Studies on the biosynthesis of gibberellins from (−)-kaurenoic acid in cultures of Gibberella Fujikuroi

Geissman, T. A.; Verbiscar, Anthony J.; Phinney, Bernard O.; Cragg, Gordon M.

doi:10.1016/s0031-9422(00)82790-9

Cited by 177 publications

(103 citation statements)

References 11 publications

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“…We have shown by disruption of P450-1 that the gene is involved in the conversion of ent-kaurenoic acid. Furthermore, by expressing P450-1 in a G. fujikuroi mutant lacking the entire gene cluster, we have demonstrated that the gene product catalyzes four steps in the metabolism of ent-kaurenoic acid to GA 14 . Our results also indicate that P450-1 is responsible for the formation of entkaurenoid-derived by-products of the GA-biosynthetic pathway.…”

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confidence: 97%

“…The identification of these genes has provided the opportunity to study the fungal enzymes and metabolic steps they catalyze, which had previously proved difficult at the protein level (2). It has been possible to demonstrate enzyme activities in vitro for only the early part of the fungal GA pathway; these activities include the formation (7) and metabolism (8) of the intermediate, ent-kaurene, the 7␤-hydroxylation of ent-kaurenoic acid (9), and the conversion of GA 12 -aldehyde to GA 14 (10). Each of these reactions after ent-kaurene is catalyzed by microsomal monooxygenases with the properties expected for cytochrome P450s.…”

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The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

Rojas

Hedden

Gaskin

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

125

106

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Recent studies have shown that the genes of the gibberellin (GA) biosynthesis pathway in the fungus Gibberella fujikuroi are organized in a cluster of at least seven genes. P450 -1 is one of four cytochrome P450 monooxygenase genes in this cluster. Disruption of the P450 -1 gene in the GA-producing wild-type strain IMI 58289 led to total loss of GA production. Analysis of the P450 -1-disrupted mutants indicated that GA biosynthesis was blocked immediately after ent-kaurenoic acid. The function of the P450 -1 gene product was investigated further by inserting the gene into mutants of G. fujikuroi that lack the entire GA gene cluster; the gene was highly expressed under GA production conditions in the absence of the other GA-biosynthesis genes. Cultures of transformants containing P450 -1 converted ent- cytochrome P450 ͉ ent-kaurenoid metabolism ͉ GA14 synthase

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confidence: 97%

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confidence: 99%

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

Rojas

Hedden

Gaskin

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

125

106

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“…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 entkaurene (1) in the mycelium. The low quantities of fujenoic acid (11), GA 15 (15), and GA 24 (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.…”

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confidence: 95%

“…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 GA 15 (15) and GA 24 (16) carried out by the mutant (Table 1) is borne in mind.…”

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Microbial Transformation of ent-Kaurenoic Acid and Its 15-Hydroxy Derivatives by the SG138 Mutant of Gibberella fujikuroi

et al. 2001

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Feeding experiments with ent-kaurenoic acid (4), 15R-hydroxy-ent-kaurenoic acid (5), 15 -hydroxy-entkaurenoic 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 7 ,15R-dihydroxykaurenolide (9) and 7 ,15R-dihydroxy-ent-kaurenoic acid (13). The incubation of 6 produced 7 ,15 -dihydroxy-ent-kaurenoic acid (7) and 7 ,15 -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 7 -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), entkaurenol (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 wildtype strain were also analyzed. 5 The results of these analyses and preliminary feeding experiments 6 suggested to us that one of these mutants (SG138) was blocked in the oxidative transformations from ent-kaurene (1) to entkaurenoic acid (4), as well as in 3 -hydroxylation, 13 -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 15 -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 stimulated 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 substances could be isolated, and their structures were confirmed by NMR techniques (see the Experimental Section for more details). A num...

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“…Meteor. Gibberellin biosynthesis is believed to proceed in the order of increasing hydroxylation (4,11,27) and GA,, a nonhydroxylated GA could therefore serve as an efficient precursor to other GAs. This, in conjunction with the fact that a compound chromatographically similar to GA, occurs in shoots of etiolated dwarf pea seedlings (Railton, Durley.…”

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Metabolism of Tritiated Gibberellin A₉ by Shoots of Dark-grown Dwarf Pea, cv. Meteor

Railton

Durley²,

Pharis³

1974

Plant Physiol.

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Tritium-labeled gibberellin A0 (3H-GA0) was metabolized by etiolated shoots of dwarf pea (Pisum sativum cv. Meteor) to GA20, GA1o, 2, 3-dihydro-GA3,, and a number of highly polar, acidic GA-like substances. Identifications were made by gasliquid radiochromatography and combined gas chromatography-mass spectrometry. Kinetic studies showed that GAao and 2,3-dihydro-GA,: were produced within 5 hours following 3H-GA9 application to pea shoots. The polar GA-like substances were produced between 5 and 10 hours after 'H-GA0 application. Levels of GALo increased with time, and since no GA1o was produced during the purification procedures, GA1o was, in all probability, produced from 3H-GA9 within the plant tissue. The radioactive interconversion products produced by pea from 3H-GA9 have chromatographic properties similar to biologically active GA-like substances present in etiolated shoots of dwarf pea. Large scale applications of 'H-GA9 with very low specific activity to etiolated pea shoots showed that the radioactivity of the interconversion products was correlated exactly with biological activity as assayed by dwarf rice (Oryza sativa cv. Tan-ginbozu).Dwarf peas contain two main fractions exhibiting gibberellin (GA)-like activity (15,17) and, in seeds of the dwarf cultivar Progress No. 9, these two GAs have been identified as GA20 and GA2-(10). Jones (14) detected the presence of two more zones of GA-like activity in buffer extracts from a tall variety of pea, cv. Alaska. One zone appeared more polar than A20, while the second was less polar than GA20. Railton and Reid (unpublished data) have confirmed the findings of Jones (14) and furthermore have detected the presence of at least six zones of GA-like activity in extracts of chloroplasts and leaves of tall peas, cv. Alaska (25,26). One of these zones has similar chromatographic properties to those of GA0.Studies have been carried out previously on the metabolism of radioactive GAs in dwarf pea (16,21). 'H-GA1 was metabolized to a more polar compound, but its identity was not established (15). Similarly, 3H-GA1 was converted to a more polar

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Studies on the biosynthesis of gibberellins from (−)-kaurenoic acid in cultures of Gibberella Fujikuroi

Cited by 177 publications

References 11 publications

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

Microbial Transformation of ent-Kaurenoic Acid and Its 15-Hydroxy Derivatives by the SG138 Mutant of Gibberella fujikuroi

Metabolism of Tritiated Gibberellin A₉ by Shoots of Dark-grown Dwarf Pea, cv. Meteor

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Studies on the biosynthesis of gibberellins from (−)-kaurenoic acid in cultures of Gibberella Fujikuroi

Cited by 177 publications

References 11 publications

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

Microbial Transformation of ent-Kaurenoic Acid and Its 15-Hydroxy Derivatives by the SG138 Mutant of Gibberella fujikuroi

Metabolism of Tritiated Gibberellin A9 by Shoots of Dark-grown Dwarf Pea, cv. Meteor

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Metabolism of Tritiated Gibberellin A₉ by Shoots of Dark-grown Dwarf Pea, cv. Meteor