Patent Application
20120083013
The present invention relates to a Forsythia koreana-derived glucosyltransferase specific to the position-4 of a furofuran-type lignan (furofuran lignan 4-O-glucosyltransferase), a polynucleotide encoding the enzyme, a vector comprising the polynucleotide, a transformant obtained using the vector; and so on.
Lignans are a class of plant secondary metabolites ubiquitously distributed in the plant kingdom (Non-Patent Literature 1: Umezawa, T. (2003) Phytochem. Rev. 2, 371-390). Some lignans have useful biological activities and are used as drugs or health foods. For example, main furofuran-type lignans contained in Sesamum indicum of the family Pedaliaceae are known to have functions including antioxidative properties, improvement of lipid metabolism, protection of liver function, etc. and are commercially available (Non-Patent Literature 2: Nakai, M. et al. (2003) J. Agri. Food Chem. 51, 1666-1670; Non-Patent Literature 3: Tsuruoka, T. et al. (2005) Biosci. Biotech. Biochem. 69, 179-188; Non-Patent Literature 4: Sirato-Yasumoto, S. et al. (2001) J. Agri. Food Chem. 49, 2647-2651). On the other hand, podophyllotoxins, which are allyltetralin lactone type anti-tumor lignans contained in the root of Berberidaceae Podophyllum peltatum (American May apple), have a cell division inhibitory activity and are used as carcinostatic agents and for development of novel carcinostatic agents (Non-Patent Literature 5: Ayres, D. C. and Loike, J. D. (1990) Lignans: Chemical Biological and Clinical Properties. Cambridge Univ. Press, Cambridge, U.K.). Also, numerous lignans are considered to be metabolized in vivo, after their uptake, into enterolactone which has a female sex hormone-like activity. Lignans are secondary metabolites to which attention has been drawn in recent years (Non-Patent Literature 6: Heinonen, S. et al. (2001) J. Agric. Food Chem. 49, 3178-3186).
Forsythia koreana (genus Forsythia) of the family Oleaceae, which is a perennial woody plant, is strong and easy to cultivate, bears yellow bright flowers and is often used as a garden shrub. On the other hand, it is known that lignans having an antioxidative or anti-inflammatory activity are abundantly accumulated in the leaves, seeds and petals of Forsythia koreana, and in Asia (especially in China and Japan) the leaves are drunk as tea (Non-Patent Literature 7: Guo, H. et al. (2007) Rapid Communications in Mass Spectrometry 21, 715-729; Non-Patent Literature 8: Chang, M-J. et al. (2008) Biosci. Biotech. Biochem. 72, 2750-2755). Unlike annual herbaceous plants such as sesame or linseed, there is no need to seed or raise seedlings of Forsythia koreana every year. It is possible to obtain functional lignans from the leaves over several years. Forsythia koreana is thus considered to be a target for molecular breeding in beneficial secondary metabolites.
Lignan biosynthesis in Forsythia koreana of the family Oleaceae starts by formation of the furofuran-type lignan of structural formula (Ia) (cf.
In general, the regioselectivity of glycosyltransferases in plants is high. The amino acid sequence between glycosyltransferases that react with the same or similar compound at the same position is conserved across the species. It is therefore possible to isolate the enzyme using sequence homology. Even if compounds are the same or similar, however, a glycosyltransferase that adds sugar to a different position has low homology in its amino acid sequence, and it is extremely difficult to isolate the same based on the sequence information of glycosyltransferase which reacts at other positions. Accordingly, it was extremely difficult to assume the structure of a glycosyltransferase that adds sugar to the other lignan compound, for example, to the position 4 and/or position 4′ of the furofuran-type lignan of structural formula (Ia), etc., based on the Sesamum indicum-derived UGT71A9 that adds sugar to the position 2 of the compound of structural formula (III) described above, and isolate the same.
Under these circumstances, it has been desired to isolate and identify a glycosyltransferase, which adds sugar to the position 4 and/or 4′ of furofuran-type lignans.
Taking the foregoing circumstances into account, the present invention has been made and provides a polynucleotide (a polynucleotide encoding a furofuran lignan 4-O-glucosyltransferase), a protein (a furofuran lignan 4-O-glucosyltransferase), a vector comprising the polynucleotide, a transformant wherein the vector is introduced, and so on.
(1) A polynucleotide of any one of (a) to (f) below:
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 5;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6;
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence wherein 1 to 15 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence represented by SEQ ID NO: 4 or 6, and having a glucosyltransferase activity against a lignan;
(d) a polynucleotide comprising a polynucleotide encoding a protein that has an amino acid sequence having a homology of at least 80% to the amino acid sequence represented by SEQ ID NO: 4 or 6, and has a glucosyltransferase activity against a lignan;
(e) a polynucleotide comprising a polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 or 5, and has a glucosyltransferase activity against a lignan; or,
(f) a polynucleotide comprising a polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6, and has a glucosyltransferase activity against a lignan.
(2) The polynucleotide of (1) described above, which is any one of (g) to (j) below:
(g) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence wherein not exceeding 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence represented by SEQ ID NO: 4 or 6, and having a glucosyltransferase activity against a lignan;
(h) a polynucleotide comprising a polynucleotide encoding a protein that has an amino acid sequence having a homology of at least 90% to the amino acid sequence represented by SEQ ID NO: 4 or 6, and has a glucosyltransferase activity against a lignan;
(i) a polynucleotide comprising a polynucleotide encoding a protein that hybridizes under high stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 or 5, and has a glucosyltransferase activity against a lignan; or,
(j) a polynucleotide comprising a polynucleotide encoding a protein that hybridizes under high stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6, and has a glucosyltransferase activity against a lignan.
In the polynucleotides of (1) and (2) described above, the glucosyltransferase activity against lignans includes, for example, a glucosyltransferase activity against a furofuran-type lignan, more specifically, a glucosyltransferase activity against the hydroxy group at position 4 and/or position 4′ of furofuran-type lignans. Herein, the furofuran-type lignan includes, for example, the lignan shown by structural formula (Ia), (Ib) or (Ic) below.
The polynucleotide of (1) described above includes, for example, a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 5 and a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6.
The polynucleotides of (1) and (2) described above include, for example, those which are DNA.
(3) A protein encoded by the polynucleotide of (1) or (2) above.
(4) A vector comprising the polynucleotide of (1) or (2) above.
(5) A transformant, wherein the polynucleotide of (1) or (2) above is introduced.
(6) A transformant, wherein the vector of (4) above is introduced.
(7) A method for producing the protein of (3) above, which comprises using the transformant of (5) or (6) above.
(8) A method for producing a glucoside, which comprises forming a glucoside from UDP-glucose and a glycosyl acceptor substrate using the protein of (3) above as a catalyst.
In the method of (8) described above, the glycosyl acceptor substrate includes, for example, a furofuran-type lignan. Herein, the furofuran-type lignan includes, for example, the lignan shown by structural formula (Ia), (Ib) or (Ic) described above.
According to the present invention, the invention can provide a glycosyltransferase capable of transferring glucose to the position 4 and/or position 4′ of furofuran-type lignans. According to the present invention, the invention can further provide a polynucleotide encoding the enzyme, a vector comprising the polynucleotide, a transformant obtained using the vector, a method for producing a glucoside using the enzyme, and so on.
According to the present invention, glucose can be transferred to the position 4 and/or position 4′ of furofuran-type lignans by introducing the gene of the invention in vitro or into a host cell, and useful lignan glycosides which can contribute to the development of new functional food materials, molecular breeding of secondary metabolism, etc. can be easily produced. In this aspect, the present invention is extremely useful.
Hereinafter, the present invention is described in detail. The scope of the present invention is not restricted to these explanations, but appropriate modifications and implementation can also be made for other examples than those which are discussed below, without departing from the gist of the invention. The specification embraces, in its entirety, Japanese Patent Application No. 2009-120637 (filed May 19, 2009), based on which the priority of this application is claimed. All publications cited in the specification, for example, prior art literatures and published patent applications, patents and other patent literatures are hereby incorporated by reference in their entirety.
The present inventors screened glucosyltransferase (UGT; UDP-glucosyltransferase)-like genes from Forsythia koreana, obtained six Forsythia koreana UGT (RengUGT) candidate genes, and isolated and identified RengUGT13 and RengUGT14 from these Escherichia coli expression proteins as glycosyltransferases having a novel activity of transferring glucose specifically to the position 4 and/or position 4′ of furofuran-type lignans represented by the lignans of structural formulae (Ia), (Ib) and (Ic).
First, the present invention provides (a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 5 (specifically, a DNA; hereinafter sometimes briefly referred to as “DNA”); and (b) a polynucleotide comprising a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 4 or 6. The DNA intended in the present invention is not limited to the DNA encoding the furofuran lignan 4-O-glucosyltransferases (e.g., RengUGT13 and RengUGT14) described above, but includes other DNAs encoding proteins functionally equivalent to this protein.
The functionally equivalent protein is, for example, (c) a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6, in which 1 to 15 amino acids are deleted, substituted, inserted and/or added and having a glucosyltransferase activity against lignans. Such a protein includes, for example, a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6 wherein 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid(s) is/are deleted, substituted, inserted and/or added and having the glucosyltransferase activity against lignans. In general, the smaller the number of deletions, substitutions, insertions, and/or additions of the amino acid residues is, the more preferable it is.
The functionally equivalent protein also includes, for example, (d) a protein having an amino acid sequence having at least 80% homology to the amino acid sequence represented by SEQ ID NO: 4 or 6 and having a glucosyltransferase activity against lignans. Such a protein includes a protein having an amino acid sequence having a homology of approximately 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher, to the amino acid sequence represented by SEQ ID NO: 4 or 6, and having the glucosyltransferase activity against lignans. In general, the higher the above homology percentage is, the more preferable it is.
As used herein, the term “glucosyltransferase activity against lignans” is used to mean an activity of catalyzing the reaction that involves the transfer of glucose to furofuran-type lignans as a glycosyl acceptor substrate, dependently on a glycosyl donor UDP-glucose to produce a glucoside. For example, the term means a furofuran lignan 4-O-glucosyltransferase (e.g., RengUGT13 and RengUGT14) activity, which catalyzes the reaction of transferring glucose to the hydroxy group at the position 4 and/or position 4′ of furofuran-type lignans dependently on UDP-glucose to form a glucoside. As used herein, the enzymes shown by the term “furofuran lignan 4-O-glucosyltransferases” are capable of transferring glucose to the hydroxy group at the position 4 and/or position 4′, as described above, but are not limited to the enzymes which transfer glucose only to the hydroxy group at the position 4. Herein, the position 4 and/or position 4′ of furofuran-type lignans are intended to mean the position 4 and/or position 4′ as defined by the IUPAC nomenclature.
The glucosyltransferase activity against lignans can be assayed, for example, by reacting UDP-glucose with the glycosyl acceptor substrate furofuran-type lignan (e.g., the lignans represented by structural formulae (Ia), (Ib) and (Ic), etc.) in the presence of an enzyme to be assayed and analyzing the resulting reaction product by HPLC, etc. (cf., EXAMPLES later described for more details).
The present invention further includes (e) a polynucleotide comprising a polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 or 5 and has the glucosyltransferase activity against lignans.
The present invention also includes (f) a polynucleotide comprising a polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6 and has the glucosyltransferase activity against lignans.
As used herein, the term “polynucleotide” is used to mean a DNA or RNA, preferably a DNA.
As used herein, the term “polynucleotide that hybridizes under stringent conditions” refers to, for example, a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 or 5; or a polynucleotide obtained by colony hybridization, plaque hybridization, Southern hybridization or the like, using as a probe all or part of a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence of a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 4 or 6. The hybridization method which may be used includes methods described in, for example, “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001,” “Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997,” etc.
As used herein, the term “stringent conditions” may be any of low stringent conditions, moderate stringent conditions and high stringent conditions. The “low stringent conditions” are, for example, the conditions of 5×SSC, 5× Denhardt's solution, 0.5% SDS, 50% formamide and 32° C. The “moderate stringent conditions” are, for example, the conditions of 5×SSC, 5× Denhardt's solution, 0.5% SDS, 50% formamide and 42° C. The “high stringent conditions” are, for example, the conditions of 5×SSC, 5× Denhardt's solution, 0.5% SDS, 50% formamide and 50° C. Under these conditions, as the temperature is higher, a DNA with higher homology is expected to be obtained efficiently at higher temperature, although multiple factors are involved in the hybridization stringency including temperature, probe concentration, probe length, ionic strength, time, salt concentration and the like. A person skilled in the art may achieve a similar stringency by appropriately choosing these factors.
In the case that a commercially available kit is used for the hybridization, for example, Alkphos Direct Labeling Reagents (manufactured by Amersham Pharmacia) can be used. In this case, according to the attached protocol, a membrane is incubated with a labeled probe overnight, the membrane is washed with a primary wash buffer containing 0.1% (w/v) SDS at 55° C. and the hybridized DNA can then be detected.
In addition to those described above, other polynucleotides that can be hybridized include DNAs having a homology of approximately 60% or higher, approximately 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher. 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher or 99.9% or higher, to a DNA encoding the nucleotide sequence of SEQ ID NO: 3 or a DNA encoding the amino acid sequence represented by SEQ ID NO: 4, as calculated by homology search software, such as FASTA and BLAST using default parameters.
Homology between amino acid sequences or nucleotide sequences can be determined by using algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, vol. 87, p. 2264-2268, 1990; Proc. Natl. Acad. Sci. USA, vol. 90, p. 5873, 1993). The programs called BLASTN and BLASTX based on BLAST algorithm have been developed (Altschul S F, et al., J. Mol. Biol., vol. 215, p. 403, 1990). When a nucleotide sequence is sequenced using BLASTN, the parameters are set at, for example, score=100 and word length=12. When an amino acid sequence is sequenced using BLASTX, the parameters are set at, for example, score=50 and word length=3. When BLAST and Gapped BLAST programs are used, default parameters for each of the programs are employed.
The polynucleotide of the present invention described above can be acquired by known genetic engineering techniques or known synthetic techniques.
In yet another embodiment, the present invention also provides the protein encoded by the polynucleotide of the present invention described above. The protein in an embodiment of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6. The protein in another embodiment of the present invention is a protein having the amino acid sequence represented by SEQ ID NO: 4 or 6.
The protein in yet another embodiment of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 6, in which 1 to 15 amino acids are deleted, substituted, inserted and/or added and having the glucosyltransferase activity against lignans. Such a protein includes a protein having the amino acid sequence having the homology described above to the amino acid sequence represented by SEQ ID NO: 4 or 6 and having the glucosyltransferase activity against lignans. These proteins may be obtained by using site-directed mutagenesis described in “Sambrook & Russell, Molecular Cloning; A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001,” “Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997,” “Nuc. Acids Res., vol. 10, p. 6487, 1982,” “Proc. Natl. Acad. Sci. USA, vol. 79, p. 6409, 1982,” “Gene, vol. 34, p. 315, 1985”, “Nuc. Acids Res., vol. 13, p. 4431, 1985,” “Proc. Natl. Acad. Sci. USA, vol. 82, p. 488, 1985,” etc.
In the amino acid sequence for the protein of the present invention, the deletion, substitution, insertion and/or addition of one or more (e.g., 1 to 15, preferably 10 or less) amino acid residues is intended to mean that one or a plurality of amino acid residues are deleted, substituted, inserted and/or added at one or a plurality of positions in the same amino acid sequence. Two or more types of deletion, substitution, insertion and addition may occur concurrently.
Examples of amino acid residues which are mutually substitutable are given below. Amino acid residues in the same group are mutually substitutable. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine and cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid and 2-aminosuberic acid; Group C: asparagine and glutamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid and 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline and 4-hydroxyproline; Group F: serine, threonine and homoserine; and Group G: phenylalanine and tyrosine.
The protein of the present invention may also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method), the tBoc method (t-butyloxycarbonyl method), or the like. In addition, peptide synthesizers available from Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corp., etc. may also be used for the chemical synthesis.
Herein, the protein of the present invention is the furofuran lignan 4-O-glucosyltransferase. The “glucosyltransferase” catalyzes the reaction of transferring a glucose residue from a glycosyl donor to a glycosyl acceptor substrate to form a glucoside. In the present invention, the glycosyl acceptor substrate is, for example, a furofuran-type lignan, and the glycosyl donor is, e.g., UDP-glucose. In an embodiment of the present invention, the protein catalyzes the reaction of transferring a glucose residue from UDP-glucose to the position 4 and/or position 4′ of a glycosyl acceptor substrate furofuran-type lignan (e.g., the lignans of structural formulae (Ia), (Ib) and (Ic), etc.) to form the glucoside and UDP.
The furofuran-type lignans which are glycosyl acceptor substrates include, for example, the lignans of structural formulae (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih), etc. Among others, the lignans of structural formulae (Ia), (Ib) and (Ic) are preferred, more preferably the lignans of structural formulae (Ia) and (Ic), and most preferably the lignan of structural formula (Ia).
In yet another embodiment, the present invention provides an expression vector comprising the polynucleotide of the present invention. The vector of the present invention comprises the polynucleotide of the present invention (e.g., any one of the polynucleotides (a) to (j) described above). Preferably, the expression vector of the present invention comprises any one of the polynucleotides (g) to (j) described above. More preferably, the expression vector of the present invention comprises a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 5, or a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4 or 6.
The vector of the present invention is generally constructed to contain an expression cassette comprising as the constituents (i) a promoter that can be transcribed in a host cell, (ii) the polynucleotide of the present invention linked to the promoter above (e.g., any one of the polynucleotides described in (a) to (j) above), and (iii) a signal that functions in a host cell with respect to the transcription termination and polyadenylation of RNA molecule. The vector thus constructed is introduced into a host cell. Methods for construction of the expression vector include those using a plasmid, phage, cosmid, etc. but are not particularly limited thereto.
Specific types of the vector are not particularly limited, and vectors capable of expressing in a host cell can be suitably chosen. That is, a suitable promoter sequence may be chosen depending upon the type of the host cell to reliably express the polynucleotide of the present invention, and a vector obtained by incorporating this sequence and the polynucleotide of the present invention into various plasmids or the like may be used as an expression vector.
The expression vector of the present invention contains an expression control region (for example, a promoter, a terminator, and/or a replication origin, etc.) depending on the type of a host to be introduced. A conventional promoter (for example, trc promoter, tac promoter, lac promoter, etc.) is used as a promoter for a bacterial expression vector. As a promoter for yeast, there are used, for example, a glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc. As a promoter for fungi there are used, for example, amylase, trpC, etc. Additionally, a viral promoter (e.g., SV40 early promoter, SV40 late promoter, etc.) is used as a promoter for animal-derived host cell.
The expression vector preferably contains at least one selective marker. Such a marker that is available includes an auxotrophic marker (ura5, niaD), a drug-resistant marker (hygromycin, zeocin), a geneticin-resistant marker (G418r), a copper-resistant gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, vol. 81, p. 337, 1984), a cerulenin resistant gene (fas2m, PDR4) (Junji Inokoshi et al., Biochemistry, vol. 64, p. 660, 1992; Hussain et al., Gene, vol. 101, p. 149, 1991, respectively), and the like.
The present invention provides a transformant in which the polynucleotide of the present invention (for example, the polynucleotide described in any one of (a) to (j) above) is introduced.
A method of preparing (method of producing) the transformant is not particularly limited and includes, for example, a method which comprises introducing the recombinant vector described above into a host followed by transformation. The host cell as used herein is not particularly limited and various known cells may be preferably used. Specific examples are bacteria such as Escherichia coli, etc., yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematode (Caenorhabditis elegans), oocyte of African clawed frog (Xenopus laevis), etc. Culture media and conditions suitable for the host cells above are well known in the art. The organism to be transformed is not particularly limited, and includes various microorganisms, plants and animals given as examples of the host cells above.
For transformation of the host cell, there may be used generally known methods. For example, methods that can be used include, but not limited to, the electroporation method (Mackenxie D. A. et al., Appl. Environ. Microbiol., vol. 66, p. 4655-4661, 2000), the particle delivery method (method described in JPA 2005-287403 “Method of Breeding Lipid-Producing Fungus”), the spheroplast method (Proc. Natl. Acad. Sci. USA, vol. 75, p. 1929, 1978), the lithium acetate method (J. Bacteriology, vol. 153, p. 163, 1983), and methods described in Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc.
In another embodiment of the present invention, the transformant can be a plant transformant. The plant transformant in accordance with the embodiment can be acquired by introducing a recombinant vector bearing the polynucleotide in accordance with the present invention into a plant in such a manner that the polypeptide encoded by the said polynucleotide can be expressed.
Where a recombinant expression vector is used, the recombinant expression vector used to transform the plant is not particularly limited as far as the vector is capable of expressing the polynucleotide in accordance with the present invention in the said plant. Examples of such vectors include a vector bearing a promoter capable of constitutively expressing the polynucleotide in plant cells (e.g., a 35S promoter of cauliflower mosaic virus) in plant cells, and a vector inducibly activated by external stimulation.
Plants which are to be the target of transformation in the present invention may be any of entire plant bodies, plant organs (e.g., leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, palisade tissues, spongy tissues, etc.) or plant culture cells, or various types of plant cells (e.g., suspension culture cells), protoplasts, leaf slices, calli, and the like. Specific examples of plant species which are used for the transformation include, but are not limited to, those belonging to the Monocotyledoneae or the Dicotyledoneae.
In the introduction of genes into plants, conventional transformation methods known to those skilled in the art (e.g., the Agrobacterium method, gene gun, the PEG method, the electroporation method, etc.) are used. For example, the Agrobacterium-mediated method and the method of directly introducing into plant cells are well known. When the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium strain (e.g., Agrobacterium tumefaciens), followed by infection of aseptically cultured leaf discs with this strain according to the leaf disc method (Hirobumi Uchimiya, Manuals for Plant Gene Manipulation (1990), 27-31, Kodansha Scientific Co., Ltd., Tokyo). Thus, the transgenic plant can be obtained. In addition, the method of Nagel, et al. (Microbiol. Lett., 67, 325 (1990)) may also be used. This method involves introducing first, e.g., an expression vector into Agrobacterium and then introducing the transformed Agrobacterium into plant cells or plant tissues according to the method described in Plant Molecular Biology Manual (S. B. Gelvin, et. al., Academic Press Publishers). Herein, the “plant tissue” includes callus obtained by culturing plant cells. When the transformation is carried out using the Agrobacterium method, binary vectors (pBI121, pPZP202, etc.) can be used.
For direct transfer of genes into plant cells or plant tissues, the electroporation method and the gene gun method are known. When the gene gun is used, entire plant bodies, plant organs or plant tissues per se may be used, or may be used after preparation of protoplasts. The samples thus prepared can be bombarded using a gene transfer apparatus (e.g., PDS-1000 (BIO-RAD, Inc.), etc.). Bombardment conditions vary depending upon type of the plant or sample. Normally, the sample is bombarded under a pressure of about 450 to 2000 psi at a distance of about 4 to 12 cm.
The cells or plant tissues, in which the gene is introduced, are first selected by chemical resistance such as a hygromycin resistance, etc. and then regenerated into plant bodies in a conventional manner. Regeneration of plant bodies from the transformant cells can be performed by methods known to one skilled in the art, depending upon kind of plant cells.
When a plant culture cell is used as a host, transformation is performed by introducing the recombinant vector into the culture cell by means of gene gun, the electroporation method, etc. Calli, shoots, hairy roots or the like resulting from the transformation can be used for cell culture, tissue culture or organ culture as they are. Alternatively, these tissues or cells can be allowed to regenerate into a plant by any known conventional method for culturing plant tissues, which involves administering a plant hormone (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).
Whether or not the gene is introduced into a plant can be confirmed by PCR, Southern hybridization, northern hybridization or the like. For example, a DNA is prepared from transformed plants, DNA-specific primers are then designed, and PCR is subsequently performed. PCR can be performed under the same conditions as used for preparing the plasmids described above. Subsequently, the transformation can be confirmed by applying the amplified product to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis or the like, staining the product with an appropriate dye, such as ethidium bromide, SYBR Green solution, etc. and then detecting the amplified product as a single band. Furthermore, the amplified products can also be detected by performing PCR using primers labeled with suitable labels, e.g., a fluorescent dye. Other methods that can also be employed herein involve binding the amplified product to a solid phase, such as a microplate, and then confirming the amplified product by fluorescence, an enzyme reaction or the like.
Once a transformed plant wherein the polynucleotide in accordance with the present invention is introduced into the genome is obtained, it is possible to acquire descendants from that plant body by sexual or vegetative propagation. Alternatively, the plant can be mass-produced from, for example, seeds, fruits, ears, tubers, tubercles, tubs, calli, protoplasts, etc., which are obtained from the plant or its descendants or their clones. Accordingly, the present invention also includes plant bodies in which the polynucleotide in accordance with the present invention is expressibly introduced, and descendants having the same properties as those of the plant bodies as well as tissues derived therefrom.
Methods for transformation of various plants have already been reported. Examples of the transgenic plants in accordance with the present invention include, but are not limited to, Forsythia koreana, Sesamum indicum, rice plant, tobacco, barley, wheat, rapeseed, potato, tomato, poplar, banana, eucalyptus, sweet potato, soy, alfalfa, lupinus, corn, cauliflower, rose, chrysanthemum, carnation, snapdragon, cyclamen, orchid, Eustome russellianum, freesia, gerbera, gladiolus, Grypsophila elegans, kalanchoe, lily, pelargonium graveolen, geranium, petunia, torenia, tulip, Arabidopsis thaliana, grapevine, Lotus japonicus, and the like.
In yet another embodiment, the present invention provides a method of producing the protein of the present invention using the transformants described above.
Specifically, the protein of the present invention may be obtained by isolating and purifying the protein of the present invention from the culture of the transformants described above. As used herein, the culture is intended to mean any of culture broth, cultured bacteria or cultured cells, and the homogenate of cultured bacteria or cultured cells. Conventional methods may be used to isolate and purify the protein of the present invention.
Specifically, when the protein of the present invention accumulates within cultured bacteria or within cultured cells, a crude extract of the protein of the present invention may be obtained by culturing the bacteria or cells, then disrupting the bacterial or cells using a conventional technique (e.g., ultrasonication, lysozymes, freezing and thawing, etc.) and applying a conventional method (e.g., centrifugation or filtration, etc.). When the protein of the present invention is accumulated in culture broth, the culture supernatant containing the protein of the present invention can be obtained, after completion of the incubation, by separating the bacteria or cells from the culture supernatant in a conventional manner (e.g., centrifugation, filtration, etc.).
Purification of the protein of the present invention contained in the extract or culture supernatant obtained as described above can be performed by a conventional method of separation and purification. The separation and purification methods including, e.g., ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reversed phase high performance liquid chromatography, dialysis, ultrafiltration, etc. may be used singly or in a suitable combination.
The present invention further provides a method of producing a glucoside using the protein of the present invention.
The protein of the present invention catalyzes the reaction of transferring glucose from the glycosyl donor UDP-glucose to the glycosyl acceptor substrate furofuran-type lignan (in detail, which is the reaction of transferring glucose to the hydroxy group at the position 4 and/or position 4′ of furofuran-type lignans). Accordingly, the glucoside can be produced from the glycosyl acceptor substrate and the glycosyl donor as the starting materials by using the protein of the present invention. The glycosyl acceptor substrate includes various furofuran-type lignans such as those of structural formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), etc. Among others, the lignans of structural formulae (Ia), (Ib) and (Ic) are preferred, more preferably the lignans of structural formulae (Ia) and (Ic), and most preferably the lignan of structural formula (Ia).
The glucoside can be produced, for example, by preparing a solution containing 1 mM of the glycosyl acceptor substrate, 2 mM of the glycosyl donor, 50 mM of calcium phosphate buffer (pH 7.5) and 20 μM of the protein of the present invention and reacting them at 30° C. for 30 minutes. The glucoside can be isolated/purified from the solution by known methods. Specifically, e.g., ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reversed phase high performance liquid chromatography, dialysis, ultrafiltration, etc. can be used alone or in an appropriate combination.
Hereinafter, the present invention is described more specifically with reference to EXAMPLES below but is not deemed to be limited thereto.
The molecular biological techniques used in this EXAMPLE were performed according to the methods described in Molecular Cloning (Sambrookra, Cold Spring Harbour Laboratory Press, 2001), unless otherwise described in detail.
Total RNA was extracted from the leaves of Forsythia koreana of the family Oleaceae using an RNeasy Plant Mini kit (QIAGEN). Subsequently, PolyA(+) RNA was isolated using an Oligotex-dT mRNA kit (TaKaRa Bio). Using 4 μg of this polyA(+) RNA, a cDNA library was prepared using a lambda ZAP II directional cDNA library synthesis kit (Stratagen) by the method as recommended by the manufacturer.
The above cDNA library of approximately 500,000 pfu was screened by plaque hybridization using the UGT71C1 gene of Arabidopsis thaliana (GenBank Accession No.: NM—12852) as a screening probe. The probe was labeled by PCR with a non-radioactive isotope DIG-Nucleic Acid detection system (Roche) under the conditions as recommended by the manufacturer. In this case, the PCR solution (50 μl) was composed of cDNA derived from the root of Arabidopsis thaliana, 1× Taq buffer (TakaRa Bio), 0.2 mM dNTPs, 0.2 pmol each/μl of gene-specific primers (SEQ ID NOs: 1 and 2) and 1.25 U Taq polymerase. PCR was performed, after reacting at 94° C. for 5 minutes, by repeating 30 cycles in total, each cycle consisting of 94° C. for 1 minute, 52° C. for 1 minute and 72° C. for 2 minutes, and finally treating at 72° C. for 5 minutes. The PCR product that the primer and unreacted dNTP were removed through a Mini QuickSpin column (Roche) was used as the probe.
Screening of the library and detection of positive clones were conducted by the method as recommended by the manufacturer, using a non-radioisotope DIG-Nucleic Acid detection system (Roche Diagnostics). Hybridization was performed in 5×SSC containing 30% formamide at 37° C. overnight, and the membrane was washed in 4×SSC and 1% SDS at 55° C. for 20 minutes. The positive clones obtained by screening of approximately 400,000 plaques were sequenced on a DNA Sequencer Model 3100 (Applied Biosystems) by primer walking using synthetic oligonucleotide primers to obtain cDNA sequences. The cDNA sequences obtained were subjected to homology analysis with Blastx program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to obtain Forsythia koreana UGT-like genes (RengUGT).
Clone 13 (RengUGT13) shown in SEQ ID NOs: 3 and 4 and clone 14 (RengUGT14) shown in SEQ ID NOs: 5 and 6 had 90% sequence homology to each other on an amino acid level, and showed approximately 40% homology to UGT71C1 of Arabidopsis thaliana used as the probe for the Blastx analysis and approximately 56% homology to Sesamum indicum UGT71A9 (glycosyltransferase specific for the position 2 of the lignan of structural formula (III)), but no clear function could be inferred. Therefore, the clones were expressed in Escherichia coli to analyze their function.
The full length ORFs of RengUGT13 and RengUGT14 were amplified by PCR using the primers with added restriction enzyme sites (SEQ ID NOs: 7 and 8), which are given below.
This set of primers share a common sequence, which permits to amplify the two RengUGT clones. The underlined nucleotide sequences in the primers are the restriction enzyme recognition sequences added to the primers.
The PCR solution (50 μl) was composed of 1 μl of template DNA (phage solution after secondary screening), 1× ExTaq buffer (TaKaRa Bio), 0.2 mM dNTPs, 0.4 pmol each/μl of the primers and 2.5U of ExTaq polymerase. PCR was performed, after reacting at 94° C. for 3 minutes, by repeating total 30 cycles for amplification, each cycle consisting of 94° C. for 1 minute, 50° C. for 1 minute and 72° C. for 2 minutes. The PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide to give the amplified band at about 1.4 kb predicted from each template DNA.
These PCR products were subcloned into pENTR-TOPO Directional vector (Invitrogen) according to the method as recommended by the manufacturer. The clones were sequenced on a DNA Sequencer Model 3100 (Applied Biosystems) by primer walking using synthetic oligonucleotide primers to verify that any mutation by PCR was not revealed in the inserted fragments.
Approximately 1.4 kb fragments of RengUGT13 and RengUGT14 were excised using the restriction enzyme sites of NdeI and BglII added to the primers and ligated to the NdeI and BglII site of Escherichia coli expression vector pET15b (Novagen) to give the Escherichia coli expression vector of this enzyme gene. The open reading frame of His tag upstream of the NdeI site of this vector was matched with that of the two RengUGT genes; a design was intended to express a chimeric protein of the two RengUGTs fused to the His tag.
To clarify the biochemical functions of the enzymes, the two enzymes were expressed in Escherichia coli. Using the plasmid for two RengUGT Escherichia coli expression obtained above, Escherichia coli BL21 (DE3) strain was transformed in a conventional manner. The resulting transformants were shake cultured in 4 ml of LB medium (10 g/l tryptone peptone, 5 g/l yeast extract, 1 g/l NaCl) containing 50 μg/ml of ampicillin at 37° C. overnight. When the cells reached the stationary phase, 4 ml of the culture broth was inoculated onto 80 ml of fresh medium of the same composition, followed by shake culture at 37° C. At the point when the cell turbidity (OD 600) reached approximately 0.5, IPTG in a final concentration of 0.5 mM was added to the cells, followed by shake culture at 18° C. for 20 hours.
The following procedures were all carried out at 4° C. The transformants cultured were collected by centrifugation (5,000×g, 10 mins.) and suspended by adding 1 ml/g cell of Buffer S (20 mM HEPES buffer (pH 7.5), 20 mM imidazole and 14 mM (3-mercaptoethanol). Subsequently, the suspension was ultrasonicated (15 secs×8 times) and then centrifuged (15,000×g, 15 mins.). The supernatant obtained was recovered as a crude enzyme solution. The crude enzyme solution was loaded onto His SpinTrap (GE Healthcare), which had been equilibrated with Buffer S, and centrifuged (70×g, 30 secs.). After washing with the buffer, the proteins bound to the column were eluted stepwise with 5 ml each of Buffer S containing 100 mM and 500 mM imidazole. In each of the eluted fractions, the buffer was replaced through a Microcon YM-30 (Amicon) by 20 mM HEPES buffer (pH 7.5)/14 mM β-mercapto ethanol (magnification of dialysis, ×1000).
As a result of the SDS-PAGE analysis, in the fraction eluted with 200 mM imidazole, the protein was confirmed at approximately 50 KDa of the estimated molecular weight for the two RengUGTs. This fraction was used for the enzyme analysis (
Conditions for the standard enzyme reaction are as follows. The reaction solution (1 mM UDP-glucose, 100 μM glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5) and 25 μl of purified enzyme solution) was prepared into a 50 μl solution with distilled water and reacted at 30° C. for an hour. The reaction was stopped by adding 100% acetonitrile containing 50 μl of 0.1% trifluoroacetic acid (TFA), followed by analysis on reversed phase HPLC.
Lc-2010c (Shimadzu Corp.) was used for LC; eluent A: 0.1% TFA and eluent B: 0.1% TFA/90% acetonitrile as the moving phase; Develosil-C30-UG5 (Nomura Chemical, 4.6 mm×150 mm) as the column at 40° C. After the column was equilibrated, elution was performed at a flow rate of 1 ml/min in a linear density gradient of B5%→B 100% for 20 minutes and B100% for 5 minutes. Detection was performed by measuring the absorption at 280 nm. Based on the analysis that the spectra from 220 nm to 330 nm were measured using SPD-10AV (Shimadzu Corp.), search was made for substances having two absorption maxima (230 nm and 280 nm) inherent to lignans. Under the conditions, the lignan of structural formula (Ia) was detected at approximately 11.6 minutes, the lignan of structural formula (Ib) at approximately 12.2 minutes, the lignan of structural formula (Ic) at approximately 13.8 minutes, and the lignan of structural formula (V) at approximately 9.8 minutes.
The reaction solution of the lignan of structural formula (Ia) and RengUGT13 or RengUGT14 was analyzed for HPLC under the conditions described above. As a result, the peak eluted at about 8.7 minutes was newly detected (
Subsequently, the peak was fractionated and provided for MS analysis under the conditions described below.
For LC-MS, a Develosil C30-UG-3 column (Nomura Chemical, 3.0 mm×150 mm) was used in a LCMS-IT-TOF system (Shimadzu Corp.); the moving phase used was water containing 0.1% formic acid as eluent A and 100% acetonitrile containing 0.1% formic acid as eluent B. Elution was performed in a linear density gradient (eluent B 5%→100%) for 20 minutes, followed by isocratic elution with eluent B 100% for 5 minutes (flow rate: 0.2 ml/min, column oven: 40° C.). Detection was performed using a photodiode array detector (SPD-M10A, Shimadzu Corp.) to collect data at 230 to 500 nm and measure the chromatogram at A280 nm. The measurement condition of MS was the measurement in a negative mode (ionization: ESI, interface voltage: −3.5 kV).
Under the conditions, the peak was assumed to be a monoglucoside which gave a molecular ion of 519.16391 [M-H]— (
The expression level of RengUGT14 recombinant protein was high in Escherichia coli (
In order to identify the functional domains of RengUGT13 and RengUGT14 genes, the expression profile of the gene from Forsythia koreana in each organ was analyzed by RT-PCR in a similar manner to the method as described in “Ono, E., et al. (2006) Proc. Natl. Acad. Sci. USA 103, 10116-10121.”
Total RNA was extracted from each organ of Forsythia koreana (leaf, young bud, petal and culture cell (derived from callus)) in a manner similar to EXAMPLE 1, and 0.5 μg of the total RNA was subjected to reverse transcription (RT) using a Random Oligo-dT primer to acquire cDNA derived from each organ. The cDNA was used as a template for PCR.
The gene-specific primers (SEQ ID NOs: 9 to 12) used for RT-PCR were designed to be able to distinguish between the RengUGT13 and RengUGT14 genes. Forsythia koreana ribosomal RNA (Forsythia koreana rRNA; GenBank Accession No.: AF534808) was taken as an internal standard gene and amplified using the following gene-specific primers (SEQ ID NOs: 13 and 14).
<Primers for RengUGT14 Gene>
<Primers for Forsythia koreana rRNA>
The PCR solution (50 p1) was composed of 1 μl of template cDNA, 1× ExTaq buffer (TaKaRa Bio), 0.2 mM dNTPs, 0.4 pmol each/μl of the primers and 2.5U ExTaq polymerase. PCR was performed, after reacting at 94° C. for 3 minutes, by repeating total 20 to 35 cycles for amplification, each cycle consisting of 94° C. for 1 minute, 50° C. for 1 minute and 72° C. for 2 minutes, and the conditions that gene expression was not saturated were set up for each gene.
The PCR product was electrophoresed on 1% agarose and stained with ethidium bromide for detection (
The foregoing results indicate that RengUGT13 and RengUGT14 are the enzymes, which are similar in structure, function and expression domain.
The genes for the enzymes which catalyze the transfer of glucose to the position 4 and/or position 4′ of furofuran-type lignans could be identified. By using the enzymes, glucose can be artificially transferred in vitro specifically to the position 4 and/or position 4′ of furofuran-type lignans. Accordingly, the enzymes can contribute to the development of new functional food materials, molecular breeding of secondary metabolism, etc.
Therefore, the present invention can be used over a wide range, including agriculture, food industry, drug industry and other industries related thereto and is extremely beneficial in this regard.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-120637 | May 2009 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2010/058352 | 5/18/2010 | WO | 00 | 12/15/2011 |
