KR20010071027A - Recombinant Dehydrodiconiferyl Alcohol Benzylic Ether Reductase and Methods of Use - Google Patents

Abstract

An isolated DNA sequence is provided that encodes the expression of dihydrodiconiferyl alcohol benzyl ether reductase. In another aspect, a dihydrodiconiferyl alcohol benzyl ether reductase or a dihydrodiconiferyl alcohol benzyl ether reductase that can hybridize with it is a replicable that encodes a base sequence that is sufficiently complementary to at least a portion of DNA or RNA Provide a recombinant cloned vehicle. In addition, another aspect of the invention provides a modified host cell that has been transformed, transfected, infected and / or injected with a DNA sequence encoding a recombinant cloning carrier and / or a dihydrodiconiferyl alcohol benzyl ether reductase. to provide. Thus, the method provides recombinant expression of dehydrodiconiferyl alcohol benzyl ether reductase in host cells such as plant cells.

Description

Recombinant Dehydrodiconiferyl Alcohol Benzylic Ether Reductase and Methods of Use}

Dihydrodiconiferyl alcohol (DDC) is the three major compounds by radical bonding of E-coniperyl alcohol in vitro, promoted by peroxidases and laccases such as those provided by lignans and 8 and 5 'bound lignans and woodlation. One of the products. (Freudenberg, K. Bull. Soc. Chim. France, 1748-1753 (1959); Freudenberg, k & Neish, AC Constitution and Biosynthesis of Lignin 1-123 (Springer-Verlag, New York, NY, (1968)) DDC Pinus taeda (Nose, M, et al. Phytochemisry 39; 71-79 (1995) and Tobacco tabacum (Binns, A, N, Chen, R, H, Wood, H, N & Lynn) , D, F. Proc. Natl. Acad. Sci. USA 84: 980-984 (1987)), which are found in plants and are ubiquitous in the plant kingdom. It is also shown to include a '-(alky bond) reduced derivative, dihydrodihydrodicophenyl alcohol (DDDC) (Nose, M., et al. Phytochemistry 39: 71-79 (1995)), however, its formation Enzymes involved in and subsequent metabolism have not been described previously.

Summary of the Invention

As described above, cDNA encoding dihydrodiconiferyl alcohol benzyl ether reductase from Pinus teada and Cryptomeria japonica was isolated and sequenced and the corresponding amino acid sequence was inferred. Thus, the present invention provides a sequence designated SEQ ID NO: 1 encoding dihydrodiconiferyl alcohol benzyl ether reductase (SEQ ID NO: 2) from Pinus taeda and dihydrodiconiferyl alcohol benzyl ether reduct from Cryptomeria japonica. Codes for the expression of a dihydrodiconiperyl alcohol benzyl ether reductase such as the sequences named SEQ ID NO: 3 and SEQ ID NO: 5, which encodes the enzymes (SEQ ID NO: 4 and SEQ ID NO: 6, respectively) To an isolated DNA sequence. In another aspect, the invention relates to at least one portion of a DNA or RNA encoding, for example, a dihydrodiconiferyl alcohol benzyl ether reductase or a dihydrodiconiferyl alcohol benzyl ether reductase that can hybridize with it. DNA or DNA molecules encoding sufficiently complementary base sequences (e.g., dihydrodiconiperyl alcohol benzyl ether reductase available as polymerase chain reaction primers, such as probes for any of the aforementioned reductases or related genes). A replicable recombinant cloned carrier comprising a nucleic acid sequence, such as a DNA sequence encoding a complementary antisense RNA or DNA fragment). Moreover, another aspect of the invention provides a modified host cell that has been transformed, transfected, infected and / or injected with a recombinant cloned carrier and / or a DNA sequence of the invention. Accordingly, the present invention provides for the expression of dihydrodiconiferyl alcohol benzyl ether reductase recombinants and the inventive concept provides for the use of recombinant dihydrodiconiferyl alcohol benzyl ether reductase (or primary enzyme products thereof) for subsequent use. It can be used to promote significant amounts of production, separation and purification, and can be used to obtain expression or increased expression of dihydrodiconiferyl alcohol benzyl ether reductase in plants, microorganisms or animals, or alternatively, reductase Or in the environment in which control or expression of dihydrodiconiferyl alcohol benzyl ether reductase is desired for the production of enzyme products thereof or derivatives thereof.

In another aspect, the invention relates to a method of increasing or otherwise modifying expression of dihydrodiconiferyl alcohol benzyl ether reductase in a suitable host cell, such as a plant cell.

Related applications

This invention claims the benefit of priority from US Provisional Patent Application Serial No. 60 / 094,012, filed on July 24, 1998.

Technical Field

The present invention provides an isolated dihydrodiconiferyl alcohol benzyl ether reductase protein, a nucleic acid sequence encoding a dihydrodiconiferyl alcohol benzyl ether reductase protein, and a vector comprising the sequence, a host cell comprising the sequence, and A method for producing a recombinant dihydrodiconiferyl alcohol benzyl ether reductase protein and mutants thereof.

The foregoing and many additional advantages of the present invention will be more readily appreciated and will be better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.

FIG. 1A shows HPLC separations of the following standard: peak 1: tetrahydrodihydrodiconiferyl alcohol (TDDC), peak 2: isodihydrodihydrodiconiperyl alcohol (IDDDC), peak 3: dihydrodihydrodi Coniferyl Alcohol (DDDC), Peak 4: Dihydrodiconiferyl Alcohol (DDC).

1B shows an HPLC chromatogram in which DDC is reduced to IDDDC.

1C shows reduced HPLC chromatogram of DDDC to TDDC by benzyl ether reductase (SEQ ID NO: 2).

1d shows the mass spectrum of the DDC (substrate).

FIG. 1E shows the mass spectrum of IDDDC (product of DDC reduction with benzyl ether reducing agent (SEQ ID NO: 2)).

1F shows the mass spectrum of TDDC (Product of DDDC Reduction with Benzyl Ether Reducing Agent (SEQ ID NO: 2)).

Detailed Description of the Preferred Embodiments

As used herein, the term “amino acid” refers to any naturally occurring L-α-amino acid or residue thereof. Amino acids are identified by single-letter or three-letter naming.

Asp D Aspartic Acid Ile I Isoleucine

Thr T threonine Leu L Leucine

Ser S Serine Tyr Y Tyrosine

Glu G Glutamic Acid Phe F Phenylalanine

Pro P Proline His H Histidine

Gly G Glycine Lys K Lysine

Ala A Alanine Arg R Arginine

Cys C Cysteine Trp W Tryptophan

Val V valine Gln Q glutamine

Met M Methionine Asn N Asparagine

As used herein, the term “nucleotide” refers to a monomer unit of RNA comprising a heterocyclic base containing DNA or a sugar moiety (pentose sugar), phosphorus and nitrogen. The base is bound to the sugar moiety by glycoside carbon (1 'carbon of pentose sugar) and the combination of base and sugar is called nucleoside. Bases are characterized by four bases of DNA: adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). Inosine ("I") is a synthetic base that can be used to substitute any of the four naturally occurring bases (A, C, G or T). The four RNA bases are A, G, C and uracil ("U"). Nucleotide sequences described herein include a line array connected by phosphodiester bonds between the 3 'and 5' carbons of adjacent pentose sugars.

"Oligonucleotide" means a short or single stranded sequence of deoxyribonucleotides linked by phosphodiester bonds. Oligonucleotides are chemically synthesized by known methods and purified, for example, on polyacrylamide gels.

As used herein, the term "dihydrodiconiferyl alcohol benzyl ether reductase" refers to dihydroconiferyl alcohol as 7-O-4'-isodihydrodiconiperyl alcohol as determined in the assay described in Experiment 3. Means an enzyme that can be converted.

The terms "conversion", "amino acid sequence conversion", "variant" and "amino acid sequence variant" refer to dihydrodiconiferyl alcohol benzyl, which differs slightly in amino acid sequence compared to the corresponding natural dihydrodiconiferyl alcohol benzyl ether reductase. Ether reductase molecule. Typically, the variant has at least about 70% homology with the corresponding natural dihydrodiconiferyl alcohol benzyl ether reductase, preferably at least about 80% homology with the corresponding natural dihydrodiconiferyl alcohol benzyl ether reductase. I will have Amino acid sequence variants of the dihydrodiconiperyl alcohol benzyl ether reductase within the scope of the present invention include substitutions, deletions, and / or insertions at certain positions. The sequence of variants of dihydrodiconiperyl alcohol benzyl ether reductase can be used to achieve the desired improved or reduced enzymatic activity, modified regiochemistry or stereochemistry, or converted substrate utilization or product distribution.

Substituted dihydrodiconiferyl alcohol benzyl ether reductase variants are those having at least one amino acid residue in the corresponding dihydrodiconiferyl alcohol benzyl ether reductase sequence inserted at the same position. Substitutions may be those in which only one amino acid is substituted in the molecule, or a plurality in which two or more amino acids are substituted in the same molecule. Significant changes in the dihydrodiconiperyl alcohol benzyl ether reductase molecular activity can be obtained by amino acid substitutions with side chains that are noticeably different from the structure and / or charge of natural amino acids. This type of substitution can be expected to affect the structure of the polypeptide backbone and / or the charge or hydrophobicity of the molecule in the vicinity of the substitution.

Suitable changes in the activity of the dihydrodiconiperyl alcohol benzyl ether reductase molecule are expected by amino acid substitutions having side chains similar to the charge and / or structure of the natural molecule. This type of substitution, which means conservative substitutions, is not expected to significantly alter the structure or structure of the polypeptide backbone or the charge or hydrophobicity of molecules near the substitution.

Insertion dihydrodiconiferyl alcohol benzyl ether reductase variants are those having one or more amino acids inserted closest to an amino acid at a specific position in the natural dihydrodiconiferyl alcohol benzyl ether reductase molecule. Closest to an amino acid means bound to the α-carboxy or α-amino functional group of the amino acid. Insertion may be one or more. Typically, the insertion will consist of one or two conservative amino acids. An amino acid that is similar in charge and / or structure to an amino acid close to the insertion position is defined as conservative. Alternatively, the present invention encompasses the insertion of amino acids having charges and / or structures that differ significantly from amino acids close to the insertion site.

Deletion variants are those in which one or more amino acids of natural dihydrodiconiferyl alcohol benzyl ether reductase are removed. Typically, the deletion variant will have one or two amino acids deleted in certain regions of the dihydrodiconiferyl alcohol benzyl ether reductase molecule.

The term “antisense” or “antisense RNA” or “antisense nucleic acid” means herein a nucleic acid molecule that is complementary to all or part of a messenger RNA molecule. Antisense nucleic acid molecules are typically used to inhibit the expression of complementary expressed messenger RNA molecules in vivo.

Amino acid sequence variants of the dihydrodiconiferyl alcohol benzyl ether reductase may have the desired modified biological activity, including, for example, modified reaction kinetics, substrate utilization, production distribution or other features such as regiochemistry and stereochemistry. have.

The terms "DNA sequence coding", "DNA coding" and "nucleic acid coding" refer to the sequence or sequence of deoxyribonucleotides along the strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the amino acid order of the translated polypeptide chains. Thus, the DNA sequence encodes an amino acid sequence.

The terms "vector", "replicable expression vector" and "expression vector" refer to a generally double stranded DNA fragment (insertion DNA) that can be inserted into a piece of DNA. The vector is used to carry the insert DNA into a suitable host cell. Once inside the host cell, the vector can replicate independently or simultaneously with the host chromosomal DNA, and several copies of the vector and the DNA inserted therein can be produced. Additionally, expression vectors (including replicable expression vectors) contain the necessary elements to translate the insert DNA into a polypeptide. Thus, many polypeptide molecules encoded by insert DNA can be rapidly synthesized.

The terms "transformed host cell", "transformed" and "transformed" mean the introduction of DNA into a cell. The cell is referred to as a "host cell" and it can be a prokaryotic or eukaryotic cell. Prokaryotic host cells typically include various strains of E. coli. Typical eukaryotic host cells are plant cells such as corn cells, yeast cells, insect cells or animal cells. The introduced DNA is generally in the form of a vector containing the inserted DNA fragment. The introduced DNA sequence can be a species such as a host cell or a species other than the host cell, or can be a hybrid DNA sequence comprising foreign DNA and any DNA derived from the host species.

According to the present invention, a cDNA molecule (SEQ ID NO: 1) encoding dehydrodiconiferyl alcohol benzyl ether reductase is a gamma phage made from RNA extracted from Pinus taeda cell suspension culture cells grown in 2,4-D medium. Is separated from the cDNA library based on. This cDNA library was selected using a 5 ′ terminal fragment (SEQ ID NO: 7) from cDNA (PLR-Fi1), which encodes a pinorezinol-lasiriresinol reductase in Forsythia intermedia. Twenty positive phage plaques were purified and cDNA insertions representing the putative reductase clones were sequenced. The β-galactosidase fusion protein encoded by this clone lacked pinorerenolol-larisirezinol activity. The cDNA (SEQ ID NO: 1) encoding the putative reductase was cloned into the expression plasmid pSBETa. As a result, natural protein (SEQ ID NO: 2) lacking the β-galactosidase domain was purified from crude E.coli extract, resulting in pinorezinol-larishrezinol activity and The ability to reduce dihydrodiconiferyl alcohol was analyzed.

Purified P. taeda reductase (SEQ ID NO: 2) was used to convert dihydrodiconiferyl alcohol to 7-O-4 '(iso) dihydrodihydrodiconiperyl alcohol. Reduction of benzyl ether bonds was carried out.

Additionally, two cDNA molecules encoding dihydrodiconiferyl alcohol benzyl ether reductase were isolated from the Cryptomeria japonica cDNA library in the following manner. C.japonica cDNA libraries were selected using Pinus taeda dihydrodiconiferyl alcohol benzyl ether reductase cDNA (SEQ ID NO: 1) increased by 5 ng PCR. Approximately 300,000 pfu of the C.japonica amplified cDNA library was selected and two pCj found to encode dihydrodiconiferyl alcohol benzyl ether reductase (SEQ ID NO: 4 and SEQ ID NO: 6, respectively) Twenty positive plaques of -PCBER1 (SEQ ID NO: 3) and pCj-PCBER2 (SEQ ID NO: 5) were obtained.

Separation of cDNA encoding dihydrodiconiferyl alcohol benzyl ether reductase enables the development of efficient expression systems for functional enzymes and provides a useful tool for investigating the developmental regulation of lignan biosynthesis and Allows separation of peryl alcohol benzyl ether reductase. Separation of the dihydrodiconiperyl alcohol benzyl ether reductase cDNA also allows the transformation of a wide range of organics to enhance or modify lignan biosynthesis.

By way of non-limiting example, the proteins and nucleic acids of the invention can be used to increase or otherwise modify the level of lignans in food items, including plant species, and materials derived from genetically modified plants. have. Antisense nucleic acid fragments complementary to all or part of a nucleic acid sequence encoding dihydrodiconiferyl alcohol benzyl ether reductase, or a nucleic acid sequence encoding dihydrodiconiferyl alcohol benzyl ether reductase, include, but are not limited to, It can be introduced to any plant species for a variety of purposes, including but not limited to: alteration or improvement of color tone, permanence and pest-resistance of fabrics, tree tissues, especially heartwood tissues; of formation of lignans and / or lignin in plant species Reducing or modifying; reducing or modifying the lignan / lignin content of plant species used in pulp and paper making, making pulp and paper production easier and cheaper; by increasing the production of protective lignans or lignin, to carnivores and pathogens. Improvement of the defense ability of plants against; other mediated by lignans or lignin Modification of ecological interactions; introduction, increase or inhibition of the production of dihydrodiconiferyl alcohol benzyl ether reductase or product thereof. Nucleic acid sequences encoding dihydrodiconiferyl alcohol benzyl ether reductase can be introduced into any organic material for a variety of purposes, including but not limited to the following: Production Introduction of Dihydrodiconiferyl alcohol benzyl ether reductase , Increasing or inhibiting or producing an enzymatic product or derivative thereof.

N-terminal transport sequences well known in the art (eg, von Heijne, G. et al., Eur. J. Biochem 180: 535-545 (1989); Styer, Biochemistry WH Freeman and Company, New York, NY , p. 769 (1988)) can be used to direct dihydrodiconiferyl alcohol benzyl ether reductase protein to various locations intracellularly or extracellularly.

Sequence variants of wild-type dihydrodiconiferyl alcohol benzyl ether reductase clones that can be produced by deletions, substitutions, mutations and / or insertions are intended to be within the scope of the invention, except as limited in the prior art. Dihydrodiconiferyl alcohol benzyl ether reductase amino acid sequence variants are prepared by mutagenesis of DNA sequences encoding wild type dihydrodiconiferyl alcohol benzyl ether reductase using techniques commonly referred to as site-directed mutagenesis. Can be. Various polymerase chain reaction (PCR) methods currently well known in the art, such as two primer systems such as the transformant specific site mutagenesis kit at Clontech, can be used for this purpose.

Following denaturation of the target plasmid in the system, two primers are annealed simultaneously to the plasmid; One of the primers contains the desired site mutation. Another includes mutations at another point in the plasmid, resulting in the removal of restriction sites. At that time, the two mutations are strongly bound to produce a second strand synthesis, and the resulting plasmid is transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria and limited to the relevant restriction enzymes (which thereby linearize the non-mutant plasmid), which then retransforms E. coli. This system enables the generation of direct mutations in expression plasmids without the need for the production of aclones or single stranded phagemids. Tight binding of both mutations and subsequent linearization of nonmutant plasmids show high mutation efficiency and allow minimal selection. Following the synthesis of the first restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than preparing a mutant separately at each position, a series of "designed degenerate" oligonucleotide primers can be synthesized to introduce all of the desired mutations at the same time at the receiving site. Transformants may be selected by sequencing of plasmid DNA over the entire site mutated for identification and classification of mutant clones. At that time, each mutant DNA could be restricted and by electrophoresis on a Mutation Detection Enhancement gel (JTBaker) to ensure that no transformation occurred within the sequence (by band shift relative to the non-mutated control). Can be analyzed.

Confirmed mutant duplexes can be cloned into this form of vector if they have not already been cloned into a replicable expression vector, and the resulting expression preparation can be used for the production of high levels of mutant protein and subsequent purification thereof. It can be used to transform E. coli such as (DE3) pLysS strain. The method of FAB-MS mapping can be used to quickly check the fidelity of mutation expression. This technique provides sequencing fragments for all of the proteins and provides the confidence needed for sequencing. In this type of mapping experiment, the protein is degraded by the protease (the choice depends on the specific site to be modified, since the fragment is the most important and the remaining map is the same as the map of the unmutated protein). Fragments are sorted by microbore HPLC (again, inverted phage or ion exchange depending on the particular region to be modified) to provide several peptidase in each fragment and the molecular weight of the peptidase is determined by FAB-MS. Is determined. At that time, the mass is compared with the molecular weight of the peptide expected from the degradation of the predicted sequence and the calibration of the sequence is quickly confirmed. Because mutations close to protein modifications are indicated, sequencing of the converted peptides may not be necessary if the MS does not agree with the prediction. If it is necessary to confirm the changed residues, CAD-tandem MS / MS can be used to sequence the peptides of the mixture or purified target peptides for carboxypeptidase Y degradation depending on the subtracted Edman degradation or modification site.

In the design of specific site specific mutations, it is generally desirable to first make non-conservative substitutions (eg, to Ala for Cys, His or Glu) and determine whether the activity is significantly reduced. The nature of a mutated protein whose changes in binding and / or catalysis can be deduced in comparison with natural enzymes on its own is an indicator sensitive to modified function, with special attention to the kinetics of km and kcat as reaction rate variables. Is investigated. If this means that the residues are important by activity impairment or knockout, then, for example, to substitute Glu for Asp, Cys for Ser, or His for Arg to change the side chain length, The same conservative substitution can be made. Alkyl side chains can also be substituted with aromatics, but most of the size hydrophobic segments can be converted. Changes in general product distribution can indicate which steps in the reaction sequence have been modified by mutations.

Other site specific mutagenesis techniques can also be used for the nucleotide sequences of the present invention. For example, subsequent restriction endonuclease digestion of DNA is described in Sambrook et al., Paragraph 15.3 (Molecular Cloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press, New York, NY (1989)). As described, it can be used to generate dihydrodiconiferyl alcohol benzyl ether reductase deletion variants. Similar strategies can be used to design insertional variants as described in Sambrook et al., Supra, paragraph 15.3.

Oligonucleotide specific mutagenesis can be used to prepare substitutional variants of the invention. It can also be used to readily prepare for deletion and insertion variants of the invention. This technique is described in Adelman et al. It is well known in the art as described in (DNA 1: 183 (1983)). Generally, at least 25 nucleotides of oligonucleotides in length are used to insert, delete or substitute two or more nucleotides of the dihydrodiconiferyl alcohol benzyl ether reductase gene. The optimal nucleotide will have 12 to 15 nucleotides perfectly linked to either side of the nucleotide encoding the mutation. To mutate wild type dihydrodiconiferyl alcohol benzyl ether reductase, the oligonucleotides are annealed to single-stranded DNA template molecules under appropriate hybridization conditions. Generally, DNA polymer forming enzymes, such as the clouno fragment of E. coli DNA polymerase I, are added. This enzyme uses oligonucleotides as primers to complete the synthesis of DNA strands containing mutations. Thus, one strand of DNA encodes a wild type dihydrodiconiferyl alcohol benzyl ether reductase to be inserted into the vector, and the second strand of DNA mutagens the dihydrodiconiferyl alcohol benzyl ether reductase to be inserted into the same vector. Heterodimeric molecules are formed that darken the isolated form. The heterodimeric molecule is then transformed into the appropriate host cell.

Mutants with one or more amino acids substituted can be generated by several methods. If the amino acids are all located close to the polypeptide chain, they can be mutated simultaneously using one oligonucleotide encoding the desired amino acid substitution. However, if the amino acids are located some distance from each other (eg separated by more than 10 amino acids), it is much more difficult to produce a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods can be used.

In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed simultaneously to the single-stranded template DNA and the second strand of DNA synthesized from the template will encode all of the desired amino acid substitutions.

Alternative methods include two or more mutagenesis to produce the desired mutation. The first cycle, as described for a single mutation: wild-type dihydrodiconiferyl alcohol benzyl ether reductase DNA is used as a template, an oligonucleotide encoding the first desired amino acid substitution is annealed to this template, and a hetero duplex DNA molecules are then generated. The second time of mutation uses the mutated DNA, which was used as a template in the first mutagenesis. Therefore, this template already contains one or more mutations. Oligonucleotides encoding additional desired amino acid substitutions are then annealed to this template, and the resulting DNA strands now encode mutations from both the first and second mutagenesis. As a result, the DNA can be used as a template for the third mutagenesis and continued in this way.

Eukaryotic expression systems can be used for the production of dihydrodiconiperyl alcohol benzyl ether reductase as they can perform any necessary post-translational modifications and transport the enzymes to the appropriate intramembrane position. Representative eukaryotic expression systems for this purpose include recombinant baculoviruses, ie Autographa californica nuclear polyhydrin virus (AcNPV; MD Summers and GESmith, A) for the expression of the dihydrodiconiferyl alcohol benzyl ether reductase of the present invention. Manual of Methods for Baculovirus (1986); Luckow et al., Bio-technology 6: 47-55 (1987)). Infection with recombinant baculovirus of insect cells (such as cells of Spodoptera frugiperda species) enables the production of large amounts of dehydrodiconiferyl alcohol benzyl ether reductase protein. In addition, the baculovirus system has another important advantage in the production of recombinant dihydrodiconiferyl alcohol benzyl ether reductase. For example, baculovirus is not infected by humans and therefore can be safely handled in large quantities. In baculovirus systems, DNA constructs are prepared comprising DNA fragments and vectors encoding dehydrodiconiferyl alcohol benzyl ether reductase. The vector allows the replication of the construct within the polyhedrin gene promoter region of baculovirus, the baculovirus conformation sequence essential for correct crossover during recombination (the sequence contains 200 to 300 base pairs adjacent to the promoter) and bacteria. Include bacterial replication origins. The vector is (i) the DNA sequence is located adjacent (or operably linked or "downstream" or "under control") of the polyhedrin gene promoter, and (ii) the promoter / dihydrodiconiperyl alcohol benzyl ether reductase The combination is constructed to be abut on both sides by 200-300 base pairs of baculovirus DNA (adaptation sequence).

In order to produce the dihydrodiconiferyl alcohol benzyl ether reductase DNA construct, cDNA encoding dihydrodiconiferyl alcohol benzyl ether reductase without deletion is obtained using the method as described herein. The DNA construct contacts the appropriate baculovirus DNA (the DNA of a baculovirus of the same species as the promoter encoded within the construct) in the host cell under conditions in which recombination is induced. The resulting recombinant baculovirus encodes the entire dihydrodiconiferyl alcohol benzyl ether reductase. For example, insect host cells can be transfected or transformed separately into DNA constructs and functional baculoviruses. The resulting recombinant baculovirus can then be isolated and used to infect cells to induce dihydrodiconiferyl alcohol benzyl ether reductase production. Host insect cells include, for example, Spodopetra frugiperda cells. Insect host cells infected with the recombinant baculovirus of the invention are then cultured under conditions that allow for baculovirus-encoded dihydrodiconiferyl alcohol benzyl ether reductase expression. The recombinant protein thus produced is then extracted from the cells using methods known in the art.

Other eukaryotic microorganisms such as yeast are also used for the practice of the present invention. Several other strains are available, but baker's yeast Saccharomyces cerevisiae is the commonly used yeast. Plasmid YPp7 (Stinchcomb et al., Nature 282: 39 (1979); Kingsman et al., Gene 7: 141 (1979); Tschemper et al., Gene 10: 157 (1980)) is generally an expression vector in Saccharomyces. Used. This plasmid is strain ATCC No. 44,076 and PEP4-1 (Jones, Genetics 85: 12 (1977)), including the trp1 gene, which provides a selection marker for yeast mutant strains lacking the ability to grow under tryptophan. The presence of trp1 lesions as a feature of yeast host cells now provides an effective environment for transform detection through proliferation in the absence of tryptophan. Yeast host cells are typically transformed using the polyethyleneglycol method as described by Hinnen (Proc. Natl. Acad. Sci. USA 75: 1929 (1978)). Additional yeast transformation procedures are described in Gietz et al., N.A.R. 20 (17): 1425 (1992); Reeves et al., FEMS 99: 193-197 (1992).

Promote sequences in suitable yeast vectors are either promoters of 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073 (1980)) or enolase, glyceraldehyde-3-phosphate dehydro Ginase, hexokinase, pyruvate decarboxylase, phosphoflactokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, trios-phosphate isomerase, phosph Promoters of other glucose degrading enzymes such as poglucose isomerase, and glucokinase (Hess et al., J. Adv. Enzyme Reg. 7: 149 (1968); Holland et al., Biochemistry 17: 4900 (1978)). Include. In constructing suitable expression plasmids, the termination sequences associated with these genes are also linked into the expression vector 3 'desired to be expressed to provide for polymerisation and termination of the mRNA. Other promoters with the additional advantage that transcription is regulated by growth conditions include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes associated with the nitrogen mechanism and glyceraldehyde-3-phosphate di Promoter region of hydroginase and enzymes responsible for maltose and galactose use. Any plasmid vector comprising a yeast-compatible promoter, point of replication and termination sequence is suitable.

Cell-derived cell cultures, such as plants, are used as hosts for the practice of the present invention. Transformed plants include, for example, plasmids encoding dihydrodiconiferyl alcohol benzyl ether reductase and screening marker genes, such as the kac gene encoding kanamycin resistance, Hoeckema et al., Nature 303: 179-. Grafted into Agrobacterium tumifaciens containing a helper Ti plasmid as described in 181 (1983), and the Agrobacterium cells were transformed with the leaf fragments to be transformed as described by An et al., Plant Physiology 81: 301-305 (1986). Can be obtained by culturing. Transformation of cultured plant host cells is normally done via Agrobacterium tumifaciens as described above. Mammalian host cells and host cell cultures that do not have other robust cell membrane barriers are usually transformed with calcium phosphate methods and originally described by Graham and Van der Eb (Virology 52: 546 (1978)) and Sections 16.32-16.37 of Sambrook et al., supra. However, polybrene (Kawai and Nishizawa, Mol. Cell. Biol. 4: 1172 (1984)), protoplast fusion (Schaffner, Proc. Natl. Acad. Sci. USA 77: 2163 (1980)), electroporation (Neumann et al., EMBO J. 1: 841 (1982)), and other intracellular DNA transduction methods such as direct microinjection into the nucleus (Capecchi, Cell 22: 479 (1980)). Figures are also summarized in Monastersky GM and Robl, JM, Strategies in Transgenic Animal Science, ASM Press, Washington, DC (1995) Transformed plant coli can induce cells, for example kanamycin, and an appropriate amount of callus and germination. It is grown on plant hormone containing media such as naphthalene acetic acid and benzyladenin for selection to selectable markers.The plant cells are then regenerated and the resulting plants are transferred to the soil by techniques well known to those skilled in the art.

Additionally, nucleic acid sequences encoding dihydrodiconiferyl alcohol benzyl ether reductase can be inserted into the plant with the necessary inducible promoter. In an embodiment of the invention, a promoter that responds only to a specific external or internal stimulus is fused to the target cDNA. Thus, the gene will not be transcribed without responding to a specific stimulus. Unless a gene is transcribed, that gene product is not produced.

An illustrative example of a reactive promoter system that can be used in the practice of the present invention is a corn glutathione-S-transferase (GST) system. GST is a family of enzymes capable of decoding many hydrophobic electrophilic compounds, often used as pre-emergency herbicides (Weigand et al., Plant Molecular Biology 7: 235-243 (1986)). The study demonstrated that GST is directly involved in inducing this enhanced herbicide tolerance. This activity is mediated by specific 1.1 kb mRNA transcription products. In summary, maize has naturally occurring, preexisting potential genes that can respond to external stimuli and be induced to produce gene products. This gene has been previously identified and cloned. Thus, in an embodiment of the invention, the promoter is removed from the GST reactive gene and attached to the dihydrodiconiferyl alcohol benzyl ether reductase gene from which the natural promoter has been previously removed. This engineered gene is a combination of a promoter responsible for the successful production of a dihydrodiconiferyl alcohol benzyl ether reductase protein in response to external chemical stimulation.

In addition to the foregoing methods, several methods are known in the art, including jimnosperm, angiospor, monocoats and dicots, which transfer cloned DNA to a wide variety of plant species (Glick and Thompson, eds. , Methods in Plant Molecular Biology, CRC Press, Boca Raton, Florida (1993). Representative examples include electroporation-promoting DNA uptake by protoplasts (Rhodes et al., Science 240 (4849): 204-207 (1988)); Polyethylene glycol treatment of protoplasts (Lyznik et al., Plant Molecular Biology 13: 151-161 (1989)); And cell bombardment with micropropellant loaded DNA (Klein et al., Plant Physiol. 91: 440-444 (1989) and Boynton et al., Science 240 (4858): 1534-1538 (1988)). For example, numerous methods of transformation of cereals currently exist (McKinnon, GE and Henry, RJ, J. Cereal Science 22 (3): 203-210 (1995); Mendel, RR and Teeri, TH, Plant and Microbial Biothecnology Research Series, 3: 81-98, Cambridge University Press (1995); McElroy, D. and Brettell, RIS, Trends in Biotechnology, 12 (2): 62-68 (1994); Christou et al., Trendsin Biotechnology 10 (7): 239-246 (1992); Christou, P. and Ford, TL, Annuals of Botan, 75 (5): 449-454 (1995); Park et al., Plant Molecular Biolog, 32 (6) : 1135-1148 (1996); see Altpeter et al., Plant Cell Report, 16: 12-17 (1996)). In addition, plant transformation strategies and techniques are described in Forest et al., Exp. Agric. 33: 15-33 (1997). Minor variations make this technology applicable to a wide range of plant species.

Each of these techniques has advantages and disadvantages. In each technique, plasmid free DNA is genetically engineered to include selectable and searchable marker genes as well as the gene of interest. Selectable genes are used only to select cells with inserted plasmids of the inserted copy (therefore their constructs are transferred with the gene of interest and selectable and searchable genes in one unit). Searchable genes provide yet another confirmation of successful culture of cells only containing the gene of interest. A commonly used searchable marker gene is neomycin phosphotransferase II (NPTII). This gene delivers kanamycin resistance, which can be added directly on the growth medium on which cells grow. Plant cells are normally susceptible to kanamycin and die as a result. The presence of the NPTII gene overcomes the effects of kanamycin and each cell with this gene survives. Another selectable marker gene that can be employed in the practice of the present invention is a gene with herbicide glufosinate (Bast) resistance. A commonly used searchable gene is the β-glucuronidase gene (GUS). The presence of this gene is characterized using a histochemical reaction in which the cell sample suspected of transformation is treated with a GUS assay solution. After proper incubation, the cells containing the GUS gene turn blue. Preferably, the plasmids comprise both selectable and searchable marker genes.

Plasmids containing one or more such genes are introduced into plant protoplasts or callus cells by any of the techniques described above. If the marker gene is a selectable marker, only cells into which the DNA package is inserted survive the selection of suitable phytotoxic agents. Once the appropriate cells are identified and propagated, the plant is regenerated. Transformed plant-derived progeny should be tested to confirm that the DNA package was successfully inserted into the plant genome.

Mammalian host cells are also used in the practice of the present invention. Examples of suitable mammalian host cell lines include monkey kidney CVI strains transformed with SV40 (COS-7, ATTCC CRL 1651); Human embryonic kidney strain 293S (Graham et al., J. Gen. Virol. 36: 59 (1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (Urlab and Chasin, Proc. Natl Acad.Sci USA 77: 4216 (1980)); mouse cetriocytes (TM4, Mather, Biol. Reprod. 23: 243 (1980)); monkey kidney cells (CVI-76, ATCC CCL 70); African green monkey Kidney cells (VERO-70, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); calf kidney cells (MDCK, ATCC CCL 34); buffalo mouse liver cells (BRL 3A, ATCC CRL 1442); human lungs Cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary cells (MMT 060562, ATCC CCL 51); mouse liver cancer cells (HTC, MI.54, Bauumann et al., J. Cell Biol. 85: 1 (1980); and TRI cells (Mather et al., Annals NYAcad. Sci. 383: 44 (1982)) Expression vectors for these cells (if required) are usually time of replication. , A promoter, ribosome attachment site, located in front of the gene to be expressed, DNA sequences for RNA splice sites, polymerized adenylation sites, and transcription termination.

Promoters used in mammalian expression vectors are often derived from viruses. Such viral promoters are usually derived from the polyoma virus, adenovirus 2, and most commonly Simian Virus 40 (SV40). The SV40 virus contains two promoters named early and late promoters. Both such promoters are particularly useful as they can easily be obtained from a virus as a DNA fragment, which also has a point of virus replication (Fiers et al., Nature 273: 113 (1978)). Smaller or larger SV40 DNA fragments can also be used provided they contain approximately 250 bp sequences ranging from the HindIII site located in the viral replication origin to BglII.

Alternatively, a promoter (homologous promoter) naturally associated with a foreign gene can be used, as long as it is compatible with the host cell line selected for transformation.

The point of replication is obtained from exogenous origin such as SV40 or other viruses (eg Polyoma, Adeno, VSV, BPV) and inserted into cloning vectors. Alternatively, the point of replication is provided from a host cell chromosome replication mechanism. If a vector containing a foreign gene is inserted into the host chromosome, the latter is often sufficient.

The use of secondary DNA coding sequences can enhance the level of production of dihydrodiconiferyl alcohol benzyl ether reductase in the transformed cell line. Secondary DNA coding sequences typically include the enzyme dihydrofolate reductase (DHFR). The DHFR wild type is normally inhibited by the chemical methotrexate (MTX). DHFR expression levels will vary depending on the amount of MTX added to the cultured host cell. An additional feature of DHFR that makes DHFR particularly useful as a secondary sequence is that it can be used as a selectable marker to identify transformed cells. Two forms of DHFR, wild type DHFR and MTX-resistant DHFR, are available for secondary sequence use. The type of DHFR used for a particular host cell depends on whether the host cell lacks DHFR (if lacking it produces endogenously low levels of DHFR or does not produce functional DHFR). DHFR-deficient cell lines, such as the CHO cell line described by Urlaud and Chasin, supra., Are transformed with the coding sequence of wild type DHFR. After transformation, these DHFR-deficient cell lines express functional DHFR and can be grown in culture media lacking nutrient hypoxanthine, glycine, and thymidine. Non-transformed cells will not survive in this medium.

MTX-resistant DHFR can be used as a means of screening for transformed host cells in host cells that endogenously produce a normal amount of functional DHFR that is MXT sensitive. CHO-KL cell line (ATCC No. CL 61) has this feature and is therefore a useful cell line for this purpose. The addition of MXT to cell culture medium allows only cells transformed with DNA encoding MTX-resistant DHFR. Non-transformed cells will not survive in this medium.

Prokaryotes are used as host cells in the initial cloning step of the present invention. Prokaryotes are particularly useful for the production of fast, large amounts of DNA for the production of single-stranded DNA templates for use in site mutagenesis, for the retrieval of many mutations simultaneously, and for the DNA sequencing of the resulting mutations. Suitable prokaryotic host cells include E. coli strain 294 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli X1776 (ATCC No. 31,537), and E. coli B; However, many other E. coli strains, such as HB101, JM101, NM522, NM538, NM539, and prokaryotes of many different species or genera, including the bacilli, Baillius subtilis, enterobacteria such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species. Can be used as Prokaryotic host cells or host cells with other rigid cell walls are preferably transformed using the calcium chloride method, as described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation is used for the transformation of these cells. Prokaryotic transformation techniques are described in Dower, W.J., Genetic Engineering, Principles and Methods, 12: 275-296, Plenum Publishing Corp. (1990); Hanahan et al., Meth. Enxymol., 204: 63 (1991).

As a representative example, a cDNA sequence encoding dihydrodiconiferyl alcohol benzyl ether reductase is transferred to a commercially available (His) ₆ · Tag pET vector (from Novagen) for overexpression in E. coli as a heterologous host. This pET expression plasmid has several advantages in high level heterologous expression systems. The desired DNA insert is linked to a plasmid vector sequence frame, which encodes six histidines followed by a high specific protease recognition site (thrombin) and is linked to the amino terminal code of the target protein. Histidine “fragments” of expressed fusion proteins promote very strong adhesion to immobilized metal ions and allow for the rapid purification of recombinant proteins by immobilized metal ion affinity chromatography. Histidine leader sequences are then cleaved at specific proteolytic sites by thrombin treatment of the purified protein, and the dihydrodiconiperyl alcohol benzyl ether reductase protein is eluted. The overexpression-purification system has a high capacity, excellent resolution, is fast, and has very few chances of E. coli protein contamination with similar adhesion tendencies (before and after thrombin proteolysis).

As will be apparent to those skilled in the art, any plasmid vector comprising replicon and regulatory sequences derived from species compatible with the host cell may also be used in the practice of the present invention. Vectors usually have a polymerization linker site comprising a replication site, a marker gene that provides for transgenic intracellular selection, one or more promoters, and some restriction sites for insertion of foreign DNA. Plasmids typically used for E. coli transformation include pBR322, pUC18, pUC19, pUC118, pUC119, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., Supra. However, many other suitable vectors are also available. These vectors include genes encoding ampicillin and / or tetracycline resistance that allow cells transformed with the vector to grow in the presence of these antibiotics.

The most commonly used promoters in prokaryotic vectors are β-lactamase (penicillinase) and lactose promoter system (Chang et al. Nature 375: 615 (1978); Itakura et al., Science 198: 1056 (1977); Goeddel et al., Nature 281: 544 (1979)) and tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8: 4057 (1980); EPO Appl. Publ. No. 36,776), and eggs Carin phosphatase systems. These are most commonly used, but other microbial promoters have also been used, and details of their nucleotide sequences have been published so that the skilled person can maintain the function of these promoters and link them into the plasmid vector (Siebenlist et al., Cell 20: 269 ( 1980).

Many eukaryotic proteins are normally secreted from cells that contain intrinsic secretion signal sequences as part of the amino acid sequence. Thus, a protein normally found in cells can be a target by which a signal sequence is linked to and secreted from the protein. This is accomplished immediately by linking the DNA encoding the signal sequence to the 5 'end of the DNA encoding the protein and then expressing this fusion protein in a suitable host cell. DNA encoding a signal sequence is obtained as a restriction fragment from any gene encoding a protein having a signal sequence. Thus, signal sequences of prokaryotes, yeasts, and eukaryotes may be used herein depending on the host cell type used to practice the invention. For example, DNA and amino acid sequences encoding signal sequence portions of several eukaryotic genes, including human growth hormone, pre-insulin, and pre-albumin, are known (Stryer, Biochemistry WH Freeman and Company, New York, NY, p. 769 (1988)), which can be used as a signal sequence for a suitable eukaryotic host cell. Yeast signal sequences are used to induce secretion from yeast host cells, for example as acid phosphatase (Arima et al., Nucleic Acids Res. 11: 1657 (1983)), alpha-factors, alkaline phosphatase and invertase do. For example, prokaryotic signal sequences derived from LamB or OmpF (Wong et al., Gene 68: 193 (1988)), MalE, PhoA, or beta-lactamase, as well as other genes, target proteins from prokaryotes into the culture medium. It can be used to

Plant, animal and microbial derived traffic sequences can be employed in the practice of the present invention to target gene products for transport to, or spread into, the cytoplasm, endoplasmic reticulum, mitochondria or other intracellular organelles. This consideration applies to the indication of intracellular or intact organ expression such that dihydrodiconiferyl alcohol benzyl ether reductase overexpression and gene products can function at any desired site.

Construction of a suitable vector comprising a DNA that encodes a replication sequence, a regulatory sequence, a transgenic gene and a dihydrodiconiferyl alcohol benzyl ether reductase DNA of interest is prepared using standard recombinant DNA procedures. Separated plasmids and DNA fragments are cleaved, cut, and linked together in a particular order to produce the desired vector (see, eg, Sambrook et al., Supra.), As is well known in the art.

As noted above, the dihydrodiconiferyl alcohol benzyl ether reductase variant is preferably produced by mutations generated using site specific mutagenesis methods. This method requires the synthesis and use of the desired mutations, and a sufficient number of contiguous nucleotides, specific oligonucleotides encoding both sequences, to allow stable hybridization of the oligonucleotide to the DNA template.

The former is more fully understood in connection with the representative examples below, where "Plasmid" is represented by a lowercase p followed by an alphanumeric notation. Starting plasmids used in the present invention can be made commercially available, unlimited common use, or by published procedures from such available plasmids. In addition, other equivalent plasmids are known in the art and will be apparent to those of ordinary skill in the art.

"Disassembly", "cutting" or "cutting" of DNA refers to catalytic cleavage of DNA by enzymes that act only at specific positions in the DNA. Such enzymes are referred to as restriction endonucleases and sites along the DNA sequence that each enzyme cuts are referred to as restriction sites. Restriction enzymes used in the present invention are commercially available and used according to the instructions supplied by the manufacturer (see Section 1.60-1.61 of Sambrook et al., Supra.).

"Recovery" or "separation" of a given DNA fragment, derived from restriction digestion, refers to polyacrylamide gel or agar gel phase separation by electrophoresis of the resulting DNA, identification of fragments of interest through comparison of motility with marker DNA of known molecular weight, Removal of gel fragments containing the desired fragments, and gel separation from DNA. This procedure is generally known. See, for example, Lawn et al. (Nucleic Acids Res. 9: 6103-6114 (1982)) and Goeddel et al. See Nucleic Acids Res., Supra.

Example 1

Cloning of Dehydrodiconiferyl Alcohol Benzyl Ether Reductase Protein from Pinus taeda

Unless otherwise stated, the following materials, methods and apparatuses were used in Example 1 and all subsequent examples.

Plant material-Pinus taeda cell suspension culture as described above (van Heerden, PS, Towers, GHN & Lewis, NG J. Biol. Chem. 271, 12350-12355 (1996)), 2,4-dichlorophenoxy It was maintained in a medium containing acetic acid (2,4-D). Seven days after transfer to fresh medium, cells were obtained by filtration and frozen in liquid nitrogen and stored at -80 ° C.

General Methods—Unless otherwise described below, all molecular biological techniques were performed according to standard methods (Sambrook, J., Fritsch, EF & Maniatis, T. Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1994); Ausubel, FM, et al., Current Protocols in Molecular Biology, 2 volumes (Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons, NY, 1991).

Substance-All solvents and chemicals used were reagent or HPLC grade. Taq thermostable DNA polymerase was obtained from Promega. Competitive NovaBlue cells were purchased from Novagen and radiolabeled nucleotides ([α ^-32 P] dCTP) were purchased from DuPont NEN. pCRII TA cloning kit was purchased from Invitrogen. Restriction endonuclease NdeI was purchased from New England Biolabs.

Oligonucleotide primers for polymerase chain reaction (PCR) and sequencing were synthesized by Gibco BRL Life Technologies. GENECLEAN II for purification of PCR fragments Kit (BIO 101 Inc.) was used, wherein the gel purified DNA concentration was measured by comparing it to a low DNA mass ladder (Gibco BRL) in a 1.5% agarose gel.

Instrumentation-UV (including RNA and DNA measurements at OD ₂₆₀ ) spectra were read on a lambda 6 UV / VIS spectrometer. All PCR amplifications were used with the Temptronic II thermocycler (Thermolyne). Purification of the plasmid DNA for sequencing involves the QIA Well Plus Plasmid Purification System (Qiagen) followed by PEG precipitation or Wizard. A Plus SV Minipreps DNA Purification System (Promega) was used, where DNA sequences were determined using Applied Biosystems Model 373A Auto Sequencer. All high performance liquid chromatography (HPLC) separations were performed on Millenium (Waters, Inc.) or Alliance (Waters, Inc.) instruments and the effluent was monitored at 280 nm.

Pinus taeda cDNA Library Synthesis—Total RNA (100 μg / g new weight) was obtained from frozen tea grown as described above using the method of Dong and Dunsten (Dong, JZ & Dunstan, DI Plant Cell Reports 15 (1996)). Obtained from Pinus taeda cells. Pinus taeda cDNA library contains 5 μg of purified poly (A) ⁺ mRNA (Oligotex-dT ^™ suspension, Qiagen) and ZAP-cDNA. Synthesis kit, ZAP ^TM XR vector Uni, and Gigapack II Gold packaging extract (Stratagene) was used and the titer for the primary library was 1 × 10 ⁶ pfu. Amplified libraries (1 × 10 ⁹ pfu / ml, 120 ml total) were used for screening (Dinkova-Kostova, AT, et al., J. Biol. Chem. 271, 29473-29482 (1996)).

DNA Probe Synthesis—Pinoresinol-Larisirresinol Reductase cDNA (PLR-Fil) (Dinkova-Kostova, AT, et al., J. Biol , previously isolated, having the nucleic acid sequence shown in SEQ ID NO: 7) The 5'-end of Chem. 271: 29473-29482 (1996) was used as a probe to screen the Pinus taeda cDNA library for similar / homologous enzymes. Probes were constructed as follows: 10 ng of purified pBSPLR-Fil plasmid (PLR-Fil contained in cloning plasmid pBluescript SK [-]) was added to 5 100 μl of PCR reaction solution (10 mM Tris-HCl [pH]). 9.0], 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl ₂ , 0.2 mM of each dNTP and 2.5 units of Taq DNA polymerase), with the primer PLRNT1 (AT (A / T / C) AT (A / T / C) GGI GGI ACI GGI TA) (SEQ ID NO: 8) (100 pmol) and PLRI5R (TC (T / C) TCI A (A / G) I GTI AC (T / C) TTI CC) (SEQ ID NO: 9) (100 pmol). PCR amplification was performed in a thermocycler as follows: 35 cycles for 1 minute at 94 ° C., 2 minutes at 50 ° C., 3 minutes at 72 ° C .; And hold at 72 ° C. for 5 minutes after the last cycle and then hold at 4 ° C. for an indefinite time. Concentrate the five reaction solutions (Microcon 30, Amicon Inc.), wash with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA; 2 × 200 μl), then continue to PCR product Recovered in buffer (2 × 50 μl). PLR-Fil 5′-end reaction product (˜400 base pair bands) was resolved on a 1.5% agarose gel for separation and GENECLEAN II Purified from agarose using kit (BIO 101 Inc.). Gel purified PLR-Fil 5′-end fragment (SEQ ID NO: 7) (50 ng), following the kit instructions, ^T7 QuickPrime from Pharmacia Radiolabeled probes were prepared (in 0.1 ml) using the kit and [a- ³² P] dCTP and purified on a BioSpin 6 column (Bio-Rad) to carry carrier DNA (0.9 ml of 0.5 mg / ml sheared). Salmon sperm DNA [Sigma]).

Library Screening-600,000 pfu of Pinus taeda amplified cDNA libraries were plated for primary screening following the instructions of Stratagene. Plaques were blotted onto Magna nylon membrane circles (Micron Separations Inc.) and then left to dry in air. Only two hits of membrane Placed between 3MM Chr paper. cDNA library phage DNA was immobilized on the membrane and denatured by autoclaving at 100 ° C. for 2 minutes in one step and then evacuating rapidly. Membranes were washed in 6 × standard saline citrate (SSC) and 0.1% SDS at 37 ° C. for 30 minutes, preheated 6 × SSC on crystallization dish (190 × 75 mm) at 45 ° C. for 5 hours, Prehybridization was followed by gentle shaking in 0.5% SDS and 5 × Denhardt's reagent (hybridization solution, 300 ml). [ ³² P] Radioactive labeled probe (SEQ ID NO: 7) was denatured (boiled for 10 minutes), rapidly cooled (ice, 15 minutes) and then preheated fresh hybridization on crystallization dish (150 × 75 mm) To solution (60 ml, 45 ° C.). The hybridized membrane was then added to this dish and covered with plastic wrap over it. Hybridization was performed with gentle shaking at 45 ° C. for 18 hours. The membrane was washed at room temperature for 5 minutes in 4 × SSC and 0.5% SDS, then transferred to 2 × SSC and 0.5% SDS (room temperature) and wrapped in plastic wrap to prevent drying for 20 minutes at 45 ° C. Incubation with gentle shaking for a while, and finally exposed to Kodak X-OMAT AR film with increasing screen at -80 ° C for 24 hours. Twenty positive plaques were purified by adding two more rounds of hybridization and screening as described above.

In Vivo Excision and Sequencing of Putative Reductase Protein cDNA-Containing Phagemids—Purified cDNA clones were freed from phage following Stratazine's in vivo excision protocol. Fully sequenced using sequencing primers that overlap two strands of several different cDNAs encoding genes homologous to PLR-Fil. Two cDNAs were identified and they differed only in their 5'- and 3'-untranslated regions at the site inserted into the pBluescript SK [-] cloning plasmid. The difference between these two cDNAs was a cloning artifact, so that only one gene homologous to F.intermedia pinoresinol-larisilesinol reductase was cloned from this Pinus taeda cell suspension culture cDNA library. The nucleotide sequence of this reductase gene is shown in SEQ ID NO: 1, which indicates a sequence common to both of the aforementioned reductase cDNAs.

Sequencing-DNA and amino acid sequencing can be found in the Unix-based GCG Wisconsin Package (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, 1994); Rice, P. (The Sanger Center, Hinxton Hall, Cambridge, England (1996) ) And ExPASy World Wide Web Molecular Biology Server (Geneva University Hospital and University of Geneva, Geneva, Switzerland).

Example 2

Expression of Dehydrodiconiferyl Alcohol Benzyl Ether Reductase Protein as a Fusion Protein in E. Coli

Expression as a fusion protein of dehydrodiconiferyl alcohol benzyl ether reductase protein in Escherichia coli-An open reading frame of the putative reductase obtained from Pinus taeda was placed in frame with the β-galactosidase gene α-offset particles of pBluescript. . Thus its purified plasmid DNA was transformed in NovaBlue cells according to Novagen's instructions. Transformed cells (5 ml of culture) were shaken at 37 ° C. until reaching intermediate log phase (OD ₆₀₀ = 0.5) in LB medium containing 12.5 μg / ml tetracycline and 50 μg / ml ampicillin (225 rpm) was grown. IPTG (isopropyl β-D-thioglucopyranoside) was then added at a final concentration of 10 mM and left to grow for 2 hours. Cells were collected by centrifugation and then resuspended in 500 μl (per 5 ml of culture tube) buffer (20 mM Tris-HCl, pH 8.0, 5 mM dithiothritol). Lysozyme (5 μl of 0.1 mg / ml, Research Organics, Inc.) was then added and incubated for 10 minutes before lysing the cells (3 × 15 seconds). After centrifugation at 14,000 × g for 10 minutes at 4 ° C., the supernatant was taken and analyzed for the activity of pinoresinol-lasirishinol reductase and dehydrodiconiperyl alcohol reductase, as described herein and in Example 3. The assay was as described in (210 μl of supernatant used for assay). Even if the assay was allowed to incubate for 24 hours, the same system was used to generate catalytically active (+)-pinoresinol / (+)-larishrecinol reductase from F. intermedia . (Dinkova-Kostova, AT, et al., J. Biol. Chem. 271: 29473-29482 (1996)), in E. coli extracts expressing the protein as a fusion with the β-galactosidase gene α-complementary particles No pinoresinol-larishresinol reductase activity was observed.

Pinot Lecinol was evaluated by monitoring the Pinot Lecinol the reductase activity ^[3 H] La when Lecinol and ^[3 H] Seko formation of Ra receiver Lecinol-La when the radioactive chemical assay for Lecinol reductase activity. Each assay for pinoresinol reductase activity consisted of 20 mM Bis-Tris propane, pH 7.0, 0.4 mM (±) -pinoresinol (added to 20 μl MeOH) and enzyme preparation (ie, from E. coli). Total extract, 210 μl). Enzyme reaction was started by adding 0.8 mM [4 R ^-3 H] NADPH (6.79 MBq / mmol). After incubation with shaking at 30 ° C. for 3 hours, the assay mixture was used as a radiochemical carrier to contain (±) -larisicylenol (20 μg) and (±) -secoisolarishresinol (20 μg). 500 μl). After centrifugation (17,000 × g, 5 min), EtOAc solubles were taken and the extraction process was continued with 500 μl EtOAc. EtOAc solubles were combined for each assay and aliquots (100 μl) were taken to determine their radioactivity using liquid scintillation counting. The remainder of the combined EtOAc solubles was evaporated to dryness and reconstituted in MeOH / H ₂ O (3: 7, 100 μl) followed by reverse phase HPLC. Larrislecinol reductase activity was assessed by monitoring the formation of [ ³ H] secoisolariciresinol. The assay was carried out exactly as described above, except that 0.4 mM of (±) -larishlesinol was used as the substrate and (±) -secoisorariciresinol (20 μg) was added as a radiochemical carrier. HPLC was performed as described above (Dinkova-Kostova, AT, et al., J. Biol. Chem. 271: 29473-29482 (1996)) to separate the lignan substrate and the product. Briefly stated, reversed phase column chromatography consists of a Nova-pak C ₁₈ column (3.5 mm x 150 mm, Waters) and MeOH: 3% acetic acid (in H2O) (30:70) at a flow rate of 0.5 ml / min. An isocratic solvent system was used. 0.5 ml fractions were collected and subjected to scintillation counting to determine the level of integration into the ³ H assay product.

Non-Radioactive Assay for Pinoresinol-Larisirresin Reductase Activity—Pinoresinol reductase activity was further performed as described above with the following exception assay: total volume was 150 μL ; 4 mM of NADPH (non-radioactive) was used; 2 mM of (±) -pinoresinol or (±) -larishrecinol was used as substrate; No (±) -laryricinol or (±) -secoisolaryricininol was added as a radiochemical carrier; The reaction was stopped by boiling for 3 minutes; 50 μl of MeOH was then added to 30% concentration and the assay mixture was centrifuged (17,000 × g, 3 min); 150 μl of the resultant mixture was directly injected onto the HPLC to determine whether any of the larisirecinol and the secoisolariciresinol were formed.

As a result, when the assay lasted for 24 hours (ie, all available substrates and F. intermedia reductase [PLR-Fil] (Dinkova-Kostova, AT, et al., J. Biol. Chem. 271: 29473- 29482 (1996)), was found to be not reduced to either larysiresinol or secoisorarishrecinol, respectively, under the conditions that the depletion) was either pinoressinol or larislesilenol.

Example 3

Expression of Natural Dehydrodiconiferyl Alcohol Benzyl Ether Reductase Protein (SEQ ID NO: 2) in Escherichia Coli

Delivery of putative reductase into pSBETa—overexpressed plasmid pSBETa has two restriction sites that can be utilized for sub-cloning (BamHI for expression with small fusions with NdeI for natural expression), and the argU gene (rare And a pET3a expression cassette containing for the production of AGA-Arg tRNA _arg4 ) and kanamycin resistance (for high plasmid stability during liquid culture). Since the putative reductase obtained from Pinus taeda does not have an internal NdeI site, a primer for introducing NdeI into starting methionine (primer PT-ATG-NdeI: TTC AGG GCC CAT ATGGGA AGC AGG AGC AGG ATA CTC) (SEQ ID NO: 10) and primers for introducing the NdeI site into the 3'-end untranslated region (primer PT-Rev-NdeI: TGT CGA ATA CAT ATG AAA GGC GAT AAC CAA CAA TTT) (SEQ ID NO: 11) . These two primers (SEQ ID NO: 10) and (SEQ ID NO: 11) (5 pmol each) are cDNA (SEQ ID NO: 2) encoding the putative Pinus taeda reductase (SEQ ID NO: 2) as described above. : 1) Five PCR reaction solutions containing 10 ng were used and sub-cloned into pCRII plasmid according to Invitrogen's instructions. The resulting pCRII containing the estimated reductase was purified and digested with NdeI to purify the resulting 1 kb fragment gel, ligated into pSBETa previously digested with NdeI, and then transduced in competing NovaBlue cells. Switched. The resulting pSBET construct containing the putative putative reductase was purified and the expression region containing the desired cDNA was fully sequenced for both strands, demonstrating that no mutation occurred during PCR.

General Procedure for Enzyme Purification—The protein purification procedure was performed at 4 ° C. and the chromatographic effluent was monitored at 280 nm. Protein concentration was measured using BioRad's protein measurement kit. Polyacrylamide gel electrophoresis was performed using gradients (4-15%, linear gradient, BioRad) under denaturing and reducing conditions in the Laemry buffer system, followed by visualization of the proteins by silver staining.

Overexpression of natural enzyme (SEQ ID NO: 2) in E. coli -B834 (DE3) Escherichia coli for expression of the resulting pSBETa plasmid containing putative reductase (SEQ ID NO: 2) from Pinus taeda Cells were transformed. High level expression of putative reductase (SEQ ID NO: 2) was inoculated with 2-4 ml of 10 ml culture grown overnight in 50 ml / ml of kanamycin in 1 liter of LB broth. By doing so. The cells were then grown at 37 ° C. with 250 rpm until a density of OD ₆₀₀ = 0.65 was achieved, at which point the growth conditions were changed to 20 ° C. and 265 rpm. The production of reductase (SEQ ID NO: 2) was induced by adding IPTG to a final concentration of 1 mM and the cells reached a temperature of about 22 ° C. (about 30 minutes after the incubator temperature changed). Cells were then grown for 21 hours, then cells were harvested by centrifugation in a 4 × 250 ml centrifuge bottle at 3000 × g for 25 minutes, and then the pellet was frozen at −80 ° C. for at least 2 hours. Accelerated.

Crude protein preparation—4 pellets of E. coli cells obtained in the previous step were thawed at room temperature and then 10 ml of Buffer A (20 mM Tris-HCl, pH 8.0; 2 mM ethylenediamine tetraacetic acid [EDTA]; 1 mM Phenylmethylsulfonyl fluoride [PMSF]; (Pare, PW, Wang, H.-B., Davin, LB & Lewis, NG Tetrahedron Lett. 35: 4731-4734 (1994)) 5 mM dithiothitol [ DTT]), respectively, were resuspended, and then combined and sonicated for 3 x 30 seconds. The sonicates were centrifuged at 20,000 × g for 30 minutes to pellet cell debris and filtered through a 0.2 μm syringe filter. The resulting filtrate (290 ml) was precipitated with 40-70% ammonium sulfate saturated cut using ammonium sulfate, then centrifuged at 20,000 × g for 30 minutes and then PD-10 desalting column (Pharmacia ) And desalted with buffer A again.

Affinity Column (Affi-Blue Gel) Tablets—Affi-Gel Blue Gel (BioRad) columns, 1.6 cm in diameter and 11.5 cm (23 ml bed volume) in length, were pre-equilibrated with Buffer A. Desalted 40-70% cut (15 ml, 513 mg) was then applied to the column. After washing the column with 300 ml of Buffer A (1 ml / min), run a linear gradient of 500 ml of 0 to 100% Buffer B (Buffer A + 5 M NaCl) and 100% Buffer for 60 ml. After holding B, the reductase (SEQ ID NO: 2) was eluted at 30 ml by returning from 100% buffer B to 100% buffer A in 30 ml. Fractions of 4 ml were collected and putative reductase (SEQ ID NO: 2) eluted in fractions 33-43. These fractions were collected and concentrated to about 5 mg / ml in a Centricon 10 microconcentrator (Amicon, Inc.), desalted on a PD100 column and assayed for activity.

Anion exchange chromatography-The resulting enzyme solution (118 mg) was taken to a POROS 20 QE perfusion anion exchange column previously equilibrated with buffer C (50 mM Bis-Tris propane, pH 6.8, 5 mM DTT) to the next step. Most of the contaminating E. coli proteins remained applied and bound to the anion exchange column under these conditions while eluting under load (ie not bound to the column).

Cation exchange chromatography-The resulting enzyme solution (59 mg) was taken to the next step in a POROS 20 SP perfusion cation exchange column previously equilibrated with buffer C (50 mM Bis-Tris propane, pH 6.8, 5 mM DTT). Most of the remaining contaminating E. coli proteins remained bound to the cation exchange column under these conditions while being applied and eluted under load (ie, not bound to the column). This gave 36 mg of purified putative reductase protein (SEQ ID NO: 2).

Enzyme Characterization—Optimal temperature and pH were determined using standard (non-radioactive) assay conditions as described above. However, the concentration of the buffer was changed to 19 mM and the protein (30 μl) after the Affi-Blue column chromatography step was used. Product formation analysis was investigated by reverse phase HPLC analysis, with the exception of the following: For temperature optimization, incubation was performed at different pH (7.0) at varying temperatures (6 to 58 ° C.); For pH optimization, the pH was varied (5.5-9.5) at constant temperature (30 ° C.). Initial rate kinetics were analyzed by assaying protein activity under standard (non-radioactive) conditions at pH 7.0, but performed at 11 different lignan substrate concentrations (0.167-2.5 mM) and 6 hours at 22 ° C., while NADPH concentrations were 5 It was kept constant at mM. In order to determine whether the 4 R -or 4 S -hydride of the NADPH cofactor is used for enzymatic catalyzed reduction, radiochemical assays were specifically labeled (4 R- [ ³ H] NADPH or 4 S- [ ³ H] NADPH) and analyzed for radiochemical integration into 7- O- 4 '-(iso) dihydrodehydrodiconiferyl alcohol product.

Radiochemical Assays for Dehydrodiconiferyl Alcohol Reductase Activity—Each 150 μl of assay is 19 mM MES: Bis-Tris Propane, pH 6.5, 20 μl protein solution of corresponding purity grade, 5 mM DTT, It consisted of 2.5 mM (±) -dehydrodiconiperyl alcohol and 5 mM of 4 R- [ ³ H] -NADPH (14.2 x 10 ³ kBq / mmol). After incubation at 22 ° C. for 6 hours, the assay mixture was extracted with EtOAc (2 × 500 μl). The EtOAc soluble fraction was evaporated to dryness and reconstituted in 100 μl of CH ₃ CN: 3% acetic acid (1: 9), followed by reversed phase HPLC as described above, both UV and radiochemical detection It was. Fractions of 1 ml were collected and each aliquot (100 μl) was taken and scintillated counted to determine the level of integration of ³ H into the assay product. A control assay was performed using denatured enzyme (5 min, 100 ° C.) or without (±) -dehydrodiconiferyl alcohol substrate.

Non-Radioactive Assay for Dehydrodiconiferyl Alcohol Reductase Activity—Each 150 μl of assay is 22 mM MES: Bis-Tris Propane, pH 6.5, 20 μl protein solution of corresponding purity grade, 5 mM DTT , 2 mM (±) -dehydrodiconiferyl alcohol and 4 mM NADPH. After incubation at 30 ° C. for 3 hours, the assay mixture is boiled for 3 minutes, centrifuged (17,000 × g, 3 minutes), followed by reverse phase HPLC on aliquots (125 μl) and then 16.6 μl of CH ₃ CN was added. Quantification of the formation of 7- O- 4 '-(iso) dihydrodehydrodiconiferyl alcohol was measured using a standard curve prepared in advance. Control assays were also performed using denatured enzyme (5 min, 100 ° C.) or without (±) -dehydrodiconiferyl alcohol substrate.

HPLC separation of dehydrodiconiferyl alcohol and reduced products thereof-dehydrodiconiferyl alcohol, dihydrodehydrodiconiperyl alcohol (reduced 7 ', 8'-allyl bond), 7- O- 4'-(iso Separation of dihydrodehydrodiconiferyl alcohol and 7 ', 8', 7-O-4 ''-tetrahydrodehydrodiconiferyl alcohol was carried out in a reversed phase column (Symmetry Shield RP ₈ , 3.9 mm x 150 mm, Waters, Inc.). On.) Using acetonitrile: 3% acetic acid (in H ₂ O) solvent system as follows. The column was equilibrated in CH ₃ CN: 3% acetic acid (A: B, 1: 9). After injecting the sample to be analyzed, three compounds were eluted using the following elution profile: A: B (1: 9) for 5 minutes followed by A: B (25:75) linear over 30 minutes. Gradient, and finally the linear gradient over 25 minutes to 100% A, flow rate was 1 ml / min.

Assay for 7 ', 8'-dihydrodehydrodiconiferyl alcohol reductase activity-Assays are performed using both unlabeled and radiolabeled substrates as described for (±) -dehydrodiconiperyl alcohol. 7 ', 8'-dihydrodehydrodiconiferyl alcohol was added instead as a substrate.

As reductase (SEQ ID NO: 2) did not show the ability to reduce pinorescinol or larisirecinol when expressed as a β-galactosidase fusion protein, native expression was attempted. PSBETa, an overexpressed plasmid, has not only two restriction sites (NdeI for natural expression and BamHI for expression with small fusions) that can be utilized for sub-cloning, but also kanamycin resistance (for high plasmid stability in liquid culture). It contains the included pET3a expression cassette. Sub-cloning into pSBETa was relatively accurate because the putative reductase obtained from Pinus taeda did not have an internal NdeI site. To introduce the NdeI site into the starting methionine of the putative reductase (SEQ ID NO: 2) (primer PT-ATG-NdeI) (SEQ ID NO: 10) and to introduce the NdeI site into the 3'-end untranslated region (Primer PT-Rev-NdeI) SEQ ID NO: 11) Two primers were used for PCR reaction. The resulting PCR fragment was subcloned into a PCR cloning plasmid (pCRII, Invitrogen) and cut out by cleavage with NdeI. A fragment of about 1 kb containing the putative reductase (SEQ ID NO: 2) was gel purified and ligated to pSBETa previously digested with NdeI. PSBET constructs containing the resulting putative reductase (SEQ ID NO: 2) were transformed into and purified from competitive E. coli cells, and then the expression region containing the desired cDNA was completely sequenced for both strands. To demonstrate that no mutations were introduced during PCR.

Introducing the putative reductase (SEQ ID NO: 2) in the pSBET overexpression system transformed B834 (DE3) expressing cells with pSBET constructs and then 1 liter in LB broth with 50 μg / ml kanamycin added. Four cultures were grown until the density of the OD ₆₀₀ reached 0.6 or approximately 0.6, cooled to approximately 22 ° C. and inoculated to a final concentration of 1 mM using IPTG. After growing at about 20-22 ° C. for 21 hours, cells were obtained by centrifugation.

Purifying the heterologously expressed native enzyme (SEQ ID NO: 2) to demonstrate homogeneity consisted of four chromatography steps followed by cell lysis (as described herein) and cell debris removal by centrifugation. . The first step uses ammonium sulphate precipitation, where the desired protein is pelleted at 40-70% saturated cuts. This protein fraction (513 mg) was desalted with Buffer A and applied to an Affinity (Affi Blue gel) column and eluted using a linear salt gradient. The resulting protein (118 mg) was again desalted with Buffer A followed by subsequent anion exchange (POROS 20 QE) and cation exchange (POROS 20 SP) chromatography. Both of these were performed under conditions that bound the contaminating E. coli protein while the desired protein did not bind (pH 6.8), thereby removing the E. coli protein. In addition, after each step, a reductase (SEQ ID NO: 2) was assayed for pinoresinol-larishlesinol reductase activity, but none was detected.

Moreover, even after the reductase (SEQ ID NO: 2) was purified almost homogeneously, it was still not possible to reduce the pinoresinol or larisirecinol. Since this putative reductase obtained from Pinus taeda was closely aligned with the so-called isoflavone / pinoresnol-larishresinol reductase "homolog" based on the deduced amino acid sequence similarity, alternative functions were considered next. . In this respect, because dehydrodiconiferyl alcohol contains a structure capable of forming a combined enone, and because Pinus taeda may contain significant levels of dehydrodiconiferyl alcohol, and its metabolites, The inventors then evaluated the reductase (SEQ ID NO: 2) for its ability to reduce dehydrodiconiperyl alcohol at its allyl position or at the benzyl ether position, the latter being the pinoresinol-larishrecinol reduction. Similar to that catalyzed by enzymes (Gang, DR, Fujita, M., Davin, LB & Lewis, NG ACS Symp . Ser . 697: 389-421 (1998)).

Before such reactions can be analyzed, dehydrodiconiferyl alcohol, 7 ', 8'-dihydrodehydrodiconiferyl alcohol (reduced allyl bond), 7-O-4'-(iso) dihydrodehydrodicony Conditions for HPLC separation of peryl alcohol (reduced benzyl ether), and 7 ', 8', 7-O-4'-tetrahydrodehydrodiconiperyl alcohol (reduced allyl bond mill benzyl ether) had to be developed. This was done using a gradient acetonitrile: 3% acetic acid (in water) solvent system as described above. This solvent system provided nearly baselin separation of all four lignans, as shown in FIG. 1A.

Assays for putative reductase (SEQ ID NO: 2) for dehydrodiconiperyl alcohol reductase activity (either allyl or benzyl ether) were performed as described herein. As shown in FIG. 1B, the purified Pinus taeda reductase affects the reduction of the benzyl ether bonds of dehydrodiconiferyl alcohol and converts it to 7-O-4 '-(iso) dihydrodehydrodiconiperyl alcohol. Switch. The activity towards the dehydrodiconiperyl alcohol of the putative reductase (SEQ ID NO: 2) of Pinus taeda in all purification schemes is listed in Table 1.

Purification Scheme for Benzyl Ether Reductase (SEQ ID NO: 2) from Pinus taeda Purification steps Protein (mg) Inactive (mmol / h / mg) Melt 1052 11 (NH ₄ ) ₂ SO ₄ Precipitation 513 31 Affi-blue 118 72 Anion Exchange (POROS-QE) 59 121 Cation Exchange (POROS-SP) 36 134

Reliability of the substrate and product was measured using LC-MS (FIGS. 1D and 1E). Thus, the so-called isoflavone / pinoresinol-larishresinol reductase "homolog" is actually a phenylcoumarane benzyl ether reductase (which can reduce dehydrodiconylperyl alcohol at the 7-O-4 'position. PCBER). As shown in Table 2 below, the initial characterization of the enzyme (SEQ ID NO: 2) showed a pH optimum plateau of 6.5 to 7.0 and a temperature optimum of 49 ° C. In addition, it is a type A reductase in which the hydride delivery of the NADPH cofactor occurs almost exclusively from the 4 R -position on the nicotinamide ring (Table 2).

Properties of Benzyl Ether Reductase (SEQ ID NO: 2) from Pinus taeda DDC Substrate DDDC K _M (mM) 0.61 ± 0.03 1.95 ± 0.1 V _max (nmol / h / mg) 104.2 ± 10.8 55.87 ± 2.79 ³ H-radioisotope effect (V ¹ H / V ³ H) 14.4 4.0 Hydride Separation from NADPH 4 pro - R (> 99%) - pH optimal 6.4-7.0 - Temperature optimum (℃) 49 -

When 7 ', 8'-dihydrodehydrodiconiperyl alcohol was used as the substrate, reduction of benzyl ether was also made as evidenced by the formation of tetrahydrodehydrodiconiperyl alcohol (FIGS. 1C and 1F).

Initial rate studies were performed to determine whether Pinus taeda benzyl ether reductase (SEQ ID NO: 2) favored one of these substrates over others. The results are shown in Table 2. As can be seen from the table, it is 2-fold higher for dehydrodiconiferyl alcohol as substrate than 7 ', 8'-dihydrodehydrodiconiferyl alcohol. In addition, formaldehyde Lodi Coney perylene K _M for the alcohol reduction 7 ', 8'-dihydro-formaldehyde Lodi Coney perylene three times slower than the K _M for the reduction of alcohol. Interestingly, the observed dynamic radioisotope effect (V ¹ H / V ³ H) (which indicates that the hydride transfer step of the reaction contributes to the rate limiting step (s) of the reaction) is less than that of dehydrodiconiperyl alcohol reduction. Considerably higher. When dehydrodiconiperyl alcohol is the substrate, hydride delivery contributes significantly (as indicated by the high dynamic radioisotope effect, V ¹ H / V ³ H = 14.4) to the rate limiting step of the enzyme catalyzed reaction. However, when 7 ', 8'-dihydrodehydrodiconiferyl alcohol is used as the substrate, the dynamic radioisotope effect is reduced to V ¹ H / V ³ H = 4.0, which means that hydride delivery is a step in the rate limiting step of the reaction. Indicates no more important contribution. This is also supported by a significant reduction in the K _M value for 7 ', 8'-dihydrodehydrodiconiferyl alcohol reduction, which indicates that binding of the substrate is much less effective for this lignan than for dehydrodiconiferyl alcohol. Point. These results indicate that Pinus taeda benzyl alcohol reductase (SEQ ID NO: 2) has a significantly higher affinity for dehydrodiconiperyl alcohol as a substrate.

Example 4

Cloning of Dehydrodiconiferyl Alcohol Reductase cDNA (SEQ ID NO: 3) and (SEQ ID NO: 5) from Cryptomeric Japonica

Cryptomeric Japonica cDNA Library Synthesis-Total RNA (200 μg / g fresh weight) was grown in greenhouses using the method of Dong and Dunstan (Dong, JZ & Dunstan, DI Plant Cell Reports 15 (1996)) Obtained from the leaves of the Plumeria japonica tree. Cryptomeric japonica cDNA library was prepared using 5 μg of purified poly (A) ⁺ mRNA (Oligotex-dT ^™ suspension, Qiagen). Synthesis kit, ZAP ^TM XR vector Uni, and Gigapack It was constructed using with II Gold packaging extract (Stratagene) and the titer for the primary library was 2.2 × 10 ⁶ pfu. Amplified library (8.3 × 10 ⁹ pfu / ml; 175 ml total) was used for screening (Dinkova-Kostova, AT, et al., J. Biol. Chem. 271, 29473-29482 (1996)).

DNA Probe Synthesis-PCR reaction of 5 100 μl of pSBETa plasmid containing Pinus taeda dehydrodiconiferyl alcohol reductase cDNA (5 ng) (SEQ ID NO: 1) (10 mM Tris-HCl [pH 9.0] , 50 mM KCl, 0.1% Triton X-100, 2.5 mM of each dNTP and 2.5 units of Taq DNA polymerase) as a template and primer Cj-PCBERNT (100 pmol) (SEQ ID NO: 12) And Cj-PCBERCT (100 pmol) (SEQ ID NO: 13) were also used. PCR amplification was performed in a thermocycler as follows: 35 cycles of 1 minute at 94 ° C., 2 minutes at 50 ° C., and 3 minutes at 72 ° C .; After the last cycle it was left at 72 ° C. for 5 minutes and held at 4 ° C. indefinitely. Reactions using only one primer, template only, and primers were performed as controls.

Concentrate the five reaction solutions (Microcon 30, Amicon Inc.), wash with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA; 2 × 200 μl), and then continue to PCR product. Recovered to buffer (2 × 50 μl). PCR product (-980 bp band) was dissolved in a 1.0% agarose gel for separation, GENECLEAN II Purified from agarose using kit (BIO 101 Inc.). Gel-purified PCR product (40 ng) was transferred to ^T7 QuickPrime from Pharmacia. Radiolabeled probes (in 0.1 ml) were prepared using the kit and [α- ³² P] dCTP according to the kit instructions, which were purified on a BioSpin 6 column (Bio-Rad) to provide carrier DNA (0.5 mg / ml). Sheared salmon sperm DNA [Sigma], 0.9 ml).

Library Screening—300,000 pfu of Cryptomeric Japonica amplified cDNA library was plated for primary screening, according to the instructions of Stratagene. Plaques were blotted onto Magna nylon membrane circles (Micron Separations Inc.) and then dried in air. Membrane only 2 layers of membrane Placed between 3MM Chr paper. The cDNA library phage DNA was fixed on the membrane and denatured by autoclaving and rapid evacuation at 100 ° C. for 2 minutes in one step. Membranes were washed in 6 × standard saline citrate (SSC) and 0.1% SDS at 37 ° C. for 30 minutes and then preheated 6 × SSC in a crystallization dish (190 × 75 mm) with gentle shaking at 49 ° C. for 5 hours, Prehybridization in 0.5% SDS and 5 × Denhardt's reagent (hybridization solution, 300 ml). [ ³² P] Radiolabeled probe is denatured (boiled, 10 minutes), rapidly cooled (ice, 15 minutes) and then preheated fresh hybridization solution (60 ml, 49 ° C.) in crystallization dish (150 × 75 mm) )). The prehybridized membrane was then added to this dish and covered with a plastic wrap. Hybridization was performed with gentle shaking at 49 ° C. for 18 hours. Membranes were washed at room temperature for 5 minutes at 4 × SSC and 0.5% SDS, transferred to 2 × SSC and 0.5% SDS (at room temperature), then wrapped in plastic wrap and softened at 49 ° C. for 20 minutes to prevent drying. Shaking was incubated and finally exposed to Kodak X-OMAT AR film with increasing screen at −80 ° C. for 24 hours. Twenty positive plaques were purified by performing two more rounds of screening with hybridization conditions as described above, two of which were found to encode the expected enzyme.

In Vivo Excision and Sequencing of pCj-PCBER1 and pCJ-PCBER2 Phagemids-Purified cDNA clones were recovered from phagemid following the in vivo excision protocol of stratazine. Two different cDNAs (pCj-PCBER1 (SEQ ID NO: 3) and pCJ-PCBER2 (SEQ ID NO: 5)) were completely sequenced using overlapping sequencing primers.

Example 5

Properties of Presently Preferred Dehydrodiconiferyl Alcohol Benzyl Ether Reductase Proteins of the Invention

The presently preferred dehydrodiconiferyl alcohol benzyl ether reductase proteins of the present invention have a molecular weight of about 33 kDa to about 34 kDa, a pH optimum in the range of about 6.0 to about 7.0, and a pI value (computer generated) of about 6.0 NADPH dependent reductase that is from about 7.0. In addition, the present most preferred dehydrodiconiferyl alcohol benzyl ether reductase protein of the present invention has a V _max of 104.2 ± 10.8 nmol / h / mg and a K _m of 0.61 ± 0.03 mM. The presently preferred dehydrodiconiferyl alcohol benzyl ether reductase proteins (and nucleic acids encoding them) of the present invention are those obtained from the bark or paja plant species.

While the preferred embodiments of the invention have been illustrated and described, it will be apparent that various changes may be made herein without departing from the scope and spirit of the invention.

Claims (33)

An isolated nucleic acid molecule encoding a dihydrodiconiferyl alcohol benzyl ether reductase protein. The isolated nucleic acid molecule of claim 1, which encodes a gymnosperm dihydrodiconiferyl alcohol benzyl ether reductase protein. The isolated nucleic acid molecule of claim 1, which encodes a dihydrodiconiferyl alcohol benzyl ether reductase protein derived from the Pinus genus. The isolated nucleic acid molecule of claim 3, which encodes Pinus taeda-derived dihydrodiconiferyl alcohol benzyl ether reductase protein. The isolated nucleic acid molecule of claim 4, comprising the nucleotide sequence of SEQ ID NO: 1. 6. The isolated nucleic acid molecule of claim 1, which encodes a protein consisting of the amino acid sequence of SEQ ID NO: 2. 8. The isolated nucleic acid molecule of claim 1, which encodes a dihydrodiconiferyl alcohol benzyl ether reductase protein derived from the genus Cryptomeria. 8. The isolated nucleic acid molecule of claim 7, which encodes a dihydrodiconiferyl alcohol benzyl ether reductase protein derived from Cryptomeria japonica. The isolated nucleic acid molecule of claim 8, comprising SEQ ID NO: 3 nucleotide sequence. The isolated nucleic acid molecule of claim 8, comprising SEQ ID NO: 5 nucleotide sequence. The isolated nucleic acid molecule of claim 1, which encodes a protein consisting of SEQ ID NO: 4 amino acid sequence. The isolated nucleic acid molecule of claim 1, which encodes a protein consisting of SEQ ID NO: 6 amino acid sequence. Isolated Dihydrodiconiferyl Alcohol Benzyl Ether Reductase Protein. The isolated angiosperm dihydrodiconiferyl alcohol benzyl ether reductase protein of claim 13. 14. The isolated jimnosperm dihydrodiconiferyl alcohol benzyl ether reductase protein of claim 13. 14. The pinus dihydrodiconiferyl alcohol benzyl ether reductase protein of claim 13, wherein the protein is isolated. 14. The pinus taeda dihydrodiconiferyl alcohol benzyl ether reductase protein of claim 13 isolated. The isolated dihydrodiconiferyl alcohol benzyl ether reductase protein of claim 13, comprising SEQ ID NO: 2 amino acid sequence. The protein of claim 13, wherein the Cryptomeria dihydrodiconiferyl alcohol benzyl ether reductase protein is isolated. The isolated Cryptomeria japonica dihydrodiconiferyl alcohol benzyl ether reductase protein of claim 13. The isolated dihydrodiconiferyl alcohol benzyl ether reductase protein of claim 13, comprising SEQ ID NO: 4 amino acid sequence. The isolated dihydrodiconiferyl alcohol benzyl ether reductase protein of claim 13, comprising SEQ ID NO: 6 amino acid sequence. A replicable expression vector comprising a nucleotide sequence encoding a dihydrodiconiferyl alcohol benzyl ether reductase protein. 24. The replicable expression vector of claim 23, comprising a nucleotide sequence encoding a jimnoscum dihydrodiconiferyl alcohol benzyl ether reductase protein. The replicable expression vector of claim 23 comprising a nucleotide sequence encoding angiosporm dihydrodiconiferyl alcohol benzyl ether reductase protein. 24. The replicable expression vector of claim 23 comprising a nucleotide sequence encoding Pinus dihydrodiconiferyl alcohol benzyl ether reductase protein. The replicable expression vector of claim 23 comprising a nucleotide sequence encoding Cryptomeria dihydrodiconiferyl alcohol benzyl ether reductase protein. Expression of the dihydrodiconiferyl alcohol benzyl ether reductase protein in a suitable host cell comprising introducing into the host cell an expression vector comprising a nucleotide sequence encoding the dihydrodiconiferyl alcohol benzyl ether reductase protein. How to enhance. 29. The method of claim 28, wherein the host cell is a plant cell. Dehydrodiconiferyl alcohol benzyl ether reductase in a suitable host cell comprising introducing into an host cell an expression vector comprising a nucleotide sequence expressing RNA capable of hybridizing to the cDNA molecule set forth in SEQ ID NO: 1 How to modify protein expression. 31. The method of claim 30, wherein the host cell is a plant cell. Dehydrodiconiperyl alcohol benzyl ether reductase in a suitable host cell comprising introducing into an host cell an expression vector comprising a nucleotide sequence expressing RNA capable of hybridizing to the cDNA molecule set forth in SEQ ID NO: 3 How to modify protein expression. 33. The method of claim 32, wherein the host cell is a plant cell.