HK1059800A - High level production of p-hydroxybenzoic acid in green plants - Google Patents

Description

High level production of para-hydroxybenzoic acid in green plants

This application claims the benefit of U.S. provisional application No. 60/209,854, filed on 2/6/2000.

Technical Field

The present invention relates to the field of plant gene expression and molecular biology and microbiology. More specifically, a method is provided for the production of para-hydroxybenzoic acid (pHBA) in green plants, relying on expression of a unique expression cassette comprising a gene encoding chorismate pyruvate lyase operably linked to a specific chloroplast targeting sequence.

Background

Para-hydroxybenzoic acid (pHBA) is a Liquid Crystal Polymer (LCP) -Zenite^TMThe main monomer component (. about.65% by weight). LCP has characteristics superior to conventional resins, such as high strength/stiffness, low melt viscosity, excellent environmental resistance, retention of characteristics at higher temperatures, and low gas permeability. However, the cost of the current synthetic method for synthesizing pHBA (Kolbe-Schmitt reaction (Kolbe and Lautemann, Ann. 113: 125(1869)) is prohibitive, and the inexpensive route to synthesize LCP monomers will be exploited for many new applications in the automotive, electronics and other industries.

pHBA has been produced in microbial systems. For example, JP 06078780 discloses the preparation of pHBA by culturing benzoic acid in the presence of a microorganism, preferably Aspergillus, which oxidizes benzoic acid to pHBA. In addition, Enterobacter (Enterobacter) strains capable of converting p-cresol into pHBA have been isolated from soil (JP 05328981). Furthermore, JP05336980 and JP 05336979 disclose isolates of Pseudomonas putida (Pseudomonas putida) capable of producing pHBA from p-cresol. Also, commonly owned WO 9856920 discloses a method for producing pHBA from toluene using a Pseudomonas mendocina (Pseudomonas mendocina) mutant incapable of expressing a parahydroxybenzoate hydroxylase (pHBH). Finally, U.S.6030819 discloses the production of pHBA in genetically engineered escherichia coli (e.coli) expressing the Chorismate Pyruvate Lyase (CPL) gene.

Despite these successes, the ability to produce commercially useful quantities of pHBA on a microbial platform has been hampered by the use of toxic feedstocks and limited biomass. A method for pHBA production that solves these problems is required.

Coincidentally, pHBA is naturally present in almost all plants, animals and microorganisms, albeit in trace amounts. In many bacteria, the production of pHBA occurs via the chorismate pathway, which is an important branch point intermediate in the synthesis of many aromatic compounds, including phenylalanine, tyrosine, p-aminobenzoic acid, and ubiquinone. In E.coli, chorismate itself undergoes 5 different enzymatic reactions, yielding 5 different products, and the enzyme ultimately responsible for the synthesis of pHBA is chorismate pyruvate lyase, known as CPL. The latter is the product of the E.coli ubiC gene, which was cloned independently by two different research groups (Siebert et al, FEBS Lett 307: 347-350 (1992); Nichlols et al, J.Bacteriol 174: 5309-5316 (1992)). The enzyme is a 19kDa monomeric protein and does not require known cofactors or energies. CPL by eliminating C of its sole substrate₃The side chain of enol pyruvyl can catalyze the direct conversion of 1mol of branched acid into 1mol of pyruvic acid and 1mol of pHBA. Recombinant CPL has been overexpressed in E.coli, purified to homogeneity, and partially characterized biochemically and kinetically (Siebert et al, Microbiology 104: 897-904; Nichlols et al, J.Bacteriol 174: 5309-5316 (1992)). In addition, detailed mechanisms for the enzymatic reaction of CPL have also been proposed (Walsh et al, ChemRev.90: 1105-1129).

The fact that pHBA plants have all the necessary enzymatic machinery for the synthesis of pHBA has been found in plants, already in carrot tissue (Schnitzler et al, Planta, 188, 594, (1992)), in many grasses and crops (Lydon et al, j.agric. food. chem., 36, 813, (1988)), in poplar lignin (Terashima et al, Phytochemistry, 14, 1991, (1972)), and in many other plant tissues (Billek et al, oesterr. chem., 67, 401, (1966)) suggests that they may be a useful platform for the production of such monomers. Despite the obvious benefits of using plants as a tool for producing pHBA, the high level production of the monomer is still not clear.

One difficulty to overcome is the metabolic fate of chorismate in plant tissues. In fact, the production of pHBA from chorismate is much more complex in higher plants than microorganisms, since the former lacks enzymes functionally equivalent to CPL. For example, the biosynthetic pathway for the production of pHBA in Lithospermum erythrorhizon (Lithospermum erythrorhizon) is thought to consist of up to 10 consecutive reactions (Loscher and Heide, Plant physiol.106: 271-279(1992)), presumably all catalyzed by different enzymes. In addition, most of the enzymes catalyzing these reactions have not been identified, nor have their genes cloned. Even little information is available about how pHBA is synthesized in other plant species. For a more complex problem, those enzymes known to be involved in plant pHBA production span two different pathways, which are differentially regulated and located in different cellular compartments. Chorismic acids are therefore intermediates of the shikimate pathway which are mainly restricted to chloroplasts and other types of plastids (Siebert et al, Plant physiol.112: 811-819 (1996)); sommer et al, Plant CellPhysiol.39 (11): 1240-1244(1998)), and all intermediates downstream of phenylalanine belong to the phenylpropanoid (phenylpropanoid) pathway that occurs in the cytosol and endoplasmic reticulum.

Although there is little understanding of how plants normally synthesize pHBA and the enzymes involved in this process, transgenic plants that accumulate significantly higher levels of pHBA than wild-type plants have been described. For example, Kazufumi Yazaki, (Baiosaiensis to Inductori (1998), 56(9), 621-622) discusses introducing a CPL-encoding gene into tobacco to produce pHBA in an amount sufficient to impart insect resistance. Also, Siebert et al, (Plant Physiol.112: 811-819(1996)) have demonstrated that tobacco plants (Nicotiana tabacum) transformed with a constitutively expressing chloroplast-targeted form of E.coli CPL (referred to as "TP-Ubic) have elevated pHBA levels at least 3 orders of magnitude higher than wild-type plants (WO 96/00788, granted DE 4423022). Interestingly, the genetically modified tobacco plants contained only minor amounts of free pHBA. Virtually all of the compound (. about.98%) was converted to two glucose conjugates-a phenolic glucoside and an ester glucoside-present in a ratio of about 3: 1(Siebert et al, Plant Physiol.112: 811-819 (1996); Li et al, Plant Cell Physiol.38 (7): 844-850 (1997)). Both glucose conjugates are 1- β -D-glucosides with a single glucose residue covalently linked to the hydroxyl or carboxyl group of pHBA. The best transgenic plants identified in this study had a total pHBA glucoside content of about 0.52% (dry weight) when leaf tissue was analyzed. The actual amount of pHBA produced in transgenic tobacco plants was only about half of this value, corrected for the relevant glucose residues.

In a recent study, the same artificial fusion protein was expressed in transformed tobacco Cell cultures using both a constitutive promoter (Sommer et al, Plant Cell Physiol.39 (11): 1240-1244(1998)) and an inducible promoter (Sommer et al, Plant Cell Reports 17: 891-896 (1998)). Although the accumulation of pHBA glucoside was slightly higher than in the original study with whole plants, the level did not exceed 0.7% (dry weight) in both cases. In contrast, when TP-Ubic was examined in the hairy root culture of Lithospermum erythrorhizon (Sommer et al, Plant Molecular Biology 39: 683-693(1999)), the pHBA glucoside content reached a level as high as 0.8% (dry weight) after correction based on the endogenous level of the untransformed control culture.

Although these studies demonstrate the feasibility of using genetic engineering to increase pHBA levels in higher plants, the above TP-Ubic artificial fusion protein does not produce commercially useful amounts of the compound. Such efforts would require increasing the pHBA content of agronomically suitable plants to levels 10-20 fold previously reported. Thus, there is a need for one or more modifications to the current systems to achieve these levels. Since the substrate chorismate of CPL is synthesized in plastids, one potential aspect for improvement may be to design better chloroplast targeting sequences to achieve higher levels of enzymatic activity in the cellular compartment of interest. Indeed, in several of the above studies, a positive correlation was evident between CPL enzyme activity and pHBA glucoside accumulation (Siebert et al, Plant physiol.112: 811-819 (1996); Sommer et al, Plant Cell physiol.39 (11): 1240-1244 (1998); Sommer et al, Plant Cell Reports 17: 891-896 (1998)). Furthermore, in these studies, there was no evidence that the CPL enzyme activity of the system would be saturated using the TP-UbiC artificial fusion protein.

It is well known that most naturally occurring chloroplast proteins are nuclear-encoded, synthesized as larger molecular weight precursors, with a cleavable N-terminal polypeptide extension called a transit peptide. It is also generally accepted that transit peptides contain all the information necessary for transit to the chloroplast. Although the mechanistic details of protein import remain to be elucidated, several important facts have emerged: (a) precursor uptake occurs post-translationally (Chua and Schmidt, Proc Natl.Acad.Sci.75: 6110-6114 (1978); Highfield and Ellis, Nature 271: 420-424(1978)) and is mediated by proteinaceous receptors present in the chloroplast envelope (Cline et al, J.biol.chem.260: 369132696 (1985)); (b) ATP hydrolysis is the only driving force for transport (Grossman et al, Nature 285: 625-628 (1980); Cline et al, J.biol.chem.260: 3691-3696 (1985)); (c) fusion of transit peptides to foreign proteins is sometimes, but not always, in vivo (Van den Broeck et al, Nature 313: 358-362 (1985)); schreier et al, EMBO J.4: 25-32(1985)) and in vitro (Wasmann et al, mol.Gen.Genet.205: 446-453(1986)) is sufficient to trigger uptake into chloroplasts; finally, (d) following chloroplast import, the transit peptide is proteolytically removed from the precursor protein, thereby producing the "mature" polypeptide. Although the complete sequence of thousands of transit peptides is now known, manipulation of these sequences to achieve optimal targeting of foreign proteins and expression in the plant chlorophyll compartment remains the subject of trial and error. However, it is well established that simply attaching a transit peptide to a foreign protein does not necessarily ensure efficient uptake or correct processing by the chloroplast. Even when The same targeting sequence is fused to different proteins, The results are completely unpredictable (Lubben et al, The Plant Cell 1: 1223-1230(1989)), with different transport efficiencies for different passenger proteins. The reason for this is not clear, however, it has been suggested that chloroplast uptake and transit peptide removal are somehow coupled and that certain artificial fusion proteins are either not processed or are processed inefficiently. For example, it has been shown that even very subtle changes near the natural cleavage site of Rubisco small subunit precursors can lead to processing abnormalities (Robinson and Ellis, Eur.J.biochem.142: 342-346 (1984); Robinson and Ellis, Eur.J.biochem.152: 67-73(1985)) and reduced chloroplast uptake (Wasmann et al, J.biol.chem.263: 617-619 (1988)).

Some improvement in this regard can be achieved by including in the chloroplast targeting sequence not only the transit peptide and scissile bond, but also a small portion of the mature N-terminus of the transit peptide donor. In fact, this approach has worked both in vivo and in vitro for another bacterial protein, neomycin phosphotransferase II (NPT-II) (Van den Broeck et al, Nature 313: 358-362 (1985); Schreier et al, EMBO J.4: 25-32 (1985); Wasmann et al, mol. Gen. Genet.205: 446-453 (1986); Herrera-Estralla et al, EP 0189707; U.S.5,728,925; U.S.5,717,084). Thus, the uptake by chloroplasts of the chimeric protein consisting of the transit peptide of the Rubisco small subunit precursor plus the first 22 residues of mature Rubisco fused to the N-terminus of NPT-II is much better than for a similar construct containing only the transit peptide and scissile bond. However, this strategy is not very simple and still correlates with a high unpredictability that the linkage to the passenger protein is not releasable. This is most readily seen in the literature attempting to target CPL to chloroplasts. For example, Sommer et al, Plant Cell Physiol.39 (11): 1240-1244(1998) describes a similar artificial fusion protein comprising the CPL gene product fused at its N-terminus to the transit peptide and the first 21 amino acid residues of the Rubisco small subunit (e.g., "TP 21 Ubic"). While this modification would be expected to improve chloroplast uptake and processing, cells containing the original construct TP-UbiC had much higher levels of both CPL enzyme activity and pHBA glucoside. Thus, the use of the Wasmann et al disclosure (mol. Gen Genet.205: 446-453(1986)) has a deleterious effect on a different protein.

Therefore, the problem to be solved is to provide a method for producing pHBA in plants at a commercially useful level using a chemical reaction catalyzed by the bacterial protein CPL. This is a particularly unusual goal, since, on top of all the complications mentioned above, it is clear from said document that certain N-terminal modifications of E.coli CPL may lead to considerable loss of enzymatic activity (Siebert et al, Plant Physiol.112: 811-819 (1996)). Thus, it is not only necessary to identify artificial fusion proteins that are efficiently imported into chloroplasts, but also that are processed by proteases to produce unmodified CPL or CPL variants with N-terminal extensions that do not interfere with enzymatic activity. The prior art does not address the solution to this problem. The applicant has solved the problem by constructing a novel artificial fusion protein that enables CPL enzyme activity to be expressed in chloroplasts at sufficiently high levels to accumulate commercially useful levels of pHBA.

Summary of The Invention

The present invention provides a method for producing pHBA in a green plant, the method comprising:

a) providing a green plant having an endogenous source of chorismate and containing a chorismate pyruvate lyase expression cassette having the structure:

P-T-C-D-CPL

wherein:

p is a promoter suitable for driving expression of a chorismate pyruvate lyase gene;

t is a nucleic acid molecule encoding a ribulose-1, 5-bisphosphate carboxylase (Rubisco) chloroplast transit peptide;

c is a nucleic acid molecule encoding a Rubisco chloroplast transit peptide cleavage site;

d is a nucleic acid molecule encoding about 4-20 contiguous amino acids of the N-terminal portion of the Rubisco chloroplast transit peptide donor polypeptide; and

CPL is a nucleic acid molecule encoding a mature chorismate pyruvate lyase protein; wherein P, T, C, D and CPL are each operably linked such that expression of the cassette results in translation of a chimeric protein comprising a chloroplast targeting sequence fused to the N-terminus of a mature chorismate pyruvate lyase protein;

b) cultivating the plant under conditions such that the chimeric protein can be expressed and transported to chloroplasts for conversion of chorismic acid to paraben glucosides and paraben derivatives;

c) recovering para-hydroxybenzoic acid and para-hydroxybenzoic acid derivatives from said plant; and

d) processing the paraben glucoside and paraben derivative into free paraben.

In particular, the method of the invention produces p-hydroxybenzoic acid glucoside in plants at a concentration higher than 2% of the dry weight of the plant biomass, preferably at a concentration higher than 10%.

In addition, the present invention provides a chorismate pyruvate lyase expression cassette comprising a chimeric gene having a nucleic acid molecule encoding a chloroplast-targeting sequence derived from the small subunit of ribulose-1, 5-bisphosphate carboxylase, the chloroplast-targeting sequence having the amino acid sequence of SEQ ID NO: 15 operably linked to a sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and a chorismate pyruvate lyase of the amino acid sequence shown in figure 4.

Brief description of the figures and sequence descriptions

Figure 1 shows a primary amino acid sequence alignment of two different chloroplast-targeted forms of CPL. Both of these forms are artificial fusion proteins. The form in line 3 corresponds to TP-Ubic used in the previous studies (Siebert et al, Plant physiol.112: 811-819(1996) Sommer et al, Plant Cell physiol.39 (11): 1240-1244 (1998); Sommer et al, Plant Cell Reports 17: 891-896 (1998); Sommer et al, Plant molecular biology 39: 683-693(1999)), while the form in line 2 corresponds to TP-CPL developed in this study. Coli (e.coli) CPL (line 4) and tomato Rubisco small subunit precursor to rbcS2 (line 1) are also included in this alignment. The amino acid residues corresponding to the "mature" Rubisco small subunit are shown in bold. The N-terminal chloroplast transit peptide of Rubisco small subunit precursor is shown in plain text. The primary amino acid sequence of E.coli CPL is shown in italics. Arrows indicate highly conserved Cys-Met junctions (Mazur et al, Nuc Acids Res.13: 2373-2386 (1985); Berry-Lowe et al, J.mol.and appl.Gen.1, 483-498(1982)), where transit peptide cleavage occurs normally, yielding mature Rubisco small subunits.

FIG. 2 shows a schematic representation (circular diagram) of the intermediate plasmid "TP-CPL-pML 63" and the relevant restriction sites.

FIG. 3 shows a schematic representation (circled) of a binary vector plant expression construct "TP-CPL-pZBL 1" for transformation of tobacco and Arabidopsis after introduction of Agrobacterium.

FIG. 4 shows a representative HPLC profile of leaf tissue extracts prepared from a TP-CPL expressing transgenic tobacco plant (transformant #5) compared to wild type plants.

FIG. 5 shows the total pHBA-glucoside content of 15 different transgenic tobacco plants expressing TP-CPL. This analysis was performed on fresh leaf material obtained 5 weeks after transfer of the primary transformants to soil.

FIG. 6 shows age-dependent accumulation of total pHBA glucoside in transgenic tobacco plants expressing TP-CPL. This analysis was performed on leaf tissue obtained from primary transformants at various developmental stages. Total pHBA glucoside is expressed as a dry weight percentage.

FIG. 7 shows Western blots of wild type tobacco plants (lane 9) and transgenic tobacco plants expressing TP-CPL (lanes 1-7). This analysis was performed on leaf tissue obtained from primary transformants at 5 weeks of age. Lane 8 contains 20ng of purified recombinant Δ TP-CPL (e.g., predicted chloroplast cleavage product of TP-CPL). After SDS-PAGE, the proteins were transferred to nitrocellulose and probed with a 1: 200 dilution of anti-CPL antiserum.

The present invention may be understood more fully from the following detailed description and the accompanying sequence descriptions which form a part of this application.

Applicants provide 16 sequences which are in accordance with 37C.F. R.1.821-1.825 ("Requirements for Patent Applications relating to Nucleotide sequences and/or Amino Acid Sequence sequences-the Sequence Rules"), and with World Intellectual Property Organization (PO) Standard ST.25(1998) and Sequence listing Requirements for EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the advanced instruments constructs). The symbols and formats used for nucleotide and amino acid sequence data comply with the rules of 37c.f.r. § 1.822.

SEQ ID NO: 1 is a 5' primer useful for introducing E.coli CPL having Genbank accession number M96268 into E.coli expression vector pET-24a (+) (Novagen).

SEQ ID NO: 2 is a 3' primer useful for introducing E.coli CPL having Genbank accession number M96268 into E.coli expression vector pET-24a (+) (Novagen).

SEQ ID NO: 3 is the nucleotide sequence of ORF of Escherichia coli CPL with Genbank accession number M96268 in Escherichia coli expression vector pET-24a (+) (Novagen).

SEQ ID NO: 4 is the primary amino acid sequence of ORF of E.coli CPL with Genbank accession number M96268 in E.coli expression vector pET-24a (+) (Novagen).

SEQ ID NO: 5 is a 5' primer that can be used to amplify chloroplast targeting sequences of tomato Rubisco small subunit precursors for expression of TP-CPL in E.coli.

SEQ ID NO: 6 is a 3' primer that can be used to amplify chloroplast targeting sequences of tomato Rubisco small subunit precursors for expression of TP-CPL in E.coli.

SEQ ID NO: 7 is the nucleotide sequence of the ORF of the chloroplast targeting CPL fusion protein (TP-CPL) in the Escherichia coli expression vector pET-24a (+) (Novagen).

SEQ ID NO: and 8 is the primary amino acid sequence of ORF of chloroplast targeting CPL fusion protein (TP-CPL) in the Escherichia coli expression vector pET-24a (+) (Novagen).

SEQ ID NO: 9 is a 5' primer that can be used to amplify the predicted chloroplast cleavage product of TP-CPL (Δ TP-CPL) and insert it into the E.coli expression vector pET-24d (+) (Novagen).

SEQ ID NO: 10 is a 3' primer that can be used to amplify the predicted chloroplast cleavage product (Δ TP-CPL) of TP-CPL and insert it into the E.coli expression vector pET-24d (+) (Novagen).

SEQ ID NO: 11 is a 5' primer that can be used to amplify and modify TP-CPL without altering its primary amino acid sequence for insertion into the in vitro transcription/translation vector pCITE4a (+) (Novagen).

SEQ ID NO: 12 is a 3' primer that can be used to amplify and modify TP-CPL without altering its primary amino acid sequence for insertion into the in vitro transcription/translation vector pCITEAa (+) (Novagen).

SEQ ID NO: 13 is a 5 'primer useful for amplifying a truncated form of the 3' NOS termination sequence using plasmid pMH40 as a template.

SEQ ID NO: 14 are truncated forms of the 3 'primers that can be used to amplify the 3' NOS termination sequences using plasmid pMH40 as a template.

SEQ ID NO: 15 is a chloroplast targeting sequence derived from the small subunit of tomato ribulose-1, 5-bisphosphate carboxylase.

SEQ ID NO: 16 is a processed chloroplast targeting CPL fusion protein (TP-CPL).

Detailed Description

The present invention provides a method for producing p-hydroxybenzoic acid (pHBA) at a commercially useful level in green plants at a high level. pHBA is used as a monomer in liquid crystal polymers used in the automobile industry, the electronics industry and other industries.

The method relies on the efficient expression of a gene encoding a modified form of the enzyme Chorismate Pyruvate Lyase (CPL), which catalyzes the direct conversion of 1mol chorismate to 1mol pyruvate and 1mol pHBA. Introducing said CPL variant into a green plant in the form of an expression cassette comprising said CPL coding sequence operably linked to a suitable promoter capable of driving protein expression in a plant. In addition, the expression cassette contains a DNA segment immediately upstream and adjacent to the CPL coding sequence, encoding the chloroplast transit peptide, its natural cleavage site, and a small portion of the transit peptide donor polypeptide. The function of the transit peptide is to target the chimeric protein encoded by the expression cassette to the chloroplast and enable it to be taken up into the organelle responsible for the synthesis of chorismic acid, which is the substrate of CPL, converted into pHBA. The cleavage site is unique to the original transit peptide donor, and cleavage of the artificial protein encoded by the cassette at this site releases a novel polypeptide comprising at its N-terminus the mature CPL enzyme of a small portion of the transit peptide donor.

In this specification, a number of terms and abbreviations are used. The following definitions are provided.

"polymerase chain reaction" is abbreviated PCR.

"chorismate pyruvate lyase," abbreviated CPL, refers to a gene encoding an enzyme that catalyzes the conversion of chorismate to pyruvate and pHBA.

"parahydroxybenzoic acid (Para-hydroxybenzoic acid)" or "parahydroxybenzoic acid (P-hydroxybenzoic acid)" is abbreviated as pHBA.

The term "p-hydroxybenzoic acid glucoside" or "pHBA glucoside" refers to a conjugate comprising pHBA and a glucose molecule.

The term "pHBA derivative" refers to any conjugate of pHBA that can be formed in plants due to the catalytic activity of the CPL enzyme.

The term "transit peptide" or "chloroplast transit peptide", which will be abbreviated as "TP", refers to the N-terminal portion of a chloroplast precursor protein that targets the chloroplast precursor protein to the chloroplast and is subsequently cleaved off by a chloroplast processing protease.

The term "chloroplast targeting sequence" refers to any polypeptide extension linked to the N-terminus of a foreign protein so as to be transported into the chloroplast. In the case of naturally occurring chloroplast precursor proteins, transit peptides are considered chloroplast targeting sequences, although optimal uptake and protease processing may depend in part on the "mature" portion of the chloroplast protein.

The term "transit peptide donor sequence" refers to the portion of the chloroplast targeting sequence derived from the "mature" portion of the chloroplast precursor protein. The transit peptide donor sequence is always located downstream of the transit peptide cleavage site and immediately adjacent to the transit peptide cleavage site that separates the transit peptide from the mature chloroplast protein.

The term "chloroplast processing protease" refers to a protease that is capable of cleaving a frangible bond between a transit peptide and a mature chloroplast protein.

The term "transit peptide cleavage site" refers to the site in the chloroplast targeting sequence between two amino acids acted on by the chloroplast processing protease.

As used herein, an "isolated nucleic acid fragment" is a polymer of RNA or DNA that is single-or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.

"Gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding (5 'non-coding sequences) and following (3' non-coding sequences) the coding sequence. "native gene" refers to a gene found in nature with its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different than that found in nature. "endogenous gene" refers to a native gene that is in its natural location in the genome of an organism. "foreign" gene refers to a gene that does not normally exist in a host organism but is introduced into the host organism by gene transfer. The foreign gene may comprise a native gene inserted into a non-native organism, or a chimeric gene. A "transgene" is a gene that has been introduced into the genome by transformation.

A "synthetic gene" can be assembled from oligonucleotide building blocks that can be chemically synthesized using methods known to those skilled in the art. These constructs are ligated and annealed to form gene segments, which are then enzymatically assembled to construct the complete gene. "chemical synthesis" in relation to a DNA sequence means that the component nucleotides will be assembled in vitro. Artificial chemical synthesis of DNA can be accomplished by well-established methods or can be automated using one of a number of commercially available machines. Thus, the gene can be tailored for optimal gene expression based on optimization of the nucleotide sequence to reflect host cell codon bias. The skilled artisan will recognize that successful gene expression may be achieved if codon usage is biased towards codons preferred by the host. Determination of preferred codons can be based on available sequence information, a survey of genes derived from the host cell.

"coding sequence" refers to a DNA sequence that encodes a particular amino acid sequence. "suitable regulatory sequences" refer to nucleotide sequences located upstream (5 'non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and which affect transcription, RNA processing or stability or translation of the linked coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures.

"promoter" refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, a coding sequence is located 3' to a promoter sequence. Promoter sequences consist of proximal and more distal upstream elements, the latter elements often being referred to as enhancers. Thus, an "enhancer" is a nucleotide sequence that can stimulate the activity of a promoter, either an intrinsic element of the promoter or a heterologous element inserted to increase the level or tissue specificity of the promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide segments. One skilled in the art will recognize that different promoters may direct gene expression in different tissues or cell types, or at different stages of development or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types most of the time are commonly referred to as "constitutive promoters". Various types of novel promoters are frequently found useful in plant cells, a large number of examples being found in Okamuro and Goldberg editors (1989) Biochemistry of Plants 15: 1-82. It will further be appreciated that nucleic acid fragments of different lengths may have the same promoter activity, since in most cases the actual boundaries of the regulatory sequences have not yet been fully defined.

"3' non-coding sequence" refers to a DNA sequence located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. Polyadenylation signals are generally identified by affecting the addition of polyadenylic acid stretches at the 3' end of the mRNA precursor.

The term "operably linked" refers to the linkage of nucleic acid sequences on a single nucleic acid fragment such that the function of one sequence is affected by the other sequence. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to the regulatory sequence in sense or antisense orientation.

The term "expression" as used herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

"mature" protein refers to a polypeptide that is post-translationally processed; i.e., a polypeptide from which any propeptide or propeptide present in the primary translation product has been removed. By "precursor" protein is meant the primary translation product of the mRNA, i.e., the propeptide and propeptide are still present. The propeptides and propeptides may be, but are not limited to, intracellular localization signals.

"transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing such transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

The terms "plasmid", "vector" and "cassette" refer to an extra-chromosomal element, which often carries a moiety that is not metabolized in the center of the cell, usually in the form of a circular double-stranded DNA molecule. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular single-or double-stranded DNA or RNA, derived from any source, many of which have been ligated or recombined into a single construct capable of introducing into a cell the DNA sequences of the promoter fragment and the gene product of choice, as well as appropriate 3' untranslated sequences. "transformation cassette" refers to a specific vector that contains an exogenous gene and has the ability to facilitate transformation of a specific host cell in addition to the exogenous gene. "expression cassette" refers to a specific vector containing a foreign gene and having elements other than the foreign gene that allow for enhanced expression of the gene in a foreign host.

Standard recombinant DNA techniques and molecular cloning techniques used herein are well known in the art and are described in Sambrook, j., Fritsch, e.f. and maniotis, t.,Molecular Cloning：A Laboratory Manual，Second Editioncold Spring Harbor laboratory Press, Cold Spring Harbor, NY (1989) (hereinafter referred to as "Maniatis"); and Silhavy, t.j., Bennan, m.l. and Enquist, l.w.,Experiments with Gene Fusionscold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and Ausubel, f.m., etc.,Current Protocols in Molecular Biologypublished by Greene Publishing Assoc. and Wiley-Interscience (1987).CPL expression cassette

The present invention provides an expression cassette useful for expressing a modified form of Chorismate Pyruvate Lyase (CPL) with full activity and targeting the polypeptide to chloroplasts of a host plant. Typically, the expression cassette will comprise (1) a cloned CPL gene under the transcriptional control of 5 'and 3' regulatory sequences and (2) a dominant selectable marker. The expression cassettes of the invention may also contain a promoter regulatory region (e.g.a promoter regulatory region conferring inducible or constitutive, environmental or developmental regulation, or cell or tissue specific/selective expression), a transcription initiation site, a ribosome binding site, an RNA processing signal, a transcription termination site and/or a polyadenylation signal. In a preferred embodiment, the cassette of the invention will additionally contain a sequence encoding a transit peptide and a sequence encoding a portion of the transit peptide donor which contains a transit peptide cleavage site suitable for processing by a host plant cell chloroplast processing protease. The cassettes of the invention optionally may also contain one or more introns to facilitate CPL expression.

The CPL gene encodes an enzyme that converts 1mol chorismate to 1mol pyruvate and 1mol pHBA. The most well characterized CPL gene has been isolated from E.coli under GenBank accession number M96268.

Promoters useful for driving the CPL gene of the present invention are numerous and well known in the art. Suitable promoters will be those that function in plants, typically derived from a plant host harboring the CPL expression cassette. Any combination of any promoter and any terminator capable of inducing expression of said CPL gene can be used in the cassette of the invention. Some suitable examples of promoters and terminators include promoters and terminators derived from nopaline synthase (nos), octopine synthase (ocs), and cauliflower mosaic virus (CaMV) genes. One type of useful plant promoter that can be used is a high level plant promoter. Such a promoter, operably linked to a genetic sequence of the invention, should be capable of promoting expression of the gene product of the invention. High level plant promoters that may be used in the present invention include, for example, the promoters of the small subunit of ribulose-1, 5-bisphosphate carboxylase (ss) from soybean (Berry-Lowe et al, J.molecular and App.Gen., 1: 483-4981982)), and the promoters of chlorophyll a/b binding proteins. Both promoters are known to be light-inducible in plant cells (see, e.g., forGenetic Engineering of Plants，and Agricultural PerspectiveCashmore, Plenum, New York (1983), pages 29-38; coruzzi, G. et al, The Journal of Biological Chemistry, 258: 1399(1983), and Dunsmuir, P. et al, Journal of Molecular and applied genetics, 2: 285(1983)).

In the present invention, where polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3' end of the CPL coding region. The polyadenylation region may be derived from a variety of plant genes or from T-DNA. The 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or from another plant gene, or less preferably from any other eukaryotic gene.

An intron sequence may be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the maturation message that accumulates in the cytosol. The inclusion of a spliceable intron in the transcription unit of plant and animal expression constructs has been shown to increase gene expression at both the mRNA level and the protein level, up to 1000-fold. Buchman and Berg, mol.cell biol.8: 4395-4405 (1988); callis et al, Genes Dev.1: 1183-1200(1987). Such intron enhancement of gene expression is generally greatest when placed near the 5' end of the transcription unit. The use of the maize intron Adh1-S introns 1,2 and 6 as well as the Bronze-1 intron are known in the art. See generally The Maize Handbook, Chapter 116, Freeling and Walbot eds, Springer, New York (1994).

In a preferred embodiment, it would be useful to direct the CPL protein to chloroplasts and other plastids. Typically, this is achieved by introducing a chloroplast transit peptide which targets the expressed protein to the plastid and also facilitates its transport into the organelle. A number of chloroplast transit peptides are known and may be used in the expression cassettes of the invention, including but not limited to chloroplast transit peptides derived from: the transit peptides from a variety of plants of the genus Pisum (Pisum) (Esturorera et al, JP 1986224990; E00977), carrot (Luo et al, Plant mol. biol., 33(4), 709-722 (1997; Z33383), Nicotiana (Nicotiana) (Bowler et al, EP 0359617; A09029), Oryza (Oryza) (de Pater et al, Plant mol. biol., 15(3), 399-406 (1990); X51911), and synthetic sequences such as provided in Herrera-Estralla et al, EP 0189707; U.S.5,728,925; U.S.5,717,084(A10396 and A10398.) the small subunit transit peptides from chloroplast of ketose-1, 5-bisphosphate carboxylase (Rubisco) from any Plant are well characterized in the present invention, wherein the small subunit of the transit peptides from any Plant of the genus Leucospora (e.g. Quiltra et al, Shiratra, Potentilla) (see, U.S.5, Bruch. 35; Shinetsu et al; see, Shinetsu et al, Shinetsu 8760; see, Shinetsu et al, Shinetsu, et al, U.7, et al, U.g. 7, U.7, U., AI 563260); nicotiana (Appleby et al, Heredity (1997), 79(6), 557-563); alfalfa (Khoudi et al, Gene (1997), 197(1/2), 343-351); potatoes and tomatoes (Fritz et al, Gene (1993), 137(2), 271-4); wheat (Galili et al, the or. appl. gene. (1991), 81(1), 98-104); and rice (Xie et al, Sci.sin., Ser.B (Engl. Ed.) (1987), 30(7), 706-19). For example, the transit peptide may be derived from a Rubisco small subunit isolated from plants including, but not limited to, soybean, canola, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oat, sorghum, rice, Arabidopsis (Arabidopsis), sugar beet, sugarcane, canola, millet, bean, pea, rye, flax, and grasses. Preferred for use in the present invention is the tomato Rubisco small subunit precursor protein.

The chloroplast-targeting sequence not only targets the desired protein to the chloroplast, but also aids in its transport to the organelle. This is accomplished by excision of the transit peptide from the mature polypeptide or protein by the chloroplast's native chloroplast processing protease at the appropriate transit peptide cleavage site. Thus, the chloroplast-targeting sequence of the invention comprises a suitable cleavage site for correct processing of the proprotein to the active mature polypeptide contained in the chloroplast. In the present invention, the chloroplast targeting sequence of the tomato Rubisco small subunit precursor protein is preferred, which has a cleavage site between the naturally occurring Cys and Met residues, separating the transit peptide from the mature polypeptide.

A suitable plant host is transformed with a functional CPL expression cassette so that CPL is expressed in chloroplasts and pHBA glucoside is produced. In fact, any plant host capable of supporting the expression of the CPL gene will be suitable, however various crops are preferred because of their ease of harvesting and large biomass. Suitable plant hosts will include, but are not limited to, monocots and dicots, such as soybean, oilseed rape (Brassica napus), canola (b. campestris), sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), wheat (Triticum sp), barley (Hordeum vulgare), oats (Avena sativa, L), Sorghum (Sorghum bicolor), rice (Oryza sativa), arabidopsis, sugar beet, sugarcane, canola, millet, beans, peas, rye, flax, and gramineous grasses.

A wide variety of techniques for introducing constructs into plant cell hosts are available and known to those skilled in the art. These techniques include transformation with DNA such as agrobacterium tumefaciens (a. tumefaciens) or agrobacterium rhizogenes (a. rhizogenes) as transformation factors, electroporation, particle acceleration, and the like. [ see, for example, EP 295959 and EP 138341]. One suitable method involves the use of binary vectors of Ti and Ri plasmids of Agrobacterium spp. Ti-derived vectors transform a wide variety of higher plants, including monocots and dicots, such as soybean, cotton, canola, tobacco, and rice [ Pacciotti et al (1985) Bio/Technology 3: 241, a first electrode and a second electrode; byrne et al, (1987) Plant Cell, Tissue and Organ Culture 8: 3; sukhapinda et al, (1987) Plant mol. biol. 8: 209-216; lorz et al, (1985) mol.Gen Genet.199: 178; portrykus (1985) mol.gen.gene.199: 183; park et al, J.plant Biol. (1995), 38(4), 365-71; hiei et al, Plant J. (1994), 6: 271-282]. Transformation of plant cells with T-DNA has been extensively studied and has been extensively described [ EP 120516; hoekema, carried byThe Binary Plant Vector SystemOffset-drakkerij Kanters b.v.; alblaserdam (1985), Chapter V, Knauf et al, Genetic Analysis of Host Range Expression by Agrobacterium, supported byMolecular Genetics of the Bacteria-Plant InteractionPuhler, A. eds., Springer-Verlag, New York, 1983, p.245; and An et al, EMBO J. (1985) 4: 277-284]. For introduction into plants, the chimeric genes of the invention can be inserted into binary vectors as described in the examples.

Other transformation methods are available to the person skilled in the art, such as direct uptake of the foreign DNA construct [ see EP 295959], electroporation techniques [ see Fromm et al (1986) Nature (London) 319: 791] or ballistic bombardment with metal microparticles coating the nucleic acid construct [ see Kline et al (1987) Nature (London) 327: 70, and see U.S. patent No.4,945,050 ]. Once transformed, the skilled artisan can regenerate the cells. Particularly suitable are the recently introduced methods for transforming foreign genes into commercially important crops, such as oilseed rape [ De Block et al, (1989) Plant physiol.91: 694-701], sunflower [ Everett et al, (1987) Bio/Technology 5: 1201], soybean [ McCabe et al (1988) Bio/Technology 6: 923; hinche et al, (1988) Bio/Technology 6: 915; chee et al, (1989) Plant physiol.91: 1212-1218; christou et al, (1989) proc.natl.acad.sci USA 86: 7500-7504; EP 301749], rice [ Hiei et al, Plant J. (1994), 6: 271-282] and maize [ Gordon-Kamm et al, (1990) Plant Cell 2: 603-618; fromm et al, (1990) Biotechnology 8: 833-839].

The transgenic plant cells are then placed in an appropriate selection medium to select for transgenic cells, which are then grown into callus. Shoots were grown from the callus and plantlets were produced from the shoots by growth in rooting medium. Each construct is typically linked to a marker for selection in plant cells. Conveniently, the marker may be biocide (particularly antibiotic, e.g. kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, etc.) resistance. The specific marker used will allow selection of transformed cells compared to cells lacking the introduced DNA. The components of the DNA construct comprising the transcription cassette of the invention may be prepared from sequences that are native (endogenous) or foreign (exogenous) to the host. By "exogenous" is meant a sequence that is not present in the wild-type host into which the construct is introduced. The heterologous construct will contain at least one region that is not native to the gene from which the transcriptional initiation region is derived. To confirm the presence of the transgene in transgenic cells and plants, one canSouthern blot analysis was performed using methods known to those skilled in the art.CPL is translocated into chloroplasts and subsequently processed

The present invention relies on novel manipulation of chloroplast targeting sequences to achieve transport of the CPL gene product into chloroplasts with sufficient enzymatic activity to produce commercially useful amounts of pHBA. Applicants have found that an important aspect of the invention is to include not only the transit peptide, but also a naturally occurring chloroplast cleavage site and a small portion of the mature N-terminus of the transit peptide donor. It is reasonable to improve chloroplast uptake and process the foreign protein to obtain higher conversion rates of chorismate to pHBA. However, after uptake into the organelle, the transit peptide is proteolytically removed by chloroplast processing enzymes, resulting in a CPL variant with a small polypeptide extension attached to its N-terminus. Unexpectedly, these additional amino acid residues do not interfere with CPL enzymatic activity, and transformed plants expressing the chimeric proteins of the invention accumulate significantly higher amounts of pHBA derivatives than previously reported. The need for this type of specificity has not been recognized in the art with respect to the production of pHBA.

The only examples of attempts reported to express CPL in chloroplasts of live plants are described in Siebert et al, Plant physiology.112: 811-819(1996). However, there are a number of important differences between the chimeric proteins of the invention (e.g., TP-CPL) and the chloroplast-targeted forms of E.coli CPL (e.g., TP-Ubic) described in Siebert et al (supra). For example, the invention of the chimeric includes a chloroplast targeting sequence, the sequence has a well defined cleavage site, for effective removal of the transit peptide. In addition, removal of the transit peptide at this specific site resulted in the addition of 5 additional amino acids in the N-terminal region of the mature CPL polypeptide. In contrast, TP-Ubic, described in Siebert et al (supra), lacks a well-defined cleavage site and additionally contains a 9 amino acid sequence stretch inserted between the putative transit peptide cleavage site of E.coli CPL and the initiator methionine residue. These differences are further illustrated in fig. 1.

FIG. 1 shows an amino acid sequence alignment of the intact tomato Rubisco small subunit precursor (line 1), TP-CPL (line 2), TP-Ubic (line 3), and E.coli CPL (line 1) with their transit peptides. The chimeric protein of the invention (line 2) consists of the chloroplast transit peptide of the tomato Rubisco small subunit precursor (green residue) fused to the initiator Met residue of e.coli CPL plus the first 4 amino acid residues of the "mature" Rubisco. Thus, TP-CPL contains not only the entire transit peptide, but also a highly conserved cleavage site where transit peptide removal normally occurs (e.g., between Cys and Met residues as indicated by the arrow). Assuming that TP-CPL also cleaves at this position in chloroplasts, the resulting protein will be a CPL variant with 5 additional amino acid residues at its N-terminus. Applicants have expressed the predicted chloroplast cleavage product of TP-CPL in E.coli, purified it to homogeneity, and shown it to be fully functional in enzyme activity. Applicants have also demonstrated that protease processing does occur at the Cys-Met junction by purifying the "mature" polypeptide from transgenic tobacco plants expressing the chimeric protein of the invention and Edman degradation of its N-terminus.

In contrast, as shown in line 3 of FIG. 1, TP-Ubic (Siebert et al, supra) does not contain a cleavage site that normally occurs to remove transit peptide from Rubisco small subunit precursors or any amino acid residue belonging to a mature Rubisco polypeptide (Mazur et al, Nuc Acids Res.13: 2373-2386 (1985); Berry-Lowe et al, J.mol.and appl.Gen.1, 483-498 (1982)). In fact, the Met residue, which constitutes part of the highly conserved scissile bond in most plant species, has been replaced by an Ala residue, which may or may not be recognized by chloroplast processing enzymes. In addition, Tp-Ubic contained a stretch of 9 additional amino acid residues juxtaposed between the Cys residue and the initiator Met residue of the putative cleavage site of E.coli CPL (shown in bold letters) (FIG. 1). These additional amino acids were introduced as cloning artifacts during the construction of TP-Ubic artificial fusion proteins (Siebert et al, supra) and their potentially detrimental effects on chloroplast import and/or protease processing were not investigated. In any event, even if, as suggested, cleavage of the transit peptide occurs at the Cys-Ala junction, the resulting "mature" protein will contain 9 additional amino acid residues at its N-terminus that may have deleterious effects on CPL enzyme activity (see table I, lines 2 and 4 of Siebert et al, supra).

Examples

The invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.General procedure

Standard recombinant DNA techniques and Molecular Cloning techniques used in the examples are well known in the art and are described in Sambrook, j., Fritsch, e.f. and manitis, t., Molecular Cloning: a Laboratory Manual; cold Spring Harbor laboratory Press: cold Spring Harbor, (1989) (Maniatis) and T.J.Silhavy, M.L.Bennan and L.W.Enquist, Experiments with Gene Fusions, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1984) and Ausubel, F.M. et al, Current protocols in Molecular Biology, Green Publishing asset and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples can be found in the following documents:Methods for General Bacteriology(Phillipp Gerhardt, R.G.E.Murray, Ralph N.Costilow, Eugene W.Nester, Willis A.Wood, Noel R.Krieg and G.Briggs Phillips, eds.), American Society for Microbiology, Washington, DC (1994)) or Thomas D.Brock,Biotechnology：A Textbook of Industrial Microbiology，Second Edition，Sinauer associates, Inc., Sunderland, MA (1989). All reagents, restriction enzymes and materials used for bacterial cell growth and maintenance were obtained from Aldrich Chemica1s (Milwaukee, Wis.), DIFCO Laboratories (Detroit, MI), GIBCO/BRL (Gaithersburg, Md.) or Sigma Chemical Company (St. Louis, Mo.) unless otherwise noted.

Manipulation of genetic sequences is accomplished using the program set available from the Genetics Computer Group inc (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI). When using the GCG program "Pileup", the used gap creation (gap creation) default value is 12 and the gap extension (gap extension) default value is 4. When using the CGC "Gap" or "Bestfit" programs, the default Gap creation penalty used is 50 and the default Gap extension penalty is 3. When there are no GCG program parameter hints in these GCG programs or any other GCG program, the default values are used.

The abbreviations have the following meanings: "h" means hours, "min" means minutes, "sec" means seconds, "d" means days, "ml" means milliliters, "L" means liters.

Example 1

PCR cloning of E.coli CPL

The E.coli ubiC gene was amplified from genomic DNA using two PCR primers, while a single restriction site was added to its flanking region for subsequent ligation into a high copy number plasmid. This gene encodes chorismate pyruvate lyase, hereinafter referred to as CPL. The primers used for this purpose are based on the published DNA sequence of the E.coli ubic gene (GenBank accession M96268), consisting of the following nucleotides: primer 1- (SEQ ID NO: 1):

5′-CTA CTC ATT Tca tat gTC ACA CCC CGC GTT AA-3' primer 2- (SEQ ID NO: 2):

5′-CAT CTT ACT aga tct TTA GTA CAA CGG TGA CGC Cthe 3' underlined bases hybridized to the target gene, while the lower case letters indicated restriction sites (NdeI or BglII) added to the ends of the PCR primers.

Amplification of the E.coli ubic gene was accomplished using primers 1 and 2 and genomic DNA from E.coli strain W3110(Campbell et al, Proc. Natl. Acad. Sci.75: 2276-2284 (1978)). Primer 1 hybridizes at the beginning of the gene and introduces an NdeI site at the start codon of the protein, while primer 2 hybridizes at the opposite end, providing a BglII site immediately after the stop codon. A100. mu.l PCR reaction contained about 100ng of genomic DNA, with a final concentration of 0.5. mu.M for both primers. Other reaction components were provided by the GeneAmp PCR Kit (GeneAmp PCR Reagent Kit) (Perkin Elmer) following the manufacturer's protocol. Amplification was performed in a DNA thermal cycler 480(Perkin Elmer) for 22 cycles, each cycle comprising 94 ℃ for 1 minute, 55 ℃ for 1 minute, and 72 ℃ for 1 minute. After the last cycle, extension was carried out for 7 minutes at 72 ℃.

The PCR product was cut with NdeI and BglII, and the resulting fragment was ligated into E.coli expression vector pET-24a (+) (Novagen) which had been digested with NdeI and BamHI. Coli DH10B electrocompetent cells (GibcoBRL) were transformed with the ligation reaction mixture using BTX Transactor 100(Biotechnologies and Experimental Research Inc.), following the manufacturer's protocol; the growth was selected in LB medium containing kanamycin (50. mu.g/ml). Transformants containing the plasmid with the CPL insert were identified by PCR reactions using primers 1 and 2 and each resuspended colony as template source; therefore, this technique is simply referred to as "colony PCR". Isolating plasmid DNA from representative colonies that produce PCR products of the correct size, and performing full sequencing of the complete insert corresponding to the CPL to check for PCR errors; no errors were found. The plasmid chosen for further manipulation is hereinafter referred to as "pET 24 a-CPL". The nucleotide sequence of the ORF of CPL in the pET24a escherichia coli expression construct and its predicted primary amino acid sequence are described in SEQ ID NO: 3 and SEQ ID NO: 4. note that the coding region is identical to the ORF given in GenBank accession number M96268.

Example 2

Overexpression, purification and characterization of recombinant E.coli CPL

To generate sufficient CPL for enzyme characterization and antibody production, pET24a-CPL was introduced into E.coli BL21(DE 3). This was done by electroporation with BTX Transactor 100(Biotechnologies and Experimental Research Inc.), following the manufacturer's protocol. The growth was selected on LB medium containing kanamycin (50. mu.g/ml) and one colony was selected for further manipulation. To produce the recombinant protein, the strain containing the plasmid was grown in liquid culture in the above medium at 30 ℃ and cells were induced with 0.15mM IPTG at A600nm of about 0.8. After an induction period of 4.5 hours under the same growth conditions, the cells were harvested by centrifugation and stored at-80 ℃ until use. The subsequent steps are carried out at 0-4 ℃.

The frozen cell pellet was resuspended in about 3 volumes of 0.1M Tris-HCl (pH7.7), 5mM MgSO₄1mM dithiothreitol, 0.03mg/ml DNase I, 0.5mM phenylmethanesulfonyl fluoride, and passed through a French press twice at 20,000 psi. Debris was removed by centrifugation (43,0000Xg, 1h), and cell-free extracts containing about 30mg protein/mL were supplemented with glycerol (5%) and stored at-80 ℃ until use. Protein concentration was determined using the method of Lowry et al (Lowry et al, J.biol chem.193: 265-275(1951)) using BSA as a standard. SDS-PAGE analysis of cell-free extracts showed that the recombinant protein was well expressed in E.coli BL21(DE3) under the growth conditions, at an expression level of more than 15% of total soluble protein. However, only about 25% of the recombinant protein was recovered in the soluble fraction of the extract crushed in a French press, and this material was used for purification as described below.

The first step of purification requires anion exchange chromatography. An aliquot (1.0mL) of the cell-free extract of e.coli containing recombinant CPL was quickly thawed to room temperature, diluted 1: 1 with deionized water, and then filtered through a 0.2 μm Acrodisc filter (Gelman Sciences, cat. No. 4192). All samples were loaded onto a Mono Q HR5/5 column (Pharmacia Biotech Inc) and developed at 25 ℃ with buffer Q (50mM Tris-HCI, pH7.7, 10mM sodium sulfite, 1mM EDTA) at a flow rate of 1 ml/min. Under these conditions, recombinant CPL was not adsorbed onto the anion exchange resin and was eluted from the column isocratically during the first few minutes of development. The column flow-through was collected in a tube, supplemented with 5% (w/v) glycerol, and concentrated to a final volume of 450. mu.l in Centricon-10(Amicon Inc.) at 4 ℃. After this simple procedure, the purity of the recombinant protein was approximately 90%, as judged by SDS-PAGE (Laemmli U., Nature 227: 680-685(1970)) and Coomassie blue staining. In the next step, 200. mu.l of the concentrated sample were applied to a 7.5X 600mm TSK G3000SW gel filtration column (TOSOH Corp.) pre-equilibrated with buffer Q containing 0.3M NaCl. The column was expanded at a flow rate of 1.0mL/min (25 ℃) and the highly purified recombinant CPL eluted between 19.7-21 min. The latter was kept on ice, while the remaining half of the sample was processed in the same manner. The peak fractions from both gel filtration columns were combined, supplemented with glycerol (5%), concentrated to about 12mg protein/mL, and stored at-80 ℃ until use. The yield of purified protein was about 3.7mg, corresponding to about 12% of the total protein present in the cell-free extract. Visual inspection of the overloaded coomassie stained gel indicated that the final recombinant protein preparation was more than 98% pure.

Subjecting the purified recombinant CPL to Edman degradation revealed that the initiator Met residue of the protein was removed in e. However, in addition to this minor post-translational modification, the first 13 amino acids of recombinant CPL are identical to SEQ ID NO: 4 (e.g.ORF of a true E.coli protein) are identical at residues 2-14. The protomer molecular weight of the purified recombinant CPL was 18644.6 daltons as determined by electrospray ionization mass spectrometry. This value corresponds well to the molecular weight (18645.49 daltons) predicted from the DNA sequence excluding the initiator Met residue. From these observations, it is reasonable to conclude that: the initiator Met residue was also excised from the native e.coli protein, since the nucleotide sequence of the latter was identical to that of recombinant CPL.

A continuous spectrophotometric assay was developed to evaluate the catalytic activity of the purified recombinant protein. The assay is based on the increase in absorbance at 246nm due to the formation of an aromatic ring in the latter, with conversion of the chorismate to pHBA. Measuring the rate of initial product formation in quartz cuvettes containing 90mM Tris-HCl (pH7.6), 0.2M NaCl, 100. mu.M barium chorismate (Sigma) and various amounts of purified recombinant CPL at 25 ℃; the reaction is initiated with an enzyme. 11,220M at 246nm with pHBA^-1The extinction coefficient of (2) was calculated from the change in absorbance. Measuring absorbance under the same conditions at pHBA at a concentration ranging from 5. mu.M to 100. mu.M; the absorbance is proportional to the concentration of pHBA. Based on the above determination, the conversion number of the purified recombinant CPL at 25 ℃ was about 36min^-1. Two other preparations of the same recombinant protein purified on a much larger scale, under the same conditions, gave slightly higher turnover numbers (e.g. 41 min)^-1And 42min^-1). The only value available in the literature for this enzyme is 49min^-1(Nichols et al, J.Bacteriol.174: 5309-5316(1992)), but the assay was performed at 37 ℃. Assuming that the CPL enzymatic reaction is characterized by a Q10 (temperature coefficient) of at least 2, these observations indicate that the purified recombinant protein described above has full activity.

Example 3

CPL chloroplast-targeted forms: construction of TP-CPL

Chorismic acids are physiological substrates of CPL and are important branch point intermediates in the synthesis of a variety of aromatic compounds including the amino acids phenylalanine and tyrosine. In plants, chorismic acid is produced in the shikimate pathway located in chloroplasts and other types of plastids (Siebert et al, Plant physiol.112: 811-819 (1996)). Therefore, it is necessary to provide CPL with an N-terminal chloroplast targeting sequence that is capable of efficiently targeting foreign proteins to the chloroplast,i.e. the site of production of the chorismic acid. This is accomplished by constructing a chimeric protein consisting of chloroplast targeting sequences derived from tomato Rubisco small subunit precursor protein fused to the initiator Met residue of CPL; the resulting fusion protein is hereinafter referred to as "TP-CPL". To generate a DNA fragment corresponding to the first 4 amino acid residues of Rubisco small subunit transit peptide and "mature" Rubisco, PCR was used. The target of the amplification was the plasmid pTSS1-91- (#2) -IBI (Siebert et al, Plant physiol.112: 811-819(1996)) which contained a full-length cDNA clone of the tomato Rubisco small subunit precursor of rbcS2 (Sugita et al, Mol Gen Genet.209: 247-256 (1987); Siebert et al, Plant physiol.112: 811-819 (1996)). The following primers were used for the reaction:primer 35′-CTA CTC ACT TAG ATC Tcc atg gCT TCC TCT GTC ATT TCT- 3′′(SEQ ID NO：5)Primer 45′-CAT CTT ACT cat at g CCA CAC CTG CAT GCA GC-3′(SEQ ID NO：6)

The underlined part of primer 3 hybridizes to the first 21 nucleotides of Rubisco small subunit precursor and introduces an NcoI site (lower case) at the initiator Met residue at the beginning of the chloroplast-targeting sequence. As shown, this primer also contains a BglII site (bold letters) at its 5' end, immediately upstream of the NcoI site. Primer 4 hybridizes to nucleotides 167-184 of the ORF of the Rubisco small subunit precursor at the other end of the chloroplast-targeting sequence. A single NdeI site was engineered into the primer (lower case) to allow the PCR fragment containing the chloroplast targeting sequence to be ligated to the NdeI site at the CPL start codon in the pET-24a expression construct. A100. mu.l PCR reaction contained about 75ngpTSS1-91- (#2) -IBI and primers 3 and 4 at a final concentration of about 0.9. mu.M. Amplification was performed in a DNA thermal cycler 480(Perkin Elmer) for 25 cycles, each cycle comprising 94 ℃ for 1 minute, 55 ℃ for 1 minute, and 72 ℃ for 1 minute; an extension period of 7 minutes at 72 ℃ was carried out after the last cycle. The PCR product was digested with BglII and NdeI and ligated into pET24a-CPL containing only the vector sequence including the T7 promoter, which had been cut with the same restriction enzyme to remove a small DNA fragment (106 bp). The ligation reaction mixture was introduced into E.coli DH10B by electroporation, and the growth was selected on LB medium containing kanamycin (50. mu.g/mL). Transformants harboring the plasmid with the inserted chloroplast targeting sequence were identified by colony PCR using primers 2 and 3. Selecting a representative plasmid that produces a PCR product of the correct size for further manipulation; this plasmid is hereinafter referred to as "pET 24 a-TP-CPL". To confirm the absence of PCR errors, the plasmid region corresponding to the amplified chloroplast-targeting sequence was fully sequenced with custom primers. The nucleotide sequence of the ORF of TP-CPL and its predicted primary amino acid sequence are described in SEQ ID NO: 7 and SEQ ID NO: 8.

example 4

Prediction of TP-CPL chloroplast cleavage product having full Activity

A DNA fragment corresponding to the amino acid sequence of the predicted chloroplast cleavage product of TP-CPL (e.g., MQVWH-CPL) was generated by PCR using the insert in plasmid pet24a-TP-CPL as a template. The following primers were used for the reaction:primer 55′CTA CTC ATT Tga aga cTG CAT GCA GGT GTG GCA T-3′(SEQ ID NO：9)：Primer 65′-CAT CTT ACT gtc gac TTT AGT ACA ACG GTG ACG C-3′(SEQ ID NO：10)

The underlined portion of primer 5 was bound to the 5' end of the TP-CPL gene insert and a single BBSI site (lower case) was introduced just upstream of the predicted chloroplast cleavage product (hereinafter "Δ TP-CPL") initiator Met residue. Primer 6 hybridizes to the opposite end of the gene insert, providing a single SalI site (lower case) immediately after the stop codon. The PCR product was cut with BBSI (which left NcoI-matched "sticky ends") and SalI, and the resulting fragment was ligated into the E.coli expression vector pET-24d (+) (Novagen) digested with NcoI and SalI. Coli DH10B electrocompetent cells (GibcoBRL) were transformed with the ligation reaction mixture using BTX Transactor 100(Biotechnologies and Experimental Research Inc.), following the manufacturer's protocol; the growths were selected in LB medium containing kanamycin (50. mu.g/ml). Transformants containing the plasmid with the Δ TP-CPL insert were identified by colony PCR using appropriate primers. A representative plasmid (e.g., pET24a- Δ TP-CPL) was isolated from colonies that produced the correct size PCR product, and the insert corresponding to Δ TP-CPL was fully sequenced to confirm that there were no PCR errors.

To express the recombinant protein for purification and kinetic analysis, pET24 a-. DELTA.TP-CPL was introduced into E.coli BL21(DE3) using electroporation. Transformed cells were plated on LB medium containing kanamycin (50. mu.g/ml) and a representative colony was selected for further manipulation. 300ml of the culture was grown in the above medium at 30 ℃. At about 0.8 of A600nm, IPTG was added to a final concentration of 0.15 mM. After 4.5 hours of induction under the same conditions, the cells were harvested by centrifugation and stored at-80 ℃. The subsequent steps are carried out at 0-4 ℃ unless otherwise stated.

The frozen cell pellet was resuspended in 2.5ml of a medium containing 0.1M Tris-HCl (pH7.7), 5mM MgSO₄1mM dithiothreitol, 0.03mg/ml DNase I, 0.5mM phenylmethanesulfonyl fluoride, was passed twice through a French press at 20,000 psi. The cell-free extract was centrifuged (43,000Xg, 25min), the supernatant (4.5ml) was carefully removed, supplemented with 5% glycerol and stored at-80 ℃ until use. The purification scheme for Δ TP-CPL was essentially the same as described above for unmodified recombinant E.coli CPL. Briefly, all of the above samples were thawed and concentrated to a final volume of 2.5ml using Centriprep-10(Amicon Inc). The sample was then exchanged into buffer Q using a PD-10 gel filtration column (Pharmacia Biotech Inc) pre-equilibrated with buffer Q, according to the manufacturer's protocol. In Centriprep-10, the volume was reduced to 2ml, and the entire sample was loaded onto a Mono Q HR 10/10 column (Pharmacia Biotech Inc) and developed with buffer Q at 4ml/min (25 ℃). The material eluted between 2-3 minutes was collected and glycerol was added to a final concentration of 5% (v/v). The sample was concentrated to 200. mu.l in Centricon-10(Amicon Inc), loaded to 7.5 for extraction600mm TSK G3000SW gel filtration column (TOSOH Corp.). The column was developed with 1ml/min of buffer Q (25 ℃) containing 0.3M NaCl and the recombinant. DELTA.TP-CPL was eluted between 20.7 and 22 min. The fractions containing the purified recombinant protein were supplemented with 5% glycerol, concentrated to about 0.7mg protein/ml and stored at-80 ℃ until use.

The enzymatic activity of the purified recombinant Δ TP-CPL was determined at 25 ℃ using the spectrophotometric assay described in example 2. Under these conditions, the turnover number was 40.7min^-1. This value is practically identical to that obtained with the purified recombinant E.coli CPL without N-terminal extension (for example 36-42 min)^-1). This observation clearly shows that the 5 additional amino acid residues fused to the N-terminus of Δ TP-CPL do not impair enzyme activity, and also suggests that the predicted chloroplast cleavage product of TP-CPL may have full activity.

Example 5

In vitro protein input: import of TP-CPL into isolated chloroplasts

Before introducing TP-CPL into higher plants, it was important to show that it might be taken up by chloroplasts. This was done by synthesizing the radioactive form of the artificial fusion protein and performing a typical chloroplast protein import assay. The first step is to produce a polypeptide which can be used³⁵S]Methionine radiolabeled the DNA constructs of the proteins for transport experiments. To this end, the sequence encoding TP-CPL was modified for insertion into the MscI and BglII sites of the in vitro transcription/translation vector pCITE4a (+) (Novagen) using primers 7 and 8, and the insert from plasmid pet24A-TP-CPL as a template for PCR amplification.Primer 75′-CTA CTC ATT tgg cca G CT CTG TCA TTT CTT CAG CAG C-3′(SEQ ID NO：11)Primer 85′-CAT CTT ACT a ga tct TTA GTA CAA CGG TGA C-3′(SEQ ID NO：12)

Primer 7 hybridizes to the nucleotide sequence stretch immediately after the TP-CPL start codon (underlined region) and adds a single MscI site (in lower case letters) at the initiator Met residue. Primer 8 binds to the other end of the gene insert and introduces a single BglII site immediately after the stop codon. Neither primer introduces any amino acid changes in the artificial fusion protein. The resulting PCR fragment was digested with MscI and BglII, ligated into pCITE4a (+) cleaved with MscI and BamHI; BglII and BamHI produced matched "sticky ends". The ligation reaction mixture was introduced into E.coli DH10B by electroporation, and the transformed cells were plated on LB medium containing ampicillin (100. mu.g/ml). One representative colony with the plasmid with the correct insert was selected (identified by colony PCR using appropriate primers) for further manipulation. Plasmid DNA was fully sequenced to confirm that there were no PCR errors.

Next, the above plasmid constructs were transcribed/translated in vitro using [35S ] methionine and the "Single Tube Protein System 2, T7" kit (Novagen) according to the commercial protocol. The reaction was stopped with 2 Xinput buffer containing 60mM unlabeled methionine (Viitanen et al, J.biol.chem.263: 15000-15007 (1988)). Chloroplasts were isolated from 14-day-old young pea seedlings (Pisum sativum) and assayed in vitro with radiolabeled TP-CPL (Viitanen et al, J.biol. chem.263: 15000-15007 (1988)). Protease post-treatment was used to identify binding and import polypeptides (Cline et al, J.biol.chem.260: 3691-3696 (1985)). The intact plastids were then re-purified by centrifugation through a Percoll pad, resuspended in 150. mu.l of 2X gel sample buffer, and analyzed by SDS-PAGE/fluorography as previously described (Viitanen et al, J.biol.chem.263: 15000-15007 (1988)).

The in vitro transcription/translation of TP-CPL results in the synthesis of a radioactive polypeptide with an apparent molecular weight of about 25kDa (mobility based on SDS-PAGE (Laemmli, U.K., Nature 227: 680-685(1970)), which is consistent with the value predicted from its DNA sequence (25188 Da). in the presence of ATP, this polypeptide is taken up by chloroplasts and processed to a smaller size, which appears to co-migrate with Coomassie-stained purified recombinant Δ TP-CPL (e.g., the predicted chloroplast cleavage product of TP-CPL).

In contrast, when chloroplasts were incubated under conditions that did support protein import (e.g., in the dark, without ATP), no uptake and processing of TP-CPL was observed. Under the condition of no energy supply, the only radioactive band recovered with the intact plastid is the full-length fusion protein TP-CPL. Furthermore, the radioactive band corresponding to the latter disappeared completely after treatment with protease, demonstrating that it was not imported, but bound only to the chloroplast outer membrane. Taken together, these results clearly demonstrate that the chloroplast targeting sequence linked to the N-terminus of TP-CPL is able to direct the artificial fusion protein to the chloroplast and that, following uptake into organelles, protease processing occurs in the expected manner.

Example 6

Construction of expression plasmids for transformation of tobacco and Arabidopsis

After establishing that TP-CPL is efficiently taken up by chloroplasts (example 5) and cleaved into a novel protein with high CPL activity (example 4), it was decided to introduce it into plants. To generate constructs that can be used for constitutive expression in tobacco and arabidopsis, a DNA fragment corresponding to the full length TP-CPL fusion protein was subcloned into the modified form of plasmid pML 63. The latter was derived from pML40 and contained the following genetic elements: a CaMV35S promoter, a cab leader sequence, a uidA coding region, and a NOS polyadenylation signal sequence. Briefly, the CaMV35S promoter is a 1.3kb DNA fragment that extends 8 base pairs after the transcription start site (Odell et al, (1985) Nature 303: 810-812). A cab leader sequence was operably linked at its 3' end, which is a60 bp untranslated double stranded DNA fragment obtained from the chlorophyll a/b binding protein gene 22L (Harpster et al (1988) mol. Gen. Genet. 212: 182-190). The uidA gene encoding the protein β -glucuronidase (e.g., "GUS") was fused at the 3' end of the cab leader sequence (Jefferson et al (1987) EMBO J6: 3901). Finally, the 3' end of the GUS gene was ligated with an 800bp DNA fragment containing a polyadenylation signal sequence from the nopaline synthase (e.g. "NOS") gene (Depicker et al (1982) J.mol.appl.Genet.1: 561-564). These DNA fragments, which contained the 35S-GUS chimeric gene in combination, were inserted into the vector pGEM9Zf (-) (Promega; Madison Wis.) by standard cloning techniques to give the plasmid pMH 40.

Plasmid pML63 was essentially identical to pMH40, but with a truncated form of the 3' NOS termination sequence, produced in the following manner. First, pMH40 was digested with SalI and the two resulting 4.03kb and 2.9kb DNA fragments were religated to give a plasmid with the 35S promoter/cab 22 leader sequence/GUS gene/3' NOS termination cassette in reverse orientation. The resulting construct was then digested with Asp718I and HindIII to release a 770bp fragment containing the 3' NOS termination sequence. The latter was discarded and replaced with the shorter version generated by PCR using pMH40 as template and primers 9 and 10.Primer 9:5′-CCC GGG GGT ACC TAA AGA AGG AGT GCG TCG AAG-3′(SEQ ID NO：13)：primer 10:5′-GAT ATC AAG CTT TCT AGA GTC GAC ATC GAT CTA GTA ACA TAG ATG A 3′(SEQ ID NO：14)：

the PCR product was digested with HindIII and Asp718I, resulting in a 298 bp fragment containing 279bp of the 3' NOS stop sequence, nucleotide 1277(TAA stop codon) starting with the published sequence (Depicker et al, J. mol Appl Genet (1982) 1: 561-574) and ending at nucleotide 1556. This PCR fragment was ligated into pML3, resulting in plasmid pML 63.

pML63 contained the GUS coding region under the control of the 35S promoter and a truncated form of the 3' NOS terminator as described above. Thus, it contains all the transcriptional information necessary for constitutive expression of GUS in plants. To generate a similar construct of TP-CPL, plasmid pML63 was digested with NcoI and EcoRI. This procedure releases only the GUS gene insert, leaving the regulatory flanking sequences and the remainder of the complete vector. Plasmid pet24a-TP-CPL was also treated with NcoI and EcoRI, which released the entire coding region of the TP-CPL fusion protein. The small DNA fragment (693bp) corresponding to the latter was purified by agarose gel electrophoresis and subjected to standard ligation reaction with the large vector fragment (4.63bp) obtained by cleaving pML63 with NcoI and EcoRI. The ligation reaction mixture was introduced into E.coli DH10B by electroporation and the growth was selected on LB medium containing ampicillin (100. mu.g/ml). Transformants harboring the plasmid containing the inserted TP-CPL coding sequence were identified by colony PCR using primers 2 and 3. One representative plasmid that produced the correct size PCR product was selected for further manipulation. A schematic of the final construct, hereinafter referred to as "TP-CPL-pML 63", is shown in FIG. 2.

The binary vector used for Agrobacterium-mediated transformation of tobacco leaf discs was plasmid pZBL1, deposited at ATCC at 24.6.1997 with accession number 209128. pZBL1 contained: an origin of replication derived from pBR 322; bacterial nptI kanamycin resistance gene; the replication and stability regions of the Pseudomonas aeruginosa (Pseudomonas aeruginosa) plasmid pVS1(Itoh et al, 1984); the T-DNA border (border) described by van den Elzen et al, 1985, in which the OCS enhancer (extending from-320 to-116 of the OCS promoter) was removed as part of the right border segment (Greve et al, 1983, J.mol.appl.Genet.1: 499-511); and a NOS/P-nptII-OCS 3' gene as a kanamycin-resistant plant selection marker. To express TP-CPL, plasmid pZBL1 was digested with SalI, which cleaves at a single site between the right and left borders, a position ideal for insertion of a foreign gene and stable integration into the plant genome. To minimize the possibility of religation without insert, the cleaved vector was dephosphorylated with bovine small intestine alkaline phosphatase (GibcoBRL) as recommended by the manufacturer. To obtain a fragment that could be inserted into the binary vector, the plasmid TP-CPL-pML63 was also digested with SalI. This treatment released the entire transcription unit of the TP-CPL fusion gene (e.g., 35S promoter/cab 22 leader sequence/TP-CPL/3' NOS terminator) as a 2.4kb DNA fragment. The latter was purified by agarose gel electrophoresis and subjected to standard ligation with the dephosphorylated 11.0kb fragment obtained from pZBL1 as described above. The ligation reaction mixture was introduced into E.coli DH10B by electroporation and the growth was selected on LB medium containing kanamycin (50. mu.g/ml). Transformants harboring the plasmid containing the TP-CPL fusion gene were identified by colony PCR using primers 2 and 3, and the orientation of the insert was determined by restriction enzyme digestion analysis with KpnI. In the plasmid chosen for further manipulation, hereinafter referred to as "TP-CPL-pZBL 1", the start codon of TP-CPL is adjacent to the right border segment of the T-DNA, as illustrated in FIG. 3. This expression construct was used to transform tobacco and Arabidopsis thaliana for overproduction of pHBA, as described below.

Example 7

Generation of transgenic tobacco plants

The plasmid TP-CPL-pZBL1 was introduced into Agrobacterium tumefaciens strain LBA4404(Hoekema et al, Nature 303: 179-180(1983)) by the freeze-thaw transformation method (Holsters et al, mol. Gen. Genet. 163: 181-187). Cells were plated at 28 ℃ on YEP medium (10 g tryptone, 10g yeast extract and 5g NaCl per liter) also containing kanamycin (1000. mu.g/ml) and rifampicin (20. mu.g/ml), and colonies with the binary construct were identified by PCR using appropriate primers.

Potted tobacco plants (Nicotiana tabacum cv. xanthhi) for leaf disc infestation were grown in a growth chamber with a 14hr 21 ℃ light, 10hr 18 ℃ dark cycle, relative humidity of approximately 80%, using a mixed cold white fluorescent and incandescent lamp. Agrobacterium-mediated transformation of leaf discs essentially follows De Blaere et al, meth.enzymol.153: 277-292 with the following modifications. Leaf discs 8mm in diameter were prepared from whole leaves using a sterile paper drill and 4-6 week old plants. Inoculation was performed by submerging leaf discs in concentrated Agrobacterium solution with TP-CPL-pZBL1 resuspended to an OD600 of-Z in Murashige Minamal Organics medium for 30 minutes. Inoculated leaf discs were placed directly on medium containing (per liter) 30g sucrose, 1mg 6-Benzylaminopurine (BAP), 0.1mg naphthalene acetic acid, 8g agar and 1 pack of Murashige's Minimal organic medium from GibcoBRL (cat. # 23118-029). After 3 days of incubation at 28 ℃ under light, the leaf discs were transferred to fresh medium of the same composition also containing kanamycin (300. mu.g/ml) and cefotaxime (500. mu.g/ml) in order to select according to the growth of the transformed tobacco cells and to eliminate the residual Agrobacterium. Leaf discs were grown under the above growth conditions for 3 weeks and then transferred to fresh medium of the same composition at 3-week intervals until the optimal bud size for root induction was obtained. Shoots were rooted on medium containing (per liter) 1 pack of Murashige's Minimal Organics medium, 8g agar and 10g sucrose. After about 4 weeks, the plants were transferred to soil and allowed to grow to maturity in a growth chamber under the conditions described above.

Example 8

Chemical Synthesis of pHBA glucoside Standard

To synthesize pHBA ester glucoside, 110mmol of 4-hydroxybenzoic acid was mixed with 1L of benzene containing 55mmol of bis (tributyltin) oxide. The mixture was heated in situ to reflux with an azeotropic apparatus under nitrogen for 16 hours. Benzene was removed under reduced pressure to give a clear oil, predominantly tributyltin 4-hydroxybenzoate (Ogawa et al, (1982) Tetrahedron 36: 2641-2648). Next, 1.2L1, 2-dichloroethane containing 25mmol of acetyl bromide-a-D-glucose was added to 25mmol of tributyltin 4-hydroxybenzoate intermediate, followed by 12.5mmol of tetrabutylammonium bromide. The mixture was heated to reflux under nitrogen for 3 hours and the progress of the reaction was monitored by TLC, by detection by charring with sulfuric acid. The solvent was removed under reduced pressure and the acetyl protected pHBA ester glucoside purified on silica gel eluting with a 1: 1 mixture of ethyl acetate and hexane. Then, the acetyl protecting group was selectively saponified with 1 equivalent of potassium carbonate in 10% aqueous methanol for 3 hours. The solvent was removed under reduced pressure and the pHBA ester glucoside was triturated cleanly with methanol. The latter was removed by filtration and the resulting white powder exhibited a melting point of 209-210 ℃. By passing¹HNMR confirmed the pHBA ester dextranChemical structure of glycoside.

To synthesize pHBA acylglucoside, 16.4mmol of methyl 4-hydroxybenzoate and 14.6mmol of acetyl bromide-a-D-glucose were dissolved in 7.0ml of anhydrous pyridine, followed by addition of 23.3mmol of 99.99% silver oxide. The reaction was stirred at room temperature under nitrogen for 3 hours. The insoluble silver salt was collected by filtration, washed with pyridine, and the combined filtrate and washing solution were concentrated under reduced pressure and poured into a mixture of ice-cold water. The dark brown solid was collected, washed with water, dissolved in a 1: 1 mixture of chloroform and dichloromethane and subsequently dried with sodium carbonate as a drying agent. The solution was filtered through celite and the solvent removed under reduced pressure. The hydroxyl-linked methyl benzoate acetyl protected glucoside is then purified by silica gel chromatography (Durkee et al, (1979) Carbohydrate Research 77: 252-254); the column was eluted with a 1: 2 mixture of ethyl acetate and hexane. The purified compound was dissolved in 40ml of methanol, and 1.5mmol of sodium methoxide was added. After 4.5 hours, the solution turned yellow and the solvent was removed under reduced pressure; the resulting residue was dissolved in 25ml of water. Concentrating the solution to about 5ml and allowing it to crystallize to provide said hydroxy-linked methyl benzoate glucoside; the crystals were collected and then dried under high vacuum. To selectively saponify the methyl ester groups, 2.5mmol of hydroxyl-linked methyl benzoate glucoside were dissolved in 25ml of water, and 2.5ml of 1M NaOH was added. After stirring overnight at room temperature, the solution was neutralized, concentrated to about 5ml and allowed to crystallize to give the desired pHBA acylglucoside. The melting point of the compound was found to be 108-110 ℃ by¹HNMR confirmed its chemical structure.

Example 9

Preparation of tobacco leaf samples for pHBA glucoside analysis

From the top third of the tobacco plant stem, a healthy leaf was selected measuring about 15cm along the midvein. 1/3 of the tissue (100mg fresh weight) from the distal end of the leaf was quickly cut with scissors and placed in a Biopulverizer H-tube (cat. No.6570-201 or 6540-401) containing ceramic beads, both of which were obtained from BIO 101(Joshua Way, Vista, Calif.). After addition of 1ml of methanol, the tube was capped and mechanically stirred for 40 seconds with a Savant FastPrep FP120 tissue disruption device operating at a speed of 5 m/s. Next, the tube was placed on a rotary shaker and vigorously stirred at 400rpm for 1 hour at room temperature. The extract was clarified by centrifugation (10,000Xg, 10min) using a conventional bench-top microcentrifuge, and the supernatant containing both pHBA glucosides was carefully removed and transferred to an empty tube. The remaining insoluble leaf material was re-extracted with 0.5ml methanol at room temperature for 30 minutes using a rotary shaker and the conditions described above. The supernatant from the second extraction was combined with the supernatant from the first extraction and the samples were stored at-20 ℃ for subsequent processing. The mass was converted to volume using the analytical balance and density of methanol, and the volume of methanol added to each sample of leaf material and the final volume recovered after extraction and centrifugation were measured gravimetrically.

The samples were further processed for HPLC analysis as follows. All steps were performed at room temperature unless otherwise indicated. An aliquot of the methanol extract was transferred to a microcentrifuge tube and its actual volume was measured as described above. Heating was started in a Speed-Vac (Savant Instruments) and the solvent was removed in vacuo to completely dry the sample. The dry residue was dissolved in 100. mu.l of 0.2N HCl and 0.7mL of water saturated ether was added. After vigorous vortex mixing and centrifugation, the ether phase was carefully removed and discarded and the sample re-extracted with ether as described above. An aliquot of the remaining aqueous phase (50. mu.l) was then filtered through a 0.22um cellulose acetate filter (Costar EZ-spin) and injected into a Vydac 218TP54 PROTEIN AND PEPTIDEC18 column pre-equilibrated with 90% buffer A (0.1% aqueous formic acid) and 10% buffer B (methanol) at 1 ml/min. Immediately after injection of the sample, the column was developed with a linear gradient to a final concentration of 50% buffer B generated during 20 min. The flow rate was 1 ml/min. The elution of phenolic and ester pHBA glucosides was monitored spectrophotometrically at 254 nm. FIG. 4 shows representative HPLC profiles of a TP-CPL expressing tobacco plant (transformant #5) and a wild-type plant.

The retention time of the HPLC separation was corrected using the true pHBA glucoside standard (see above) and the extinction coefficients of both compounds were accurately measured under the HPLC conditions used. Thus, the peak areas were integrated using the software provided by H/P Chemstation, and the values obtained using known amounts of appropriate standards were used to quantify the microgram amount of pHBA glucoside per injection. After accounting for the dilution and percentage of the original methanol extract injected onto the column, the values were corrected to reflect the overall recovery from the leaf sample analyzed. This is coupled with individual measurements of the plant tissue analyzed (e.g., dry weight from the same plant on the same day), enabling pHBA-glucoside to be expressed as a percentage of dry weight.

Example 10

Isolation of kanamycin resistance in first selfed progeny

Seeds of primary tobacco transformant #34 produced by selfing were passed through a 10% bleaching solution [ containing 5.25% Na (OCl) also containing 0.1% SDS₂Clorox (C) of^]The surface was sterilized by immersion for 30 minutes at room temperature with gentle stirring. The germination rate of 200 seeds selected without antibiotics was 97.5%. In contrast, of 500 seeds inoculated onto germination medium also containing kanamycin (300. mu.g/ml), about 20% exhibited the recessive phenotype (e.g., 1: 4 kanamycin-sensitive to kanamycin-resistant seeds). Since the segregation ratio for transformant #34 was very close to the theoretical ratio for the single-gene dominant trait of 1: 3 (e.g., as compared to the 1: 16 ratio characteristic of a double locus event), it can be concluded that the selectable marker and TP-CPL gene expression construct were stably integrated into a single locus of the genome.

Example 11

Measurement of CPL enzyme Activity in tobacco leaf extract

Leaf tissue extracts from wild-type and transgenic tobacco plants were prepared and analyzed for CPL enzyme activity as previously described (Siebert et al, plant Physiol.112: 811-819(1996)) with minor modifications. Leaf samples (2g wet weight) were homogenized in an ice-cold mortar in 2.6ml of a solution containing 50mM Tris-HCl (pH7.5), 0.1% beta-mercaptoethanol, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride and 75mg/ml polyvinylpyrrolidone. All subsequent steps were carried out at 0-4 ℃ unless otherwise stated. After removal of insoluble material by low speed centrifugation, the samples were buffer exchanged for 50mM Tris-HCl (pH8.0), 10mM EDTA and 200mM NaCl using a PD-10 gel filtration column (Pharmacia Biotech Inc) according to the manufacturer's recommendations. The concentration of the protein was measured using the Bio-Rad (Bradford) protein assay.

The CPL enzyme assay was performed as follows. The basic reaction mixture (final volume 500. mu.l) contained 50mM Tris pH8.0 (at 37 ℃), 10mM EDTA, 200mM NaCl and 150. mu.M purified barium chorenate (Siebert et al Microbiology 140: 897-904 (1994)). After incubation at 37 ℃ for 5 minutes, the reaction was initiated with tobacco leaf extract containing 50. mu.g of protein. After 2 minutes at 37 ℃ the reaction was stopped with 0.3ml of 0.75M sodium acetate (pH4) and the amount of pHBA produced in the reaction was determined. To monitor product recovery, each tube received 9,500dpm of [14C ]]Labeled pHBA (55mCi/mmol) as internal standard. The mixture was diluted with 1ml of H₂O-saturated ethyl acetate, the organic phase was collected and dried. The amount of pHBA was then quantified by reverse phase HPLC using exactly the same column and conditions as described in example 9. The peak corresponding to pHBA was collected and the amount of radioactivity was measured by liquid scintillation counting. The values reported below for CPL enzyme activity are expressed in pkats per mg of protein and have been corrected for the recovery of the internal standard and the small amount of pHBA produced from the chorismate by spontaneous decomposition (Siebert et al, Plant Physiol.112: 811-819 (1996)).

Example 12

Analysis of transgenic tobacco plants expressing TP-CPL

TP-CPL was introduced into tobacco (Nicotiana tabacum) using Agrobacterium-mediated leaf disc transformation, as described above, to determine its effect on pHBA glucoside accumulation. From the data shown in FIG. 5, it is evident that this artificial fusion protein is actually superior to other chloroplast-targeted forms of E.coli CPL that have been previously used to elevate pHBA levels in plants (Siebert et al, Plant physiol.112: 811-819 (1996); Sommer et al, Plant Cell Reports 17: 891-896 (1998)). This analysis was performed with leaf tissue from 15 tobacco plants (primary transformants) produced by different transformation events. Note that sampling was only done 5 weeks after the plants were transferred to soil. As expected, the primary transformants exhibited various levels of pHBA glucoside, varying from 0 to 2.3% of total dry weight. This type of variation is commonly observed in almost all plant transformation experiments, presumably reflecting different levels of gene expression due to so-called "location" effects (e.g., stable integration of the trait gene at different locations in the genome) and transgene copy number. A similar phenomenon also occurred in this study, supported by western blot analysis of tobacco transformants with antiserum against purified recombinant e. For example, although most of the plants (e.g., 14/15) have immunologically detectable levels of the foreign protein, there is still considerable variation in the expression levels. However, in general, there is a positive correlation between Western blot signal intensity and accumulation of pHBA glucoside, in contrast to previous observations (Siebert et al, Plant Physiol.112: 811-819 (1996)); sommer et al, Plant Cell Physiol.39 (11): 1240-1244 (1998); sommer et al, Plant CellReports 17: 891-896 (1998)).

On a dry weight basis, the mean pHBA glucoside content of 5 week old tobacco plants was 1.12% (+/-0.186%), with the numbers in parentheses being the standard error of the mean. More importantly, the pHBA glucoside levels of 3 transformants (#13, #19 and #37) in primary transformants alone were less than 0.52%, the highest level obtained in a similar study with TP-Ubic artificial fusion protein (Siebert et al, Plant physiol.112: 811-819 (1996)). In addition, the three best plants (#8, #34, and #39) in this study had a pHBA glucoside content of at least 2% dry weight.

To check the stability of the desired phenotype, 3 of the transgenic tobacco plants were monitored over an extended period of time until the seed formation period (#4, #5, and # 34). It is possible that the plant may not be able to maintain such high levels of pHBA glucoside while the plant continues to develop. However, as shown in fig. 6, this is not the case. When the plant became old, its leaf pHBA glucoside content increased significantly. For example, in transformant #5, total pHBA glucoside levels were 0.5%, 1.6%, 7.2% and 10% of total dry weight when analyzed 1 week, 5 weeks, 11 weeks and 13 weeks after the plants were transferred to soil. The value at 13 weeks represents an almost 20-fold increase over the result obtained with TP-Ubic, corresponding to a pHBA content of about 4.5% after correction of the mass of the associated glucose molecule. Despite these very high levels of secondary metabolites, the transgenic tobacco plants appear completely normal, morphologically indistinguishable from wild-type plants.

To follow the fate of the foreign gene and the associated phenotype of pHBA accumulation in the next generation, transformant #34 was selected for further analysis. As a primary transformant at 13 weeks of age, the pHBA glucoside content of this plant was 8% of the total dry weight (fig. 6). As described in example 10, when seeds obtained by selfing were germinated in the presence of kanamycin and examined for segregation of the antibiotic resistance phenotype, a ratio of 1 (sensitivity): 4 (resistance) was observed. This suggests that integration of the selectable marker with TP-CPL occurs at a single location in the genome, rather than a two locus event where kanamycin resistance segregation would be 1: 15. Theoretically, kanamycin resistant plants consist of two populations present in a 2: 1 ratio-a heterozygous population and a homozygous population. Assuming no co-suppression, the CPL enzyme activity of homozygous plants would be expected to be twice that of heterozygous plants, perhaps accumulating even higher levels of pHBA glucoside. To solve this problem, 5 of kanamycin-resistant seedlings (hereinafter referred to as #34A-34E) were grown to mature plants, and CPL enzyme activity and pHBA glucoside thereof were analyzed. The plants were sampled at 15 weeks of age and the results of this study are shown in table I below.

TABLE I

Plant 34 CPL enzyme Activity Total pHBA-glucoside

Sister plants (pkat/mg protein) (dry weight percentage)

34A 927 4.8

34B 991 5.9

34C 1048 5.0

34D 784 5.0

34E 3563.2 As expected, all kanamycin-resistant seedlings also exhibited CPL enzyme activity and accumulated pHBA glucoside. Therefore, the gene of the artificial fusion protein TP-CPL was stably transmitted to the next generation. Although the number of plants examined was small, it appeared to be two different populations. The CPL enzyme activities of 4 strains in the progeny (e.g., #34A-34D) were very similar, ranging from 784 to 1,048pkat per mg of protein. It is noted that the average CPL activity of this group (e.g., 938pkat/mg) is about 4.5 times higher than the optimal value obtained with TP-Ubic when examining live tobacco plants (Siebert et al, Plant Physiol.112: 811-819 (1996)). Also these 4 sister strains have comparable levels of pHBA glucoside. With an average of about 5.2% dry weight, the values are very aggregated (e.g., 4.8% -5.9%).

In contrast, the CPL enzyme activity and pHBA glucoside levels were very low for 1 of the plants (e.g., # 34E). Although we have speculated that this sister plant is heterozygous and the other 4 plants are homozygotes, this conclusion is too early. First, based on the segregation pattern of the kanamycin resistance phenotype obtained, only 1/3 of the plants were expected to be homozygous instead of the 80% of the population observed. Second, it is conceivable that homozygous states with duplicate copies of the trait gene could result in co-suppression, resulting from conflicting low levels of CPL enzyme activity and pHBA glucoside. Experiments are currently being conducted to try to solve this problem. In any event, it is interesting to note that the level of accumulation of pHBA glucoside in the second generation plants was not exactly the same as that of the primary transformants.

Example 13

Protease processing of TP-CPL occurs at predicted cleavage sites in vivo

As shown in FIG. 7, the whole leaf extract of transgenic tobacco plants expressing the artificial fusion protein TP-CPL contained only one polypeptide that cross-reacted with antiserum against purified recombinant E.coli CPL. Furthermore, the size of the cross-reactive polypeptide, which is not present in wild-type plants, is much smaller than the original fusion protein introduced into tobacco, as measured by SDS-PAGE. In fact, it appears to co-migrate exactly with purified recombinant Δ TP-CPL, which is the predicted chloroplast cleavage product of TP-CPL (example 4), and also the radioactive band observed after in vitro chloroplast import experiments (example 5). Nevertheless, to provide a clear demonstration that removal of the chloroplast-targeting sequence from the artificial fusion protein did indeed occur at the predicted cleavage site in vivo, the protein was purified from leaf tissue obtained from tobacco transformant #34 and its N-terminal amino acid residue determined by Edman degradation.

Leaf tissue (6.9g wet weight) was homogenized with an ice-cold solution (triturate buffer) containing 50mM Tris-HCl (pH7.5), 1mM EDTA, 0.1% beta-mercaptoethanol, 1mM phenylmethylsulfonyl fluoride and 75mg/ml polyvinyl polypyrrolidone using a mortar and pestle. All subsequent steps were performed at 0-4 ℃ unless otherwise stated, and the leaf extract was centrifuged for 30 minutes(40,000Xg) insoluble matter was removed, and the resulting supernatant was supplemented with solid (NH)₄)₂SO₄To a final concentration of 80% (w/v). The solution was gently stirred for 30 minutes and then centrifuged at 20,000Xg for 10 minutes to precipitate most of the protein. The supernatant was discarded and the resulting pellet resuspended in 2.0ml of grinding buffer without polyvinylpolypyrrolidone but supplemented with 8% (v/v) glycerol and at a protein concentration of 14.3mg/ml as measured by Bio-Rad (Bradford) protein assay.

Then, an aliquot of the above sample (0.5ml) was exchanged for buffer Q (example 2) using a PD-10 gel filtration column (Pharmacia Biotech Inc). After the sample completely entered the resin, the column was washed once with 2.2ml of buffer Q, and the effluent was discarded. After addition of another 1.1ml of the same buffer, the material eluted in the external water volume was collected. All samples were then loaded onto a MonoQ HR5/5 column equilibrated at room temperature with buffer Q. The column was developed with the same buffer at a flow rate of 1.0ml/min, and fractions (1.0ml per fraction) were collected from the time of sample injection. The fraction containing the chloroplast cleavage product of TP-CPL was identified by Western blot analysis using antiserum against purified recombinant E.coli CPL. Virtually all cross-reactive material was eluted in fractions #3 and #4, and only species detected with the antiserum co-migrated with purified recombinant Δ TP-CPL as previously described. Column fractions #3 and #4 were pooled, supplemented with 7.5% glycerol, 0.3M NaCl and 0.01% Tween20 (Bio-Rad cat. #170-6531), and concentrated to a final volume of about 200. mu.l with Centricon 10 (Amicon). All samples were then loaded onto a 7.5X 600mM TSK G3000SW gel filtration column (TOSOH Corp.) pre-equilibrated at room temperature with 50mM Tris-HCl (pH7.2), 0.3M NaCl and 0.01% Tween 20. The column was developed with the same buffer at 1.0ml/min (25 ℃), and fractions eluted between 21.5-23min containing the authentic TP-CPL chloroplast cleavage product were pooled and concentrated to a final volume of 55. mu.l with Microcon 10 (Amicon). The concentrated material was diluted 1: 1 with sample buffer and analyzed by SDS-PAGE to assess the degree of purification. The TP-CPL chloroplast cleavage product is a major protein species that is well separated from other contaminants, although many other bands are evident in Coomassie blue stained gels. Analysis of the N-terminus of the polypeptide corresponding to this band (after electrophoretic transfer to polyvinylidene fluoride membrane and 6 cycles of Edman degradation) confirmed that proteolytic processing of the artificial fusion protein occurred at the predicted cleavage site, e.g., at the Cys-Met junction shown in FIG. 1. From this observation and the enzymatic activity data described in example 4, it can be concluded that the polypeptide responsible for converting chorismate to pHBA in the chloroplasts of TP-CPL expressing tobacco plants is a fully active CPL variant with 5 additional amino acid residues attached to its N-terminus.

Example 14

Generation and analysis of transgenic Arabidopsis plants expressing TP-CPL

The artificial fusion protein TP-CPL was introduced into Arabidopsis thaliana, and the pHBA glucoside level was determined. The binary vector carrying the CaMV35S-CPL expression cassette (e.g., TP-CPL-pZBL1) was transformed by electroporation into the Agrobacterium tumefaciens strain C58C 1 Rif (also known as strain GV3101) carrying the non-oncogenic (disarmed) Ti (virulence) plasmid pMP90(Koncz, C. and Schell, J. (1986) mol.Gen.Gene.204: 383-396) using available protocols (Meyer et al (1994) Science 264: 1452-1455). Arabidopsis thaliana (Arabidopsis thaliana) ecotype Columbia plants with a genetic background of wild type fahl-2(Chapple et al, Plant Cell 4: 1413-1424(1992)), sngl-1(Lorenzen et al, Plant Physiology 112: 1625-1630(1996)) were transformed with MP90 strain carrying a binary vector with a CPL expression construct using a published vacuum infiltration protocol (Clough S.J., Bent A.F (1998) Plant J.16 (6): 735-43). Transgenic seedlings were identified by selection with kanamycin (50. mu.g/ml) on standard plant growth medium under sterile conditions. Kanamycin-resistant seedlings were transferred to soil and in a soil/perlite (soil/perlite) mixture at 12 hours light/12 hours dark photoperiod at 100Em^-2s^-1Incubate at 18 ℃ (dark) and 21 ℃ (light). In this way, a transformation event derived from an independent transformation event is generatedA population of 301 primary transformants. Transgenic arabidopsis plants were analyzed for pHBA glucoside 6 weeks after transfer to soil using reverse phase HPLC as described below.

Freshly cut leaf material was homogenized in 50% MeOH (5. mu.l/mg wet weight) and the resulting extract was clarified by low speed centrifugation. An aliquot of the leaf extract was then applied to a Nova-Pak C18 column (60 angstrom pore size, 4 μm particle size) using an acetonitrile gradient (6% -48%) containing 1.5% phosphoric acid. pHBA phenolic and ester glucosides were detected by UV absorption at 254nm and quantified using the extinction coefficients obtained from authentic chemical standards (see example 8). Of the 272 transgenic arabidopsis plants analyzed, 239 (or about 88%) contained detectable levels of both glucose conjugates, and these conjugates were present in approximately equal amounts. The optimal overproducer had a total pHBA glucoside content of 10.73% dry weight, which is very similar to the highest level observed with tobacco using the same construct. The average value for the entire transgenic Arabidopsis plant population was 3.35% (+/-0.13%); the numbers in parentheses are the standard error of the mean.

Taken together, these results clearly demonstrate that the chimeric protein-TP-CPL of the present invention is capable of producing high levels of pHBA glucoside not only in tobacco, but also in other plant species.

Sequence Listing <110> Naveldu DuPont de Nemours and Company <120> description of the high level production of P-hydroxybenzoic acid <130> BC1015 PCT <140> <141> <160>16<170> MICROSOFT OFFICE 97<210>1<211>32<212> DNA <213> Artificial sequence <220> <223> in Green plants: description of the primer <400>1ctactcattt catatgtcac accccgcgtt aa 32< 32 >2<211>34<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of the primers <400>2catcttacta gatctttagt acaacggtga cgcc 34<210>3<211>495<212> DNA <213> unknown organism <220> <223> unknown organism: description of escherichia coli (e.coli) <400>3atgtcacacc ccgcgttaac gcaactgcgt gcgctgcgct attgtaaaga gatccctgcc 60ctggatccgc aactgctcga ctggctgttg ctggaggatt ccatgacaaa acgttttgaa 120cagcagggaa aaacggtaag cgtgacgatg atccgcgaag ggtttgtcga gcagaatgaa 180atccccgaag aactgccgct gctgccgaaa gagtctcgtt actggttacg tgaaattttg 240ttatgtgccg atggtgaacc gtggcttgcc ggtcgtaccg tcgttcctgt gtcaacgtta 300agcgggccgg agctggcgtt acaaaaattg ggtaaaacgc cgttaggacg ctatctgttc 360acatcatcga cattaacccg ggactttatt gagataggcc gtgatgccgg gctgtggggg 420cgacgttccc gcctgcgatt aagcggtaaa ccgctgttgc taacagaact gtttttaccg 480gcgtcaccgt tgtac 495<210>4<211>165<212> PRT <213> unknown organism <220> <223> unknown organism: coli (e.coli) <400>4Met Ser His Pro Ala Leu Thr Gln Leu Arg Ala Leu Arg Tyr Cys Lys 151015 Glu Ile Pro Ala Leu Asp Pro Gln Leu Leu Asp Trp Leu Leu Leu Glu

20 25 30Asp Ser Met Thr Lys Arg Phe Glu Gln Gln Gly Lys Thr Val Ser Val

35 40 45Thr Met Ile Arg Glu Gly Phe Val Glu Gln Asn Glu Ile Pro Glu Glu

50 55 60Leu Pro Leu Leu Pro Lys Glu Ser Arg Tyr Trp Leu Arg Glu Ile Leu65 70 75 80Leu Cys Ala Asp Gly Glu Pro Trp Leu Ala Gly Arg Thr Val Val Pro

85 90 95Val Ser Thr Leu Ser Gly Pro Glu Leu Ala Leu Gln Lys Leu Gly Lys

100 105 110Thr Pro Leu Gly Arg Tyr Leu Phe Thr Ser Ser Thr Leu Thr Arg Asp

115 120 125Phe Ile Glu Ile Gly Arg Asp Ala Gly Leu Trp Gly Arg Arg Ser Arg

130 135 140Leu Arg Leu Ser Gly Lys Pro Leu Leu Leu Thr Glu Leu Phe Leu Pro145 150 155 160Ala Ser Pro Leu Tyr

165<210>5<211>39<212> DNA <213> Artificial sequence <220> <223> description of the Artificial sequence: description of the primer <400>5ctactcactt agatctccat ggcttcctct gtcatttct 39<210>6<211>32<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of the primers <400>6catcttactc atatgccaca cctgcatgca gc 32< 32 >7<211>684<212> DNA <213> Artificial sequence <220> <223> description of the Artificial sequence: description of the synthetic CPL <400>7atggcttcct ctgtcatttc ttcagcagct gttgccacac gcagcaatgt tacacaagct 60agcatggttg cacctttcac tggtctcaaa tcttcagcca ctttccctgt tacaaagaag 120caaaaccttg acatcacttc cattgctagc aatggtggaa gagttagctg catgcaggtg 180tggcatatgt cacaccccgc gttaacgcaa ctgcgtgcgc tgcgctattg taaagagatc 240cctgccctgg atccgcaact gctcgactgg ctgttgctgg aggattccat gacaaaacgt 300tttgaacagc agggaaaaac ggtaagcgtg acgatgatcc gcgaagggtt tgtcgagcag 360aatgaaatcc ccgaagaact gccgctgctg ccgaaagagt ctcgttactg gttacgtgaa 420attttgttat gtgccgatgg tgaaccgtgg cttgccggtc gtaccgtcgt tcctgtgtca 480acgttaagcg ggccggagct ggcgttacaa aaattgggta aaacgccgtt aggacgctat 540ctgttcacat catcgacatt aacccgggac tttattgaga taggccgtga tgccgggctg 600tgggggcgac gttcccgcct gcgattaagc ggtaaaccgc tgttgctaac agaactgttt 660ttaccggcgt caccgttgta ctaa 684<210>8<211>227<212> PRT <213> artificial sequence <220> <223> artificial sequence: synthetic CPL <400>8Met ala Ser Ser Val Ile Ser Ser Ala Ala Val Ala Thr Arg Ser Asn 151015 Val Thr Gln Ala Ser Met Val Ala Pro Phe Thr Gly Leu Lys Ser Ser

20 25 30Ala Thr Phe Pro Val Thr Lys Lys Gln Asn Leu Asp Ile Thr Ser Ile

35 40 45Ala Ser Asn Gly Gly Arg Val Ser Cys Met Gln Val Trp His Met Ser

50 55 60His Pro Ala Leu Thr Gln Leu Arg Ala Leu Arg Tyr Cys Lys Glu Ile65 70 75 80Pro Ala Leu Asp Pro Gln Leu Leu Asp Trp Leu Leu Leu Glu Asp Ser

85 90 95Met Thr Lys Arg Phe Glu Gln Gln Gly Lys Thr Val Ser Val Thr Met

100 105 110Ile Arg Glu Gly Phe Val Glu Gln Asn Glu Ile Pro Glu Glu Leu Pro

115 120 125Leu Leu Pro Lys Glu Ser Arg Tyr Trp Leu Arg Glu Ile Leu Leu Cys

130 135 140Ala Asp Gly Glu Pro Trp Leu Ala Gly Arg Thr Val Val Pro Val Ser145 150 155 160Thr Leu Ser Gly Pro Glu Leu Ala Leu Gln Lys Leu Gly Lys Thr Pro

165 170 175Leu Gly Arg Tyr Leu Phe Thr Ser Ser Thr Leu Thr Arg Asp Phe Ile

180 185 190Glu Ile Gly Arg Asp Ala Gly Leu Trp Gly Arg Arg Ser Arg Leu Arg

195 200 205Leu Ser Gly Lys Pro Leu Leu Leu Thr Glu Leu Phe Leu Pro Ala Ser

210215220 Pro Leu Tyr225<210>9<211>34<212> DNA <213> Artificial sequence <220> <223> description of the Artificial sequence: description of the primer <400>9ctactcattt gaagactgca tgcaggtgtg gcat 34<210>10<211>34<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of the primer <400>10catcttactg tcgactttag tacaacggtg acgc 34<210>11<211>37<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of the primer <400>11ctactcattt ggccagctct gtcatttctt cagcagc 37< 37 >12<211>31<212> DNA <213> Artificial sequence <220> <223> description of the Artificial sequence: description of the primers <400>12 cathttaeta gatctttagt acaacggtga c 31<210>13<211>33<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: description of the primer <400>13cccgggggta cctaaagaag gagtgcgtcg aag 33<210>14<211>46<212> DNA <213> Artificial sequence <220> <223> description of the Artificial sequence: description of the primer <400>14gatatcaagc tttctagagt cgacatcgat ctagtaacat agatga 46<210>15<211>62<212> PRT <213> Artificial sequence <220> <223> Artificial sequence: synthetic CPL <400>15Met Ala Ser Ser Val Ile Ser Ser Ala Ala Val Ala Thr Arg Ser Asn 151015 Val Thr Gln Ala Ser Met Val Ala Pro Phe Thr Gly Leu Lys Ser Ser

20 25 30Ala Thr Phe Pro Val Thr Lys Lys Gln Asn Leu Asp Ile Thr Ser Ile

35 40 45Ala Ser Asn Gly Gly Arg Val Ser Cys Met Gln Val Trp His

505560 <210>16<211>170<212> PRT <213> Artificial sequence <220> <223> description of the Artificial sequence: synthesis of CPL <400>16Met Gln Val Trp His Met Ser His Pro Ala Leu Thr Gln Leu Arg Ala 151015 Leu Arg Tyr Cys Lys Glu Ile Pro Ala Leu Asp Pro Gln Leu Leu Asp

20 25 30Trp Leu Leu Leu Glu Asp Ser Met Thr Lys Arg Phe Glu Gln Gln Gly

35 40 45Lys Thr Val Ser Val Thr Met Ile Arg Glu Gly Phe Val Glu Gln Asn

50 55 60Glu Ile Pro Glu Glu Leu Pro Leu Leu Pro Lys Glu Ser Arg Tyr Trp65 70 75 80Leu Arg Glu Ile Leu Leu Cys Ala Asp Gly Glu Pro Trp Leu Ala Gly

85 90 95Arg Thr Val Val Pro Val Ser Thr Leu Ser Gly Pro Glu Leu Ala Leu

100 105 110Gln Lys Leu Gly Lys Thr Pro Leu Gly Arg Tyr Leu Phe Thr Ser Ser

115 120 125Thr Leu Thr Arg Asp Phe Ile Glu Ile Gly Arg Asp Ala Gly Leu Trp

130 135 140Gly Arg Arg Ser Arg Leu Arg Leu Ser Gly Lys Pro Leu Leu Leu Thr145 150 155 160Glu Leu Phe Leu Pro Ala Ser Pro Leu Tyr

165 170

Claims (19)

1. A method for producing para-hydroxybenzoic acid in a green plant, comprising:

a) providing a green plant having an endogenous source of chorismate and containing a chorismate pyruvate lyase expression cassette having the structure:

P-T-C-D-CPL

wherein:

p is a promoter suitable for driving expression of a chorismate pyruvate lyase gene;

t is a nucleic acid molecule encoding a ribulose-1, 5-bisphosphate carboxylase (Rubisco) chloroplast transit peptide;

c is a nucleic acid molecule encoding a Rubisco chloroplast transit peptide cleavage site;

d is a nucleic acid molecule encoding about 4-20 contiguous amino acids of the N-terminal portion of the Rubisco chloroplast transit peptide donor polypeptide; and

CPL is a nucleic acid molecule encoding a mature chorismate pyruvate lyase protein; wherein P, T, C, D and CPL are each operably linked such that expression of the cassette results in translation of a chimeric protein comprising a chloroplast targeting sequence fused to the N-terminus of a mature chorismate pyruvate lyase protein;

b) cultivating the plant under conditions such that the chimeric protein can be expressed and transported to chloroplasts for conversion of chorismic acid to paraben glucosides and paraben derivatives;

c) recovering para-hydroxybenzoic acid and para-hydroxybenzoic acid derivatives from said plant; and

d) processing the paraben glucoside and paraben derivative into free paraben.

2. A method according to claim 1, wherein said Rubisco transit peptide is derived from a plant selected from the group consisting of: soybean, canola, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, Arabidopsis (Arabidopsis), sugar beet, sugarcane, canola, millet, beans, peas, rye, flax and grasses.

3. A method according to claim 1, wherein the promoter is selected from the group consisting of: 35S promoter, nopaline synthase promoter, octopine synthase promoter, cauliflower mosaic virus promoter, ribulose-1, 5-bisphosphate carboxylase promoter, and chlorophyll a/b binding protein promoter.

4. A method according to claim 1, wherein the chorismate pyruvate lyase consists of seq id NO: 3, or a nucleic acid sequence as set forth in seq id No. 3.

5. A method of claim 1 wherein said chimeric protein comprising a chloroplast targeting sequence fused to the N-terminus of said mature chorismate pyruvate lyase protein has the amino acid sequence of SEQ ID NO: 8, or a pharmaceutically acceptable salt thereof.

6. A method of claim 5 wherein the chimeric protein comprising a chloroplast targeting sequence fused to the N-terminus of the mature chorismate pyruvate lyase protein is processed to the amino acid sequence of SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof.

7. A method according to claim 1 wherein the pHBA glucoside is produced at a concentration of at least 2% of the paraben glucoside by dry weight of the plant biomass.

8. A method according to claim 1, wherein the glucosinolates of para-hydroxybenzoic acid are produced at a concentration of at least 10% glucosinolates of para-hydroxybenzoic acid by dry weight of plant biomass.

9. A method according to claim 1, wherein the green plant containing the chorismate pyruvate lyase expression cassette is selected from the group consisting of: soybean, canola, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, arabidopsis, sugarbeet, sugarcane, canola, millet, beans, peas, rye, flax, and grasses of the Gramineae family.

10. A method according to claim 1, wherein the para-hydroxybenzoic acid is produced at a concentration greater than 4.5% pHBA by dry weight of plant biomass.

11. A chorismate pyruvate lyase expression cassette comprising a chimeric gene having a nucleotide sequence identical to a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, encoding a nucleic acid molecule having the amino acid sequence shown in SEQ id no: 15, or a nucleic acid molecule of a ribulose-1, 5-bisphosphate carboxylase small subunit chloroplast targeting sequence.

12. A chorismate pyruvate lyase expression cassette according to claim 11, wherein the chimeric gene encodes the amino acid sequence of SEQ ID NO: 8.

13. A plant comprising the CPL expression cassette of claim 11.

14. The plant of claim 13, selected from the group consisting of: soybean, canola, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, arabidopsis, sugarbeet, sugarcane, canola, millet, beans, peas, rye, flax, and grasses of the Gramineae family.

15. A chimeric protein comprising a sequence identical to a sequence having SEQ ID NO: 8, and (b) a chloroplast targeting sequence fused to the N-terminus of the mature chorismate pyruvate lyase protein.

16. An isolated nucleic acid fragment encoding a chimeric protein comprising a nucleotide sequence identical to a nucleotide sequence having the sequence of SEQ ID NO: 15, and (b) a chloroplast targeting sequence fused to the N-terminus of the mature CPL protein of the amino acid sequence set forth in fig. 15.

17. An isolated nucleic acid fragment according to claim 16, having the sequence of SEQ id no: 7, or a sequence shown in seq id no.

18. The chloroplast cleavage product of claim 15, wherein said cleavage product has the amino acid sequence of SEQ id no: 16, or a pharmaceutically acceptable salt thereof.

19. A nucleic acid fragment encoding the processed protein of claim 18.