From the
Westfälische
Wilhelms-Universität Münster, Institut für Botanik,
Schlo
garten 3, D-48149 Münster, Germany,
¶Laboratorio de Bioorgánica, Departamento
de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653,
Santiago, Chile, and ||Rothamsted Research at Long
Ashton, Long Ashton, Bristol BS41 9AF, United Kingdom
Received for publication, February 24, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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GAs are diterpenoids and are synthesized in G. fujikuroi via the mevalonate pathway (1). Most of the genes of the early isoprenoid pathway have been cloned from G. fujikuroi, viz 3-hydroxy-3-methylglutaryl-CoA reductase (2), farnesyl diphosphate synthase (3), and two geranylgeranyl diphosphate (GGPP) synthase genes, one of which (ggs1) is involved in general isoprenoid biosynthesis (4) and a second (ggs2) of which is specific for GA biosynthesis (5). Biosynthesis of the major metabolite GA3 (gibberellic acid) from GGPP requires 13 steps (Fig. 1). GGPP is converted to ent-kaurene via ent-copalyldiphosphate in a two-step cyclization reaction (6). ent-Kaurene is metabolized to GAs in G. fujikuroi by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenases, whereas in plants, P450 monooxygenases and 2-oxoglutarate-dependent dioxygenases are involved (7). In contrast to plants in which cyclization of GGPP is catalyzed by two enzymes, ent-copalyl diphosphate synthase and ent-kaurene synthase, in the fungi G. fujikuroi and Phaeosphaeria, both steps are catalyzed by a bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase enzyme (810).
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Recently, six genes of the GA-biosynthetic pathway in G. fujikuroi comprising the GA-specific GGPP synthase (ggs2), ent-kaurene synthase (cps/ks), and four cytochrome P450 monooxygenase genes (P4501 to P4504) were shown to be closely linked in a gene cluster (5, 11). P4504 encodes ent-kaurene oxidase, catalyzing the three oxidation steps between ent-kaurene and ent-kaurenoic acid (Fig. 1) (12), while P4501 encodes a highly multifunctional monooxygenase, which catalyzes four steps involving oxidation at two carbon atoms, in the main pathway from ent-kaurenoic acid to GA14 via GA12-aldehyde as well as producing kaurenolides and fujenoic acids as byproducts (Fig. 1) (13). P4502 was shown recently to encode a GA 20-oxidase, which converts GA14 to GA4 by removal of C-20 (Fig. 1) (14).
In this paper, we characterize the remaining two genes of the cluster and demonstrate that they are responsible for the last two steps in the biosynthesis of GA3. We describe the isolation and functional characterization of the seventh gene, orf3, which is located at the left border of the cluster. By gene knock-out and expression in the absence of the other GA-biosynthetic genes, we show that orf3 encodes the desaturase that converts GA4 to GA7. Furthermore, we demonstrate that the fourth P450 monooxygenase gene, P4503, encodes the 13-hydroxylase that converts GA7 to the end product, GA3. The identification of the last two GA-biosynthetic genes and the production of single or double deletion mutants have enabled us to construct strains producing GA7, GA1 or the commercially important GA4. Expression studies with the desaturase and P4503 genes in combination with biochemical analysis revealed significant differences in their regulation by nitrogen.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and PlasmidsEscherichia coli strain Top10 (Invitrogen) was used for plasmid propagation. Vector pUC19 was used to clone DNA fragments carrying the G. fujikuroi orf3 gene or parts of it. The plasmid porf3-Sal carries a 3.4-kb fragment with the entire gene. For inactivation of orf3, an internal BamHI/XbaI fragment of plasmid porf3-Sal, was replaced by the hygromycin resistance cassette from pGPC1 (17). For inactivation of P4503, three different gene disruption vectors, pP4503-GD1, pP4503-GD2, pP4503-GD3, were constructed by cloning internal fragments of the gene into the vectors pUCH28 (18) or pAN71 (19), both of which carry the hygromycin B resistance cassette. The internal fragments of P4503 were first amplified by PCR, cloned into the PCR cloning vector pPCR2.1 (Invitrogen), cut with KpnI/XhoI (pP4503-GD2 and pP4503-GD3), and then cloned into pUCH28. For pP4503-GD1, the fragment was cut with HindIII/XbaI and cloned into pAN71. For gene expression of orf3 in the GA-deficient mutant SG139, the 3.4-kb SalI fragment from plasmid porf3-Sal was cloned into pUCH28, resulting in plasmid porf3-GC. For expression of P4503 in SG139, a 2.3-kb XbaI fragment with the entire gene was cloned into pAN71, resulting in the vector p4503-GC. In addition, the cosmid clone, cos5 carrying the entire P4503 gene and 40 kb to the right of P4503 was used for complementation of SG139. The Uni-Zap XR vector of the orf3 cDNA clones allowed in vivo excision and recircularization of cloned inserts to form a phagemid in pBluescript SK() carrying the insert. This subcloning step was performed according the manufacturer's protocol (Stratagene, La Jolla, CA).
PCRConditions were as described previously (14). The primers for amplifying the internal fragments of P4503 were as follows: pP4503-GD1 (P4503-F1, 5'-TGGAGCATGAAGACAAGTTTCAGG-3', and P4503-R1, 5'-CTTTGGTATCCAGCCGAGCCG-3'); pP4503-GD2 (P4503-F2, 5'-TAGCGGATCAGACGGAAGGGG-3', and P4503-R1, 5'-CTTTGGTATCCAGCCGAGCCG-3'); and pP4503-GD3 (P4503-F1, 5'-TGGAGCATGAAGACAAGTTTCAGG-3', and P4503-R2, 5'-GCATGCATGCTTCCATGG-3'). The mutant allele of P4503 from strain 6314 was amplified with the forward primer P4503-MF in combination with the reverse primers P4503-MR and P4503/1 as follows: P4503-MF (5'-CCTTCTTGGCTGGATCACG-3'); P4503-MR (5'-AGCCTCCATCAGTATTCTGG-3'); and P4503/1 (5'-CTAGCGGATCAGACGGAAGGGC-3'). To identify the transformants of SG139 in which orf3 and P4503 had been integrated correctly, the following PCR-primers were used: orf3-F1 (5'-ATCGTGGCTCTAACAAACTTCGCG-3'); orf-R1 (5'-TCTCACTTCCTCCTTCTCAGTTCC-3'); P4503-F3 (5'-AACCGACCTTGCCATACCCTG-3'); and P4503-R3 (5'-ATCACTCTAGATGCTGTGCGC-3'). All of the PCR primers were synthesized by MWG-Biotech (München, Germany).
Southern and Northern Blot AnalysisDNA and RNA isolation and Southern and Northern analyses were as described previously (14). The G. fujikuroi small subunit of rDNA was used as a control for RNA transfer.
Screening of G. fujikuroi cDNA and Genomic EMBL3
Libraries The expression library (UniZapTMXR vector,
Stratagene) was constructed from RNA isolated from mycelium grown under
optimal conditions for GA3 formation
(4). Approximately 30,000
recombinant phages were plated at
7,500 plaques/150-mm diameter Petri
dishes and transferred to nylon membranes. For screening of the genomic
library (20),
40,000
recombinant phages were plated and transferred to membranes. The hybridization
was performed at high stringency (65 °C), and the blots were washed at the
same temperature in 2x SSC, 0.1% SDS, followed by 0.1x SSC, 0,1%
SDS. Positive recombinant clones were used for a second round of plaque
purification.
Sequence AnalysisDNA sequencing of recombinant plasmid clones was accomplished by the dideoxy chain termination method (21) using an automatic sequencer "LI-COR 4000" (MWG-Biotech, München, Germany).
Transformation of G. fujikuroiPreparation of protoplasts
and transformations were carried out as described previously
(20). 107
protoplasts (100 µl) of the strain IMI 58289 or the mutant 6314 were
transformed with 10 µgofthe SalI-fragment from the gene
replacement vector pORF3-GR or one of the circular disruption vectors
pP4503-GD1, pP4503-GD2, and pP4503-GD3. Transformed
protoplasts were regenerated at 28 °C in a complete regeneration agar (0.7
M sucrose, 0.05% yeast extract, 0.1%
(NH4)2SO4 containing 120 µg/ml hygromycin
B (Calbiochem)) for 67 days. Single conidial cultures were established
from hygromycin B-resistant transformants and used for DNA isolation and
Southern blot analysis. For expression of orf3 and P450-3 in
the GA-deficient mutant SG139, 107 protoplasts were transformed
with 10 µg of the complementation vectors porf3-GC, pP4503-GC, or
the cosmid clone GA-cos5, carrying the entire P4503 gene and
an 40-kb genomic region to the right of P4503 probably
not involved in GA biosynthesis.
Gibberellin AnalysisFor analysis of GA formation, the wild-type strain IMI 58289 and orf3-disrupted mutants were cultivated in 100-ml Erlenmeyer flasks containing 100 ml of OPM medium. The cultures were incubated for 710 days on a rotary shaker (200 rpm) at 28 °C. GA3,GA1,GA4, and GA7 were analyzed by HPLC according to Barendse et al. (22) using a Merck HPLC system with a UV detector and a LiChrospher 100 RP-18 column (5-µm particle size; 250 x 4 mm; Merck Eurolab GmbH, Darmstadt, Germany). GA3, GA4, and GA7 were also analyzed by thin layer chromatography using a mixture of ethyl acetate/chloroform/acetic acid (60:40:5). GC-MS analysis of total culture filtrates or HPLC-purified fractions was performed on derivatized samples (11) using a Trio2A mass spectrometer (Micromass, Manchester, United Kingdom) connected to an Agilent 5890 gas chromatograph. Samples in hexane (1 µl) were injected splitless into a 25 m x 0.2 mm x 0.25 µm (film thickness) BPX-5 WCOT column (Scientific Glass Engineering) at 50 °C. After 2 min, the oven temperature was increased at 13 °C·min1 to 180 °C and then at 3 °C·min1 to 300 °C. The He inlet pressure was 90 kPa, and the injector, transfer line, and MS source temperatures were 280, 280, and 200 °C, respectively. Spectra were obtained at 70 eV, scanning at 2 Hz from 75045 atomic mass unit. GAs were identified by comparison of their mass spectra with published data (23).
Incubations with Isotopically Labeled
Substrates[17-14C1]GA4 was
obtained from [17-14C1]GA9 using a
recombinant GA 3-hydroxylase from Arabidopsis thaliana
(24), and
[1,7,12,18-14C4]GA4 (5.77 TBq
mol1) was obtained from
[14C4]GA14 by incubation with cultures of
SG139-P4502 transformants of G. fujikuroi
(14).
[14C]4GA7 (6.22 TBq
mol1) was obtained from
[14C4]GA12-aldehyde by incubation with
cultures of the 6314 mutant of G. fujikuroi.
[14C4]GA9 (5.51 TBq
mol1) was prepared from
[14C4]GA12 by incubation with cultures of
SG139-P4502 transformants
(14). For incubations in high
nitrogen conditions, pre-cultivation was carried out for 3 days in 100% ICI
medium (16) followed by
transfer to fresh 100% ICI medium buffered at pH 3.0 after harvest and washing
of the mycelia with the same solution. For low nitrogen conditions,
pre-cultivation in 40% ICI medium (3 days) was followed by transfer to 0% ICI
medium buffered at pH 3.0. The substrates
[14C4]GA4 (3,364 Bq) or
[14C4]GA7 (2,523 Bq) were applied to the
P4502 disruption mutants in both high and low nitrogen
conditions and incubated for an additional 2 days at 28 °C. Products were
extracted from the culture fluid as already described
(13) and analyzed by
reversed-phase HPLC on a C18 column (Symmetry, Waters) with a
linear gradient of 60100% MeOH/H2O, pH 3.0, over 30 min at 1
ml/min. Radioactivity was measured in aliquots of the eluate by liquid
scintillation counting, and labeled products were further analyzed by GC-MS as
described above. Incubations of SG139 orf-3 transformants with
[14C4]GA9 or
[14C1]GA4 as well as incubations of SG139
P4503 transformants with
[14C4]GA7 or
[14C4]GA4 were carried out under low nitrogen
conditions in cultures buffered at pH 3.0.
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RESULTS |
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Functional Analysis of orf3A gene replacement vector pORF3-GR was obtained by replacing the central BamHI/XbaI fragment of plasmid porf3-Sal carrying the 3.4-kb SalI fragment of orf3 (Fig. 2B) with the hygromycin B resistance cassette from the vector pGPC1 (17). The wild-type strain IMI58289 was transformed with the 4.3-kb SalI-fragment from pORF3-GR. TLC and HPLC analysis revealed that 5 of 42 transformants (T18, T21, T23, T55, and T60) were not able to produce GA3, the final product of the GA-biosynthetic pathway in G. fujikuroi, or its immediate precursor, GA7. Southern blot analysis confirmed that the transformants have lost the 6.3-kb PstI wild-type band because of homologous integration of the replacement cassette into the orf3 locus (data not shown). GC-MS analysis of culture filtrates of transformant T23 and wild-type indicated that GA1 rather than GA3 was the major C19-GA in the orf3-deficient line (Fig. 3). Fujenoic acid and GA13 (Fig. 1) were major metabolites in both lines. Three of the transformants were shown by HPLC analysis to contain GA1 and GA4 in ratios from 3:1 to 5:1 after cultivation in the synthetic 20% ICI medium or in OPM for 10 days (Table I). Since the mutants produced no GA3 or GA7 and accumulated GA4 and GA1, the mutation appears to block the 1,2-desaturation of GA4 to GA7 (Fig. 1), indicating that orf3 encodes GA4 desaturase.
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To confirm the function of the orf3 expression product, we
transformed the deletion mutant SG139, which has lost the entire GA gene
cluster, with the gene complementation vector porf3-GC carrying the wild-type
orf3 gene. From 20 analyzed transformants, 12 were shown by PCR to
contain a 2.1-kb fragment expected after correct integration of the gene into
the SG139 genome. Analysis of five transformants by Northern hybridization
confirmed that the gene was expressed in strain SG139 despite the loss of the
entire GA gene cluster (data not shown). Four strains SG139-T1, SG139-T2,
SG139-T4, and -T5, which expressed orf3 at levels comparable with the
wild-type, were incubated with the radiolabeled substrates
[14C]GA4 (3-hydroxylated) or
[14C]GA9 (non-hydroxylated) in medium buffered at pH
3.0. GC-MS analysis of the HPLC-purified product from incubation of
[14C]GA4 with transformants T1, T2, T4, and T5
identified [14C]GA7 as sole product (shown for T1 in
Table II), whereas SG139 did
not metabolize this substrate (data not shown). Thus, the desaturase gene is
expressed as an active enzyme in SG139. In addition, the orf3
transformants efficiently utilized the non-hydroxylated substrate
[14C]GA9, producing two more polar fractions of
radioactivity on HPLC in 93% yield. The more polar of these (65%) was found by
GC-MS to contain [14C]GA40
(2
-hydroxyGA9), whereas the less polar fraction (28%)
contained GA120 (1,2-didehydroGA9)
(Table II and
Fig. 4) and three isomers of a
diene dicarboxylic acid derivative assumed to be formed from GA120
by rearrangement during work-up and derivatization. The enzyme will thus
accept both 3
-hydroxylated and non-hydroxylated substrates, although in
the latter case, there is an apparent loss of regiospecificity.
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Functional Analysis of P4503To determine the
function of the remaining gene of the cluster, P4503, the
wild-type strain IMI58289 was transformed with the gene disruption vector
pP4503-GD1, which contained the hygromycin-resistant gene, hph
(Fig. 2C). After
analysis of 100 hygromycin-resistant transformants by TLC, all were found to
produce high amounts of GAs after 7 days of cultivation in OPM but three
lines, T52, T59, and T61, did not produce GA3, although its
precursors, GA4 and GA7, were present. To be sure that
this change in GA profile was the result of P4503 gene
inactivation, we constructed two additional disruption vectors
pP4503-GD2 and pP4503-GD3. In pP4503-GD3, a mutation in
the heme-binding domain was introduced via PCR
(Fig. 2C, primer
P4503-R3). We analyzed 100 hygromycin-resistant transformants
for both vectors by TLC. One of the transformants from pP4503-GD2 (T92)
and three of the transformants from pP4503-GD3 (T11, T49, and T55)
produced GA7 as final product instead of GA3, which was
confirmed by HPLC. GC-MS analysis of the culture fluid from transformant
P4503-T55 confirmed that GA7 was the major
product and that no GA3 was produced
(Fig. 3). Southern blot
analysis (data not shown) revealed that transformants with all of the three
vectors had lost the 4.3-kb EcoRI wild-type band for
P4503 and produced smaller hybridizing bands. Therefore, the
loss of the ability to produce GA3 in these transformants is due to
the disruption of gene P4503, suggesting that it encodes the
13-hydroxylase catalyzing the conversion of GA7 to GA3.
Furthermore, the transformants produced no GA1, another
13-hydroxylated GA, which is produced in small amounts (
1.3% total GAs)
in the wild-type. Thus, P4503 is responsible for 13-hydroxylation of
both GA7 and GA4
(Fig. 4).
To confirm the function of P4503, complementation of SG139 with the complementation vector pP4503-GC and cosmid clone cos5, both carrying the entire gene P4503, was performed. However, Northern analysis indicated that none of the resulting transformants expressed the gene despite the correct integration of the gene into the SG139 genome (data not shown). Furthermore, none of the transformants from both complementation vectors were able to metabolize [14C]GA7 or [14C]GA4 to radiolabeled GA3 and GA1, respectively. Several intermediates of the GA-biosynthetic pathway were tested as possible inductors of P4503 expression by adding them to cultures of the P4503 transformants at 350500 µM. We found that ent-kaurenoic acid, GA14, GA4, or GA7 were ineffective in inducing GA 13-hydroxylase activity in the SG139-P4503 transformants, which remained unable to convert [14C]GA7 or [14C]GA4 under these conditions. The lack of P4503 expression in the SG139 + P4503 transformants is in contrast with the efficient expression of P4501 (13), P4502 (14), P4504 (12), and des in SG139. Interestingly, complementation of SG139 with a cosmid carrying the entire gene cluster (cos1) fully restored 13-hydroxylase activity.
Characterization of the 13-Hydroxylase Mutant 6314 The
identification of P4503 as the 13-hydroxylase that catalyzes the
conversion of GA7 to GA3 allowed us to classify the
GA7-overproducing mutant 6314 as a putative P4503
mutant. This strain was isolated after mutagenesis of the highly
GA3-producing wild-type strain m567 and screening for
GA-overproducing
mutants.2 To identify
the nature of the mutation, the coding region of the P4503
gene from the mutant was amplified by PCR using the primer pairs
P4503-MF and P4503-MR as well as P4503-MF and
P4503/1. Nine independent clones were sequenced from both strands. A
point mutation was found at position 844, resulting in an amino acid
substitution from arginine to tryptophan at position 221
(Fig. 5). This substitution
results in a dramatic change in the GA product spectrum, indicating greatly
reduced 13-hydroxylase activity, whereas the wild-type strain m567 produced
84% GA3, 12% GA4 and
14%
GA7. As determined by HPLC, the mutant 6314 produced 3%
GA3, 3% GA4, and 94% GA7 under the same
culture conditions (Fig. 6).
GA1, which was found in small amounts in strain m567, was not
detectable in the mutant. Complementation of mutant 6314 with the
P4503 gene restored GA3 synthesis as demonstrated
by GC-MS analysis of the culture fluid (data not shown), thus confirming that
the mutation was in the 13-hydroxylase. Interestingly, the
P4503 gene was expressed efficiently in 6314, which contains
all of the GA-biosynthesis genes, in contrast to its lack of expression in
SG139 in which the gene cluster is missing.
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Creation of a des/P4503 Double
MutantWild-type strains of G. fujikuroi produce mainly
GA3, whereas certain horticultural applications require
specifically GA4
(27). Therefore, strains that
produce GA4 in the absence of GA3 and GA7
would have considerable utility. To this end, we prepared double mutants
lacking both desaturase and 13-hydroxylase activities by transforming the
mutant strain 6314 with the SalI fragment of vector pORF3-GR. HPLC
analysis of 35 hygromycin B-resistant colonies by HPLC revealed that three,
6314ORF3-T1, 6314
ORF3-T2, and 6314
ORF3-T8, had lost their
ability to produce GA7 and instead accumulated large amounts of
GA4 (Fig. 6 and
Table I). In contrast to the
des mutants isolated from the wild-type IMI58289, they do not produce
detectable amounts of GA1, confirming that P4503 catalyzes
the 13-hydroxylation of GA4 as well as GA7. Thus, the
targeted mutation of des, P4503, or both allows the creation
of strains producing high amounts of GA1, GA7, or
GA4, respectively.
Expression of P4503 Is Not Repressed by NitrogenAll
genes of the GA-biosynthetic gene cluster analyzed so far are highly expressed
under conditions of nitrogen limitation, whereas only a low level of
transcripts was detected in media with high amounts of ammonium or glutamine
(5,
12,
13). Expression of the newly
isolated gene des showed the same pattern. Transcription of
des was investigated in mycelia grown for 15, 24, 38, 48 and 60 h in
media with a high N (100% ICI) or low N (10% ICI) content
(Fig. 7A). Northern
blot analysis of total RNA revealed a single band of 1.0 kb in 10% ICI
medium, the transcript level increasing in abundance with culture time in a
similar manner to P4504
(Fig. 7A),
cps/ks and ggs2 (data not shown). No transcript was
observed for des or P4504 in mycelia grown in 100%
ICI medium. In contrast to the other six genes of the cluster,
P4503 appears to be expressed more highly in medium with high
N than with low N (Fig. 7A,
C). After 15 h in culture in 10% ICI medium, when some
nitrogen still remains, only very low levels of expression of des,
P4504 (Fig.
7A) and the other four genes (data not shown) can be
detected, whereas P4503 is highly expressed. With depletion of
nitrogen, transcript abundance for des and P4504
increases, whereas P4503 transcript levels decrease
(Fig. 7A). In
addition, transcript abundance for P4503 is not reduced in
areA mutants confirming that AREA does not control expression of
P4503, whereas des expression
(Fig. 7B) and that of
the other genes (data not shown) require the active nitrogen regulator, AREA.
The pH of the culture medium in the range 3.5 to 8.0 does not affect the
expression of des, P4503
(Fig. 7C) or any of
the other GA-biosynthetic genes (data not shown).
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To confirm that the differential regulation of des and
P4503 expression can be observed at the biochemical level, we
investigated metabolism of [14C]GA4 or
[14C]GA7 in P4502 disruption mutants.
These mutants are blocked for the oxidative removal of C-20 from
GA14 and are therefore not able to produce GA4,
GA7, GA1 and GA3
(Fig. 1)
(14). The experiments with the
gene-disruption mutants P4502-T35 and T39, demonstrated clear
differential inhibition of the desaturase and 13-hydroxylase activities by N.
Under high N conditions (100% ICI), [14C]GA4 was mainly
converted to [14C]GA1, together with a small amount of
[14C]GA3 (Table
III), while incubations in ICI medium with no N gave almost
complete conversion of [14C]GA4 to
[14C]GA3 (Table
III). On the other hand, [14C]GA7 was
converted efficiently to [14C]GA3 in both high and low N
conditions, thus confirming that the 13-hydroxylase shows no significant
inhibition by high N concentration. In contrast, the above results demonstrate
that the desaturase is strongly inhibited by N, in agreement with the
expression analysis on the des and P4503 genes
described above (Fig.
7A). The product distribution obtained from
[14C]GA4 under high N conditions, is consistent with the
spectrum of GAs found in des disruption mutants, which accumulate
mainly GA1 (Table
I).
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DISCUSSION |
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In plants, oxidation of ring A of the GAs is catalyzed by
2-oxoglutarate-dependent dioxygenases, many of which are multifunctional,
catalyzing desaturation, and hydroxylation reactions
(29). For example, a GA
3-oxidase from Phaseolus vulgaris catalyzes dehydrogenation of
GA20 at C-2,3 and 2-hydroxylation in addition to its major
3
-hydroxylase activity
(30). Furthermore, the same
multifunctional dioxygenase is probably responsible for GA3
synthesis from the 2,3-didehydro intermediate (GA5) by
rearrangement of the double bond from C-2,3 to C-1,2 followed by
3
-hydroxylation (31).
Thus, in this case, desaturation at C-1,2, which results in the loss of the
1
,2
-H atoms (32),
is a side reaction of the main pathway to GA1. In contrast, in
G. fujikuroi direct 1,2-desaturation by loss of the 1
,
2
-H atoms (33) is the
major reaction and is accompanied by 2
-hydroxylation. It is of interest
that immature seeds of Prunus persica contain a number of 3-deoxy
1,2-didehydro GAs including GA120 that are probably formed by
direct 1,2-desaturation (34).
The seeds also contain 1
-hydroxy GAs, which could be formed as
byproducts of the desaturation. Nothing is known regarding the nature of the
putative desaturase/1
-hydroxylase in this case.
We have also shown by gene disruption that P4503, the fourth cytochrome P450 monooxygenase gene in the GA gene cluster, encodes the 13-hydroxylase, which catalyzes the final reaction in the biosynthesis of GA3. The same enzyme catalyzes also the 13-hydroxylation of GA4 to form GA1. However, little GA1 (23% total GA content) is produced in these two wild-type strains of G. fujikuroi, suggesting either that the fungal cultures contain much more GA4 desaturase than 13-hydroxylase activity or that GA4 has a higher affinity for the desaturase than for the 13-hydroxylase. Removal of desaturase activity by disruption of the des gene resulted in approximately a 100-fold accumulation of GA1 but only a 2-fold accumulation of GA4. The mutant strain could thus provide the means to produce large quantities of GA1 for agricultural or experimental purposes. Similarly, the double mutant lacking both desaturase and 13-hydroxylase activities could be used for the production of the commercially important GA4.
There was an unexpected difference in the regulation of the des and P4503 genes. Expression of des is high under conditions of low N that promote GA production but very low in the growth phase when the N content is high. These expression patterns correspond to those of ggs2, cps/ks, P4501, P4502, and P4504, which are all under control of the major nitrogen regulator, AREA (35, 36). In contrast, the expression pattern of P4503 differs from that of the other six GA genes. Northern analysis with mycelia grown in medium with a high or low N content showed that P4503 is expressed under both conditions, and in fact, more transcript was present under high N conditions. Thus, P4503 is the only gene in the GA cluster for which transcript is detectable in 100% ICI medium, which contains 4.8 g·liter1 NH4NO3. These results are in agreement with those from incubations of 14C-labeled GA4 and GA7 with P4502 knock-out mutants, which do not contain these GAs and later metabolites because the conversion of GA14 to GA4 is blocked (14). In cultures growing in low N, both desaturase and 13-hydroxylase activities were present so that GA3 was the major product formed from GA4, whereas in high N, more GA1 was produced than GA3 due to low desaturase activity (Table III). Furthermore, GA7 was converted to GA3 regardless of the N content of the medium. The lack of repression of P4503 expression by N is consistent with the absence of the double GATA sequence elements in its promoter. These elements, which bind the AREA transcriptional regulator (37), are present in the promoters of the other six GA-biosynthesis genes.
In addition to its different regulation by N, P4503 is the only gene in the cluster that cannot be expressed in the deletion mutant SG139, which lacks the gene cluster. In contrast, it was effectively expressed in 6314, which has a point mutation in P4503, as well as in SG139 complemented with cosmid 1 containing the entire GA gene cluster. We have not yet investigated how many of the other genes need to be present and active for P4503 to be expressed but have addressed the possibility that its expression requires induction by a GA-biosynthetic intermediate by applying precursors to SG139-P4503 transformants. However, the presence of ent-kaurenoic acid, GA14,GA4,orGA7, which are the end products of P4504, P4501, P4502, and DES, respectively, did not improve expression. The fusion of the P4503 gene to a strong and/or N-repressible promoter will determine whether or not the low expression of the gene under N-limiting conditions is responsible for the failure to complement P4503 in SG139 or whether other elements of the gene cluster are needed for gene expression and enzyme activity.
The functional characterization of des and P4503 completes the analysis of the GA gene cluster in G. fujikuroi. Disruption of smt (25) and an alcohol dehydrogenase gene2 at the left border of the cluster and of orf1 and orf2 at the right of P45032 did not affect the production of GA3 or its regulation. Thus, the biosynthesis of the final product, GA3, in 13 steps beginning with the formation of GGPP by the pathway-specific geranylgeranyl-diphosphate synthase GGS2 requires only seven enzymes, many of which are multifunctional. In contrast to many other fungal secondary metabolite gene clusters, e.g. the aflatoxin gene cluster in Aspergillus parasiticus (38) and the trichothecene gene cluster in Fusarium sporotrichoides (39), the GA gene cluster does not contain a pathway-specific regulatory gene. We have evidence that, in addition to the general regulator AREA, a regulatory gene that controls expression of all of the seven GA-biosynthesis genes exists and must be located elsewhere in the genome.
We are now able to compare GA biosynthesis in G. fujikuroi with
that in higher plants at the chemical, biochemical, and gene levels. Although
the fungus and plants produce structurally identical GAs and the early
enzymatic steps up to the formation of GA12-aldehyde are similar,
the pathways thereafter differ fundamentally as do the character of enzymes
involved and the regulation of their genes. An important difference is that
the early 3-hydroxylation and 20-oxidation steps in the fungus are
catalyzed by cytochrome P450 monooxygenases, whereas soluble dioxygenases are
responsible for these reactions in higher plants. Another major difference is
that 13-hydroxylation occurs early (at the stage of GA12) in
plants, whereas it is the last step in the fungus. Even in the first part of
the pathways in which cytochrome P450s participate in the fungus and plants,
the equivalent enzymes in G. fujikuroi and plants have low levels of
amino acid identity, e.g. only 10% for ent-kaurene oxidase
of the fungus (P4504) and A. thaliana
(40).
In higher plants, GAs play a key role in development and their concentration is maintained at a very low level by a number of endogenous and environmental factors including feedback regulation (7, 41). In contrast, wild-type strains of G. fujikuroi produce approximately 1 g·liter1 GA3 under laboratory conditions. The absence of feedback regulation in the fungus (14) suggests that GAs do not have an important role in growth and differentiation in this species. On the other hand, as discussed above, the production of fungal GAs in common with that of many fungal secondary metabolites is regulated by N repression. However, the molecular mechanism of the AREA-mediated regulation including N sensing, signal transduction, and modulation of activity of the transcription factor AREA is still to be investigated.
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FOOTNOTES |
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* This work was supported by AGTROL (Houston, TX), the Deutsche
Forschungsgemeinschaft (Tu101-7) and the Deutscher Akademischer
Austausdidienst/Consejo Nacional de Ciencia y Tecnología Cooperation
Program and Fondo Nacional de Desarrollo Científico y
Tecnológico (Grant 1020140). Rothamsted Research receives grant-aided
support from the Biotechnology and Biological Sciences Research Council of the
United Kingdom. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-251-83224801; Fax:
49-251-8323823; E-mail:
Bettina.Tudzynski{at}uni-muenster.de.
1 The abbreviations used are: GA, gibberellin; GC-MS, gas chromatography-mass
spectrometry; GGPP, geranylgeranyl diphosphate; HPLC, high performance liquid
chromatography; ORF, open reading frame; OPM, optimized GA3
production medium; des, desaturase; ICI, Imperial Chemical Industries
Ltd.
2 B. Tudzynski, unpublished data.
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ACKNOWLEDGMENTS |
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