Faa2 (Knoll et al. 1994; van Roermund et al.
2000). Ant1 is an adenine nucleotide transporter, which exchanges AMP by ATP across
the peroxisomal membrane, thereby providing
the energy for the acyl-CoA activation of
the free FA (van Roermund et al. 2001). To
decrease b-oxidation of specifically shortchain FA, a strain with three knockouts
(Dfaa2Dant1Dpex11) was generated, which displayed a much stronger increase in hexanoic
and octanoic acid levels compared to the
“full” b-oxidation-deficient Dfaa2Dpxa1Dpox1
mutant (Leber et al. 2016). This observation
was attributed to the disruption of the AcCoA
recycle mechanism in the latter strain (Leber
et al. 2016). Overall, the ideal combination of
fatty acyl-synthetase and b-oxidation gene
knockouts must be evaluated carefully, depending on the desired chain length of the final FA.
Nevertheless, most reported gene knockouts of
this pathway have turned out to be beneficial
for increasing FA titers (Buijs et al. 2015; Zhou
et al. 2016b).
2. Disruption of Triacylglycerol and Steryl Ester
Synthesis
A competing pathway that can decrease FA
levels is the incorporation into storage lipids,
primarily triacylglycerols (TAGs) or steryl
esters (SEs). In S. cerevisiae, TAGs are synthesized from diacylglycerols and fatty acyl-CoAs
by the acyltransferases Dga1 and Lro1 (Oelkers
et al. 2000, 2002). Steryl ester synthesis of sterols and fatty acyl-CoAs is catalyzed by the acylCoA:sterol acyltransferases Are1 and Are2
(Yang et al. 1996). In engineered yeast strains,
the knockout of one or several of these four
TAG/SE genes has led to increased production
of FA derivatives such as 1-hexadecanol (Tang
and Chen 2015), fatty alcohols (d’Espaux et al.
2017; Tang et al. 2017), and FAEEs (ValleRodrı ´guez et al. 2014). In another strain optimized for FA production (by disruption of boxidation and acyl-CoA-activating enzymes),
TAG synthesis was increased by overexpressing
Dga1. This was combined with an overexpression of the lipid recycle via the triacylglycerol
lipase Tgl3, leading to 2.2 g L
À1 extracellular
free FA (Leber et al. 2015).
B. Engineering Yeast Product Tolerance and
Excretion
Some FA as well as some of their derivatives are
toxic to S. cerevisiae. In general, yeast is a
robust cell factory that can sense stress and
adapt its metabolism accordingly. However, to
avoid loss of cell viability and ensure economically competitive yields, yeast strains are
needed, which are tolerant to high product concentrations even at low pH (Deparis et al. 2017).
Over the last years, much progress has been
made to understand the underlying principles
of the toxicity of these compounds, and random
as well as rational approaches have been
applied to improve yeast robustness and
thereby product yields.
1. Toxicity Mechanisms and Transport of Fatty
Acids and Derivatives
FAs that are toxic to S. cerevisiae include hexanoic, octanoic, and decanoic acids (LafonLafourcade et al. 1984; Viegas et al. 1989; Liu
et al. 2013). At acidic pH, which is the common
fermentation condition, the undissociated FA
can enter the cells by passive diffusion (Viegas
1997). In the neutral cytosol, they dissociate,
thereby causing a decrease of the intracellular
pH and an accumulation of the toxic anions
(Viegas et al. 1989, 1998; Viegas 1997). Furthermore, they disturb the plasma membrane
integrity, causing changes in membrane composition, permeability, and fluidity (Alexandre
et al. 1996; Legras et al. 2010; Liu et al. 2013). To
generate strains with higher tolerance, it is
important to ensure a rapid transport/secretion of FA out of the cell, as well as to avoid
their re-entrance into the cells. A transcriptome
analysis of octanoic- and decanoic acidstressed cells, respectively, revealed a partly
overlapping response mechanism, which was
similar to an oxidative stress response, but it
also showed a compound-specific activation of
352
L. Baumann et al.
2000). Ant1 is an adenine nucleotide transporter, which exchanges AMP by ATP across
the peroxisomal membrane, thereby providing
the energy for the acyl-CoA activation of
the free FA (van Roermund et al. 2001). To
decrease b-oxidation of specifically shortchain FA, a strain with three knockouts
(Dfaa2Dant1Dpex11) was generated, which displayed a much stronger increase in hexanoic
and octanoic acid levels compared to the
“full” b-oxidation-deficient Dfaa2Dpxa1Dpox1
mutant (Leber et al. 2016). This observation
was attributed to the disruption of the AcCoA
recycle mechanism in the latter strain (Leber
et al. 2016). Overall, the ideal combination of
fatty acyl-synthetase and b-oxidation gene
knockouts must be evaluated carefully, depending on the desired chain length of the final FA.
Nevertheless, most reported gene knockouts of
this pathway have turned out to be beneficial
for increasing FA titers (Buijs et al. 2015; Zhou
et al. 2016b).
2. Disruption of Triacylglycerol and Steryl Ester
Synthesis
A competing pathway that can decrease FA
levels is the incorporation into storage lipids,
primarily triacylglycerols (TAGs) or steryl
esters (SEs). In S. cerevisiae, TAGs are synthesized from diacylglycerols and fatty acyl-CoAs
by the acyltransferases Dga1 and Lro1 (Oelkers
et al. 2000, 2002). Steryl ester synthesis of sterols and fatty acyl-CoAs is catalyzed by the acylCoA:sterol acyltransferases Are1 and Are2
(Yang et al. 1996). In engineered yeast strains,
the knockout of one or several of these four
TAG/SE genes has led to increased production
of FA derivatives such as 1-hexadecanol (Tang
and Chen 2015), fatty alcohols (d’Espaux et al.
2017; Tang et al. 2017), and FAEEs (ValleRodrı ´guez et al. 2014). In another strain optimized for FA production (by disruption of boxidation and acyl-CoA-activating enzymes),
TAG synthesis was increased by overexpressing
Dga1. This was combined with an overexpression of the lipid recycle via the triacylglycerol
lipase Tgl3, leading to 2.2 g L
À1 extracellular
free FA (Leber et al. 2015).
B. Engineering Yeast Product Tolerance and
Excretion
Some FA as well as some of their derivatives are
toxic to S. cerevisiae. In general, yeast is a
robust cell factory that can sense stress and
adapt its metabolism accordingly. However, to
avoid loss of cell viability and ensure economically competitive yields, yeast strains are
needed, which are tolerant to high product concentrations even at low pH (Deparis et al. 2017).
Over the last years, much progress has been
made to understand the underlying principles
of the toxicity of these compounds, and random
as well as rational approaches have been
applied to improve yeast robustness and
thereby product yields.
1. Toxicity Mechanisms and Transport of Fatty
Acids and Derivatives
FAs that are toxic to S. cerevisiae include hexanoic, octanoic, and decanoic acids (LafonLafourcade et al. 1984; Viegas et al. 1989; Liu
et al. 2013). At acidic pH, which is the common
fermentation condition, the undissociated FA
can enter the cells by passive diffusion (Viegas
1997). In the neutral cytosol, they dissociate,
thereby causing a decrease of the intracellular
pH and an accumulation of the toxic anions
(Viegas et al. 1989, 1998; Viegas 1997). Furthermore, they disturb the plasma membrane
integrity, causing changes in membrane composition, permeability, and fluidity (Alexandre
et al. 1996; Legras et al. 2010; Liu et al. 2013). To
generate strains with higher tolerance, it is
important to ensure a rapid transport/secretion of FA out of the cell, as well as to avoid
their re-entrance into the cells. A transcriptome
analysis of octanoic- and decanoic acidstressed cells, respectively, revealed a partly
overlapping response mechanism, which was
similar to an oxidative stress response, but it
also showed a compound-specific activation of
352
L. Baumann et al.