Characterization of the Final Two Genes of the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi

Recently, six genes of the gibberellin (GA) biosynthesis gene cluster in Gibberella fujikuroi were cloned and the functions of five of these genes were determined. Here we describe the function of the sixth gene, P450–3, and the cloning and functional analysis of a seventh gene, orf3, located at the left border of the gene cluster. We have thereby defined the complete GA biosynthesis gene cluster in this fungus. The predicted amino acid sequence of orf3 revealed no close homology to known proteins. High performance liquid chromatography and gas chromatography-mass spectrometry analyses of the culture fluid of knock-out mutants identified GA1 and GA4, rather than GA3 and GA7, as the major C19-GA products, suggesting that orf3 encodes the GA4 1,2-desaturase. This was confirmed by transformation of the SG139 mutant, which lacks the GA biosynthesis gene cluster, with the desaturase gene renamed des. The transformants converted GA4 to GA7, and also metabolized GA9 (3-deoxyGA4) to GA120 (1,2-didehydroGA9), but the 2α-hydroxylated compound GA40 was the major product in this case. We demonstrate also by gene disruption that P450-3, one of the four cytochrome P450 monooxygenase genes in the GA gene cluster, encodes the 13-hydroxylase, which catalyzes the conversion of GA7 to GA3, in the last step of the pathway. This enzyme also catalyzes the 13-hydroxylation of GA4 to GA1. Disruption of the des gene in an UV-induced P450–3 mutant produced a double mutant lacking both desaturase and 13-hydroxylase activities that accumulated high amounts of the commercially important GA4. The des and P450–3 genes differ in their regulation by nitrogen metabolite repression. In common with the other five GA biosynthesis genes, expression of the desaturase gene is repressed by high amounts of nitrogen in the culture medium, whereas P450-3 is the only gene in the cluster not repressed by nitrogen. Recently, six genes of the gibberellin (GA) biosynthesis gene cluster in Gibberella fujikuroi were cloned and the functions of five of these genes were determined. Here we describe the function of the sixth gene, P450–3, and the cloning and functional analysis of a seventh gene, orf3, located at the left border of the gene cluster. We have thereby defined the complete GA biosynthesis gene cluster in this fungus. The predicted amino acid sequence of orf3 revealed no close homology to known proteins. High performance liquid chromatography and gas chromatography-mass spectrometry analyses of the culture fluid of knock-out mutants identified GA1 and GA4, rather than GA3 and GA7, as the major C19-GA products, suggesting that orf3 encodes the GA4 1,2-desaturase. This was confirmed by transformation of the SG139 mutant, which lacks the GA biosynthesis gene cluster, with the desaturase gene renamed des. The transformants converted GA4 to GA7, and also metabolized GA9 (3-deoxyGA4) to GA120 (1,2-didehydroGA9), but the 2α-hydroxylated compound GA40 was the major product in this case. We demonstrate also by gene disruption that P450-3, one of the four cytochrome P450 monooxygenase genes in the GA gene cluster, encodes the 13-hydroxylase, which catalyzes the conversion of GA7 to GA3, in the last step of the pathway. This enzyme also catalyzes the 13-hydroxylation of GA4 to GA1. Disruption of the des gene in an UV-induced P450–3 mutant produced a double mutant lacking both desaturase and 13-hydroxylase activities that accumulated high amounts of the commercially important GA4. The des and P450–3 genes differ in their regulation by nitrogen metabolite repression. In common with the other five GA biosynthesis genes, expression of the desaturase gene is repressed by high amounts of nitrogen in the culture medium, whereas P450-3 is the only gene in the cluster not repressed by nitrogen. Gibberellins (GAs) 1The 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. are plant hormones that are produced by all higher plants and some fungi. The rice pathogen Gibberella fujikuroi, mating population C (anamorph Fusarium fujikuroi), produces high amounts of gibberellic acid (GA3) and some other GAs and is used for commercial production of these agriculturally important compounds. GAs are diterpenoids and are synthesized in G. fujikuroi via the mevalonate pathway (1Birch A.J. Richards R.W. Smith H. Proc. Chem. Soc. 1958; : 192-193Google Scholar). Most of the genes of the early isoprenoid pathway have been cloned from G. fujikuroi, viz 3-hydroxy-3-methylglutaryl-CoA reductase (2Woitek S. Unkles S.E. Kinghorn J.R. Tudzynski B. Curr. Genet. 1997; 31: 38-47Crossref PubMed Scopus (36) Google Scholar), farnesyl diphosphate synthase (3Homann V. Mende K. Arntz C. Ilardi V. Macino G. Morelli G. Böse G. Tudzynski B. Curr. Genet. 1996; 30: 232-239Crossref PubMed Scopus (45) Google Scholar), and two geranylgeranyl diphosphate (GGPP) synthase genes, one of which (ggs1) is involved in general isoprenoid biosynthesis (4Mende K. Homann V. Tudzynski B. Mol. Gen. Genet. 1997; 255: 96-105Crossref PubMed Scopus (57) Google Scholar) and a second (ggs2) of which is specific for GA biosynthesis (5Tudzynski B. Hölter K. Fungal Genet. Biol. 1998; 25: 157-170Crossref PubMed Scopus (164) Google Scholar). 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 (6MacMillan J. Nat. Prod. Rep. 1997; 14: 221-244Crossref Scopus (117) Google Scholar). 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 (7Hedden P. Phillips A.L. Trends Plant Sci. 2000; 5: 523-530Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). 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 (8Kawaide H. Imai R. Sassa T. Kamiya Y. J. Biol. Chem. 1997; 272: 21706-21712Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 9Toyomasu T. Kawaide H. Ishizaki A. Shinoda S. Otsuka M. Mitsuhashi W. Sassa T. Biosci. Biotechnol. Biochem. 2000; 64: 660-664Crossref PubMed Scopus (57) Google Scholar, 10Tudzynski B. Kawaide H. Kamiya Y. Curr. Genet. 1998; 34: 234-240Crossref PubMed Scopus (77) Google Scholar). 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 (P450–1 to P450–4) were shown to be closely linked in a gene cluster (5Tudzynski B. Hölter K. Fungal Genet. Biol. 1998; 25: 157-170Crossref PubMed Scopus (164) Google Scholar, 11Linnemannstöns P. Voss T. Hedden P. Gaskin P. Tudzynski B. Appl. Environ. Microbiol. 1999; 65: 2558-2564Crossref PubMed Google Scholar). P450–4 encodes ent-kaurene oxidase, catalyzing the three oxidation steps between ent-kaurene and ent-kaurenoic acid (Fig. 1) (12Tudzynski B. Hedden P. Carrera E. Gaskin P. Appl. Environ. Microbiol. 2001; 67: 3514-3522Crossref PubMed Scopus (82) Google Scholar), while P450–1 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) (13Rojas M.C. Hedden P. Gaskin P. Tudzynski B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). was shown to a GA which GA14 to GA4 by of (Fig. 1) B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this we the two genes of the cluster and demonstrate that are for the last two steps in the biosynthesis of We describe the and functional of the seventh gene, orf3, which is located at the left border of the cluster. gene knock-out and expression in the of the other GA-biosynthetic genes, we that orf3 encodes the desaturase that GA4 to we demonstrate that the P450 monooxygenase gene, P450–3, encodes the 13-hydroxylase that GA7 to the The of the last two GA-biosynthetic genes and the production of double mutants have to producing GA7, GA1 the commercially important GA4. with the desaturase and P450–3 genes in with analysis revealed in their regulation by nitrogen. Fungal and fujikuroi a from was by the Fungal producing GA7 as main product was by of The G. fujikuroi and the mutant SG139 E. M. 1999; Scopus Google Scholar) were by J. SG139 the GA-biosynthetic gene cluster as by and for for were as B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). analysis of GA the was in the optimized GA production medium, B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), in A.J. G. 5: Scopus Google Scholar) for at a and was used for was used to the G. fujikuroi orf3 gene of The a with the of orf3, an of was by the from J. Microbiol. Scopus Google Scholar). of P450–3, three gene disruption were by cloning of the gene the Mol. Gen. Genet. Scopus (164) Google Scholar) C. PubMed Scopus Google Scholar), both of which the The of P450–3 were by cloned the cloning with and and cloned the was with and cloned gene expression of orf3 in the mutant the from was cloned in expression of P450–3 in a with the gene was cloned in the In the the P450–3 gene and to the of P450–3 was used for of The of the orf3 in and of cloned to a in the This step was the were as B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The for the of P450–3 were as and and and and The mutant of P450–3 from was with the in with the and as and the transformants of SG139 in which orf3 and P450–3 been the were and of the were synthesized by and and and and analyses were as B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The G. fujikuroi of was used as a for of G. fujikuroi and The expression was from from for GA3 (4Mende K. Homann V. Tudzynski B. Mol. Gen. Genet. 1997; 255: 96-105Crossref PubMed Scopus (57) Google Scholar). were at and to of the B. Mende K. Kinghorn J.R. Unkles S.E. 1996; PubMed Scopus Google Scholar), were and to The was at high and the were at the in by were used for a second of of was by the S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) an of G. of and were as B. Mende K. Kinghorn J.R. Unkles S.E. 1996; PubMed Scopus Google Scholar). of the the mutant were with from the gene one of the disruption and were at in a complete for were from transformants and used for and expression of orf3 and P450-3 in the mutant were with of the the the P450–3 gene and an to the of P450–3 not involved in GA analysis of GA the and mutants were in of The were for a at and GA7 were by to J. Scopus Google Scholar) a with a and a GA3, GA4, and GA7 were also by chromatography a of acid analysis of culture was P. Voss T. Hedden P. Gaskin P. Tudzynski B. Appl. Environ. Microbiol. 1999; 65: 2558-2564Crossref PubMed Google Scholar) a to an gas in were a at the was at 13 to and at to The was and the and were and were at at from GAs were identified by of their with P. J. of the Gibberellins and and a of Scholar). with was from a GA from J. Phillips A.L. Gaskin P. Hedden P. Plant 1998; PubMed Scopus Google Scholar), and was from by with of transformants of G. fujikuroi B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was from by with of the mutant of G. was from by with of transformants B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in high nitrogen was for in A.J. G. 5: Scopus Google Scholar) by to at and of the with the nitrogen in was by to at The were to the disruption mutants in both high and nitrogen and for an at were from the culture fluid as (13Rojas M.C. Hedden P. Gaskin P. Tudzynski B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar) and by a with a of at was in of the by liquid and were by as of SG139 transformants with as well as of SG139 P450–3 transformants with were nitrogen in at and of (Fig. of the left of the gene cluster, from the gene P450–4 (12Tudzynski B. Hedden P. Carrera E. Gaskin P. Appl. Environ. Microbiol. 2001; 67: 3514-3522Crossref PubMed Scopus (82) Google Scholar) to the gene T. J. Tudzynski B. Curr. Genet. 2001; PubMed Scopus Google Scholar), was analysis revealed a open reading in the as The sequence that the open reading is not by The orf3 gene is the of the for revealed only a homology with of the from P. J. PubMed Google Scholar), of function in GA In the of orf3, for of the major nitrogen were that expression of orf3 be by nitrogen as are of the other GA-biosynthetic genes (5Tudzynski B. Hölter K. Fungal Genet. Biol. 1998; 25: 157-170Crossref PubMed Scopus (164) Google Scholar, B. Hedden P. Carrera E. Gaskin P. Appl. Environ. Microbiol. 2001; 67: 3514-3522Crossref PubMed Scopus (82) Google Scholar, M.C. Hedden P. Gaskin P. Tudzynski B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). of gene was by the of the of orf3 (Fig. with the from the J. Microbiol. Scopus Google Scholar). The was with the from and analysis revealed that of transformants and were not to GA3, the product of the GA-biosynthetic pathway in G. fujikuroi, analysis confirmed that the transformants have the of of the the orf3 not analysis of culture of and that GA1 rather than GA3 was the major C19-GA in the (Fig. acid and (Fig. 1) were major in both of the transformants were shown by analysis to GA1 and GA4 in from to in the in for the mutants produced no GA3 GA7 and accumulated GA4 and the to the of GA4 to GA7 (Fig. that orf3 encodes GA4 of GAs produced by the and mutant and in mutants from both of in in a the function of the orf3 expression we the mutant which the GA gene cluster, with the gene the orf3 were shown by to a of the gene the SG139 of five transformants by confirmed that the gene was in SG139 the of the GA gene cluster not and which orf3 at with the were with the in at analysis of the product from of with transformants and identified as product for in whereas SG139 not this not the desaturase gene is as an enzyme in In the orf3 transformants the producing two of in The of these was by to whereas the GA120 and and three of a acid to be from GA120 by and The enzyme both and in the is an of of from of and with SG139 des conversion in and from from conversion in from in a of the function of the gene of the cluster, P450–3, the was with the gene disruption which the gene, (Fig. analysis of transformants by all were to high amounts of GAs of in but three and not GA3, GA4 and GA7, were be that this in GA was the of P450–3 gene we two disruption and In a in the was via (Fig. We transformants for both by of the transformants from and three of the transformants from and produced GA7 as product of GA3, which was confirmed by analysis of the culture fluid from confirmed that GA7 was the major product and that no GA3 was produced (Fig. analysis not revealed that transformants with all of the three the for P450–3 and produced the of the to GA3 in these transformants is to the disruption of gene P450–3, suggesting that encodes the 13-hydroxylase catalyzing the conversion of GA7 to the transformants produced no GA, which is produced in amounts in the P450–3 is for 13-hydroxylation of both GA7 and GA4 (Fig. the function of P450–3, of SG139 with the and both the gene P450–3, was analysis that of the transformants the gene the of the gene the SG139 not of the transformants from both were to to GA3 and of the GA-biosynthetic pathway were as of P450–3 expression by to of the P450–3 transformants at We that ent-kaurenoic GA4, GA7 were in GA 13-hydroxylase in the which to these The of P450–3 expression in the SG139 P450–3 transformants is in contrast with the expression of P450–1 (13Rojas M.C. Hedden P. Gaskin P. Tudzynski B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar), B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), P450–4 (12Tudzynski B. Hedden P. Carrera E. Gaskin P. Appl. Environ. Microbiol. 2001; 67: 3514-3522Crossref PubMed Scopus (82) Google Scholar), and des in of SG139 with a the gene cluster 13-hydroxylase of the of P450–3 as the 13-hydroxylase that catalyzes the conversion of GA7 to GA3 to the mutant as a P450–3 This was of the highly and for the of the the of the P450–3 gene from the mutant was by the and as well as and were from both was at in an amino acid from to at (Fig. This in a in the GA product 13-hydroxylase whereas the produced GA3, GA4 and by HPLC, the mutant produced GA3, GA4, and GA7 the culture (Fig. which was in amounts in was not in the of mutant with the P450–3 gene GA3 as by analysis of the culture fluid not that the was in the the P450–3 gene was in which all of the genes, in contrast to of expression in SG139 in which the gene cluster is analysis of culture of mutant mutant and a double mutant The was at and were the of of a of G. fujikuroi GA3, whereas GA4 P. G. of Plant and Scholar). that GA4 in the of GA3 and GA7 have this we double mutants lacking both desaturase and 13-hydroxylase activities by the mutant with the of analysis of by revealed that and their to GA7 and accumulated amounts of GA4 (Fig. and In contrast to the des mutants from the not amounts of that P450–3 catalyzes the 13-hydroxylation of GA4 as well as the of des, P450–3, both the of producing high amounts of GA7, GA4, of P450–3 by genes of the GA-biosynthetic gene cluster are highly of nitrogen whereas only a of was in with high amounts of (5Tudzynski B. Hölter K. Fungal Genet. Biol. 1998; 25: 157-170Crossref PubMed Scopus (164) Google Scholar, B. Hedden P. Carrera E. Gaskin P. Appl. Environ. Microbiol. 2001; 67: 3514-3522Crossref PubMed Scopus (82) Google Scholar, M.C. Hedden P. Gaskin P. Tudzynski B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). of the gene des the of des was in for and in with a high (Fig. analysis of revealed a of in medium, the in with culture in a to P450–4 (Fig. and not was for des P450–4 in in In contrast to the other six genes of the cluster, P450–3 to be highly in with high than with (Fig. in culture in medium, some nitrogen only of expression of des, P450–4 (Fig. and the other four genes not be whereas P450–3 is highly of for des and P450–4 whereas P450–3 (Fig. In for P450–3 is not in mutants that not expression of P450–3, whereas des expression (Fig. and that of the other genes not the nitrogen The of the culture in the to not the expression of des, P450–3 (Fig. of the other GA-biosynthetic genes not that the regulation of des and P450–3 expression be at the we of in disruption mutants are for the of from GA14 and are not to GA4, GA7, GA1 and GA3 (Fig. 1) B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The with the mutants and of the desaturase and 13-hydroxylase activities by high was converted to with a of while in with no complete conversion of to the other was converted to in both high and that the 13-hydroxylase no by high In the demonstrate that the desaturase is by in with the expression analysis the des and P450–3 genes (Fig. The product from high is with the of GAs in des disruption which GA1 of nitrogen of the culture desaturase and 13-hydroxylase activities in Gibberella fujikuroi disruption in the culture by from the of the at and in the culture A.J. G. 5: Scopus Google Scholar), by from the of the at and in a We describe the cloning of the seventh and gene of the GA biosynthesis gene cluster in G. fujikuroi and demonstrate that encodes GA4 desaturase The gene is located between the identified gene T. J. Tudzynski B. Curr. Genet. 2001; PubMed Scopus Google Scholar) and the ent-kaurene gene P450–4 (12Tudzynski B. Hedden P. Carrera E. Gaskin P. Appl. Environ. Microbiol. 2001; 67: 3514-3522Crossref PubMed Scopus (82) Google Scholar) at the left of the cluster. The gene is not to other GA-biosynthetic gene from G. fujikuroi from higher plants, but homology with in the C cluster of P. J. PubMed Google Scholar). This of the which both and no in and not in the function of which was by a of gene and of the SG139 mutant the des gene in the of the other GA biosynthesis genes converted to and also metabolized the to GA40 was the major product from that is an of the This enzyme for the of 2α-hydroxylated GAs in G. fujikuroi J. J. Plant Scopus Google Scholar). 2α-hydroxylated were in of the des transformants with the GA4, suggesting that the of the in of the of GAs in J. J. Plant Scopus Google Scholar) that of GA4 at a we have no the of the the of a product a for In plants, oxidation of of the GAs is catalyzed by 2-oxoglutarate-dependent of which are catalyzing and reactions P. 1997; Google Scholar). a GA from catalyzes of at and in to major Gaskin P. J. Plant PubMed Scopus Google Scholar). the multifunctional is for GA3 from the by of the double from to by M. Y. Gaskin P. J. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). in this at which in the of the Gaskin P. J. Plant PubMed Scopus Google Scholar), is a reaction of the main pathway to GA1. In in G. fujikuroi by of the R. J.R. J. Chem. Soc. : Scholar) is the major reaction and is by is of that of a of GAs GA120 that are by M. M. H. H. B. P. T. 2001; PubMed Scopus Google Scholar). The also which be as byproducts of the is known the of the in this case. We have also shown by gene disruption that P450–3, the cytochrome P450 monooxygenase gene in the GA gene cluster, encodes the 13-hydroxylase, which catalyzes the reaction in the biosynthesis of The enzyme catalyzes also the 13-hydroxylation of GA4 to GA1. GA1 GA is produced in these two of G. fujikuroi, suggesting that the GA4 desaturase than 13-hydroxylase that GA4 a higher for the desaturase than for the of desaturase by disruption of the des gene in a of GA1 but only a of GA4. The mutant the to of GA1 for the double mutant lacking both desaturase and 13-hydroxylase activities be used for the production of the commercially important GA4. was an in the regulation of the des and P450–3 of des is high of that GA production but in the the is expression to of and which are all of the major nitrogen M. Homann V. Tudzynski B. Mol. Microbiol. PubMed Scopus Google Scholar, B. Homann V. B. Mol. Gen. Genet. 1999; PubMed Scopus Google Scholar). In the expression of P450–3 from that of the other six GA analysis with in with a high that P450–3 is both and in was high P450–3 is the only gene in the GA cluster for which is in medium, which are in with from of GA4 and GA7 with knock-out which not these GAs and the conversion of GA14 to GA4 is B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In in both desaturase and 13-hydroxylase activities were that GA3 was the major product from GA4, whereas in high GA1 was produced than GA3 to desaturase GA7 was converted to GA3 of the of the The of of P450–3 expression by is with the of the double sequence in which the B. T. S. R.W. J. PubMed Scopus Google Scholar), are in the of the other six In to regulation by P450–3 is the only gene in the cluster that be in the mutant which lacks the gene cluster. In was in which a in P450–3, as well as in SG139 with the GA gene cluster. We have not of the other genes to be and for P450–3 to be but have the that expression requires by a GA-biosynthetic by to the of ent-kaurenoic which are the of and not The of the P450–3 gene to a not the expression of the gene is for the to P450–3 in SG139 other of the gene cluster are for gene expression and enzyme The functional of des and P450–3 the analysis of the GA gene cluster in G. Disruption of T. J. Tudzynski B. Curr. Genet. 2001; PubMed Scopus Google Scholar) and an at the left border of the cluster and of and at the of P450–3 not the production of GA3 the biosynthesis of the GA3, in 13 steps with the of GGPP by the synthase requires only enzymes, of which are In contrast to other metabolite gene the gene cluster in M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) and the gene cluster in Fusarium and M. to Scholar), the GA gene cluster not a We have in to the general a gene that expression of all of the genes and be located in the We are to GA biosynthesis in G. fujikuroi with that in higher plants at the and gene the and plants GAs and the early steps to the of GA12-aldehyde are the differ as the of involved and the regulation of their important is that the early and steps in the are catalyzed by cytochrome P450 monooxygenases, whereas dioxygenases are for these reactions in higher major is that 13-hydroxylation early the of in plants, whereas is the last step in the fungus. in the of the in which cytochrome in the and plants, the in G. fujikuroi and plants have of amino acid only for ent-kaurene of the and A. P. Phillips A.L. M.C. Carrera E. Tudzynski B. J. Plant Scopus Google Scholar). In higher plants, GAs a in and their is at a by a of and regulation (7Hedden P. Phillips A.L. Trends Plant Sci. 2000; 5: 523-530Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, S. Kamiya Y. Plant 2000; PubMed Scopus Google Scholar). In of G. fujikuroi GA3 The of regulation in the B. M.C. Gaskin P. Hedden P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that GAs not have an important in and in this the other as the production of GAs in common with that of is by repression. the of the regulation and of of the is to be We for and for the