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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

Definitions

• the present invention relates to isolated dirigent proteins and pinoresinol/lariciresinol reductases from Forsythia intermedia, Tsuga heterophylla and Thuja plicata, to nucleic acid sequences which code for dirigent proteins and pinoresinol/lariciresinol reductases from Forsythia intermedia, Tsuga heterophylla and Thuja plicata, and to vectors containing the sequences, host cells containing the sequences and methods of producing recombinant pinoresinol/lariciresinol reductases, recombinant dirigent protein and their mutants.
• Lignans are a large, structurally diverse, class of vascular plant metabolites having a wide range of physiological functions and pharmacologically important properties (Ayres, D.C., and Loike, J.D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge
• lignans are present in "primitive" plants, such as the fern Blechnum orientale (Wada, H. et al., Chem. Pharm. Bull. 40:2099-2101 (1992)) and the hornworts, e.g., Dendroceros japonicus and Megaceros flagellaris (Takeda, R. et al., in Bryophytes. Their Chemistry and Chemical Taxonomy, Vol. 29 (Zinsmeister, H.D. and Mues, R. eds) pp. 201-207, Oxford University Press: New York, NY (1990); Takeda, R. et al., Tetrahedron Lett.
• lignans can contribute extensively to heartwood formation/generation by enhancing the resulting heartwood color, quality, fragrance and durability.
• lignans also have important pharmacological roles.
• podophyllotoxin as its etoposide and teniposide derivatives, is an example of a plant compound that has been successfully employed as an anticancer agent (Ayres, D.C., and Loike, J.D.
• lignans are considered to be derived from lignans such as matairesinol and secoisolariciresinol (Boriello et al., J Applied Bacteriol., 58:37-43 (1985)).
• Bimolecular phenoxy radical coupling reactions such as the stereoselective coupling of two achiral coniferyl alcohol molecules to afford the furofuran lignan (+)-pinoresinol, are involved in numerous biological processes. These are presumed to include lignin formation in vascular plants (M. Nose et al., Phytochemistry 39:71 (1995)), lignan formation in vascular plants (N.G. Lewis and L.B. Davin, ACS Symp. Ser. 562:202 (1994); P. W. Pare et al., Tetrahedron Lett. 35:4731 (1994)), suberin formation in vascular plants (M.A. Bernards et al., J Biol. Chem.
• (+)- ⁇ inoresinol the product of the stereospecific coupling of two E-coniferyl alcohol molecules, undergoes sequential reduction to generate (+)-lariciresinol and then (-)-secoisolariciresinol (Katayama, T. et al., Phytochemistry 32:581-591 (1993); Chu, A. et al., J. Biol. Chem. 268:27026-27033 (1993)) (FIGURE 1).
• (-)-Matairesinol is subsequently formed via dehydrogenation of (-)-secoisolariciresinol, further metabolism of which presumably affords lignans such as the antiviral (-)-trachelogenin in Ipomoea cairica and (-)-podophyllotoxin in Podophyllum peltatum.
• (+)-pinoresinol and the subsequent reductive steps giving (+)-lariciresinol and (-)-secoisolariciresinol are pivotal points in lignan metabolism, since they represent entry into the furano, dibenzylbutane, dibenzylbutyrolactone and aryltetrahydronaphthalene lignan subclasses. Additionally, it should be noted that while lignans are normally optically active, the particular enantiomer present may differ between plant species.
• (-)-pinoresinol occurs in Xanthoxylum ailanthoides (Ishii et al., Yakugaku Zasshi, 103:279-292 (1983)), and (-)-lariciresinol is present in Daphne tangutica (Lin-Gen, et al, Planta Medica, 45:172-176 (1982)).
• the optical activity of a particular lignan may have important ramifications regarding biological activity.
• (-)-trachelogenin inhibits the in vitro replication of HIV-1, whereas its (+)-enantiomer is much less effective (Schroder et al., Naturforsch. 45c: 1215-1221(1990)).
• a 78-kD dirigent protein is involved in conferring stereospecificity in 8,8'-linked lignan formation.
• This protein has no detectable catalytically active oxidative center and apparently serves only to bind and orient coniferyl alcohol-derived free radicals, which then undergo stereoselective coupling to form (+)-pinoresinol.
• the formation of free-radicals in the first instance, requires the oxidative capacity of either a nonspecific oxidase or even a non-enzymatic electron oxidant.
• pinoresinol/lariciresinol reductase catalyzes the conversion of pinoresinol to lariciresinol and then to secoisolariciresinol.
• one aspect of the invention relates to isolated dirigent proteins and to isolated pinoresinol/lariciresinol reductases, such as, for example, those from Forsythia intermedia, Thuja plicata and Tsuga heterophylla.
• cDNAs encoding dirigent protein from Forsythia intermedia (SEQ ID Nos: 12 and 14), Thuja plicata (SEQ ID Nos:20,22,24,26,28,30,32 and 34) and Tsuga heterophila (SEQ ID Nos: 16 and 18) have been isolated and sequenced, and the corresponding amino acid sequences have been deduced.
• cDNAs encoding pinoresinol/lariciresinol reductase from Forsythia intermedia (SEQ ID Nos:47,49,51,53,55 and 57), Thuja plicata (SEQ ID Nos:61, 63,65 and 67) and Tsuga heterophila (SEQ ID Nos:69 and 71) have been isolated and sequenced, and the corresponding amino acid sequences have been deduced.
• the present invention relates to isolated proteins and to isolated DNA sequences which code for the expression of dirigent protein or pinoresinol/- lariciresinol reductase.
• the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence which codes for a pinoresinol/lariciresinol reductase or for a dirigent protein.
• the present invention is also directed to a base sequence sufficiently complementary to at least a portion of a pinoresinol/lariciresinol reductase DNA or RNA, or to at least a portion of a dirigent protein DNA or RNA, to enable hybridization therewith.
• the aforesaid complementary base sequences include, but are not limited to: antisense pinoresinol/lariciresinol reductase RNA; antisense dirigent protein RNA; fragments of DNA that are complementary to a pinoresinol/lariciresinol reductase DNA, or to a dirigent protein DNA, and which are therefore useful as polymerase chain reaction primers, or as probes for pinoresinol/lariciresinol reductase genes, dirigent protein genes, or related genes.
• modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention.
• the present invention provides for the recombinant expression of pinoresinol/lariciresinol reductases and dirigent proteins in plants, animals, microbes and in cell cultures.
• inventive concepts described herein may be used to facilitate the production, isolation and purification of significant quantities of recombinant pinoresinol/lariciresinol reductase or dirigent protein, or of their enzyme products, in plants, animals, microbes or cell cultures.
• FIGURE 1 shows the stereospecific conversion of £-coniferyl alcohol to (+)-pinoresinol in Forsythia intermedia.
• the stereoselectivity of this reaction is controlled by dirigent protein.
• (+)-Pinoresinol is then sequentially converted to (+)-lariciresinol and (-)-secoisolariciresinol by (+)-pinoresinol/(+)-lariciresinol reductase.
• (+)-pinoresinol, (+)-lariciresinol and (-)-secoisolariciresinol are the precursors of the furofuran, furano and dibenzylbutane families of lignans, respectively.
• amino acid and “amino acids” refer to all naturally occurring L- ⁇ -amino acids or their residues.
• the amino acids are identified by either the single-letter or three-letter designations:
• nucleotide means a monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate and a nitrogenous heterocyclic base.
• the base is linked to the sugar moiety via the glycosidic carbon (V carbon of pentose) and that combination of base and sugar is called a nucleoside.
• the base characterizes the nucleotide with the four bases of DNA being adenine ("A”), guanine (“G”), cytosine (“C”) and thymine (“T”).
• Inosine is a synthetic base that can be used to substitute for any of the four, naturally-occurring bases (A, C, G or T).
• RNA bases are A,G,C and uracil ("U").
• the nucleotide sequences described herein comprise a linear array of nucleotides connected by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
• %I percent identity
• percent similarity is a statistical measure of the degree of relatedness of two compared protein sequences. The percent similarity is calculated by a computer program that assigns a numerical value to each compared pair of amino acids based on chemical similarity (e.g., whether the compared amino acids are acidic, basic, hydrophobic, aromatic, etc.) and/or evolutionary distance as measured by the minimum number of base pair changes that would be required to convert a codon encoding one member of a pair of compared amino acids to a codon encoding the other member of the pair. Calculations are made after a best fit alignment of the two sequences has been made empirically by iterative comparison of all possible alignments. (Henikoff, S.
• Oligonucleotide refers to short length single or double stranded sequences of deoxyribonucleotides linked via phosphodiester bonds.
• the oligonucleotides are chemically synthesized by known methods and purified, for example, on polyacrylamide gels.
• pinoresinol/lariciresinol reductase is used herein to mean an enzyme capable of catalyzing two reduction reactions: the reduction of pinoresinol to lariciresinol, and the reduction of lariciresinol to secoisolariciresinol.
• the products of these reactions, lariciresinol and secoisolariciresinol can be either the (+)- or (-)-enantiomers.
• dirigent protein is used herein to mean a protein capable of guiding a bimolecular phenoxy radical coupling reaction thereby determining the stereochemistry and regiochemistry of the product of the reaction and/or its polymeric derivatives.
• alteration refers to dirigent protein or pinoresinol/lariciresinol reductase molecules with some differences in their amino acid sequences as compared to the corresponding native dirigent protein or pinoresinol/lariciresinol reductase.
• the variants will possess at least about 70% homology with the corresponding, native dirigent protein or pinoresinol/lariciresinol reductase, and preferably they will be at least about 80% homologous with the corresponding, native dirigent protein or pinoresinol/lariciresinol reductase.
• the amino acid sequence variants of dirigent protein or pinoresinol/lariciresinol reductase falling within this invention possess substitutions, deletions, and/or insertions at certain positions. Sequence variants of dirigent protein or pinoresinol/lariciresinol reductase may be used to attain desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution.
• Substitutional dirigent protein variants or pinoresinol/lariciresinol reductase variants are those that have at least one amino acid residue in the corresponding native dirigent protein sequence or pinoresinol/lariciresinol reductase sequence removed and a different amino acid inserted in its place at the same position.
• the substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
• Substantial changes in the activity of the dirigent protein or pinoresinol/lariciresinol reductase molecule may be obtained by substituting an amino acid with a side chain that is significantly different in charge and/or structure from that of the native amino acid. This type of substitution would be expected to affect the structure of the polypeptide backbone and/or the charge or hydrophobicity of the molecule in the area of the substitution.
• Moderate changes in the activity of the dirigent protein or pinoresinol/- lariciresinol reductase molecule would be expected by substituting an amino acid with a side chain that is similar in charge and/or structure to that of the native molecule. This type of substitution, referred to as a conservative substitution, would not be expected to substantially alter either the structure of the polypeptide backbone or the charge or hydrophobicity of the molecule in the area of the substitution.
• Insertional dirigent protein variants or pinoresinol/lariciresinol reductase variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the native dirigent protein or pinoresinol/- lariciresinol reductase molecule.
• insertion may be one or more amino acids. Ordinarily, the insertion will consist of one or two conservative amino acids. Amino acids similar in charge and/or structure to the amino acids adjacent to the site of insertion are defined as conservative. Alternatively, this invention includes insertion of an amino acid with a charge and/or structure that is substantially different from the amino acids adjacent to the site of insertion.
• Deletional variants are those where one or more amino acids in the native dirigent protein or pinoresinol/lariciresinol reductase molecule have been removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the dirigent protein or pinoresinol/lariciresinol reductase molecule.
• antisense or “antisense RNA” or “antisense nucleic acid” is used herein to mean 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, in vivo, of complementary, expressed messenger RNA molecules.
• biological activity refers to the ability of the pinoresinol/lariciresinol reductase molecule to reduce pinoresinol and lariciresinol to yield lariciresinol and secoisolariciresinol, respectively, as measured in an enzyme activity assay, such as the assay described in Example 8 below.
• biological activity when used with reference to a dirigent protein refers to the ability of the dirigent protein to guide a bimolecular phenoxy radical coupling reaction thereby determining the stereochemistry and regiochemistry of the product of the reaction and of its polymeric derivatives.
• Amino acid sequence variants of dirigent protein or pinoresinol/lariciresinol reductase may have desirable altered biological activity including, for example, altered reaction kinetics, substrate utilization, product distribution or other characteristics such as regiochemistry and stereochemistry.
• DNA sequence encoding refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the translated polypeptide chain. The DNA sequence thus codes for the amino acid sequence.
• replicable expression vector and "expression vector” refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA.
• Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host.
• the vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of or coincidentally with the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated.
• the vector contains the necessary elements that permit translating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized.
• transformed host cell refers to the introduction of DNA into a cell.
• the cell is termed a "host cell”, and it may be a prokaryotic or a eukaryotic cell.
• Typical prokaryotic host cells include various strains of E. coli.
• Typical eukaryotic host cells are plant cells, such as maize cells, yeast cells, insect cells or animal cells.
• the introduced DNA is usually in the form of a vector containing an inserted piece of DNA.
• the introduced DNA sequence may be from the same species as the host cell or from a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign DNA and some DNA derived from the host species.
• cDNAs encoding dirigent protein and pinoresinol/lariciresinol reductase from Forsythia intermedia, Thuja plicata and Tsuga heterophylla were isolated, sequenced and expressed in the following manner.
• an empirically-determined purification protocol was developed to isolate the Forsythia dirigent protein. This procedure yielded at least six isoforms of the dirigent protein. Amino acid sequencing of the amino terminus of each of these isoforms revealed that the sequence of each isoform was identical.
• Sequencing of the N-terminus of a mixture of these isoforms yielded a 28 amino acid sequence (SEQ ID No:l). Tryptic digestion of a mixture of these isoforms yielded six peptide fragments which were purified in sufficient quantity to permit sequencing SEQ ID Nos:2-7.
• a primer designated PSINT1 was synthesized based on the sequence of amino acids 9 to 15 of the N-terminal peptide (SEQ ID No: l).
• a primer designated PSI1R was synthesized based on the sequence of amino acids 3 to 9 of the internal peptide sequence set forth in (SEQ ID No:2).
• a primer designated PSI2R was synthesized based on the sequence of amino acids 13 to 20 of the internal peptide sequence set forth in (SEQ ID No:2).
• a primer designated PSI7R was synthesized based on the sequence of amino acids 6 to 12 of the internal peptide sequence set forth in (SEQ ID No:3).
• RNA was isolated by means of a protocol adapted from a method specifically designed for woody tissues which contain a large concentration of polyphenols.
• Poly A+ RNA was isolated and a cDNA library constructed using standard means.
• a PCR reaction utilizing primers PSINT1 (SEQ ID No:8) and one of PSI7R, (SEQ ID No:l l) PSI2R (SEQ ID No:10) or PSI1R (SEQ ID No:9), together with an aliquot of Forsythia cDNA as substrate, each yielded a single cDNA band of -370 bp, -155 bp and -125 bp, respectively.
• the -370 bp product of the PSINT1 (SEQ ID NO:8)-PSI7R (SEQ ID No:l 1) reaction was amplified by PCR and utilized as a probe to screen approximatley 600,000 PFU of a Forsythia intermedia cDNA library.
• Two distinct cDNAs were identified, called pPSDFil (SEQ ID No: 12) and pPSDFi2 (SEQ ID No: 14).
• the cDNA insert encoding dirigent protein was excised from plasmid pPSDFil and cloned into the baculovirus transfer vector pBlueBac4. The resulting construct was used to transform Spodoptera frugiperda from which functional dirigent protein was purified.
• the Forsythia cDNAs were used as probes to isolate two dirigent protein clones from Tsuga heterophylla (SEQ ID Nos: 16, 18), and eight dirigent protein cDNA clones from Thuja plicata (SEQ ID Nos:20, 22, 24, 26, 28, 30, 32,
• a primer designated PLRN5 (SEQ ID No:44) was synthesized based on the sequence of amino acids 7 to 13 of the N-terminal peptide (SEQ ID No: 36).
• a primer designated PLR14R (SEQ ID No:45) was synthesized based on the sequence of amino acids 2 to 8 of the internal peptide sequence set forth in SEQ ID No:37.
• a primer designated PLR15R (SEQ ID No:46) was synthesized based on the sequence of amino acids 9 to 15 of the internal peptide sequence set forth in SEQ ID No:37.
• sequence of amino acids 9 to 15 of the internal peptide sequence set forth in SEQ ID No:37, upon which the sequence of primer PLR15R (SEQ ID No:46) was based also corresponded to the sequence of amino acids 4 to 10 of the cyanogen bromide-generated, internal fragment set forth in SEQ ID No:41.
• Forsythia total RNA was isolated by means of a protocol adapted from a method specifically designed for woody tissues which contain a large concentration of polyphenols.
• Poly A+ RNA was isolated and a cDNA library constructed using standard means.
• a PCR reaction utilizing primers PLRN5 (SEQ ID No:44) and either PLR14R (SEQ ID No:45) or PLR15R (SEQ ID No:46), together with an aliquot of Forsythia cDNA as substrate, yielded two, amplified bands of 380 bp and 400 bp.
• One 400 bp cDNA insert was utilized as a probe with which to screen the Forsythia cDNA library.
• the 400 bp probe corresponded to bases 22 to 423 of SEQ ID No:47.
• Six cDNA clones were isolated and sequenced (SEQ ID Nos:47, 49, 51, 53, 55, 57). The clones shared a common coding region, many had a different 5'-untranslated region and the 3'-untranslated region of each terminated at a different point.
• One of these cDNAs (SEQ ID No:47), expressed as a ⁇ -galactosidase fusion protein in E. coli, catalyzed the same enantiomer-specific reactions as the native plant protein.
• (+)-pinoresinol/(+)-lariciresinol reductase With respect to the cloning of (+)-pinoresinol/(+)-lariciresinol reductase and
• the cDNA insert of clone plr-Tpl was used to screen the T. plicata cDNA library and identified an additional, unique clone, designated plr-Tp2, (SEQ ID No:63).
• plr-Tp2 has high homology to plr-Tpl but encodes a (+)-pinoresinol/(+)-lariciresinol reductase.
• the cDNA insert of clone plr-Tpl was used to screen the T. plicata cDNA library and identify an additional two pinoresinol/lariciresinol reductase cDNAs (SEQ ID Nos: 65, 67).
• Tsuga heterophylla Two cDNAs encoding pinoresinol/lariciresinol reductases from Tsuga heterophylla (SEQ ID Nos:69, 71) were isolated by screening a Tsuga heterophylla cDNA library with the plr-Tpl cDNA insert.
• the isolation of cDNAs encoding dirigent proteins, (+)-pinoresinol/- (+)-lariciresinol reductase and (-)-pinoresinol/(-)-lariciresinol reductase permits the development of an efficient expression system for these functional enzymes; provides useful tools for examining the developmental regulation of lignan biosynthesis and permits the isolation of other dirigent proteins and pinoresinol/lariciresinol reductases.
• the isolation of the dirigent protein and pinoresinol/lariciresinol reductase cDNAs also permits the transformation of a wide range of organisms in order to enhance or modify lignan biosynthesis.
• the proteins and nucleic acids of the present invention can be utilized to predetermine the stereochemistry, regiochemistry, or both, of the products of bimolecular phenoxy coupling reactions, such as the furofuran, furano and dibenzylbutane lignans.
• the proteins and nucleic acids of the present invention can be utilized to: elevate or otherwise alter the levels of health-protecting lignans, such as podophyllotoxin, in plant species, including but not limited to vegetables, grains and fruits, and to food items incorporating material derived from such genetically altered plants; genetically alter plant species to provide an abundant, natural supply of lignans useful for a variety of purposes, for example as neutriceuticals and dietary supplements; to genetically alter living organisms to produce an abundant supply of optically pure lignans having desirable biological properties, for example (-)-arctigenin which possesses antiviral properties.
• characterization of the dirigent protein binding site and mechanism of action permits the development of synthetic proteins consisting of an array of dirigent protein binding sites which serve as templates for stereochemically- controlled polymeric assembly.
• N-terminal transport sequences well known in the art (see, e.g., von Heijne, G. et al., Eur. J. Biochem 180:535-545 (1989); Stryer, Biochemistry W.H. Freeman and Company, New York, NY, p. 769 (1988)) may be employed to direct the dirigent protein or pinoresinol/lariciresinol reductase to a variety of cellular or extracellular locations.
• Sequence variants of wild-type dirigent protein clones and pinoresinol/- lariciresinol clones that can be produced by deletions, substitutions, mutations and/or insertions are intended to be within the scope of the invention except insofar as limited by the prior art.
• Dirigent protein or pinoresinol/lariciresinol reductase amino acid sequence variants may be constructed by mutating the DNA sequence that encodes wild-type dirigent protein or wild-type pinoresinol/lariciresinol reductase, such as by using techniques commonly referred to as site-directed mutagenesis.
• two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site.
• Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli.
• This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids.
• the verified mutant duplexes can be cloned into a replicable expression vector, if not already cloned into a vector of this type, and the resulting expression construct used to transform E. coli, such as strain E. coli BL21(DE3)pLysS, for high level production of the mutant protein, and subsequent purification thereof.
• the method of FAB-MS mapping can be employed to rapidly check the fidelity of mutant expression. This technique provides for sequencing segments throughout the whole protein and provides the necessary confidence in the sequence assignment. In a mapping experiment of this type, protein is digested with a protease (the choice will depend on the specific region to be modified since this segment is of prime interest and the remaining map should be identical to the map of unmutagenized protein).
• the set of cleavage fragments is fractionated by microbore HPLC (reversed phase or ion exchange, again depending on the specific region to be modified) to provide several peptides in each fraction, and the molecular weights of the peptides are determined by FAB-MS.
• the masses are then compared to the molecular weights of peptides expected from the digestion of the predicted sequence, and the correctness of the sequence quickly ascertained. Since this mutagenesis approach to protein modification is directed, sequencing of the altered peptide should not be necessary if the MS agrees with prediction.
• CAD-tandem MS/MS can be employed to sequence the peptides of the mixture in question, or the target peptide purified for subtractive Edman degradation or carboxypeptidase Y digestion depending on the location of the modification.
• a non-conservative substitution e.g., Ala for Cys, His or Glu
• the properties of the mutagenized protein are then examined with particular attention to the kinetic parameters of K m and k cat as sensitive indicators of altered function, from which changes in binding and/or catalysis per se may be deduced by comparison to the native enzyme. If the residue is by this means demonstrated to be important by activity impairment, or knockout, then conservative substitutions can be made, such as Asp for Glu to alter side chain length, Ser for Cys, or Arg for His. For hydrophobic segments, it is largely size that will be altered, although aromatics can also be substituted for alkyl side chains. Changes in the normal product distribution can indicate which step(s) of the reaction sequence have been altered by the mutation.
• a non-conservative substitution e.g., Ala for Cys, His or Glu
• site directed mutagenesis techniques may also be employed with the nucleotide sequences of the invention.
• restriction endonuclease digestion of DNA followed by ligation may be used to generate dirigent protein or pinoresinol/lariciresinol reductase deletion variants, as described in Section 15.3 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York, NY (1989)).
• a similar strategy may be used to construct insertion variants, as described in Section 15.3 of Sambrook et al., supra.
• Oligonucleotide-directed mutagenesis may also be employed for preparing substitution variants of this invention. It may also be used to conveniently prepare the deletion and insertion variants of this invention. This technique is well known in the art as described by Adelman et al. (DNA 2:183 (1983)). Generally, oligonucleotides of at least 25 nucleotides in length are used to insert, delete or substitute two or more nucleotides in the dirigent protein gene or pinoresinol/- lariciresinol reductase gene. An optimal oligonucleotide will have 12 to 15 perfectly matched nucleotides on either side of the nucleotides coding for the mutation.
• the oligonucleotide is annealed to the single-stranded DNA template molecule under suitable hybridization conditions.
• a DNA polymerizing enzyme usually the Klenow fragment of E. coli DNA polymerase I, is then added. This enzyme uses the oligonucleotide as a primer to complete the synthesis of the mutation-bearing strand of DNA.
• a heteroduplex molecule is formed such that one strand of DNA encodes the wild-type dirigent protein or pinoresinol/lariciresinol reductase inserted in the vector, and the second strand of DNA encodes the mutated form of dirigent protein or pinoresinol/lariciresinol reductase inserted into the same vector.
• This heteroduplex molecule is then transformed into a suitable host cell.
• Mutants with more than one amino acid substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.
• An alternative method involves two or more rounds of mutagenesis to produce the desired mutant.
• the first round is as described for the single mutants: wild-type dirigent protein or pinoresinol/lariciresinol reductase DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated.
• the second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template.
• this template already contains one or more mutations.
• the oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.
• This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
• Eukaryotic expression systems may be utilized for dirigent protein or pinoresinol/lariciresinol reductase production since they are capable of carrying out any required posttranslational modifications and of directing the enzyme to the proper membrane location.
• a representative eukaryotic expression system for this purpose uses the recombinant baculovirus, Autographa calif ornica nuclear polyhedrosis virus (AcNPV; M.D. Summers and G.E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures (1986); Luckow et al., Bio-technology 6:47-55 (1987)) for expression of the dirigent protein or pinoresinol/lariciresinol reductases of the invention.
• baculovirus system Infection of insect cells (such as cells of the species Spodoptera frugiperda) with the recombinant baculoviruses allows for the production of large amounts of the dirigent protein or pinoresinol/lariciresinol reductase protein.
• the baculovirus system has other important advantages for the production of recombinant dirigent protein or pinoresinol/lariciresinol reductase.
• baculoviruses do not infect humans and can therefore be safely handled in large quantities.
• a DNA construct is prepared including a DNA segment encoding dirigent protein or pinoresinol/lariciresinol reductase and a vector.
• the vector may comprise the polyhedron gene promoter region of a baculovirus, the baculovirus flanking sequences necessary for proper cross-over during recombination (the flanking sequences comprise about 200-300 base pairs adjacent to the promoter sequence) and a bacterial origin of replication which permits the construct to replicate in bacteria.
• the vector is constructed so that (i) the DNA segment is placed adjacent (or operably-linked or "downstream” or "under the control of) to the polyhedron gene promoter and (ii) the promoter/pinoresinol/lariciresinol reductase, or promoter/- dirigent protein, combination is flanked on both sides by 200-300 base pairs of baculovirus DNA (the flanking sequences).
• a cDNA clone encoding a full length dirigent protein or pinoresinol/lariciresinol reductase is obtained using methods such as those described herein.
• the DNA construct is contacted in a host cell with baculovirus DNA of an appropriate baculovirus (that is, of the same species of baculovirus as the promoter encoded in the construct) under conditions such that recombination is effected.
• the resulting recombinant baculoviruses encode the full dirigent protein or pinoresinol/lariciresinol reductase.
• an insect host cell can be cotransfected or transfected separately with the DNA construct and a functional baculovirus. Resulting recombinant baculoviruses can then be isolated and used to infect cells to effect production of dirigent protein or pinoresinol/lariciresinol reductase.
• Host insect cells include, for example, Spodoptera frugiperda cells.
• Insect host cells infected with a recombinant baculovirus of the present invention are then cultured under conditions allowing expression of the baculovirus-encoded dirigent protein or pinoresinol/lariciresinol reductase.
• Recombinant protein thus produced is then extracted from the cells using methods known in the art.
• Other eukaryotic microbes such as yeasts may also be used to practice this invention.
• yeast Saccharomyces cerevisiae is a commonly used yeast, although several other strains are available.
• the plasmid YRp7 (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al, Gene 10:157 (1980)) is commonly used as an expression vector in Saccharomyces.
• This plasmid contains the trpl gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1 (Jones, Genetics 85: 12 (1977)).
• the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Yeast host cells are generally transformed using the polyethylene glycol method, as described by Hinnen (Proc. Natl Acad. Sci. USA 75:1929 (1978)). Additional yeast transformation protocols are set forth in Gietz et al., N.A.R. 20(17):1425 (1992); Reeves et al., FEMS 99:193-197 (1992).
• Suitable promoting sequences in yeast vectors include the promoters for
• the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
• Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
• Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.
• Transgenic plants can be obtained, for example, by transferring plasmids that encode pinoresinol/lariciresinol reductase, and/or dirigent protein, and a selectable marker gene, e.g., the kan gene encoding resistance to kanamycin, into Agrobacterium tumifaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature 303:179-181 (1983) and culturing the Agrobacterium cells with leaf slices of the plant to be transformed as described by An et al., Plant Physiology 81:301-305 (1986).
• a selectable marker gene e.g., the kan gene encoding resistance to kanamycin
• Transformation of cultured plant host cells is normally accomplished through Agrobacterium tumifaciens, as described above.
• Cultures of mammalian host cells and other host cells that do not have rigid cell membrane barriers are usually transformed using the calcium phosphate method as originally described by Graham and Van der Eb (Virology 52:546 (1978)) and modified as described in Sections 16.32-16.37 of Sambrook et al., supra.
• other methods for introducing DNA into cells such as Polybrene (Kawai and Nishizawa, Mol. Cell. Bio I. 4:1172 (1984)), protoplast fusion (Schaffner, Proc. Natl. Acad. Sci. USA 77:2163 (1980)), electroporation (Neumann et al., EMBO J.
• Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, e.g., kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.
• a gene regulating pinoresinol/lariciresinol reductase production, or dirigent protein production can be incorporated into the plant along with a necessary promoter which is inducible.
• a promoter that only responds to a specific external or internal stimulus is fused to the target cDNA.
• the gene will not be transcribed except in response to the specific stimulus. As long as the gene is not being transcribed, its gene product is not produced.
• GSTs are a family of enzymes that can detoxify a number of hydrophobic electrophilic compounds that often are used as pre-emergent herbicides (Weigand et al., Plant Molecular Biology 7:235-243 (1986)). Studies have shown that the GSTs are directly involved in causing this enhanced herbicide tolerance. This action is primarily mediated through a specific 1.1 kb mRNA transcription product. In short, maize has a naturally occurring quiescent gene already present that can respond to external stimuli and that can be induced to produce a gene product.
• the promoter is removed from the GST responsive gene and attached to a pinoresinol/lariciresinol reductase gene, or a dirigent protein gene, that previously has had its native promoter removed.
• This engineered gene is the combination of a promoter that responds to an external chemical stimulus and a gene responsible for successful production of pinoresinol/lariciresinol reductase or dirigent protein.
• Representative examples include electroporation-facilitated DNA uptake by protoplasts (Rhodes et al, Science 240(4849):204-207 (1988)); treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant Molecular Biology 13:151-161 (1989)); and bombardment of cells with DNA laden microprojectiles (Klein et al., Plant Physiol. 91:440-444 (1989) and Boynton et al, Science 240(4858): 1534-1538 (1988)).
• Numerous methods now exist, for example, for the transformation of cereal crops see, e.g., McKinnon, G.E. and Henry, R.J., J.
• DNA from a plasmid is genetically engineered such that it contains not only the gene of interest, but also selectable and screenable marker genes.
• a selectable marker gene is used to select only those cells that have integrated copies of the plasmid (the construction is such that the gene of interest and the selectable and screenable genes are transferred as a unit).
• the screenable gene provides another check for the successful culturing of only those cells carrying the genes of interest.
• a commonly used selectable marker gene is neomycin phosphotransferase II (NPT II). This gene conveys resistance to kanamycin, a compound that can be added directly to the growth media on which the cells grow.
• Plant cells are normally susceptible to kanamycin and, as a result, die.
• the presence of the NPT II gene overcomes the effects of the kanamycin and each cell with this gene remains viable.
• Another selectable marker gene which can be employed in the practice of this invention is the gene which confers resistance to the herbicide glufosinate (Basta).
• a screenable gene commonly used is the ⁇ -glucuronidase gene (GUS). The presence of this gene is characterized using a histochemical reaction in which a sample of putatively transformed cells is treated with a GUS assay solution. After an appropriate incubation, the cells containing the GUS gene turn blue.
• the plasmid will contain both selectable and screenable marker genes.
• the plasmid containing one or more of these genes is introduced into either plant protoplasts or callus cells by any of the previously mentioned techniques. If the marker gene is a selectable gene, only those cells that have incorporated the DNA package survive under selection with the appropriate phytotoxic agent. Once the appropriate cells are identified and propagated, plants are regenerated. Progeny from the transformed plants must be tested to insure that the DNA package has been successfully integrated into the plant genome.
• Mammalian host cells may also be used in the practice of the invention.
• suitable mammalian cell lines include monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 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 sertoli cells (TM4, Mather, Biol. Reprod.
• monkey kidney cells CVI-76, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); rat hepatoma cells (HTC, MI.54, Baumann et al., J. Cell Biol. 85:1 (1980)); and TRI cells (Mather et al consent Annals N. Y.
• Expression vectors for these cells ordinarily include (if necessary) DNA sequences for an origin of replication, a promoter located in front of the gene to be expressed, a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription terminator site.
• Promoters used in mammalian expression vectors are often of viral origin. These viral promoters are commonly derived from polyoma virus, Adenovirus 2, and most frequently Simian Virus 40 (SV40).
• the SV40 virus contains two promoters that are termed the early and late promoters. These promoters are particularly useful because they are both easily obtained from the virus as one DNA fragment that also contains the viral origin of replication (Fiers et al., Nature 273: 113 (1978)). Smaller or larger SV40 DNA fragments may also be used, provided they contain the approximately 250-bp sequence extending from the Hindlll site toward the Bgll site located in the viral origin of replication.
• promoters that are naturally associated with the foreign gene may be used provided that they are compatible with the host cell line selected for transformation.
• An origin of replication may be obtained from an exogenous source, such as SV40 or other virus (e.g., Polyoma, Adeno, VSV, BPV) and inserted into the cloning vector.
• the origin of replication may be provided by the host cell chromosomal replication mechanism. If the vector containing the foreign gene is integrated into the host cell chromosome, the latter is often sufficient.
• the use of a secondary DNA coding sequence can enhance production levels of pinoresinol/lariciresinol reductase or dirigent protein in transformed cell lines.
• the secondary coding sequence typically comprises the enzyme dihydrofolate reductase (DHFR).
• DHFR dihydrofolate reductase
• the wild-type form of DHFR is normally inhibited by the chemical methotrexate (MTX).
• MTX chemical methotrexate
• the level of DHFR expression in a cell will vary depending on the amount of MTX added to the cultured host cells.
• An additional feature of DHFR that makes it particularly useful as a secondary sequence is that it can be used as a selection marker to identify transformed cells. Two forms of DHFR are available for use as secondary sequences, wild-type DHFR and MTX-resistant DHFR.
• DHFR-deficient cell lines such as the CHO cell line described by Urlaub and Chasin, supra, are transformed with wild-type DHFR coding sequences. After transformation, these DHFR-deficient cell lines express functional DHFR and are capable of growing in a culture medium lacking the nutrients hypoxanthine, glycine and thymidine. Nontransformed cells will not survive in this medium.
• the MTX-resistant form of DHFR can be used as a means of selecting for transformed host cells in those host cells that endogenously produce normal amounts of functional DHFR that is MTX sensitive.
• the CHO-K1 cell line (ATCC No. CL 61) possesses these characteristics, and is thus a useful cell line for this purpose.
• the addition of MTX to the cell culture medium will permit only those cells transformed with the DNA encoding the MTX-resistant DHFR to grow. The nontransformed cells will be unable to survive in this medium.
• Prokaryotes may also be used as host cells for the initial cloning steps of this invention.
• Suitable prokaryotic host cells include E. coli K12 strain 294 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli XI 776 (ATCC No. 31,537), and E. coli B; however many other strains of E.
• coli such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts.
• Prokaryotic host cells or other host cells with rigid cell walls are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells.
• Prokaryote transformation techniques are set forth in Dower, W. J., in Genetic Engineering, Principles and Methods, 12:275-296, Plenum Publishing Corp. (1990); Hanahan et al, Meth. Enxymol, 204:63 (1991).
• cDNA sequences encoding dirigent protein or pinoresinol/lariciresinol reductase may be transferred to the (His) 6 » Tag pET vector commercially available (from Novagen) for overexpression in E. coli as heterologous host.
• This pET expression plasmid has several advantages in high level heterologous expression systems.
• the desired cDNA insert is ligated in frame to plasmid vector sequences encoding six histidines followed by a highly specific protease recognition site (thrombin) that are joined to the amino terminus codon of the target protein.
• the histidine "block" of the expressed fusion protein promotes very tight binding to immobilized metal ions and permits rapid purification of the recombinant protein by immobilized metal ion affinity chromatography.
• the histidine leader sequence is then cleaved at the specific proteolysis site by treatment of the purified protein with thrombin, and the dirigent protein or pinoresinol/lariciresinol reductase eluted.
• This overexpression-purification system has high capacity, excellent resolving power and is fast, and the chance of a contaminating E. coli protein exhibiting similar binding behavior (before and after thrombin proteolysis) is extremely small.
• any plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell may also be used in the practice of the invention.
• the vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA.
• Plasmids typically used for transformation of E. coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and Bluescript Ml 3, all of which are described in Sections 1.12-1.20 of Sambrook et al., supra.
• pBR322 pUC18, pUC19, pUCI18, pUC119
• Bluescript Ml 3 all of which are described in Sections 1.12-1.20 of Sambrook et al., supra.
• Many other suitable vectors are available as well. These vectors contain genes coding for ampicillin and/or tetracycline resistance which
• the promoters most commonly used in prokaryotic vectors include the ⁇ -lactamase (penicillinase) and lactose promoter systems (Chang et al. Nature 375:615 (1978); Itakura et al., Science 198: 1056 (1977); Goeddel et al., Nature 281:544 (1979)) and a tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); EPO Appl. Publ. No. 36,776), and the alkaline phosphatase systems.
• proteins normally secreted from the cell contain an endogenous secretion signal sequence as part of the amino acid sequence.
• proteins normally found in the cytoplasm can be targeted for secretion by linking a signal sequence to the protein. This is readily accomplished by ligating DNA encoding a signal sequence to the 5' end of the DNA encoding the protein and then expressing this fusion protein in an appropriate host cell.
• the DNA encoding the signal sequence may be obtained as a restriction fragment from any gene encoding a protein with a signal sequence.
• prokaryotic, yeast, and eukaryotic signal sequences may be used herein, depending on the type of host cell utilized to practice the invention.
• the DNA and amino acid sequence encoding the signal sequence portion of several eukaryotic genes including, for example, human growth hormone, proinsulin, and proalbumin are known (see Stryer, Biochemistry W.H. Freeman and Company, New York, NY, p. 769 (1988)), and can be used as signal sequences in appropriate eukaryotic host cells.
• Yeast signal sequences as for example acid phosphatase (Arima et al., Nucleic Acids Res. 11: 1657 (1983)), alpha-factor, alkaline phosphatase and invertase may be used to direct secretion from yeast host cells.
• Prokaryotic signal sequences from genes encoding, for example, LamB or OmpF (Wong et al., Gene 68:193 (1988)), MalE, PhoA, or beta-lactamase, as well as other genes, may be used to target proteins from prokaryotic cells into the culture medium. Trafficking sequences from plants, animals and microbes can be employed in the practice of the invention to direct the gene product to the cytoplasm, endoplasmic reticulum, mitochondria or other cellular components, or to target the protein for export to the medium. These considerations apply to the overexpression of pinoresinol/lariciresinol reductase or dirigent protein, and to direction of expression within cells or intact organisms to permit gene product function in any desired location.
• suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the dirigent protein DNA or pinoresinol/lariciresinol reductase DNA of interest are prepared using standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Sambrook et al., supra).
• pinoresinol/lariciresinol reductase variants, or dirigent protein variants are preferably produced by means of mutation(s) that are generated using the method of site-specific mutagenesis.
• This method requires the synthesis and use of specific oligonucleotides that encode both the sequence of the desired mutation and a sufficient number of adjacent nucleotides to allow the oligonucleotide to stably hybridize to the DNA template.
• a dirigent protein gene and/or pinoresinol/lariciresinol reductase gene, or an antisense nucleic acid fragment complementary to all or part of a dirigent protein gene or pinoresinol/lariciresinol reductase gene may be introduced, as appropriate, into any plant species for a variety of purposes including, but not limited to: altering or improving the color, texture, durability and pest-resistance of wood tissue, especially heartwood tissue; reducing the formation of lignans and/or lignins in plant species, such as corn, which are useful as animal fodder, thereby enhancing the availability of the cellulose fraction of the plant material to the digestive system of animals ingesting the plant material; reducing the lignan lignin content of plant species utilized in pulp and paper production, thereby making pulp and paper production easier and cheaper; improving the defensive capability of a plant against predators and pathogens by enhancing the production of defensive lignans or lignins; the alteration of other
• a dirigent protein and/or pinoresinol/lariciresinol reductase gene may be introduced into any organism for a variety of purposes including, but not limited to: introducing, enhancing or inhibiting the production of dirigent protein and/or pinoresinol/lariciresinol reductase, or the production of pinoresinol or lariciresinol and their derivatives.
• the foregoing may be more fully understood in connection with the following representative examples, in which "Plasmids" are designated by a lower case p followed by an alphanumeric designation.
• the starting plasmids used in this invention are either commercially available, publicly available on an unrestricted basis, or can be constructed from such available plasmids using published procedures.
• other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.
• “Digestion”, “cutting” or “cleaving” of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at particular locations in the DNA. These enzymes are called restriction endonucleases, and the site along the DNA sequence where each enzyme cleaves is called a restriction site.
• the restriction enzymes used in this invention are commercially available and are used according to the instructions supplied by the manufacturers. (See also Sections 1.60-1.61 and Sections 3.38-3.39 of Sambrook et al., supra.)
• Recovery or "isolation" of a given fragment of DNA from a restriction digest means separation of the resulting DNA fragment on a polyacrylamide or an agarose gel by electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA.
• This procedure is known generally. For example, see Lawn et al. (Nucleic Acids Res. 9:6103-6114 (1982)), and Goeddel et al. (Nucleic Acids Res., supra).
• Forsythia intermedia plants were either obtained from Bailey's Nursery (var. Lynwood Gold, St., Paul, MN), and maintained in Washington State University greenhouse facilities, or were gifts from the local community.
• the insoluble residue was consecutively extracted, with continuous agitation at 250 rpm, as follows: with chilled (-20°C) re-distilled acetone (4 liters, 3 x 30 min); 0.1 M KH 2 PO4-K2HPO4 buffer (pH 6.5) containing 0.1% ⁇ -mercaptoethanol (solution A, 8 liters, 30 min); solution A containing 1% Triton XI 00 (8 liters, 4 hours) and finally solution A (8 liters, 16 hours). Between each extraction, the residue was filtered through one layer of Miracloth (Calbiochem). Solubilization of the (+)-pinoresinol forming system was achieved by mechanically stirring the residue in solution A containing 1 M NaCl (8 liters, 4 hours).
• the homogenate was decanted and the resulting solution consecutively filtered through Miracloth (Calbiochem) and glass fiber (G6, Fisher Sci.).
• the filtrate was concentrated in an Amicon cell (Model 2000, YM 30 membrane) to a final volume of -800 ml, and subjected to (NH 4 ) 2 S ⁇ 4 fractionation. Proteins precipitating between 40 and 80% saturation were recovered by centrifugation (15,000g, 30 min) and the (NH 4 ) 2 SO 4 pellet stored at -20°C until required.
• Mono S Column Chromatography Purification of 78-kD dirigent protein and partial purification of oxidase.
• the ammonium sulfate pellet (obtained from 2 kg of F intermedia stems) was reconstituted in 40 mM MES [2-(N-Morpholino)ethanesulfonic acid] buffer, adjusted to pH 5.0 with 6 M NaOH (solution B, 30 ml), the slurry being centrifuged (3,600g, 5 min), and the supernatant dialyzed overnight against solution B (4 liters). The dialyzed extract was filtered (0.22 ⁇ m) and the sample (35 to 40 mg proteins) was applied to a MonoS HR5/5 (50 mm by 5 mm) column equilibrated in solution B at 4°C.
• MES 2-(N-Morpholino)ethanesulfonic acid
• protease inhibitors that is, phenyl- methanesulfonyl fluoride (0.1 mmol ml" 1 ), EDTA (0.5 nmol ml - 1 ), pepstatin A (1 ⁇ g ml" 1 ), and antipain (1 ⁇ g ml” 1 )] were added during the solubilization and all subsequent purification stages, no differences were observed in the elution profiles of fractions I, II, III, and IV.
• each of these bands was electroblotted onto a polyvinylidene fluoride (PVDF) membrane and subjected to amino terminal sequencing, which established that all had similar sequences indicating a series of isoforms.
• the ultraviolet-visible spectrum of the protein had only a characteristic protein absorbance at 280 nm with a barely perceptible shoulder at -330 nm.
• ICP Inductively coupled plasma
• Fraction I (containing dirigent protein) had very little (+)-pinoresinol-forming activity ( ⁇ 5% of total activity loaded onto the POROS SP-M column), whereas fraction III catalyzed nonspecific oxidative coupling to give ( ⁇ )-dehydrodiconiferyl alcohols, ( ⁇ )-pinoresinols, and ( ⁇ )-erythro/fhreo guaiacylglycerol 8-0-4'-coniferyl alcohol ethers. Thus, Fraction III appeared to contain an endogenous plant oxygenating protein.
• E-coniferyl alcohol coupling assay E-[9- 3 H]Coniferyl alcohol (4 ⁇ mol ml" 1 , 29.3 kBq) was incubated with a 120-kD laccase (previously purified from Forsythia intermedia stem tissue) over a 24-hour period, in the presence and absence of the dirigent protein, as follows. Each assay consisted of E-[9- 3 H]coniferyl alcohol
• EtOAc ethyl acetate
• Pinoresinol-containing fractions were redissolved in methanol (100 ⁇ l) and subjected to chiral column chromatography (Daicel, Chiralcel OD, 50 mm by 4.6 mm) with a solution of hexanes and ethanol (1 :1) as the mobile phase (flow rate 3 ml min -1 cm -2 ), whereas dehydrodiconiferyl alcohol fractions were subjected to Chiralcel OF (250 mm by 4.6 mm) column chromatography eluted with a solution of hexanes and isopropanol (9:1) as the mobile phase (flow rate 2.4 ml min - 1 cm" 2 ), the radioactivity of the eluent being measured with a flow-through detector (Radiomatic, Model A 120).
• the reaction mixture was extracted with ⁇ tOAc, but with the addition of an internal standard and radiochemical carriers omitted.
• reversed-phase column chromatography the enzymatically formed pinoresinol was collected, freeze-dried, redissolved in methanol (100 ⁇ l) and subjected to chiral column chromatography (Daicel, Chiralcel OD, 50 mm by 4.6 mm) with detection at 280 nm and analysis by mass spectral fragmentation in the El mode (Waters, Integrity System).
• auxiliary one-electron oxidants can also facilitate stereoselective coupling with the dirigent protein.
• Ammonium peroxydisulfate readily undergoes homolytic cleavage (A. Usaitis, R. Makuska, Polymer 35:4896 (1994)) and is routinely used as an one-electron oxidant in acrylamide polymerization. Ammonium peroxydisulfate was first incubated with E-[9- 3 H]coniferyl alcohol (4 ⁇ mol ml" 1 , 29.3 kBq) for 6 hours using the E-coniferyl alcohol coupling assay procedure described above.
• Nonspecific bimolecular radical coupling was observed, to afford predominantly ( ⁇ )-dehydrodiconiferyl alcohols as well as the other racemic lignans (Table 1).
• the dirigent protein was added, the stereoselectivity of coupling was dramatically altered to give primarily (+)-pinoresinol at both concentrations of oxidant, together with small amounts of racemic lignans.
• an inorganic oxidant such as ammonium peroxydisulfate, could promote (+)-pinoresinol synthesis in the presence of the dirigent protein, even if it was not oxidatively as selective toward the monolignol as was the fraction III oxidase or laccase.
• preliminary kinetic data support the concept of free-radical capture based on the formal values of Michaelis constant (K m ) and maximum velocity (V max ) characterizing the conversion of E-coniferyl alcohol into (+)-pinoresinol, with the dirigent protein alone and in the presence of the various oxidases or oxidants.
• EXAMPLE 4 Kinetic Characterization of the Conversion of E-Coniferyl Alcohol to (+)-pinoresinol in the Presence of Dirigent Protein and an Oxygenating Agent.
• the K m (mM) and V max (mol s" 1 mol” 1 enzyme) were as follows: with respect to the laccase, 0.200 ⁇ 0.001 and 3.9 ⁇ 0.2 for ( ⁇ )-erythro/threo guaiacylglycerol 8 -O-4' -coniferyl alcohol ethers, 0.3000 ⁇ 0.0003 and 13.1 ⁇ 0.6 for ( ⁇ )-dehydrodiconiferyl alcohols, and 0.300 ⁇ 0.002 and 7.54 ⁇ 0.50 for ( ⁇ )-pinoresinols; with respect to the fraction III oxidase (estimated to have a native molecular weight of 80 kDa), 2.2 ⁇ 0.3 and 0.20 ⁇ 0.03 for ( ⁇ )-erythro/threo guaiacylglycerol 8-0-4'- coniferyl alcohol ethers, 2.2 ⁇ 0.2 and 0.7 ⁇ 0.1 for ( ⁇ )-dehydrodiconiferyl alcohol
• Taq thermostable DNA polymerase was obtained from Promega, whereas restriction enzymes were from Gibco BRL (Haelll), Boehringer Mannheim (Sau3a) and
• pT7Blue T-vector and competent NovaBlue cells were purchased from Novagen and radiolabeled nucleotide ([ " 32 P]dCTP) was from DuPont NEN.
• Oligonucleotide primers for polymerase chain reaction (PCR) and sequencing were synthesized by Gibco BRL Life Technologies. GENECLEAN II® kits
• DNA concentrations determined by comparison to a low DNA mass ladder (Gibco BRL) in 1.5% agarose gels.
• the dirigent protein N-terminal amino acid sequence (SEQ ID No:l) was obtained from the purified protein using an Applied Biosystems protein sequencer with on-line HPLC detection.
• the purified enzyme 150 pmol
• the purified enzyme was suspended in 0.1 M Tris-HCl (50 ⁇ l, pH 8.5, Boehringer Mannheim, sequencing grade), with urea added to give a final concentration of 8 M in 77.5 ⁇ l.
• the mixture was incubated for 15 min at 50°C, following which 100 mM iodoacetamide (2.5 ⁇ l) was added, with the whole kept at room temperature for 15 min.
• RNA 300 ⁇ g/g fresh weight was obtained (Dong, Z.D., and Dunstan, D.I. (1996) Plant Cell Reports 15:516-521) from young green stems of greenhouse-grown Forsythia intermedia plants (var. Lynwood Gold).
• a Forsythia intermedia stem cDNA library was constructed using 5 ⁇ g of purified poly A + mRNA (Oligotex-dTTM Suspension, QIAGEN) with the ZAP-cDNA® synthesis kit, the Uni-ZAPTM XR vector and the Gigapack® II Gold packaging extract (Stratagene), with a titer of 1.2 x 10 6 PFU for the primary library.
• a portion (30 ml) of the amplified library (1.2 x 10 10 PFU/ml; 158 ml total) (Sambrook, J.
• PCR amplification was carried out in a thermocycler as follows: 35 cycles of 1 min at 94°C, 2 min at 50°C and 3 min at 72°C; with 5 min at 72°C and an indefinite hold at 4°C after the final cycle. Single-primer, template-only and primer-only reactions were performed as controls. PCR products were resolved in 1.5%) agarose gels, where a single band (-370-, -155- or ⁇ 125-bp, respectively) was observed for each reaction.
• Restriction analyses were performed to determine whether all inserts from the reactions utilizing each of the foregoing primer pairs were the same, as follows: to 20 ⁇ l each of a 100 ⁇ l PCR reaction (insert of interest amplified with R20mer(SEQ ID No:74) and U19mer(SEQ ID No:75) primers) were added 4 units Haelll, 1.5 units Sau3a or 5 units T ⁇ ql restriction enzyme. Restriction digestions were allowed to proceed for 60 min at 37°C for H ⁇ elll and S ⁇ u3A and at 65°C for T ⁇ ql reactions. Restriction products were resolved in 1.5%o agarose gels giving one restriction group for each insert tested.
• pT7PSIl-pT7PSI5 Five recombinant plasmids from PSINT1 (SEQ ID No:8) +PSI7R (SEQ ID No:l 1) (called pT7PSIl-pT7PSI5) and 2 recombinant plasmids from PSINT1 (SEQ ID No:8) +PSI2R (SEQ ID No: 10) (called pT7PSI6 and pT7PSI7) PCR products were selected for DNA sequencing; all contained the same open reading frame (ORF) (SEQ ID No:69).
• the dirigent protein probe was next constructed as follows: five 100 ⁇ l PCR reactions were performed as above with 10 ng pT7PSIl DNA (SEQ ID No:69) with primers PSINT1 (SEQ ID No:8) and PSI7R (SEQ ID No:l 1).
• Gel-purified pT7PSIl insert 50 ng was used with Pharmacia's T7 QuickPrime ® kit and [ ⁇ - 32 P]dCTP, according to kit instructions, to produce a radiolabeled probe (in 0.1 ml), which was purified over BioSpin 6 columns (Bio-Rad) and added to carrier DNA (0.5 mg/ml sheared salmon sperm DNA [Sigma], 0.9 ml).
• the membranes were washed for 30 min at 37°C in 6X standard saline citrate (SSC) and 0.1%) SDS and prehybridized for 5 h with gentle shaking at 57-58°C in preheated 6X SSC, 0.5% SDS and 5X Denhardt's reagent (hybridization solution, 300 ml) in a crystallization dish (190 x 75 mm).
• the [ 32 P]radiolabeled probe was denatured (boiling, 10 min), quickly cooled (ice, 15 min) and added to a preheated fresh hybridization solution (60 ml, 58°C) in a crystallization dish (150 x 75 mm).
• the prehybridized membranes were next added to this dish, which was then covered with plastic wrap. Hybridization was performed for 18 h at 57-58°C with gentle shaking. The membranes were washed in 4X SSC and 0.5%> SDS for 5 min at room temperature, transferred to 2X SSC and 0.5%> SDS (at room temperature) and incubated at 57-58°C for 20 min with gentle shaking, wrapped with plastic wrap to prevent drying and finally exposed to Kodak X-OMAT AR film for 24 h at -80°C with intensifying screens. Twenty positive plaques were purified through two more rounds of screening with hybridization conditions as above.
• This 1.2 kb fragment was directionally subcloned into these same restriction sites in the multiple cloning site of the baculovirus transfer vector pBlueBac4 (Invitrogen, San Diego, CA). This produced the 6.0 kb construct pBB4/PSD which generates a non- fusion dirigent protein with translation being initiated at the dirigent protein cDNA start codon.
• This construct was then co-transfected with linearized Bac-N-Blue DNA (Invitrogen) into Spodoptera frugiperda Sf9 cells by the technique of cationic liposome mediated transfection to produce, by means of homologous recombination, the recombinant A utographa calif ornica nuclear polyhedrosis viral (AcMNPV) DNA Bac-N-Blue dirigent protein (BB/PSD) which was purified from plaques according to procedures described by Invitrogen.
• the final recombinant AcMNPV-BB/PSD contains the PSD gene under the polyhedrin promoter control and the essential sequence needed for replication of the recombinant virus.
• EXAMPLE 7 Isolation of Dirigent Protein Clones from Thuja plicata and Tsusa heterophylla
• Two dirigent protein cDNAs were isolated from Tsuga heterophylla (SEQ ID Nos: 16, 18), and eight dirigent protein cDNAs were isolated from Thuja plicata (SEQ ID Nos:20, 22, 24, 26, 28, 30, 32, 34).
• Forsythia intermedia plants were either obtained from Bailey's Nursery (var. Lynwood Gold, St., Paul, MN), and maintained in Washington State University greenhouse facilities, or were gifts from the local community. Materials. All solvents and chemicals used were reagent or HPLC grade.
• Affi-Gel Blue Gel (100-200 mesh) and Bio-Gel HT Hydroxyapatite were purchased from Bio-Rad, whereas Phenyl Sepharose CL-4B, MonoQ HR 5/5, MonoP HR 5/20, Superose 6, Superose 12, Superdex 75, PD-10 columns, molecular weight standards and Polybuffer 74 were obtained from Pharmacia LKB Biotechnology, Inc. Adenosine 2',5'-diphosphate Sepharose and Reactive Yellow 3 Agarose were from Sigma Chemical Co.
• Pinoresinol and lariciresinol reductase activities were assayed by monitoring the formation of (+)-[ Hjlariciresinol and (-)-[ H] secoisolariciresinol (Chu, A. et al., J. Biol Chem. 268:27026-27033 (1993)).
• each assay for pinoresinol reductase activity consisted of ( ⁇ )-pinoresinols (5 mM in MeOH, 20 ⁇ l), the enzyme preparation at the corresponding stage of purity (100 ⁇ l), and buffer (20 mM Tris-HCl, pH 8.0, 110 ⁇ l).
• the enzymatic reaction was initiated by addition of [4R- H] ⁇ ADPH (10 mM, 6.79 kBq/mmol in 20 ⁇ l of double-distilled H 2 O).
• the assay mixture was extracted with EtOAc (500 ⁇ l) containing ( ⁇ )-lariciresinols (20 ⁇ g) and ( ⁇ )-secoisolariciresinols (20 ⁇ g) as radiochemical carriers. After centrifugation (13,800 x g, 5 min), the EtOAc solubles were removed and the extraction procedure was repeated. For each assay, the EtOAc solubles were combined with an aliquot (100 ⁇ l) removed for determination of its radioactivity using liquid scintillation counting.
• Lariciresinol reductase activity was assayed by monitoring the formation of (-)-[ H]secoisolariciresinol. These assays were carried out exactly as described above, except that ( ⁇ )-lariciresinols (5 mM in MeOH, 20 ⁇ l) were used as substrates, with ( ⁇ )-secoisolariciresinols (20 ⁇ g) added as radiochemical carriers.
• the resulting supernatant was fractionated with (NH4)2SO , with proteins precipitating between 40 and 60%) saturation recovered by centrifugation (10,000 x g, 1 h).
• the pellet was next reconstituted in a minimum amount of Tris-HCl buffer (20 mM, pH 8.0), containing 5 mM dithiothreitol (buffer A) and desalted using prepacked PD-10 columns (Sephadex G-25 medium) equilibrated with buffer A.
• Affinity (Affi Blue Gel) Chromatography The crude enzyme preparation (191 mg in buffer A, 5 nmol h "1 mg "1 ) was applied to an Affi Blue Gel column (2.6 x 70 cm) equilibrated in buffer A. After washing the column with 200 ml of buffer A, pinoresinol/lariciresinol reductase was eluted with a linear NaCl gradient (1.5-5 M in 300 ml) in buffer A at a flow rate of 1 ml min " . Active fractions were stored (-80°C) until needed.
• NAD+ (up to 3 mM) did not elute pinoresinol/lariciresinol reductase activity.] Because of the interference of the absorbance of the NADP+, it was not possible to directly monitor the eluent at 280 nm. Protein concentrations for each fraction were determined spectrophotometrically according to Bradford (Bradford, M.M., Anal Biochem. 72:248-254 (1976)).
• Pinoresinol/lariciresinol reductase was eluted with a linear NaCl gradient (0-2.5 M in 100 ml) at a flow rate of 1 ml min "1 .
• Pinoresinol/lariciresinol reductase was eluted with 12.8 ml of the mobile phase.
• the active fractions which coincided with the UV profile (absorbance at 280 nm) were pooled (20 ⁇ g, 15,300 nmol h "1 mg "1 ) and desalted (PD-10 prepacked columns).
• (+)-pinoresinol/(+)-lariciresinol reductase As for many of the enzymes involved in phenylpropanoid metabolism, the protein was in very low abundance, i.e. 20 kg F. intermedia stems yielded only -20 ⁇ g of the purified ( ⁇ )-pinoresinol/-
• the column was washed with 10 ml of buffer A and pinoresinol/lariciresinol reductase activity eluted using a linear NaCl gradient (0-500 mM in 50 ml) in buffer A at a flow rate of 0.5 ml min "1 .
• Aliquots (30 ⁇ l) of the collected fractions were analyzed by SDS polyacrylamide gel electrophoresis, using a gradient (4-15% acrylamide) gel. Proteins were visualized by silver staining. Active fractions 34 through 37 (27,760 nmol h '1 mg "1 ) and 38 through 41 (30,790 nmol h " mg " ) were pooled separately and immediately used for characterization.
• the two protein bands thus resolved under denaturing conditions had apparent molecular masses of -36 and -35 kDa, respectively.
• Each of the two reductase forms had a pl ⁇ 5.7.
• Example 8 except that the buffer was replaced with 50 mM Bis-Tris Propane buffer in the pH range of 6.3 to 9.4.
• the pH optimum was found to be pH 7.4.
• the temperature optimum of pinoresinol/lariciresinol reductase was examined in the range between 4°C and 80°C under standard assay conditions (Example 8) utilizing the enzyme preparation from the gel filtration step (Example 8). At optimum pH, the temperature optimum for the reductase activity was established to be -30°C.
• the kinetic parameters were essentially the same for both the 35 kDa and the 36 kDa forms of the enzyme (i.e., Km for pinoresinol: 27 ⁇ 1.5 ⁇ m for the 35 kDa form of the enzyme, and 23 ⁇ 1.3 ⁇ M for the 36 kDa form of the enzyme; Km for lariciresinol: 121 ⁇ 5.0 ⁇ M for the 35 kDa form of the enzyme and 123 ⁇ 6.0 ⁇ M for the 36 kDa form of the enzyme).
• the EtOAc soluble fraction was combined, washed with saturated NaCl (50 ml), dried (Na 2 SO4), and evaporated to dryness in vacuo.
• the resulting extract was reconstituted in a minimum amount of EtOAc, applied to a silica gel column (0.5 x 7 cm), and eluted with EtOAc/hexanes (1:2). Fractions containing the enzymatic product were combined and evaporated to dryness.
• the enzymatic product was established to be (+)-[7'R- H]laric ⁇ res ⁇ nol, as evidenced by the disappearance of the 7'-proR proton at ⁇ 2.51 ppm due to its replacement by deuterium and by its molecular ion at (m/z) 361 (M++1) corresponding to the presence of one deuterium atom at C-7.
• (+)-pinoresinol/(+)-lariciresinol reductase N-terminal amino acid sequence was obtained from each of the purified proteins, and a mixture of both, using an Applied
• Biosystems protein sequencer with on-line HPLC detection was the same for both isoforms (SEQ ID No:36).
• EXAMPLE 11 Cloning of Pinoresinol/Lariciresinol Reductase from Forsythia intermedia Plant Materials.
• Forsythia intermedia plants were either obtained from Bailey's Nursery (var. Lynwood Gold, St., Paul, MN), and maintained in Washington State University greenhouse facilities, or were gifts from the local community. Materials. All solvents and chemicals used were reagent or HPLC grade.
• UV RNA and DNA determinations at OD 260 were obtained on a Lambda 6 UVNIS spectrophotometer.
• a Temptronic II thermocycler (Thermolyne) was used for all PCR amplifications.
• Taq thermostable D ⁇ A polymerase was obtained from Promega, whereas restriction enzymes were from Gibco BRL (Haelll), Boehringer Mannheim (Sau3a) and Promega (Taql).
• pT7Blue T-vector and competent ⁇ ovaBlue cells were purchased from ⁇ ovagen and radiolabeled nucleotides ([ ⁇ - 32 P]dCTP and [ ⁇ - 32 P]ATP) were from DuPont ⁇ E ⁇ .
• Oligonucleotide primers for polymerase chain reaction (PCR) and sequencing were synthesized by Gibco BRL Life Technologies.
• GE ⁇ ECLEA ⁇ II® kits (BIO 101 Inc.) were used for purification of PCR fragments, with the gel-purified D ⁇ A concentrations determined by comparison to a low D ⁇ A mass ladder (Gibco BRL) in 1.5% agarose gels.
• Forsythia intermedia stem cDNA Library Synthesis was obtained from young green stems of greenhouse-grown Forsythia intermedia plants (var. Lynwood Gold) (Dong, Z.D., and Dunstan, D.I., Plant Cell Reports 15:516-521 (1996)).
• a Forsythia intermedia stem cD ⁇ A library was constructed using 5 ⁇ g of purified poly A+ mR ⁇ A (Oligotex-dTTM Suspension, QIAGEN) with the ZAP-cDNA® synthesis kit, the Uni-ZAPTM XR vector and the Gigapack® II Gold packaging extract (Stratagene), with a titer of 1.2xl0 6 PFU for the primary library.
• a portion (30 ml) of the amplified library (1.2x10 PFU/ml; 158 ml total) was used to obtain pure cDNA library DNA for PCR (Sambrook, J.
• the N-terminal and internal peptide amino acid sequences were used to construct the degenerate oligonucleotide primers.
• the primer PLRN5 (SEQ ID No:44) was based on the sequence of amino acids 7 to 13 of the N-terminal peptide (SEQ ID No:36).
• the primer PLR14R (SEQ ID No:45) was based on the sequence of amino acids 2 to 8 of the internal peptide sequence set forth in (SEQ ID No:37).
• the primer PLR15R (SEQ ID No:46) was based on the sequence of amino acids 9 to 15 of the internal peptide sequence set forth in (SEQ ID No: 37).
• sequence of amino acids 9 to 15 of the internal peptide sequence set forth in SEQ ID No: 37, upon which the sequence of primer PLR15R (SEQ ID No:46) was based also corresponded to the sequence of amino acids 4 to 10 of the cyanogen bromide-generated, internal fragment set forth in SEQ ID No:41.
• PCR amplification was carried out in a thermocycler as follows: 35 cycles of 1 min at 94°C, 2 min at 50°C and 3 min at 72°C; with 5 min at 72°C and an indefinite hold at 4°C after the final cycle. Single-primer, template-only and primer-only reactions were performed as controls. PCR products were resolved in 1.5% agarose gels.
• the combination of primers PLRN5 (SEQ ID No:44) and PLRI4R (SEQ ID No:45) yielded a single band of 380-bp corresponding to bases 22 to 393 of SEQ ID No:47.
• the combination of primers PLRN5 (SEQ ID No:44) and PLRI5R (SEQ ID No:46) yielded a single band of 400-bp corresponding to bases 22 to 423 of SEQ ID No:47.
• Restriction analysis was performed to determine whether all inserts for each combination of primers and template were the same. Restriction analysis was carried out as follows: each of the inserts was amplified by PCR utilizing the R20 (SEQ ID No:74) and U19 (SEQ ID No:75) primers. To 20 ⁇ l each of a 100 ⁇ l PCR reaction were added 4 units Haelll, 1.5 units Sau3a or 5 units Taql restriction enzyme. Restriction digestions were allowed to proceed for 60 min at 37°C for Haelll and Sau3A and at 65°C for Taql reactions. Restriction products were resolved in 1.5%) agarose gels giving one restriction group for all inserts tested.
• pT7PLRl- pT7PLR3 The inserts from three of the recombinant plasmids (called pT7PLRl- pT7PLR3) were generated by a combination of primers PLRN5 (SEQ ID No:44) and PLRI5R (SEQ ID No:46) with the 400 bp PCR product as substrate.
• the inserts from the remaining two recombinant plasmids (called pT7PLR4 and pT7PLR5) were generated from a combination of primers PLRN5 (SEQ ID No:44) and PLRI4R (SEQ ID No:45) and the 380 bp PCR product as substrate. All of the five, sequenced PCR products contained the same open reading frame.
• (+)-pinoresinol/(+)-lariciresinol reductase probe was constructed as follows: five, 100 ⁇ l PCR reactions were performed as described above with 10 ng pT7PLR3 DNA with primers PLRN5 (SEQ ID No:44) and PLRI5R (SEQ ID No:46).
• Gel-purified pT7PLR3 cDNA insert 50 ng was used with Pharmacia's T7QuickPrime® kit and [ ⁇ - P]dCTP, according to kit instructions, to produce a radiolabeled probe (in 0.1 ml), which was purified over BioSpin 6 columns (Bio-Rad) and added to carrier DNA (0.9 ml of 0.5 mg/ml sheared salmon sperm DNA obtained from Sigma).
• the membranes were washed for 30 min at 37°C in 6X standard saline citrate (SSC) and 0.1%) SDS and prehybridized for 5 h with gentle shaking at 57-58°C in preheated 6X SSC, 0.5%) SDS and 5X Denhardt's reagent (hybridization solution, 300 ml) in a crystallization dish (190x75 mm).
• SSC 6X standard saline citrate
• the [ Pjradiolabeled probe was denatured (boiling, 10 min), quickly cooled (ice, 15 min) and added to a preheated fresh hybridization solution (60 ml, 58°C) in a crystallization dish (150x75 mm).
• the prehybridized membranes were next added to this dish, which was then covered with plastic wrap. Hybridization was performed for 18 h at 57-58°C with gentle shaking. The membranes were washed in 4X SSC and 0.5%.
• plr-Fil -plr-Fi6 Phagemids The six purified cDNA clones were rescued from the phage following Stratagene's in vivo excision protocol. Both strands of the six different cDNAs (plr-Fil to plr-Fi6) that coded for ( ⁇ )-pinoresinol/ (+)-lariciresinol reductase were completely sequenced using overlapping sequencing primers.
• RNA gel blot analysis For RNA gel blot analysis, total RNA (30 ⁇ g per lane) from F. intermedia stem tips was separated by size by denaturing agarose gel electrophoresis. The RNA was transferred to charged nylon membranes (GeneScreen Plus®, Dupont NEN), cross-linked to the membrane (Stratalinker from Stratagene), prehybridized, hybridized with the same probe used to screen the cDNA library during cDNA cloning and washed according to the manufacturer's instructions for aqueous hybridization conditions. The membrane was then exposed to Kodak X- OMAT film for 48 hr at -80°C with intensifying screens.
• EXAMPLE 12 Expression of (+)-Pinoresinol/f+)-Lariciresinol Reductase cDNA plr-Fil in E. coli Expression in Escherichia coli.
• the putative (+)-pinoresinol/(+)-lariciresinol reductase cDNAs encoded functional (+)- pinoresinol/(+)-lariciresinol reductase the cDNAs putatively encoding (+)-pinoresinol/(+)-lariciresinol reductase were heterologously expressed in E. coli. Heterologous expression was also necessary in order to obtain sufficient protein to enable the systematic study of the precise biochemical mechanism of (+)-pinoresinol/(+)-lariciresinol reductase at a future date.
• IPTG isopropyl ⁇ -D-thioglucopyranoside
• (+)-lariciresinol reductase plr-Fil contained all of the peptide sequences determined by Edman degradation of digest fragments.
• the single ORF predicts a polypeptide of 312 amino acids (SEQ ID No:48) with a calculated molecular mass of 34.9 kDa, in close agreement with the value (-35 or -36 kDa) estimated previously by SDS-PAGE for the two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase.
• An equal number of acidic and basic residues are also present, with a theoretical isoelectric point (pi) of 7.09, in contrast to that experimentally obtained by chromatofocussing (pi -5.7).
• the amino acid composition reveals seven methionine residues.
• the N-terminus of the plant-purified enzyme lacks the initial methionine, this being the most common post-translational protein modification known. Consequently, the first methionine in the cDNA can be considered to be the site of translational initiation.
• the sequence analysis also reveals a possible N- glycosylation site at residue 215 (although no secretory targeting signal is present), and seven possible protein phosphorylation sites at residues 50 and 228 (protein kinase C-type), residues 228, 250, 302 and 303 (casein kinase Il-type ) and residue 301 (tyrosine kinase type).
• Regions of the pinoresinol/lariciresinol polypeptide chain were also identified that contained conserved sequences associated with NADPH binding (J ⁇ rnvall, H., in Dehydrogenases Requiring Nicotinamide Coenzymes (Jeffery, J., ed) pp. 126-148, Birkhauser Verlag, Basel (1980); Branden, C, and Tooze, J., Introduction to Protein Structure, pp. 141-159, Garland Publishing, Inc., New York and London (1991); Wierenga, R.K. et al., J. Mol. Biol. 187:101-108 (1986)).
• invariant amino acids in the sequences of different reductases which are viewed as indicative of NADPH binding sites. These include three conserved glycine residues with the sequence G-X-G-X-X-G (SEQ ID No:76), where X is any residue, and six conserved hydrophobic residues. The glycine-rich region is considered to play a central role in positioning the NADPH in its conect conformation.
• isoflavone reductases catalyze the reduction of ⁇ , ⁇ -unsaturated ketones during isoflavonoid formation.
• the Medicago sativa L. isoflavone reductase catalyzes the stereospecific conversion of 2'-hydroxy- formononetin to (3R)-vestitone in the biosynthesis of the phytoalexin, (-)-medicarpin (Paiva, N.L. et al, Plant Mol. Biol 17:653-667 (1991)).
• sequence analysis establishes significant homology between (+)-pinoresinol/(+)-lariciresinol reductase, isoflavone reductases and putative isoflavone reductase "homologs" which do not possess isoflavone reductase activity.
• Taq thermostable DNA polymerase and restriction enzymes (Sad and Xbal) were obtained from Promega.
• pT7Blue T-vector and competent NovaBlue cells were purchased from Novagen and radiolabeled nucleotide ([ - 32 P]dCTP) was purchased from DuPont NEN.
• RNA (6.7 ⁇ g/g fresh weight) was obtained from young green leaves (including stems) of greenhouse-grown western red cedar plants (Thuja plicata) according to the method of Lewinsohn et al (Lewinsohn, E., et al., Plant Mol Biol Rep. 12:20-25 (1994)).
• a Tplicata cDNA library was constructed using 3 ⁇ g of purified poly(A)+ mRNA (Oligotex-dTTM Suspension, Qiagen) with the ZAP-cDNA® synthesis kit, the Uni ZAPTM XR vector, and the Gigapack® II Gold packaging extract (Stratagene), with a titer of 1.2 X 10 5 pfu for the primary library.
• the amplified library (7.1 X 10 pfu /ml; 28 ml total) was used for screening (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1994)).
• T. plicata (-)-pinoresinol/(-)-lariciresinol reductase cDNA was obtained from mRNA by a reverse transcription-polymerase chain reaction (RT-PCR) strategy (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1994)).
• RT-PCR reverse transcription-polymerase chain reaction
• mRNA 150 ng was mixed with linker-primer (1.4 ⁇ g) from ZAP-cDNA® synthesis kit (Stratagene), heated to 70°C for 10 min, and quickly chilled on ice. The mixture of denatured mRNA template and linker-primer was then mixed with First Strand Buffer (Life Technologies), 10 mM DTT, 0.5 mM each dNTP, and 200 units of Super ScriptTMII (Life Technologies) in a final volume of 20 ⁇ l. The reaction was carried out at 42°C for 50 min and then stopped by heating (70°C, 15 min). E. coli RNase H (1.5 units, 1 ⁇ l) was added to the solution and incubated at 37°C for 20 min.
• the first-strand reaction (2 ⁇ l) was next used as the template in 100- ⁇ l PCR reactions (10 mM Tris-HCl, pH 9.0, 50 mM KC1, 0.1 % Triton X-100, 1.5 mM MgCl 2 , 0.2 mM each dNTP, and 5 units of Taq DNA polymerase) with primer CR6- NT (5'GCACATAAGAGTATGGATAAG3')(SEQ ID No:60) (10 pmol) and primer XhoI-Poly(dT) (5'GTCTCGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
• PCR amplification was carried out in a thermocycler as described in (Dinkova-Kostova, A.T., et al., J Biol. Chem. 271:29473-29482 (1996)) except for the annealing temperature at 52°C.
• PCR products were resolved in 1.3 %> agarose gels, where at least two bands possessing the expected length (about 1 ,200-bp) were observed. The bands were extracted from the gel.
• the gel-purified PCR products 56 ng
• the size and orientation of the inserted cDNAs were determined using the rapid boiling lysis and PCR technique, following the manufacturer's (Novagen's) instructions, with the following primer combinations: R20-mer(SEQ ID No: 74) with U19-mer (SEQ ID No:75); R20-mer (SEQ ID No:74) with CR6-NT (SEQ ID No:60); U19-mer (SEQ ID No:75) with CR6-NT (SEQ ID No:60).
• the CR6-NT primer end of the inserted DNAs was located next to the U19-mer primer site of the T-vector.
• the T-vectors containing the inserted cDNAs were purified with Wizard® Plus SV Minipreps DNA Purification System.
• plr-Tpl The longest cDNA, designated plr-Tpl, (SEQ ID No:61) was used for detection of enzyme activity using the pBluescript expression system.
• each assay for pinoresinol reductase activity consisted of ( ⁇ )-pinoresinols (5 mM in MeOH, 20 ⁇ l) and the enzyme preparation (i.e., total protein extract from E. coli, 210 ⁇ l).
• the enzymatic reaction was initiated by addition of [4R- 3 H]NADPH (10 mM, 6.79 kBq/mmol in distilled H 2 O, 20 ⁇ l).
• the assay mixture was extracted with EtOAc (500 ⁇ l) containing ( ⁇ )-lariciresinols (20 ⁇ g) and ( ⁇ )-secoisolariciresinols (20 ⁇ g) as radiochemical carriers. After centrifugation (13,800 x g, 5 min), the EtOAc solubles were removed and the extraction procedure was repeated. For each assay, the EtOAc solubles were combined with an aliquot (100 ⁇ l) removed for determination of its radioactivity using liquid scintillation counting. The remainder of the combined EtOAc solubles was evaporated to dryness in vacuo, reconstituted in MeOH/H 2 O (30:70, 100 ⁇ l) and subjected to reversed phase and chiral column HPLC.
• Lariciresinol reductase activity was assayed by monitoring the formation of (+)-[ Hjsecoisolariciresinol. These assays were carried out exactly as described above, except that ( ⁇ )-lariciresinols (5 mM in MeOH, 20 ⁇ l) were used as substrates, with ( ⁇ )-secoisolariciresinols (20 ⁇ g) added as radiochemical carriers.
• plr-Tpl in E. coli -
• ORF open reading frame
• plr-Tpl was excised out of pT7Blue T-vector with Sad and Xbal, gel-purified, and then ligated into the expression vector digested with these same enzymes.
• This plasmid, pPCR-Tpl was transformed into NovaBlue cells according to Novagen's instructions.
• the cells were next collected by centrifugation (1000 x g, 10 min) and resuspended in fresh LB medium supplemented with 10 mM IPTG (isopropyl ⁇ -D-thioglucopyranoside) and 50 ⁇ g ml " carbenicillin to an absorbance of 0.6 (at 600 nm).
• the cells allowed to grow overnight, were collected by centrifugation and resuspended in 500-700 ⁇ l of (per 5 ml culture tube) of buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 5 mM DTT). Next, the cells were lysed by sonication (5 x 45 s) and after centrifugation (17500 x g, 4°C, 10 min) the supernatant was removed and assayed for (-)-pinoresinol/(-)-lariciresinol reductase activity as described above.
• plr-Tpl can use both (-)-pinoresinol and ( ⁇ )-pinoresinol as substrates, with the former being converted via (-)-lariciresinol completely to (+)-secoisolariciresinol, and the latter being converted much more slowly to (+)-lariciresinol, but not further to (-)-secoisolariciresinol.
• plr-Tp2 in E. coli.
• the plr-Tp2 cDNA was found to be in frame with the ⁇ -galactosidase gene ⁇ -complementation particle in pBluescript SK(-).
• plr-Tp2 was found to possess the same substrate specificity and product formation as the original Forsythia intermedia reductase (Dinkova-Kostova, A.T., et al, J. Biol. Chem. 271:29473-29482 (1996)) except that a small amount of (-)-lariciresinol was also detected.
• plr-Tp2 has a higher sequence similarity to plr-Tpl than it does to the Forsythia reductase. All the above observations were confirmed using deuterolabeled substrates
• Two additional pinoresinol/lariciresinol reductases were cloned from a Thuja plicata young stem cDNA library as described in Example 15 for the cloning of plr- Tp2.
• the two additional pinoresinol/lariciresinol reductases were designated plr-Tp3 (SEQ ID No:65) and plr-Tp4 (SEQ ID No:67).
• Two additional pinoresinol/lariciresinol reductases were cloned from a Tsuga heterophylla young stem cDNA library as described in Example 15 for the cloning of plr-Tp2.
• the two additional pinoresinol/lariciresinol reductases from Tsuga heterophylla were designated plr-Tp3 (SEQ ID No:69) and plr-Tp4 (SEQ ID No:71).
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTI-SENSE NO
• FRAGMENT TYPE N-terminal
• ORGANISM Forsythia intermedia dirigent protein N-terminal sequence
• ORGANISM Forsythia intermedia clone psd-fil
• MOLECULE TYPE protein: Forsythia intermedia PSD-Fil protein
• MOLECULE TYPE Forsythia intermedia cDNA PSD-Fi2
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE Tsuga heterophylla dirigent protein cDNA PSD-Thl
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE Tsuga heterophylla dirigent protein PSD-Th2 cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE Thuja plicata dirigent protein PSD-Tpl cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE Thuja plicata dirigent protein PSD-Tp3 cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE Thuja plicata dirigent protein PSD-Tp4 cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE Thuja plicata dirigent protein PSD-Tp5 cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• GTCTAATTGA GAGAAAATTC CAATAATTTT TTACCAATAG CA ATG AAA GCC ATT 54
• MOLECULE TYPE Thuja plicata dirigent protein PSD-Tp6 cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• CTCAGTCTAA TTGAGAGAAA ATTCCAATAA TTTTTTCCCA ATAGCA ATG AAA GCC 55
• GCC AAT CTC ACC ATT ATG ACT GGT AAT AAC CAT TTT GGG AAT CTT GCT 295 Ala Asn Leu Thr He Met Thr Gly Asn Asn His Phe Gly Asn Leu Ala 260 265 270 GTG TTT GAT GAT CCT ATT ACT CTT GAC AAC AAT CTC CAC TCT CCT CCT 343 Val Phe Asp Asp Pro He Thr Leu Asp Asn Asn Leu His Ser Pro Pro Pro 275 280 285

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• 1997
  • 1997-11-07 EP EP97946908A patent/EP0948602A1/en not_active Withdrawn
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  • 1997-11-07 WO PCT/US1997/020391 patent/WO1998020113A1/en not_active Application Discontinuation

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