Cyclofarnesoids and methylhexanoids produced from β-carotene in Phycomyces blakesleeanus

Article abstract of DOI:10.1016/j.phytochem.2016.01.013

The oxidative cleavage of β-carotene in the Mucorales produces three fragments of 18, 15, and 7 carbons, respective heads of three families of apocarotenoids: The methylhexanoids, the trisporoids, and the cyclofarnesoids (named after their 1,6-cyclofarnes

Full text of DOI:10.1016/j.phytochem.2016.01.013

Phytochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cyclofarnesoids and methylhexanoids produced from b-carotene in
Phycomyces blakesleeanus
Eugenio Alcalde a,1, Humberto R. Medina a, M. Mar Herrador b, Alejandro F. Barrero b,
Enrique Cerdá-Olmedo a,
⇑
a Departamento de Genética, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain
b Departamento de Química Orgánica, Instituto de Biotecnología, Universidad de Granada, Fuente Nueva s/n, E-18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The oxidative cleavage of b-carotene in the Mucorales produces three fragments of 18, 15, and 7 carbons,
respective heads of three families of apocarotenoids: the methylhexanoids, the trisporoids, and the cyclo-
farnesoids (named after their 1,6-cyclofarnesane skeleton). The apocarotenoids are easily recognized
because they are absent in white mutants unable to produce b-carotene. In cultures of Phycomyces bla-
kesleeanus we detected thirty-two apocarotenoids by LC, UV absorbance, and MS. With additional IR
and NMR we identified two methylhexanoids and the eight most abundant cyclofarnesoids. Four of them
were previously-unknown natural compounds, including 4-dihydrocyclofarnesine S, the most abundant
cyclofarnesoid in young cultures. We arranged the apocarotenoids of the Mucorales in a scheme that
helps classifying and naming them and suggests possible metabolites and biosynthetic pathways. We
propose specific biosynthetic pathways for cyclofarnesoids and methylhexanoids based on structural
comparisons, the time course of appearance of individual compounds, and the bioconversion of b-apo-
12-carotenol, an early precursor, to three more oxygenated cyclofarnesoids by the white mutants.
Some of the reactions occur spontaneously in the increasingly acidic culture media. Mating increased
the contents of methylhexanoids and cyclofarnesoids by ca. threefold in young cultures and ca. twelve-
fold in old ones (five days); cyclofarnesine S, the most abundant cyclofarnesoid in old cultures, increased
over one hundredfold. We found no differences between the sexes and no activity as sexual pheromones,
but we suggest that methylhexanoids and cyclofarnesoids could mediate species-specific physiology and
behavior.
Received 1 November 2015
Received in revised form 11 January 2016
Accepted 20 January 2016
Available online xxxx
Keywords:
Phycomyces blakesleeanus
Phycomycetaceae
Mucorales
Bioconversion
Cyclofarnesoids
Methylhexanoids
b-Carotene
Apocarotenoids
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
they are missing in mutants that cannot synthesize b-carotene
(Sutter, 1975).
The sexual cycle of Phycomyces blakesleeanus Bgff., Blakeslea tris-
pora Thaxter, and other fungi of the order Mucorales (Blakeslee,
1904) begins when hyphae of different sex, (+) and (ꢀ), grow
together under appropriate conditions (‘‘mated cultures”). Sex-
specific diffusible signals induce nearby hyphal tips of the opposite
sex to develop into zygophores, the initial structures of the sexual
cycle. This was the first case of a pheromone exchange reported for
any organism (Burgeff, 1924). The active molecules are believed to
be apocarotenoids, derived from fragments of b-carotene, because
Sexual interaction is accompanied in many strains by an
increased accumulation of b-carotene that is exploited by industry
(Cerdá-Olmedo, 1989; Ciegler, 1965; Iturriaga et al., 2000). Trispo-
ric acid C, a metabolite of b-carotene found in mated cultures of
Blakeslea, stimulates carotene production in single cultures
(Austin et al., 1970; Caglioti et al., 1966). It was the first of the tris-
poroids, a family of C18 compounds and their C19 methyl esters
(Schimek and Wöstemeyer, 2009; Sutter, 1987; van den Ende,
1979).
The discovery in Phycomyces of the cleavage of b-carotene into
C18, C15, and C7 fragments justified the recognition of two
⇑
additional families of apocarotenoids: the cyclofarnesoids (with
the carbon skeleton of 1,6-cyclofarnesane) and the methylhex-
anoids (Polaino et al., 2010). An oxygenase, the product of gene
carS, first cleaves b-carotene into C15 and C25 fragments (Medina
et al., 2011; Tagua et al., 2012), and another one, the product of
Corresponding author.
E-mail addresses: eugenalc@gmail.com (E. Alcalde), hurmedina@gmail.com
(H.R. Medina), mmar@ugr.es (M.M. Herrador), afbarre@ugr.es (A.F. Barrero), eco@
us.es (E. Cerdá-Olmedo).
1
Present address: School of Biological Sciences, Royal Holloway University of
London, Egham, Surrey TW20 0EX, UK.
http://dx.doi.org/10.1016/j.phytochem.2016.01.013
0031-9422/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto-
chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013

2
E. Alcalde et al. / Phytochemistry xxx (2016) xxx–xxx
gene acaA, cleaves the C25 fragment into C18 and C7 fragments
(Medina et al., 2011). This discovery has been extended to Blakeslea
(Barrero et al., 2011; Sahadevan et al., 2013), Mucor mucedo
(Sahadevan et al., 2013), and Mucor circinelloides (our unpublished
work).
The dedicated b-carotene biosynthetic pathway of Phycomyces
depends on two structural genes, carB for phytoene dehydrogenase
and carRA for phytoene synthase and lycopene cyclase (Arrach
et al., 2001; Eslava and Cerdá-Olmedo, 1974; Torres-Martínez
et al., 1980). The carA mutants are white and deficient in all carote-
nes, the carB mutants are white and rich in phytoene, and the carR
mutants are red and rich in lycopene. The carS mutants (Murillo
and Cerdá-Olmedo, 1976; Tagua et al., 2012) accumulate large con-
centrations of b-carotene, which makes them deep yellow, instead
of light yellow, as the wild type. Mutants of these genes lack apoc-
arotenoids (Tagua et al., 2012) and are unable to stimulate their
sexual partners (Murillo et al., 1978; Sutter, 1975).
cultures of the wild types; nineteen were found in mated cultures
only; and two were not found in mated cultures (Medina, 2013).
For convenience we designate the compounds detected as Nt or
At, where N stands for neutral extracts; A, for acid extracts; and
t, for the elution time in tens of seconds. Thus, the (2S, 6E, 8E)
and (2S, 6Z, 8E) isomers of cyclofarnesa-4,6,8-triene-2,10,11-triol
(10, 11) will be called N121 and N122 for short, since we did not
set up a column for them in Fig. 1.
In our liquid chromatography with trifluoroacetic acid added to
the mobile phase, the methylhexanoids eluted rapidly, around min.
12, the cyclofarnesoids, more slowly, min. 15–20, and the trispor-
oids thereafter. The known cyclofarnesoids absorb shorter wave-
lengths (kmax < 320 nm) than trisporic acids and other trisporoids.
All the cyclofarnesoids of Phycomyces were found in the neutral
extracts, but compounds (5), (7), and (8) were polar enough to be
present in the acid extracts as well.
Comparisons of the extracts of wild-type and carB-mutant cul-
tures of Phycomyces revealed an unforeseen diversity of apoc-
arotenoids (Medina, 2013; Polaino et al., 2012). We set out to
describe the cyclofarnesoids and the methylhexanoids in single
and mated cultures of wild-type and mutant strains under various
conditions. Fortunately, the carB mutants that make no b-carotene
retain the ability to bioconvert apocarotenoids.
2.3. Identification and chemical diversity of cyclofarnesoids and
methylhexanoids
The structures (Fig. 1) of the two methylhexanoids (1, 2) and
the eight cyclofarnesoids (4–11) that gave the highest UV absorp-
tion have been deduced from their 1H and 13C NMR spectra, UV
absorption spectra, and MS molecular formulas (see Table S1,
Figs. S1 and S2, and Text S1 in Supplementary Materials). The
structures of compounds (2), (6), (10), and (11) are supported by
previous syntheses (González-Delgado et al., 2013; Polaino et al.,
2010). Our results on compounds (1), (2), (6), (7), (10), and (11)
coincide with those on patrons isolated from nature in previous
work (Barrero et al., 2011; Polaino et al., 2012).
2. Results
2.1. A network of apocarotenoids
The apocarotenoids of the Mucorales have unpractical system-
atic names and they are so numerous and diverse that giving them
arbitrary trivial names would be untoward. A convenient nomen-
clature can be suggested on the basis of a scheme ordered as a net-
work (Fig. 1), in which contiguous compounds could be derived
from each other by simple metabolic steps. Additionally, this
scheme can be seen as a set of possible biosynthetic pathways.
Trisporic acids were named, as they were discovered, with the
letters A through E to indicate the substitutions at their C-2, C-3,
and C-13 atoms. These letters can be used to define extended sub-
families that include the neutral trisporoids. For the cyclofarne-
soids we define here subfamilies with additional capital letters
that indicate the substitutions at their C-2, C-8, and C-14 atoms.
We arrange both the trisporoids and the cyclofarnesoids in col-
umns for the subfamilies and in rows according to the substitu-
tions at C-4 and at the b carbon linked to C-1. The proposed
semisystematic names consist of the roots ‘‘trispor” or ‘‘cyclofar-
nes”, the suffixes and prefixes of the rows and the capital letters
of the columns.
4-Dihydrocyclofarnesine S (4), 4-dihydrocyclofarnesine T (5),
trans-cyclofarnesine U (8), and cyclofarnesine T (9) are natural
products that had not been described previously. The structure of
(4) was deduced from its formula, C15H24O3, its IR absorption
bands at 3409 and 1010 cmꢀ1, its UV absorption spectrum, which
pointed out to a cyclofarnesa-5,7,9-triene, and its 1H and 13C
NMR spectra. The slight differences (Table S1) in the NMR spectra
of (4) and a synthetic compound (12) (González-Delgado et al.,
2013), involve the arrangement of the OH groups on C-2 and C-4
(Fig. 2) and identify these compounds as trans- and cis-4-dihydro-
cyclofarnesine S, respectively.
4-Dihydrocyclofarnesine T (5), with the molecular formula
C15H24O4 and a UV spectrum characteristic of a conjugate triene,
is the most polar of the cyclofarnesoids and only partially extracted
in basic medium. It contains one more hydroxyl group than (4),
tentatively located on C-16 on the basis of the proposed conversion
of (4) to cis-cyclofarnesine U (7) (see subsection 2.5). trans-Cyclo-
farnesine U (8), C15H23O3, must be the trans isomer of (7)
(Polaino et al., 2012), as indicated by its polarity and its UV
absorption.
Compound (9), C15H23O4, with UV spectrum characteristic of a
conjugated trienone (Grasselli and Selva, 1970), is very similar to
cyclofarnesine S, but has an additional hydroxyl group (Supple-
mentary Text S1). Compound (9) could be either cyclofarnesine T
or cyclofarnesol S, but the last is unlikely, because it would be
the only cyclofarnesoid from Phycomyces hydroxylated at the b car-
bon linked to C-1.
The scheme in Fig. 1 generalizes the semisystematic names
already used for some trisporoids and can be widened to include
new variations to be discovered in the future. We have left cyclo-
farnesa-4,6,8-triene-2,10,11-triol (10, 11, isomers 2S, 6E, 8E and
2S, 6Z, 8E, respectively) out of the scheme, but it could be placed
in a new column in the future.
2.2. Putative apocarotenoids
We analyzed by LC/MS and LC/UV the neutral (pH 8) and acidic
(pH 2) extracts of single and mated cultures of strains NRRL1555
and A56, two largely isogenic wild types of opposite sex. Putative
apocarotenoids were immediately recognized as compounds that
were absent in similar extracts of the white carB mutant strains,
which completely lack b-carotene. There were thirty-two such
compounds in five-days cultures, disregarding those with very
low absorbance. Eleven of them were present in single and mated
2.4. Variations with age and sex
The total amounts of methylhexanoids and cyclofarnesoids
increased at least from the second to the fifth day in cultures
grown from spores (Table 1). Judging from their molecular struc-
tures, the apocarotenoids of each family are expected to have very
similar molar absorption coefficients, thus allowing the use of the
absorption in the chromatograms for quantitative comparisons.
Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto-
chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013

E. Alcalde et al. / Phytochemistry xxx (2016) xxx–xxx
3
Fig. 1. Apocarotenoids of the Mucorales, both known and hypothetical. Trisporoids and cyclofarnesoids are named by the respective roots ‘‘trispor” and ‘‘cyclofarnes”, the
prefixes and suffixes given on the right, and the letters given below. Known compounds are boxed. Bold numbers in the boxes indicate the natural apocarotenoids mentioned
in this work. Numbers in brackets indicate references: 1, Medina et al., 2011; 2, Polaino et al., 2010; 3, Cainelli et al., 1967; 4, Sutter and Whitaker, 1981; 5, Sutter and
Zawodny, 1984; 6, Polaino et al., 2012; 7, Sutter, 1986; 8, Bu’lock et al., 1972; 9, Prisbylla et al., 1979; 10, Barrero et al., 2011; 11, Sutter et al., 1989; 12, Austin et al., 1970; 13,
Miller and Sutter, 1984; 14, Bu’lock et al., 1976.
The mix of cyclofarnesoids varied considerably with time: 4-dihy-
drocyclofarnesine S (4) accounted for more than 90% in two-days
single cultures, but decreased as more oxidized compounds were
produced. The concentration of 4-dihydrocyclofarnesine S in the
culture medium of two-days mated cultures was 3.9 mg/l, as mea-
sured following purification from their neutral extracts.
The two methylhexanoids were present throughout. The ratio of
the 5-methyl isomer to the 2-methyl isomer remained at 2.3 0.6
Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto-
chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013

4
E. Alcalde et al. / Phytochemistry xxx (2016) xxx–xxx
Fig. 2. Conformational equilibrium of the trans (4) and cis (12) isomers of
4-dihydrocyclofarnesine S.
(mean and standard deviation of its distribution). The concentra-
tion of both isomers in the culture medium of five-days mated
cultures was 41.5 mg/l, as measured following purification from
acid extracts (Polaino et al., 2010, Supplementary information).
Mating increased the contents of methylhexanoids and cyclo-
farnesoids to about the same extent. The ratio of the concentra-
tions in mated cultures to the mean of the corresponding single
cultures was 2.4-fold, on the average, in two and three-days cul-
tures, and 11.7-fold in five days cultures (Table 1). The different
cyclofarnesoids did not increase equally. Cyclofarnesine S (6)
surged over 100-fold in the older mated cultures to become the
predominant cyclofarnesoid. A previously-unknown compound
detected in mated cultures was identified tentatively as cyclofar-
nesine T (9).
Fig. 3. Synthesis of b-apo-12-carotenol (3). See Supplementary Text S2 for details.
2.5. Bioconversion of cyclofarnesoids by white mutants
The loss of phytoene dehydrogenase in the white carB mutants
renders them unable to produce b-carotene and apocarotenoids,
but does not necessarily deprive them of other enzyme activities.
b-Apo-12-carotenol (3) has not been reported from any organism,
but is most likely an early natural intermediate in the biosynthesis
of cyclofarnesoids. We synthesized it by condensation of b-ionone
with diethylcyanomethyl phosphonate, followed by a double
reduction (Fig. 3). Young cultures of carB mutants readily metabo-
lized it to three natural products found in the wild-type strains:
4-dihydrocyclofarnesines S (4) and T (5) and cyclofarnesine S (6)
(Fig. 4 and Table 2). Cyclofarnesine S (6) was not bioconverted to
other known apocarotenoids (Table 2).
Fig. 4. Bioconversion of b-apo-12-carotenol (3). HPLC fractionations of the neutral
extracts from agar media where the substrate had been incubated either alone
(above) or with a two-days culture of the white mutant strain C5, carB (ꢀ), (below).
3. Discussion
depends to a large extent on their stability. Cyclofarnesine S (6)
was very unstable in cell-free medium, but less so in the presence
of mycelia (Table 2), suggesting that it is protected by interaction
with the cell walls.
The observed mixtures vary within the Mucorales. Seven of the
eight cyclofarnesoids from Phycomyces reported here do not coin-
cide with those reported from B. trispora (references in Fig. 1).
Prominent apocarotenoids of B. trispora, such as cyclofarnesol R
3.1. Chemical diversity
Cyclofarnesine S (6), the most abundant cyclofarnesoid in old
(five days) mated cultures, has long been known as apotrisporine
E (Sutter, 1986), a name based on an error in the determination
of its structure and on the conjecture that it derived from a trispor-
ine by the loss of three carbon atoms. The cyclofarnesoids are now
a diversified group of apocarotenoids. The observed mixture
Table 1
Amounts of methylhexanoids and cyclofarnesoids. Single and mated cultures of the (+) A56 and the (ꢀ) NRRL1554 wild type strains were grown for 2 to 5 d before extraction. The
values are the total absorbance (200–900 nm peak area in absorbance units ꢁ s) in HPLC chromatographs of the material in 1 ml of culture medium. The numbers in parentheses
identify the chemical structures in Fig. 1.
2 d
3 d
5 d
(ꢀ)
(+)
(ꢀ) ꢁ (+)
(ꢀ)
(+)
(ꢀ) ꢁ (+)
(ꢀ)
(+)
(ꢀ) ꢁ (+)
6-Hydroxy-5-methylhexa-2,4-dienoic acid
6-Hydroxy-2-methylhexa-2,4-dienoic acid
(1)
(2)
25
10
19
5.9
59
26
25
8.7
47
20
47
34
13
6.3
22
11
219
122
4-Dihydrocyclofarnesine S
N121
N122
4-Dihydrocyclofarnesine T
Cyclofarnesine U
Cyclofarnesine U
(4)
20
0
0
1.4
0
0
28
0
0
2.7
0
0
42
0
0
9.0
0
0
45
3.3
1.2
6.3
6.9
4.7
0.8
0
72
1.5
1.4
10
2.6
2.0
1.1
0
63
2.9
0
19
1.8
11
101
10
17
9.6
3.2
1.5
8.4
8.7
2.1
0
45
2.4
0.9
2.7
5.7
3.2
0.8
0
41
78
21
26
(10)
(11)
(5)
(7)
(8)
19
126
250
26
Cyclofarnesine S
Cyclofarnesine T or Cyclofarnesol S
(6)
(9)
0
0
0
0
8.9
2.4
Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto-
chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013

E. Alcalde et al. / Phytochemistry xxx (2016) xxx–xxx
5
Table 2
Bioconversion of cyclofarnesoids by white mutants of Phycomyces. The substrate (1 mg) was incubated for one day on agar plates
with or without mycelia pregrown for 2 d. Peak-area absorbance at 328 nm (cyclofarnesine S) or at 254 nm (the others) in HPLC
chromatographs. Single cultures of strains C5, carB10 (ꢀ), and S342, carB10 (+), and their mated cultures. The numbers in
parentheses identify the chemical structures in Fig. 1.
(ꢀ)
(+)
(ꢀ) ꢁ (+)
Medium
Substrate:
Products:
b-Apo-12-carotenol
b-Apo-12-carotenol
4-Dihydrocyclofarnesine S
4-Dihydrocyclofarnesine T
Cyclofarnesine S
(3)
(3)
(4)
(5)
(6)
1400
28.5
0
0
0
0
69
16.6
15.2
100.8
0
0
68.2
23
18.3
109.5
85.6
23.1
10.2
118.9
Total
28.5
Substrate:
Product:
Cyclofarnesine S
Cyclofarnesine S
(6)
(6)
231
4.2
15.4
15.6
16.8
and cyclofarnesoic acid R and three of its five methylhexanoids
(Barrero et al., 2011), seem to be absent from Phycomyces.
Heterocyclic apocarotenoids, such as cyclofarnesine U (7, 8), the
second most abundant cyclofarnesoid of Phycomyces, are a novelty,
except for a very minor compound of Blakeslea (Barrero et al.,
2012).
3.2. Bioconversion of cyclofarnesoids by mutants that do not
synthesize them
The white carB mutants, defective in the four dehydrogenations
that are needed to convert phytoene to lycopene, converted exter-
nally supplied b-apo-12-carotenol (3) to the 4-dehydrocyclofar-
nesines S (4) and T (5) and cyclofarnesine S (6) (Table 2). This
indicates that the enzymes for apocarotenoid synthesis involved
in these reactions are constitutive, and not induced by the presence
of b-carotene. The bioconversion yield in mated mutant cultures
was the same as in single cultures. This indicates that the mated
mutants did not have the pheromones needed to interact sexually,
nor formed them from the precursor supplied.
The bioconversions support that b-apo-12-carotenol (3) is actu-
ally an intermediate in the biosynthesis of cyclofarnesoids from the
initial fragment, b-apo-12-carotenal, whose bioconversions are dif-
ficult to test because of its instability. The three early reactions for
cyclofarnesoid biosynthesis, reduction of C-11 and oxidation of C-2
and C-4, must be very effective, since no intermediates have been
found, and are likely to be carried out in any order, as the early
reactions for the production of the trisporoids (Sahadevan et al.,
2013).
The failure to find (7), (8), (10) and (11) among the products of
b-apo-12-carotenol (3) does not prove the absence of the corre-
sponding activities in the white mutants because only small
amounts of these compounds were found in three-days cultures
of the wild type and the bioconversion experiments were limited
to a single day.
Fig. 5. Biosynthetic pathway of the cyclofarnesoids in Phycomyces. Straight arrows,
enzymatic reactions known or very likely; dashed arrows, possible spontaneous
reactions; in boxes, compounds identified in this work; in dashed box, hypothetical
intermediary used in bioconversions.
3.3. Biosynthesis of cyclofarnesoids
The proposed pathway (Fig. 5) is based on structural compar-
isons of the cyclofarnesoids, the time course of their appearance,
the results of bioconversions, and parsimony in the total number
of reactions involved. All the known cyclofarnesoids (Fig. 1) have
a hydroxyl group at C-11, the end of the polyene chain. Subfamilies
R, S, and T are oxidized in the ring at C-4 and are distinguished by
other oxidations.
The absence of the R subfamily in Phycomyces (Fig. 1) leaves
4-dihydrocyclofarnesine S (4) as the common precursor of all its
cyclofarnesoids. This compound, here described for the first time,
is the most abundant in young cultures. As it dwindles with time,
all the other cyclofarnesoids increase and cyclofarnesine S (6)
becomes the most abundant. We have not found any oxidation of
C-12 in Phycomyces, although this occurs in Blakeslea (Barrero
et al., 2011; Sutter and Whitaker, 1981).
The 4-dihydrocyclofarnesines S (4) and T (5) are inherently
unstable because their hydroxyl group on C-4, secondary and
allylic, is prone to nucleophilic displacement reactions (Fig. 6). In
fact, the conversion of 4-dihydrocyclofarnesine S (4) to the isomers
(10, 11) was proposed (Polaino et al., 2012) and used as the
last step in the synthesis of these compounds (González-Delgado
et al., 2013). In a similar way, 4-dihydrocyclofarnesine T (5)
could produce the heterocyclic cyclofarnesine
U (7, 8) by
Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto-
chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013

6
E. Alcalde et al. / Phytochemistry xxx (2016) xxx–xxx
2008). They include genes carB and carRA for carotene synthesis
(Almeida and Cerdá-Olmedo, 2008) and carS and acaA for b-caro-
tene cleavage (Medina et al., 2011). Sexual activation of transcrip-
tion thus offers the basis for the increased amounts of
apocarotenoids reported here, for the increased synthetic activity
in cell-free extracts (Salgado et al., 1991), and for the increased car-
otene content (Ciegler, 1965; Kuzina and Cerdá-Olmedo, 2007;
Murillo and Cerdá-Olmedo, 1976). Strains of opposite sex of differ-
ent species of the Mucorales, including very distant ones, exhibit
incomplete sexual reactions (Blakeslee, 1904; Blakeslee and
Cartledge, 1927). This suggests that the complex sexual process
is initiated by the same or similar sex-specific pheromones and
that its progression requires additional species-specific factors.
The cyclofarnesoids and the methylhexanoids offer excellent can-
didates for this role. The enormous increase in cyclofarnesine S
(6) in the mature mated cultures could be due to a specific regula-
tion for its synthesis and may suggest a distinct role for this com-
pound in the sexual process.
Fig. 6. (Above) Conversion of 4-dihydrocyclofarnesine S (4) to the apocarotenoids
(10, 11) by a nucleophilic attack of water on C-10 with loss of the hydroxyl group on
C-4 via a SN20 mechanism. (Below) Heterocyclization of 4-hydroxycyclofarnesine T
(5) to cyclofarnesine U (7, 8) with concomitant hydroxyl elimination at the ring
following a SN20 mechanism.
Carotenes have various roles in Phycomyces and other fungi
(Avalos and Limón, 2015). They and the apocarotenoids derived
from them could protect against free radicals created by radiation
and by oxygen poisoning, but no such effect could be detected in
careful comparisons of various car mutants and the wild type
(Cerdá-Olmedo et al., 1996; Martín-Rojas et al., 1996).
heterocyclization. Both reactions are supported by the rapid
decrease of the dihydrocyclofarnesine substrates as the proposed
products increase (Table 2). The stereoisomers of cyclofarnesine
U (7, 8) will result from the attack of an OH group on either prochi-
ral face of the olefin; their presence in similar amounts argues
against an enzyme catalysis. The conversion of 4-dihydrocyclofar-
nesine S may be helped by an enzyme.
Whatever functions they may have, the apocarotenoids are
much more abundant than b-carotene in Phycomyces (Polaino
et al., 2010, Supplementary information; Alcalde, 2014). In ‘‘pulse
and chase experiments” (Murillo et al., 1981; Bejarano and
Cerdá-Olmedo, 1992), most of the radioactivity incorporated into
b-carotene in Phycomyces cultures grown in the presence of
radioactive mevalonate remained in the same compound upon fur-
ther incubation in non-radioactive medium. The apparent conclu-
sion was that most of the b-carotene was not metabolized
further. The current conclusion would be that most of the b-caro-
tene extracted from mycelia is inert material transferred to lipid
globules and other inert cellular structures. A small amount serves
as pathway intermediate at any given moment and sustains the
main flow of the pathway to the large amounts of apocarotenoids
that are exported to the culture medium.
3.4. Biosynthesis of methylhexanoids
The two methylhexanoids of Phycomyces (1, 2) differ from the
original C7 fragment of b-carotene (Fig. 1) in the reduction of one
end and the oxidation of the other, and from each other in the posi-
tion of the methyl group. Blakeslea produces the same regioisomers
in the same proportion of ca. 2:1, but contains three additional
methylhexanoids; the most abundant one has hydroxyl groups at
both ends and the others have hydroxyl and aldehyde ends, again
as 5-methyl and 2-methyl isomers (Barrero et al., 2011).
The biosynthesis of these methylhexanoid requires two activi-
ties, oxidation and reduction, carried out on two substrates, the
asymmetric ends of the original C7 fragment. This might involve
four enzymes, but an oxidase and a reductase would suffice if each
acts on either end. The two methylhexanoids of Phycomyces would
result from a close association of both activities, be it in a hetero-
dimer or in a single bifunctional enzyme, with a preference for the
substrate with the methyl group close to the reductase. Blakeslea
would have the same association of oxidase and reductase with
the same preference, but would allow the oxidation to occur often
without the reduction.
4. Experimental
4.1. Strains and culture conditions
NRRL1555 is the standard (ꢀ) wild-type strain of P. blakesleea-
nus and A56 is a (+) strain largely isogenic with it (Álvarez and
Eslava, 1983). The white mutant strains C5, carB10 (ꢀ) (Eslava
and Cerdá-Olmedo, 1974), and S342, carB10 nicA101 (+), are unable
to produce b-carotene. The last one is a nicotinic-acid auxotroph
from the crosses [(UBC21 ꢁ C2) ꢁ S102] ꢁ S265, involving the
strains UBC21, wild type (+); C2, carA5 (ꢀ); S102, nicA101 (ꢀ);
and S265, a spontaneous mutant of C5. Strains C2, C5, and S102
are mutants derived from spores of NRRL1555 exposed to
N-methyl-N0-nitro-N-nitrosoguanidine (Eslava et al., 1975;
Meissner and Delbrück, 1968). The infertility of S265 was circum-
vented by the heterokaryon method (Cerdá-Olmedo, 1975). About
7/8 of the genetic background of S342 comes from NRRL1555.
Cultures (Cerdá-Olmedo, 1987; Polaino et al., 2012) were
3.5. Possible functions
We did not find any sex-specific methylhexanoids or cyclofar-
nesoids and we found no positive results when we tested the avail-
able ones for induction of zygophores on single cultures of
Phycomyces of either sex. It is thus very unlikely that these com-
pounds act as sexual pheromones in this fungus.
The two sexes, (+) and (ꢀ), of the Mucorales depend on sex-
specific allelomorphs, DNA sequences with little homology to each
other that occupy the same place in the genome and behave in
crosses as single allelic markers. Each allelomorph contains a single
gene, sexP and sexM, respectively, whose protein products are
putative sex-specific transcription factors (Idnurm et al., 2008;
Camino et al., 2015). The transcripts of hundreds of genes are more
abundant in mated cultures than in single ones (Kuzina et al.,
grown at 22 °C in the dark on minimal agar (glucose, L-asparagine,
thiamine, and mineral salts), enriched with yeast extract (2 g/l).
4.2. Extraction, fractionation, and identification
The agar cultures were frozen overnight at ꢀ80 °C, thawed at
room temperature, and pressed to release an exudate that was
Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto-
chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013

E. Alcalde et al. / Phytochemistry xxx (2016) xxx–xxx
7
filtered through a nitrocellulose filter (Millipore HA, 0.45
lm). The
4.4. Bioconversion assay
volume of the exudate was very close to one half of that of the orig-
inal medium, so that we estimated the amounts in the cultures as
twice the actual measurements. The mycelia did not contain
appreciable apocarotenoids. Neutral and acid extracts of the exu-
dates were subjected to reverse phase HPLC (Polaino et al.,
2012). All manipulations were done under dim light.
The test compound (1 mg) was dissolved in methanol (0.1 ml),
mixed with distilled water (2 ml), and spread on a culture previ-
ously grown for 2 days. After a further 1-day incubation, the cul-
ture was frozen at ꢀ80 °C before extraction and LC analysis.
For LC/MS the samples (5
fractionated in the same way in a liquid chromatographer (Agilent
HPLC 1200), but trifluoroacetic acid (100 l/l) was added to the
ll of extract in methanol, 15 g/l) were
Acknowledgements
l
We appreciate the technical help and discussions of Lola P.
Camino. Fundación Juan Miguel Villar Mir, Junta de Andalucía
(P08-CVI-03901), and the Government of Spain (CTQ2010-16818,
BIO2012-38520, and BIO2012-39716) provided economic support
and did not intervene further.
mobile phase and absorption spectra, 200–900 nm, were obtained
with a set of diodes. Molecular masses were determined with a
coupled mass spectrometer (Bruker Daltonics, maXis HR-QTOF).
The eluting solvent was divided with a static splitter and ionized
with the standard maXis ESI source adjusted to a drying gas flow
of 18 ml/s at 200 °C and a nebulizer pressure of 0.38 MPa. The cap-
illary voltage was 4000 V. Mass spectra of 50–700 m/z were col-
lected in positive mode.
In addition, 4-dihydrocyclofarnesine S (4) was purified by semi-
preparative chromatography and subjected to MS as described
(Polaino et al., 2012), but ethyl acetate (20 g/l) was the solvent
and ethyl acetate–methanol (10:1 v/v) was the mobile phase.
NMR spectra were recorded with an Agilent (Varian) NMR spec-
trometer (1H 600 MHz/13C 150 MHz). Infrared spectra were mea-
sured as thin-films between NaCl plates using a Matson Satellite
FTIR spectrometer. All reactions sensitive to air or water were per-
formed in flame-dried flasks under an argon atmosphere. The sol-
vents used were purified according to standard literature
techniques and stored under an argon atmosphere.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2016.
01.013.
References
Alcalde, E., 2014. Doctoral Thesis, Universidad de Sevilla, Seville.
Almeida, E.R.A., Cerdá-Olmedo, E., 2008. Gene expression in the regulation of
carotene biosynthesis in Phycomyces. Curr. Genet. 53, 129–137.
Álvarez, M.I., Eslava, A.P., 1983. Isogenic strains of Phycomyces blakesleeanus suitable
for genetic analysis. Genetics 105, 873–879.
Arrach, N., Fernández-Martín, R., Cerdá-Olmedo, E., Avalos, J., 2001. A single gene for
lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in
Phycomyces. Proc. Natl. Acad. Sci. U.S.A. 98, 1687–1692.
4-Dihydrocyclofarnesine
S
(4)—tR = 18.9 min. UV: kmax =
Avalos, J., Limón, M.C., 2015. Biological roles of fungal carotenoids. Curr. Genet. 61,
309–324.
247 nm. MS: calcd. for C30H48O6Na 527.3342 [2M+Na]+, found
527.3346. Other peaks were observed at m/z 235.1683
[M+HꢀH2O]+ (calcd.: 235.1692) and m/z 217.1584 [M+Hꢀ2H2O]+
(calcd.: m/z 217.1592).
Austin, D.J., Bu’Lock, J.D., Drake, D., 1970. Biosynthesis of trisporic acids from b-
carotene via retinal and trisporol. Experientia 26, 348–349.
Barrero, A.F., Herrador, M.M., Arteaga, P., Gil, J., González, J.A., Alcalde, E., Cerdá-
Olmedo, E., 2011. New apocarotenoids and Ò-carotene cleavage in Blakeslea
trispora. Org. Biomol. Chem. 9, 7190–7195.
Barrero, A.F., Herrador, M.M., Arteaga, P., González, J.A., Arteaga, J.F., 2012. A minor
dihydropyran apocarotenoid from mated cultures of Blakeslea trispora.
Molecules 17, 12553–12559.
Bejarano, E.R., Cerdá-Olmedo, E., 1992. Independence of the carotene and sterol
pathways of Phycomyces. FEBS Lett. 306, 209–212.
Blakeslee, A.F., 1904. Sexual reproduction in the Mucorineae. Proc. Am. Acad. Arts
Sci. 40, 206–319.
Colorless sirup (5 mg) isolated by semi-preparative HPLC
(tR = 12.2–12.5 min) from the neutral extract (59 mg) of exudate
(1.5 l) of two-days mated culture. IR (film):
m
3409, 2958, 2924,
2870, 1735, 1718, 1645, 1010, 734 cmꢀ1
.
1H NMR (600 MHz,
(CD3)2CO), 1H NMR (600 MHz, (CD3)2CO + D2O), and 13C NMR
(150 MHz, (CD3)2CO): see Table S1. MS: calcd. for C15H24O3Na
[M+Na]+ 275.1623, found 275.1629.
Blakeslee, A.F., Cartledge, J.L., 1927. Sexual dimorphism in Mucorales. II.
Interspecific reactions. Bot. Gaz. (Chicago) 84, 51–57.
Boutagy, J., Thomas, R., 1974. Olefin synthesis with organic phosphonate
carbanions. Chem. Rev. 74, 87–99.
4-Dihydrocyclofarnesine T (5)—tR = 16.2 min. UV: kmax = 248
nm. MS: calcd. for C15H25O4 [M+H]+ 269.1980, found 269.1973.
Other peaks were observed at m/z 251.1629 [M+HꢀH2O]+ (calcd.:
m/z 251.1642) and m/z 233.1531 [M+Hꢀ2H2O]+ (calcd.: m/z
233.1536).
Cyclofarnesine U (8), a previously-unknown stereoisomer—
tR = 19.7 min. UV: kmax = 239 nm. MS: calcd. for C15H23O3
251.1642 [M+H]+, found 251.1615. Other peaks were observed at
m/z 233.1516 [M+HꢀH2O]+ (calcd.: 233.1536) and m/z 268.1888
[M+NH4]+ (calcd.: m/z 268.1907).
Cyclofarnesine T (9)—tR = 16.0 min. UV: kmax = 230 nm, 307
nm. MS: calcd. for C15H23O4 267.1591 [M+H]+, found 267.1584.
Other peaks were observed at m/z 249.1481 [M+HꢀH2O]+
(calcd.: 249.1485) and m/z 231.1372 [M+Hꢀ2H2O]+ (calcd.:
231.1379).
Bu’lock, J.D., Drake, D., Winstanley, D.J., 1972. Specificity and transformations of the
trisporic acid series of fungal sex hormones. Phytochemistry 11, 2011–2018.
Bu’lock, J.D., Jones, B.E., Winskill, N., 1976. The apocarotenoid system of sex
hormones and prohormones in Mucorales. Pure Appl. Chem. 47, 191–202.
Burgeff, H., 1924. Untersuchungen über sexualität und parasitismus bei
Mucorineen. Bot. Abh. 4, 1–135.
Caglioti, L., Cainelli, G., Camerino, B., Mondelli, R., Prieto, A., Quilico, A., Salvatori, T.,
Selva, A., 1966. The structure of trisporic C acid. Tetrahedron Suppl. 7, 175–187.
Cainelli, G., Grasselli, P., Selva, A., 1967. Struttura dell’acido trisporico B. Chim. Ind.
49, 628–629.
Camino, L.P., Idnum, A., Cerdá-Olmedo, E., 2015. Diversity, ecology, and evolution in
Phycomyces. Fungal Biol. 119, 1007–1102.
Cerdá-Olmedo, E., 1975. The genetics of Phycomyces blakesleeanus. Genet. Res. 25,
285–296.
Cerdá-Olmedo, E., 1987. Standard growth conditions and variations. In: Cerdá-
Olmedo, E., Lipson, E.D. (Eds.), Phycomyces. Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York, NY, pp. 337–339.
Cerdá-Olmedo, E., 1989. Production of carotenoids in fungi. In: Vandamme, E.J.
(Ed.), Biotechnology of Vitamins, Pigments and Growth Factors. Elsevier,
London, pp. 27–42.
Cerdá-Olmedo, E., Martín-Rojas, V., Cubero, B., 1996. Causes of cell death following
ultraviolet B and C exposures and the role of carotenes. Photochem. Photobiol.
64, 547–551.
Ciegler, A., 1965. Microbial carotenogenesis. Adv. Appl. Microbiol. 7, 1–34.
Eslava, A.P., Álvarez, M.I., Burke, P.V., Delbrück, M., 1975. Genetic recombination in
sexual crosses of Phycomyces. Genetics 80, 445–462.
Eslava, A.P., Cerdá-Olmedo, E., 1974. Genetic control of phytoene dehydrogenation
in Phycomyces. Plant Sci. Lett. 2, 9–14.
4.3. Synthesis of b-apo-12-carotenol (3)
Our synthesis (Fig. 3 and Supplementary Text S2) was based in
well-known reactions (Boutagy and Thomas, 1974; van Wijk et al.,
2003). The spectroscopic data of the synthetic product and the
intermediates (Supplementary Fig. S3) were consistent with those
previously reported (Barrero et al., 2011; Polaino et al., 2012).
Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto-
chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013

8
E. Alcalde et al. / Phytochemistry xxx (2016) xxx–xxx
González-Delgado, J.A., Arteaga, J.F., Herrador, M.M., Barrero, A.F., 2013. First total
synthesis of (+)-apotrisporin and (+)-apotrientriols A-B: cyclization
approach to apocarotenoids. Org. Biomol. Chem. 11, 5404–5408.
Prisbylla, M.P., Takabe, K., White, J.D., 1979. Stereospecific synthesis of (.+-.)-
trisporol B, a prohormone of Blakeslea trispora, and a facile synthesis of (.+-.)-
trisporic acids. J. Am. Chem. Soc. 101, 762–763.
E
a
Grasselli, P., Selva, A., 1970. Structure of desoxytrisporone, a novel sesquiterpenic
substance from Choanephora trispora. Chim. Ind. 52, 584–585.
Idnurm, A., Walton, F.J., Floyd, A., Heitman, J., 2008. Identification of the sex genes in
an early diverged fungus. Nature 451, 193–196.
Sahadevan, Y., Richter-Fecken, M., Kaerger, K., Voigt, K., Boland, W., 2013. Early and
late trisporoids differentially regulate Ò-carotene production and gene
transcript levels in the mucoralean fungi Blakeslea trispora and Mucor mucedo.
Appl. Environ. Microbiol. 79, 7466–7475.
Iturriaga, E.A., Velayos, A., Eslava, A.P., 2000. The structure and function of the genes
involved in the biosynthesis of carotenoids in the Mucorales. Biotechnol.
Bioprocess Eng. 5, 263–274.
Salgado, L.M., Avalos, J., Bejarano, E.R., Cerdá-Olmedo, E., 1991. Correlation between
in vivo and in vitro carotenogenesis in Phycomyces. Phytochemistry 30, 2587–
2591.
Kuzina, V., Cerdá-Olmedo, E., 2007. Ubiquinone and carotene production in the
Mucorales Blakeslea and Phycomyces. Appl. Microbiol. Biotechnol. 76, 991–999.
Kuzina, V., Ramírez-Medina, H., Visser, H., van Ooyen, A.J.J., Cerdá-Olmedo, E., van
den Berg, J.A., 2008. Genes involved in carotene synthesis and mating in
Blakeslea trispora. In: Curr. Genet. 54, 143–152.
Martín-Rojas, V., Gómez-Puerto, A., Cerdá-Olmedo, E., 1996. Lack of protection by
carotenes against gamma-radiation damage in Phycomyces. Radiat. Environ.
Biophys. 35, 193–197.
Medina, H.R., 2013. Doctoral Thesis, Universidad de Sevilla, Seville.
Medina, H.R., Cerdá-Olmedo, E., Al-Babili, S., 2011. Cleavage oxygenases for the
biosynthesis of trisporoids and other apocarotenoids in Phycomyces. Mol.
Microbiol. 82, 199–208.
Schimek, C., Wöstemeyer, J., 2009. Carotene derivatives in sexual communication of
zygomycete fungi. Phytochemistry 70, 1867–1875.
Sutter, R.P., 1975. Mutations affecting sexual development in Phycomyces
blakesleeanus. Proc. Natl. Acad. Sci. U.S.A. 72, 127–130.
Sutter, R.P., 1986. Apotrisporin E: a new sesquiterpenoid isolated from Phycomyces
blakesleeanus and Blakeslea trispora. Exp. Mycol. 10, 256–258.
Sutter, R.P., 1987. Sexual development. In: Cerdá-Olmedo, E., Lipson, E.D. (Eds.),
Phycomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 317–
336.
Sutter, R.P., Dadok, J., Bothner-By, A.A., Smith, R.R., Mishra, P.K., 1989. Cultures of
separated mating types of Blakeslea trispora make D and E forms of trisporic
acids. Biochemistry 28, 4060–4066.
Meissner, G., Delbrück, M., 1968. Carotenes and retinal in Phycomyces mutants.
Plant Physiol. 43, 1279–1283.
Sutter, R.P., Whitaker, J.P., 1981. Sex pheromone metabolism in Blakeslea trispora.
Naturwiss 68, 147–148.
Miller, M.L., Sutter, R.P., 1984. Methyl trisporate E. A sex pheromone in Phycomyces
blakesleeanus. J. Biol. Chem. 259, 6420–6422.
Sutter, R.P., Zawodny, P.D., 1984. Apotrisporin: a major metabolite of Blakeslea
trispora. Exp. Mycol. 8, 89–92.
Murillo, F.J., Calderón, I.L., López-Díaz, I., Cerdá-Olmedo, E., 1978. Carotene-
superproducing strains of Phycomyces. Appl. Environ. Microbiol. 36, 639–642.
Murillo, F.J., Cerdá-Olmedo, E., 1976. Regulation of carotene synthesis in
Phycomyces. Mol. Gen. Genet. 148, 19–24.
Tagua, V.G., Medina, H.R., Martín-Domínguez, R., Eslava, A.P., Corrochano, L.M.,
Cerdá-Olmedo, E., Idnurm, A., 2012. A gene for carotene cleavage required for
pheromone biosynthesis and carotene regulation in the fungus Phycomyces
blakesleeanus. Fungal Genet. Biol. 49, 398–404.
Murillo, F.J., Torres-Martínez, S., Aragón, C.M.G., Cerdá-Olmedo, E., 1981. Substrate
transfer in carotene biosynthesis in Phycomyces. Eur. J. Biochem. 119,
511–516.
Torres-Martínez, S., Murillo, F.J., Cerdá-Olmedo, E., 1980. Genetics of lycopene
cyclization and substrate transfer in b-carotene biosynthesis in Phycomyces.
Genet. Res. 36, 299–309.
Polaino, S., González-Delgado, J.A., Arteaga, P., Herrador, M.M., Barrero, A.F., Cerdá-
Olmedo, E., 2012. Apocarotenoids in the sexual interaction of Phycomyces
blakesleeanus. Org. Biomol. Chem. 10, 3002–3009.
van den Ende, H., 1979. Sexual morphogenesis in the Phycomycetes. In: Smith, J.E.,
Berry, D.R. (Eds.), The Filamentous Fungi, vol. 3. John Wiley, New York, NY, pp.
256–274.
Polaino, S., Herrador, M.M., Cerdá-Olmedo, E., Barrero, A.F., 2010. Splitting of b-
carotene in the sexual interaction of Phycomyces. Org. Biomol. Chem. 8, 4229–
4231.
van Wijk, A.A.C., van de Weerd, M.B., Lugtenburg, J., 2003. Synthetic scheme for the
preparation of 13C-labeled 3,4-didehydro-retinal, 3-hydroxyretinal, and
4-hydroxyretinal up to uniform 13C-enrichment. Eur. J. Org. Chem., 863–868
Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto-
chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013