| |

Received: 13 July 2021    Revised: 11 October 2021    Accepted: 12 October 2021

DOI: 10.1111/jam.15335

ORIGINAL ARTICLE

Effect of ethanol on astaxanthin and fatty acid production

in the red yeast Xanthophyllomyces dendrorhous

Hiroshi Kikukawa1,2   | Chisato Shimizu1  | Yoko Hirono-­Hara1  | Kiyotaka Y. Hara1,2

1
Department of Environmental and
Life Sciences, School of Food and
Nutritional Sciences, University of
Shizuoka, Shizuoka, Japan
2
Graduate Division of Nutritional and
Environmental Sciences, University of
Shizuoka, Shizuoka, Japan
Correspondence
Kiyotaka Y. Hara, Department of
Environmental and Life Sciences,
School of Food and Nutritional
Sciences, University of Shizuoka, 52-­1
Yada, Suruga-­ku, Shizuoka 422-­8526,
Japan.
Email: k-hara@u-shizuoka-ken.ac.jp
Funding information
Marine Biotechnology Program of
Marine Open Innovation Institute;
JST-­Mirai Program, Grant/Award
Number: JPMJMI17EJ; Grant-­in-­Aid for
Scientific Research from Japan Society
for the Promotion of Science (JSPS),
Grant/Award Number: JP21H03645,
JP19K15729 and JP21K14774
Abstract
Aim: The effects of detergent, ethanol and ethanol with plant meadowfoam oil on
the growth of the red heterobasidomycete Xanthophyllomyces dendrorhous and on
the production of astaxanthin (3,3′-­dihydroxy-­β,β-­carotene-­4,4′-­dione) and fatty
acids in this red yeast were investigated.
Methods and Results: Ethanol supplementation at a final concentration of 0.8%
(v/v) caused an increase in the growth, astaxanthin production and fatty acid
production of treated X. dendrorhous compared with untreated X. dendrorhous.
Supplementation of meadowfoam oil with 0.8% ethanol further improved the growth
and astaxanthin production of X. dendrorhous. Fatty acid compositions following
supplementation with various concentrations of ethanol and oil were also analysed.
With 0.8% ethanol supplementation, the ratio of linoleic acid (C18:2) and α-­linolenic
acid (C18:3ω3, ALA) decreased. Conversely, with 1.8% ethanol supplementation, the
ALA ratio increased.
Conclusions: Ethanol can serve as a promoting factor for coproduction of astaxan-
thin and fatty acids in X. dendrorhous, whereas simultaneous supplementation of
ethanol and meadowfoam oil can cause further astaxanthin production.
Significance and Impact of Study: Astaxanthin is widely used in various functional
products because of its antioxidant activity. This study shows that X. dendrorhous
can coproduce astaxanthin and functional fatty acids at high levels following sup-
plementation with ethanol.

KEYWORDS
astaxanthin, ethanol induction, fatty acid, meadowfoam oil, Xanthophyllomyces dendrorhous

I N T RO DU CT ION is widely used not only in aquaculture products as a co-

Carotenoids are yellow to orange-­red pigments with iso-
prenoid structures that are used commercially as colour-
ing agents in the aquaculture industry and as dietary
supplements. The carotenoid astaxanthin (3,3′-­dihydroxy
-­β,β-­carotene-­4,4′-­dione; C40H52O4) has high antioxidant
activity and acts as an antioxidative agent in vivo (Satoh
et al., 2009; Tripathi & Jena, 2009). Therefore, astaxanthin
louring agent for seafood species but also in dietary sup-
plements, cosmetics and pharmaceuticals (Miao et al.,
2011).
Recent demand for astaxanthin has led to an increase
in studies of carotenoid production methods, especially
those that use marine organisms (Davinelli et al., 2018).
Although astaxanthin is naturally found in many animals,
including marine animals and birds such as flamingos,

| 2021 The Society for Applied Microbiology

2034    © wileyonlinelibrary.com/journal/jam J Appl Microbiol. 2022;132:2034–2041.
KIKUKAWA et al.
these animals do not synthesize astaxanthin but acquire it
from their diets, which contain natural astaxanthin produc-
ers including microalgae (e.g. Haematococcus pluvialis and
Chlorella zofingiensis), yeast (e.g. the heterobasidiomycet-
ous red yeast Xanthophyllomyces dendrorhous, also known
as Phaffia rhodozyma) and bacteria (e.g. Paracoccus spp.
and Brevundimonas spp.) (Asker, 2017; Han et al., 2013;
Ide et al., 2012; Visser et al., 2003; Zhang, 2018; Zhang
et al., 2020). Most commercial astaxanthin is synthesized
biologically by the green alga H. pluvialis because it has
the ability to biosynthesize relatively high levels of astax-
anthin content (Ambati et al., 2014); however, production
of astaxanthin by H. pluvialis is notoriously slow.
The red yeast X. dendrorhous is one of the most promis-
ing producers of astaxanthin because it maximizes the pro-
duction process to biosynthesize astaxanthin at a faster rate
than can be achieved with H. pluvialis (Barredo et al., 2017;
Kothari et al., 2019; Melillo et al., 2013; Schmidt et al., 2011;
Visser et al., 2003); however, astaxanthin production by X.
dendrorhous results in lower amounts than can be pro-
duced by H. pluvialis. Therefore, researchers are striving to
improve the production of astaxanthin by X. dendrorhous
through methods such as culture engineering and genetic
metabolic engineering (Hara et al., 2021; Hara, Morita,
Endo, et al., 2014; Hara, Morita, Mochizuki, et al., 2014;
Yamamoto et al., 2016). Previously, coproduction of both
fatty acids and carotenoids by carotenoid-­producing mi-
croorganisms was reported (Saenge et al., 2011; Wu et al.,
2011). Indeed, we have recently reported on the capability
of X. dendrorhous to coproduce astaxanthin and fatty acids
(Kikukawa et al., 2021), revealing that X. dendrorhous is
potentially unique as a yeast species capable of producing
both astaxanthin and a functional α-­linolenic acid (ALA)
(D'Angelo et al., 2020), which is an ω3-­fatty acid.
The use of oils as a carbon source and lipid substrate
for both oil-­ and lipid-­producing Rhodotorula yeast has
previously been reported (Zhang et al., 2011). We screened
plant oils that could promote growth, metabolism, and/or
astaxanthin and fatty acid production in X. dendrorhous
and identified meadowfoam oil as a growth-­stimulating
factor (Kikukawa et al., 2021). It has been reported that
ethanol supplementation promotes astaxanthin accumu-
lation in H. pluvialis (Liu et al., 2019). In the present study,
in order to achieve high coproductivity of astaxanthin and
fatty acid, we analysed their production by X. dendrorhous
when the red yeast was supplemented with detergent,
meadowfoam oil, and ethanol. Moreover, we tested the
optimal meadowfoam oil and ethanol supplementation
conditions by which to enhance fatty acid and astaxanthin
coproduction by X. dendrorhous. Overall, our study indi-
cated that not only meadowfoam oil but also ethanol pro-
motes astaxanthin and functional fatty acid production in
X. dendrorhous.
     |  2035
MATERIALS AND METHODS
Strain and culture conditions
Xanthophyllomyces dendrorhous NBRC 10129 was used
in this study. It was cultured in 10  ml of rich yeast–­
malt (YM) medium (5  g  L−1 tryptone, 3  g  L−1 yeast ex-
tract, 3 g L−1 malt extract and 10 g L−1 d-­glucose) as the
base medium. The media were autoclaved at 121°C and
0.2  MPa for 20  min. When required, the medium was
supplemented with tergitol type NP-­40 (Sigma-­Aldrich
Co.) as a commonly used detergent for yeasts at a final
concentration of 1% (w/v) (Choudhary & Mishra, 2021)
and meadowfoam oil (FUJIFILM Wako Pure Chemical
Corporation) at a final concentration of 0.01% (w/v). Yeast
and malt extracts were purchased from Becton Dickinson.
The X. dendrorhous strain was grown on a YM agar
plate at 22°C for 3  days. Three colonies were inocu-
lated into 10 ml of liquid YM medium in a 50-­ml baffled
Erlenmeyer flask and cultivated at 22°C with agitation
at 200 rpm (0.56 g) for 24 h. Each culture was inoculated
into 10 ml of fresh medium to achieve initial OD600 values
of 0.15 without pH adjustment. Cells were then grown at
22°C with agitation at 200 rpm (0.56 g) for 72 h.
Fatty acid analysis
The total fatty acids extracted in the form of methyl ester
were analysed using previously reported methods with
minor modifications (Kikukawa et al., 2016). Following
cultivation, yeast cells were harvested from 5  ml of cul-
ture broth by centrifugation at 3260 g and 4°C for 20 min,
after which they were dried at 100°C for 2.5 h. The dried
cell weight was measured and then the dried cells were di-
rectly transmethylated with 10% methanolic HCl at 55°C
for 2.5  h. Tricosanoic acid (C23:0) was used as an inter-
nal standard. The resultant fatty acid methyl esters were
extracted with n-­hexane, before being concentrated and
then analysed by gas chromatography.
Astaxanthin analysis
Extraction and measurement of astaxanthin was conducted
using previously described methods and conditions (Hara
et al., 2021). To extract the intracellular carotenoid content
of the isolated strains, yeast cells harvested from 2-­ml cul-
ture broth by centrifugation (4500 g and 4°C for 10 min)
were suspended in 1-­ml acetone and broken using a bead
shaker (Shake Master NEO; BMS) with 0.6-­mm-­diameter
zirconia beads. The resulting cell extract was centrifuged
at 5900 g and 4°C for 10 min, and the acetone layer was
 |
2036       EFFECT OF ETHANOL ON X. DENDRORHOUS
filtrated with a 0.2-­μm regenerated cellulose membrane
filter. The concentration of astaxanthin was determined
using a high-­performance liquid chromatography system
equipped with a Develosil ODS-­HG-­5 column (Nomura
Chemical). The separation was performed at 25°C with
acetonitrile/methanol/2-­propanol [85%/10%/5% (v/v/v)]
as the mobile phase (flow rate: 0.8 mL min−1). Detection
was performed at 470  nm with a SPD-­20A UV detector.
Astaxanthin production (mg  L−1, i.e. mg of astaxanthin
per litre of broth) and productivity (mg g−1, i.e. mg of asta-
xanthin per gram of dried cell weight) were evaluated.
R E S U LT S
Effect of tergitol on astaxanthin and fatty
acid production in X. dendrorhous
The effects of supplementation with tergitol type NP-­40,
a well-­known detergent used to emulsify oil during the
cultivation of a variety of yeasts, on astaxanthin and fatty
acid production in X. dendrorhous were evaluated (Table
1). Tergitol did not affect the growth of X. dendrorhous
(data not shown) or astaxanthin production with mead-
owfoam oil at a concentration of 0.01% (w/v). However,
tergitol affected the amount of total fatty acids, which de-
creased by about 6% to 219.82  mg  L−1. The composition
of ALA also decreased from 6.77% to 5.10% with tergitol
supplementation.
Effect of ethanol supplementation on
astaxanthin and fatty acid production
To evaluate the effects of ethanol on the growth, astaxan-
thin production, and/or fatty acid production of X. den-
drorhous, the red yeast was first cultured in YM liquid
medium individually supplemented with 0%–­1.8% of eth-
anol (Figure 1, Table 2). Remarkably, by supplementation
with ethanol at a final concentration of 0.8%, the growth
T A B L E 1   Effect of the detergent tergitol with meadowfoam
oil on the production of astaxanthin and fatty acids in
Xanthophyllomyces dendrorhous
Astaxanthin Total fatty
(mg L−1) acids (mg L−1)
Control 0.49 207.33
Meadowfoam oil 0.83 234.31
Meadowfoam 0.83 219.82
oil + tergitol
Abbreviation: ALA, α-­linolenic acid.
a
ALA, C18:3, 9cis,12cis,15cis-­octadecatrienoic acid.
of X. dendrorhous increased by about 1.8-­fold (7.0 g L−1)
relative to its growth without ethanol supplementation
(Figure 1a). Correspondingly, astaxanthin and fatty acid
production by X. dendrorhous were promoted by supple-
mentation with 0.8% ethanol: astaxanthin increased by
about 2.2-­fold (reaching about 9.0  mg  L−1) (Figure 1b)
and astaxanthin productivity reached about 0.13  mg  g−1
relative to its production without ethanol (Figure 1c);
total fatty acid production increased by about 3.2-­fold
(reaching about 1.0 g L−1) relative to its production with-
out ethanol (Figure 1d). By supplementation with etha-
nol, the degree of saturation also increased (Table 2), with
the composition of C18:1 and C18:2 found to be especially
increased and decreased respectively. Furthermore, with
ethanol supplementation at 0.8%, the composition of
ALA decreased to <4.0% of total fatty acids. In contrast,
increasing the supplementation of ethanol to 1.8% led to
the highest ratio and amount of ALA produced (about 12%
and 80 mg L−1 respectively). Thus, a higher concentration
of ethanol seems to promote the production of the polyun-
saturated fatty acid ALA.
Effect of meadowfoam oil and ethanol
supplementation on astaxanthin and fatty
acid production
From our previous study (Kikukawa et al., 2021) and the
present study, meadowfoam oil and ethanol were selected
as candidates for promoting factors of growth with both
astaxanthin and fatty acid production. Simultaneous sup-
plementation with meadowfoam oil and ethanol was in-
vestigated with the aim of further improving astaxanthin
and fatty acid production (Figure 2, Table 3). With mead-
owfoam oil or ethanol supplementation, X. dendrorhous
growth was improved by around 3-­fold (Figure 2a).
Moreover, with simultaneous supplementation of both
meadowfoam oil and ethanol, growth further improved by
up to 5.3-­fold compared with growth in the control condi-
tion. As shown in Figure 2b, astaxanthin production was
also improved with meadowfoam oil and ethanol supple-
mentation relative to its production with ethanol supple-
mentation alone: production reached 1.05 mg L−1, that is,
a 3.5-­fold increase compared with production under con-
trol conditions. In contrast, the astaxanthin productivity
ALAa
(mg g−1) was reduced when X. dendrorhous was supple-
(%)
mented with both meadowfoam oil and ethanol (Figure
4.68 2c). In addition, although the production of total fatty
6.77 acids with meadowfoam oil and ethanol supplementa-
5.10 tion showed an 8.2-­fold increase relative to production in
the control group, total fatty acid production was actually
lower than that achieved with ethanol supplementation
alone (Figure 2d). However, fatty acid composition was
KIKUKAWA et al.      |  2037

(a) (b)
8·0 1·2
**
7·0
1·0 *
6·0
0·8

Astaxanthin (mg/L)
Growth (gDCW/L)

5·0

4·0 0·6

3·0
0·4
2·0
0·2
1·0

0 0
0 0·4 0·8 1·2 1·6 1·8 0 0·4 0·8 1·2 1·6 1·8
Ethanol concentration (%) Ethanol concentration (%)

(c) (d)
0·14
** 1,200
*
0·12
Astaxanthin productivity (mg/gDCW)

1,000
0·10
Total fatty acid (mg/L)

800
0·08
600
0·06

400
0·04

0·02 200

0·00 0
0 0·4 0·8 1·2 1·6 1·8 0 0·4 0·8 1·2 1·6 1·8
Ethanol concentration (%) Ethanol concentration (%)

F I G U R E 1   Effect of ethanol supplementation on the growth (a), astaxanthin production (b), astaxanthin productivity (c), and fatty acid
production (d) of the red yeast Xanthophyllomyces dendrorhous. Data represent the means ± standard deviation (n = 3). ** and * indicate
p < 0.01 and p < 0.05 respectively. DCW, dried cell weight

T A B L E 2   Effects of ethanol supplementation on fatty acid composition in Xanthophyllomyces dendrorhous

Fatty acid compositionb (%)/ethanol supplementation

Fatty acida 0%c 0.4% 0.8% 1.2% 1.6% 1.8%

C16:0 9.61 ± 0.10 11.82 ± 0.05 11.03 ± 0.11 10.97 ± 0.06 10.34 ± 0.09 9.84 ± 0.34
C16:1 0.19 ± 0.01 0.15 ± 0.00 0.09 ± 0.03 0.13 ± 0.01 0.15 ± 0.00 0.15 ± 0.01
C18:0 2.88 ± 0.06 6.10 ± 0.12 9.27 ± 0.15 9.28 ± 1.02 7.89 ± 0.10 7.81 ± 0.30
C18:1 27.41 ± 0.26 39.09 ± 0.40 44.01 ± 0.15 42.03 ± 0.46 37.44 ± 0.18 36.39 ± 0.29
C18:2 46.81 ± 0.15 33.99 ± 0.44 28.08 ± 0.09 28.91 ± 0.98 31.00 ± 0.36 30.94 ± 0.78
ALA 9.08 ± 0.15 4.14 ± 0.04 3.87 ± 0.05 5.78 ± 0.54 10.06 ± 0.19 12.03 ± 0.52
Others 4.03 ± 0.23 4.71 ± 0.16 3.65 ± 0.11 2.89 ± 0.11 3.12 ± 0.00 2.84 ± 0.38
Abbreviation: ALA, α-­linolenic acid.
a
C16:0, hexadecanoic acid; C16:1, 9cis-­hexadecenoic acid; C18:0, octadecanoic acid; C18:1, 9cis-­octadecenoic acid, OA; C18:2, 9cis,12cis-­octadecadienoic acid,
LA; C18:3, 9cis,12cis,15cis-­octadecatrienoic acid, ALA. Others include C14:0, C15:0, C17:0, C20:0, C22:0, and a small amount of unidentified fatty acids.
b
Data represent the mean ± standard deviation (n = 3).
c
These data appeared in a previous report (Kikukawa et al., 2021).
2038      | EFFECT OF ETHANOL ON X. DENDRORHOUS

(a) (b)
25 1·6
***
20 ***
** **
Growth (gDCW/L)

1·2

Astaxanthin (mg/L)
15
0·8
10

0·4
5

0 0
Meadowfoam

Meadowfoam

Meadowfoam
Meadowfoam
Control

Ethanol

Ethanol
Control
+ ethanol

+ ethanol
(c) (d)
Astaxanthin productivity (mg/gDCW)

0·16 1,200
*
**
Total fatty acid (mg/L)
0·12
800

0·08

400
0·04

0 0 Meadowfoam
Meadowfoam

Meadowfoam

Meadowfoam
Control

Ethanol

Ethanol
Control
+ ethanol

+ ethanol
F I G U R E 2   Effects of simultaneous supplementation of ethanol and meadowfoam oil on the growth (a), astaxanthin production
(b), astaxanthin productivity (c), and fatty acid production (d) of the red yeast Xanthophyllomyces dendrorhous. Data represent the
means ± standard deviation (n = 3). ***, ** and * indicate p < 0.001, p < 0.01 and p < 0.05 respectively. DCW, dried cell weight

T A B L E 3   Fatty acid composition in Xanthophyllomyces dendrorhous when supplemented with 0.01% (w/v) meadowfoam oil and 1%
(v/v) ethanol

Fatty acid compositionb (%)

Meadowfoam
Fatty acida Control Meadowfoam oil Ethanol oil + ethanol
C16:0 9.34 ± 0.07 9.06 ± 0.17 11.26 ± 0.20 11. 17 ± 0.16
C16:1 0.19 ± 0.01 0.18 ± 0.01 0.12 ± 0.01 0.12 ± 0.00
C18:0 2.60 ± 0.04 2.46 ± 0.03 9.53 ± 0.89 8.70 ± 0.32
C18:1 27.45 ± 0.20 25.75 ± 0.48 43.64 ± 0.67 39.40 ± 0.75
C18:2 47.55 ± 0.16 46.11 ± 0.39 27.57 ± 1.07 29.82 ± 0.72
ALA 8.52 ± 0.04 8.20 ± 0.17 4.44 ± 0.51 4.21 ± 0.15
Others 4.30 ± 0.09 8.22 ± 0.80 3.44 ± 0.34 6.58 ± 1.24
Abbreviation: ALA, α-­linolenic acid.
a
C16:0, hexadecanoic acid; C16:1, 9cis-­hexadecenoic acid; C18:0, octadecanoic acid; C18:1, 9cis-­octadecenoic acid, OA; C18:2, 9cis,12cis-­octadecadienoic acid,
LA; C18:3, 9cis,12cis,15cis-­octadecatrienoic acid, ALA. Others include C14:0, C15:0, C17:0, C20:0, C22:0, and a small amount of unidentified fatty acids.
b
Data represent the mean ± standard deviation (n = 3).
KIKUKAWA et al.
similar whether X. dendrorhous was supplemented with
both the oil and ethanol or with ethanol alone (Table 3).
DI S C US S I O N
Previously, we had shown that X. dendrorhous grew well
on media supplemented with meadowfoam oil, but the
cells did not produce substantial amounts of astaxanthin
(Kikukawa et al., 2021). Here, we show that ethanol sup-
plementation led to higher growth, astaxanthin produc-
tion, and fatty acid production than were observed in
yeast cultured without ethanol (Figure 1). Considering
the results in Figure 1, ethanol can be considered a cheap
and easy to use supplement for the cultivation of red yeast
that, in particular, is a useful promoting factor for astax-
anthin and fatty acid production. In another prior study
on ethanol supplementation to H. pluvialis, a final con-
centration of 0.4% led to the highest increase in astaxan-
thin content (Liu et al., 2019). In contrast, in the present
study, 0.8% ethanol led to the highest increase in not only
astaxanthin production but also fatty acid production and
the growth X. dendrorhous. Moreover, 0.8% ethanol led
to a decrease in the total ratio of unsaturated fatty acids
(83.49% vs. 76.05%) (Table 2), whereas saturated fatty
acids (C16:0 and C18:0) and C18:1 increased. Considering
these results, we suggest that the increase in C18:0, which
is a substrate for Δ9 desaturase, led to the increase in
C18:1 and/or ethanol supplementation led to the promo-
tion of Δ9 desaturase gene expression. Previously, 0.2%
ethanol was shown to induce transcriptional regulation
in red yeast (Marcoleta et al., 2011). Specifically, glucose
was found to cause transcriptional repression of three
genes (crtYB, crtI and crtS) involved in the synthesis of
astaxanthin from geranylgeranyl pyrophosphate in X.
dendrorhous; however, the addition of ethanol caused
the induction of crtYB and crtS gene expression and pro-
moted de novo synthesis of carotenoids. Similar to the
findings of this previous report, our results suggest that
astaxanthin synthesis is promoted in X. dendrorhous by
supplementation with 0.8% ethanol. Ethanol supplemen-
tation can also potentially promote astaxanthin produc-
tion under other reported optimized culture conditions
because it upregulates the gene expression related to asta-
xanthin biosynthesis (Marcoleta et al., 2011).
Tergitol type NP-­40 is commonly used for emulsifica-
tion of oils during cultivation of yeasts because of its excel-
lent detergency, wetting, solubilization and emulsification
activity (Choudhary & Mishra, 2021). In the current study,
tergitol supplementation decreased both the amount of
total fatty acids and the composition of ALA, which is a
ω3-­fatty acid that plays a functional role in human health
(Table 1). From these results, it was concluded that a
     |  2039
representative tergitol, that is, type NP-­40, should not be
used as a detergent during the culture of X. dendrorhous
with meadowfoam oil.
With simultaneous supplementation of both ethanol
and meadowfoam oil (Figure 2b), the growth of X. den-
drorhous increased by approximately 2.4-­fold relative
to the growth achieved when ethanol or meadowfoam
oil was used separately, and astaxanthin production in-
creased with the combined treatment relative to its pro-
duction with ethanol supplementation alone. In contrast,
the astaxanthin productivity (mg  g−1) and production
of total fatty acid was reduced with the simultaneous
supplementation, and ALA production did not change
substantially (around 30  mg  L−1). Based on the results
presented in Figure 2, it can be concluded that ethanol is
an excellent promoting factor for the growth, astaxanthin
production, and fatty acid production of X. dendrorhous,
whereas ethanol and meadowfoam oil combined can im-
prove the growth and astaxanthin production of the red
yeast.
In conclusion, we revealed that 0.8% ethanol supple-
mentation substantially increases the growth rate of the
red yeast X. dendrorhous while promoting considerable
astaxanthin and fatty acid production. The increasing ef-
fect of ethanol on the growth and astaxanthin production
of X. dendrorhous was also enhanced by the combinato-
rial addition of meadowfoam oil. Moreover, ethanol sup-
plementation was shown to alter fatty acid composition.
In summary, these results show that X. dendrorhous can
coproduce astaxanthin and functional fatty acids at high
levels via supplementation with ethanol.
ACKNOWLEDGEMENTS
This work was supported by Seeds Creation Research
centered on Marine Biotechnology Program of Marine
Open Innovation Institute, the JST-­Mirai Program (No.
JPMJMI17EJ), and Grant-­in-­Aid for Scientific Research
from Japan Society for the Promotion of Science (JSPS)
(no. JP21H03645, JP19K15729 and JP21K14774). The au-
thors thank Enago (www.enago.jp) for editing a draft of
this manuscript.
CONFLICT OF INTEREST
No conflict of interest declared.
DATA AVAILABILITY STATEMENT
Research data are not shared.
ORCID
Hiroshi Kikukawa  https://orcid.
org/0000-0002-1869-4827
Kiyotaka Y. Hara  https://orcid.
org/0000-0002-4477-6594
 |
2040       EFFECT OF ETHANOL ON X. DENDRORHOUS
REFERENCES
Ambati, R.R., Phang, S.M., Ravi, S. & Aswathanarayana, R.G. (2014)
Astaxanthin: sources, extraction, stability, biological activities
and its commercial applications–­a review. Marine Drugs, 12(1),
128–­152. https://doi.org/10.3390/md120​10128
Asker, D. (2017) Isolation and characterization of a novel, highly
selective astaxanthin-­producing marine bacterium. Journal of
Agriculture and Food Chemistry, 65(41), 9101–­9109. https://doi.
org/10.1021/acs.jafc.7b03556
Barredo, J.L., García-­Estrada, C., Kosalkova, K. & Barreiro, C.
(2017) Biosynthesis of astaxanthin as a main carotenoid in
the heterobasidiomycetous yeast Xanthophyllomyces dendror-
hous. Journal of Fungi, 3(3), 44. https://doi.org/10.3390/jof30​
30044
Choudhary, A.K. & Mishra, G. (2021) Functional characterization
and expression profile of microsomal FAD2 and FAD3 genes
involved in linoleic and α-­linolenic acid production in Leucas
cephalotes. Physiology and Molecular Biology of Plants, 27,
1233–­1244. https://doi.org/10.1007/s1229​8-­021-­01016​-­z
D'Angelo, S., Motti, M.L. & Meccariello, R. (2020) ω-­3 and ω-­6 poly-
unsaturated fatty acids, obesity and cancer. Nutrients, 12(9),
2751. https://doi.org/10.3390/nu120​92751
Davinelli, S., Nielsen, M.E. & Scapagnini, G. (2018) Astaxanthin
in skin health, repair, and disease: a comprehensive review.
Nutrients, 10, 522. https://doi.org/10.3390/nu100​40522
Han, D., Li, Y. & Hu, Q. (2013) Astaxanthin in microalgae: pathways,
functions and biotechnological implications. ALGAE, 28(2),
131–­147. https://doi.org/10.4490/algae.2013.28.2.131
Hara, K.Y., Kageyama, Y., Tanzawa, N., Hirono-­Hara, Y., Kikukawa,
H. & Wakabayashi, K. (2021) Development of astaxanthin pro-
duction from citrus peel extract using Xanthophyllomyces den-
drorhous. Environmental Science and Pollution Research, 28(10),
12640–­12647. https://doi.org/10.1007/s1135​6-­020-­11163​-­7
Hara, K.Y., Morita, T., Endo, Y., Mochizuki, M., Araki, M. & Kondo,
A. (2014) Evaluation and screening of efficient promoters to
improve astaxanthin production in Xanthophyllomyces den-
drorhous. Applied Microbiology and Biotechnology, 98(15),
6787–­6793. https://doi.org/10.1007/s0025​3-­014-­5727-­2
Hara, K.Y., Morita, T., Mochizuki, M., Yamamoto, K., Ogino, C.,
Araki, M. et al. (2014) Development of a multi-­gene expres-
sion system in Xanthophyllomyces dendrorhous. Microbial Cell
Factories, 13, 175. https://doi.org/10.1186/s1293​4-­014-­0175-­3
Ide, T., Hoya, M., Tanaka, T. & Harayama, S. (2012) Enhanced pro-
duction of astaxanthin in Paracoccus sp. strain N-­81106 by using
random mutagenesis and genetic engineering. Biochemical
Engineering Journal, 65, 37–­43. https://doi.org/10.1016/j.
bej.2012.03.015
Kikukawa, H., Sakuradani, E., Ando, A., Okuda, T., Shimizu, S. &
Ogawa, J. (2016) Microbial production of dihomo-­γ-­linolenic
acid by Δ5-­desaturase gene-­disruptants of Mortierella alpina
1S–­4. Journal of Bioscience and Bioengineering, 122(1), 22–­26.
https://doi.org/10.1016/j.jbiosc.2015.12.007
Kikukawa, H., Shimizu, C., Hirono-­Hara, Y. & Hara, K.Y. (2021)
Screening of plant oils promoting growth of the red yeast
Xanthophyllomyces dendrorhous with astaxanthin and fatty
acid production. Biocatalysis and Agricultural Biotechnology,
35, 102101. https://doi.org/10.1016/j.bcab.2021.102101
Kothari, D., Lee, J.H., Chon, J.W., Seo, K.H. & Kim, S.K. (2019)
Improved astaxanthin production by Xanthophyllomyces
dendrorhous SK984 with oak leaf extract and inor-
ganic phosphate supplementation. Food Science and
Biotechnology, 28(4), 1171–­1176. https://doi.org/10.1007/
s1006​8-­019-­00604​-­w
Liu, Y.H., Alimujiang, A., Wang, X., Luo, S.W., Balamurugan, S.,
Yang, W.D. et al. (2019) Ethanol induced jasmonate pathway
promotes astaxanthin hyperaccumulation in Haematococcus
pluvialis. Bioresource Technology, 289, 121720. https://doi.
org/10.1016/j.biort​ech.2019.121720
Marcoleta, A., Niklitschek, M., Wozniak, A., Lozano, C., Alcaíno, J.,
Baeza, M. et al. (2011) Glucose and ethanol-­dependent tran-
scriptional regulation of the astaxanthin biosynthesis pathway
in Xanthophyllomyces dendrorhous. BMC Microbiology, 11, 190.
https://doi.org/10.1186/1471-­2180-­11-­190
Melillo, E., Setroikromo, R., Quax, W.J. & Kayser, O. (2013)
Production of α-­cuprenene in Xanthophyllomyces dendror-
hous: a step closer to a potent terpene biofactory. Microbial Cell
Factories, 12(1), 13. https://doi.org/10.1186/1475-­2859-­12-­13
Miao, L., Chi, S., Tang, Y., Su, Z., Yin, T., Guan, G. et al. (2011)
Astaxanthin biosynthesis is enhanced by high carotenogenic
gene expression and decrease of fatty acids and ergosterol in a
Phaffia rhodozyma mutant strain. FEMS Yeast Research, 11(2),
192–­201. https://doi.org/10.1111/j.1567-­1364.2010.00705.x
Saenge, C., Cheirsilp, B., Suksaroge, T.T. & Bourtoom, T. (2011)
Efficient concomitant production of lipids and carotenoids by
oleaginous red yeast Rhodotorula glutinis cultured in palm oil
mill effluent and application of lipids for biodiesel production.
Biotechnology and Bioprocess Engineering, 16(1), 23–­33. https://
doi.org/10.1007/s1225​7-­010-­0083-­2
Satoh, A., Tsuji, S., Okada, Y., Murakami, N., Urami, M., Nakagawa,
K. et al. (2009) Preliminary clinical evaluation of toxicity and
efficacy of a new astaxanthin-­rich Haematococcus pluvialis ex-
tract. Journal of Clinical Biochemistry and Nutrition, 44(3), 280–­
284. https://doi.org/10.3164/jcbn.08-­238
Schmidt, I., Schewe, H., Gassel, S., Jin, C., Buckingham, J.,
Hümbelin, M. et al. (2011) Biotechnological production of
astaxanthin with Phaffia rhodozyma/Xanthophyllomyces den-
drorhous. Applied Microbiology and Biotechnology, 89(3), 555–­
571. https://doi.org/10.1007/s0025​3-­010-­2976-­6
Tripathi, D.N. & Jena, G.B. (2009) Intervention of astaxanthin against
cyclophosphamide-­induced oxidative stress and DNA damage:
a study in mice. Chemico-­Biological Interactions, 180(3), 398–­
406. https://doi.org/10.1016/j.cbi.2009.03.017
Visser, H., van Ooyen, A.J.J. & Verdoes, J.C. (2003) Metabolic
engineering of the astaxanthin-­biosynthetic pathway of
Xanthophyllomyces dendrorhous. FEMS Yeast Research, 4(3),
221–­231. https://doi.org/10.1016/s1567​-­1356(03)00158​-­2
Wu, S., Zhao, X., Shen, H., Wang, Q. & Zhao, Z.K. (2011) Microbial
lipid production by Rhodosporidium toruloides under sulfate-­
limited conditions. Bioresource Technology, 102(2), 1803–­1807.
https://doi.org/10.1016/j.biort​ech.2010.09.033
Yamamoto, K., Hara, K.Y., Morita, T., Nishimura, A., Sasaki, D.,
Ishii, J. et al. (2016) Enhancement of astaxanthin production
in Xanthophyllomyces dendrorhous by efficient method for the
complete deletion of genes. Microbial Cell Factories, 15(1), 155.
https://doi.org/10.1186/s1293​4-­016-­0556-­x
Zhang, C. (2018) Biosynthesis of carotenoids and apocarotenoids by
microorganisms and their industrial potential: progress in carot-
enoid research. London: IntechOpen. https://doi.org/10.5772/
intec​hopen.79061
KIKUKAWA et al.
Zhang, C., Chen, X. & Too, H.P. (2020) Microbial astaxanthin biosyn-
thesis: recent achievements, challenges, and commercializa-
tion outlook. Applied Microbiology and Biotechnology, 104(13),
5725–­5737. https://doi.org/10.1007/s0025​3-­020-­10648​-­2
Zhang, J., Saerens, K.M., Van Bogaert, I.N. & Soetaert, W. (2011)
Vegetable oil enhances sophorolipid production by Rhodotorula
bogoriensis. Biotechnology Letters, 33(12), 2417–­2423. https://
doi.org/10.1007/s1052​9-­011-­0703-­8
     |  2041
How to cite this article: Kikukawa, H., Shimizu, C.,
Hirono-­Hara, Y. & Hara, K.Y. (2022) Effect of ethanol
on astaxanthin and fatty acid production in the red
yeast Xanthophyllomyces dendrorhous. Journal of
Applied Microbiology, 132, 2034–­2041. https://doi.
org/10.1111/jam.15335