of b-oxidation, involving FAA2, PEX11,
and ANT1 (Leber et al. 2016). Pex11 is thought
to transport short- and medium-chain FA into
the peroxisomes, where they are activated by
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
351