compartmentalization into peroxisomes by a
peroxisomal targeting of pathway enzymes
(Sheng et al. 2016; Zhou et al. 2016a). Similarly,
blocking competing pathways through deletion
of specific genes is a common strategy. For
instance, the deletion of HFD1 was shown to
be a crucial step for alkane and alcohol biosynthesis (Buijs et al. 2015; Zhou et al. 2016b).
HFD1 encodes an aldehyde dehydrogenase
involved in sphingolipid degradation and coenzyme Q biosynthesis and catalyzes the oxidation of fatty aldehydes to FA (Zhu et al. 2017a).
Additionally, many engineering strategies for
increasing the supply of the precursor AcCoA
and free FA also led to an increase in the production of FA-derived products (Zhou et al.
2016b; Teixeira et al. 2017).
VI. Optimization of Fermentation
Conditions
Yeast FA titers keep rising through a plethora of
metabolic engineering efforts; however, general
production process optimization is just as
important to unravel the full potential of producer strains. In oleaginous yeasts, like Y. lipolytica, lipid overproduction starts with the
exhaustion of a primary nutrient, i.e., when
entering stationary growth (Beopoulos et al.
2009). Consequently, nitrogen and/or glucose
limitation has been utilized to increase lipid
production in S. cerevisiae (Thompson and
Trinh 2014; Yu et al. 2018). Such limiting conditions led to high lipid titers in a highly engineered S. cerevisiae strain, producing 33.4 g L
À1
free FA in a fed-batch fermentation—the highest reported free FA titer by microbial fermentation to date (Yu et al. 2018). These results also
emphasize the importance of the transition
from flask cultivation to fed-batch cultivation
in a fermenter. The latter permits a tight control
of cultivation parameters, such as pH, aeration,
and nutrient supply, thereby enabling higher
titers of FA and derivatives (Thompson and
Trinh 2014; Zhou et al. 2016b; Yu et al. 2018).
In a very recent study, the effect of “forced”
FA synthesis on the metabolism and physiology
of an engineered S. cerevisiae strain was
analyzed (Gossing et al. 2018). Such a systematic characterization could help to determine
the crucial parameters for improving yeast
lipid production. Besides the expected increase
in b-oxidation and storage lipids, the analyzed
strain also showed higher levels of oxidative
stress and decreased amino acid levels (Gossing
et al. 2018). Therefore, a viable approach could
be an adjustment of media composition, e.g.,
by providing higher amounts of amino acids, or
overexpressing genes to improve the amino
acid uptake or synthesis.
Several previous studies have demonstrated a positive
effect of amino acid supplementation on lipid accumulation. The addition of methionine led to increased
palmitoleic acid production (Kamisaka et al. 2015),
whereas high leucine levels led to increased lipid accumulation in a Dsnf2 strain (Kamisaka et al. 2007). The
additional supplementation of several other amino
acids has been demonstrated to contribute to ethanol
tolerance in yeast (Hirasawa et al. 2007; Sekine et al.
2007; Pham and Wright 2008; Yoshikawa et al. 2009).
Nevertheless, it is also important to consider the
genetic background of the FA producing strain at
hand, as working with auxotrophic strains can decrease
growth (Baganz et al. 1997; C ¸ akar et al. 1999; Basso et al.
2010). Additionally, for some strain series, such as the
BY strains, the importance of sufficient amino acid
supply for optimal growth has been emphasized
(Hanscho et al. 2012).
Vitamins, such as biotin and pantothenate,
also play pivotal roles for normal yeast growth
and are essential in FA synthesis (Suomalainen
and Kera ¨nen 1963; Tehlivets et al. 2007). The
addition of pantothenate, for example, was
used as a metabolic switch to regulate the synthesis of b-farnesene, an AcCoA-derived sesquiterpene (Sandoval et al. 2014). This method
could possibly be transferred to FA production
processes, as they, similarly to b-farnesene,
depend on CoA intermediates.
The fermentation temperature has a major
influence not only on yeast growth but also on
FA tolerance and production (Piper 1995; Viegas and Sa ´-Correia 1995; Viegas 1997). An engineered S. cerevisiae produced more palmitoleic
acid at low temperatures (20–25
C) (Kamisaka
et al. 2015), and the oleaginous yeast Metschnikowia pulcherrima was shown to produce
high lipid levels at low temperatures and pH
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
359
peroxisomal targeting of pathway enzymes
(Sheng et al. 2016; Zhou et al. 2016a). Similarly,
blocking competing pathways through deletion
of specific genes is a common strategy. For
instance, the deletion of HFD1 was shown to
be a crucial step for alkane and alcohol biosynthesis (Buijs et al. 2015; Zhou et al. 2016b).
HFD1 encodes an aldehyde dehydrogenase
involved in sphingolipid degradation and coenzyme Q biosynthesis and catalyzes the oxidation of fatty aldehydes to FA (Zhu et al. 2017a).
Additionally, many engineering strategies for
increasing the supply of the precursor AcCoA
and free FA also led to an increase in the production of FA-derived products (Zhou et al.
2016b; Teixeira et al. 2017).
VI. Optimization of Fermentation
Conditions
Yeast FA titers keep rising through a plethora of
metabolic engineering efforts; however, general
production process optimization is just as
important to unravel the full potential of producer strains. In oleaginous yeasts, like Y. lipolytica, lipid overproduction starts with the
exhaustion of a primary nutrient, i.e., when
entering stationary growth (Beopoulos et al.
2009). Consequently, nitrogen and/or glucose
limitation has been utilized to increase lipid
production in S. cerevisiae (Thompson and
Trinh 2014; Yu et al. 2018). Such limiting conditions led to high lipid titers in a highly engineered S. cerevisiae strain, producing 33.4 g L
À1
free FA in a fed-batch fermentation—the highest reported free FA titer by microbial fermentation to date (Yu et al. 2018). These results also
emphasize the importance of the transition
from flask cultivation to fed-batch cultivation
in a fermenter. The latter permits a tight control
of cultivation parameters, such as pH, aeration,
and nutrient supply, thereby enabling higher
titers of FA and derivatives (Thompson and
Trinh 2014; Zhou et al. 2016b; Yu et al. 2018).
In a very recent study, the effect of “forced”
FA synthesis on the metabolism and physiology
of an engineered S. cerevisiae strain was
analyzed (Gossing et al. 2018). Such a systematic characterization could help to determine
the crucial parameters for improving yeast
lipid production. Besides the expected increase
in b-oxidation and storage lipids, the analyzed
strain also showed higher levels of oxidative
stress and decreased amino acid levels (Gossing
et al. 2018). Therefore, a viable approach could
be an adjustment of media composition, e.g.,
by providing higher amounts of amino acids, or
overexpressing genes to improve the amino
acid uptake or synthesis.
Several previous studies have demonstrated a positive
effect of amino acid supplementation on lipid accumulation. The addition of methionine led to increased
palmitoleic acid production (Kamisaka et al. 2015),
whereas high leucine levels led to increased lipid accumulation in a Dsnf2 strain (Kamisaka et al. 2007). The
additional supplementation of several other amino
acids has been demonstrated to contribute to ethanol
tolerance in yeast (Hirasawa et al. 2007; Sekine et al.
2007; Pham and Wright 2008; Yoshikawa et al. 2009).
Nevertheless, it is also important to consider the
genetic background of the FA producing strain at
hand, as working with auxotrophic strains can decrease
growth (Baganz et al. 1997; C ¸ akar et al. 1999; Basso et al.
2010). Additionally, for some strain series, such as the
BY strains, the importance of sufficient amino acid
supply for optimal growth has been emphasized
(Hanscho et al. 2012).
Vitamins, such as biotin and pantothenate,
also play pivotal roles for normal yeast growth
and are essential in FA synthesis (Suomalainen
and Kera ¨nen 1963; Tehlivets et al. 2007). The
addition of pantothenate, for example, was
used as a metabolic switch to regulate the synthesis of b-farnesene, an AcCoA-derived sesquiterpene (Sandoval et al. 2014). This method
could possibly be transferred to FA production
processes, as they, similarly to b-farnesene,
depend on CoA intermediates.
The fermentation temperature has a major
influence not only on yeast growth but also on
FA tolerance and production (Piper 1995; Viegas and Sa ´-Correia 1995; Viegas 1997). An engineered S. cerevisiae produced more palmitoleic
acid at low temperatures (20–25
C) (Kamisaka
et al. 2015), and the oleaginous yeast Metschnikowia pulcherrima was shown to produce
high lipid levels at low temperatures and pH
14 Engineering Saccharomyces cerevisiae for Production of Fatty Acids and Their Derivatives
359