From the
The Ascomycete Gibberella fujikuroi synthesizes
gibberellins, fujenal, carotenoids, and other terpenoids. Twelve gib mutants, isolated through the modified gibberellin
fluorescence of their culture media, were subjected to chemical and
biochemical analyses. Two mutants were specifically defective in the
hydroxylation of carbon 13; their total gibberellin production was
normal, but their main gibberellin was GA
The gibberellins are terpenoid hormones that regulate plant
growth and development (1, 2) and have found many
applications in agriculture and
brewing(3, 4, 5) . They are present at low
concentrations in plant tissues and are obtained industrially from the
culture media of the fungus Gibberella fujikuroi ssp. fujikuroi(6, 7) .
Gibberella synthesizes about 20 different gibberellins(8, 9) ,
of which the most abundant is gibberellic acid (GA
Compounds were identified by comparison of mass spectra with
those previously described (17) and by using our own
standards(18) . The amounts of the different gibberellins were
calculated by reference to known amounts of GA
The alkaline extracts were dissolved in
a mixture of pyridine and N,O-bis-(trimethylsilyl)-trifluoroacetamide (10 µl each
per mg of extract), heated at 100 °C for 30 min in a closed vial,
and analyzed by gas chromatography and mass spectrometry. The
quantitative results were deduced from the peak area given by the
detector and the total weight of the extracts.
Mycelia were washed,
dried, ground in a mortar, and extracted with ethyl acetate for 24 h.
The extracts (containing abundant triglycerides) were saponified with 2 M KOH in methanol (10 µl per mg of extract) for 12 h,
diluted with water, extracted with tert-butylmethylether and
analyzed as the alkaline extracts above.
Mycelial pigments were
extracted with acetone; the dried extracts were partitioned three times
between methanol/water (30:1) and petroleum ether (b.p. 40-60
°C). The bikaverins were retained in the hypophase. The dried
petroleum ether extract was redissolved in n-hexane for
spectrophotometry. The total amounts of colored carotenoids
(predominantly neurosporaxanthin) were estimated using an absorbance
coefficient (1-cm optical path) of 2000 for a 1% (w/v) solution. The
absence of phytoene in strain SG127 was confirmed by
chromatography(19) .
In Vitro Terpenoid Biosynthesis-Washed mycelia from
500-ml cultures grown for 8 days in 1-liter flasks were lyophilized,
extracted, and incubated as described(20, 21) , except
that the buffer included 20% (v/v) glycerol and the cofactor mixture
did not contain NADH or NADPH. Incubation mixtures (0.5 ml) contained
1.2-2.0 mg of protein and 18,500 Bq of sodium
3R-[2-
The mutants used in this study showed the growth pattern of
the wild type and had no special growth requirements. The slower growth
of three mutants in comparison with the wild type () may be
due, not to the gib mutations, but to other mutations
introduced into the same genome by the high dose of mutagen.
Four mutants (SG139, SG121, SG138, and SG136) produced no
gibberellins, fujenal, or kaurenolides, or only very small amounts,
indicating a virtually complete block in a step before kaurenoic acid,
the last common precursor of the absent isoprenoids. As expected,
therefore, negligible kaurenoic acid (less than 0.05 mg/liter) was
detected in these mutants, while wild type cultures produced 1.3
mg/liter.
Strains SG139 and SG121 produced very little kaurene (less
than 1 mg/liter in 14-day cultures), in contrast to the wild type level
of 22 mg/liter, of which 70% was in the mycelium. The defect was
confirmed by the low level of kaurene biosynthesis from labeled
mevalonate in vitro (Fig. 4). SG139 was the tightest of
the gib mutants, as it contained no detectable gibberellins,
fujenal, or kaurenolides.
The partial blocks found in six
strains (SG128, SG129, SG124, SG127, SG122, and SG135) led to
gibberellin and fujenal contents one-fifth to one-third of those of the
wild type, each compound being reduced to about the same extent. An
exception to this was the GA
The main defect in strains SG128 and SG129 was probably the
conversion of kaurenoic acid to 7-hydroxykaurenoic acid, since they
produced more kaurenolides than would be expected from the overall
leakiness of their mutations (). Strains SG124 and SG127
contained early blocks in the pathway, since they did not produce
kaurene.
Strain SG127 produced no carotenoids, whether in the light
or dark, in liquid media or on agar. Other strains deficient in both
gibberellins and carotenoids could not be obtained. A total of 29 new
white mutants were derived from strain SG22, but they produced
approximately normal amounts of gibberellins, as shown by the
fluorescence assay(14) .
The gibberellin-containing culture
media were red in color, due to the presence of bikaverin, as judged
from the solubility of the pigment and its absorption
spectrum(22, 23) . The red color was absent in strain
SG121 and less pronounced in strain SG127 than in the wild type.
This report represents the most exhaustive description of
gibberellin mutants of Gibberella to date. The gibberellins
produced by 12 mutants have been analyzed and compared with those
produced by the wild type. The results confirm the usefulness of the
fluorescence method (14) for the isolation of mutants with
quantitative and qualitative changes in gibberellin production.
Gibberellins do not seem to carry out any important function in the
life of Gibberella as a saprophyte, in contrast to higher
plants, where the characteristic phenotypic changes associated with
decreased gibberellin contents (e.g. dwarfism) reflect the
physiological role of these hormones(24) .
One pair of
mutants (SG123 and SG133), defective in the hydroxylation of carbon 13
of the gibberellin molecule, are similar to two natural Gibberella strains(25, 26) . The phenotype of these mutants
suggests that the same 13-hydroxylase acts on different substrates,
such as GA
Other mutants (SG139 and
SG121) are defective in kaurene synthesis. Since they accumulate normal
carotenoid concentrations, they must produce geranylgeranyl
pyrophosphate, and are therefore presumably defective in kaurene
synthetase, which catalyzes in two steps the four cyclizations needed
to convert geranylgeranyl pyrophosphate into kaurene(27) .
Alternatively, they could be regulatory mutants, unable to carry out
all the reactions in the pathway.
The oxidations of kaurene to
kaurenoic acid (28) and the subsequent hydroxylation to
7-hydroxykaurenoic acid (29) are catalyzed by monooxygenases
dependent on cytochrome P-450. The mutant B1-41a (10) made
gibberellic acid and other compounds when incubated with kaurenoic
acid, but not when incubated with earlier intermediates(11) .
Strain SG138 is also deficient in the oxidation of kaurene, but
different from B1-41a. The structure of the minor gibberellins in
SG138 implies additional defects in the hydroxylation at carbon 3 and
in the loss of carbon 20. This suggests that a common gene product
participates in three seemingly different biochemical modifications.
Under this interpretation, strain SG138 would not be expected to
produce gibberellic acid from kaurenoic acid.
Strain SG136 is a
clean mutant which produces very small amounts of gibberellins very
late in the growth cycle. There are several possible explanations for
the contradiction between the absence of kaurene in this strain in
vivo and its ability to produce it in vitro (Fig. 4). For example, the mutant may be unable to target
its kaurene synthetase to the correct subcellular compartment or the
enzyme may be subject to an inhibition in vivo that is lost in vitro.
The six strains with partial early blocks in the
gibberellin pathway do not lend themselves to detailed biochemical
analyses. Two of them, SG128 and SG129, seem to be defective in the
hydroxylation of kaurenoic acid at carbon 7. Their existence confirms
that such a hydroxylation is not required for kaurenolide biosynthesis,
which begins with the introduction of a double bond between carbons 6
and 7(30, 31) .
The white, carotenoid-less phenotype
of SG127 could be due to a partial loss of a specific prenyl
transferase that converts farnesyl pyrophosphate to geranylgeranyl
pyrophosphate, as it occurs in the al-3 mutants of Neurospora crassa(32) . We failed to isolate such
mutants by screening white Gibberella mutants for gibberellin
production. The alternative hypothesis, that SG127 is a rare double
mutant, was not confirmed because of the lack of suitable strains to
cross with our Gibberella strain.
Mutants exhibiting very
small decreases in gibberellin production are easily obtained (33, 34, 35) and many are probably unspecific.
The competition for a common substrate (geranylgeranyl pyrophosphate)
explains why mutants with increased or decreased carotenoid content
exhibit a small variation of opposite sign in gibberellin
content(14) . In addition, the reduced biosynthesis of
gibberellins in mutants isolated for resistance to
pefurazoate(36) , an inhibitor of sterol biosynthesis, suggests
that some gene products may act on both the gibberellin and the sterol
pathways.
Most of the chemical modifications needed to produce
gibberellins are not specifically blocked in any of the gib mutants, and none is blocked in more than two mutants. This
indicates that the present collection is incomplete and that repeated
application of the fluorescence test should yield new mutant types.
Some steps may be refractory to mutational analysis for various
reasons, including a similar fluorescence of the accumulated
gibberellins in the mutants and the wild type.
The results in this
paper support the existence of ``gene-saving devices'' in the
development and diversification of secondary metabolism(37) .
Foremost is the versatility of enzymes to carry out similar reactions
on different substrates, so that the same enzyme may be used repeatedly
for successive reactions and different enzymes may act in any order
along the pathway. Some reactions, particularly those yielding minor
side products, may occur spontaneously during the week-long
incubations.
Mycelial dry weight (g/liter), gibberellins, fujenal,
and carotenoids (mg/liter) after 8 (upper value) and 14 (lower value)
days growth in low-nitrogen minimal broth. Values shown are the average
of two independent experiments.
Kaurenolides (mg/liter) in culture media after
14-days growth in low-nitrogen minimal broth. Values shown are the
average of two independent experiments Kde; ent-6
R. F.-M. thanks Prof. John R. Bowyer (Department of
Biochemistry, Royal Holloway and Bedford New College) for his
hospitality.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
instead of
GA
. Four mutants were blocked in the early reactions
between geranylgeranyl pyrophosphate and 7-hydroxykaurenoic acid; two
of them could not synthesize kaurene and another one was blocked in
several oxidative steps. Six mutants had partial defects in early
reactions, leading to the production of one-fifth to one-third of the
wild type amounts of gibberellins and fujenal. Two of these produced
considerable amounts of kaurenolides due to a defect in the conversion
of kaurenoic acid to 7-hydroxykaurenoic acid. Another one produced no
carotenoids, but attempts to isolate mutants of reactions shared by the
carotenoid and gibberellin pathways failed. The gib mutations
did not modify the ability of the fungus to live as a saprophyte.
) as well
as other related metabolites, such as fujenal and the kaurenolides. The
biosynthetic pathway (Fig. 1) has been deduced from the
determination of chemical structures, the use of labeled precursors,
and the analysis of biotransformations(10, 11) . A major
role in this work was played by a mutant called B1-41a, which is
unable to synthesize kaurenoic acid,
(
)but can
convert it to gibberellins. This mutant was isolated after testing
50,000 survivors of ultraviolet exposure in a bioassay with barley
seeds.
Figure 1:
Main steps in the biosynthesis of
gibberellins in G. fujikuroi. Dotted arrows represent
two or more reactions.
Ease of growth in the laboratory, availability of active
cell-free systems for biochemical analysis, and suitability for the
isolation of recessive mutations make Gibberella an attractive
organism for the investigation of gibberellin
biosynthesis(12, 13) . A simplified method to detect
changes in gibberellin production led to the isolation of 14 gib mutants from about 4,000 colonies tested(14) . We have now
characterized these mutants to gain information on the biosynthetic
pathway.
Strains
The wild type IMI58289 of G.
fujikuroi, ssp. fujikuroi came from the Commonwealth
Mycological Institute, Kew, United Kingdom. The 12 gib mutants
defective in gibberellin biosynthesis (14) listed in and the carotenoid-superproducing mutant SG22 (15) were derived from IMI58289 after exposure of its spores to N-methyl-N`-nitro-N-nitrosoguanidine (12).
The same mutagenesis procedure was used to obtain white mutants of
SG22. For chemical and biochemical analyses, low-nitrogen (0.48 g/liter
NHNO
) minimal liquid medium (16) was
inoculated with spores grown on sporulation agar (13) and
incubated in the dark at 30 °C in an orbital shaker (150 rpm).
Analysis of Gibberellins and Related
Compounds
Aliquots (21 ml) of culture media from 200-ml
cultures, grown in 500-ml Erlenmeyer flasks, were separated from the
mycelia by filtration and brought to pH 8 with NaOH. They were
extracted three times with ethyl acetate, brought to pH 2 with HCl, and
extracted three more times with ethyl acetate. The dried acid extracts
were methylated with diazomethane, mixed with 40 µl of
tetrahydrofuran and 40 µl of N,O-bis-(trimethylsilyl)-trifluoroacetamide and heated at 60
°C for 30 min to form methyltrimethylsilyl derivatives for gas
chromatography (Hewlett-Packard 5890A; capillary column HP-1 of
cross-linked methylsilicone gum, 25 m 0.2 mm
0.33-µm film thickness; programmed temperature increase from 120 to
220 °C at 5 °C/min and from 220 to 280 °C at 3 °C/min;
injector temperature 260 °C; flame ionization detector at 290
°C; carrier gas N
at 25 ml/min). The gas chromatograph
was coupled to a Hewlett-Packard 5988A mass spectrometer operating at
70 eV.
subjected to
the same procedures. The amounts of GA
and GA
include those of their isomers marked with * and iso (Fig. 2), formed during the extraction procedure. The
gibberellins and fujenal (isolated in its diacid form) were identified
as follows: GA
(retention time 29.57 min): 330
(M
, 14), 298(71) , 270(69) ,
243(57) , 227(54) , 226(60) ; GA
(Rt 31.40): 430 (M
, 6), 370 (16),
311(51) , 281(71) , 221(100) ; GA
(Rt
32.69): 404 (M
, 0), 372(17) , 312(60) ,
284(100) , 253(9) , 225(80) ; GA
(Rt
32.80): 374 (M
, 3), 314(84) , 286 (62),
285(40) , 226(100) , 225(95) ; iso-GA
(Rt 33.84): 416 (M
, 11),
384(27) , 356(65) , 223(72) , 222(100); GA
(Rt 34.02): 418 (M
, 15), 283 (35),
284(100) , 225(91) , 224(81) ; GA
(Rt
34.54): 416 (M
10), 384(50) , 356(70) ,
223(63) , 222(100) ; Fujenal diacid (Rt 34.89): 376
(M
, 0.4), 345(3) , 344(2) , 316 (5),
227(37) , 195(86) , 167(64) , 107(100) ;
GA
(Rt 35.01): 518 (M
, 3), 418
(15), 399(9) , 369(19) , 309(11) ,
257(32) , 227(27) ; GA
(Rt 36.06): 492
(M
, 2), 477(5) , 436(9) ,
400(21) , 310 (33), 282(25) ; iso-GA
(Rt 36.92): 504 (M
, 100), 489(12) ,
370(23) , 347(18) , 208(27) ; GA
(Rt
37.30): 506 (M
, 19), 416(19) , 390 (100),
357(37) , 360(31) , 340(26) , 300(39) ;
GA
(Rt 37.54): 506 (M
, 100),
491(10) , 448(18) , 377(22) , 313 (17); GA
(Rt 38.05): 504 (M
, 100), 489(9) ,
370(11) , 347(2) , 208(57) .
Figure 2:
GA and GA
isomers
formed during the extraction procedure.
The high peaks at m/e 73 in the spectra of GA, GA
,
GA
, iso-GA
, GA
, iso-GA
, and GA
were not included when
calculating ion abundances.
C]mevalonate (2.0 TBq/mol),
prepared from the lactone purchased from Amersham (Bucks, United
Kingdom). After stopping the reaction with methanol, the mixture was
extracted three times with petroleum ether. The radioactivity in
aliquots of the extract was used to estimate total terpenoid
biosynthesis. The rest of the extract was reduced to about 0.1 ml for
chromatography on Silica Gel G thin layers (20
5 cm) developed
with 15% toluene in petroleum ether. The kaurene band (R
0.7) was scraped off and
radioassayed(21) . The results are expressed as geometrical
means and their standard errors in two independent experiments, with
two determinations in each (four independent experiments in the case of
the wild type).
The Gibberellin Pathway
The wild type and the mutants
differed in the production of gibberellins, fujenal, kaurenolides, and
their precursors (Fig. 3, Tables I and II). The acid extracts of
the wild type contained considerable amounts of gibberellic acid
(GA) and fujenal, and smaller amounts of various compounds,
among which GA
, GA
, and GA
were
prominent. The alkaline extracts contained kaurenolides and kaurene,
but negligible quantities of gibberellins.
Figure 3:
Analysis of acid extracts of G.
fujikuroi culture media. Gas chromatograms of the wild type and
two gib mutants grown for 8 days in low-nitrogen minimal
medium.
Strain SG123 accumulated
GA at the expense of GA
and showed a drastic
decrease in GA
. The ratio of GA
to GA
in 14-day-old cultures was 17 times larger in this mutant than in
the wild type, although both strains were similar in their overall
production of gibberellins and in their content of other terpenoids.
Therefore, the effect of the mutation in strain SG123 was an impairment
of the ability to hydroxylate the carbon at position 13 (Fig. 1).
The same phenotype, quantitatively less marked, was found in strain
SG133.
Figure 4:
In vitro kaurene biosynthesis.
Incorporation of 3R-[2-C]mevalonic acid
into terpenoids (radioactivity in petroleum ether extracts) and kaurene
(relative to incorporation into terpenoids) in crude extracts of the
wild type and several gib mutants.
Strain SG138 made kaurene in vitro and in vivo (13 mg/liter, of which 55% was in the
mycelium), indicating a defect in the oxidation of kaurene to kaurenoic
acid. This strain was slightly leaky and produced small amounts of
GA and GA
. The gibberellins hydroxylated at
carbon 3 and those with 19 carbons, which predominate in the wild type,
were completely absent. A similar strain, SG136, synthesized kaurene in vitro (Fig. 4).
content of strains SG129,
SG128, and SG124, which was closer to that of the wild type, thus
leading to higher GA
/GA
ratios than in the
latter.
Other Metabolites
With the exception of strain
SG127, the carotenoid contents of the mutants were very similar to that
of the wild type. The variations (about double in SG122, about half in
SG121, and smaller variations in others), are possibly indirect
effects.
and GA
.
Table: Terpenoid production in the wild type and
the mutants
Table: Kaurenolide production in the wild type
and the mutants
,
7
-dihydroxykaur-16-en-19-oic acid 19,6
lactone(7
-hydroxykaurenolide).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.