WO2002055725A2

WO2002055725A2 - Novel nucleic acids, methods and transformed cells for the modulation of gibberellin production

Info

Publication number: WO2002055725A2
Application number: PCT/US2001/043992
Authority: WO
Inventors: Bettina Tudzynski
Original assignee: Phibro-Tech, Inc., Doing Business As Agtrol International
Priority date: 2000-11-14
Filing date: 2001-11-14
Publication date: 2002-07-18
Also published as: AU2002243235A1; EP1341926A2; JP2004517625A; WO2002055725A9; WO2002055725A3

Abstract

The present invention provides for nucleic acids, vectors comprising nucleic acids, transformed cells, methods for modulating gibberellin biosynthesis, and methods for identifying modulators of gibberellin biosynthesis.

Description

NOVEL NUCLEIC ACIDS, METHODS AND TRANSFORMED CELLS FOR THE MODULATION OF GIBBERELLIN PRODUCTION

Field of the Invention

The present invention relates generally to the field of gibberellin production from transformed cells, using novel nucleic acids and recombinant DNA technology, modulation of gibberellin biosynthesis, and the identification of inhibitors of biosynfhetic enzymes. Background of the Invention

Gibberellins (GAs) are a large family of isoprenoid plant hormones, some of which are bioactive growth regulators controlling seed germination, stem elongation, and flowering. The rice pathogen Gibberella fujikuroi (mating population C) is able to produce large amounts of GAs, especially the bioactive compounds gibberellic acid (GA₃) and its precursors, gibberellin A₄ (GA₄) and gibberellin A₇ (GA₇). (Tudzynski, B., 1999, "Biosynthesis of gibberellins in Gibberella fujikuroi: biomolecular aspects. " Appl. Microbiol. Biotechnol. 52(3) -298-310). One of the pathogenic effects of Gibberellins is that G. fujikuori gibberellin production induces super elongation (bakanae) disease of rice. The gibberellin biosynthetic pathway for GA₃ in the ascomycetous fungus

Gibberella fujikuroi involves the conversion of the precursor gibberellin A₁₄ (GA₁₄) into the intermediary GA₄, then GA₇, before finally generating GA₃ as the main product. A secondary end-product from an alternative conversion of GA₄ into gibberellin A_λ (GA_X) also takes place at the same time. A 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the first specific enzyme of the isoprenoid pathway and is involved in the production of mevalonate, the precursor for essential sterol and quinone biosynthesis and secondary metabolites such as gibberellins. The structural gene of HMG-CoA reductase was isolated from G. fujikuroi and characterized. (Woitek, S. et al., 1997, "3-Hydroxy-3-methylglutaryl-CoA reductase gene of Gibberella fujikuroi: isolation and characterization. " Curr. Genetics 31(1): 38-47). Gibberellins are diterpenoid compounds which are synthesized via the isoprenoid biosynthetic pathway. Geranylgeranyl diphosphate synthase (GGDS) is a key enzyme in isoprenoid biosynthesis. The GGDS enzyme generates geranylgeranyl diphosphate (GGDP). The GGDS gene of G. fujikuroi was isolated and characterized as a single copy gene which is not linked to the farnesyl diphosphate synthase gene. (Mende, K. et al. , 1997, "The geranylgeranyl diphosphate synthase gene of Gibberella fujikuroi: isolation and expression. " Mol. Gen. Genet. 255(1):96-105).

The first gene of the gibberellin pathway is the copalyl diphosphate synthase (CPDS) gene, a terpene cyclase which catalyzes the first specific step of the gibberellin pathway as it branches off from the general isoprenoid biosynthetic pathway at geranylgeranyl diphosphate (GGDP). This gene was isolated from G. fujikuroi and characterized by comparison with other known plant CPSs. G. fujikuroi CPDS is highly enhanced under conditions for optimized gibberellin biosynthesis, and is reduced when high amounts of ammonium are present in the medium. (Tudzynski, B. et al. , 1998, "Gibberellin biosynthesis in Gibberella fujikuroi: cloning and characterization of the copalyl diphosphate synthase gene." Curr. Genetics 34(3): 234-240).

Differential screening of a Gibberella fujikuroi cDNA library was used to clone and identify a gene having the conserved heme-binding motif of cytochrome P450 monooxygenases. Subsequent screening of a genomic library with a homologous probe revealed that a second gene was closely linked to the first gene. Further chromosome walking localized a putative GGDS gene, a CPDS gene, and a third P450 gene. The G. fujikuroi P450-1, P450-2 and P450-3 gene product, a 13-Hydroxylase, were all implicated in the gibberellic acid biosyntheic pathway. (Tudzynski, B. , Holter, K., 1998, "Gibberellin biosynthetic pathway in Gibberella fujikuroi: evidence for a gene cluster." Fungal Genet. Biol. 25(3): 157-70).

Currently, the most common method for obtaining gibberellins utilizes isolation by purification from cell cultures which are derived mainly from Gibberella fujikuroi. Improvements in culture media, reaction conditions, or yield of gibberellin are desirable and useful for the economical commercial production of gibberellin in large quantities.

To this end, ammonium and nitrate has been identified as an inhibitor to gibberellin production. (Tudzynski, B. et al., 1999, "Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi" ; Sanchez-Fernandez, R. et al. , 1997, "Inhibition of gibberellin biosynthesis by nitrate in Gibberella fujikuroi. " FEBS Letters 413(l):35-9). Higher aeration was reported to increase growth and production of bikaverin and gibberellins. (Giordano, W., Domenech, CE., 1999, "Aeration affects acetate destination in

Gibberella fujikuoήX FEMS Microbiol. Lett. 180(l): lll-6). The use of fluidized bioreactors was reported to result in gibberellic acid production that is three-times greater than reported for submerged and solid fermenters. The impact of pH and temperature, as well as Carbon to Nitrogen ratio was also studied. (Escamilla, EM. et al., 2000, "Optimization of gibberellic acid production by immobilized Gibberella fujikuroi mycelium in fluidized bioreactors." Biotechnol. 76(2-3): 147-55).

New and useful methods, constructs, cells, the identification of a new enzyme component of the biosynthetic pathway, and manipulation of the genetics of the biosynthetic pathway utilizing recombinant nucleic acids are taught by the present invention. Summary of the Invention

The present invention provides a new gene, herein called orfS, encoding for an enzyme involved in the gibberellin biosynthetic pathway. The o gene product is an enzyme which converts GA₄ to GA₇ by introducing a double-bond between carbon atoms 1 and 2 of GA₄ (Figure 1), and is herein called GA₄- desaturase (GA₄D). It should be understood that "GA₄-desaturase" and "gibberellin A₄-desaturase" are hereafter regarded as equivalent terms.

The present invention provides for an isolated nucleic acid having a nucleotide sequence which corresponds to the nucleic acid sequence depicted as SEQ ID. NO. : l (Figure 2). Thus, the present invention encompasses an isolated nucleic acid comprising a nucleotide sequence which corresponds to the protein coding domain segment (CDS) of the nucleic acid sequence depicted in Figure 2 as SEQ ID. NO. :l. In particular, the present invention provides for an isolated nucleic acid having a nucleotide sequence which corresponds to the nucleic acid sequence depicted as nucleic acid residues 1-1026 in Figure 2 (nucleic acid residues 984 to 2009 inclusive, of the nucleic acid sequence depicted in SEQ ID. NO.: l). Thus, the present invention encompasses a nucleic acid having the nucleotide sequence depicted as residues 984-2009 of that depicted as SEQ ID NO. : 1.

The present invention also provides for a GA₄D protein. In a preferred embodiment, the GA₄D protein is encoded for by a nucleic acid of the invention. Thus, the present invention encompasses a protein comprising an amino acid residue sequence which corresponds to that depicted as SEQ ID NO.: 2 (Figure 2). In particular, the present invention encompasses a 342 amino acid length protein having an amino acid sequence corresponding to the amino acid residue sequence depicted as SEQ ID NO. : 2 (FIGURE 2). It is also envisioned that the GA₄D of the present invention encompasses biologically active fragments of the GA₄D protein which have less than the entire amino acid sequence depicted as SEQ ID NO. :2, yet still retains at least a portion of the enzymatic GA₄- desaturase activity of the whole protein.

The present invention encompasses fusion proteins which combine the GA₄D protein of the invention with one or more other polypeptides or proteins. Other polypeptide or proteins may include and are not limited to marker proteins for visualization of the fusion protein product, such as betagalactosidase, green fluorescent protein and the like. Other polypeptides or proteins may also include and are not limited to polypeptides or proteins useful for isolation of the fusion protein product, such as for example, and not limited to, biotin/avidin, protein A or other immuno-conjugates, laminin binding domains, and the like. The present invention also provides for nucleic acid vectors or constructs, comprising at least a portion of a nucleic acid of the present invention.

In particular, the present invention provides for a nucleic acid vector which operably joins an intact, expressible nucleic acid of the invention with a regulatory nucleic acid sequence.

The present invention also encompasses nucleic acid vectors which comprise a disrupted oφ gene.

Alternatively, the nucleic acid vectors of the invention can comprise an oφ gene which contains a mutation in coding sequence which negatively impacts the enzymatic activity of the resultant protein, or contains a mutation in coding sequence which negatively impacts the expression of the resultant protein, or the like.

Thus, the present invention encompasses nucleic acid vectors which express an inhibited GA₄D protein, or exhibit inhibited expression of GA₄D protein.

By inhibited GA₄D protein, it is meant that the GA₄D protein has less than about 50% of the activity of the wild-type protein when compared under similar culture conditions.

By inhibited expression of GA₄D protein, it is meant that the GA₄D protein is expressed in an amount less than about 50% of that found in an uninhibited GA₄D expressing cell when compared under similar culture conditions.

The present invention provides for a method of selectively modifying the gibberellin biosynthetic pathway of a cell comprising modulating the expression of the endogenous oφ gene. The method and nucleic acid constructs used for such modulation are dictated by the desired effect.

In a first instance, the present invention provides for methods wherein the desired modulation is an increase in oφ gene expression which results in greater production of GA₄D protein, and thus increased GA₇ production. Such modulation can be accomplished by the transfection of the target cell with a nucleic acid vector of the invention which can express an intact GA₄D protein. Increased oφ gene expression can be accomplished by introducing multiple copies of the gene into the target cell in this manner. Further increase in gene expression can be accomplished by using higher activity promoters or other such regulatory elements. In another instance, the present invention provides for methods wherein the desired modulation is a decrease in orf3 gene expression which results in less or no production of GA₄D protein, and thus decreased GA₇ production and increased GA₄ accumulation. Such modulation can be accomplished by the transfection of the target cell with a nucleic acid vector of the invention which can disrupt the oφ gene by homologous recombination. In this method, most if not all of the endogenous intact gene of the target cell is replaced with a disrupted oφ gene from the transfected nucleic acid vector.

The present invention also provides for methods wherein the desired modulation is the expression of an inhibited GA₄D protein which results in less GA₄D protein activity in the target transformed cell, and thus decreased GA₇ production and increased GA₄ accumulation. Such modulation can be accomplished by the transfection of the target cell with a nucleic acid vector of the invention which comprises an oφ gene encoding for an inhibited GA₄D protein, by homologous recombination. In this method, most if not all of the endogenous intact gene of the target cell is replaced with the oφ gene from the transfected nucleic acid vector.

In a preferred embodiment, the present invention provides for a method for modulating the production of gibberellins, and in particular the amount of GA₄, GA₇ or GA₃, by manipulating the gibberellin biosynthetic pathway to reduce or block the enzymatic conversion of precursor molecules into subsequent products. In particular, the present invention encompasses a method of modulation which comprises the disruption or deletion of the oφ gene in a gibberellin producing cell. In a most preferred embodiment, the present invention encompasses a method of modulation which comprises the disruption or deletion of the endogenous oφ gene by replacement with an exogenous disrupted oφ gene, in a gibberellin producing cell.

In further preferred embodiment, the present invention encompasses a method of modulation which comprises the disruption or deletion of the oφ gene in a gibberellin producing cell which has a disrupted or inhibited P450-3 gene. Disrupting the expression of the P450-3 gene, disruption was caused to the 13-hydroxylations of GA₄ to GA , and GA₇ to GA₃ resulting in an enhanced output of GA₇. Combining mutations in the oφ gene with a mutated P 450-3 gene resulted in enhanced production of GA₄. In a most preferred embodiment, the present invention encompasses a method of modulation which comprises the disruption or deletion of the endogenous oφ gene by replacement with an exogenous disrupted oφ gene, in a gibberellin producing cell which has a disrupted or inhibited P450-3 gene.

In one aspect, disrupted or inhibited P450-3 gene is one where the gene encoding a 13-hydroxylase, is expressed at a lower level than in a wild-type gibberellin producing cell, under similar conditions. In addition, a disrupted or inhibited P450-3 gene can be one where the gene expresses a modified P450-3 protein that has lower enzymatic activity than in a wild-type P450-3 enzyme, under similar conditions. As discussed with regard to oφ, a disrupted P450-3 gene can comprise a mutation of the nucleic acid coding sequence, as in a single point mutation, multiple point mutations, an insertion, or a deletion which alters the expressibility of a functional P450-3 protein, or the enzymatic activity of the resulting protein.

The present invention provides for a transformed cell, modified to increase production of GA₄ by disrupting the endogenous oφ gene, such that the cell will have disrupted GA₄D protein expression.

The present invention also provides for a transformed cell modified for increased production of GA₇ by transfection with at least one additional expressible oφ gene. Suitable cells for use in the methods of the present invention are those which produce endogenous gibberellins, or cells which are transformed with separate genes encoding for proteins essential to produce gibberellins. Suitable cells can be bacterial, fungal, or mammalian. It is preferred that the gibberellin producing cells are fungal cells. In one preferred embodiment, the gibberellin producing cells are Gibberella fujikori derived cells. In a preferred embodiment, the cells are derived from the cell strains identified as SI (IMI58289), 6314, 6314Δorf3-Tl, 6314Δorf3-T2, 6314Δorf3-T8, SlΔorβ- T23, SlΔorf3-T33, or SlΔorf3-T55.

Similarly, the present invention also provides for a transformed cell modified for increased production of GA₇ by transfection with an oφ gene which is regulated by a higher activity regulatory nucleic acid sequence such that more GA₄D protein is expressed. The present invention also provides for a transformed cell wherein the endogenous oφ gene has been replaced, entirely or in part, by an oφ gene modified for increased production of GA₄D by regulation by a higher activity regulatory nucleic acid sequence, wherein such replacement is by homologous recombination with a transfected vector, and such that more GA₄D protein is expressed.

In a most preferred embodiment, the present invention provides for a transformed cell modified for increased production of GA₄ by the disruption of the endogenous oφ gene of the cell. In one embodiment of a preferred method, the disruption of the endogenous gene is accomplished by the homologous recombination with a transfection vector with a disrupted oφ gene, resulting in the replacement of the endogenous gene with the exogenous disrupted gene of the transfection vector.

In a most preferred embodiment, the present invention provides for a gibberellin producing cell comprising a disrupted oφ gene and a disrupted

P450-3 gene.

The constructs and transformed cells of the present invention allow for methods for the detection, characterization and screening for modulators of gibberellin biosynthesis, in situ, in vitro, and in vivo. The present invention provides for the use of isolated GA₄D protein for the detection of chemical inhibitors of the GA₄D protein activity. Chemical inhibitors can be small chemical molecules, complex compounds, protein, antibodies or the like. Methods for using GA₄D protein to screen candidate substances for inhibition of GA₄D enzyme activity can be performed as in vivo cellular assays, or as in vitro cell-free assays utilizing lipid bi-layer reaction vessels such as liposomes or red blood cell ghosts, and/or scaled reaction chambers or the like. Such methods will allow for the identification of inhibitors of GA₄D enzymatic activity.

The transformed cells of the present invention allow for the screening for modulators of gibberellin biosynthesis in the presence of absence of oφ gene activity.

The methods of the invention will allow for the screening of candidate substances for the ability to modulate the biological activity of GA₄D, that of a gibberellin GA₄-desaturase enzyme.

A chemical substance identified as an inhibitor of GA₄D enzyme activity can thus be characterized and selected. Such methods are easily adaptable for screening combinatorial chemistry libraries of compounds, and the repeated serial limiting screening of mixtures of these compounds for the identification of suitable GA₄D inhibiting pools of chemical substances. Thus, the present invention also provides for chemical substances identified by such screening methods, identifiable by such, or corresponding to a chemical identified thereby.

A further aspect of the present invention encompasses the specific gibberellins produced by the cells of the claimed invention, or produced by cell generated by the methods of transformation of the claimed invention. Specifically, the present invention encompasses the gibberellin Al, A4 and A7, isolated from cells of the present invention, or by cells that have been transformed by the methods of the present invention to modulate gibberellin production. Gibberellin proteins can be isolated and purified from gibberellin producing cells using methods known in the art.

The present invention also provides for an optimized gibberellin producing culture media for producing gibberellins in cultures of Gibberella fujikori, wherein said media comprises a plant oil medium containing 15 g/L corn steep solid; 1.5 g/L (NH₄)₂SO₄; 60 g/L sunflower oil and 1 g/L KH₂PO₄.

Description of the Drawings

The present invention and its embodiments are better understood with reference to the drawings in which: Figure 1 is a diagram of the gibberellin biosynthetic pathway.

Figure 2 is a depiction of the nucleic acid sequence of a nucleic acid encoding for the oφ gene (SEQ ID NO. : 1), and the translated GA₄D protein (SEQ ID

NO. :2) encoded for by the protein coding domain segment (CDS) of the depicted nucleic acid. Figure 3 is a diagrammatic representation of the physical map of the Gibberella fujikuroi GA₄-desaturase encoding gene oφ, and the construction of a gene replacement vector.

Figure 4 graphically depicts the results of a typical Southern blot analysis of protein production from gibberellin producing fungi. Depicted are the wild-type SI (IMI58289), oφ disrupted transformed SI strains S1-T23 and S1-T55, P450-

3 inhibited strain 6314, oφ disrupted transformed 6314 strains 6314-T2 and

6314-T8.

Figure 5 depicts a HPLC-chromatogram analysis (GA-spectrum) of the wild-type

(SI), the GA₇ producing mutant 6314, and an oφ knock-out mutant 6314Δorf3- TI. The peaks are identified by labeled arrows (GA_l5 GA₃, GA₄, and GA₇).

Detailed Description of the Invention

The present invention provides for nucleic acids and methods for modulating the production of gibberellins in a cell which normally produces gibberellins, by manipulating the gibberellin biosynthetic pathway in the cell. In a related embodiment, the present invention provides for nucleic acid vectors, and host cells transformed by such vectors for the modulation of gibberellin production.

As used herein and above, the term "regulatory nucleic acid sequence", is meant to encompass nucleic acid sequences which regulate the expression of the expressible nucleic acid. Such regulatory nucleic acid sequences include and are not limited to enhancers, promoters, promoter binding sequences, and the like, where the regulation can be either inducible or constitutive.

By "operably joins", it is meant that the reading frame of the expressible nucleic acid corresponds with the function of the regulatory nucleic acid sequence such that the expression of the nucleic acid is controlled by the operation of the regulatory nucleic acid sequence.

By "intact, expressible", it is meant that the nucleic acid of the invention can be expressed and result in a functional GA₄D protein or fragment thereof.

A "disrupted" gene comprises a nucleic acid of the invention wherein the coding domain segment open reading frame is altered. For example and not limited thereby, the disrupted gene is altered such that it contains a deletion of nucleic acid residues, and/or contains an insertion of nucleic acid residues, or a point mutation which negatively effects the enzymatic activity of the expressed protein. Thus, a disrupted gene can result in no expressible protein, or the expression of an inhibited or inactive enzyme or fragment thereof.

Generally, techniques as used herein for the manipulation of nucleic acids and the production of vectors using recombinant DNA techniques are well known in the art. Many of the methods and materials for carrying out the basic molecular biology manipulations described are known in the art, and can be found in such references as Sambrook et al. , Molecular Cloning, 2nd edition,

Cold Spring Harbor Laboratory Press (1989); Berger et al., Guide to Molecular Cloning Techniques, Methods in Enz mology, Vol. 152, Academic Press, Inc., (1987); Davis et al. , Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc. (1986); Ausubel et al., Short Protocols in Molecular Biology. 2nd ed., John Wiley & Sons, (1992); Goeddel Gene Expression Technology. Methods in Enzymology, Vol. 185, Academic Press, Inc., (1991); Guthrie et al. , Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, Inc. , (1991); McPherson et al., PCR Volume 1, Oxford University Press, (1991); McPherson et al. , PCR Volume 2, Oxford University Press, (1995); Richardson, CD. ed., Baculovirus Expression

Protocols, Methods in Molecular Biology, Vol. 39, Humana Press, Inc. (1995).

It should be understood that the taxonomic or nomenclature term "Gibberella fujϊkon" , as used in the present application, encompasses all variations or equivalent nomenclature terms as might be used elsewhere in the art. A review of the Gibberella fujikori species complex, including alternative nomenclature terms, is found in O'Donnel et al., 1998, "Molecular Systematics and Phylogeography of the Gibberella-Fujikuroi Species Complex", Mvcologia, 90(3), 465-493; this review is incorporated herein by reference in its entirety. Modulation of Gibberellin Production Referring to the gibberellin biosynthetic pathway depicted in Figure 1, the isolation and characterization of the oφ gene allows for the manipulation and selective increased production of particular gibberellin intermediates such as GA₄ or GA₇, at the expense of the production of the normal major end product GA₃. As can be seen from Figure 1, the enzymatic step between GA₄ and GA_X (the minor end-product) is regulated by the activity of the P450-3 gene product.

The enzymatic step between GA₇ and the major end-product GA₃ is also regulated by the activity of the P450-3 gene product, which catalyzes both, the 13-hydroxylation of GA₇ and of GA₄.

Blockade of the biosynthetic pathway at the oφ regulated step, i.e. disruption of GA₄D activity, results in the accumulation of GA₄, and the minor end-product GAj. Blockade of the oφ regulated step in combination with blockade of the P450-3 regulated conversion of GA₄ to GA_j will further increase the yield of GA₄ at the expense of the production of GA_j. Blockade of the P450-3 regulated conversion in a cell, in the absence of oφ blockade, will result in the accumulation of GA₇, as both GAj and GA₃ production are inhibited.

Blockade of oφ regulated gibberellin biosynthetic step in a wild-type cell A wild-type Gibberella fujikuroi strain, SI (Accession No. IMI 58289,

IMI, Egham, Surrey, UK) producing GA₃ was obtained, and the oφ gene replaced with a disrupted oφ. From the transformants, 3 strains were selected, isolated, and cultivated in 20% ICI medium for 10 days, for further characterization, SlΔorf3-T23, SlΔorf3-T33, and SlΔorf3-T55. Table 1 below describes the results of analysis of the isolated oφ disrupted SI strains for gibberellin production. The data is reported as mg/L of the particular gibberellin product.

Table 1.

Strain GA3 GA1 GA4 GA7

SI 26 0 2 11

SlΔorf3-T23 0 498 37 0

SlΔorf3-T33 0 119 55 0

SlΔorf3-T55 0 162 62 0

Thus, blockade of the oφ regulated step results in an accumulation of GAj and GA₄ in the transformed cell.

Blockade of orf3 regulated gibberellin biosynthetic step in a P450-3 point mutation cell

A wild-type Gibberella fujikuroi strain, m567 (Fungal Culture Collection Weimar, Germany) producing GA₃ was obtained, and subjected to UV irradiation. The treated cells were screened and selected for a deficient P450-3 activity. The isolated strain, 6314 has a point mutation in the P450-3 gene which gives the expressed enzyme a low enzymatic activity. Cells of the 6314 strain thus produce large amounts of GA₇, (up to about 1000 mg/L GA₇ and only about 5-10% GA₃) due to the inhibition of the P450-3 regulated gibberellin biosynthetic steps. Taking cells of the 6314 strain, the endogenous oφ gene was replaced with a disrupted oφ using the replacement vector, as with the SI cells. From the transformants, 3 strains were selected, isolated, and cultivated in 20% ICI medium for 10 days, for further characterization, 6314Δorf3-Tl, 6314Δorf3-T2, and 6314Δorf3-T8.

Table 2 below describes the results of analysis of the isolated oφ disrupted 6314 strains for gibberellin production. The data is reported as mg/L of the particular gibberellin product.

Table 2.

Strain GA3 GA1 GA4 GA7

6314 18 0 34 414

6314Δorf3-T 0 146 165 0

6314Δorf3-T2 0 22 267 0

6314Δorf3-T8 0 8 381 0 Thus, blockade of the oφ regulated step in combination with an inhibition of the P450-3 regulated step results in an accumulation of GA_j and GA₄, and predominantly GA₄ in a selected transformed cell. Optimization of GA^ production from transformed cells

Modification of the culture media was undertaken to optimize production of gibberellins. It was found that a plant oil medium containing 15 g/L corn steep solid; 1.5 g/L (NH₄)₂SO₄; 60 g/L sunflower oil and 1 g/L KH₂PO₄ gave the best results.

Thus, culturing transformed cells of the invention under the appropriate gibberellin producing conditions can be optimized by further manipulation of the culture media.

Table 3 below summarizes the results of using the optimized medium, with yield in mg/L gibberellin. The data indicate that relatively pure isolated gibberellin GA₄ can be produced and isolated from cells, transformed with a disrupted oφ gene and having P450-3 of low activity, under optimized culture conditions. Table 3.

Strain GA3 GA1 GA4 GA7

6314 28 n.d.* 39 767

52 n.d. 28 813

6314Δorf3-Tl 0 n.d. 780 0

0 n.d. 740 0

6314ΔorO-T2 0 n.d. 737 0

0 n.d. 587 0

6314Δorf3-T8 0 n.d. 666 0

0 n.d. 642 0

*n.d. = not detectable

Construction of Replacement Vector

In order to disrupt the gene oφ, encoding the gibberellin desaturase (GA₄D), a gene replacement vector was constructed. As depicted graphically in Figure 3, the nucleic acid segment encoding for oφ and part of P450-4, which is located to the right of oφ, was cloned into vector pUC-19 (New England Biolabs; Yanisch-Perron, C , Vieira, J. and Messing, J., 1985, Gene 33: 103-119), and the new vector called pORF3-GR. An internal BamHI/Xbal-fragment was replaced by a hygromycin resistance cassette from the vector pGPCl (Desjardins AE, et al. 1992, "Detoxification of sesquiterpene phytoalexins by Gibberella pulicaris (Fusarium sambucinum) and its importance for virulence on potato tubers. J. Indust. Microbiol. 9:201-211. The insertion of this antibiotic resistance marker results in the disruption of the oφ gene open reading frame, and at the same time provides a selection marker for screening for successful transformants.

The Sα//-fragment with the mutated oφ gene (from pORF3-GR) was transformed into the Gibberella strains SI and 6314. This restriction enzyme cut fragment comprises the disrupted oφ gene, and the inserted antibiotic resistance marker. Other suitable fungal selection markers are known in the art (such as phleomycin, benomyl, nourseothricin, and BASTA (glufosinate)) and can be used in place of the specific hygromycin antibiotic resistance cassette illustrated by the example herein.

After expansion and selection under antibiotic control, 100 hygromycin resistant transformants were isolated and analyzed by Southern blot analysis for homologous integration of the disrupted oφ gene. The selected transformants were selected for loss of oφ activity by TLC and HPLC.

Figure 4 graphically depicts the results of a typical Southern blot analysis of protein production from gibberellin producing fungi. Depicted are the wild- type SI (IMI 58289), oφ disrupted transformed SI strains S1-T23 and S1-T55, P450-3 inhibited strain 6314, oφ disrupted transformed 6314 strains 6314-T2 and 6314-T8. In all of the oφ disrupted strains, the approximately 7 kb band corresponding to the wild-type copy of the gene oφ is missing.

All strains were purified by single spore isolation and cultivation in gibberellin production medium (20% ICI). The production of the various species of gibberellins can be monitored by HPLC-chromatography for the GA-spectrum of protein products. Figure 5 depicts a representative HPLC-chromatogram showing wild-type m567 at the top, mutant 6314 middle, and a transformed 6314 oφ disrupted strain at bottom. The peaks are identified by labeled arrows. Transformation of Gibberellin fujikuori Preparation of protoplasts

In 0.8% NaCl-solution as stabilizer was added the following enzymes: 1 % snail enzyme (Sigma Aldrich Chemical, St. Louis, MO), 0.5% novozyme 234 (Novo Nordisk), 0.1 % driselase (Sigma), and 0.1 % lyticase (Sigma). The enzyme solution was centrifuged and the supernatant filter sterilized. The fungus was grown overnight in CM-medium for 16-18 hours, about

0.5g of the filtered and 0.8% NaCl washed mycelium was incubated in 10 ml of the enzyme solution for 2-3 hours at 30°C on a shaker (100 rpm). The protoplasts were separated from the mycelium by filtration through glass filters (G2) and washed with 0.8% NaCl and then resuspended in 0.5-1.0 ml of STC buffer (1.3 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl₂). Approximately 10⁷ protoplasts (50 μl) of the fungus were transformed with approximately 10 μg of the prepared transformation vector. Transformation

To mixtures containing 50 μl protoplasts, at a concentration of about 5xl0⁸ per ml in STC buffer, 10 μg DNA transformant vector (the 2.7 kb Sall- fragment containing the mutated oφ and the hygromycin resistance selection marker cassette) and 50 μl polyethylene glycol solution (PEG solution; 25% PEG 6000 in STC) were added. The transformation mixture was incubated on ice for 25 min, and then an additional 2 ml of PEG solution was added. The mixture was mixed and kept at room temperature for 10 min before 4 ml of STC buffer was added. The transformation mixture was then added to 100 ml liquified regeneration medium (0.05% yeast extract (Difco, Detroit, MI), 0.7 M sucrose, and 2% agar) containing 125 μg/ml hygromycin B (Calbiochem, Bad Soden, Germany). Individual transformants appeared after about 3-4 days incubation at

28 °C. Selected transformants were transferred to CM-agar supplemented with 125 μg/ml hygromycin B. For further purification and isolation, single microconidia colonies were isolated and tested again for hygromycin resistance.

Biological Deposit Under the Budapest Treaty

The following specific strains have been deposited with the international depository Bureau of Microbiology at Health Canada (BMHC), Federal

Laboratories for Health Canada, Room H5190, 1015 Arlington Street,

Winnipeg, Manitoba, Canada R3E 3R2, under the Budapest Treaty. Table 4.

Strain Ace. No. Strain Ace. No. .

6314

6314Δorf3-Tl SlΔorf3-T23

6314Δorf3-T2 SlΔorf3-T33 6314Δorf3-T8 SlΔorf3-T55 The present invention provides for nucleic acids, vectors comprising nucleic acids, transformed cells, methods for modulating gibberellin biosynthesis, and methods for identifying modulators of gibberellin biosynthesis which are novel and useful for the controlled production of gibberellins from transformed cell cultures.

The invention, having been fully described in many of its aspects and claimed herein can be made and executed without undue experimentation by one of skill in the art according to the teaching herein. While the compositions and methods of this invention have been described by way of example above, it will be apparent to those of skill in the art that many variations and modifications can be applied to the compositions and methods described herein without departing from the concept, spirit and scope of the invention.

Claims

What is Claimed is:

I. An isolated nucleic acid comprising a gene encoding for a gibberellin A₄- desaturase enzyme which has an amino acid residue sequence corresponding to that depicted as SEQ ID NO. : 2. _* 2. A nucleic acid of claim 1 isolated from a Gibberella species.

3. A nucleic acid of claim 2 wherein said Gibberella species is Gibberella fujikori.

4. A nucleic acid of claim 1 having a nucleotide sequence corresponding to the nucleotide sequence depicted as SEQ ID NO. : l. 5. A nucleic acid of claim 4 having a nucleotide sequence corresponding to residues 984-2009 of the nucleotide sequence depicted as SEQ ID NO. : l. 6. An isolated gibberellin A₄-desaturase enzyme protein having an amino acid residue sequence corresponding to the amino acid residue sequence depicted as SEQ ID NO. : 2. 7. A protein of claim 6 isolated from Gibberella species.

8. A protein of claim 7 wherein said Gibberella species is Gibberella fujikori.

9. A protein of claim 6 produced by recombinant DNA methodology in a transformed host cell. 10. A protein of claim 9, wherein said host cell is a fungi.

II. A protein of claim 9 wherein said host cell is a bacteria.

12. A protein of claim 9 wherein said host cell is a mammalian cell.

13. A protein of claim 6 expressed as a fusion protein.

14. A biologically active fragment of a protein of claim 6, wherein said biological activity is a gibberellin A₄-desaturase activity.

15. An immunogenic fragment of a protein of claim 6.

16. A protein of claim 14 expressed as a fusion protein.

17. A protein of claim 15 expressed as a fusion protein.

18. An antibody specific for a protein of claim 6.

19. A nucleic acid vector comprising a nucleic acid having a gibberellin GA₄- desaturase encoding nucleic acid sequence corresponding to residues 984-2009 of the nucleic acid depicted as SEQ ID NO. : l.

20. A nucleic acid vector of claim 19 further comprising a nucleic acid regulatory element.

21. A nucleic acid vector of claim 20 wherein said regulatory element is a promoter.

22. A nucleic acid vector of claim 19 which can replicate in a transformed host cell. 23. A nucleic acid vector of claim 22, wherein said host cell is a bacteria.

24. A nucleic acid vector of claim 23, wherein said host cell is a fungi.

25. A nucleic acid vector of claim 24, wherein said host cell is mammalian.

26. A nucleic acid vector of claim 19, further comprising a selectable marker.

27. A nucleic acid vector of claim 19, wherein said vector can homologously recombine with a genomic nucleic acid of a transformed host cell, resulting in the exchange of an exogenous nucleic acid segment from said vector, with an endogenous nucleic acid segment from said transformed host cell genomic nucleic acid.

28. A nucleic acid vector of claim 27, further comprising a selectable marker in the exchanged nucleic acid segment.

29. A nucleic acid vector of claim 28, which is a restriction enzyme cut fragment of a larger nucleic acid.

30. A nucleic acid vector of claim 29, which is a Sail-fragment of vector pORF3-GR. 31. A nucleic acid vector of claim 19, wherein said gibberellin GA₄- desaturase encoding nucleic acid is disrupted.

32. A nucleic acid vector of claim 31, wherein said disruption is by point mutation.

33. A nucleic acid vector of claim 31, wherein said disruption is by altering the open reading frame of said gibberellin GA₄-desaturase encoding nucleic acid.

34. A nucleic acid vector of claim 33, wherein said disruption is by insertion of exogenous nucleic acid.

35. A nucleic acid vector of claim 34, wherein said exogenous nucleic acid encodes for a selectable marker. 36. A nucleic acid vector of claim 35, wherein said selectable marker is an antibiotic resistance gene.

37. A nucleic acid vector of claim 36, identified as pORF3-GR.

38. A nucleic acid vector of claim 33, wherein said disruption is by deletion of a portion of the nucleic acid. 39. A nucleic acid vector of claim 34, wherein said disruption is by incorporating inhibitory regulatory elements.

40. A host cell transformed with a nucleic acid vector of claim 19.

41. A cell of claim 40 identified as SlΔorf3-T23.

42. A cell of claim 40 identified as S lΔorf3-T33. 43. A cell of claim 40 identified as S lΔorf3-T55.

44. A cell of claim 40 that further comprises a disrupted P450-? encoding gene.

45. A cell of claim 44 wherein said disruption results in a P450-3 enzyme with lower enzymatic activity when compared with a wild-type P450-3 enzyme under similar conditions.

46. A cell of claim 40 that is a bacterial cell.

47. A cell of claim 40 that, is a fungal cell.

48. A cell of claim 47 identified as 6314Δorf3-Tl .

49. A cell of claim 47 identified as 6314Δorf3-T2. 50. A cell of claim 47 identified as 6314Δorf3-T8.

51. A host cell of claim 40 that is a nr-mmalian cell.

52. A method for modulating gibberellin biosynthesis in a cell producing gibberellins, said method comprising transforming said cell with a nucleic acid, vector of claim 19, and culturing said transformed cell under gibberellin producing conditions.

53. A method of claim 52, wherein said cell is identified as SI (Accession No. IMI 58289).

54. A method of claim 52, wherein said cell producing gibberellins further comprises a disrupted P450-3 encoding gene. 55. A method of claim 54, wherein said disruption comprises a point mutation that results in expression of a P450-3 enzyme that has lower activity than a wild-type P450-3 enzyme under similar conditions.

56. A method of claim 55, wherein said cell is identified as 6314.

57. A method for identifying inhibitors of the enzymatic activity of isolated GA₄D protein said method comprising contacting GA₄D protein with a candidate inhibitor under conditions suitable for enzymatic activity of GA₄D protein, and detecting any decrease in said enzymatic activity.

58. A method of claim 57 wherein said candidate inhibitor is selected from the group consisting of small chemical molecules, complex compounds, protein, and antibodies.

59. A method of claim 57 performed in vitro.

60. A method of claim 57 performed in a cell.

61. A method of claim 57 performed in a liposome or red blood cell ghost.

62. A candidate inhibitor identified by the method of claim 57. 63. A candidate inhibitor of claim.62 identified by screening a combinatorial chemistry library of compounds.

64. An inhibitor corresponding to that of claim 62.

65. An inhibitor identifiable by the method of claim 57.

66. A gibberellin A_l5 produced by a host cell of claim 40. 67. A gibberellin A_l5 produced by a host cell of claim 41.

68. A gibberellin A_1; produced by a host cell of claim 42.

69. A gibberellin A_l5 produced by a host cell of claim 43.

70. A gibberellin A₄, produced by a host cell of claim 40.

71. A gibberellin A₄, produced by a host cell of claim 41. 72. A gibberellin A₄, produced by a host cell of claim 42.

73. A gibberellin A₄, produced by a host cell of claim 43.

74. A gibberellin A₄, produced by a host cell of claim 48.

75. A gibberellin A₄ of claim 74 wherein the cell was cultured in an optimized media. 76. A gibberellin A₄ of claim 75, wherein said media comprises a plant oil medium containing 15 g/L corn steep solid; 1.5 g/L (NH₄)₂SO₄; 60 g/L sunflower oil and 1 g/L KH₂PO₄.

77. A gibberellin A₄, produced by a host cell of claim 49.

78. A gibberellin A₄ of claim 77 wherein the cell was cultured in an optimized media.

79. A gibberellin A₄ of claim 78, wherein said media comprises a plant oil medium containing 15 g/L corn steep solid; 1.5 g/L (NH₄)₂SO₄; 60 g/L sunflower oil and 1 g/L KH₂PO₄.

80. A gibberellin A₄, produced by a host cell of claim 50. 81. A gibberellin A₄ of claim 80 wherein the cell was cultured in an optimized media. 82. A gibberellin A₄ of claim 81, wherein said media comprises a plant oil medium containing 15 g/L corn steep solid; 1.5 g/L (NH₄)₂SO₄; 60 g/L sunflower oil and 1 g/L KH₂PO₄. 83. An isolated gibberellin A_l9 produced by a host cell generated by the method of claim 52.

84. An isolated gibberellin A_1; produced by a host cell produced by the method of claim 55.

85. An isolated gibberellin A_1; produced by a host cell produced by the method of claim 56.

86. An isolated gibberellin A₄, produced by a host cell generated by the method of claim 52.

87. An isolated gibberellin A₄, produced by a host cell produced by the method of claim 55.

88. An isolated gibberellin A₄, produced by a host cell produced by the method of claim 56.

89. A gibberellin A₄ of claim 88 wherein the cell was cultured in an optimized media. 90. A gibberellin A₄ of claim 89, wherein said media comprises a plant oil medium containing 15 g/L corn steep solid; 1.5 g/L (NH₄)₂SO₄; 60 g/L sunflower oil and 1 g/L KH₂PO₄. 91. A culture media, wherein said media comprises a plant oil medium containing 15 g/L corn steep solid; 1.5 g/L (NH₄)₂SO₄; 60 g/L sunflower oil and 1 g/L KH₂PO₄.