Microbial transformation of ent-kaurenoic acid and its 15-hydroxy derivatives by the SG138 mutant of Gibberella fujikuroi

Article abstract of DOI:10.1021/np0003239

Feeding experiments with ent-kaurenoic acid (4), 15αc-hydroxy-ent-kaurenoic acid (5), 15β-hydroxy-ent-kaurenoic acid (6), and mixtures of 4 plus 5 and 4 plus 6 were conducted using the SG138 mutant of Gibberella fujikuroi, to gain information about the phenotype of this unique strain. The biotransformation of 5 gave 7β15α-dihydroxykaurenolide (9) and 7β,15α-dihydroxy-ent-kaurenoic acid (13). The incubation of 6 produced 7β, 15β-dihydroxy-ent-kaurenoic acid (7) and 7β,15β-dihydroxykaurenolide (10). No 15-hydroxylated gibberellins were detected in any of these experiments. The results indicated that a hydroxy group at C-15 does not inhibit 7β-hydroxylase activity but in the SG138 strain obstructs the enzymatic ring-B contraction of ent-kaurenoids to gibberellins. Exogenous 4 stimulated both the excretion of ent-kaurene and the fungal metabolism of 5 and 6.

Full text of DOI:10.1021/np0003239

222
J . Nat. Prod. 2001, 64, 222-225
Micr obia l Tr a n sfor m a tion of en t-Ka u r en oic Acid a n d Its 15-Hyd r oxy Der iva tives
by th e SG138 Mu ta n t of Gibber ella fu jiku r oi
Alejandro F. Barrero,*,† J . Enrique Oltra,† Enrique Cerda´-Olmedo,‡ J avier AÄ valos,‡ and J ose´ J usticia†
Departamento de Quı´mica Orga´nica, Instituto de Biotecnologı´a, Facultad de Ciencias, Universidad de Granada,
Granada, Spain, and Departamento de Gene´tica, Universidad de Sevilla, Sevilla, Spain
Received J une 30, 2000
Feeding experiments with ent-kaurenoic acid (4), 15R-hydroxy-ent-kaurenoic acid (5), 15â-hydroxy-ent-
kaurenoic acid (6), and mixtures of 4 plus 5 and 4 plus 6 were conducted using the SG138 mutant of
Gibberella fujikuroi, to gain information about the phenotype of this unique strain. The biotransformation
of 5 gave 7â,15R-dihydroxykaurenolide (9) and 7â,15R-dihydroxy-ent-kaurenoic acid (13). The incubation
of 6 produced 7â,15â-dihydroxy-ent-kaurenoic acid (7) and 7â,15â-dihydroxykaurenolide (10). No
15-hydroxylated gibberellins were detected in any of these experiments. The results indicated that a
hydroxy group at C-15 does not inhibit 7â-hydroxylase activity but in the SG138 strain obstructs the
enzymatic ring-B contraction of ent-kaurenoids to gibberellins. Exogenous 4 stimulated both the excretion
of ent-kaurene and the fungal metabolism of 5 and 6.
Gibberellins (GAs) are terpenoid phytohormones which
play an important role in the regulation of plant growth
and development. Apart from plants some fungi also
produce these terpenoids, particularly Gibberella fujikuroi
(Hypocreale). GAs have many applications in agriculture
and the brewing industry.1 Therefore, their abundant
production by some strains of G. fujikuroi has led to the
study of their biosynthesis by this fungus in considerable
detail over the last three decades.2 Some aspects of the
biogenesis of the gibberellins via ent-kaurene (1), ent-
kaurenol (2), ent-kaurenal (3), and ent-kaurenoic acid (4)
are fully described,2 but many of the biochemical and
physiological aspects of fungal gibberellin biosynthesis are
still poorly understood. As part of our research into the
biotechnology of G. fujikuroi we have investigated the
metabolites produced by the GA-producer IMI58289 strain.3,4
Subsequently, the kaurenoids and GAs synthesized by
several gib-mutants developed from the IMI58289 wild-
type strain were also analyzed.5 The results of these
analyses and preliminary feeding experiments6 suggested
to us that one of these mutants (SG138) was blocked in
the oxidative transformations from ent-kaurene (1) to ent-
kaurenoic acid (4), as well as in 3â-hydroxylation, 13â-
hydroxylation, and the loss of C-20 by the GAs. These
unusual features prompted us to make further feeding
experiments with strain SG138 to gain more information
about its unusual phenotype. We describe here the results
of incubations with ent-kaurenoic acid (4), the “unnatural”
substrates 15R-hydroxy-ent-kaurenoic acid (5) and 15â-
hydroxy-ent-kaurenoic acid (6), and mixtures of 4 plus 5
and 4 plus 6. Throughout these experiments it was
observed that ent-kaurenoic acid (4) substantially stimu-
lated the metabolism and/or excretion of 1, 5, and 6.
The results of incubations using the SG138 mutant of
G. fujikuroi, both with and without ent-kaurenoic acid (4),
are summarized in Table 1. All compounds included in this
table were identified by means of GC-MS. Several sub-
stances could be isolated, and their structures were con-
firmed by NMR techniques (see the Experimental Section
for more details). A number of blank experiments (with no
exogenous substrate) were made and always gave similar
results; their arithmetical means are included in Table 1.
In the usual culture conditions for GA production, minimal
medium with no nitrogen source (blank, Table 1), the
mutant SG138 accumulated a high proportion of ent-
kaurene (1) in the mycelium. The low quantities of fujenoic
acid (11), GA15 (15), and GA24 (16) found in the culture
medium may be ascribed to the leaky blockage in the
metabolic steps from 1 to 4 of this mutant. When the
fungus was fed with ent-kaurenoic acid (4), this compound
was taken up into the mycelium, and the proportions of
11, 15, and 16 excreted were much higher. Kaurenolide
(8) was also formed, but neither the C19 gibberellins nor
the C-3 or C-13 hydroxylated GAs were detected, confirm-
ing the multiple metabolic blockage of the SG138 mutant.
Moreover, ent-kaurenoic acid (4) stimulated the excretion
of ent-kaurene (1) into the medium. This observation
prompted us to add ent-kaurenoic acid (4) to the incuba-
tions in combination with other kaurenoids in order to
obtain information about its effect on the metabolism and
excretion of these compounds.
The results of the feeding experiments with grandifloric
acid (5) and with a mixture of 5 plus 4 are included in
Table 1. When the fungus was fed with 5 in the absence
of exogenous 4, low proportions of the dihydroxylated
metabolites 9 and 13 were synthesized by the mutant. The
previously undescribed acid 13 was tentatively identified
(GC-MS analysis) on the basis of the mass spectrum of
its methyl ester bis-trimethylsilyl ether, closely related to
that reported for the corresponding derivative of 7â,15â-
dihydroxy-ent-kaurenoic acid.7 When G. fujikuroi mutant
SG138 was fed with 5 in the presence of exogenous 4,
enhanced amounts of 9 and 13 were obtained, suggesting
that ent-kaurenoic acid stimulated the metabolism of 5.
Additionally, acid 13 could be isolated as its methyl ester
13a (the culture broth extract was methylated before
chromatography). Its HRMS showed a C21H32O4 molecular
1
formula. In the H NMR spectrum several signals (H-13,
H-17a, H-17b, H-18, and H-20) closely related to those of
5 were observed (see Experimental Section). Moreover,
apart from the signal of a methoxy group (3.63 ppm), those
of two hydroxylated methines appeared at 4.09 and 3.94
* To whom correspondence should be addressed. Tel and Fax: 34-958
24 33 18. E-mail: afbarre@goliat.ugr.es.
† Universidad de Granada.
‡ Universidad de Sevilla.
10.1021/np0003239 CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/05/2001

Notes
J ournal of Natural Products, 2001, Vol. 64, No. 2 223
Ta ble 1. Kaurenoids (1-11, 13) and GAs (15, 16) Found after the Feeding Experimentsa
blank
4b 5b 4 + 5b
6b
4 + 6b
Mc
Bd
Mc
Bd
Mc
Bd
Mc
Bd
Mc
Bd
9.2
trace
0.2
Mc
9.4
Bd
1
2
3
4
5
46.4
3.8
8.9
62.6
0.9
1.2
9.3
trace
45.6
19.7
30.0
12.0
1.1
3.5
3.5
10.2
0.9
trace
27.7
trace
4.0
70.0
trace
trace
24.0
0.7
2.0
6
7
98.7
4.4
4.5
1.9
52.0
8
9
5.5
7.3
3.7
26.8
10
11
13
15
16
0.7
6.7
1.9
trace
1.8
trace
trace
6.7
48.0
3.3
7.9
1.3
3.9
20.6
18.0
trace
trace
1.7
1.5
a
b
d
Proportions are in mg/L of culture broth. Exogenous substrate. c M: Products found in the mycelium. B: Products found in the
culture broth.
Ch a r t 1. Chemical Structures of Compounds 1-16
able substrate specificity of the enzymatic system respon-
sible for the kaurenoid ring B contraction by the SG138
mutant was already observed when feeding experiments
with 3â-hydroxy-ent-kaurenoic acid were performed in our
laboratory.6 This specificity can be attributed to a lack of
enzymatic recognition and not to transport difficulties,
because 13 was synthesized in situ by the fungus itself.
Another explanation, based on an enzyme deactivation
derived from the mutagenesis process, seems less probable
if the transformation of 4 into GA15 (15) and GA24 (16)
carried out by the mutant (Table 1) is borne in mind.
The results of incubations with 15â-hydroxy-ent-kaur-
16-en-19-oic acid (6), and with 6 plus 4, are summarized
in Table 1. With no exogenous 4, incubation with 6 gave
low proportions of the dihydroxylated metabolites 7 and
10. 15â-Hydroxy derivatives with a GA skeleton were not
detected, confirming that a 15-hydroxy group inhibits the
kaurenoid ring B contraction in the G. fujikuroi mutant
SG138. Subsequently, when SG138 was fed with 6 plus 4,
the peaks derived from 7 in the GC-MS chromatogram of
the culture broth rose sharply, confirming the stimulating
effect of 4 on the 7â-hydroxylation of the “unnatural” 15-
hydroxy-ent-kaurenoic acids. This chromatogram also
showed a considerable increase in the peak corresponding
to the kaurenolide 10. It is known that 7â-hydroxy-ent-
kaurenolide (8) is biosynthesized from ent-kaurenoic acid
(4) via ent-kaur-6,16-dienoic acid.9 Thus, the enhanced
proportions of kaurenolides 9 and 10 (Table 1) suggest that
exogenous 4 may also stimulate the 6,7-dehydrogenation
processes of 5 and 6. Finally, it is interesting to note that,
while diol 7 was found (besides low proportions of 15â-
hydroxy gibberellins) in previously reported biotransfor-
mations of 6,10,11 diol 13 was not detected in previous
incubations of 5 with the ACC917 wild-type strain8 and
with the mutant B1-41a12 of G. fujikuroi.
In this work and in a previous publication6 we have seen
that the G. fujikuroi mutant SG138 is very capable of
taking kaurenoids from the culture broth, transforming
them, and returning the transformed products to the
culture medium. This capacity makes the SG138 strain a
valuable biological tool for feeding experiments. This kind
of experiment with “unnatural” kaurenoid and gibberellin
analogues has proved to be a useful device for studying the
substrate specificity of G. fujikuroi enzymes.2,13 Most of
these experiments were made under conditions lacking ent-
kaurenoic acid biogenesis, either with a specific mutant
(such as B1-41a) or a wild-type strain in the presence of a
specific inhibitor (such as AMO-1618). In many cases the
quantities of biotransformed products obtained were very
ppm. The broad singlet at 4.09 ppm was attributed to H-15,
which is on an allylic position, while the signal at 3.94 ppm
(t, J ) 3 Hz) was assigned to H-7. Its multiplicity and
coupling constant indicated that H-7 was in an R-equatorial
position, and thus the â-axial arrangement of the C-7
hydroxy group was established. The 13C NMR spectrum
confirmed the structure methyl 7â,15R-dihydroxy-ent-kaur-
16-en-19-oate (13a ). Biotransformation products from 5
with a GA skeleton were not detected in any of these
experiments. These results tend to confirm that a 15R-
hydroxyl group does not inhibit ent-kaurenoic acid hy-
droxylase activity, as previously suggested by Fraga et al.8
On the other hand, the lack of 15-oxygenated GAs indicates
that a 15R-hydroxy group obstructs kaurenoid ring B
contraction in the G. fujikuroi SG138 mutant. The notice-

224 J ournal of Natural Products, 2001, Vol. 64, No. 2
Notes
(see above). The extracts were then chromatographed on a Si
gel column eluting with solvent mixtures of increasing polarity.
Compounds 1, 4-7, 9, 12, and GA15 (15), together with the
methyl ester derivative 13a of the natural acid 13, were
isolated. Thus, the identification of 1,25,26 7,11,17 9,8 12,27,28 and
1529 could be confirmed by NMR analysis. The chemical
structure of the hitherto unknown compound 13 was estab-
lished in the same way.
Meth yl 7â,15r-Dih yd r oxy-en t-k a u r -16-en -19-oa te (13a ):
oil; [R]25D -2.8° (c 0.38, MeOH); IR (film) νmax 3426 (OH), 1723
(CO) cm-1; 1H NMR (CDCl3, 300 MHz) δ 5.21 (1H, br s, H-17a),
5.09 (1H, br s, H-17b), 4.09 (1H, br s, H-15), 3.94 (1H, t, J )
3 Hz, H-7), 3.63 (3H, s, OMe), 2.78 (1H, br s, H-13), 1.24 (3H,
s, H-18), 0.81 (3H, s, H-20); 13C NMR (CDCl3, 100 MHz) δ 170.0
(s, C-19), 159.8 (s, C-16), 108.7 (t, C-17), 81.1 (d, H-15), 72.7
(d, C-7), 57.5 (q, OMe), 48.0 (d, C-9), 47.3 (d, C-5), 43.5 (s, C-8),
42.9 (d, C-13), 41.9 (s, C-4), 40.4 (t, C-1), 39.2 (s, C-10), 38.0
(t, C-3), 35.3 (t, C-14), 33.0 (t, C-12), 28.6 (q, C-18), 28.2 (t,
C-6), 19.2 (t, C-2), 17.8 (t, C-11), 15.3 (q, C-20); HRFABMS
m/z 371.2197 (calcd for C21H32O4Na, 371.2198); EIMS (TMS-
ether) m/z 420 [M]+ (2), 405 (5), 345 (13), 330 (77), 315 (35),
297 (28), 270 (42), 253 (62), 213 (10), 197 (17), 173 (14), 121
(42), 91 (53), 73 (100); (bis-TMS-ether) m/z 492 [M]+ (9), 402
(48), 387 (7), 343 (4), 312 (4), 297 (5), 253 (14), 221 (7), 181
(10), 156 (13), 147 (25), 91 (16), 73 (100).
low, allowing only a tentative identification on the basis
of GC-MS analysis. Our findings concerning the stimulat-
ing effect of 4 could be useful in future feeding experiments
of the SG138 mutant with “unnatural” substrates, allowing
an enhanced production of biotransformation compounds.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. These procedures
were described previously.14
F u n ga l Ma ter ia l. The development of the G. fujikuroi
SG138 mutant from the IMI58289 wild-type strain has been
reported elsewhere.5,15 The fungus was stored on Saboureau
agar slants at 4 °C.
en t-Ka u r en oic Acid (4) a n d Its 15-Hyd r oxy Der iva tives
(5 a n d 6). ent-Kaur-16-en-19-oic acid (4) was generously
donated to us by Prof. M. Grande, Department of Organic
Chemistry, University of Salamanca, Spain. Its spectroscopic
properties, including optical rotation, matched those re-
ported.16-18 15R-Hydroxy-ent-kaur-16-en-19-oic acid (5) was
isolated from Helichrysum foetidum,19 with [R]D -119.8° (c
25
1.32, CHCl3); its 1H and 13C NMR spectra matched those
described elsewhere.17,20 15â-Hydroxy-ent-kaur-16-en-19-oic
acid (6) was obtained in the following manner. Methyl 15â-
hydroxy-ent-kaur-16-en-19-oate (500 mg), donated to us by
Prof. M. Grande, was treated with sodium propanethiolate
(1.44 g) in DMF (27 mL). The mixture was stirred for 45 h at
50 °C under an inert atmosphere. Water (50 mL) and 2 N HCl
were added to attain pH 2. The mixture was then extracted
with t-BuOMe, with the organic layer dried over anhydrous
Na2SO4 and the solvent removed. Flash chromatography21
(hexane/t-BuOMe, 7:3) of the residue yielded 323 mg of 6 in
the form of white needles, mp 211-212 °C, [R]D25 -89° (c 0.77,
Qu a n tita tive An a lysis. Proportions of compounds 1, 4-6,
9, and 13, as stated in Table 1, were determined on the basis
of the peak area of the GC-MS chromatograms, with the aid
of calibration curves worked out using solutions of a known
concentration. Proportions of the remaining compounds in
Table 1 were determined directly from the peak area of the
chromatograms.
1
CHCl3) (lit.22 mp 204-206 °C, [R]D25 -95°). Its H and 13C NMR
Ack n ow led gm en t. This research was financed by the
Spanish CICYT and the European Community (FEDER pro-
gramme), project 1FD97-1346-C02-01. We thank Professor
Manuel Grande for providing us samples of ent-kaurenoic acid
and methyl 15â-hydroxy-ent-kaurenoate and our colleague Ms.
A. Tate for her comments and revision of our English text.
spectra matched those reported elsewhere.17,22
In cu ba tion P r oced u r e. Fragments of mycelium taken
from the stored agar slants were transferred to Petri dishes
containing sporulation agar23 and incubated at 28 °C for 6
days. Suspensions (1 mL) of mycelial fragments and spores,
obtained by washing the surface of each agar plate with 15
mL of sterile distilled water, were used to inoculate 500 mL
Erlenmeyer flasks containing 50 mL of minimal liquid me-
dium24 with 50% of the nitrogen source (2.4 g/L NH4NO3). The
flasks were incubated in an orbital shaker at 200 rpm at 28
°C for 4 days. Identical flasks with 50 mL of fresh minimal
medium with a 50% nitrogen source were inoculated with 1
mL from the previously mentioned flasks and incubated at 200
rpm at 28 °C for 4 further days. The mycelia were then filtered
off and resuspended in a minimal liquid medium24 without
NH4NO3 (50 mL per 500 mL flask). Immediately afterward, 8
mg of an exogenous substrate, 4, 5, or 6 alone, or the 4 plus 5
(8 mg of each) or 4 plus 6, was added per flask. In all
experiments two flasks were incubated without substrate
addition as a control for endogenous metabolites (blank). The
biotransformations were monitored using thin-layer chroma-
tography, taking daily samples of the culture broth. When the
exogenous substrate spot was no longer visible, the incubation
was stopped (usually after 4 or 5 days of incubation).
Extr a ction a n d Id en tifica tion P r oced u r es. At the end
of each incubation the mycelia were separated from the
medium by vacuum filtration, lyophilized, ground with the aid
of glass beads, and extracted with EtOAc. The culture broth
was also extracted with EtOAc. Aliquots from both extracts
were treated with ethereal CH2N2 and either bis(trimethylsi-
lyl)trifluoroacetamide (BSTFA) and pyridine or SIGMA-SIL-A
(SIGMA) and were analyzed by GC-MS under the following
conditions: He as the carrier gas; flow rate of 0.6 mL/min;
temperature from 120 to 220 °C increasing at a rate of 5 °C/
min, from 220 to 280 °C increasing at 3 °C/min, and kept at
280 °C for 10 min; injector temperature 260 °C; detector
temperature 280 °C. In this way, compounds 1-12, GA15 (15),
GA24 (16), and palmitic, stearic, oleic, and linoleic acids were
identified initially on the basis of the mass spectra of their
derivatives (methyl esters and/or TMS-ethers).7 The identifica-
tion of compounds 4-6 was confirmed using our own standards
Su p p or tin g In for m a tion Ava ila ble: Chromatograms of culture
broth extracts in feeding experiments with 6 and 6 plus 4. This
information is available free of charge via the Internet at http://
pubs.acs.org.
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NP0003239