CN101283089A

CN101283089A - Nucleic acids encoding modified cytochrome P450 enzymes and methods of use thereof

Info

Publication number: CN101283089A
Application number: CNA2006800373195A
Authority: CN
Inventors: M·C·-Y·常; R·伊切斯; D·-K·罗; 吉国靖雄; J·D·基斯林
Original assignee: University of California
Current assignee: University of California
Priority date: 2005-10-07
Filing date: 2006-10-05
Publication date: 2008-10-08
Also published as: ZA200803368B

Abstract

The present invention provides nucleic acids comprising nucleotide sequences encoding modified cytochrome P450 enzymes; as well as recombinant vectors and host cells comprising the nucleic acids. The present invention further provides methods of producing a functionalized compound in a host cell genetically modified with a nucleic acid comprising nucleotide sequences encoding a modified cytochrome P450 enzyme.

Description

The coding nucleic acid of modified cytochrome P 450 enzymes and its application method

Cross reference

The right of priority of the U.S. Provisional Patent Application that the application requires to submit on October 7th, 2005 U.S. Provisional Patent Application is submitted to number on January 27th, 60/724,525 and 2006 number 60/762,700, it is for referencial use to include these applications in this paper in full.

Invention field

The present invention relates to isoprenoid (isoprenoid) compound production field, specifically is the host cell with the coding nucleic acid genetic modification of isoprenoid precursor modifying enzyme.

Background of invention

Isoprenoid has constituted very big and various natural product type, and they have identical biosynthesizing source, and promptly a kind of metabolic precursor thereof bisphosphate isoamyl-1-alkene ester (isopentenyl diphosphate, IPP).The isoprenoid compound is also referred to as " terpene " or " terpenoid ".Described and surpassed 40,000 kinds of isoprenoids.By definition as can be seen, isoprenoid (C5) is made up of the unit so-called isopentene (isoprene).The quantity of the C atom that exists in the isoprenoid generally can be divided exactly (C5, C10, C15, C20, C25, C30 and C40) by 5, but has also reported irregular isoprenoid and polyterpene.The isoprenoid compound is also referred to as " terpene " or " terpenoid ".The important member of isoprenoid comprises carotenoid, sesquiterpenoids, diterpenoids and hemiterpene.Carotenoid for example comprises: Lyeopene, many materials as antioxidant such as β-Hu Luobusu.Sesquiterpenoids for example comprises: a kind of compound Artemisinin with antimalarial active.Diterpenoids for example comprises: a kind of cancer chemotherapy medicine taxol.

Isoprenoid comprises most species and the different natural product family of structure.In this family, the terpenoid that separates from plant and other natural origin is used as commodity seasonings and flavor compounds, and antimalarial drug and anticarcinogen.The most of terpenoids that use are natural product or derivatives thereofs at present.The organism that many these natural products are originated (as tree, marine invertebrate) both can't have been cultivated on a large scale to produce the commercial quantity (product) that is fit to, and also was not suitable for genetic manipulation and derived with the output that improves these compounds or to these compounds.Therefore, must be by analogue semi-synthetic or with synthetic these natural products of conventional chemical synthetic method.And many natural products have complex construction, and therefore, synthetic at present these natural products are uneconomical or impossible.These natural products must extract from natural origin such as tree, sponge, coral and marine microorganism; Or synthetic or semi-synthetic generation by abundanter precursor.Extract the operability that natural product is subject to natural origin by natural origin; Synthetic or semi-synthetic production natural product may run into low-yield and/or expensive puzzlement.The limitation of these production problems and natural origin can limit the commerce and the clinical development of this product.

(as in the multicellular organisms of genetic modification) biosynthesizing isoprenoid natural product can inspire the commercial or treatment potential that these natural origins fail to realize in external in the host cell of engineered (genetic modification) (as in fermentation system) or the body, and produces cheap and more widely used fine chemicals and medicine.An obstacle that produces isoprenoid or isoprenoid precursor compound in the genetic modification host is that effective production can be modified the polyisoamylene precursor of isoprenoid compound or the enzyme of modification isoprenoid precursor.

Most important class of enzymes is Cytochrome P450 (P450) superfamily in the biochemical conversion of many natural product target spots, and it has participated in the various widely metabolic reactions of the utmost point.In a surprising example, in about 20 steps by precursor tetra-sodium geranyl geranyl ester biological taxol biosynthesis, the catalysis of P450 energy is 8 steps wherein.

This area needs improved generation isoprenoid or produces the host cell of isoprenoid precursor, so that high level produces the isoprenoid compound.The invention solves this demand and associated advantages is provided.

Reference

U.S. Patent Publication No. 2004/005678; U.S. Patent Publication No. 2003/0148479; Martin etc. (2003) Nat.Biotech.21 (7): 796-802; Polakowski etc. (1998) Appl.Microbiol.Biotechnol.49:67-71; Wilding etc. (2000) J Bacteriol 182 (15): 4319-27; U.S. Patent Publication No. 2004/0194162; Donald etc. (1997) Appl.Env.Microbiol.63:3341-3344; Jackson etc. (2003) Organ.Lett.5:1629-1632; U.S. Patent Publication No. 2004/0072323; U.S. Patent Publication No. 2004/0029239; U.S. Patent Publication No. 2004/0110259; U.S. Patent Publication No. 2004/0063182; U.S. Patent number 5,460,949; U.S. Patent Publication No. 2004/0077039; U.S. Patent number 6,531,303; U.S. Patent number 6,689,593; Hamano etc. (2001) Biosci.Biotechnol.Biochem.65:1627-1635; T.Kuzuyama. (2004) Biosci.Biotechnol.Biochem.68 (4): 931-934; T.Kazuhiko. (2004) Biotechnology Letters.26:1487-1491; Brock etc. (2004) Eur J.Biochem.271:3227-3241; Choi etc. (1999) Appl.Environ.Microbio.654363-4368; Parke etc. (2004) Appl.Environ.Microbio.70:2974-2983; Subrahmanyam etc. (1998) J.Bact.180:4596-4602; Murli etc. (2003) J.Ind.Microbiol.Biotechnol.30:500-509; Starai etc. (2005) J.Biol.Chem.280:26200-26205; With (2004) J.Mol.Biol.340:1005-1012 such as Starai; Chem.Biol.2004 such as Jennewein, 11,379-387; Org.Biomol.Chem.2005 such as Sowden, 3,57-64; Plant such as Luo J.2001,28,95-104; Phytochem.2003 such as Carter, 64,425-433; Appl.Environ.Microbiol.2003 such as Craft, 69,5983-5991; Proc.Natl.Acad.Sci.USA such as Barnes 1991,88,5597-5601; Plant Physiol.2003 such as Schoch, 133,1198-1208; Science such as Roosild 2005,307,1317-1321.

Summary of the invention

The invention provides the nucleic acid of the nucleotide sequence that contains the modified cytochrome P 450 enzymes of encoding; And the recombinant vectors and the host cell that comprise this nucleic acid.The present invention also provides in the host cell of the nucleic acid genetic modification of using the nucleotide sequence that contains the modified cytochrome P 450 enzymes of encoding, produces the method for functionalized compounds.

Brief Description Of Drawings

Fig. 1 is the synoptic diagram of biosynthesizing 8-hydroxyl-δ-cadinene in intestinal bacteria (E.coli).

Fig. 2 is gas chromatography-mass spectrum (GC-MS) figure by the organic layer that extracts in the intestinal bacteria of expressing the CadOH biosynthetic pathway.

Fig. 3 is the GC-MS figure of the organic layer that extracts in the intestinal bacteria by the expression CadOH biosynthetic pathway of the mevalonic acid of feeding and a part of mevalonate pathway (pMBIS).

Fig. 4 A and 4B have described the various N-terminal that CadH is carried out and have modified (Fig. 4 A); Produce the time-histories figure of CadOH with the intestinal bacteria of various CadH construction genetic modifications.

Fig. 5 has described the aminoacid sequence of Mi Sidi albumen (mistic).

Fig. 6 describes the aminoacid sequence of limonene hydroxylase.

Fig. 7 has described the aminoacid sequence of aristolochine (aristolochene) two hydroxylases.

Fig. 8 A-D has described the cadinene hydroxylase (Fig. 8 A) that contains natural membrane spaning domain (underscore); The cadinene hydroxylase (Fig. 8 B) that contains allos membrane spaning domain (runic); The cadinene hydroxylase (Fig. 8 C) that contains solubilising structural domain (runic); Aminoacid sequence with the cadinene hydroxylase (Fig. 8 D) that contains secretory structure territory and allos membrane spaning domain (runic).

Fig. 9 A and 9B have described the aminoacid sequence of Japanese yew diene (taxadiene) hydroxylase.

Figure 10 has described the oxidasic aminoacid sequence of ent-kaurene.

Figure 11 A has described the nucleotide sequence (representing initial atg with runic) of coding cadinene hydroxylase; Figure 11 B has described according to express the variation nucleotide sequence that carries out codon optimized coding cadinene hydroxylase in prokaryotic cell prokaryocyte.

Figure 12 A has described the aminoacid sequence of the cytochrome P450 reductase (CPR) of Ramulus et folium taxi cuspidatae (Taxus cuspidata) northeast; Figure 12 B has described the aminoacid sequence from the CPR of Oidium tropicale (Candida tropicalis); Figure 12 C has described the aminoacid sequence from the CPR (ATR1) of Arabidopis thaliana (Arabidopsis thaliana); Figure 12 D has described the aminoacid sequence of the CPR (ATR2) from Arabidopis thaliana; Figure 12 E has described the variation ATR2 aminoacid sequence that lacks chloroplast targeted sequence.

Figure 13 is the synoptic diagram of two kinds of protoheme biosynthetic pathways.

Figure 14 is the synoptic diagram of the exemplary isoprenoid product of biosynthesizing taxol, Artemisinin and menthol.

Figure 15 is the synoptic diagram that produces the reaction scheme of exemplary isoprenoid compound.

Figure 16 is the synoptic diagram of isoprenoid pathways metabolism, and this pathways metabolism is produced isoprenoid biosynthetic pathway intermediate bisphosphate many isopentene ester (polyprenyl diphosphate), bisphosphate geranyl ester (GPP), bisphosphate method ester (FPP) and bisphosphate geranyl geranyl ester (GGPPP) by bisphosphate isoamyl-1-alkene ester (IPP) and bisphosphate dimethyl allyl ester (DMAPP).

Figure 17 is the synoptic diagram that produces mevalonic acid (MEV) approach of IPP.

Figure 18 is the synoptic diagram that is used to produce the DXP approach of IPP and dimethyl allyl pyrophosphate (DMAPP).

Figure 19 A-C has described the aminoacid sequence of the P450 enzyme of various modified biological alkali approach intermediates.

Figure 20 A-C has described the aminoacid sequence of the P450 enzyme of various modification phenylpropionic acid approach intermediates.

Figure 21 A and 21B have described the aminoacid sequence of the P450 enzyme of various modification polyketide approach intermediates.

Figure 22 is the synoptic diagram of various AMORPHADIENE oxydase (AMO) construction.(1) nAMO separates the natural A MO sequence from Herba Artemisiae annuae (Artemisia annua); 2) sAMO carries out codon optimized synthetic AMO gene according to escherichia coli expression; 3) A13-AMO has replaced the synthetic AMO gene that wild-type is striden the film sequence with the A13N end sequence of Oidium tropicale (C.tropicalis); 4) A17-AMO has replaced the synthetic AMO gene that wild-type is striden the film sequence with the A17N end sequence of Oidium tropicale (C.tropicalis); 5) Bov-AMO has replaced the synthetic AMO gene that wild-type is striden the film sequence with ox microsome N-terminal sequence.

Figure 23 A and B have described in intestinal bacteria by various AMO construction oxidation AMORPHADIENE.

Figure 24 A and B have described the nucleotide sequence of encoding wild type AMO.

Figure 25 has described the amino acid sequence translation figure of nucleotide sequence shown in Figure 24.

Figure 26 and 27 has described the nucleotide sequence of coding A13-AMO and amino acid sequence translation figure respectively.

Figure 28 and 29 has described the nucleotide sequence of coding A17-AMO and amino acid sequence translation figure respectively.

Figure 30 and 31 has described the nucleotide sequence of coding ox-AMO and amino acid sequence translation figure respectively.

Figure 32 has described except that the nucleotide sequence that contains coding CadOH, CPR and CadS, also contains the CadOH that produces in the intestinal bacteria of whole mevalonate pathways.

Figure 33 be comparison express whole AMORPHADIENE approach and contain the pDUET-ctAACPR-A13AMO plasmid or the intestinal bacteria of pCWori-A17AMO-ctAACPR plasmid in the GC-MS gas phase spectrum and the mass spectrum of the arteannuinic acid that produces.

Figure 34 has described in the intestinal bacteria of the nucleic acid of using coding mevalonate pathway enzyme and AMORPHADIENE synthase and pCWori-A17AMO-ctAACPR plasmid genetic modification, artemisinol is oxidized to the GC-MS gas phase spectrogram of sweet wormwood aldehyde.

Figure 35 A and 35B have described the nucleotide sequence of the HMGR (tHMGR) of coding Acetoacetyl-CoA thiolase (" atoB "), HMGS and brachymemma.

Figure 36 A-D has described the nucleotide sequence of pMBIS.

Definition

Term " isoprenoid ", " isoprenoid compound ", " terpene ", " terpene compound ", " terpenoid " and " terpenoid Compound " be used interchangeably. The isoprenoid compound is by so-called iso-amylene (C5) the unit group of varying number Become. The carbon atom number that exists in the isoprenoid generally can 5 be divided exactly (as C5, C10, C15, C20, C25, C30 and C40). Once reported irregular isoprenoid and polyterpene, they are also included within " class isoamyl Alkene " definition in. The isoprenoid compound includes but not limited to: monoterpene, sequiterpene, triterpene, polyterpene and Diterpene.

Term used herein " bisphosphate isopentene ester (prenyl diphosphate) " is used interchangeably with " tetra-sodium isopentene ester (prenyl pyrophosphate) ", comprise the bisphosphate list isopentene ester (as IPP and DMAPP) that contains an isopentene group, and many isopentene of the bisphosphate ester that contains two or more isopentene groups.Bisphosphate list isopentene ester comprises tetra-sodium isoamyl-1-alkene ester (IPP) and its isomer dimethyl allyl pyrophosphate (DMAPP).

Term used herein " diterpene synthase " refers to and can zymetology modify IPP, DMAPP or many isopentene of tetra-sodium ester, so that produce any enzyme of terpene precursor compound.Term " diterpene synthase " comprises that energy catalysis bisphosphate isopentene ester is converted into the enzyme of isoprenoid or isoprenoid precursor.

In this article, term " tetra-sodium " can exchange with " bisphosphate " and use.Therefore, for example, term " bisphosphate isopentene ester " and " tetra-sodium isopentene ester " are interchangeable; Term " tetra-sodium isoamyl-1-alkene ester " and " bisphosphate isoamyl-1-alkene ester " are interchangeable; Term " bisphosphate method ester " and " farnesyl pyrophosphate " are interchangeable; Or the like.

Term used herein " mevalonate pathway " or " MEV approach " refer to acetyl-CoA is changed into the biosynthetic pathway of IPP.Mevalonate pathway comprises the enzyme of catalysis following steps: (a) make two acetyl-CoA molecule be condensed into acetoacetyl CoA; (b) make acetoacetyl CoA and acetyl-CoA condensation form HMG-CoA; (c) HMG-CoA is converted into mevalonic acid; (d) mevalonic acid phosphoric acid is turned to mevalonic acid 5-phosphoric acid; (e) mevalonic acid 5-phosphoric acid is converted into mevalonic acid 5-tetra-sodium; (f) mevalonic acid 5-tetra-sodium is converted into tetra-sodium isoamyl-1-alkene ester.Figure 17 has schematically illustrated mevalonate pathway." upper part " of mevalonate pathway refers to be responsible for by MEV approach intermediate acetyl-CoA being changed into the enzyme of mevalonic acid.

Term used herein " 1-deoxy-D-xylulose 5-bisphosphate approach " or " DXP approach " refer to change glyceraldehyde-3-phosphate and pyruvic acid the approach of IPP and DMAPP into by DXP approach intermediate, and wherein the DXP approach comprises the enzyme of the reaction that catalysis Figure 18 schematically illustrates.

Term used herein " prenyltransferase " is used interchangeably with term " bisphosphate isopentene ester synthase " and " many isopentene groups synthase " (as " GPP synthase ", " FPP synthase ", " OPP synthase " etc.), referring to can catalysis bisphosphate isoamyl-1-alkene ester and continuous 1 '-4 condensations of allyl group initial substrate (primer substrate), causes forming the enzyme of the bisphosphate isopentene ester of various chain lengths.

Term " polynucleotide " and " nucleic acid " are used interchangeably in this article, refer to the Nucleotide of any length of polymerized form, comprise ribonucleotide or deoxynucleotide.Therefore, this term includes but not limited to: strand, two strands or multichain DNA or RNA, genomic dna, cDNA, DNA RNA hybrid, or contain the polymkeric substance of purine and pyrimidine bases or other non-natural or deutero-nucleotide base natural, chemistry or biochemical modification.

Term " peptide ", " polypeptide " and " protein " are used interchangeably in this article, the amino acid that refers to any length of polymerized form, can comprise coding and noncoding amino acid, chemistry or biochemical modification or deutero-amino acid, and the polypeptide that contains the peptide main chain of modification.

Term used herein " natural generation " can be applicable to nucleic acid, cell or organism, refers to nucleic acid, cell or the organism found under natural situation.For example, can be natural generation by natural origin separation and the middle polypeptide or the polynucleotide sequence that exists of organism (comprising virus) of having a mind to modify without lab assistant.

Term used herein " separation " refers to and natural polynucleotide, polypeptide or the cell that exists in the different environment of environment of polynucleotide, polypeptide or cell.The host cell of isolating genetic modification can be present in the population mixture of host cell of genetic modification.

Term used herein " exogenous nucleic acid " refers under the common or natural situation less than the nucleic acid of finding and/or producing in given natural bacteria, organism or cell.Term used herein " endogenous nucleic acid " refers to the nucleic acid of discovery and/or generation in given natural bacteria, organism or cell usually." endogenous nucleic acid " is also referred to as " natural acid " or for the nucleic acid of given bacterium, organism or cell " natural ".For example, the nucleic acid of coding HMGS, Mevalonic kinase and Phosphomevalonic kinase is represented the exogenous nucleic acid of intestinal bacteria (E.coli).(Sacchromyces cerevisiae) clones these mevalonate pathway nucleic acid by yeast saccharomyces cerevisiae.In yeast saccharomyces cerevisiae, the gene order of encode on the karyomit(e) HMGS, MK and PMK is " endogenous " nucleic acid.

Term used herein " heterologous nucleic acids " refers at least to satisfy the nucleic acid of following a kind of condition: (a) nucleic acid is given host microorganism or (" exogenous ") beyond the host cell (promptly not being natural discovery); (b) nucleic acid is included in (as " endogenous ") nucleotide sequence (comprising host microorganism or host cell endogenous nucleotide sequence as nucleic acid) of natural discovery in given host microorganism or the host cell, but the output of this nucleic acid in cell is non-natural amount (as greater than expected volume or greater than the output under the natural situation); Perhaps the sequence of this nucleic acid is different with the endogenous nucleotide sequence, so that produces the same proteins encoded (aminoacid sequence is identical or basic identical) of the interior Feed Discovery of non-natural amount (as greater than expected volume or greater than the output under the natural situation) in cell; (c) nucleic acid is included in different two or more nucleotide sequences or the sections of mutual relationship under the natural situation, is recombinant nucleic acid as nucleic acid.

Term used herein " heterologous polypeptide " refers under the natural situation and the irrelevant polypeptide of given polypeptide.For example, the isoprenoid precursor modifying enzyme that contains " allos membrane spaning domain " refers to contain under the natural situation irrelevant usually (as not adjoining usually with isoprenoid precursor modifying enzyme; Usually in same polypeptide chain, do not find) the isoprenoid precursor modifying enzyme of membrane spaning domain.Similarly, the isoprenoid precursor modifying enzyme that contains one or more " allos secretory structure territories ", " allos film insert polypeptide " and " allos solubilising structural domain " is to contain under one or more natural situations to have nothing to do usually (as not adjoining usually with isoprenoid precursor modifying enzyme; Usually in same polypeptide chain, do not find) secretory structure territory, film insert the isoprenoid precursor modifying enzyme of polypeptide and solubilising structural domain.

Term used herein " reorganization " refers to that concrete nucleic acid (DNA or RNA) is the product of the various combinations of clone, restriction enzyme digestion and/or Connection Step, and these combination results have the structural coding that can differentiate with the endogenous nucleic acid found in the natural system or the construction of non-coding sequence.Usually, the dna sequence dna of coding structure encoding sequence can be by cDNA fragment and short oligonucleotide joint or by a series of synthetic oligonucleotides assemblings, so that the nucleic acid that can be expressed by cell or cell-free transcription and the contained reorganization transcriptional units of translation system to be provided.Can do not provided this sequence by the form of the open reading frame of inner non-translated sequence or intron (generally being present in the eukaryotic gene) interruption.The genomic dna that comprises correlated series also can be used for forming recombination or transcriptional units.The non-translation DNA sequence may reside in 5 ' or 3 ' end of open reading frame, and these sequences are not disturbed the operation or the expression of coding region, and in fact can be used for regulating with various mechanism the generation (referring to following " DNA regulating and controlling sequence ") of required product.

Therefore, for example, term " reorganization " polynucleotide or " reorganization " nucleic acid refer to not to be natural generation, for example by human polynucleotide or the nucleic acid of getting involved two kinds of isolating sequence sections preparations of artificial combination.Usually realize this artificial combination by chemical synthesis process or by the isolating nucleic acid sections of manual operation (as gene engineering).Usually doing like this is for the sub codon that replaces of the redundant code of or conservative amino acid identical with coding, generally introduces or remove the recognition sequence site.Perhaps, carry out this method and have the nucleic acid sections of required function, produce required function combinations with connection.Usually realize this artificial combination by chemical synthesis process or by the isolating nucleic acid sections of manual operation (as gene engineering).

Similarly, term " reorganization " polypeptide refers to not to be natural generation, but (for example) makes up the polypeptide that two isolating aminoacid sequence sections prepare artificially by artificial intervention.Therefore, for example, containing the allogeneic amino acid polypeptide of sequence is recombinant polypeptide.

" construction " or " carrier " refers to recombinant nucleic acid, recombinant DNA normally, and the purpose that produces recombinant DNA is to express and/or breed specific nucleotide sequence, or makes up other recombinant nucleotide sequence with it.

Term used herein " operon " and " single transcriptional units " are used interchangeably, and refer to be subjected to the coordinately regulated two or more coding regions (encoding gene product such as RNA or proteinic nucleotide sequence) of adjoining of one or more controlling elementss (as promotor).Term used herein " gene product " refers to the RNA (vice versa) of dna encoding or the protein of RNA or dna encoding, wherein gene generally comprises one or more nucleotide sequences of coded protein, also can comprise intron and other non-coding nucleotide sequence.

Term " DNA regulating and controlling sequence ", " controlling elements " and " controlling element " are used interchangeably herein, refer to transcribe or translate control sequence, as promotor, enhanser, polyadenylation signal, terminator, proteolytic degradation signal etc., they provide and/or the modulate host cell in the expression of encoding sequence and/or the generation of coded polypeptide.

Term " conversion " is used interchangeably with " genetic modification " herein, guides the permanent or instantaneous inductive cytogenetics in new nucleic acid (being the exogenous DNA of cell) back to change.Can be by new DNA be mixed the host cell gene group, or by new DNA is instantaneous or stably be maintained the outer element of losing and realize hereditary change (" modification ").When cell is eukaryotic cell, realize permanent hereditary change by DNA being introduced cellular genome usually.In prokaryotic cell prokaryocyte, can will forever change introducing karyomit(e) or, with help they be maintained in the recombinant host cell but extra-chromosomal element can comprise one or more selective markers by extra-chromosomal element such as plasmid and expression vector introducing.The appropriate method of genetic modification comprises virus infection, transfection, joint, protoplastis fusion, electroporation, gene gun technology, calcium phosphate precipitation, direct microinjection etc.To the selection of method depend on cell type to be transformed usually and the environment that take place to transform (promptly external, exsomatize or body in).Overall discussion to these methods can be referring to Ausubel etc., Short Protocols in Molecular Biology (molecular biology simple method), the 3rd edition, Wei Sen Publishing Group (Wiley and Sons), 1995.

" operability connection " refers to side by side, and the mutual relationship of wherein said assembly allows them to work in required mode.For example, transcribe or express if promotor influences certain encoding sequence, this promotor operability is connected in this encoding sequence so.The promotor that does not link to each other under term used herein " allogeneic promoter " and " allos control region " reason of making a comment or criticism condition and other control region with natural concrete nucleic acid.For example, " with the allogenic transcripting controling area in coding region " is the transcripting controling area that does not link to each other with natural coding region under the normal circumstances.

" host cell " used herein refers in the body or external eukaryotic cell, prokaryotic cell prokaryocyte or from multicellular organisms (as clone) but cultivate and be the cell of unicellular entity, wherein eucaryon or prokaryotic cell prokaryocyte can be used as or as nucleic acid acceptor the expression vector of the nucleotide sequence of coding one or more biosynthetic pathway gene products such as mevalonate pathway gene product (as comprise), it comprises the offspring of the initiating cell of using the nucleic acid genetic modification.The offspring who should be understood that individual cells is not necessarily identical with the form of original parent or genomic dna or a whole set of DNA, because have natural, accidental or have a mind to sudden change." recombinant host cell " (being also referred to as " host cell of genetic modification ") is the host cell of having introduced heterologous nucleic acids such as expression vector.For example, because heterologous nucleic acids is introduced suitable prokaryotic host cell, described prokaryotic host cell is the prokaryotic host cell (as bacterium) of genetic modification, wherein said heterologous nucleic acids is the exogenous nucleic acid of for example (finding under the natural situation) beyond this prokaryotic host cell, or the recombinant nucleic acid of not finding in this prokaryotic host cell usually; Because heterologous nucleic acids has been introduced suitable eukaryotic host cell, described eukaryotic host cell is the eukaryotic host cell of genetic modification, wherein for example exogenous nucleic acid beyond this eukaryotic host cell or the recombinant nucleic acid do not found in this eukaryotic host cell usually of heterologous nucleic acids.

The amino-acid residue that has similar side chain in term " conservative amino acid replacement " the finger protein matter is interchangeable.For example, one group of amino acid with aliphatic lateral chain is made up of glycine, L-Ala, Xie Ansuan, leucine and Isoleucine; One group of amino acid with aliphatic series-hydroxyl side chain is made up of Serine and Threonine; One group of amino acid with amide containing side chain is made up of l-asparagine and glutamine; One group of amino acid with aromatic side chains is made up of phenylalanine, tyrosine and tryptophane; One group of amino acid with basic side chain is made up of Methionin, arginine and Histidine; One group of amino acid with sulfur-containing side chain is made up of halfcystine and methionine(Met).Exemplary conservative amino acid substituting group is: Val-Leu-Isoleucine, phenylalanine-tyrosine, Methionin-arginine, L-Ala-Xie Ansuan and l-asparagine-glutamine.

" nucleic acid " can be by oligonucleotide structure piece (building block) assembling with the method known to those skilled in the art chemosynthesis.Connect and these block structures of annealing,, assemble these sections with enzyme afterwards and make up whole gene to form gene segment.When mentioning dna sequence dna, " chemosynthesis " refers to that its composition amino acid is in assembled in vitro.Can be with the manual chemical synthesising DNA of the method set up, or one of available many available machinery robotics synthetic DNA.But the nucleotide sequence of modification of nucleic acids is with according to optimizing the codon bias of nucleotide sequence with the reflection host cell (principle) optimization expression.It will be understood by those skilled in the art that if codon uses the codon of being partial to be fit to the host, just can improve the possibility of successful expression.Can determine preferred codon according to gene studies available from the host cell that can obtain sequence information.

Polynucleotide or polypeptide and another kind of polynucleotide or polypeptide have certain percentage ratio " sequence homogeny " and are meant, when comparison, the base or the amino acid of this percentage ratio are identical, and when comparing two sequences, the base of this percentage ratio or amino acid are in identical relative position.Available many different modes are measured sequence similarity.In order to measure the sequence homogeny, methods availalbe and computer program aligned sequences, computer program comprises the BLAST that can be obtained by internet address ncbi.nlm.nih.gov/BLAST.Referring to for example, Altschul etc. (1990), J.Mol.Biol.215:403-10.Another kind of alignment algorithm is FASTA, can be from genetics calculating group (GCG) bag (Oxford Molecular Group company (Oxford MolecularGroup, wholly-owned subsidiary the Inc.)) acquisition of Wisconsin, USA Madison.Other comparison technology is referring to " Enzymology method " (Methods inEnzymology), the 266th volume: the computer approach of macromole sequential analysis (Computer Methods forMacromolecular Sequence Analysis) (1996), Doolittle compiles, (the Academic Press of academic press company, Inc.), (the Harcourt Brace﹠amp of haab company; Co.) branch office, San Diego, CA, USA.What cherish a special interest is to allow sequence comparison program jaggy.Smith-Waterman allows a kind of algorithm types jaggy in the sequence alignment.Referring to Meth.Mol.Biol.70:173-187 (1997).Simultaneously, adopt Needleman﹠amp; The GAP program of Wunsch comparison method can be used for aligned sequences.Referring to J.Mol.Biol.48:443-453 (1970).

When under temperature and solution ion strength appropriate condition, the single stranded form of nucleic acid can be annealed when another nucleic acid, and this nucleic acid and another nucleic acid (as cDNA, genomic dna or RNA) " can be hybridized ".Hybridization and wash conditions are known, referring to for example Sambrook, J., Fritsch, E.F. and Maniatis, T. " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual), second edition, press of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press), cold spring port (1989), particularly wherein Chapter 11 and table 11.1; And Sambrook, J. and Russell, W., " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual), the third edition, press of cold spring harbor laboratory, cold spring port (2001).Temperature and ionic strength conditions have determined hybridization " preciseness ".Can regulate the preciseness condition screening the homologous sequence of the less organism of medium similar fragment such as dependency, to the gene of highly similar fragment as the functional enzyme that duplicates closely related organism.Hybridization conditions and post-hybridization washing can be used for obtaining the decisive rigorous condition of required hybridization.One group profile post-hybridization washing is a series of washings, beginning is 6 * SSC (SSC is 0.15M NaCl and 15mM citrate buffer), 0.5%SDS, following 15 minutes of room temperature is used 2 * SSC then, 0.5%SDS, 45 ℃ of repeated washings 30 minutes are used 0.2 * SSC, 0.5%SDS then, 50 ℃ were washed 30 minutes, and repeated twice.Obtain other rigorous condition with comparatively high temps, wherein washing is identical with above-mentioned washing, and except last twice usefulness 0.2 * SSC, 30 minutes temperature of 0.5%SDS washing is brought up to 60 ℃.0.1 * SSC, 0.1%SDS, 65 ℃ are adopted in last twice washing of the highly rigorous condition of another group.Another example of rigorous hybridization conditions is to hybridize under 50 ℃ or higher temperature and among 0.1 * SSC (15mM sodium-chlor/1.5mM Trisodium Citrate).Another example of rigorous hybridization conditions is 42 ℃ of overnight incubation in solution, used solution is: the salmon sperm DNA that 50% methane amide, 5 * SSC (150mMNaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5 * Denhardt solution, 10% T 500 and 20 μ g/ml sex change are sheared, and use 0.1 * SSC at about 65 ℃ of washing nozzles then.Rigorous hybridization conditions and post-hybridization washing condition be at least with same rigorous hybridization conditions and the post-hybridization washing condition of above-mentioned representative of conditions.

Hybridization needs two kinds of nucleic acid to contain complementary sequence, but according to the preciseness of hybridizing, has base mispairing.The suitable preciseness of nucleic acid hybridization depends on length nucleic acid and complementary degree, and they are variablees well known in the art.Similarity or homology degree between two nucleotide sequences are big more, and the melting temperature(Tm) (Tm) with nucleic acid hybrids of these sequences is high more.The relative stability of nucleic acid hybridization (corresponding to higher Tm) reduces in the following order successively: RNA:RNA, DNA:RNA, DNA:DNA.For the crossbred of length greater than 100 Nucleotide, produced the equation that calculates Tm (referring to Sambrook etc., the same, 9.50-9.51).For shorter nucleic acid is the hybridization of oligonucleotide, and the mispairing position becomes more important, the length of oligonucleotide determine its specificity (referring to Sambrook etc., the same, 11.7-11.8).Usually, but the length of hybrid nucleic acid is at least about 10 Nucleotide.But the exemplary minimum length of hybrid nucleic acid is: at least about 15 Nucleotide; At least about 20 Nucleotide; At least about 30 Nucleotide.And one skilled in the art will appreciate that can be according to such as factor attemperation and washing soln salt concn such as probe length.

Before further describing the present invention, should be understood that the present invention is not limited only to described embodiment, they also can change certainly.Should be understood that also term used herein just to describing embodiment, is not intended to restriction, therefore, the scope of the invention only is subjected to the restriction of appended claims.

When the scope of the value of providing, should be understood that to the present invention includes between this scope bound to be each intervening value (not being like this) and any other value of pointing out at interval or to point out intervening value in the scope unless spell out in the literary composition with lower limit unit 1/10th.In these bounds more among a small circle can be included in more independently, and also belong to the present invention, also can in pointing out scope, get rid of limit value especially.When described scope comprised one or two limit value, the present invention also comprised the scope of getting rid of one or two limit value.

Except as otherwise noted, all scientific and technical terminologies used herein are identical with the implication of one skilled in the art's common sense of the present invention.Though implement or test the present invention also can adopt similar or is equivalent to any method and the material of content described herein, described preferable methods and material now.With described herein all deliver thing to include this paper in for referencial use so that deliver thing disclosure and description method and/or material together with what quote.

Must be noted that singulative " ", " a kind of " and " this " used in this paper and the appended claims comprise plural implication, are not like this unless spell out in the literary composition.Therefore, for example, mention " a kind of cytochrome P 450 enzymes " and comprise multiple this fermentoid, mention " a kind of cytochrome P450 reductase " and comprise one or more cytochrome P450 reductases well known by persons skilled in the art and its equivalent etc.Note also, can make claims get rid of any optional member.Therefore, this statement is intended to as the prerequisite basis of removing property term as " separately ", " only " etc. and the coupling of claim part, or adopts " bearing " restriction.

It only is because they are open before the application's the applying date that the thing of delivering described herein is provided.Should not be construed as and admit to make the present invention not have qualification prior to this publication owing to formerly inventing.And open day that provides may be different with open day of reality, may need independent affirmation.

Detailed Description Of The Invention

The present invention also provides the nucleic acid of the nucleotide sequence that contains coding isoprenoid precursor modifying enzyme; And the recombinant vectors and the host cell that comprise this nucleic acid.The invention provides the method that in host cell, produces the isoprenoid precursor modifying enzyme that enzymic activity is arranged.The present invention also provides in the host cell of the nucleic acid genetic modification of using the nucleotide sequence that contains coding isoprenoid precursor modifying enzyme, produces the method for isoprenoid compound.

Nucleic acid, carrier and host cell

The invention provides the nucleic acid of the nucleotide sequence that contains the modified cytochrome P 450 enzymes of encoding; And the recombinant vectors and the host cell that comprise this nucleic acid.The invention provides the nucleic acid of the nucleotide sequence that contains coding isoprenoid precursor modifying enzyme; And the recombinant vectors and the host cell that comprise this nucleic acid.

Term used herein " modified cytochrome P 450 enzymes " refers to modify the enzyme of the intermediate of (as " functionalized ") biosynthetic pathway.Following one or more reactions of modified cytochrome P 450 enzymes energy catalysis of nucleic acid encoding of the present invention: hydroxylation, oxidation, epoxidation, dehydration, dehydrogenation, dehalogenation, isomerization, pure oxidation, formoxy-ization, dealkylation and C-C cleavage reaction.In this article this class reaction is generically and collectively referred to as " biosynthetic pathway intermediate modification reaction ".These reactions are referring to for example, ((1996) Chem.Rev.96:2841-2887 such as Sono; Referring to for example, Fig. 3 of Sono etc., the synoptic diagram of this class reaction).

In some embodiments, modified cytochrome P 450 enzymes is an isoprenoid precursor modifying enzyme.Term used herein " isoprenoid precursor modifying enzyme " is used interchangeably with " isoprenoid modifying enzymes ", they refer to modify the isoprenoid precursor compound, for example make the enzyme of substrate, following one or more reactions of described isoprenoid precursor modifying enzyme energy catalysis: hydroxylation, epoxidation, oxidation, dehydration, dehydrogenation, dehalogenation, isomerization, pure oxidation, formoxy-ization, dealkylation and C-C cleavage reaction with the isoprenoid precursor compound.In this article this class reaction is generically and collectively referred to as " isoprenoid precursor modification reaction ".These reactions are referring to for example, and ((1996) are the same for Sono etc.; Referring to for example, Fig. 3 of Sono etc., the synoptic diagram of this class reaction).In many embodiments, isoprenoid precursor modifying enzyme is a cytochrome P 450 enzymes.Referring to for example, Sono etc. (1996) are the same.

The substrate of modified cytochrome P 450 enzymes

As mentioned above, the substrate of modified cytochrome P 450 enzymes is the intermediate of biosynthetic pathway.Exemplary intermediate includes but not limited to: the isoprenoid precursor; Alkaloid precursors; The phenylpropionic acid precursor; The flavonoid precursor; The steroid precursor; The polyketide precursor; The macrolide precursor; The sugar alcohol precursor; Phenolic compound precursor etc.Referring to for example, ((2003) Appl.Environ.Microbiol.69:2699-2706 such as Hwang; ((2004) TRENDS Plant Sci.9:116 such as Facchini.

Interested biosynthetic pathway product includes but not limited to: isoprenoid compound, alkaloid compound, phenylpropionic acid compound, flavonoids, sterid, polyketide compound, Macrocyclic lactone compounds, sugar alcohol, phenolic compound etc.

Alkaloid compound is the diversified natural product of finding in about 20% floristics of a big class.Normally there is nitrogen-atoms in their definition in the heterocycle under the state of oxidation.Alkaloid compound comprises bisindole alkaloid compound, indole alkaloid compound, morphinane alkaloid compound etc.Alkaloid compound comprises monocycle alkaloid compound, dicyclo alkaloid compound, three ring alkaloid compounds, Fourth Ring alkaloid compound and the alkaloid compound with cage structure.Alkaloid compound comprises: 1) pyridines: piperine, coniine, trigonelline, arecaidine, guvacine, pilocarpine, Cytisine, Tocosamine, pelletierine; 2) pyrrolidines: hygrine, nicotine, cuskhygrine; 3) tropine bases: coromegine, Cocaine, tropine carboxylic acid, pelletierine, Scopolamine; 4) quinoline: quinine, hydroquinine, Quinidine, dihydrochinidin, strychnine, brucine and veratrum alkaloid (as veratrine, cevadine); 5) iloquinoline derivative: morphine, morphine monomethyl ether, thebaine, Papaverine, opianine, narceine, hydrastine and berberine; 6) phenylethylamine class: metamfetamine, mescaline, ephedrine; 7) indoles: tryptamines class (as BMLH, Psilocybine, serotonin), ergoline class (as ergine, Ergotamine, sphacelic acid etc.) and β-Ka Lin class (as harmine, Yohimbine, serpentine, ipecamine); 8) purine class: xanthine (as caffeine, Theobromine, theophylline); 9) terpene: aconite alkaloid (as napelline) and steroid (as solanine, samandarine); 10) betaines: (quaternary ammonium compound :) as muscarine, choline, neurine; And 11) pyrazoles: pyrazoles, fomepizole.Exemplary alkaloid compound is morphine, berberine, vinealeucoblastine(VLB), vincristine(VCR), Cocaine, Scopolamine, caffeine, nicotine, coromegine, Papaverine, ipecamine, quinine, serpentine, morphine monomethyl ether, serotonin etc.Referring to for example, Facchini etc. ((2004) Trends Plant Science 9:116).

The isoprenoid modifying enzymes substrate

Term " isoprenoid precursor compound " is used interchangeably with " isoprenoid precursor substrate ", and they refer to the compound as diterpene synthase reaction product on many isopentene of bisphosphate ester.Diterpene synthase (being also referred to as " terpene cyclase ") reaction product is so-called " terpene skeleton ".In some embodiments, the terpene skeleton is modified in isoprenoid modifying enzymes catalysis, or its downstream product.Therefore, in some embodiments, the isoprenoid precursor is the terpene skeleton.The isoprenoid precursor substrate of isoprenoid precursor modifying enzyme comprises monoterpene, diterpene, triterpene and sesquiterpene.

The monoterpene substrate of the isoprenoid modifying enzymes of nucleic acid encoding of the present invention includes but not limited to: the oxidation products of generation is any monoterpene substrate of the intermediate of monoterpene or the biosynthetic pathway that produces monoterpene.Exemplary monoterpene substrate includes but not limited to: the monoterpene substrate that belongs to following family: acyclic monoterpene, dimethyl octane, menthane, irregular monoterpenoid, eucalyptol, camphane, isocamphane, monocyclic monoterpene, pinane, fenchane, thujane, carane, ionone, iris alkane (Iridanes) and hemp (Cannabanoids).Exemplary monoterpene substrate, intermediate and product include but not limited to: limonene, geraniol (citranellol), Mang geraniol, menthol, perillyl alcohol, phantol and peaceful ketone.

The diterpene substrate of the isoprenoid modifying enzymes of nucleic acid encoding of the present invention includes but not limited to: the oxidation products of generation is any diterpene substrate of the intermediate of diterpene compound or the biosynthetic pathway that produces diterpene compound.Exemplary diterpene substrate includes but not limited to: the diterpene substrate that belongs to following family: acyclic diterpenoids, the dicyclo diterpenoids, the monocycle diterpenoids, Ladanum alkane (Labdanes), Andreas Kloeden alkane (Clerodanes), Taxan, three ring diterpenoids, the Fourth Ring diterpenoids, kaurene, beyerane (Beyerenes), a word used for translation ground alkene (Atiserenes), aphidocolin (Aphidicolins), grayanotoxin, Plant hormones regulators,gibberellins, macrocyclic diterpene and Elizabethan's three alkane (Elizabethatrianes).Exemplary diterpene substrate, intermediate and product include but not limited to: comb fiber crops element, eleutherobin (eleutherobin), taxol, the element of overgrowing (prostratin) and false pterosin.

The triterpene substrate of the isoprenoid modifying enzymes of nucleic acid encoding of the present invention includes but not limited to: the oxidation products of generation is any triterpene substrate of the intermediate of triterpenoid or the biosynthetic pathway that produces triterpenoid.Exemplary triterpene substrate, intermediate and product include but not limited to: Ah cloth's glucosides E (arbrusideE), bruceantin (bruceantin), testosterone, progesterone, cortisone and digoxigenin.

The sesquiterpene substrate of the isoprenoid modifying enzymes of nucleic acid encoding of the present invention includes but not limited to: the oxidation products of generation is any sesquiterpene substrate of the intermediate of sesquiterpenoid or the biosynthetic pathway that produces sesquiterpenoid.Exemplary sesquiterpene substrate includes but not limited to: the sesquiterpene substrate that belongs to following family: farnesane, the monocycle farnesane, monocyclic sesquiterpene, the dicyclo sesquiterpene, the dicyclo farnesane, double wave alkane (Bisbolanes), santalane, card ripple alkane (Cupranes), leaf-cutting tongue alkane (Herbertanes), lobe tongue alkane (Gymnomitranes), the mould alkane of single-ended spore, retinispora alkane (Chamigranes), daucane, acorane, An Tisating (Antisatins), cadinane, sesquiterpene ketone dextrorotation Japan stichopus japonicus terpene ketone (Oplopananes), copane (Copaanes), picrotoxin alkane (Picrotoxanes), cedrane, longipinane, cycloalkanes (Longicyclanes) comes into leaves, caryophyllane, do not get alkane (Modhephanes), Si Feiye alkane (Siphiperfolanes), humulane, full leaf alkane (Intergrifolianes), Li Piye alkane (Lippifolianes), former Yi Lu alkane (Protoilludanes), latent ring umbrella alkane (Illudanes), crinosity alkane (Hirsutanes), breast mushroom alkane (Lactaranes), Si Dibo alkane (Sterpuranes), rich numb alkane (Fomannosanes), horse traction alkane (Marasmanes), germacrane, elemane, eudesmane, Bake alkane (Bakkanes), figured woven silk material alkane (Chilosyphanes), guainane, false guainane, tricyclic sesquiterpene, patchoulane trioxane (Trixanes), aromadendrane (Aromadendranes), high each alkane (Gorgonanes), receive many alkane (Nardosinanes), brazan (Brasilanes), green paint alkane (Pinguisanes), sesquialter pinane (Sequipinane), sesquialter camphane (Sequicamphane), thujopsane, the dicyclo humulane, green onion alkane (Alliacanes), Si Dibo alkane (Sterpuranes), breast mushroom alkane, Ya Fuli alkane (Africanes), full leaf alkane, former Yi Lu alkane (Protoilludanes), aristolane and Niu Lan alkane (Neolemnanes).Exemplary sesquiterpene substrate includes but not limited to: AMORPHADIENE, Isolongifolene (alloisolongifolene), (-)-α-trans-bergapten (bergamotene), (-)-beta-elemene, (+)-germacrene A, germacrene B, (+)-γ-gurjunene, (+)-ledene, neointermedeol (neointermedeol), (+)-β-selinene and (+)-valencene.

Modify

Nucleic acid of the present invention contains the nucleotide sequence of the modified cytochrome P 450 enzymes of encoding, and in many embodiments, the modified cytochrome P 450 enzymes of nucleic acid encoding of the present invention contains non-natural (non-wild-type, or non-natural produces, or variation) aminoacid sequence.The modified cytochrome P 450 enzymes of coding contains one or more amino acid sequence modifications that can improve with the activity level of modified cytochrome P 450 enzymes in the host cell of nucleic acid genetic modification of the present invention and/or improve the given product level of the biosynthetic pathway that the host cell with nucleic acid genetic modification of the present invention produces (disappearance, add, insertion, replacement).

In some embodiments, nucleic acid of the present invention contains the nucleotide sequence that coding is modified isoprenoid precursor modifying enzyme, and in many embodiments, the isoprenoid precursor modifying enzyme of nucleic acid encoding of the present invention contains non-natural (non-wild-type, or the non-natural generation, or variation) aminoacid sequence.The isoprenoid precursor modifying enzyme of coding will contain and can improve with the activity level of isoprenoid precursor modifying enzyme in the host cell of nucleic acid genetic modification of the present invention and/or improve the one or more amino acid sequence modifications of given isoprenoid compound level that the host cell with nucleic acid genetic modification of the present invention produces (disappearance, add, insertion, replacement).In some embodiments, with respect to wild-type isoprenoid precursor modifying enzyme, the isoprenoid precursor modifying enzyme of coding will comprise one or more following modifications: a) replace natural membrane spaning domain with the non-natural membrane spaning domain; B) replace natural membrane spaning domain with the secretion signal structural domain; C) replace natural membrane spaning domain with the solubilising structural domain; D) insert the territory with film and replace natural membrane spaning domain; E) the natural membrane spaning domain of brachymemma; And f) aminoacid sequence of the natural membrane spaning domain of change.

In many embodiments, order with 5 ' to 3 ', nucleic acid of the present invention comprises the nucleotide sequence of coding first structural domain that operability connects and the nucleotide sequence that coding is modified the catalytic domain of P450 enzyme (as isoprenoid precursor modifying enzyme), and described first structural domain is selected from membrane spaning domain, secretory structure territory, solubilising structural domain or film and inserts albumen; Described first structural domain and described catalytic domain allos.In some embodiments, described first structural domain comprises secretion signal and membrane spaning domain.

The non-natural membrane spaning domain

In some embodiments, the modified cytochrome P 450 enzymes of coding (as isoprenoid precursor modifying enzyme) will comprise non-natural (as allos) membrane spaning domain.Suitable non-natural membrane spaning domain is selected from the membrane spaning domain that function is arranged usually in given host cell.In some embodiments, the non-natural membrane spaning domain is the membrane spaning domain that function is arranged in prokaryotic host cell.In other embodiments, the non-natural membrane spaning domain is the membrane spaning domain that function is arranged in eukaryotic host cell.

For example, in many embodiments, at expression in escherichia coli, the non-natural membrane spaning domain comprises one of following aminoacid sequence:

NH ₂-MWLLLIAVFLLTLAYLFWP-COOH(SEQ?ID?NO：1)；

NH ₂-MALLLAVFLGLSCLLLLSLW-COOH(SEQ?ID?NO：2)；

NH ₂-MAILAAIFALVVATATRV-COOH(SEQ?ID?NO：3)；

NH ₂-MDASLLLSVALAVVLIPLSLALLN-COOH (SEQ ID NO:4); With

NH ₂-MIEQLLEYWYVVVPVLYIIKQLLAYTK-COOH(SEQ?ID?NO：5)。

Secretion signal

In some embodiments, the modified cytochrome P 450 enzymes of coding (as isoprenoid precursor modifying enzyme) will comprise the alpha-non-natural amino acid sequence that can make the emiocytosis fusion rotein.Those skilled in the art understand this class secretory signal sequence.The secretion signal that is applicable to bacterium includes but not limited to: the secretion signal of the Blang of intestinal bacteria, serratia marcesens (S.marcescens), erwinia amylovora (E.amylosora), morganella morganii (M.morganii) and Proteus mirabilis (P.mirabilis) (Braun ' s) lipoprotein, the TraT albumen of intestinal bacteria and salmonella (Salmonella); Penicillinase (PenP) albumen of Bacillus licheniformis (B.licheniformis) and bacillus cereus (B.cereus) and streptococcus aureus (S.aureus); The amylopectin zymoprotein of Klebsiella Pneumoniae (Klebsiella pneumoniae) and aerogenesis klebsiella (Klebsiella aerogenese); Intestinal bacteria lipoprotein 1pp-28, Pal, RplA, RplB, OsmB, NIpB and Orl17; The chitinase protein of Vibrio harveyi (V.harseyi); The β-1 of Solanaceae pseudomonas (Pseudomonassolanacearum), 4-endoglucanase albumen, Pal of hemophilus influenzae (H.influenzae) and Pcp albumen; The OprI albumen of Pseudomonas aeruginosa (P.aeruginosa); MalX of streptococcus pneumoniae (S.pneumoniae) and AmiA albumen; 34kda antigen and the TpmA albumen of Treponoma palladium (Treponema pallidum); The P37 albumen of mycoplasma hyorhinis (Mycoplasma hyorhinis); The neutral protease of bacillus amyloliquefaciens (Bacillus amyloliquefaciens); The 17kda antigen of rickettsia rickettsii (Rickettsiarickettsii); The malE maltose binding protein; RbsB ribose is conjugated protein; The phoA alkaline phosphatase; With the OmpA secretion signal (referring to for example, Tanji etc. (1991) J Bacteriol.173 (6): 1997-2005).The zymic secretory signal sequence that is applicable to known in the art can adopt this sequence.Referring to for example, U.S. Patent number 5,712,113.RbsB, malE and phoA secretion signal be referring to for example, Collier (1994) J.Bacteriol.176:3013.

In some embodiments, for example, at prokaryotic host cell such as expression in escherichia coli, secretion signal will contain one of following aminoacid sequence:

NH ₂-MKKTAIAIAVALAGFATVAQA-COOH(SEQ?ID?NO：6)；

NH ₂-MKKTAIAIVVALAGFATVAQA-COOH(SEQ?ID?NO：7)；

NH ₂-MKKTALALAVALAGFATVAQA-COOH(SEQ?ID?NO：8)；

NH ₂-MKIKTGARILALSALTTMMFSASALA-COOH(SEQ?ID?NO：9)；

NH ₂-MNMKKLATLVSAVALSATVSANAMA-COOH (SEQ ID NO:10); With

NH ₂-MKQSTIALALLPLLFTPVTKA-COOH(SEQ?ID?NO：11)。

In some embodiments, the modified cytochrome P 450 enzymes of coding (as isoprenoid precursor modifying enzyme) will comprise non-natural secretory signal sequence and allos membrane spaning domain.Can adopt any combination of secretory signal sequence and allos membrane spaning domain.

As a non-limitative example, in some embodiments, the allos structural domain that contains non-natural secretory signal sequence and allos membrane spaning domain has following aminoacid sequence: NH ₂-MKKTAIAIAVALAGFATVAQA LLEYWYVVVPVLYIIKQLLAYTK-COOH (SEQ ID NO:12) wherein represents membrane spaning domain with underscore, and secretion signal is positioned at N-terminal one side of membrane spaning domain.

The solubilising structural domain

In some embodiments, the modified cytochrome P 450 enzymes of coding (as isoprenoid precursor modifying enzyme) will comprise the non-natural structural domain that makes the protein solubilising.

In some embodiments, the solubilising structural domain will contain one of following aminoacid sequence:

NH ₂-EELLKQALQQAQQLLQQAQELAKK-COOH (SEQ ID NO:13); With

NH ₂-MTVHDIIATYFTKWYVIVPLALIAYRVLDYFY-COOH(SEQ?IDNO：14)；

NH ₂-GLFGAIAGFIEGGWTGMIDGWYGYGGGKK-COOH (SEQ IDNO:15); With

NH ₂-MAKKTSSKG-COOH(SEQ?ID?NO：16)。

Film inserts the territory

In some embodiments, the modified cytochrome P 450 enzymes of coding (as isoprenoid precursor modifying enzyme) will comprise the alpha-non-natural amino acid sequence that can insert it in the film.In some embodiments, the modified cytochrome P 450 enzymes of coding is to contain the fusion polypeptide of frame endomixis in the allos fusion partners (as the albumen except that cytochrome P 450 enzymes) of aminoterminal or carboxyl terminal, and this fusion partners can make this fusion rotein insert in the microbial film.

In some embodiments, this fusion partners is Mi Sidi albumen (mistic), as contains the albumen of aminoacid sequence shown in Figure 5 (GenBank accession number AY874162).This Mi Sidi of coding is provided under the GenBank accession number AY874162 proteic nucleotide sequence.Other polypeptide that can insert in the microbial film known in the art, referring to for example, (J.Biol.Chem.276 (18): 14607), described bW such as PsbW Woolhead; And Kuhn (FEMS Microbiology Reviews 17 (1992i) 285), M12 Scott Proctor (procoat) albumen and Pf3 Scott Proctor albumen have been described.

Cytochrome P 450 enzymes

In many embodiments, the isoprenoid precursor modifying enzyme of coding is a cytochrome P 450 enzymes.The cytochrome P 450 enzymes of coding will carry out following one or more reactions: hydroxylation, epoxidation, oxidation, dehydration, dehydrogenation, dehalogenation, isomerization, pure oxidation, formoxy-ization, dealkylation and C-C cleavage reaction.In this article this class reaction is generically and collectively referred to as " biosynthetic pathway intermediate modification reaction ", perhaps in embodiment, is called " isoprenoid precursor modification reaction ".These reactions are referring to for example, and ((1996) are the same for Sono etc.; Referring to for example, Fig. 3 of Sono etc., the synoptic diagram of this class reaction).As mentioned above, in many embodiments, the modified cytochrome P 450 enzymes of coding (as isoprenoid precursor modifying enzyme) is cytochrome P 450 monooxygenases, Cytochrome P450 hydroxylase, Cytochrome P450 cyclooxygenase or Cytochrome P450 desaturase.Various cytochrome P 450 monooxygenases, hydroxylase are understood in this area, and cyclooxygenase and desaturase (being generically and collectively referred to as " P450 enzyme ") can be modified the aminoacid sequence of any known P450 enzyme or its variant according to the present invention.

The suitable source of nucleic acid of containing the nucleotide sequence of Codocyte cytochrome p 450 enzyme includes but not limited to: six boundaries, as bacterium circle (as eubacterium); Archeobacteria circle; Protista; Mycota; Vegitabilia; Cell or organism with arbitrary boundary in the animal kingdom.The suitable source of exogenous nucleic acid comprises the member who is similar to plant in the Protista, includes but not limited to: algae (as green alga, red algae, grey born of the same parents algae (glaucophytes), blue-green algae); Be similar to the member of fungi in the Protista, as Acarasiales, water mold etc.; Be similar to the member of animal in the Protista, as flagellate (as euglena), amoeba (as amoeba), sporozoite (as pushing up multiple worm (Apicomplexa), sticking protozoon, microsporidium) and ciliate (as paramecium).The suitable source of exogenous nucleic acid comprises the member of mycota, includes but not limited to: following any member: Basidiomycota (goatsbeard; As the member of Agaricus, day dog Pseudomonas, Boletus, Cantharellus (Cantherellus) etc.); Ascomycota (ascomycetes, comprise (as) yeast); Bacterium algae door (lichens); Zygomycota (engaging fungi); With the imperfect fungi door.The suitable source of exogenous nucleic acid comprises the member of vegitabilia, includes but not limited to: following any member: Bryophyta (as mosses), angle bryophyte door (as hornwort), liverwort door (Hepaticophyta) (as liverwort), Lepidophyta (as lycopsida), cuneus plant door (as Equisetales), Psilophyta (as the leaf fern), Herba Ophioglossi door, fern door (as fern), Cycadophyta, ginkgo door, Coniferae, Stem of Smalleaf Jointfir door and Magnoliophyta (as polycarpeae).The suitable source of exogenous nucleic acid comprises the member of animal kingdom, includes but not limited to: following any member: Porifera (Spongia); The placozoan door; Straight swimming worm door (parasite of marine invertebrate); Rhombus worm door; Cnidaria (coral, sea anemone, jellyfish, sea pen, sea pansy, sea wasp); Ctenophora (ctenophore); Platyhelminthes (flatworms); Nemertea (ribbon wirm); Jaw stomach animal door (Ngathostomulida) (the jaw worm is arranged); The gastrotrich door; Wheel echiuran door; The priapulid door; The kinorhynch door; The loriciferan door; Acanthocephala; Interior anus subphylum; Nemathelminthes; Nemathelminthes; Collar extension animal door; Mollusca (mollusk); Sipunculan door (Sipunculus nudus (peanut worms)); Annelita (annelid worm); Tardigrada (tardigrade); Onychophora (comb silkworm); Arthropoda (comprises following subphylum: Chelicerata, the polypody subphylum, six sufficient subphylums and crust subphylum, wherein Chelicerata for example comprises, Arachnida, Merostomata and sea spider guiding principle, the polypody subphylum for example comprises, chilopoda (chiropoda), Diplopoda (myriapods), Pauropoda (Paropoda) and Symphyla, six sufficient subphylums comprise Insecta, the crust subphylum comprises shrimp, krill, barnacle etc.; Phoronida; Ectoprocta (moss animal); Brachiopoda; Echinodermata (as starfish, extra large daisy, comatulid, sea urchin, sea cucumber, brittle star, crisp basket (brittlebaskets) etc.); Chaetognath (arrow worms); Hemichordata (jade hook worm); And Chordata.The suitable member of Chordata comprises any member of following subphylum: Urochordata (Ascidiacea; Comprise Ascidicea, cup Ascidicea and Larvuccea); Acrania (lancelet); Hagfish guiding principle (hagfish); And Vertebrata, wherein the Vertebrata member comprises following member, for example, gill eel guiding principle (lampreys), Chondrichthyes (selachian), spoke fin net-rope (spoke fin fish), the burnt guiding principle (coelacanth) of chamber sour jujube, Dipnoi (lung fish), reptilia (Reptilia, as snake, alligator, crocodile, lizard etc.), Aves (bird); And Mammalia (Mammals).Suitable plant comprises monocotyledons and dicotyledons.

Therefore, for example, suitable source comprises the cell from following organism, and described organism includes but not limited to: protozoon, plant, fungi, algae, yeast, Reptilia, Amphibians, Mammals, marine microorganism, marine invertebrate, arthropods, etc. sufficient class animal, insect, arachnid, archeobacteria and eubacterium.

Suitable protokaryon source comprises bacterium (as eubacterium) and archeobacteria.Suitable archeobacteria source comprises methanogen, extreme halophile, extreme thermophile bacterium etc.Suitable archeobacteria source includes but not limited to any member of following classification: the ancient bacterium door of spring is (as sulfolobus solfataricus (Sulfolobus solfataricus), motion sulphur reduction coccus (Defulfurococcus mobilis), hidden heat supply network bacterium (Pyrodictium occultum), rely on heated filament bacterium (Thermofflum pendens), special heat-resistant deforming bacterium (Thermoproteus tenax)), wide ancient bacterium door (is given birth to hot-bulb bacterium (Thermococcus celer) as speed, inorganic nutrients methanogen (Methanococcusthermolithotrophicus), Zhan Shi methanogen (Methanococcus jannaschii), hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum), formic acid methagen (Methanobacteriumformicicum), red-hot methane thermophile bacteria (Methanothermus fervidus), the ancient green-ball bacterium (Archaeoglobus fulgidus) of glimmering, thermoplasma acidophilum (Thermoplasma acidophilum), richly endowed salt bacterium (Haloferax volcanni), Pasteur's sarcina methanica (Methanosarcina barkeri), Methanosaetaconcilli, Hong Shi methane spirillum (Methanospririllum hungatei), motion methane germ (Methanomicrobium mobile) and first ancient bacterium door.Suitable eubacterium source includes but not limited to any member of following classification: produce the hydrogen bacillus, thermobacillus, green non-thiobacterium, abnormal cocci, cyanobacteria, purple bacteria, floating mould, spirochete, green sulfur bacteria, phagocyte bacterium and gram positive organism are (as mycobacterium (Mycobacterium sp.), micrococci (Micrococcus sp.), suis (Streptomyces sp.), lactobacillus (Lactobacillus sp.), Helicobacter pylori (Helicobacterium sp.), clostridium (Clostridium sp.), mycoplasma (Mycoplasma sp.), genus bacillus (Bacillus sp.) etc.).

In some embodiments, by the tissue of taking from organism; Separation separates P450 enzyme coding nucleic acid from the specific cells of organism or cell mass etc.For example, in some embodiments, when organism is plant, by isolated nuclei acid such as xylem, phloem, form layers, leaf, roots.In some embodiments, when organism is animal, by particular organization (as lung, liver, the heart, kidney, brain, spleen, skin, fetal tissue etc. or particular cell types (as neuronal cell, epithelial cell, endotheliocyte, stellate cell, scavenger cell, spongiocyte, island cell, T lymphocyte, bone-marrow-derived lymphocyte etc.) isolating nucleic acid.

In some embodiments, nucleic acid of the present invention comprises the nucleotide sequence of coding P450 enzyme, and described sequence is different from the wild-type of coding P450 enzyme or the nucleotide sequence of natural generation, and for example, nucleic acid of the present invention comprises the nucleotide sequence of coding variation P450 enzyme.In some embodiments, compare with the aminoacid sequence of the parent P450 enzyme of natural generation, an amino acid, two amino acid, three amino acid, four amino acid, five amino acid, six amino acid, seven amino acid, eight amino acid, nine amino acid or ten amino acid or more a plurality of amino acid difference are arranged in the aminoacid sequence of variation P450 enzyme.In some embodiments, compare have an appointment in the aminoacid sequence of variation P450 enzyme 10-15 amino acid, about 15-20 amino acid, about 20-25 amino acid, about 25-30 amino acid, about 30-35 amino acid, about 35-40 amino acid, about 40-50 amino acid or about 50-60 amino acid or more a plurality of amino acid difference with the aminoacid sequence of the parent P450 enzyme of natural generation.

In many embodiments, as mentioned above, the modified cytochrome P 450 enzymes of coding comprises the modification of parent (as wild-type or natural generation or natural sequence) N-terminal, as, the modification in the aminoacid sequence of membrane spaning domain and/or membrane spaning domain N-terminal.In some embodiments, compare, also comprise one or more amino acid sequence modifications in the enzyme catalysis part of the modified cytochrome P 450 enzymes of coding with the aminoacid sequence of wild-type cell cytochrome p 450 enzyme.

The nucleic acid of the nucleotide sequence of (as modifying) P450 enzyme that containing encodes makes a variation is nucleic acid.In some embodiments, the nucleic acid that contains the nucleotide sequence of coding variation P450 enzyme be under suitable hybridization conditions can with the nucleic acid of the nucleic acid hybridization of the nucleotide sequence of the P450 enzyme that contains the natural generation of encoding.In some embodiments, the nucleic acid that contains the nucleotide sequence of coding variation P450 enzyme be under rigorous hybridization conditions can with the nucleic acid of the nucleic acid hybridization of the nucleotide sequence of the P450 enzyme that contains the natural generation of encoding.In some embodiments, the nucleotide sequence that containing the coding variation P450 enzyme that the nucleic acid of the nucleotide sequence of coding variation P450 enzyme comprises and the nucleotide sequence homogeny of the nucleotide sequence of the natural generation P450 enzyme of coding are less than about 95%, for example, the nucleotide sequence of coding variation P450 enzyme is not more than about 90%-95% with the nucleotide sequence homogeny of the nucleotide sequence of the natural generation P450 enzyme of coding, about 85%-90%, about 80%-85%, about 75%-80%, about 70%-75%, about 65%-70%, about 60%-65%, about 55%-60% or about 50%-55%.

In some embodiments, the nucleotide sequence coded P450 enzyme of coding variation P450 enzyme and the aminoacid sequence homogeny of natural generation P450 enzyme are about 50%-55%, about 55%-60%, about 60%-65%, about 65%-70%, about 70%-75%, about 75%-80%, about 80%-85%, about 85%-90% or about 90%-95%.The aminoacid sequence of many P450 enzymes known in the art.

Can by contained nucleotide sequence in the nucleic acid of the present invention modify and the suitable P450 enzyme of coding include but not limited to: limonene-6-hydroxylase (referring to for example, Fig. 6; GenBank accession number AY281025 and AF124815); 5-table-aristolochine two hydroxylases (referring to for example, Fig. 7; GenBank accession number AF368376); δ-cadinene-8-hydroxylase (referring to for example, Fig. 8 A; GenBank accession number AF332974); Japanese yew diene-5 α-hydroxylase (referring to for example, Fig. 9 A and 9B; GenBank accession number AY289209, AY959320, and AY364469); Ent-kaurene oxydase (referring to for example, Figure 10; GenBank accession number AF047719; Referring to for example, Helliwell etc. (1998) Proc.Natl.Acad.Sci.USA 95:9019-9024).

Fig. 8 B-D has described exemplary P450 variant.Fig. 8 B has described the cadinene hydroxylase that contains the allos membrane spaning domain; Fig. 8 C has described the cadinene hydroxylase that contains the solubilising structural domain; Fig. 8 C has described the cadinene hydroxylase that contains secretory structure territory and allos membrane spaning domain.Figure 22 has described other exemplary P450 variant, comprises the AMORPHADIENE oxydase that contains various N-terminal sequences.

The cytochrome P 450 enzymes of modified biological alkali approach intermediate known in the art.Referring to for example, Facchini etc. (2004) are the same; ((1998) Plant J.13:793-801 for Pauli and Kutchan; ((2001) FEBS Lett.508:215-220 such as Collu; ((1999) FEBSLett.458:97-102 such as Schroder.Also referring to Figure 19 A-C.

The cytochrome P 450 enzymes of modification phenylpropionic acid approach intermediate known in the art.Referring to for example, ((1997) Plant Physiol.113:755-763 such as Mizutani; With ((2002) Plant Physiol.130:1536-1544 such as Gang.Also referring to Figure 20 A-C.

Figure 21 A and 21B have described the cytochrome P 450 enzymes of exemplary modification polyketide approach intermediate.Also referring to ((1999) Proc.Natl.Acad.Sci.USA 96:9509-9514 such as Ikeda; With ((2004) Antimicrob.Agents Chemother.48:4703-4712 such as Ward.

The modified cytochrome P 450 enzymes (as isoprenoid precursor modifying enzyme) of coding has enzymic activity, for example, modified cytochrome P 450 enzymes (as isoprenoid precursor modifying enzyme) has one or more in the following activity: a) by following one or more reaction modified biological route of synthesis intermediates: oxidation, hydroxylation, epoxidation, dehydration, dehydrogenation, dehalogenation, isomerization, pure oxidation, formoxy-ization, dealkylation or cleavage reaction; B) modify the isoprenoid precursor by following one or more reactions: oxidation, hydroxylation, epoxidation, dehydration, dehydrogenation, dehalogenation, isomerization, pure oxidation, formoxy-ization, dealkylation or cleavage reaction.By detecting the P450 enzyme to the reaction product of substrate and/or detect the downstream product of P450 enzyme, be not difficult to determine whether the encode cytochrome P 450 enzymes of enzymic activity of nucleic acid of the present invention to substrate reactions.For example, be not difficult to adopt suitable substrates, determine nucleic acid of the present invention whether encode the terpene oxydase or the terpene hydroxylase of enzymic activity according to the standard test of these enzymic activitys.Usually pass through the product that the chromatograph-mass spectrometer coupling enzyme analysis is modified.For example, be not difficult to determine nucleic acid of the present invention whether encode sesquiterpene oxydase or sesquiterpene hydroxylase with the standard test of these enzymic activitys.Referring to for example, U.S. Patent Publication No. 20050019882.

In some embodiments, modify the nucleotide sequence of coding modified cytochrome P 450 enzymes (as the isoprenoid precursor modifying enzyme of modifying), to reflect the preferred codon of concrete host cell.For example, in some embodiments, modify this nucleotide sequence according to the preferred codon of yeast.Referring to for example, Bennetzen and Hall (1982) J.Biol.Chem.257 (6): 3026-3031.In other embodiments, another non-limitative example is to modify this nucleotide sequence according to the preferred codon of intestinal bacteria.Referring to for example, Gouy and Gautier (1982) Nucleic Acids Res.10 (22): 7055-7074; Eyre-Walker (1996) Mol.Biol.Evol.13 (6): 864-872.Also referring to (2000) Nucleic Acids Res.28 (1) such as Nakamura: 292.A non-limitative example is that Figure 11 A has described the wild-type nucleotide sequence (representing the atg initiator codon with runic) of coding cadinene hydroxylase; Figure 11 B has described the codon optimized variant of sequence shown in Figure 11 A, and wherein basis is in prokaryotic cell prokaryocyte such as expression in escherichia coli optimizing codon.

Cytochrome P450 reductase

NADPH-Cytochrome P450 oxydo-reductase (CPR, EC 1.6.2.4) is the redox partner of many P450-monooxygenases.In some embodiments, nucleic acid of the present invention also comprises the nucleotide sequence of Codocyte cytochrome p 450 reductase enzyme (CPR).The nucleic acid of the present invention that contains the nucleotide sequence of the CPR that encodes is called " CPR nucleic acid ".The CPR of theme CPR nucleic acid encoding passes to Cytochrome P450 with electronics by NADPH.For example, in some embodiments, the CPR of theme CPR nucleic acid encoding passes to the isoprenoid modifying enzymes that theme isoprenoid modifying enzymes coding nucleic acid is encoded with electronics by NADPH, as the sesquiterpene oxydase.

In some embodiments, nucleic acid of the present invention comprises the nucleotide sequence of coding modified cytochrome P 450 enzymes (as the isoprenoid precursor modifying enzyme of modifying) and CPR.In some embodiments, nucleic acid of the present invention comprises the nucleotide sequence of the fusion rotein of encoding, and this fusion rotein comprises modified cytochrome P 450 enzymes (as the isoprenoid precursor modifying enzyme of modifying) (as mentioned above) with isoprenoid precursor modification activities and the CPR amino acid sequence of polypeptide that merges.In some embodiments, the fusion rotein of coding has formula NH ₂-A-X-B-COOH, wherein A is a modified cytochrome P 450 enzymes, and X is optional joint, and B is the CPR polypeptide.In some embodiments, the fusion rotein of coding has formula NH ₂-A-X-B-COOH, wherein A is the CPR polypeptide, and X is optional joint, and B is a modified cytochrome P 450 enzymes.

The joint peptide can contain various aminoacid sequences.Protein connects by common resilient spacer peptide, does not connect but also do not get rid of other chemistry.This joint can be to cut joint.Suitable joint sequence length is generally about 5-50 amino acid, or about 6-25 amino acid.Usually adopt peptide linker with certain extent of elasticity.Connection peptides can contain any aminoacid sequence basically, should be noted that the sequence of preferred joint should produce the overall elasticity peptide.Adopt p1 amino acid such as glycine and L-Ala, can be used for producing elastomeric peptide.Those skilled in the art can create this class sequence usually.Can buy various different joints, think that they also are applicable to the present invention.

Suitable joint peptide usually comprises the aminoacid sequence that is rich in L-Ala and proline residue, and known these two kinds of residues can be given protein structure with elasticity.Exemplary joint contains the combination of glycine, L-Ala, proline(Pro) and methionine residues, as AAAGGM (SEQ ID NO:17); AAAGGMPPAAAGGM (SEQ ID NO:18); AAAGGM (SEQ ID NO:19); And PPAAAGGM (SEQ ID NO:20).Other exemplary joint peptide comprises IEGR (SEQ ID NO:21); And GGKGGK (SEQ ID NO:22).Yet, can adopt length to be about 5-50 amino acid whose any resilient connector usually.Joint can contain any sequence that can produce the overall elasticity peptide basically, comprises the sequence that is rich in L-Ala-proline(Pro) of the above-mentioned type.

In some embodiments, nucleic acid of the present invention comprises the nucleotide sequence of coding CPR polypeptide, and the CPR amino acid sequence of polypeptide homogeny of described CPR polypeptide and known or natural generation is at least about 45%, at least about 50%, at least about 55%, at least about 57%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99%.

The coding nucleic acid of CPR polypeptide and CPR polypeptide is known in this area, and any CPR coding nucleic acid or its variant all can be used for the present invention.Appropriate C PR coding nucleic acid comprises the nucleic acid of the CPR that finds in the coded plant.Appropriate C PR coding nucleic acid comprises the nucleic acid of finding CPR in the coding fungi.The example of appropriate C PR coding nucleic acid comprises: GenBank accession number AJ303373 (common wheat (Triticum aestivum) CPR); GenBank accession number AY959320 (Ramulus et folium taxi cuspidatae (Taxus chinensis) CPR); GenBank accession number AY532374 (Da A rice (Ammi majus) CPR); GenBank accession number AG211221 (paddy rice (Oryza sativa) CPR); With GenBank accession number AF024635 (parsley (Petroselinumcrispum) CPR); Oidium tropicale (Candida tropicalis) cytochrome P450 reductase (GenBank accession number M35199); Arabidopis thaliana (Arabidopsis thaliana) cytochrome P450 reductase ATR1 (GenBank accession number X66016); With arabidopsis cell cytochrome p 450 reductase enzyme ATR2 (GenBank accession number X66017); With putidaredoxin reductase enzyme and putidaredoxin (GenBank accession number J05406).

In some embodiments, nucleic acid of the present invention comprises the nucleotide sequence of the specific C PR polypeptide of the given P450 enzyme of encoding.A non-limitative example is that nucleic acid of the present invention comprises nucleotide sequence (Figure 12 A of coding taxus chinensis in northeast CPR; GenBank AY571340).Another non-limitative example is that nucleic acid of the present invention comprises the nucleotide sequence (Figure 12 B) of coding Oidium tropicale CPR.In other embodiments, nucleic acid of the present invention comprises the nucleotide sequence of CPR polypeptide that coding can be used as the redox partner of two or more different P450 enzymes.Figure 12 C has described a kind of this class CPR (arabidopsis cell cytochrome p 450 reductase enzyme ATR1).Figure 12 D has described another kind of this class CPR (arabidopsis cell cytochrome p 450 reductase enzyme ATR2).The modification shown in Figure 12 D or the ATR2 of variation also are suitable, and the ATR2 of variation lacks chloroplast targeted sequence.

In some embodiments, the CPR of coding comprises allogeneic amino acid sequence or variant amino acid sequence (as replacing, lack, insert, adding).In some embodiments, the CPR with respect to wild-type CPR coding comprises following one or more modifications: a) replace natural membrane spaning domain with the non-natural membrane spaning domain; B) replace natural membrane spaning domain with the secretion signal structural domain; C) replace natural membrane spaning domain with the solubilising structural domain; D) insert the territory with film and replace natural membrane spaning domain; E) the natural membrane spaning domain of brachymemma; And f) aminoacid sequence of the natural membrane spaning domain of change.

In some embodiments, modify the nucleotide sequence of coding CPR polypeptide, to reflect the preferred codon of concrete host cell.For example, in some embodiments, according to the preferred codon modified nucleotide sequence of yeast.Referring to for example, Bennetzen and Hall (1982) J.Biol.Chem.257 (6): 3026-3031.In other embodiments, another non-limitative example is, according to the preferred codon modified nucleotide sequence of intestinal bacteria.Referring to for example, Gouy and Gautier (1982) Nucleic Acids Res.10 (22): 7055-7074; Eyre-Walker (1996) Mol.Biol.Evol.13 (6): 864-872.Also referring to (2000) Nucleic Acids Res.28 (1) such as Nakamura: 292.

Construction

The present invention also provides the recombinant vectors that comprises nucleic acid of the present invention (" construction ").In some embodiments, the recombinant vectors of the present invention nucleic acid of the present invention that can increase.In some embodiments, recombinant vectors of the present invention can produce the isoprenoid modifying enzymes of coding or the CPR of coding in eukaryotic cell, prokaryotic cell prokaryocyte or cell-free transcription/translation system.Suitable expression vector includes but not limited to: baculovirus vector, phage vector, plasmid, phagemid, clay, F clay (fosmid), bacterial artificial chromosome, virus vector is (as based on vaccinia virus, poliovirus, adenovirus, adeno associated virus, SV40, the virus vector of hsv etc.), artificial chromosome based on P1, yeast plasmid, the yeast artificial chromosome and to interested specific host (as intestinal bacteria (E.coli), yeast and vegetable cell) special any other carrier.

In some embodiments, recombinant vectors of the present invention comprises the Cytochrome P450 coding nucleic acid and the CPR coding nucleic acid of the present invention of modification of the present invention.In some embodiments, recombinant vectors of the present invention is the expression vector of modified cytochrome P 450 enzymes (as the isoprenoid modifying enzymes of modifying) that can produce coding in eukaryotic cell, prokaryotic cell prokaryocyte or cell-free transcription/translation system and the CPR that encodes.

Some bearer type expression cassette of the present invention that can increase.Nucleic acid of the present invention effectively introduced cell and introduce after expression need other bearer type.Can accept any carrier of nucleic acid of the present invention all can consider as recombinant vectors of the present invention.Carrier can be ring-type or the linear DNA that is incorporated in the host genome or keeps the episome form.Carrier may need additional operations or actual conditions could effectively mix (so multilist reaches plasmid) in the host cell, perhaps can be the part (as recombinant virus) of the cell-specific system that self integrates.In some embodiments, carrier has function in prokaryotic cell prokaryocyte, and this carrier is used to breed recombinant vectors and/or expresses nucleic acid of the present invention.In some embodiments, carrier has function in eukaryotic cell, and this in many cases carrier is an expression vector.

The known many suitable expression vector of those skilled in the art, many being commercially available.Provide following carrier with way of example; Be used for bacterial host cell: pBluescript (department in San Diego, California looks into column foot company (Stratagene)), pQE carrier (Kai Jie company (Qiagen)), pBluescript plasmid, pNH carrier, λ-ZAP vector (department looks into column foot company); PTrc (Amann etc., Gene, 69:301-315 (1988)); PTrc99a, pKK223-3, pDR540 and pRIT2T (Pharmacia Corp (Pharmacia)); Be used for eukaryotic host cell: pXT1, pSG5 (department looks into column foot company), pSVK3, pBPV, pMSG and pSVLSV40 (Pharmacia Corp).Yet, can adopt any other plasmid or other carrier, as long as it is compatible with host cell.In embodiment, pSP19g10L expresses in prokaryotic host cell with plasmid vector.In other embodiment, pCWori expresses in prokaryotic host cell with plasmid vector.The description of pSP19g10L and pCWori is referring to for example, Barnes ((1996) Methods Enzymol.272:1-14).

In many embodiments, nucleic acid of the present invention comprises the nucleotide sequence of the isoprenoid modifying enzymes of encoding, and the nucleotide sequence operability of the isoprenoid modifying enzymes of wherein encoding is connected in one or more and transcribes and/or translate controlling elements.In many embodiments, nucleic acid of the present invention comprises the nucleotide sequence of the CPR that encodes, and the nucleotide sequence operability of the CPR that wherein encodes is connected in one or more and transcribes and/or translate controlling elements.

In some embodiments, as mentioned above, recombinant vectors of the present invention comprises isoprenoid modifying enzymes-coding nucleic acid of the present invention and CPR-coding nucleic acid of the present invention.In some embodiments, the isoprenoid modifying enzymes nucleotide sequence of coding is connected in different transcriptional control elements with the nucleotide sequence operability of coding CPR.In other embodiments, the nucleotide sequence operability of the nucleotide sequence of coding isoprenoid modifying enzymes and coding CPR is connected in same transcriptional control element.In some embodiments, the equal operability of nucleotide sequence of the nucleotide sequence of coding isoprenoid modifying enzymes and coding CPR is connected in same inducible promoter.In some embodiments, the equal operability of nucleotide sequence of the nucleotide sequence of coding isoprenoid modifying enzymes and coding CPR is connected in same constitutive promoter.

The promotor that is applicable to prokaryotic host cell includes but not limited to: phage t7 rna polymerase promoter; The trp promotor; Lac operon promotor; Hybrid promoter is as lac/tac hybrid promoter, tac/trc hybrid promoter, trp/lac promotor, T7/lac promotor; The trc promotor; Tac promotor etc.; The araBAD promotor; Regulate promotor in the body, as ssaG promotor or promoter related (referring to for example, U.S. Patent Publication No. 20040131637), pagC promotor (Pulkkinen and Miller, J.Bacteriol., 1991:173 (1): 86-93; Alpuche-Aranda etc., PNAS, 1992; 89 (21): 10079-83), nirB promotor (Harborne etc. (1992) Mol.Micro.6:2805-2813) etc. is (referring to (1999) Infect.Immun.67:5133-5141 such as for example Dunstan; McKelvie etc. (2004) Vaccine22:3243-3255; With (1992) Biotechnol.10:888-892 such as Chatfield); σ 70 promotors are as total σ 70 promotors (referring to for example, GenBank accession number AX798980, AX798961 and AX798183); Stationary phase promotor, as dps promotor, spv promotor etc.; Promotor (referring to for example WO96/17951) derived from pathogenicity island SPI-2; ActA promotor (referring to for example, Shetron-Rama etc. (2002) Infect.Immun.70:1087-1096); The rpsM promotor (referring to for example, Valdivia and Falkow (1996), Mol.Microbiol.22:367-378); The tet promotor is (referring to for example, Hillen, W. and Wissmann, A. (1989), publish in Saenger, W. and Heinemann, the Topics inMolecular and Structural Biology that U compiled, Protein-Nucleic Acid Interaction (molecule and structure biology special topic, protein-nucleic acid interaction), London mcmillan (Macmillan), the 10th volume, 143-162 page or leaf); SP6 promotor (referring to for example, Melton etc. (1984) Nucl.Acids Res.12:7035-7056); Or the like.

The non-limitative example of suitable eukaryotic promoter comprises that CMV is early stage immediately, HSV thymidine kinase, early stage and late period SV40, from retroviral LTR and mouse metallothionein(MT)-I.In some embodiments, when for example expressing in yeast cell, suitable promotor is constitutive promoter such as ADH1 promotor, PGK1 promotor, ENO promotor, PYK1 promotor etc.; Perhaps adjustment type promotor such as GAL1 promotor, GAL10 promotor, ADH2 promotor, PHO5 promotor, CUP1 promotor, GAL7 promotor, MET25 promotor, MET3 promotor etc.Those of ordinary skills can select suitable carriers and promotor.Expression vector also can contain ribosome bind site and the transcription terminator that is useful on the startup translation.Expression vector also can contain the proper sequence that enlarges expression.

In many embodiments, recombinant vectors of the present invention contains one or more selected markers, to be provided for selecting the phenotypic characteristic of transformed host cells.Suitable selected marker includes but not limited to: Tetrahydrofolate dehydrogenase, neomycin resistance during eukaryotic cell is cultivated; With tsiklomitsin or the amicillin resistance in prokaryotic host cell such as the intestinal bacteria.

Usually, recombinant expression vector will comprise can transformed host cell replication orgin and selected marker, as colibacillary ampicillin resistance gene, yeast saccharomyces cerevisiae (S.cerevisiae) TRP1 gene etc.; With the promotor that is used to instruct encoding sequence to transcribe derived from cance high-expression gene.This class promotor can be derived from the operon of coding glycolytic ferment such as glycerol 3-phosphate acid kinase (PGK), α-factor, acid phosphatase or heat shock protein etc.

In many embodiments, the nucleotide sequence operability of coding modified cytochrome P 450 enzymes (as the isoprenoid modifying enzymes of modifying) is connected in inducible promoter.In many embodiments, the nucleotide sequence operability of coding CPR is connected in inducible promoter.Well known inducible promoter.Suitable inducible promoter includes but not limited to: the pL of lambda particles phage; Plac; Ptrp; Ptac (Ptrp-lac hybrid promoter); Sec.-propyl-β-D-sulfo-galactopyranoside (IPTG)-inducible promoter is as the lacZ promotor; Tsiklomitsin-inducible promoter; The pectinose inducible promoter is as P _BAD(referring to for example, Guzman etc. (1995) J.Bacteriol.177:4121-4130); The wood sugar inducible promoter is as Pxyl (referring to for example, Kim etc. (1996) Gene 181:71-76); The GAL1 promotor; Trp promoter; The lac promotor; Alcohol-induced type promotor is as methanol inducible promoters, ethanol-inducible promoter; Raffinose-inducible promoter; Thermal induction type promotor is as thermal induction type λ P _LPromotor, (the λ base table of preventing as CI857-reaches carrier by the promotor of temperature-sensitive repressor control; Referring to for example, Hoffmann etc. (1999) FEMS Microbiol Lett.177 (2): 327-34); Or the like.

In yeast, can adopt the many carriers that contain composing type or inducible promoter.Summary is referring to Current Protocols in Molecular Biology (newly organized molecular biology experiment guide), and the 2nd rolls up, and 1988, volumes such as Ausubel, Greene Publish.Assoc.﹠amp; Wiley Interscience (Green publishes associating group and Webster Science Press), the 13rd chapter; Grant etc., 1987, Expression and SecretionVector for Yeast (being used for zymic expresses and secretion vector), Methods in Enzymology (Enzymology method), Wu and Grossman compile, and 31987, academic press (Acad.Press), New York, the 153rd volume, 516-544 page or leaf; Glover, 1986, DNA Cloning (dna clone), II volume, IRL press, Washington D.C., the 3rd chapter; And Bitter, 1987, Heterologous Gene Expressionin Yeast (allogeneic gene expression in the yeast), Methods in Enzymology (Enzymology method), Berger and Kimmel compile, the academic press, New York, the 152nd volume, 673-684 page or leaf; With TheMolecular Biology of the Yeast Saccharomyces (molecular biology of yeast saccharomyces cerevisiae), 1982, volumes such as Strathern, cold spring port press, I and II volume.Can adopt composing type Yeast promoter such as ADH or LEU2 or inducible promoter such as GAL (Cloning in Yeast (in yeast, cloning), the 3rd chapter, R.Rothstein, publish in DNA Cloning (dna clone) the 11st volume, A PracticalApproach (hands-on approach), DM Glover compiles, and 1986, IRL press, the Washington D.C.).Perhaps, can adopt and to promote the foreign DNA sequence to be incorporated into the carrier in the yeast chromosomal.

In some embodiments, nucleic acid of the present invention or carrier of the present invention comprise and are used at vegetable cell expression promoter or other controlling element.The non-limitative example that the suitable constitutive promoter of function is arranged in vegetable cell is cauliflower mosaic virus 35S promoter, series connection 35S promoter (Kay etc., Science236:1299 (1987)), cauliflower mosaic virus 19S promotor, nopaline synthase gene promotor (Singer etc., PlantMol.Biol.14:433 (1990); An, PlantPhysiol.81:86 (1986), octopine synthase gene promotor and ubiquitin promoter.In vegetable cell, there is the suitable inducible promoter of function to include but not limited to: phenylalanine ammonia-lyase genes promotor, chalcone synthase gene promoter, pathology related protein gene promotor, copper-inducibility controlling element (Mett etc., Proc.Natl.Acad.Sci.USA90:4567-4571 (1993); Furst etc., Cell 55:705-717 (1988)); (Gatz etc., Plant are (1992) J.2:397-404 for tsiklomitsin and Uromycin-inducibility controlling element;

Deng, Mol.Gen.Genet.243:32-38 (1994); Gatz, Meth.Cell Biol.50:411-424 (1995)); Moulting hormone inducibility controlling element (Christopherson etc., Proc.Natl.Acad.Sci.USA 89:6314-6318 (1992); Kreutzweiser etc., Ecotoxicol.Environ.Safety 28:14-24 (1994)); Heat-inducible controlling element (Takahashi etc., Plant Physiol.99:383-390 (1992); Yabe etc., PlantCell Physiol.35:1207-1219 (1994); Ueda etc., Mol.Gen.Genet.250:533-539 (1996)); With lac operon element, (Wilde etc., EMBO are (1992) J.11:1251-1259 to produce (for example) IPTG-inducible expression in they and constructive expression's lac repressor coupling; Nitric acid-the inducible promoter of spinach nitrite reductase gene (Back etc., Plant Mol.Biol.17:9 (1991)); The photoinduction promotor is as the promotor relevant with the small subunit of RuBP carboxylase or LHCP gene family (Feinbaum etc., Mol.Gen.Genet.226:449 (1991); Lam and Chua, Science 248:471 (1990)); As U.S. Patent Publication No. 20040038400 described light-reactive controlling element; Induced by Salicylic Acid controlling element (Uknes etc., Plant Cell 5:159-169 (1993); Bi etc., Plant are (1995) J.8:235-245); Plant hormone induction type controlling element (Yamaguchi-Shinozaki etc., PlantMol.Biol.15:905 (1990); Kares etc., Plant Mol.Biol.15:225 (1990)); With people's hormone-induction type controlling element such as people's glucocorticosteroid response element (Schena etc., Proc.Natl.Acad.Sci.USA 88:10421 (1991).

Also can comprise plant tissue selective regulation element in nucleic acid of the present invention or the carrier of the present invention.The tissue selectivity controlling element that is applicable to ectopic expression nucleic acid in the tissue of a kind of tissue or limited quantity includes but not limited to: xylem-selective regulation element, test-tube baby-selective regulation element, fiber-selective regulation element, trichome-selective regulation element (referring to for example, Wang etc. (2002) J.Exp.Botany53:1891-1897), glandular hairs-selective regulation element etc.

The carrier that is applicable to vegetable cell known in the art, any this carrier all can be used for nucleic acid introduced plant host cell of the present invention.Suitable carrier for example comprises: the Ri of Ti-plasmids of Agrobacterium tumefaciens (Agrobacteriumtumefaciens) or Agrobacterium rhizogenes (A.rhizogenes) ₁Plasmid.After infecting edaphic bacillus (Agrobacterium), Ti or Ri ₁Plasmid is transported in the vegetable cell, and stable integration is in Plant Genome.J.Schell，Science，237：1176-83(1987)。The plant artificial chromosome in addition that is fit to adopt, as U.S. Patent number 6,900,012 is described.

Composition

The present invention also provides the composition that contains nucleic acid of the present invention.The present invention also provides the composition that contains recombinant vectors of the present invention.In many embodiments, the composition that contains nucleic acid of the present invention or expression vector of the present invention comprises following one or more materials: salt, and as NaCl, MgCl, KCl, MgSO ₄Deng; Buffer reagent, as Tris damping fluid, N-(2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino) ethyl sulfonic acid (MES), 2-(N-morpholino) ethyl sulfonic acid sodium (MES), 3-(N-morpholino) propanesulfonic acid (MOPS), N-three [methylol] methyl-3-aminopropanesulfonicacid acid (TAPS), etc.; Solubilizing agent; Stain remover is as non-ionic detergent such as tween 20 etc.; Nucleic acid inhibitor; Or the like.In some embodiments, freeze-drying nucleic acid of the present invention or recombinant vectors of the present invention.

Host cell

The invention provides the host cell of genetic modification, as host cell with nucleic acid of the present invention or recombinant vectors genetic modification of the present invention.In many embodiments, the host cell of genetic modification of the present invention is external host cell.In other embodiments, the host cell of genetic modification of the present invention is a host cell in the body.In other embodiments, the host cell of genetic modification of the present invention is the part of multicellular organisms.

In many embodiments, host cell is a unicellular organism, or as unicellular vitro culture.In some embodiments, host cell is an eukaryotic cell.Suitable eukaryotic host cell includes but not limited to: yeast cell, insect cell, vegetable cell, fungal cell and alga cells.Suitable eukaryotic host cell includes but not limited to: pichia pastoris phaff (Pichia pastoris), Finland's pichia spp (Pichiafinlandica), marine alga pichia spp (Pichia trehalophila), Ke Lan pichia spp (Pichia koclamae), Pichia membranaefaciens (Pichia membranaefaciens), general pichia spp (Pichia opuntiae) difficult to understand, heat-resisting pichia spp (Pichia thermotolerans), Sa Li pichia spp (Pichia salictaria), Ge Er pichia spp (Pichia guercuum), Pi Shi pichia spp (Pichia pijperi), pichia stipitis (Pichiastiptis), pichia methanolica (Pichia methanolica), pichia spp (Pichia sp.), yeast saccharomyces cerevisiae (Saccharomyces cereviseae), yeast (Saccharomyces sp.), multiple-shaped nuohan inferior yeast (Hansenulapolymorpha), kluyveromyces (Kluyveromyces sp.), lactose kluyveromyces (Kluyveromyceslactis), Candida albicans (Candida albicans), Aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), wood mould (Trichoderma reesei), Chrysosporium lucknowense, sickle-like bacteria (Fusarium sp.), Fusarium graminearum (Fusariumgramineum), fusarium (Fusarium venenatum), coarse chain born of the same parents bacterium (Neurospora crassa), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) etc.In some embodiments, host cell is the eukaryotic cell except that vegetable cell.

In other embodiments, host cell is a vegetable cell.Vegetable cell comprises the cell of monocotyledons and dicotyledons.

In other embodiments, host cell is a prokaryotic cell prokaryocyte.Suitable prokaryotic cell prokaryocyte includes but not limited to: the various laboratory strains of intestinal bacteria (Escherichia coli), Bacterium lacticum (Lactobacillus sp.), salmonella (Salmonella sp.), shigella (Shigella sp.) etc.Referring to for example, Carrier etc. (1992) J.Immunol.148:1176-1181; U.S. Patent number 6,447,784; With (1995) Science270:299-302 such as Sizemore.The example that can be used for salmonella bacterial strain of the present invention includes but not limited to: Salmonella typhi (Salmonella typhi) and Salmonella typhimurtum (S.typhimurium).Suitable shigella bacterial strain includes but not limited to: not shigella (Shigella flexneri), shigella sonnei (Shigella sonnei) and the gloomy shigella of enlightening (Shigella disenteriae).Usually, described laboratory strains is a non-pathogenic bacterial strains.The non-limitative example of other suitable bacteria includes but not limited to: subtilis (Bacillus subtilis), general pseudomonas (Pseudomonas pudita), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonasmevalonii, the red bacterium of class ball (Rhodobacter sphaeroides), the red bacterium of pod membrane (Rhodobactercapsulatus), Crimson rhodospirillum (Rhodospirillum rubrum), Rhod (Rhodococcus sp.) etc.In some embodiments, host cell is intestinal bacteria.

In order to produce the host cell of genetic modification of the present invention, to comprise the nucleic acid stability of the present invention or instantaneous the introducing in the parental generation host cell of the nucleotide sequence of coding modified cytochrome P 450 enzymes (as the isoprenoid modifying enzymes of modifying) with the technology of having set up, these technology include but not limited to: electroporation, calcium phosphate precipitation, the transfection of DEAE-dextran mediation, liposome-mediated transfection etc.In stable conversion, nucleic acid generally also comprises the selected marker thing, for example several selected marker things of knowing such as neomycin resistance, amicillin resistance, tetracyclin resistance, chlorampenicol resistant, kalamycin resistance etc.

In some embodiments, the host cell of genetic modification of the present invention is a vegetable cell.The vegetable cell of genetic modification of the present invention is used in and produces the isoprenoid compound of selecting in the external plant cell cultures.Can be about the guide of plant tissue culture referring to (for example): Plant Cell and Tissue Culture (vegetable cell and tissue culture), 1994, Vasil and Thorpe compile, Crewe dimension academic publishing group (Kluwer AcademicPublishers); With Plant Cell Culture Protocols (culture plant cell method) (Methods inMolecular Biology (molecular biology method) 111), 1999, Hall compiles, Xiu Man press (HumanaPress).

The host cell of genetic modification

In some embodiments, the host cell of genetic modification of the present invention comprises expression vector of the present invention, and described expression vector of the present invention comprises the nucleotide sequence of the modified cytochrome P 450 enzymes of encoding.In some embodiments, the host cell of genetic modification of the present invention comprises expression vector of the present invention, and described expression vector of the present invention comprises the nucleotide sequence of the isoprenoid precursor modifying enzyme of coding modification.

In some embodiments, the host cell of genetic modification of the present invention comprises first kind of expression vector of the present invention and second kind of expression vector of the present invention, and the nucleic acid of the present invention that described first kind of expression vector of the present invention comprises contains the nucleotide sequence of the modified cytochrome P 450 enzymes of encoding; The nucleic acid of the present invention that described second kind of expression vector of the present invention comprises contains the nucleotide sequence of the CPR that encodes.In other embodiments, the host cell of genetic modification of the present invention comprises expression vector of the present invention, and wherein said expression vector of the present invention comprises the nucleic acid of the present invention of the nucleotide sequence that contains the modified cytochrome P 450 enzymes of encoding and contains the nucleic acid of the present invention of the nucleotide sequence of the CPR that encodes.In other embodiments, the host cell of genetic modification of the present invention comprises expression vector of the present invention, and the nucleic acid of the present invention that described expression vector of the present invention comprises contains the nucleotide sequence of coding fusion polypeptide (as comprise modified cytochrome P 450 enzymes and CPR polypeptide).

In some embodiments, the host cell of genetic modification of the present invention comprises first expression vector and second expression vector, and described first expression vector comprises the nucleic acid of the present invention of the nucleotide sequence that contains the modified cytochrome P 450 enzymes of encoding; Described second expression vector comprises the nucleotide sequence of the CPR that encodes.In other embodiments, the host cell of genetic modification of the present invention comprises the theme expression vector, and wherein said theme expression vector comprises the nucleic acid of the present invention of nucleotide sequence that contains the modified cytochrome P 450 enzymes of encoding and the nucleotide sequence of encoding CPR.

In some embodiments, further the host cell of genetic modification genetic modification of the present invention makes it comprise one or more nucleic acid of the nucleotide sequence of one or more enzymes that contain coding generation cytochrome P 450 enzymes substrate.This zymoid example includes but not limited to: diterpene synthase; Prenyltransferase; Bisphosphate isoamyl-1-alkene ester isomerase; One or more enzymes in the mevalonate pathway; With one or more enzymes in the DXP approach.In some embodiments, the further host cell of genetic modification genetic modification of the present invention makes it comprise one or more nucleic acid that contain a kind of among coding diterpene synthase, prenyltransferase, IPP isomerase, acetoacetyl CoA thiolase, HMGS, HMGR, MK, PMK and the MPD, two kinds, three kinds, four kinds, five kinds, six kinds, seven kinds or eight kinds or more kinds of nucleotide sequence.In some embodiments, for example, host cell when further genetic modification genetic modification of the present invention, when making it comprise one or more nucleic acid that contain two or more nucleotide sequence among coding diterpene synthase, prenyltransferase, IPP isomerase, Acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK and the MPD, nucleotide sequence is present at least two operons, in two isolating operons, three isolating operons or four isolating operons.

Diterpene synthase

In some embodiments, the host cell of further genetic modification genetic modification of the present invention is to comprise the nucleic acid of the nucleotide sequence that contains the diterpene synthase of encoding.In some embodiments, diterpene synthase is to modify the diterpene synthase that FPP produces sesquiterpene.In other embodiments, diterpene synthase is to modify the diterpene synthase that GPP produces monoterpene.In other embodiments, diterpene synthase is to modify the diterpene synthase that GGPP produces diterpene.

Diterpene synthase acts on many isopentene of bisphosphate ester substrate, modify many isopentene of bisphosphate ester substrate by cyclisation, rearrangement or coupling, produce isoprenoid precursor (such as limonene, AMORPHADIENE, Japanese yew diene etc.), this class isoprenoid precursor is the substrate of isoprenoid precursor modifying enzyme.By the effect of diterpene synthase, produce the substrate of isoprenoid precursor modifying enzyme to many isopentene of bisphosphate ester substrate.

The nucleotide sequence of coding diterpene synthase known in the art, any known nucleotide sequence genetic modification host cell of available code diterpene synthase.For example, known and can adopt the nucleotide sequence (back is their GenBank accession number and identifies their biology) of following coding diterpene synthase: (-)-germacrene D synthase mRNA (AY438099; Populus simonii comospore subspecies (Populus balsamifera subsp.trichocarpa) * triangle poplar (Populus deltoids)); E, E-α-farnesene synthase mRNA (AY640154; Cucumber (Cucumissativus)); 1,8-cineole synthase mRNA (AY691947; Arabidopis thaliana (Arabidopsis thaliana)); Diterpene synthase 5 (TPS5) mRNA (AY518314; Corn (Zea mays)); Diterpene synthase 4 (TPS4) mRNA (AY518312; Corn); Myrcene/ocimene synthase (TPS10) (At2g24210) mRNA (NM 127982; Arabidopis thaliana); Mang geraniol synthase (GES) mRNA (AY362553; Sweet basil (Ocimum basilicum)); Firpene synthase mRNA (AY237645; Picea sitchensis); Myrcene synthase 1e20mRNA (AY195609; Common Snapdragon (Antirrhinumm majus)); (E)-β-ocimene synthase (0e23) mRNA (AY195607; Common Snapdragon); E-β-ocimene synthase mRNA (AY151086; Common Snapdragon); Diterpene synthase mRNA (AF497492; Arabidopis thaliana); (-)-amphene synthase (AG6.5) mRNA (U87910; Abies grandis (Abies grandis)); (-)-4S-limonene synthase gene (as genome sequence) (AF326518; Abies grandis); δ-selinene synthase gene (AF326513; Abies grandis); False indigo-4,11-diene synthase mRNA (AJ251751; Herba Artemisiae annuae (Artemisia annua)); E-α-bisabolene synthase mRNA (AF006195; Abies grandis); γ-Humuleno synthase mRNA (U92267; Abies grandis); δ-selinene synthase mRNA (U92266; Abies grandis); Firpene synthase (AG3.18) mRNA (U87909; Abies grandis); Myrcene synthase (AG2.2) mRNA (U87908; Abies grandis) etc.

Mevalonate pathway

In some embodiments, the host cell of genetic modification of the present invention is the host cell that does not synthesize tetra-sodium isoamyl-1-alkene ester (IPP) or mevalonic acid usually by mevalonate pathway.Mevalonate pathway comprises: (a) acetyl-CoA with two molecules is condensed into acetoacetyl-CoA; (b) make acetoacetyl-CoA and acetyl-CoA condensation form HMG-CoA; (c) HMG-CoA is converted into mevalonic acid; (d) mevalonic acid phosphoric acid is turned to mevalonic acid 5-phosphoric acid; (e) mevalonic acid 5-phosphoric acid is converted into mevalonic acid 5-tetra-sodium; (f) mevalonic acid 5-tetra-sodium is converted into tetra-sodium isoamyl-1-alkene ester.Producing the required mevalonate pathway enzyme of IPP may become because of culture condition.

As mentioned above, in some embodiments, the host cell of genetic modification of the present invention is the host cell that does not synthesize tetra-sodium isoamyl-1-alkene ester (IPP) or mevalonic acid usually by mevalonate pathway.In some embodiments, with expression vector genetic modification host cell of the present invention, described expression vector of the present invention comprises the nucleic acid of the present invention of the isoprenoid modifying enzymes of encoding; And with one or more heterologous nucleic acids genetic modification host cells, described heterologous nucleic acids comprises the nucleotide sequence of coding Acetoacetyl-CoA thiolase, hydroxymethyl glutaryl base-CoA synthase (HMGS), hydroxymethyl glutaryl base-CoA reductase enzyme (HMGR), Mevalonic kinase (MK), Phosphomevalonic kinase (PMK) and mevalonic acid tetra-sodium decarboxylase (MPD) (also having optional IPP isomerase).In many these class embodiments, with expression vector genetic modification host cell, described expression vector comprises the nucleotide sequence of the CPR that encodes.In some embodiments, with expression vector genetic modification host cell of the present invention, described expression vector of the present invention comprises the nucleic acid of the present invention of the isoprenoid modifying enzymes of encoding; With one or more heterologous nucleic acids genetic modification host cells, described heterologous nucleic acids comprises the nucleotide sequence of coding MK, PMK, MPD (also having optional IPP isomerase).In many these class embodiments, with expression vector genetic modification host cell, described expression vector comprises the nucleotide sequence of the CPR that encodes.

In some embodiments, the host cell of genetic modification of the present invention is the host cell that does not synthesize IPP or mevalonic acid usually by mevalonate pathway; With expression vector genetic modification host cell of the present invention, described expression vector of the present invention comprises the nucleic acid of the present invention of the isoprenoid modifying enzymes of encoding; And with one or more heterologous nucleic acids genetic modification host cells, described heterologous nucleic acids comprises the nucleotide sequence of coding Acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, IPP isomerase and prenyltransferase.In many these class embodiments, with expression vector genetic modification host cell, described expression vector comprises the nucleotide sequence of the CPR that encodes.In some embodiments, the host cell of genetic modification of the present invention is the host cell that does not synthesize IPP or mevalonic acid usually by mevalonate pathway; With expression vector genetic modification host cell of the present invention, described expression vector of the present invention comprises the nucleic acid of the present invention of the isoprenoid modifying enzymes of encoding; And with one or more heterologous nucleic acids genetic modification host cells, described heterologous nucleic acids comprises the nucleotide sequence of coding MK, PMK, MPD, IPP isomerase and prenyltransferase.In many these class embodiments, with expression vector genetic modification host cell, described expression vector comprises the nucleotide sequence of the CPR that encodes.

In some embodiments, the host cell of genetic modification of the present invention is the host cell that synthesizes IPP or mevalonic acid usually by mevalonate pathway, and for example, host cell is the host cell that comprises the endogenous mevalonate pathway.In some embodiments, host cell is a yeast cell.In some embodiments, host cell is a yeast saccharomyces cerevisiae.

In some embodiments, also use the host cell of one or more nucleic acid genetic modifications genetic modification of the present invention, described nucleic acid comprises the nucleotide sequence of the desaturase of encoding, and described desaturase is further modified the isoprenoid compound.The desaturase of described coding can be the desaturase of natural discovery in prokaryotic cell prokaryocyte or eukaryotic cell, perhaps can be the variant of this desaturase.In some embodiments, the invention provides the isolating nucleic acid of the nucleotide sequence of this desaturase of coding.

Mevalonate pathway nucleic acid

The nucleotide sequence of coding known in the art MEV pathway gene product can adopt the nucleotide sequence of any known coding MEV pathway gene product, to produce the host cell of genetic modification of the present invention.For example, the nucleotide sequence of coding Acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD and IDI is known in the art.Below be the non-limitative example of the known nucleotide sequence of coding MEV pathway gene product, the GenBank accession number and the organism of each MEV path enzyme of the content representation in the bracket of back: Acetoacetyl-CoA thiolase: (NC_000913 district: 2324131..2325315; Intestinal bacteria), (D49362; Paracoccus denitrificans (Paracoccus denitrificans)) and (L20428; Yeast saccharomyces cerevisiae); HMGS:(NC_001145 complement 19061..20536; Yeast saccharomyces cerevisiae), (X96617; Yeast saccharomyces cerevisiae), (X83882; Arabidopis thaliana), (AB037907; Ash kitasatosporia (Kitasatospora griseola)) and (BT007302; Homo sapiens (Homo sapiens)); HMGR:(NM_206548; Drosophila melanogaster (Drosophilamelanogaster)), (NM_204485; Hongyuan chicken (Gallus gallus)), (AB015627; Streptomycete (Streptomyces sp.) KO-3988), (AF542543; Tobacco (Nicotiana attenuata)), (AB037907; Ash kitasatosporia (Kitasatospora griseola)), (AX128213 provides the sequence of the HMGR of coding brachymemma; Yeast saccharomyces cerevisiae) and (NC_001145: complement (115734..118898; Yeast saccharomyces cerevisiae)); MK:(L77688; Arabidopis thaliana) and (X55875; Yeast saccharomyces cerevisiae); PMK:(AF429385; Para rubber tree (Hevea brasiliensis)), (NM_006556; The homo sapiens), (NC_001145, complement 712315..713670; Yeast saccharomyces cerevisiae); MPD:(X97557; Yeast saccharomyces cerevisiae), (AF290095; Faecium (Enterococcus faecium)) and (U49260; The homo sapiens); And IDI:(NC_000913,3031087..3031635; Intestinal bacteria) and (AF082326; Haematocoocus Pluvialls (Haematococcuspluvialis)).

In some embodiments, HMGR coding region coding lacks the HMGR (" tHMGR ") of clipped form of the membrane spaning domain of wild-type HMGR.The membrane spaning domain of HMGR contains the adjusting part of this enzyme, but does not have catalytic activity.

The encoding sequence that can variety of way known in the art changes any known MEV path enzyme changes the aminoacid sequence of codase with target.The amino acid of variation MEV path enzyme is similar substantially to the aminoacid sequence of any known MEV path enzyme usually, be that its difference is at least one amino acid, difference may be at least two, at least 5, at least 10 or at least 20 amino acid, but generally is no more than about 50 amino acid.It can be to replace, insert or disappearance that sequence changes.For example, as described below, can sexually revise nucleotide sequence according to the codon bias of concrete host cell.In addition, can introduce one or more nucleotide sequence differences that proteins encoded generation conservative amino acid is changed.

Prenyltransferase

In some embodiments, the host cell of genetic modification genetic modification of the present invention is to comprise the nucleic acid of the nucleotide sequence that contains the isoprenoid modifying enzymes of encoding; In some embodiments, also by genetic modification to comprise one or more nucleic acid (as mentioned above) of the nucleotide sequence that contains one or more mevalonate pathway enzymes of encoding; Nucleic acid with the nucleotide sequence of sequences encoding isopentenyl transferase.

Prenyltransferase has constituted the big fermentoid that catalysis IPP continuous condensating causes forming the bisphosphate isopentene ester of various chain lengths.Suitable prenyltransferase comprises that catalysis IPP and the condensation of allyl group initial substrate form the enzyme of isoprenoid compound, described isoprenoid compound has an isopentene unit or more, about 2 isopentene unit-6000, for example, 2 isopentene unit (tetra-sodium Mang ox ester synthase), 3 isopentene unit (farnesyl pyrophosphate synthase), 4 isopentene unit (tetra-sodium geranyl Mang ox ester synthase), 5 isopentene unit, 6 isopentene unit (tetra-sodium hexadecyl ester synthase), 7 isopentene unit, 8 isopentene unit (phytoene synthases, tetra-sodium eight isopentene ester synthases), 9 isopentene unit (tetra-sodium nine isopentene ester synthases), 10 isopentene unit (tetra-sodium ten isopentene ester synthases), an isopentene unit, about 10 isopentene unit-15, an isopentene unit, about 15 isopentene unit-20, an isopentene unit, about 20 isopentene unit-25, an isopentene unit, about 25 isopentene unit-30, an isopentene unit, about 30 isopentene unit-40, an isopentene unit, about 40 isopentene unit-50, an isopentene unit, about 50 isopentene unit-100, an isopentene unit, about 100 isopentene unit-250, an isopentene unit, about 250 isopentene unit-500, an isopentene unit, about 500 isopentene unit-1000, an isopentene unit, about 1000 isopentene unit-2000, an isopentene unit, about 2000 isopentene unit-3000, an isopentene unit, about 3000 isopentene unit-4000, an isopentene unit, about 4000 isopentene unit-5000, an or isopentene unit or more, about 5000 isopentene unit-6000.

Suitable prenyltransferase includes but not limited to: bisphosphate E-isopentene ester synthase includes but not limited to: bisphosphate Mang ox ester (GPP) synthase, bisphosphate method ester (FPP) synthase, bisphosphate geranyl Mang ox ester (GGPP) synthase, bisphosphate six isopentene ester (HexPP) synthase, bisphosphate seven isopentene ester (HepPP) synthase, bisphosphate eight isopentene ester (OPP) synthase, bisphosphate eggplant ester (SPP) synthase, bisphosphate ten isopentene ester (DPP) synthase, tuno gum synthase and gutta-percha synthase; And bisphosphate Z-isopentene ester synthase, include but not limited to: bisphosphate nine isopentene ester (NPP) synthase, bisphosphate 11 isopentene ester (UPP) synthase, bisphosphate dehydrogenation polyterpene ester (dehydrodolichyl diphosphate) synthase, bisphosphate 20 isopentene ester synthases, natural rubber synthase and other bisphosphate Z-isopentene ester synthase.

The nucleotide sequence of the multiple prenyltransferase of known various species, can be used for or modify after be used to produce the host cell of described genetic modification.The nucleotide sequence of sequences encoding isopentenyl transferase known in the art.Referring to for example, people's farnesyl pyrophosphate synthetic enzyme mRNA (GenBank accession number J05262; The homo sapiens); Bisphosphate method ester synthetic enzyme (FPP) gene (GenBank accession number J05091; Yeast saccharomyces cerevisiae); Bisphosphate list isopentene ester: bisphosphate dimethyl allyl ester isomerase gene (J05090; Yeast saccharomyces cerevisiae); Wang and Ohnuma (2000) Biochim.Biophys.Acta 1529:33-48; U.S. Patent number 6,645,747; Arabidopis thaliana farnesyl pyrophosphate synthetic enzyme 2 (FPS2)/FPP synthetic enzyme 2/ bisphosphate method ester synthase 2 (At4g17190) mRNA (GenBank accession number NM_202836); Ginkgo (Ginkgo biloba) bisphosphate geranyl Mang ox ester synthase (ggpps) mRNA (GenBank accession number AY371321); Arabidopis thaliana tetra-sodium geranyl Mang ox ester synthase (GGPS1)/GGPP synthetic enzyme/farnesyl tranfering enzyme (At4g36810) mRNA (GenBank accession number NM_119845); The bisphosphate farnesyl of elongated synechococcus (Synechococcuselongatus), geranyl geranyl, geranyl farnesyl, six isopentene groups, seven isopentene ester synthase genes (SelF-HepPS) (GenBank accession number AB016095); Deng.

Codon uses

In some embodiments, modify the nucleotide sequence of the host cell be used to produce genetic modification of the present invention, so that this nucleotide sequence reflects the preferred codon of concrete host cell.For example, in some embodiments, according to the preferred codon modified nucleotide sequence of yeast.Referring to for example, Bennetzen and Hall (1982) J.Biol.Chem.257 (6): 3026-3031.As another non-limitative example, in other embodiments, according to the preferred codon modified nucleotide sequence of intestinal bacteria.Referring to for example, Gouy and Gautier (1982) Nucleic Acids Res.10 (22): 7055-7074; Eyre-Walker (1996) Mol.Biol.Evol.13 (6): 864-872.Also referring to (2000) Nucleic Acids Res.28 (1) such as Nakamura: 292.

Other genetic modification

In some embodiments, the host cell of the genetic modification of the present invention of further genetic modification refers to the host cell through following modification: genetic modification is to comprise the nucleic acid that one or more contain the nucleotide sequence of coding modified cytochrome P 450 enzymes (as the isoprenoid modifying enzymes of modifying); And further genetic modification improving protoheme output and/or to improve terpene biosynthetic pathway intermediate output, and/or further genetic modification so that the damage of endogenous terpene biosynthetic pathway gene function.When mentioning endogenous terpene biosynthetic pathway gene, term used herein " function damage " refers to genetic modification terpene biosynthetic pathway gene, causes the output of the gene product of this genes encoding to be lower than normal level, and/or does not have function.

Improve protoheme output

In some embodiments, the host cell of genetic modification of the present invention comprises one or more other genetic modifications that can improve protoheme output, for example, compare with the host cell that does not comprise described one or more other genetic modifications, protoheme output is improved at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times or at least about 25 times, or more times.

The limited step that produces protoheme in the cell is biosynthesizing amino-laevulic acid (ALA).As shown in figure 13, two kinds of different C that comprise are arranged ₄Approach or C ₅The ALA biosynthetic pathway of approach.In some embodiments, the host cell of further genetic modification genetic modification of the present invention is with overexpression glutamy-tRNA reductase enzyme (GTR reductase enzyme).In some embodiments, the further host cell of genetic modification genetic modification of the present invention so that the GTR reductase activity level height that the comparison of GTR reductase activity level produces according to host cell at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times or at least about 25 times or more times.

Improve the activity level of GTR reductase enzyme in the cell in several ways, these modes include but not limited to: 1) improve the promotor intensity that operability is connected in the promotor of GTR reductase enzyme coding region; 2) increase the copy number of the plasmid of the nucleotide sequence contain coding GTR reductase enzyme; 3) improve the stability (when " GTR reductase enzyme mRNA " is when containing the mRNA of nucleotide sequence of coding GTR reductase enzyme) of GTR reductase enzyme mRNA; 4) codon of modifying the GTR reductase enzyme uses, to improve the translation skill of GTR reductase enzyme mRNA; 5) enzyme stability of raising GTR reductase enzyme; 6) ratio (per unit activity of proteins unit) alive of raising GTR reductase enzyme; With 7) reverse feedback that reduces the GTR reductase enzyme regulates.

In some embodiments, the genetic modification that causes GTR reductase enzyme level to improve is the genetic modification that reduces the reverse feedback adjusting of GTR reductase enzyme.In some embodiments, by reduction in N-terminal or near the KK sequence minimizing GTR reductase enzyme reverse feedback adjusting of insertion positively charged it.

In some embodiments, the host cell of further genetic modification genetic modification of the present invention is with overexpression ALA synthase.In some embodiments, the further host cell of genetic modification genetic modification of the present invention so that the comparison of ALA synthase level according to the ALA synthase activity level that produces in the host cell exceed at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times or at least about 25 times or more times.

Improve the activity level of ALA synthase in the cell in several ways, these modes include but not limited to:: 1) improve the promotor intensity that operability is connected in the promotor of ALA synthase coding region; 2) increase the copy number of the plasmid of the nucleotide sequence contain coding ALA synthase; 3) improve the stability (when " ALA synthase mRNA " is when containing the mRNA of nucleotide sequence of coding ALA synthase) of ALA synthase mRNA; 4) codon of modifying the ALA synthase uses, to improve the translation skill of ALA synthase mRNA; 5) enzyme stability of raising ALA synthase; With 6) ratio that improves the ALA synthase live (per unit activity of proteins unit).

Improve the output of endogenous terpene biosynthetic pathway intermediate

The genetic modification that improves endogenous terpene biosynthetic pathway intermediate output includes but not limited to: cause phosphotransacetylase enzyme level and/or the active genetic modification that reduces in the host cell.Increase the IC of terpene biosynthetic pathway intermediate by the IC that increases acetyl-CoA.Intestinal bacteria are secreted into acetyl-CoA in the cell of a large amount of acetic ester forms in the substratum.The disappearance of the gene of coding phosphotransacetylase (pta is responsible for acetyl-CoA and transforms first enzyme that forms acetic ester) will reduce the acetic ester secretion.The level and/or the active genetic modification that reduce phosphotransacetylase in the prokaryotic host cell are useful especially, wherein, the host cell of genetic modification is the host cell with the nucleic acid genetic modification of the nucleotide sequence that comprises one or more MEV pathway gene products of encoding.

In some embodiments, causing the genetic modification that the phosphotransacetylase enzyme level reduces in the prokaryotic host cell is the genetic mutation that makes the endogenous pta gene function damage of prokaryotic host cell coding phosphotransacetylase.Can make pta gene function damage in many ways, comprise: insert movable genetic elements (for example transposon etc.); Disappearance all or part gene, causing not forming gene product or product does not have function by brachymemma and in the process that acetyl-CoA is converted into acetic ester; Transgenation causes not forming gene product, product does not have function by brachymemma and in the process that acetyl-CoA is converted into acetic ester; The controlling elements of disappearance or one or more control pta genetic expression that suddenlys change causes not producing gene product; Or the like.

In some embodiments, lacked the endogenous pta gene of genetic modification host cell.Can use the method for any missing gene.A non-limitative example of pta genetically deficient method is to use λ Red recombination system.Datsenko and Wanner (2000) Proc Natl Acad Sci USA 97 (12): 6640-5 page or leaf.In some embodiments, pta gene disappearance in the host cell (as intestinal bacteria) of the nucleic acid genetic modification of using the nucleotide sequence that comprises encode MK, PMK, MPD and IDI.In some embodiments, pta gene disappearance in the host cell (as intestinal bacteria) of the nucleic acid genetic modification of using the nucleotide sequence that comprises encode MK, PMK, MPD and IPP.In some embodiments, pta gene disappearance in the host cell (as intestinal bacteria) of the nucleic acid genetic modification of using the nucleotide sequence that comprises encode MK, PMK, MPD, IPP and prenyltransferase.

The DXP approach of loss of function

In some embodiments, the host cell of genetic modification of the present invention is the genetically modified host cell that comprises one or more nucleic acid, and described nucleic acid comprises the nucleotide sequence of coding MEV biosynthetic pathway gene product; With the nucleotide sequence that makes the damage of endogenous DXP biosynthetic pathway gene function through further genetic modification.In other embodiments, the host cell of genetic modification of the present invention is the genetically modified host cell that comprises one or more nucleic acid, and described nucleic acid comprises the nucleotide sequence of encoding D XP biosynthetic pathway gene product; With through further genetic modification so that the nucleotide sequence of endogenous MEV biosynthetic pathway gene function damage.

In some embodiments, when the host cell of genetic modification of the present invention is prokaryotic host cell with the nucleic acid genetic modification of the nucleotide sequence that comprises one or more MEV pathway gene products of encoding, further genetic modification host cell is so that one or more endogenous DXP pathway gene function damage.One or more genes that can be comprised any following DXP gene product of encoding: 1-deoxy-D-xylulose-5-phosphoric acid ester synthase, 1-deoxy-D-xylulose-5-phosphoric acid ester reductone isomerase (reductoisomerase), 4-cytidine diphosphate (CDP) acyl-2-C-methyl D-tetrahydroxybutane synthase, 4-cytidine diphosphate (CDP) acyl-2-C-methyl D-tetrahydroxybutane kinases, 2C-methyl D-tetrahydroxybutane 2,4-ring bisphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-bisphosphate synthase by the DXP pathway gene of function damage.

Can make endogenous DXP pathway gene function damage in many ways, comprise and insert movable genetic elements (for example, transposon etc.); Disappearance all or part gene causes not forming gene product or product by brachymemma and there is not enzymic activity; Transgenation causes not forming gene product, product is by brachymemma and do not have the enzyme function; The controlling elements that disappearance or one or more controlling genes that suddenly change are expressed causes not producing gene product; Or the like.

In other embodiments, when the host cell of genetic modification of the present invention is prokaryotic host cell with the nucleic acid genetic modification of the nucleotide sequence that comprises one or more DXP pathway gene products of encoding, further genetic modification host cell is so that one or more endogenous MEV pathway gene function damage.One or more genes that can be comprised any following MEV gene product of encoding: HMGS, HMGR, MK, PMK, MPD and IDI by the endogenous MEV pathway gene of function damage.Can make endogenous MEV pathway gene function damage in many ways, comprise and insert movable genetic elements (for example, transposon etc.); Disappearance all or part gene causes not forming gene product or product by brachymemma and there is not enzymic activity; Transgenation causes not forming gene product, product is by brachymemma and do not have the enzyme function; The controlling elements that disappearance or one or more controlling genes that suddenly change are expressed causes not producing gene product; Or the like.

The composition that comprises the host cell of genetic modification of the present invention

The present invention also provides the composition that comprises genetic modification host cell of the present invention.The present composition comprises the host cell of genetic modification of the present invention; And also comprise one or more other compositions in some embodiments, the selection part of these compositions is based on the specific end use of genetic modification host cell.Appropriate ingredients includes but not limited to: salt; Buffer reagent; Stablizer; Proteinase inhibitor; Nucleic acid inhibitor; Cytolemma and/or cell walls keeping quality compound, for example glycerine, methyl-sulphoxide etc.; The nutritional medium that is fit to cell; Or the like.In some embodiments, freeze drying cell.

Transgenic plant

In some embodiments, nucleic acid of the present invention or expression vector of the present invention (as modified cytochrome P 450 enzymes nucleic acid of the present invention or comprise the expression vector of the present invention of modified cytochrome P 450 enzymes nucleic acid) are as transgenosis, to produce the transgenic plant that can produce the modified cytochrome P 450 enzymes of encoding.Therefore, the present invention also provides transgenic plant (or plant part, seed, tissue etc.), and this plant comprises the transgenosis that contains nucleic acid of the present invention, and described nucleic acid of the present invention comprises the nucleotide sequence of coding modified cytochrome P 450 enzymes (as mentioned above).In some embodiments, the genome of transgenic plant comprises nucleic acid of the present invention.In some embodiments, transgenic plant are that genetic modification isozygotys.In some embodiments, transgenic plant are genetic modification heterozygosis.

In some embodiments, compare with the control plant of same species such as the product output of non-transgenic plant (the genetically modified plant that does not comprise this polypeptide of encoding), the output of the modified cytochrome P 450 enzymes of the transgenes encoding that transgenic plant of the present invention produce and the product of modified cytochrome P 450 enzymes exceeds at least about 50%, at least about 2 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times or at least about 100 times or higher.

In some embodiments, transgenic plant of the present invention are transgenosis forms of non-transgenic control plant, the compound that the modification isoprenoid precursor modifying enzyme that the common isoprenoid compound that produces of this non-transgenic control plant is a transgenes encoding produces or the downstream product of this transgenes encoding polypeptide; Compare with the control plant of same species such as the isoprenoid compound output of non-transgenic plant (the genetically modified plant that does not comprise this polypeptide of encoding), the isoprenoid compound output of these transgenic plant exceeds at least about 50%, at least about 2 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times or at least about 100 times or higher.

Well known method with exogenous nucleic acid introduced plant cell.Think that this vegetable cell is by " conversion ", as mentioned above.Appropriate method comprises virus infection (as double-stranded DNA virus), transfection, joint, protoplastis fusion, electroporation, gene gun technology, calcium phosphate precipitation, directly microinjection, silicon carbide whisker (whisker) technology, agrobacterium-mediated conversion etc.The cell transformed type is depended in the selection of method usually and the environment that take place to transform (promptly external, exsomatize or body in).

Method for transformation based on soil bacteria Agrobacterium tumefaciens (Agrobacterium tumefaciens) is specially adapted to exogenous nucleic acid molecule is introduced vascular plant.The wild-type edaphic bacillus contains guidance produces the growth of tumorigenicity crown gall on host plant Ti (tumor inducing) plasmid.The tumor inducing type T-DNA district of Ti-plasmids is transferred to virulence gene and the T-DNA border that Plant Genome needs the Ti-plasmids coding, and they are the one group of DNA repetitions in the same way that touches off zone to be transferred.Carrier based on edaphic bacillus is the Ti-plasmids of modifying, and wherein the tumor inducing function is treated that introduced plant host's nucleotide sequence interested replaces.

Common integrative vector is adopted in agrobacterium-mediated conversion usually, perhaps preferably adopt the binary vector system, wherein the element of Ti-plasmids separates between assistant carrier and shuttle vectors, described assistant carrier for good and all is trapped among the edaphic bacillus host and carries virulence gene, and it is the gene of interest on border that described shuttle vectors contains with the T-DNA sequence.Well known various binary vector, and can be available from (for example) clone Tai Ke technology company (Clontech) (California Palo Alto).Well known (for example) is with the vegetable cell of edaphic bacillus and cultivation or the method for wound tissue such as leaf texture, root explant, hypocotyl (hypocotyledons), stem piece or stem tuber co-cultivation.Referring to for example, Glick and Thompson (volume), Methods inPlant Molecular Biology and Biotechnology (method of molecular biology of plants and biotechnology), Florida State Bai Kaladun (Boca Raton, Fla.): CRC press (CRC Press) (1993).

Agrobacterium-mediated conversion can be used for producing various transgenosis vascular plants (Wang etc., the same, 1995) and comprises that at least a eucalyptus belongs to (Eucalyptus) and leguminous forage (forage legumes) as clover (alfalfa); Birdfoot, butch clover, Stylosanthes (Stylosanthes), sieve pause beans (Lotononisbainessii) and sainfoin.

Also can adopt the conversion of particulate mediation to produce transgenic plant of the present invention.At first this method of being described by (Nature327:70-73 (1987)) such as Klein depends on particulate such as gold or the tungsten of coating required nucleic acid molecule by calcium chloride, spermidine or polyethylene glycol precipitation.With a kind of device as BIOLISTIC PD-1000 (Bole company (Biorad); The California is congratulated and is asked this (Hercules Calif.)) particulate is injected at a high speed in the angiosperm tissue.

With the mode of nucleic acid introduced plant of the present invention should be able to make this nucleic acid can by in (for example) body or stripped method enter vegetable cell." in the body " refers to by (for example) infiltration nucleic acid be entered in the plant living body." exsomatizing " refers to modify cell or explant outside plant, making this cell or neomorph then is plant.Describe the many carriers that are applicable to the stable conversion vegetable cell or set up transgenic plant, comprised Weissbach and Weissbach (1989) Methods for Plant Molecular Biology (molecular biology of plants method), the academic press; With Gelvin etc., (1990) Plant Molecular Biology Manual (plant molecular biology manual), the described carrier of Crewe dimension academic publishing group.Object lesson comprises the carrier derived from the Ti-plasmids of Agrobacterium tumefaciens, and (1983) Nature 303:209 such as Herrera-Estrella, Bevan (1984) Nucl Acid Res.12:8711-8721, the described carrier of Klee (1985) Bio/Technology 3:637-642.Perhaps, can adopt non-Ti carrier that DNA is transferred in plant and the cell by the dissociative DNA delivery technique.When adopting these methods, can produce transgenic plant such as wheat, paddy rice (Christou (1991) Bio/Technology 9:957-962) and corn (Gordon-Kamm (1990) Plant Cell 2:603-618).When adopting particle gun to carry out direct DNA delivery technique, jejune embryo also may be good monocotyledons target tissue (Weeks etc. (1993) Plant Physiol 102:1077-1084; Vasil (1993) Bio/Technolo 10:667-674; Wan and Lemeaux (1994) Plant Physiol 104:37-48; Shift referring to (Ishida etc. (1996) Nature Biotech 14:745-750) for agrobacterium-mediated DNA.The exemplary method of DNA being introduced chloroplast(id) is that biological projectile bombardment, polyoxyethylene glycol transform protoplastis and microinjection (Danieli etc., Nat.Biotechnol 16:345-348,1998; Staub etc., Nat.Biotechnol 18:333-338,2000; O ' Neill etc., Plant J.3:729-738,1993; Knoblauch etc., Nat.Biotechnol 17:906-909; United States Patent (USP) 5,451,513,5,545,817,5,545,818 and 5,576,198; International Application No. WO 95/16783; And Boynton etc., Methods inEnzymology (Enzymology method) 217:510-536 (1993), Svab etc., Proc.Natl.Acad.Sci.USA 90:913-917 (1993) and McBride etc., Proc.Natl.Acad.Sci.USA 91:7301-7305 (1994)).Any carrier that is applicable to biological projectile bombardment, polyoxyethylene glycol conversion protoplastis and micro-injection method all can be used as the targeting vector that chloroplast(id) transforms.Any double-stranded DNA carrier all can be used as conversion carrier, especially when introducing method does not adopt edaphic bacillus.

The plant that can carry out genetic modification comprises cereal, pasture crop, fruit, vegetables, oilseed crops, palm, forestry plant and vine.The object lesson of modifiable plant is as follows: corn, banana, peanut, field pea, Sunflower Receptacle, tomato, rape, tobacco, wheat, barley, oat, potato, soybean, cotton, carnation, Chinese sorghum, lupine and rice.Other example comprises Herba Artemisiae annuae, perhaps known other plant that can produce interested isoprenoid compound.

The present invention also provides the plant transformed cell, contains tissue, seed, plant and the product of plant transformed cell.Transformant of the present invention and contain the tissue of this cell and the feature of product is that nucleic acid of the present invention is incorporated in the genome and this vegetable cell can produce modified cytochrome P 450 enzymes.Recombinant plant cell of the present invention can be used as reconstitution cell colony and uses, or as uses such as tissue, seed, whole plant, stem, fruit, leaf, root, flower, stem, stem tuber, grain, animal-feed, large stretch of plants.

The present invention also provides the reproductive material of transgenic plant of the present invention, and described reproductive material comprises seed, offspring plant and asexual propagation material.

Produce the method for biosynthetic pathway product

The invention provides the method that produces the biosynthetic pathway product.This method is usually included in the host cell of cultivating genetic modification of the present invention in the suitable culture medium.The host cell of genetic modification of the present invention be with the nucleic acid of nucleotide sequence that comprises the modified cytochrome P 450 enzymes of encode carry out genetic modification, with the cell of generation modified cytochrome P 450 enzymes, described modified cytochrome P 450 enzymes operability is connected in and is selected from membrane spaning domain, secretory structure territory, solubilising structural domain or film and inserts proteic structural domain.When the biosynthetic pathway intermediate existed, the generation of modified cytochrome P 450 enzymes caused intermediate generation zymetology to modify and produce the biosynthetic pathway product.In other embodiments, this method generally includes transgenic plant of the present invention is maintained under the condition of the modified cytochrome P 450 enzymes that helps producing coding.The generation of modified cytochrome P 450 enzymes causes having produced the biosynthetic pathway product.This method is generally carried out external (for example cultivating at the external use viable cell), but also considers to produce in vivo the biosynthetic pathway product.In some embodiments, described host cell is an eukaryotic cell, as yeast cell.In other embodiments, described host cell is a prokaryotic cell prokaryocyte.In some embodiments, described host cell is a vegetable cell.In some embodiments, this method is carried out in transgenic plant of the present invention.

Compare with contrast parent host cell, the host cell of genetic modification of the present invention can improve the output of biosynthetic pathway product.Therefore, for example, compare with the product level that produces in the contrast parent host cell, the output of the biosynthetic pathway product of genetic modification host cell improved at least about 10%, at least about 20%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times or at least about 500 times or more times.Contrast parent host cell does not comprise the genetic modification that exists in the genetic modification host cell.

In some embodiments, compare with the contrast host cell, the host cell of genetic modification of the present invention has improved the output of biosynthetic pathway product.Therefore, for example, compare with the product level that contrast produces in the host cell, in the host cell of genetic modification the output of biosynthetic pathway product improve at least about 10%, at least about 20%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times or at least about 500 times or more times.In some embodiments, the contrast host cell does not contain the genetic modification that exists in the host cell of genetic modification, for example, isoprenoid modifying enzymes coding nucleic acid (as the cytochrome P 450 enzymes coding nucleic acid) operability in the contrast host cell is connected in natural membrane spaning domain, natural secretory structure territory, natural solubilising structural domain and natural membranes insert one or more in the polypeptide, and the isoprenoid modifying enzymes coding nucleic acid operability that the host cell of genetic modification comprises is connected in non-natural (as allos) membrane spaning domain, non-natural secretory structure territory, non-natural solubilising structural domain and non-natural film insert one or more in the territory.An example is, when the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification was connected in non-natural isoprenoid modifying enzymes coding nucleic acid, the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises was connected in natural membrane spaning domain.Another example is, when the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification was connected in allos secretion signal structural domain, the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises was connected in natural membrane spaning domain.Another example is, when the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification was connected in allos solubilising structural domain, the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises was connected in natural membrane spaning domain.Another example is, the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification is connected in the allos film when inserting the territory, and the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises is connected in natural membrane spaning domain.Another example is, the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification is connected in the variation membrane spaning domain (as the natural membrane spaning domain of brachymemma; Compare the membrane spaning domain that aminoacid sequence changes with the aminoacid sequence of natural membrane spaning domain), the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises is connected in natural membrane spaning domain.

The invention provides the method that produces the isoprenoid compound.This method is usually included in the host cell of cultivating genetic modification of the present invention in the suitable culture medium, wherein the host cell of genetic modification of the present invention is the cell with the nucleic acid genetic modification of the nucleotide sequence that contains coding isoprenoid precursor modifying enzyme, described isoprenoid precursor modifying enzyme operability is connected in the structural domain that is selected from down group: membrane spaning domain, secretory structure territory, solubilising structural domain and film insert albumen, to produce isoprenoid precursor modifying enzyme.In the presence of the isoprenoid precursor compound, produce isoprenoid precursor modifying enzyme and cause zymetology to modify isoprenoid precursor and generation isoprenoid compound.In other embodiments, described method is usually included under the condition of the isoprenoid precursor modifying enzyme that helps producing coding and keeps transgenic plant of the present invention.Produce isoprenoid precursor modifying enzyme and cause producing the isoprenoid compound.For example, in some embodiments, described method is usually included in the suitable culture medium host cell of cultivating genetic modification, wherein with containing coding terpene modifying enzyme as host cell as described in the nucleic acid genetic modification of the present invention of the nucleotide sequence of terpene oxydase, terpene hydroxylase etc.Produce the terpene oxydase and cause producing the isoprenoid compound.This method is generally carried out in external (as cultivating at the external use viable cell), but also considers to produce in vivo the isoprenoid compound.In some embodiments, described host cell is an eukaryotic cell, as yeast cell.In other embodiments, described host cell is a prokaryotic cell prokaryocyte.In some embodiments, described host cell is a vegetable cell.In some embodiments, this method is carried out in transgenic plant of the present invention.

Compare with contrast parent host cell, the host cell of genetic modification of the present invention can improve the output of isoprenoid compound.Therefore, for example, compare with the product level that produces in the contrast parent host cell, the isoprenoid of genetic modification host cell or isoprenoid precursor output improved at least about 10%, at least about 20%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times or at least about 500 times or more times.Contrast parent host cell does not comprise the genetic modification that exists in the genetic modification host cell.

In some embodiments, compare with the contrast host cell, the host cell of genetic modification of the present invention has improved the output of isoprenoid compound.Therefore, for example, compare with the product level that contrast produces in the host cell, in the host cell of genetic modification isoprenoid or isoprenoid precursor output improve at least about 10%, at least about 20%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times or at least about 500 times or more times.In some embodiments, the contrast host cell does not contain the genetic modification that exists in the host cell of genetic modification, for example, isoprenoid modifying enzymes coding nucleic acid (as the cytochrome P 450 enzymes coding nucleic acid) operability in the contrast host cell is connected in natural membrane spaning domain, natural secretory structure territory, natural solubilising structural domain and natural membranes insert one or more in the polypeptide, and the isoprenoid modifying enzymes coding nucleic acid operability that the host cell of genetic modification comprises is connected in non-natural (as allos) membrane spaning domain, non-natural secretory structure territory, non-natural solubilising structural domain and non-natural film insert one or more in the territory.An example is, when the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification was connected in non-natural isoprenoid modifying enzymes coding nucleic acid, the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises was connected in natural membrane spaning domain.Another example is, when the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification was connected in allos secretion signal structural domain, the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises was connected in natural membrane spaning domain.Another example is, when the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification was connected in allos solubilising structural domain, the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises was connected in natural membrane spaning domain.Another example is, the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification is connected in the allos film when inserting the territory, and the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises is connected in natural membrane spaning domain.Another example is, the isoprenoid modifying enzymes coding nucleic acid operability that comprises when the host cell of genetic modification is connected in the variation membrane spaning domain (as the natural membrane spaning domain of brachymemma; Compare the membrane spaning domain that aminoacid sequence changes with the aminoacid sequence of natural membrane spaning domain), the isoprenoid modifying enzymes coding nucleic acid operability that suitable contrast host cell comprises is connected in natural membrane spaning domain.

Therefore, in some embodiments, with each cytometer, the isoprenoid compound level that the contrast host cell of one or more genetic modifications contained with not comprising the genetic modification host cell produces is compared, and the isoprenoid compound level that the host cell of genetic modification of the present invention produces exceeds at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times or at least about 500 times or more times.Be not difficult to use well-known process, for example measure optical density(OD) (the OD) (OD of 600nm place bacterial liquid culture ₆₀₀); The bacterium colony size; Growth velocitys etc. are measured the growth of the host cell of genetic modification.

In some embodiments, the host cell of the genetic modification of the present invention recyclable amount that produces the isoprenoid compound be at least about 1mg/L, at least about 5mg/L, at least about 10mg/L, at least about 15mg/L, at least about 20mg/L, at least about 25mg/L, at least about 30mg/L, at least about 35mg/L, at least about 40mg/L, at least about 50mg/L, at least about 75mg/L, at least about 100mg/L, at least about 125mg/L, at least about 150mg/L, at least about 200mg/L, at least about 300mg/L, at least about 500mg/L, at least about 1000mg/L or at least about 5000mg/L.

In some embodiments, the recyclable amount that the host cell of genetic modification of the present invention produces the isoprenoid compound is about 1mg/L-5000mg/L, for example about 1mg/L-2mg/L, about 2mg/L-5mg/L, about 5mg/L-10mg/L, about 10mg/L-15mg/L, about 15mg/L-20mg/L, about 20mg/L-25mg/L, about 25mg/L-50mg/L, about 50mg/L-75mg/L, about 75mg/L-100mg/L, about 100mg/L-150mg/L, about 150mg/L-200mg/L, about 200mg/L-250mg/L, about 250mg/L-300mg/L, about 300mg/L-350mg/L, about 350mg/L-400mg/L, about 400mg/L-450mg/L, about 450mg/L-500mg/L, about 500mg/L-1000mg/L, about 1000mg/L-2000mg/L, about 2000mg/L-3000mg/L, about 3000mg/L-4000mg/L or about 4000mg/L-5000mg/L.Can be by substratum or host cell, for example reclaim the isoprenoid that produces by substratum or cell lysate or cell lysate component.Recovery method may depend on various factors, for example the characteristic of the concrete isoprenoid of Chan Shenging.

Figure 14 and 15 has schematically described the exemplary isoprenoid product of biosynthesizing.The conversion of many isopentene of diterpene synthase catalysis straight chain bisphosphate ester; Converted product is the substrate of isoprenoid precursor modifying enzyme (as the P450 enzyme).Then by the carbon skeleton reaction of P450 and its redox partner CPR catalyged precursor, thereby carry out concrete functionalized.

In some embodiments, with the host cell of the further genetic modification genetic modification of nucleic acid of the nucleotide sequence that contains the diterpene synthase of encoding, described diterpene synthase can be allos diterpene synthase (diterpene synthase that does not produce usually as host cell).Therefore, for example, in some embodiments, with one or more nucleic acid genetic modification host cells of the nucleotide sequence that contains coding diterpene synthase and isoprenoid modifying enzymes (as the sesquiterpene oxydase).In suitable culture medium, cultivate this class host cell and can produce diterpene synthase and isoprenoid modifying enzymes (as the sesquiterpene oxydase).For example, diterpene synthase can be modified farnesyl pyrophosphate, to produce the oxidasic sesquiterpene substrate of described sesquiterpene.

In some embodiments, use the further genetic modification host cell of nucleic acid of the nucleotide sequence that contains Codocyte cytochrome p 450 reductase enzyme (CPR).The nucleotide sequence of known various CPR can adopt any known CPR coding nucleic acid, as long as the CPR of coding has the activity by the NADPH metastatic electron.In some embodiments, CPR coding nucleic acid coding can be transferred to the isoprenoid modifying enzymes that isoprenoid modifying enzymes coding nucleic acid of the present invention is encoded from NADPH with electronics, as the oxidasic CPR of sesquiterpene.

In some embodiments, further the genetic modification host cell produces one or more enzymes in prenyltransferase and/or the biosynthetic pathway, to produce tetra-sodium isoamyl-1-alkene ester.General one of the two kinds of approach that adopt of cell produce isoprenoid or isoprenoid precursor (as IPP, many isopentene of bisphosphate ester etc.).Figure 16-18 is used to illustrate that cell is in order to produce the approach of isoprenoid compound or precursor such as many isopentene of bisphosphate ester.

Figure 16 has shown the isoprenoid approach, comprise with prenyltransferase and modify bisphosphate isopentene ester (IPP) and/or its isomer bisphosphate dimethyl allyl ester (DMAPP), produce bisphosphate many isopentene ester bisphosphate Mang ox ester (GPP), bisphosphate method ester (FPP) and bisphosphate geranyl Mang ox ester (GGPP).GPP and FPP are further modified by diterpene synthase, produce monoterpene and sesquiterpene respectively; GGPP is further modified by diterpene synthase, forms diterpene and carotenoids.IPP and DMAPP are by a kind of generation in following two kinds of approach: mevalonic acid (MEV) approach and 1-deoxy-D-xylulose-5-phosphoric acid ester (DXP) approach.

Figure 17 schematically illustrates the MEV approach, and wherein, acetyl-CoA is converted into IPP by series reaction.

Figure 18 schematically illustrates the DXP approach, and wherein, pyruvic acid and D-glyceraldehyde-3-phosphate are converted into IPP and DMAPP by series reaction.Eukaryotic cell except that vegetable cell only utilizes MEV isoprenoid approach so that acetyl-CoA (acetyl-CoA) be converted into IPP, isomery turns to DMAPP then.Plant utilizes MEV and mevalonic acid dependent/non-dependent or DXP approach to synthesize isoprenoid simultaneously.Remove some exceptions, prokaryotic organism utilize the DXP approach to produce IPP and DMAPP respectively by tapping point.

Synthesize IPP according to the substratum of cultivating host cell and host cell by DXP approach or mevalonate pathway, in some embodiments, host cell also can comprise other genetic modification.For example, in some embodiments, host cell is the host cell that does not have the endogenous mevalonate pathway, and for example, host cell is the host cell that does not synthesize IPP or mevalonic acid usually by mevalonate pathway.For example, in some embodiments, host cell is the host cell that does not synthesize IPP usually by mevalonate pathway, with one or more nucleic acid genetic modification host cells that comprise the nucleotide sequence of two or more enzymes, IPP isomerase, prenyltransferase, diterpene synthase and isoprenoid modifying enzymes (as the isoprenoid modifying enzymes of nucleic acid encoding of the present invention) in the coding mevalonate pathway.Cultivate this host cell and can produce mevalonate pathway enzyme, IPP isomerase, prenyltransferase, diterpene synthase and isoprenoid modifying enzymes (as the sesquiterpene oxydase).Produce mevalonate pathway enzyme, IPP isomerase, prenyltransferase, diterpene synthase and isoprenoid modifying enzymes (as the sesquiterpene oxydase) and then cause producing the isoprenoid compound.In many embodiments, prenyltransferase is the FPP synthase, and it can produce the oxidasic sesquiterpene substrate of sesquiterpene of nucleic acid encoding of the present invention; Generation sesquiterpene oxydase then causes the sesquiterpene substrate in the oxidation host cell.Be fit to adopt any nucleic acid of coding mevalonate pathway enzyme, IPP isomerase, prenyltransferase and diterpene synthase.For example, suitable nucleic acid is the same referring to (2003) such as for example Martin.

In some above-mentioned embodiments, during with one or more nucleic acid genetic modification host cells of the nucleotide sequence that contains two or more mevalonate pathway enzymes of encoding, described two or more mevalonate pathway enzymes comprise MK, PMK and MPD, and cultivate host cell in containing the substratum of mevalonic acid.In other embodiments, described two or more mevalonate pathway enzymes comprise acetoacetyl CoA thiolase, HMGS, HMGR, MK, PMK and MPD.

In some embodiments, host cell is usually the host cell by the synthetic IPP of mevalonate pathway not, genetic modification host cell as mentioned above, and host cell also comprises the DXP approach of function damage.

The inventive method can be used for producing various types of isoprenoid, include but not limited to: (as the sesquiterpene substrate is false indigo-4 to arteannuinic acid, during the 11-diene), Isolongifolene alcohol (when being Isolongifolene) as substrate, (E)-trans-Citrus bergamia-2,12-diene-14-alcohol (when being (-)-α-trans-bergapten) as substrate, (-)-elemi-1,3,11 (13)-triolefins-12-alcohol (when being (-)-beta-elemene) as substrate, big Mang ox three enols (germacra)-1 (10), 4,11 (13)-triolefins-12-alcohol (when being (+)-germacrene A) as substrate, germacrene B alcohol (when being germacrene B) as substrate, 5,11 (13)-guaiadienes-12-alcohol (when being (+)-γ-gurjunene) as substrate, loudspeaker enol (when being (+)-ledene) as substrate, 4 β-H-folium eucalypti-11 (13)-alkene-4,12-glycol (when being neointermedeol (neointermedeol)) as substrate, (+)-β-Radix Aucklandiae (Radix Vladimiriae) alcohol (when being (+)-β-selinene etc.) as substrate; Derivative with any above-mentioned substance.

In many embodiments, with the host cell of suitable culture medium external cultivation genetic modification of the present invention under suitable temp.The cell cultures temperature is generally about 18 ℃-40 ℃, for example about 18 ℃-20 ℃, about 20 ℃-25 ℃, about 25 ℃-30 ℃, about 30 ℃-35 ℃ or about 35 ℃-40 ℃ (37 ℃ according to appointment).

In some embodiments, with suitable culture medium (as Luria-Bertoni meat soup, optional one or more other materials that is supplemented with, for example inductor (nucleotides sequence of the isoprenoid modifying enzymes of for example encoding is listed in inducible promoter and controls following time) etc.) cultivate the host cell of genetic modification of the present invention; Cover substratum with organic solvent such as dodecane, form organic layer.The isoprenoid compound that the host cell of genetic modification produces is assigned in the organic layer, by this compound of organic layer purifying.In some embodiments, when the nucleotide sequence operability of coding isoprenoid modifying enzymes is connected in inducible promoter, inductor is added in the substratum; Behind the appropriate time, separate the isoprenoid compound by the organic layer that covers on the substratum.

In some embodiments, other product that may exist in isoprenoid compound and the organic layer is separated.Be not difficult to adopt (as) chromatographic process of standard separates other product that may exist in isoprenoid compound and the organic layer.

In some embodiments, in acellular reaction, further modify the inventive method synthetic isoprenoid compound with chemical process.For example, in some embodiments, separate arteannuinic acid, in acellular reaction, further modify arteannuinic acid, to produce Artemisinin with chemical process by substratum and/or cell lysate.

In some embodiments, the isoprenoid compound is purified, for example, purity is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or be higher than 98%, when mentioning the isoprenoid compound, " pure " refers to not contain the isoprenoid compound of other isoprenoid compound, macromole, pollutent etc.

Embodiment

Following examples are proposed, how to prepare and use complete disclosure and description of the present invention so that provide to those of ordinary skills, but be not intended to limit the invention scope that the contriver assert, they do not represent following experiment yet is all of carrying out or experiment is only arranged.Endeavour to ensure the accuracy of used numerical value (as amount, temperature etc.), but should allow some experimental errors and deviation.Except as otherwise noted, umber is parts by weight, and molecular weight is a weight-average molecular weight, and temperature is degree centigrade that pressure is normal atmosphere or nearly normal atmosphere.Can adopt standardized abbreviations, for example: bp, base pair; Kb, kilobase; Pl, the skin liter; S or sec, second; Min, minute; H or hr, hour; Aa, amino acid; Kb, kilobase; Bp, base pair; Nt, Nucleotide; I.m., intramuscular; I.p., intraperitoneal; S.c., subcutaneous; Or the like.

Embodiment 1: in intestinal bacteria, produce 8-hydroxyl-δ-cadinene

Present embodiment has been described with natural P450 high level (the highest 30mg L that participates in biosynthetic pathway ^-1) the interior substrate that produces of generation body.δ-cadinene-8-hydroxylase (CadH) be the film of plant derivation in conjunction with P450, it can become 8-hydroxyl-δ-cadinene (CadOH) with sesquiterpene δ-cadinene (cad) hydroxylation in the plant defense compound biosynthesizing gossypol.Fig. 1 schematically describes the biosynthesizing CadOH in the intestinal bacteria.Diterpene synthase CadS produces substrate (Cad) from endogenous farnesyl pyrophosphate (FPP) in intestinal bacteria.Effect by CadH and its redox partner (CPR) becomes product (CadOH) with the further hydroxylation of Cad.

The CadH expression vector comprises cytochrome P450 reductase (CPR) the redox partner's of CadH gene and coding Oidium tropicale gene.The consistency of this construction and δ-cadinene synthase (CadS) is expressed carrier be transformed in the intestinal bacteria jointly, thereby the substrate of CadH is provided.In the eutrophy substratum, cultivate this bacterial strain, in the presence of the protoheme fill-in, induced 48 hours for 20 ℃, then with this substratum of organic solvent extraction.Result shown in Figure 2 shows, measures through GC-MS (gas chromatography-mass spectrography), and the CadOH amount that produces in this system obviously can detect (～100 μ g L ^-1).

Fig. 2 is the GC-MS figure by the organic layer of the intestinal bacteria extraction of expressing the CadOH biosynthetic pathway.Illustration is the enlarged view that shows CadOH (peak 1) and infer the zone of ketone material (peak 2).Above line corresponding to the sample of expressing CadS, CadH and CPR, and a following line is corresponding to the negative control of only expressing CadS and CPR (no CadH).

In addition, also observe ketone the product ([M that infers on a small quantity ⁺]: m/z=218) (Fig. 2 illustration, above a line, peak 2), mean that same enzyme may have enough to meet the need repeatedly.In GC-MS figure, only contain CPR and the negative control plasmid that do not contain CadH does not have product peak (Fig. 2 illustration, below a line).The mass spectrum of the CadOH that is produced in vivo by intestinal bacteria with this system and the document spectrogram of CadOH are very near [4].Attempted the success of functionalized natural product that the natural P450 of use produces similar compound family in vivo in the past, illustrated that the substrate accessibility had problem.

By improve the FPP amount that the pMBIS plasmid produces in intestinal bacteria, significantly improved CadOH output, the pMBIS plasmid makes intestinal bacteria produce FPP[6 by mevalonic acid].The nucleotide sequence of pMBIS is referring to Figure 36 A-D (SEQ ID NO:62).PMBIS is also referring to U.S. Patent Publication No. 2003/0148479; With 2004/0005678; It comprises the nucleotide sequence of coding Mevalonic kinase, Phosphomevalonic kinase, tetra-sodium mevalonate decarboxylase, IPP isomerase and FPP synthase.In these researchs, with following three kinds of expression plasmid transformed into escherichia coli: (1) pMBIS, (2) CadS and (3) CadH/CPR.Induce the back to add the 20mM mevalonic acid.Compare with the cell that does not contain pMBIS, add pMBIS and can make CadOH output improve 74 times (Fig. 3).Again, negative control (no CadH) does not form product.These results show, the restriction of (as the viable cell in the vitro culture thing) substrate output in the P450 volume of the circular flow possibility acceptor.Also can in blended water-based/organic substratum, cultivate these cells; Cultivate this bacterial strain, in the presence of dodecane is obducent, induce, significantly do not change CadOH output (below～2 times).

Fig. 3 is the GC-MS figure by the organic layer of the intestinal bacteria extraction of the expression CadOH biosynthetic pathway of the mevalonic acid of feeding and pMBIS.Cad and CadOH have been represented on the figure.

Further prove, by not losing engineered P450 raising output under the specific situation of product.Compared natural gene (nCadH) and used output (Fig. 4 B) in the body of the synthetic gene of optimizing (sCadH) according to carry out codon at expression in escherichia coli.This comparison shows that synthetic gene slightly is better than natural gene.

In intestinal bacteria, there is the sequence of function to replace wild-type N-terminal membrane spaning domain (TM) (Fig. 4 A) with known.The N-terminal sequence that is detected be two derived from the CYP52A13 (A13) of the TM structural domain of terminal leads of the P450N of Oidium tropicale-do not contain prediction with contain the CYP52A17 (A17) of TM structural domain [7]-and ox microsome lead (ox) [8].

Remove (brachymemma) wild-type TM structural domain fully, insert albumen (Mi Sidi albumen) [10] with secretion label (OmpA), solubilising structural domain (PD1) [9] or film and replace.Ox-CadH surpasses about 2 times of wild-type CadH, generation～30mg L ^-1(Fig. 4 B).

Reference

1.M.Sono, M.P.Roach, E.D.Coulter and J.H.Dawson, Chem.Rev.1996,96,2841-2887.

2.S.Jennewein, R.M.Long, R.M.Williams and R.Croteau, Chem.Biol.2004,11,379-387.

3.R.J.Sowden, S.Yasmin, N.H.Rees, S.G.Bell and L.-L.Wong, Org.Biomol.Chem.2005,3,57-64.

4.P.Luo, Y.-H.Wang, G.-D.Wang, M.Essenberg and X.-Y.Chen, PlantJ.2001,28,95-104.

5.O.A.Carter, R.J.Peters and R.Croteau, Phytochem.2003,64,425-433.

6.V.J.J.Martin, D.J.Pitera, S.T.Withers, J.D.Newman and J.D.Keasling, Nature Biotech.2003,21,796-801

7.D.L.Craft, K.M.Madduri, M.Eshoo and C.R.Wilson, Appl.Environ.Microbiol.2003,69,5983-5991.

8.H.J.Barnes, M.P.Arlotto and M.R.Waterman, Proc.Natl.Acad.Sci.USA 1991,88,5597-5601.

9.G.A.Schock, R.Attias, M.Belghazi, P.M.Dansette and D.Werck-Reichart, Plant Physiol.2003,133,1198-1208.

10.T.P.Roosild, J.Greenwald, M.Vega, S.Castronovo, R.Riek and S.Choe Science 2005,307,1317-1321.

Embodiment 2: with AMORPHADIENE oxidizing ferment (AMO) oxidation AMORPHADIENE

Present embodiment has been described with the AMORPHADIENE oxidizing ferment (AMO) that separates from artemisia annua, is also referred to as CYP71AV1 is (in the living cells in the in vitro culture thing) oxidation AMORPHADIENE in vivo. Produce and inspection Survey contains the various constructions of the nucleotide sequence of the AMO that encodes, to optimize the output of oxidation product. Figure 22 Schematically describe various AMO constructions. (1) nAMO separates from the natural A MO of artemisia annua sequence. (2) sAMO is according to carry out codon optimized synthetic AMO gene at expression in escherichia coli. (3) A13-AMO, the wild type membrane spaning domain is by synthesizing that the A13N end sequence of Candida tropicalis replaces The AMO gene. (4) A17-AMO, the wild type membrane spaning domain is by the terminal order of the A17N of Candida tropicalis The synthetic AMO gene that row replace. (5) Bov-AMO, the wild type membrane spaning domain is by ox microsome N end The synthetic AMO gene that terminal sequence replaces. The nucleotides of various constructions and amino acid sequence are referring to figure 24-31.

Various AMO constructions and following material co expression: a) CPR; B) AMORPHADIENE synthase (ADS); And c) plasmid pMBIS. In the presence of mevalonic acid, observe AMORPHADIENE in the C12 position Be set up and be oxidized to correspondent alcohol. Figure 23 A shows the fractional yield of artemisinol in the body. Trusted standard with artemisinol Product have been confirmed the identity (figure below of Figure 23 A and Figure 23 B) of this product more afterwards.

Figure 23 A and 23B. are with various AMO constructions vivo oxidation AMORPHADIENE in Escherichia coli. (A) GC-MS figure shows and compares with trusted standard product (figure below), sAMO, A13-AMO in the Escherichia coli, The artemisinol output (upper figure) that A17-AMO and bov-AMO produce. (B) compare with trusted standard product (figure below), The EI-MS of the artemisinol that produces in the Escherichia coli (upper figure).

Embodiment 3: the substrate oxidation in the cell of The expressed mevalonate pathway

Also the cell of expressing the complete mevalonate pathway that begins from acetyl-CoA, carry out the substrate oxidation. The example that below produces CadOH utilized 3 kinds of plasmids: (1) contains AtoB, HMGR and HMGS PMevT, (2) pMBIS (contains coding MK, PMK, PMD, IDI (IPP isomerase) and IspA (FPP Synthase) nucleotide sequence) and (3) contain the expression vector of CadH, CPR and CadS. Added blood red 20 ℃ of cultured cells in the TB glycerine of plain additive, ALA. The CadOH that this cell produces Tire up to 60mg/L. Data are seen Figure 32.

In second example, produce Arteannuic acid with following 2 kinds of plasmids: (1) contain coding MevT (AtoB, HMGR and HMGS) (referring to Figure 35 A and B), MBIS (MK, PMK, PMD, IDI and IspA) Contain coding AMO and artemisia annua with the expression vector of the nucleotide sequence of ADS operon and (2) The expression vector of CPR (AACPR) redox partner's nucleotide sequence. Adding the ferroheme additive TB glycerine in 20 ℃ cultivate Bacillus coli cells after, observe the Arteannuic acid of trace with the T7 promoter vector (Figure 33). After carrier changed into pCWOri, AMO can be used for the 3-step oxidation of AMORPHADIENE, with In Escherichia coli, produce to tire and be the Arteannuic acid (Figure 33) of 20mg/L. In addition, be 40-80mg/L with tiring The time aldehyde that produces observe and progressively alcohol be oxidized to aldehyde (Figure 34).

Although described the present invention with reference to the specific embodiment, it will be understood by those skilled in the art that can In the situation that does not deviate from true design of the present invention and scope, carrying out various changes, and available being equal to Thing replaces. In addition, can carry out many modifications so that concrete condition, material, material composition, method, Processing step adapts to purpose of the present invention, design and scope. All such modifications should belong to appended right The scope of claim.

Sequence table

＜110〉M.C.-Y. normal (CHANG, MICHELLE CHIA-YU)

R. Yi Qiesi (EACHUS, RACHEL)

D.-K. sieve (RO, DAE-KYUN)

The quiet hero of lucky state (YOSHIKUNI, YASUO)

J.D. Ji Silin (KEASLING, JAY D.)

＜120〉coding nucleic acid of modified cytochrome P 450 enzymes and its application method

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<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>11

Met?Lys?Gln?Ser?Thr?Ile?Ala?Leu?Ala?Leu?Leu?Pro?Leu?Leu?Phe?Thr

1 5 10 15

Pro?Val?Thr?Lys?Ala

20

<210>12

<211>44

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>12

Met?Lys?Lys?Thr?Ala?Ile?Ala?Ile?Ala?Val?Ala?Leu?Ala?Gly?Phe?Ala

1 5 10 15

Thr?Val?Ala?Gln?Ala?Leu?Leu?Glu?Tyr?Trp?Tyr?Val?Val?Val?Pro?Val

20 25 30

Leu?Tyr?Ile?Ile?Lys?Gln?Leu?Leu?Ala?Tyr?Thr?Lys

35 40

<210>13

<211>24

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>13

Glu?Glu?Leu?Leu?Lys?Gln?Ala?Leu?Gln?Gln?Ala?Gln?Gln?Leu?Leu?Gln

1 5 10 15

Gln?Ala?Gln?Glu?Leu?Ala?Lys?Lys

20

<210>14

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>14

Met?Thr?Val?His?Asp?Ile?Ile?Ala?Thr?Tyr?Phe?Thr?Lys?Trp?Tyr?Val

1 5 10 15

Ile?Val?Pro?Leu?Ala?Leu?Ile?Ala?Tyr?Arg?Val?Leu?Asp?Tyr?Phe?Tyr

20 25 30

<210>15

<211>29

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>15

Gly?Leu?Phe?Gly?Ala?Ile?Ala?Gly?Phe?Ile?Glu?Gly?Gly?Trp?Thr?Gly

1 5 10 15

Met?Ile?Asp?Gly?Trp?Tyr?Gly?Tyr?Gly?Gly?Gly?Lys?Lys

20 25

<210>16

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>16

Met?Ala?Lys?Lys?Thr?Ser?Ser?Lys?Gly

1 5

<210>17

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>17

Ala?Ala?Ala?Gly?Gly?Met

1 5

<210>18

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>18

Ala?Ala?Ala?Gly?Gly?Met?Pro?Pro?Ala?Ala?Ala?Gly?Gly?Met

1 5 10

<210>19

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉joint peptide

<400>19

Ala?Ala?Ala?Gly?Gly?Met

1 5

<210>20

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉joint peptide

<400>20

Pro?Pro?Ala?Ala?Ala?Gly?Gly?Met

1 5

<210>21

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉joint peptide

<400>21

Ile?Glu?Gly?Arg

1

<210>22

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉joint peptide

<400>22

Gly?Gly?Lys?Gly?Gly?Lys

1 5

<210>23

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>23

Met?Leu?Phe?Pro?Val?Ala?Leu?Ser?Phe?Leu?Val?Ala?Ile?Leu?Gly?Ile

1 5 10 15

Ser?Leu?Trp?His?Val?Trp?Thr

20

<210>24

<211>110

<212>PRT

＜213〉subtilis (Bacillus subtilis)

<400>24

Met?Phe?Cys?Thr?Phe?Phe?Glu?Lys?His?His?Arg?Lys?Trp?Asp?Ile?Leu

1 5 10 15

Leu?Glu?Lys?Ser?Thr?Gly?Val?Met?Glu?Ala?Met?Lys?Val?Thr?Ser?Glu

20 25 30

Glu?Lys?Glu?Gln?Leu?Ser?Thr?Ala?Ile?Asp?Arg?Met?Asn?Glu?Gly?Leu

35 40 45

Asp?Ala?Phe?Ile?Gln?Leu?Tyr?Asn?Glu?Ser?Glu?Ile?Asp?Glu?Pro?Leu

50 55 60

Ile?Gln?Leu?Asp?Asp?Asp?Thr?Ala?Glu?Leu?Met?Lys?Gln?Ala?Arg?Asp

65 70 75 80

Met?Tyr?Gly?Gln?Glu?Lys?Leu?Asn?Glu?Lys?Leu?Asn?Thr?Ile?Ile?Lys

85 90 95

Gln?Ile?Leu?Ser?Ile?Ser?Val?Ser?Glu?Glu?Gly?Glu?Lys?Glu

100 105 110

<210>25

<211>496

<212>PRT

＜213〉peppermint (Mentha x gracilis)

<400>25

Met?Glu?Leu?Asp?Leu?Leu?Ser?Ala?Ile?Ile?Ile?Leu?Val?Ala?Thr?Tyr

1 5 10 15

Ile?Val?Ser?Leu?Leu?Ile?Asn?Gln?Trp?Arg?Lys?Ser?Lys?Ser?Gln?Gln

20 25 30

Asn?Leu?Pro?Pro?Ser?Pro?Pro?Lys?Leu?Pro?Val?Ile?Gly?His?Leu?His

35 40 45

Phe?Leu?Trp?Gly?Gly?Leu?Pro?Gln?His?Val?Phe?Arg?Ser?Ile?Ala?Gln

50 55 60

Lys?Tyr?Gly?Pro?Val?Ala?His?Val?Gln?Leu?Gly?Glu?Val?Tyr?Ser?Val

65 70 75 80

Val?Leu?Ser?Ser?Ala?Glu?Ala?Ala?Lys?Gln?Ala?Met?Lys?Val?Leu?Asp

85 90 95

Pro?Asn?Phe?Ala?Asp?Arg?Phe?Asp?Gly?Ile?Gly?Ser?Arg?Thr?Met?Trp

100 105 110

Tyr?Asp?Lys?Asp?Asp?Ile?Ile?Phe?Ser?Pro?Tyr?Asn?Asp?His?Trp?Arg

115 120 125

Gln?Met?Arg?Arg?Ile?Cys?Val?Thr?Glu?Leu?Leu?Ser?Pro?Lys?Asn?Val

130 135 140

Arg?Ser?Phe?Gly?Tyr?Ile?Arg?Gln?Glu?Glu?Ile?Glu?Arg?Leu?Ile?Arg

145 150 155 160

Leu?Leu?Gly?Ser?Ser?Gly?Gly?Ala?Pro?Val?Asp?Val?Thr?Glu?Glu?Val

165 170 175

Ser?Lys?Met?Ser?Cys?Val?Val?Val?Cys?Arg?Ala?Ala?Phe?Gly?Ser?Val

180 185 190

Leu?Lys?Asp?Gln?Gly?Ser?Leu?Ala?Glu?Leu?Val?Lys?Glu?Ser?Leu?Ala

195 200 205

Leu?Ala?Ser?Gly?Phe?Glu?Leu?Ala?Asp?Leu?Tyr?Pro?Ser?Ser?Trp?Leu

210 215 220

Leu?Asn?Leu?Leu?Ser?Leu?Asn?Lys?Tyr?Arg?Leu?Gln?Arg?Met?Arg?Arg

225 230 235 240

Arg?Leu?Asp?His?Ile?Leu?Asp?Gly?Phe?Leu?Glu?Glu?His?Arg?Glu?Lys

245 250 255

Lys?Ser?Gly?Glu?Phe?Gly?Gly?Glu?Asp?Ile?Val?Asp?Val?Leu?Phe?Arg

260 265 270

Met?Gln?Lys?Gly?Ser?Asp?Ile?Lys?Ile?Pro?Ile?Thr?Ser?Asn?Cys?Ile

275 280 285

Lys?Gly?Phe?Ile?Phe?Asp?Thr?Phe?Ser?Ala?Gly?Ala?Glu?Thr?Ser?Ser

290 295 300

Thr?Thr?Ile?Ser?Trp?Ala?Leu?Ser?Glu?Leu?Met?Arg?Asn?Pro?Ala?Lys

305 310 315 320

Met?Ala?Lys?Val?Gln?Ala?Glu?Val?Arg?Glu?Ala?Leu?Lys?Gly?Lys?Thr

325 330 335

Val?Val?Asp?Leu?Ser?Glu?Val?Gln?Glu?Leu?Lys?Tyr?Leu?Arg?Ser?Val

340 345 350

Leu?Lys?Glu?Thr?Leu?Arg?Leu?His?Pro?Pro?Phe?Pro?Leu?Ile?Pro?Arg

355 360 365

Gln?Ser?Arg?Glu?Glu?Cys?Glu?Val?Asn?Gly?Tyr?Thr?Ile?Pro?Ala?Lys

370 375 380

Thr?Arg?Ile?Phe?Ile?Asn?Val?Trp?Ala?Ile?Gly?Arg?Asp?Pro?Gln?Tyr

385 390 395 400

Trp?Glu?Asp?Pro?Asp?Thr?Phe?Arg?Pro?Glu?Arg?Phe?Asp?Glu?Val?Ser

405 410 415

Arg?Asp?Phe?Met?Gly?Asn?Asp?Phe?Glu?Phe?Ile?Pro?Phe?Gly?Ala?Gly

420 425 430

Arg?Arg?Ile?Cys?Pro?Gly?Leu?His?Phe?Gly?Leu?Ala?Asn?Val?Glu?Ile

435 440 445

Pro?Leu?Ala?Gln?Leu?Leu?Tyr?His?Phe?Asp?Trp?Lys?Leu?Pro?Gln?Gly

450 455 460

Met?Thr?Asp?Ala?Asp?Leu?Asp?Met?Thr?Glu?Thr?Pro?Gly?Leu?Ser?Gly

465 470 475 480

Pro?Lys?Lys?Lys?Asn?Val?Cys?Leu?Val?Pro?Thr?Leu?Tyr?Lys?Ser?Pro

485 490 495

<210>26

<211>473

<212>PRT

＜213〉tobacco (Nicotiana tabacum)

<400>26

Met?Gln?Phe?Phe?Ser?Leu?Val?Ser?Ile?Phe?Leu?Phe?Leu?Ala?Phe?Leu

1 5 10 15

Phe?Leu?Leu?Arg?Lys?Trp?Lys?Asn?Ser?Asn?Ser?Gln?Ser?Lys?Lys?Leu

20 25 30

Pro?Pro?Gly?Pro?Trp?Lys?Ile?Pro?Ile?Leu?Gly?Ser?Met?Leu?His?Met

35 40 45

Ile?Gly?Gly?Glu?Pro?His?His?Val?Leu?Arg?Asp?Leu?Ala?Lys?Lys?Tyr

50 55 60

Gly?Pro?Leu?Met?His?Leu?Gln?Leu?Gly?Glu?Ile?Ser?Ala?Val?Val?Val

65 70 75 80

Thr?Ser?Arg?Asp?Met?Ala?Lys?Glu?Val?Leu?Lys?Thr?His?Asp?Val?Val

85 90 95

Phe?Ala?Ser?Arg?Pro?Lys?Ile?Val?Ala?Met?Asp?Ile?Ile?Cys?Tyr?Asn

100 105 110

Gln?Ser?Asp?Ile?Ala?Phe?Ser?Pro?Tyr?Gly?Asp?His?Trp?Arg?Gln?Met

115 120 125

Arg?Lys?Ile?Cys?Val?Met?Glu?Leu?Leu?Asn?Ala?Lys?Asn?Val?Arg?Ser

130 135 140

Phe?Ser?Ser?Ile?Arg?Arg?Asp?Glu?Val?Val?Arg?Leu?Ile?Asp?Ser?Ile

145 150 155 160

Arg?Ser?Asp?Ser?Ser?Ser?Gly?Glu?Leu?Val?Asn?Phe?Thr?Gln?Arg?Ile

165 170 175

Ile?Trp?Phe?Ala?Ser?Ser?Met?Thr?Cys?Arg?Ser?Ala?Phe?Gly?Gln?Val

180 185 190

Leu?Lys?Gly?Gln?Asp?Ile?Phe?Ala?Lys?Lys?Ile?Arg?Glu?Val?Ile?Gly

195 200 205

Leu?Ala?Glu?Gly?Phe?Asp?Val?Val?Asp?Ile?Phe?Pro?Thr?Tyr?Lys?Phe

210 215 220

Leu?His?Val?Leu?Ser?Gly?Met?Lys?Arg?Lys?Leu?Leu?Asn?Ala?His?Leu

225 230 235 240

Lys?Val?Asp?Ala?Ile?Val?Glu?Asp?Val?Ile?Asn?Glu?His?Lys?Lys?Asn

245 250 255

Leu?Ala?Ala?Gly?Lys?Ser?Asn?Gly?Ala?Leu?Glu?Asp?Met?Phe?Ala?Ala

260 265 270

Gly?Thr?Glu?Thr?Ser?Ser?Thr?Thr?Thr?Val?Trp?Ala?Met?Ala?Glu?Met

275 280 285

Met?Lys?Asn?Pro?Ser?Val?Phe?Thr?Lys?Ala?Gln?Ala?Glu?Val?Arg?Glu

290 295 300

Ala?Phe?Arg?Asp?Lys?Val?Ser?Phe?Asp?Glu?Asn?Asp?Val?Glu?Glu?Leu

305 310 315 320

Lys?Tyr?Leu?Lys?Leu?Val?Ile?Lys?Glu?Thr?Leu?Arg?Leu?His?Pro?Pro

325 330 335

Ser?Pro?Leu?Leu?Val?Pro?Arg?Glu?Cys?Arg?Glu?Asp?Thr?Asp?Ile?Asn

340 345 350

Gly?Tyr?Thr?Ile?Pro?Ala?Lys?Thr?Lys?Val?Met?Val?Asn?Val?Trp?Ala

355 360 365

Leu?Gly?Arg?Asp?Pro?Lys?Tyr?Trp?Asp?Asp?Ala?Glu?Ser?Phe?Lys?Pro

370 375 380

Glu?Arg?Phe?Glu?Gln?Cys?Ser?Val?Asp?Phe?Phe?Gly?Asn?Asn?Phe?Glu

385 390 395 400

Phe?Leu?Pro?Phe?Gly?Gly?Gly?Arg?Arg?Ile?Cys?Pro?Gly?Met?Ser?Phe

405 410 415

Gly?Leu?Ala?Asn?Leu?Tyr?Leu?Pro?Leu?Ala?Gln?Leu?Leu?Tyr?His?Phe

420 425 430

Asp?Trp?Lys?Leu?Pro?Thr?Gly?Ile?Met?Pro?Arg?Asp?Leu?Asp?Leu?Thr

435 440 445

Glu?Leu?Ser?Gly?Ile?Thr?Ile?Ala?Arg?Lys?Gly?Asp?Leu?Tyr?Leu?Asn

450 455 460

Ala?Thr?Pro?Tyr?Gln?Pro?Ser?Arg?Glu

465 470

<210>27

<211>536

<212>PRT

＜213〉tree continuous (Gossypium arboreum)

<400>27

Met?Leu?Gln?Ile?Ala?Phe?Ser?Ser?Tyr?Ser?Trp?Leu?Leu?Thr?Ala?Ser

1 5 10 15

Asn?Gln?Lys?Asp?Gly?Met?Leu?Phe?Pro?Val?Ala?Leu?Ser?Phe?Leu?Val

20 25 30

Ala?Ile?Leu?Gly?Ile?Ser?Leu?Trp?His?Val?Trp?Thr?Ile?Arg?Lys?Pro

35 40 45

Lys?Lys?Asp?Ile?Ala?Pro?Leu?Pro?Pro?Gly?Pro?Arg?Gly?Leu?Pro?Ile

50 55 60

Val?Gly?Tyr?Leu?Pro?Tyr?Leu?Gly?Thr?Asp?Asn?Leu?His?Leu?Val?Phe

65 70 75 80

Thr?Asp?Leu?Ala?Ala?Ala?Tyr?Gly?Pro?Ile?Tyr?Lys?Leu?Trp?Leu?Gly

85 90 95

Asn?Lys?Leu?Cys?Val?Val?Ile?Ser?Ser?Ala?Pro?Leu?Ala?Lys?Glu?Val

100 105 110

Val?Arg?Asp?Asn?Asp?Ile?Thr?Phe?Ser?Glu?Arg?Asp?Pro?Pro?Val?Cys

115 120 125

Ala?Lys?Ile?Ile?Thr?Phe?Gly?Leu?Asn?Asp?Ile?Val?Phe?Asp?Ser?Tyr

130 135 140

Ser?Ser?Pro?Asp?Trp?Arg?Met?Lys?Arg?Lys?Val?Leu?Val?Arg?Glu?Met

145 150 155 160

Leu?Ser?His?Ser?Ser?Ile?Lys?Ala?Cys?Tyr?Gly?Leu?Arg?Arg?Glu?Gln

165 170 175

Val?Leu?Lys?Gly?Val?Gln?Asn?Val?Ala?Gln?Ser?Ala?Gly?Lys?Pro?Ile

180 185 190

Asp?Phe?Gly?Glu?Thr?Ala?Phe?Leu?Thr?Ser?Ile?Asn?Ala?Met?Met?Ser

195 200 205

Met?Leu?Trp?Gly?Gly?Lys?Gln?Gly?Gly?Glu?Arg?Lys?Gly?Ala?Asp?Val

210 215 220

Trp?Gly?Gln?Phe?Arg?Asp?Leu?Ile?Thr?Glu?Leu?Met?Val?Ile?Leu?Gly

225 230 235 240

Lys?Pro?Asn?Val?Ser?Asp?Ile?Phe?Pro?Val?Leu?Ala?Arg?Phe?Asp?Ile

245 250 255

Gln?Gly?Leu?Glu?Lys?Glu?Met?Thr?Lys?Ile?Val?Asn?Ser?Phe?Asp?Lys

260 265 270

Leu?Phe?Asn?Ser?Met?Ile?Glu?Glu?Arg?Glu?Asn?Phe?Ser?Asn?Lys?Leu

275 280 285

Ser?Lys?Glu?Asp?Gly?Asn?Thr?Glu?Thr?Lys?Asp?Phe?Leu?Gln?Leu?Leu

290 295 300

Leu?Asp?Leu?Lys?Gln?Lys?Asn?Asp?Ser?Gly?Ile?Ser?Ile?Thr?Met?Asn

305 310 315 320

Gln?Val?Lys?Ala?Leu?Leu?Met?Asp?Ile?Val?Val?Gly?Gly?Thr?Asp?Thr

325 330 335

Thr?Ser?Thr?Met?Met?Glu?Trp?Thr?Met?Ala?Glu?Leu?Ile?Ala?Ash?Pro

340 345 350

Glu?Ala?Met?Lys?Lys?Val?Lys?Gln?Glu?Ile?Asp?Asp?Val?Val?Gly?Ser

355 360 365

Asp?Gly?Ala?Val?Asp?Glu?Thr?His?Leu?Pro?Lys?Leu?Arg?Tyr?Leu?Asp

370 375 380

Ala?Ala?Val?Lys?Glu?Thr?Phe?Arg?Leu?His?Pro?Pro?Met?Pro?Leu?Leu

385 390 395 400

Val?Pro?Arg?Cys?Pro?Gly?Asp?Ser?Ser?Asn?Val?Gly?Gly?Tyr?Ser?Val

405 410 415

Pro?Lys?Gly?Thr?Arg?Val?Phe?Leu?Asn?Ile?Trp?Cys?Ile?Gln?Arg?Asp

420 425 430

Pro?Gln?Leu?Trp?Glu?Asn?Pro?Leu?Glu?Phe?Lys?Pro?Glu?Arg?Phe?Leu

435 440 445

Thr?Asp?His?Glu?Lys?Leu?Asp?Tyr?Leu?Gly?Asn?Asp?Ser?Arg?Tyr?Met

450 455 460

Pro?Phe?Gly?Ser?Gly?Arg?Arg?Met?Cys?Ala?Gly?Val?Ser?Leu?Gly?Glu

465 470 475 480

Lys?Met?Leu?Tyr?Ser?Ser?Leu?Ala?Ala?Met?Ile?His?Ala?Tyr?Asp?Trp

485 490 495

Asn?Leu?Ala?Asp?Gly?Glu?Glu?Asn?Asp?Leu?Ile?Gly?Leu?Phe?Gly?Ile

500 505 510

Ile?Met?Lys?Lys?Lys?Lys?Pro?Leu?Ile?Leu?Val?Pro?Thr?Pro?Arg?Pro

515 520 525

Ser?Asn?Leu?Gln?His?Tyr?Met?Lys

530 535

<210>28

<211>512

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the P450 monooxygenase of Xiu Shiing

<400>28

Met?Ala?Leu?Leu?Leu?Ala?Val?Phe?Leu?Gly?Leu?Ser?Cys?Leu?Leu?Leu

1 5 10 15

Leu?Ser?Leu?Trp?Ile?Arg?Lys?Pro?Lys?Lys?Asp?Ile?Ala?Pro?Leu?Pro

20 25 30

Pro?Gly?Pro?Arg?Gly?Leu?Pro?Ile?Val?Gly?Tyr?Leu?Pro?Tyr?Leu?Gly

35 40 45

Thr?Asp?Asn?Leu?His?Leu?Val?Phe?Thr?Asp?Leu?Ala?Ala?Ala?Tyr?Gly

50 55 60

Pro?Ile?Tyr?Lys?Leu?Trp?Leu?Gly?Asn?Lys?Leu?Cys?Val?Val?Ile?Ser

65 70 75 80

Ser?Ala?Pro?Leu?Ala?Lys?Glu?Val?Val?Arg?Asp?Asn?Asp?Ile?Thr?Phe

85 90 95

Ser?Glu?Arg?Asp?Pro?Pro?Val?Cys?Ala?Lys?Ile?Ile?Thr?Phe?Gly?Leu

100 105 110

Asn?Asp?Ile?Val?Phe?Asp?Ser?Tyr?Ser?Ser?Pro?Asp?Trp?Arg?Met?Lys

115 120 125

Arg?Lys?Val?Leu?Val?Arg?Glu?Met?Leu?Ser?His?Ser?Ser?Ile?Lys?Ala

130 135 140

Cys?Tyr?Gly?Leu?Arg?Arg?Glu?Gln?Val?Leu?Lys?Gly?Val?Gln?Asn?Val

145 150 155 160

Ala?Gln?Ser?Ala?Gly?Lys?Pro?Ile?Asp?Phe?Gly?Glu?Thr?Ala?Phe?Leu

165 170 175

Thr?Ser?Ile?Asn?Ala?Met?Met?Ser?Met?Leu?Trp?Gly?Gly?Lys?Gln?Gly

180 185 190

Gly?Glu?Arg?Lys?Gly?Ala?Asp?Val?Trp?Gly?Gln?Phe?Arg?Asp?Leu?Ile

195 200 205

Thr?Glu?Leu?Met?Val?Ile?Leu?Gly?Lys?Pro?Asn?Val?Ser?Asp?Ile?Phe

210 215 220

Pro?Val?Leu?Ala?Arg?Phe?Asp?Ile?Gln?Gly?Leu?Glu?Lys?Glu?Met?Thr

225 230 235 240

Lys?Ile?Val?Asn?Ser?Phe?Asp?Lys?Leu?Phe?Asn?Ser?Met?Ile?Glu?Glu

245 250 255

Arg?Glu?Asn?Phe?Ser?Asn?Lys?Leu?Ser?Lys?Glu?Asp?Gly?Asn?Thr?Glu

260 265 270

Thr?Lys?Asp?Phe?Leu?Gln?Leu?Leu?Leu?Asp?Leu?Lys?Gln?Lys?Asn?Asp

275 280 285

Ser?Gly?Ile?Ser?Ile?Thr?Met?Asn?Gln?Val?Lys?Ala?Leu?Leu?Met?Asp

290 295 300

Ile?Val?Val?Gly?Gly?Thr?Asp?Thr?Thr?Ser?Thr?Met?Met?Glu?Trp?Thr

305 310 315 320

Met?Ala?Glu?Leu?Ile?Ala?Asn?Pro?Glu?Ala?Met?Lys?Lys?Val?Lys?Gln

325 330 335

Glu?Ile?Asp?Asp?Val?Val?Gly?Ser?Asp?Gly?Ala?Val?Asp?Glu?Thr?His

340 345 350

Leu?Pro?Lys?Leu?Arg?Tyr?Leu?Asp?Ala?Ala?Val?Lys?Glu?Thr?Phe?Arg

355 360 365

Leu?His?Pro?Pro?Met?Pro?Leu?Leu?Val?Pro?Arg?Cys?Pro?Gly?Asp?Ser

370 375 380

Ser?Asn?Val?Gly?Gly?Tyr?Ser?Val?Pro?Lys?Gly?Thr?Arg?Val?Phe?Leu

385 390 395 400

Asn?Ile?Trp?Cys?Ile?Gln?Arg?Asp?Pro?Gln?Leu?Trp?Glu?Asn?Pro?Leu

405 410 415

Glu?Phe?Lys?Pro?Glu?Arg?Phe?Leu?Thr?Asp?His?Glu?Lys?Leu?Asp?Tyr

420 425 430

Leu?Gly?Asn?Asp?Ser?Arg?Tyr?Met?Pro?Phe?Gly?Ser?Gly?Arg?Arg?Met

435 440 445

Cys?Ala?Gly?Val?Ser?Leu?Gly?Glu?Lys?Met?Leu?Tyr?Ser?Ser?Leu?Ala

450 455 460

Ala?Met?Ile?His?Ala?Tyr?Asp?Trp?Asn?Leu?Ala?Asp?Gly?Glu?Glu?Asn

465 470 475 480

Asp?Leu?Ile?Gly?Leu?Phe?Gly?Ile?Ile?Met?Lys?Lys?Lys?Lys?Pro?Leu

485 490 495

Ile?Leu?Val?Pro?Thr?Pro?Arg?Pro?Ser?Asn?Leu?Gln?His?Tyr?Met?Lys

500 505 510

<210>29

<211>516

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the P450 monooxygenase of Xiu Shiing

<400>29

Glu?Glu?Leu?Leu?Lys?Gln?Ala?Leu?Gln?Gln?Ala?Gln?Gln?Leu?Leu?Gln

1 5 10 15

Gln?Ala?Gln?Glu?Leu?Ala?Lys?Lys?Ile?Arg?Lys?Pro?Lys?Lys?Asp?Ile

20 25 30

Ala?Pro?Leu?Pro?Pro?Gly?Pro?Arg?Gly?Leu?Pro?Ile?Val?Gly?Tyr?Leu

35 40 45

Pro?Tyr?Leu?Gly?Thr?Asp?Asn?Leu?His?Leu?Val?Phe?Thr?Asp?Leu?Ala

50 55 60

Ala?Ala?Tyr?Gly?Pro?Ile?Tyr?Lys?Leu?Trp?Leu?Gly?Asn?Lys?Leu?Cys

65 70 75 80

Val?Val?Ile?Ser?Ser?Ala?Pro?Leu?Ala?Lys?Glu?Val?Val?Arg?Asp?Asn

85 90 95

Asp?Ile?Thr?Phe?Ser?Glu?Arg?Asp?Pro?Pro?Val?Cys?Ala?Lys?Ile?Ile

100 105 110

Thr?Phe?Gly?Leu?Asn?Asp?Ile?Val?Phe?Asp?Ser?Tyr?Ser?Ser?Pro?Asp

115 120 125

Trp?Arg?Met?Lys?Arg?Lys?Val?Leu?Val?Arg?Glu?Met?Leu?Ser?His?Ser

130 135 140

Ser?Ile?Lys?Ala?Cys?Tyr?Gly?Leu?Arg?Arg?Glu?Gln?Val?Leu?Lys?Gly

145 150 155 160

Val?Gln?Asn?Val?Ala?Gln?Ser?Ala?Gly?Lys?Pro?Ile?Asp?Phe?Gly?Glu

165 170 175

Thr?Ala?Phe?Leu?Thr?Ser?Ile?Asn?Ala?Met?Met?Ser?Met?Leu?Trp?Gly

180 185 190

Gly?Lys?Gln?Gly?Gly?Glu?Arg?Lys?Gly?Ala?Asp?Val?Trp?Gly?Gln?Phe

195 200 205

Arg?Asp?Leu?Ile?Thr?Glu?Leu?Met?Val?Ile?Leu?Gly?Lys?Pro?Asn?Val

210 215 220

Ser?Asp?Ile?Phe?Pro?Val?Leu?Ala?ArgPhe?Asp?Ile?Gln?Gly?Leu?Glu

225 230 235 240

Lys?Glu?Met?Thr?Lys?Ile?Val?Asn?Ser?Phe?Asp?Lys?Leu?Phe?Asn?Ser

245 250 255

Met?Ile?Glu?Glu?Arg?Glu?Asn?Phe?SerAsn?Lys?Leu?Ser?Lys?Glu?Asp

260 265 270

Gly?Asn?Thr?Glu?Thr?Lys?Asp?Phe?Leu?Gln?Leu?Leu?Leu?Asp?Leu?Lys

275 280 285

Gln?Lys?Asn?Asp?Ser?Gly?Ile?Ser?Ile?Thr?Met?Asn?Gln?Val?Lys?Ala

290 295 300

Leu?Leu?Met?Asp?Ile?Val?Val?Gly?Gly?Thr?Asp?Thr?Thr?Ser?Thr?Met

305 310 315 320

Met?Glu?Trp?Thr?Met?Ala?Glu?Leu?IleAla?Asn?Pro?Glu?Ala?Met?Lys

325 330 335

Lys?Val?Lys?Gln?Glu?Ile?Asp?Asp?Val?Val?Gly?Ser?Asp?Gly?Ala?Val

340 345 350

Asp?Glu?Thr?His?Leu?Pro?Lys?Leu?Arg?Tyr?Leu?Asp?Ala?Ala?Val?Lys

355 360 365

Glu?Thr?Phe?Arg?Leu?His?Pro?Pro?Met?Pro?Leu?Leu?Val?Pro?Arg?Cys

370 375 380

Pro?Gly?Asp?Ser?Ser?Asn?Val?Gly?Gly?Tyr?Ser?Val?Pro?Lys?Gly?Thr

385 390 395 400

Arg?Val?Phe?Leu?Asn?Ile?Trp?Cys?Ile?Gln?Arg?Asp?Pro?Gln?Leu?Trp

405 410 415

Glu?Asn?Pro?Leu?Glu?Phe?Lys?Pro?Glu?Arg?Phe?Leu?Thr?Asp?His?Glu

420 425 430

Lys?Leu?Asp?Tyr?Leu?Gly?Asn?Asp?Ser?Arg?Tyr?Met?Pro?Phe?Gly?Ser

435 440 445

Gly?Arg?Arg?Met?Cys?Ala?Gly?Val?Ser?Leu?Gly?Glu?Lys?Met?Leu?Tyr

450 455 460

Ser?Ser?Leu?Ala?Ala?Met?Ile?His?Ala?Tyr?Asp?Trp?Asn?Leu?Ala?Asp

465 470 475 480

Gly?Glu?Glu?Asn?Asp?Leu?Ile?Gly?Leu?Phe?Gly?Ile?Ile?Met?Lys?Lys

485 490 495

Lys?Lys?Pro?Leu?Ile?Leu?Val?Pro?Thr?Pro?Arg?Pro?Ser?Asn?Leu?Gln

500 505 510

His?Tyr?Met?Lys

515

<210>30

<211>536

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the P450 monooxygenase of Xiu Shiing

<400>30

Met?Lys?Lys?Thr?Ala?Ile?Ala?Ile?Ala?Val?Ala?Leu?Ala?Gly?Phe?Ala

1 5 10 15

Thr?Val?Ala?Gln?Ala?Leu?Leu?Glu?Tyr?Trp?Tyr?Val?Val?Val?Pro?Val

20 25 30

Leu?Tyr?Ile?Ile?Lys?Gln?Leu?Leu?Ala?Tyr?Thr?Lys?Ile?Arg?Lys?Pro

35 40 45

Lys?Lys?Asp?Ile?Ala?Pro?Leu?Pro?Pro?Gly?Pro?Arg?Gly?Leu?Pro?Ile

50 55 60

Val?Gly?Tyr?Leu?Pro?Tyr?Leu?Gly?Thr?Asp?Asn?Leu?His?Leu?Val?Phe

65 70 75 80

Thr?Asp?Leu?Ala?Ala?Ala?Tyr?Gly?Pro?Ile?Tyr?Lys?Leu?Trp?Leu?Gly

85 90 95

Asn?Lys?Leu?Cys?Val?Val?Ile?Ser?Ser?Ala?Pro?Leu?Ala?Lys?Glu?Val

100 105 110

Val?Arg?Asp?Asn?Asp?Ile?Thr?Phe?Ser?Glu?Arg?Asp?Pro?Pro?Val?Cys

115 120 125

Ala?Lys?Ile?Ile?Thr?Phe?Gly?Leu?Asn?Asp?Ile?Val?Phe?Asp?Ser?Tyr

130 135 140

Ser?Ser?Pro?Asp?Trp?Arg?Met?Lys?Arg?Lys?Val?Leu?Val?Arg?Glu?Met

145 150 155 160

Leu?Ser?His?Ser?Ser?Ile?Lys?Ala?Cys?Tyr?Gly?Leu?Arg?Arg?6lu?Gln

165 170 175

Val?Leu?Lys?Gly?Val?Gln?Asn?Val?Ala?Gln?Ser?Ala?Gly?Lys?Pro?Ile

180 185 190

Asp?Phe?Gly?Glu?Thr?Ala?Phe?Leu?Thr?Ser?Ile?Asn?Ala?Met?Met?Ser

195 200 205

Met?Leu?Trp?Gly?Gly?Lys?Gln?Gly?Gly?Glu?Arg?Lys?Gly?Ala?Asp?Val

210 215 220

Trp?Gly?Gln?Phe?Arg?Asp?Leu?Ile?Thr?Glu?Leu?Met?Val?Ile?Leu?Gly

225 230 235 240

Lys?Pro?Asn?Val?Ser?Asp?Ile?Phe?Pro?Val?Leu?Ala?Arg?Phe?Asp?Ile

245 250 255

Gln?Gly?Leu?Glu?Lys?Glu?Met?Thr?Lys?Ile?Val?Asn?Ser?Phe?Asp?Lys

260 265 270

Leu?Phe?Asn?Ser?Met?Ile?Glu?Glu?Arg?Glu?Asn?Phe?Ser?Asn?Lys?Leu

275 280 285

Ser?Lys?Glu?Asp?Gly?Asn?Thr?Glu?Thr?Lys?Asp?Phe?Leu?Gln?Leu?Leu

290 295 300

Leu?Asp?Leu?Lys?Gln?Lys?Asn?Asp?Ser?Gly?Ile?Ser?Ile?Thr?Met?Asn

305 310 315 320

Gln?Val?Lys?Ala?Leu?Leu?Met?Asp?Ile?Val?Val?Gly?Gly?Thr?Asp?Thr

325 330 335

Thr?Ser?Thr?Met?Met?Glu?Trp?Thr?Met?Ala?Glu?Leu?Ile?Ala?Asn?Pro

340 345 350

Glu?Ala?Met?Lys?Lys?Val?Lys?Gln?Glu?Ile?Asp?Asp?Val?Val?Gly?Ser

355 360 365

Asp?Gly?Ala?Val?Asp?Glu?Thr?His?Leu?Pro?Lys?Leu?Arg?Tyr?Leu?Asp

370 375 380

Ala?Ala?Val?Lys?Glu?Thr?Phe?Arg?Leu?His?Pro?Pro?Met?Pro?Leu?Leu

385 390 395 400

Val?Pro?Arg?Cys?Pro?Gly?Asp?Ser?Ser?Asn?Val?Gly?Gly?Tyr?Ser?Val

405 410 415

Pro?Lys?Gly?Thr?Arg?Val?Phe?Leu?Asn?Ile?Trp?Cys?Ile?Gln?Arg?Asp

420 425 430

Pro?Gln?Leu?Trp?Glu?Asn?Pro?Leu?Glu?Phe?Lys?Pro?Glu?Arg?Phe?Leu

435 440 445

Thr?Asp?His?Glu?Lys?Leu?Asp?Tyr?Leu?Gly?Asn?Asp?Ser?Arg?Tyr?Met

450 455 460

Pro?Phe?Gly?Ser?Gly?Arg?Arg?Met?Cys?Ala?Gly?Val?Ser?Leu?Gly?Glu

465 470 475 480

Lys?Met?Leu?Tyr?Ser?Ser?Leu?Ala?Ala?Met?Ile?His?Ala?Tyr?Asp?Trp

485 490 495

Asn?Leu?Ala?Asp?Gly?Glu?Glu?Asn?Asp?Leu?Ile?Gly?Leu?Phe?Gly?Ile

500 505 510

Ile?Met?Lys?Lys?Lys?Lys?Pro?Leu?Ile?Leu?Val?Pro?Thr?Pro?Arg?Pro

515 520 525

Ser?Asn?Leu?Gln?His?Tyr?Met?Lys

530 535

<210>31

<211>502

<212>PRT

＜213〉taxus chinensis in northeast (Taxus cuspidata)

<400>31

Met?Asp?Ala?Leu?Tyr?Lys?Ser?Thr?Val?Ala?Lys?Phe?Asn?Glu?Val?Thr

1 5 10 15

Gln?Leu?Asp?Cys?Ser?Thr?Glu?Ser?Phe?Ser?Ile?Ala?Leu?Ser?Ala?Ile

20 25 30

Ala?Gly?Ile?Leu?Leu?Leu?Leu?Leu?Leu?Phe?Arg?Ser?Lys?Arg?His?Ser

35 40 45

Ser?Leu?Lys?Leu?Pro?Pro?Gly?Lys?Leu?Gly?Ile?Pro?Phe?Ile?Gly?Glu

50 55 60

Ser?Phe?Ile?Phe?Leu?Arg?Ala?Leu?Arg?Ser?Asn?Ser?Leu?Glu?Gln?Phe

65 70 75 80

Phe?Asp?Glu?Arg?Val?Lys?Lys?Phe?Gly?Leu?Val?Phe?Lys?Thr?Ser?Leu

85 90 95

Ile?Gly?His?Pro?Thr?Val?Val?Leu?Cys?Gly?Pro?Ala?Gly?Asn?Arg?Leu

100 105 110

Ile?Leu?Ser?Asn?Glu?Glu?Lys?Leu?Val?Gln?Met?Ser?Trp?Pro?Ala?Gln

115 120 125

Phe?Met?Lys?Leu?Met?Gly?Glu?Asn?Ser?Val?Ala?Thr?Arg?Arg?Gly?Glu

130 135 140

Asp?His?Ile?Val?Met?Arg?Ser?Ala?Leu?Ala?Gly?Phe?Phe?Gly?Pro?Gly

145 150 155 160

Ala?Leu?Gln?Ser?Tyr?Ile?Gly?Lys?Met?Asn?Thr?Glu?Ile?Gln?Ser?His

165 170 175

Ile?Asn?Glu?Lys?Trp?Lys?Gly?Lys?Asp?Glu?Val?Asn?Val?Leu?Pro?Leu

180 185 190

Val?Arg?Glu?Leu?Val?Phe?Asn?Ile?Ser?Ala?Ile?Leu?Phe?Phe?Asn?Ile

195 200 205

Tyr?Asp?Lys?Gln?Glu?Gln?Asp?Arg?Leu?His?Lys?Leu?Leu?Glu?Thr?Ile

210 215 220

Leu?Val?Gly?Ser?Phe?Ala?Leu?Pro?Ile?Asp?Leu?Pro?Gly?Phe?Gly?Phe

225 230 235 240

His?Arg?Ala?Leu?Gln?Gly?Arg?Ala?Lys?Leu?Asn?Lys?Ile?Met?Leu?Ser

245 250 255

Leu?Ile?Lys?Lys?Arg?Lys?Glu?Asp?Leu?Gln?Ser?Gly?Ser?Ala?Thr?Ala

260 265 270

Thr?Gln?Asp?Leu?Leu?Ser?Val?Leu?Leu?Thr?Phe?Arg?Asp?Asp?Lys?Gly

275 280 285

Thr?Pro?Leu?Thr?Asn?Asp?Glu?Ile?Leu?Asp?Asn?Phe?Ser?Ser?Leu?Leu

290 295 300

His?Ala?Ser?Tyr?Asp?Thr?Thr?Thr?Ser?Pro?Met?Ala?Leu?Ile?Phe?Lys

305 310 315 320

Leu?Leu?Ser?Ser?Asn?Pro?Glu?Cys?Tyr?Gln?Lys?Val?Val?Gln?Glu?Gln

325 330 335

Leu?Glu?Ile?Leu?Ser?Asn?Lys?Glu?Glu?Gly?Glu?Glu?Ile?Thr?Trp?Lys

340 345 350

Asp?Leu?Lys?Ala?Met?Lys?Tyr?Thr?Trp?Gln?Val?Ala?Gln?Glu?Thr?Leu

355 360 365

Arg?Met?Phe?Pro?Pro?Val?Phe?Gly?Thr?Phe?Arg?Lys?Ala?Ile?Thr?Asp

370 375 380

Ile?Gln?Tyr?Asp?Gly?Tyr?Thr?Ile?Pro?Lys?Gly?Trp?Lys?Leu?Leu?Trp

385 390 395 400

Thr?Thr?Tyr?Ser?Thr?His?Pro?Lys?Asp?Leu?Tyr?Phe?Asn?Glu?Pro?Glu

405 410 415

Lys?Phe?Met?Pro?Ser?Arg?Phe?Asp?Gln?Glu?Gly?Lys?His?Val?Ala?Pro

420 425 430

Tyr?Thr?Phe?Leu?Pro?Phe?Gly?Gly?Gly?Gln?Arg?Ser?Cys?Val?Gly?Trp

435 440 445

Glu?Phe?Ser?Lys?Met?Glu?Ile?Leu?Leu?Phe?Val?His?His?Phe?Val?Lys

450 455 460

Thr?Phe?Ser?Ser?Tyr?Thr?Pro?Val?Asp?Pro?Asp?Glu?Lys?Ile?Ser?Gly

465 470 475 480

Asp?Pro?Leu?Pro?Pro?Leu?Pro?Ser?Lys?Gly?Phe?Ser?Ile?Lys?Leu?Phe

485 490 495

Pro?Glu?Thr?Ile?Val?Asn

500

<210>32

<211>502

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the Japanese yew diene-5 α-hydroxylase of Xiu Shiing

<400>32

Met?Asp?Ala?Leu?Tyr?Lys?Ser?Thr?Val?Ala?Lys?Phe?Asn?Glu?Val?Thr

1 5 10 15

Gln?Leu?Asp?Cys?Ser?Thr?Glu?Ser?Phe?Ser?Ile?Ala?Leu?Ser?Ser?Ile

20 25 30

Ala?Gly?Ile?Leu?Leu?Leu?Leu?Leu?Leu?Phe?Arg?Ser?Lys?Arg?His?Ser

35 40 45

Ser?Leu?Lys?Leu?Pro?Pro?Gly?Lys?Leu?Gly?Ile?Pro?Phe?Ile?Gly?Glu

50 55 60

Ser?Phe?Ile?Phe?Leu?Arg?Ala?Leu?Arg?Ser?Asn?Ser?Leu?Glu?Gln?Phe

65 70 75 80

Phe?Asp?Glu?Arg?Val?Lys?Lys?Phe?Gly?Leu?Val?Phe?Lys?Thr?Ser?Leu

85 90 95

Ile?Gly?His?Pro?Thr?Val?Val?Leu?Cys?Gly?Pro?Ala?Gly?Asn?Arg?Leu

100 105 110

Ile?Leu?Ser?Asn?Glu?Glu?Lys?Leu?Val?Gln?Met?Ser?Trp?Pro?Ala?Gln

115 120 125

Phe?Met?Lys?Leu?Met?Gly?Glu?Asn?Ser?Val?Ala?Thr?Arg?Arg?Gly?Glu

130 135 140

Asp?His?Ile?Val?Met?Arg?Ser?Ala?Leu?Ala?Gly?Phe?Phe?Gly?Pro?Gly

145 150 155 160

Ala?Leu?Gln?Ser?Tyr?Ile?Gly?Lys?Met?Asn?Thr?Glu?Ile?Gln?Asn?His

165 170 175

Ile?Asn?Glu?Lys?Trp?Lys?Gly?Lys?Asp?Glu?Val?Asn?Val?Leu?Pro?Leu

180 185 190

Val?Arg?Glu?Leu?Val?Phe?Asn?Ile?Ser?Ala?Ile?Leu?Phe?Phe?Asn?Ile

195 200 205

Tyr?Asp?Lys?Gln?Glu?Gln?Asp?Arg?Leu?His?Lys?Leu?Leu?Glu?Thr?Ile

210 215 220

Leu?Val?Gly?Ser?Phe?Ala?Leu?Pro?Ile?Asp?Leu?Pro?Gly?Phe?Gly?Phe

225 230 235 240

His?Arg?Ala?Leu?Gln?Gly?Arg?Ala?Thr?Leu?Asn?Lys?Ile?Met?Leu?Ser

245 250 255

Leu?Ile?Lys?Lys?Arg?Lys?Glu?Asp?Leu?Gln?Ser?Gly?Ser?Ala?Thr?Ala

260 265 270

Thr?Gln?Asp?Leu?Leu?Ser?Val?Leu?Leu?Thr?Phe?Arg?Asp?Asp?Lys?Gly

275 280 285

Thr?Pro?Leu?Thr?Asn?Asp?Glu?Ile?Leu?Asp?Asn?Phe?Ser?Ser?Leu?Leu

290 295 300

His?Ala?Ser?Tyr?Asp?Thr?Thr?Thr?Ser?Pro?Met?Ala?Leu?Ile?Phe?Lys

305 310 315 320

Leu?Leu?Ser?Ser?Asn?Pro?Glu?Cys?Tyr?Gln?Lys?Val?Val?Gln?Glu?Gln

325 330 335

Leu?Glu?Ile?Leu?Ser?Asn?Lys?Glu?Glu?Gly?Glu?Glu?Ile?Thr?Trp?Lys

340 345 350

Asp?Leu?Lys?Ala?Met?Lys?Tyr?Thr?Trp?Gln?Val?Ala?Gln?Glu?Thr?Leu

355 360 365

Arg?Met?Phe?Pro?Pro?Val?Phe?Gly?Thr?Phe?Arg?Lys?Ala?Ile?Thr?Asp

370 375 380

Ile?Gln?Tyr?Asp?Gly?Tyr?Thr?Ile?Pro?Lys?Gly?Trp?Lys?Leu?Leu?Trp

385 390 395 400

Thr?Thr?Tyr?Ser?Thr?His?Pro?Lys?Asp?Leu?Tyr?Phe?Ser?Glu?Pro?Glu

405 410 415

Lys?Phe?Met?Pro?Ser?Arg?Phe?Asp?Gln?Glu?Gly?Lys?His?Val?Ala?Pro

420 425 430

Tyr?Thr?Phe?Leu?Pro?Phe?Gly?Gly?Gly?Gln?Arg?Ser?Cys?Val?Gly?Trp

435 440 445

Glu?Phe?Ser?Lys?Met?Glu?Ile?Leu?Leu?Phe?Val?His?His?Phe?Val?Lys

450 455 460

Thr?Phe?Ser?Ser?Tyr?Thr?Pro?Val?Asp?Pro?Asp?Glu?Lys?Ile?Ser?Gly

465 470 475 480

Asp?Pro?Leu?Pro?Pro?Leu?Pro?Ser?Lys?Gly?Phe?Ser?Ile?Lys?Leu?Phe

485 490 495

Pro?Glu?Thr?Ile?Val?Asn

500

<210>33

<211>509

<212>PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400>33

Met?Ala?Phe?Phe?Ser?Met?Ile?Ser?Ile?Leu?Leu?Gly?Phe?Val?Ile?Ser

1 5 10 15

Ser?Phe?Ile?Phe?Ile?Phe?Phe?Phe?Lys?Lys?Leu?Leu?Ser?Phe?Ser?Arg

20 25 30

Lys?Asn?Met?Ser?Glu?Val?Ser?Thr?Leu?Pro?Ser?Val?Pro?Val?Val?Pro

35 40 45

Gly?Phe?Pro?Val?Ile?Gly?Asn?Leu?Leu?Gln?Leu?Lys?Glu?Lys?Lys?Pro

50 55 60

His?Lys?Thr?Phe?Thr?Arg?Trp?Ser?Glu?Ile?Tyr?Gly?Pro?Ile?Tyr?Ser

65 70 75 80

Ile?Lys?Met?Gly?Ser?Ser?Ser?Leu?Ile?Val?Leu?Asn?Ser?Thr?Glu?Thr

85 90 95

Ala?Lys?Glu?Ala?Met?Val?Thr?Arg?Phe?Ser?Ser?Ile?Ser?Thr?Arg?Lys

100 105 110

Leu?Ser?Asn?Ala?Leu?Thr?Val?Leu?Thr?Cys?Asp?Lys?Ser?Met?Val?Ala

115 120 125

Thr?Ser?Asp?Tyr?Asp?Asp?Phe?His?Lys?Leu?Val?Lys?Arg?Cys?Leu?Leu

130 135 140

Asn?Gly?Leu?Leu?Gly?Ala?Asn?Ala?Gln?Lys?Arg?Lys?Arg?His?Tyr?Arg

145 150 155 160

Asp?Ala?Leu?Ile?Glu?Asn?Val?Ser?Ser?Lys?Leu?His?Ala?His?Ala?Arg

165 170 175

Asp?His?Pro?Gln?Glu?Pro?Val?Asn?Phe?Arg?Ala?Ile?Phe?Glu?His?Glu

180 185 190

Leu?Phe?Gly?Val?Ala?Leu?Lys?Gln?Ala?Phe?Gly?Lys?Asp?Val?Glu?Ser

195 200 205

Ile?Tyr?Val?Lys?Glu?Leu?Gly?Val?Thr?Leu?Ser?Lys?Asp?Glu?Ile?Phe

210 215 220

Lys?Val?Leu?Val?His?Asp?Met?Met?Glu?Gly?Ala?Ile?Asp?Val?Asp?Trp

225 230 235 240

Arg?Asp?Phe?Phe?Pro?Tyr?Leu?Lys?Trp?Ile?Pro?Asn?Lys?Ser?Phe?Glu

245 250 255

Ala?Arg?Ile?Gln?Gln?Lys?His?Lys?Arg?Arg?Leu?Ala?Val?Met?Asn?Ala

260 265 270

Leu?Ile?Gln?Asp?Arg?Leu?Lys?Gln?Asn?Gly?Ser?Glu?Ser?Asp?Asp?Asp

275 280 285

Cys?Tyr?Leu?Asn?Phe?Leu?Met?Ser?Glu?Ala?Lys?Thr?Leu?Thr?Lys?Glu

290 295 300

Gln?Ile?Ala?Ile?Leu?Val?Trp?Glu?Thr?Ile?Ile?Glu?Thr?Ala?Asp?Thr

305 310 315 320

Thr?Leu?Val?Thr?Thr?Glu?Trp?Ala?Ile?Tyr?Glu?Leu?Ala?Lys?His?Pro

325 330 335

Ser?Val?Gln?Asp?Arg?Leu?Cys?Lys?Glu?Ile?Gln?Asn?Val?Cys?Gly?Gly

340 345 350

Glu?Lys?Phe?Lys?Glu?Glu?Gln?Leu?Ser?Gln?Val?Pro?Tyr?Leu?Asn?Gly

355 360 365

Val?Phe?His?Glu?Thr?Leu?Arg?Lys?Tyr?Ser?Pro?Ala?Pro?Leu?Val?Pro

370 375 380

Ile?Arg?Tyr?Ala?His?Glu?Asp?Thr?Gln?Ile?Gly?Gly?Tyr?His?Val?Pro

385 390 395 400

Ala?Gly?Ser?Glu?Ile?Ala?Ile?Asn?Ile?Tyr?Gly?Cys?Asn?Met?Asp?Lys

405 410 415

Lys?Arg?Trp?Glu?Arg?Pro?Glu?Asp?Trp?Trp?Pro?Glu?Arg?Phe?Leu?Asp

420 425 430

Asp?Gly?Lys?Tyr?Glu?Thr?Ser?Asp?Leu?His?Lys?Thr?Met?Ala?Phe?Gly

435 440 445

Ala?Gly?Lys?Arg?Val?Cys?Ala?Gly?Ala?Leu?Gln?Ala?Ser?Leu?Met?Ala

450 455 460

Gly?Ile?Ala?Ile?Gly?Arg?Leu?Val?Gln?Glu?Phe?Glu?Trp?Lys?Leu?Arg

465 470 475 480

Asp?Gly?Glu?Glu?Glu?Asn?Val?Asp?Thr?Tyr?Gly?Leu?Thr?Ser?Gln?Lys

485 490 495

Leu?Tyr?Pro?Leu?Met?Ala?Ile?Ile?Asn?Pro?Arg?Arg?Ser

500 505

<210>34

<211>1896

<212>DNA

＜213〉tree continuous (Gossypium arboreum)

<400>34

ccacttcgca?gcaatattat?tgcagttcct?ggttggctac?ctctgagttt?tcaacttaaa?60

atttcttggt?tttcctcaag?aaggaagaag?atgttgcaaa?tagctttcag?ctcgtattca?120

tggctgttga?ctgctagcaa?ccagaaagat?ggaatgttgt?tcccagtagc?tttgtcattt?180

ttggtagcca?tattgggaat?ttcactgtgg?cacgtatgga?ccataaggaa?gccaaagaaa?240

gacatcgccc?cattaccgcc?gggtccccgt?gggttgccaa?tagtgggata?tcttccatat?300

cttggaactg?ataatcttca?cttggtgttt?acagatttgg?ctgcagctta?cggtcccatc?360

tacaagcttt?ggctaggaaa?caaattatgc?gtagtcatta?gctcggcacc?actggcgaaa?420

gaagtggttc?gtgacaacga?catcacattt?tctgaaaggg?atcctcccgt?ttgtgcaaag?480

attattacct?ttggcctcaa?tgatattgta?tttgattctt?acagtagtcc?agattggaga?540

atgaagagaa?aagtgctggt?acgtgaaatg?cttagccata?gtagcattaa?agcttgttat?600

ggtctaagga?gggaacaagt?gcttaaaggc?gtacaaaatg?ttgctcaaag?tgctggcaag?660

ccaattgatt?ttggtgaaac?ggcattttta?acatcaatca?atgcgatgat?gagcatgctg?720

tggggtggca?aacagggagg?agagcggaaa?ggggccgacg?tttggggcca?atttcgagat?780

ctcataaccg?aactaatggt?gatacttgga?aaaccaaacg?tttctgatat?tttcccggtg?840

cttgcaaggt?ttgacataca?gggattggag?aaggaaatga?ctaaaatcgt?taattctttc?900

gataagcttt?tcaactccat?gattgaagaa?agagagaact?ttagcaacaa?attgagcaaa?960

gaagatggaa?acactgaaac?aaaagacttc?ttgcagcttc?tgttggacct?caagcagaag?1020

aacgatagcg?gaatatcgat?aacaatgaat?caagtcaagg?ccttgctcat?ggacattgtg?1080

gtcggtggaa?ctgatacaac?atcaaccatg?atggaatgga?caatggctga?actaattgca?1140

aatcctgaag?caatgaaaaa?ggtgaagcaa?gaaatagacg?atgttgtcgg?ttcggatggc?1200

gccgtcgatg?agactcactt?gcctaagttg?cgctatctag?atgctgcagt?aaaggagacc?1260

ttccgattgc?acccaccgat?gccactcctt?gtaccccgtt?gcccgggcga?ctcaagcaac?1320

gttggtggct?atagcgtacc?aaagggcacc?agggtcttct?taaacatttg?gtgtattcag?1380

agggatccac?agctttggga?aaatccttta?gaattcaagc?ctgagaggtt?cttgactgat?1440

catgagaagc?tcgattattt?aggaaacgat?tcccggtaca?tgccgtttgg?ttctggaagg?1500

agaatgtgtg?ccggagtatc?tctcggtgaa?aagatgttgt?attcctcctt?ggcagcaatg?1560

atccatgctt?atgattggaa?cttggccgac?ggtgaagaaa?atgacttgat?tggcttattt?1620

ggaattatta?tgaagaaaaa?gaagccttta?attcttgttc?ctacaccaag?accatcaaat?1680

ctccagcact?atatgaagta?actttactat?tgtatttctt?ttataccact?ttattgcctc?1740

tttgtcatgt?ttaggcaaca?attctaagta?ataagtttgg?ctatatggtg?aacaataatg?1800

tgtttattat?acatcataag?caatgagctc?ttcccgaccc?tagggcaata?caatgatact?1860

gtgtattaag?tgaaatcaac?aaatctttta?ttctaa 1896

<210>35

<211>1677

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic polyribonucleotides

<400>35

atgctgcaga?ttgctttttc?ttcttattct?tggctgctga?ccgcttctaa?ccagaaagac?60

ggcatgctgt?tcccggtggc?gctgagcttc?ctggtggcaa?tcctgggcat?tagcctgtgg?120

cacgtgtgga?ctatccgtaa?accgaagaaa?gatatcgcac?cgctgccacc?gggtccgcgt?180

ggcctgccga?tcgttggcta?cctgccgtat?ctgggcaccg?acaacctgca?cctggtgttc?240

accgacctgg?cagccgcgta?cggtccgatc?tacaaactgt?ggctgggcaa?taaactgtgc?300

gtagttatct?cctctgctcc?tctggcgaag?gaggtggttc?gcgacaacga?catcaccttc?360

tccgaacgtg?acccaccggt?ctgtgctaaa?atcatcacct?tcggcctgaa?cgacatcgta?420

ttcgactcct?atagctctcc?tgactggcgt?atgaaacgta?aggttctggt?acgcgagatg?480

ctgtcccaca?gctccattaa?ggcatgctac?ggcctgcgtc?gcgaacaggt?actgaaaggc?540

gtacaaaacg?tagcgcagtc?cgcgggcaaa?ccgatcgatt?tcggcgaaac?ggccttcctg?600

actagcatca?acgctatgat?gtccatgctg?tggggtggta?aacagggcgg?cgagcgtaaa?660

ggcgccgacg?tatggggcca?gtttcgtgac?ctgatcaccg?aactgatggt?gattctgggc?720

aaaccgaacg?tcagcgacat?cttcccggtt?ctggctcgct?tcgacatcca?gggcctggaa?780

aaagaaatga?ccaagatcgt?caactctttc?gacaaactgt?ttaactccat?gatcgaagaa?840

cgcgaaaatt?tctctaacaa?actgagcaaa?gaagatggca?acaccgaaac?taaagatttc?900

ctgcagctgc?tgctggacct?gaaacaaaag?aacgattctg?gtatctccat?taccatgaac?960

caagtgaaag?cgctgctgat?ggacattgtt?gtgggtggta?ctgacaccac?ttctaccatg?1020

atggaatgga?cgatggcaga?actgattgct?aatccggaag?cgatgaagaa?agtgaaacaa?1080

gaaattgatg?atgtagtggg?ctctgatggt?gcggtagacg?agacgcacct?gcctaagctg?1140

cgttatctgg?acgcagccgt?gaaagaaacc?ttccgtctgc?atccgcctat?gccgctgctg?1200

gttccacgtt?gcccaggcga?ttccagcaac?gttggtggct?atagcgtacc?gaagggtacc?1260

cgtgtgttcc?tgaatatctg?gtgcattcag?cgcgacccgc?agctgtggga?aaacccgctg?1320

gagttcaaac?ctgaacgctt?cctgaccgac?catgaaaagc?tggactacct?gggcaacgat?1380

tcccgttaca?tgccgttcgg?ttctggccgt?cgtatgtgcg?caggcgtctc?cctgggcgag?1440

aaaatgctgt?actctagcct?ggctgccatg?atccacgctt?acgactggaa?cctggcagat?1500

ggtgaagaga?acgacctgat?cggcctgttc?ggcatcatta?tgaaaaagaa?aaagccgctg?1560

atcctggtgc?cgactccgcg?tccaagcaac?ctgcagcact?acatgaaact?ggtgccgcgt?1620

ggctctaaag?aaaccgctgc?tgcaaaattc?gaacgtcagc?acatggacag?ctaataa 1677

<210>36

<211>717

<212>PRT

＜213〉taxus chinensis in northeast (Taxus cuspidata)

<400>36

Met?Gln?Ala?Asn?Ser?Asn?Thr?Val?Glu?Gly?Ala?Ser?Gln?Gly?Lys?Ser

1 5 10 15

Leu?Leu?Asp?Ile?Ser?Arg?Leu?Asp?His?Ile?Phe?Ala?Leu?Leu?Leu?Asn

20 25 30

Gly?Lys?Gly?Gly?Asp?Leu?Gly?Ala?Met?Thr?Gly?Ser?Ala?Leu?Ile?Leu

35 40 45

Thr?Glu?Asn?Ser?Gln?Asn?Leu?Met?Ile?Leu?Thr?Thr?Ala?Leu?Ala?Val

50 55 60

Leu?Val?Ala?Cys?Val?Phe?Phe?Phe?Val?Trp?Arg?Arg?Gly?Gly?Ser?Asp

65 70 75 80

Thr?Gln?Lys?Pro?Ala?Val?Arg?Pro?Thr?Pro?Leu?Val?Lys?Glu?Glu?Asp

85 90 95

Glu?Glu?Glu?Glu?Asp?Asp?Ser?Ala?Lys?Lys?Lys?Val?Thr?Ile?Phe?Phe

100 105 110

Gly?Thr?Gln?Thr?Gly?Thr?Ala?Glu?Gly?Phe?Ala?Lys?Ala?Leu?Ala?Glu

115 120 125

Glu?Ala?Lys?Ala?Arg?Tyr?Glu?Lys?Ala?Val?Phe?Lys?Val?Val?Asp?Leu

130 135 140

Asp?Asn?Tyr?Ala?Ala?Asp?Asp?Glu?Gln?Tyr?Glu?Glu?Lys?Leu?Lys?Lys

145 150 155 160

Glu?Lys?Leu?Ala?Phe?Phe?Met?Leu?Ala?Thr?Tyr?Gly?Asp?Gly?Glu?Pro

165 170 175

Thr?Asp?Asn?Ala?Ala?Arg?Phe?Tyr?Lys?Trp?Phe?Leu?Glu?Gly?Lys?Glu

180 185 190

Arg?Glu?Pro?Trp?Leu?Ser?Asp?Leu?Thr?Tyr?Gly?Val?Phe?Gly?Leu?Gly

195 200 205

Asn?Arg?Gln?Tyr?Glu?His?Phe?Asn?Lys?Val?Ala?Lys?Ala?Val?Asp?Glu

210 215 220

Val?Leu?Ile?Glu?Gln?Gly?Ala?Lys?Arg?Leu?Val?Pro?Val?Gly?Leu?Gly

225 230 235 240

Asp?Asp?Asp?Gln?Cys?Ile?Glu?Asp?Asp?Phe?Thr?Ala?Trp?Arg?Glu?Gln

245 250 255

Val?Trp?Pro?Glu?Leu?Asp?Gln?Leu?Leu?Arg?Asp?Glu?Asp?Asp?Glu?Pro

260 265 270

Thr?Ser?Ala?Thr?Pro?Tyr?Thr?Ala?Ala?Ile?Pro?Glu?Tyr?Arg?Val?Glu

275 280 285

Ile?Tyr?Asp?Ser?Val?Val?Ser?Val?Tyr?Glu?Glu?Thr?His?Ala?Leu?Lys

290 295 300

Gln?Asn?Gly?Gln?Ala?Val?Tyr?Asp?Ile?His?His?Pro?Cys?Arg?Ser?Asn

305 310 315 320

Val?Ala?Val?Arg?Arg?Glu?Leu?His?Thr?Pro?Leu?Ser?Asp?Arg?Ser?Cys

325 330 335

Ile?His?Leu?Glu?Phe?Asp?Ile?Ser?Asp?Thr?Gly?Leu?Ile?Tyr?Glu?Thr

340 345 350

Gly?Asp?His?Val?Gly?Val?His?Thr?Glu?Asn?Ser?Ile?Glu?Thr?Val?Glu

355 360 365

Glu?Ala?Ala?Lys?Leu?Leu?Gly?Tyr?Gln?Leu?Asp?Thr?Ile?Phe?Ser?Val

370 375 380

His?Gly?Asp?Lys?Glu?Asp?Gly?Thr?Pro?Leu?Gly?Gly?Ser?Ser?Leu?Pro

385 390 395 400

Pro?Pro?Phe?Pro?Gly?Pro?Cys?Thr?Leu?Arg?Thr?Ala?Leu?Ala?Arg?Tyr

405 410 415

Ala?Asp?Leu?Leu?Asn?Pro?Pro?Arg?Lys?Ala?Ala?Phe?Leu?Ala?Leu?Ala

420 425 430

Ala?His?Ala?Ser?Asp?Pro?Ala?Glu?Ala?Glu?Arg?Leu?Lys?Phe?Leu?Ser

435 440 445

Ser?Pro?Ala?Gly?Lys?Asp?Glu?Tyr?Ser?Gln?Trp?Val?Thr?Ala?Ser?Gln

450 455 460

Arg?Ser?Leu?Leu?Glu?Ile?Met?Ala?Glu?Phe?Pro?Ser?Ala?Lys?Pro?Pro

465 470 475 480

Leu?Gly?Val?Phe?Phe?Ala?Ala?Ile?Ala?Pro?Arg?Leu?Gln?Pro?Arg?Tyr

485 490 495

Tyr?Ser?Ile?Ser?Ser?Ser?Pro?Arg?Phe?Ala?Pro?Ser?Arg?Ile?His?Val

500 505 510

Thr?Cys?Ala?Leu?Val?Tyr?Gly?Pro?Ser?Pro?Thr?Gly?Arg?Ile?His?Lys

515 520 525

Gly?Val?Cys?Ser?Asn?Trp?Met?Lys?Asn?Ser?Leu?Pro?Ser?Glu?Glu?Thr

530 535 540

His?Asp?Cys?Ser?Trp?Ala?Pro?Val?Phe?Val?Arg?Gln?Ser?Asn?Phe?Lys

545 550 555 560

Leu?Pro?Ala?Asp?Ser?Thr?Thr?Pro?Ile?Val?Met?Val?Gly?Pro?Gly?Thr

565 570 575

Gly?Phe?Ala?Pro?Phe?Arg?Gly?Phe?Leu?Gln?Glu?Arg?Ala?Lys?Leu?Gln

580 585 590

Glu?Ala?Gly?Glu?Lys?Leu?Gly?Pro?Ala?Val?Leu?Phe?Phe?Gly?Cys?Arg

595 600 605

Asn?Arg?Gln?Met?Asp?Tyr?Ile?Tyr?Glu?Asp?Glu?Leu?Lys?Gly?Tyr?Val

610 615 620

Glu?Lys?Gly?Ile?Leu?Thr?Asn?Leu?Ile?Val?Ala?Phe?Ser?Arg?Glu?Gly

625 630 635 640

Ala?Thr?Lys?Glu?Tyr?Val?Gln?His?Lys?Met?Leu?Glu?Lys?Ala?Ser?Asp

645 650 655

Thr?Trp?Ser?Leu?Ile?Ala?Gln?Gly?Gly?Tyr?Leu?Tyr?Val?Cys?Gly?Asp

660 665 670

Ala?Lys?Gly?Met?Ala?Arg?Asp?Val?His?Arg?Thr?Leu?His?Thr?Ile?Val

675 680 685

Gln?Glu?Gln?Glu?Ser?Val?Asp?Ser?Ser?Lys?Ala?Glu?Phe?Leu?Val?Lys

690 695 700

Lys?Leu?Gln?Met?Asp?Gly?Arg?Tyr?Leu?Arg?Asp?Ile?Trp

705 710 715

<210>37

<211>680

<212>PRT

＜213〉Oidium tropicale (Candida tropicalis)

<400>37

Met?Ala?Leu?Asp?Lys?Leu?Asp?Leu?Tyr?Val?Ile?Ile?Thr?Leu?Val?Val

1 5 10 15

Ala?Ile?Ala?Ala?Tyr?Phe?Ala?Lys?Asn?Gln?Phe?Leu?Asp?Gln?Gln?Gln

20 25 30

Asp?Thr?Gly?Phe?Leu?Asn?Thr?Asp?Ser?Gly?Asp?Gly?Asn?Ser?Arg?Asp

35 40 45

Ile?Leu?Gln?Ala?Leu?Lys?Lys?Asn?Asn?Lys?Asn?Thr?Leu?Leu?Leu?Phe

50 55 60

Gly?Ser?Gln?Thr?Gly?Thr?Ala?Glu?Asp?Tyr?Ala?Asn?Lys?Leu?Ser?Arg

65 70 75 80

Glu?Leu?His?Ser?Arg?Phe?Gly?Leu?Lys?Thr?Met?Val?Ala?Asp?Phe?Ala

85 90 95

Asp?Tyr?Asp?Phe?Glu?Asn?Phe?Gly?Asp?Ile?Thr?Glu?Asp?Ile?Leu?Val

100 105 110

Phe?Phe?Ile?Val?Ala?Thr?Tyr?Gly?Glu?Gly?Glu?Pro?Thr?Asp?Asn?Ala

115 120 125

Asp?Glu?Phe?His?Thr?Trp?Leu?Thr?Glu?Glu?Ala?Asp?Thr?Leu?Ser?Thr

130 135 140

Leu?Lys?Tyr?Thr?Val?Phe?Gly?Leu?Gly?Asn?Ser?Thr?Tyr?Glu?Phe?Phe

145 150 155 160

Asn?Ala?Ile?Gly?Arg?Lys?Phe?Asp?Arg?Leu?Leu?Gly?Glu?Lys?Gly?Gly

165 170 175

Asp?Arg?Phe?Ala?Glu?Tyr?Gly?Glu?Gly?Asp?Asp?Gly?Thr?Gly?Thr?Leu

180 185 190

Asp?Glu?Asp?Phe?Leu?Ala?Trp?Lys?Asp?Asn?Val?Phe?Asp?Ser?Leu?Lys

195 200 205

Asn?Asp?Leu?Asn?Phe?Glu?Glu?Lys?Glu?Leu?Lys?Tyr?Glu?Pro?Asn?Val

210 215 220

Lys?Leu?Thr?Glu?Arg?Asp?Asp?Leu?Ser?Gly?Asn?Asp?Pro?Asp?Val?Ser

225 230 235 240

Leu?Gly?Glu?Pro?Asn?Val?Lys?Tyr?Ile?Lys?Ser?Glu?Gly?Val?Asp?Leu

245 250 255

Thr?Lys?Gly?Pro?Phe?Asp?His?Thr?His?Pro?Phe?Leu?Ala?Arg?Ile?Val

260 265 270

Lys?Thr?Lys?Glu?Leu?Phe?Thr?Ser?Glu?Asp?Arg?His?Cys?Val?His?Val

275 280 285

Glu?Phe?Asp?Ile?Ser?Glu?Ser?Asn?Leu?Lys?Tyr?Thr?Thr?Gly?Asp?His

290 295 300

Leu?Ala?Ile?Trp?Pro?Ser?Asn?Ser?Asp?Glu?Asn?Ile?Lys?Gln?Phe?Ala

305 310 315 320

Lys?Cys?Phe?Gly?Leu?Glu?Asp?Lys?Leu?Asp?Thr?Val?Ile?Glu?Leu?Lys

325 330 335

Ala?Leu?Asp?Ser?Thr?Tyr?Ser?Ile?Pro?Phe?Pro?Asn?Pro?Ile?Thr?Tyr

340 345 350

Gly?Ala?Val?Ile?Arg?His?His?Leu?Glu?Ile?Ser?Gly?Pro?Val?Ser?Arg

355 360 365

Gln?Phe?Phe?Leu?Ser?Ile?Ala?Gly?Phe?Ala?Pro?Asp?Glu?Glu?Thr?Lys

370 375 380

Lys?Ser?Phe?Thr?Arg?Ile?Gly?Gly?Asp?Lys?Gln?Glu?Phe?Ala?Ser?Lys

385 390 395 400

Val?Thr?Arg?Arg?Lys?Phe?Asn?Ile?Ala?Asp?Ala?Leu?Leu?Phe?Ala?Ser

405 410 415

Asn?Asn?Arg?Pro?Trp?Ser?Asp?Val?Pro?Phe?Glu?Phe?Leu?Ile?Glu?Asn

420 425 430

Val?Gln?His?Leu?Thr?Pro?Arg?Tyr?Tyr?Ser?Ile?Ser?Ser?Ser?Ser?Leu

435 440 445

Ser?Glu?Lys?Gln?Thr?Ile?Asn?Val?Thr?Ala?Val?Val?Glu?Ala?Glu?Glu

450 455 460

Glu?Ala?Asp?Gly?Arg?Pro?Val?Thr?Gly?Val?Val?Thr?Asn?Leu?Leu?Lys

465 470 475 480

Asn?Ile?Glu?Ile?Glu?Gln?Asn?Lys?Thr?Gly?Glu?Thr?Pro?Met?Val?His

485 490 495

Tyr?Asp?Leu?Asn?Gly?Pro?Arg?Gly?Lys?Phe?Ser?Lys?Phe?Arg?Leu?Pro

500 505 510

Val?His?Val?Arg?Arg?Ser?Asn?Phe?Lys?Leu?Pro?Lys?Asn?Ser?Thr?Thr

515 520 525

Pro?Val?Ile?Leu?Ile?Gly?Pro?Gly?Thr?Gly?Val?Ala?Pro?Leu?Arg?Gly

530 535 540

Phe?Val?Arg?Glu?Arg?Val?Gln?Gln?Val?Lys?Asn?Gly?Val?Asn?Val?Gly

545 550 555 560

Lys?Thr?Val?Leu?Phe?Tyr?Gly?Cys?Arg?Asn?Ser?Glu?Gln?Asp?Phe?Leu

565 570 575

Tyr?Lys?Gln?Glu?Trp?Ser?Glu?Tyr?Ala?Ser?Val?Leu?Gly?Glu?Asn?Phe

580 585 590

Glu?Met?Phe?Asn?Ala?Phe?Ser?Arg?Gln?Asp?Pro?Thr?Lys?Lys?Val?Tyr

595 600 605

Val?Gln?Asp?Lys?Ile?Leu?Glu?Asn?Ser?Ala?Leu?Val?Asp?Glu?Leu?Leu

610 615 620

Ser?Ser?Gly?Ala?Ile?Ile?Tyr?Val?Cys?Gly?Asp?Ala?Ser?Arg?Met?Ala

625 630 635 640

Arg?Asp?Val?Gln?Ala?Ala?Ile?Ala?Lys?Ile?Val?Ala?Lys?Ser?Arg?Asp

645 650 655

Ile?His?Glu?Asp?Lys?Ala?Ala?Glu?Leu?Val?Lys?Ser?Trp?Lys?Val?Gln

660 665 670

Asn?Arg?Tyr?Gln?Glu?Asp?Val?Trp

675 680

<210>38

<211>692

<212>PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400>38

Met?Thr?Ser?Ala?Leu?Tyr?Ala?Ser?Asp?Leu?Phe?Lys?Gln?Leu?Lys?Ser

1 5 10 15

Ile?Met?Gly?Thr?Asp?Ser?Leu?Ser?Asp?Asp?Val?Val?Leu?Val?Ile?Ala

20 25 30

Thr?Thr?Ser?Leu?Ala?Leu?Val?Ala?Gly?Phe?Val?Val?Leu?Leu?Trp?Lys

35 40 45

Lys?Thr?Thr?Ala?Asp?Arg?Ser?Gly?Glu?Leu?Lys?Pro?Leu?Met?Ile?Pro

50 55 60

Lys?Ser?Leu?Met?Ala?Lys?Asp?Glu?Asp?Asp?Asp?Leu?Asp?Leu?Gly?Ser

65 70 75 80

Gly?Lys?Thr?Arg?Val?Ser?Ile?Phe?Phe?Gly?Thr?Gln?Thr?Gly?Thr?Ala

85 90 95

Glu?Gly?Phe?Ala?Lys?Ala?Leu?Ser?Glu?Glu?Ile?Lys?Ala?Arg?Tyr?Glu

100 105 110

Lys?Ala?Ala?Val?Lys?Val?Ile?Asp?Leu?Asp?Asp?Tyr?Ala?Ala?Asp?Asp

115 120 125

Asp?Gln?Tyr?Glu?Glu?Lys?Leu?Lys?Lys?Glu?Thr?Leu?Ala?Phe?Phe?Cys

130 135 140

Val?Ala?Thr?Tyr?Gly?Asp?Gly?Glu?Pro?Thr?Asp?Asn?Ala?Ala?Arg?Phe

145 150 155 160

Ser?Lys?Trp?Phe?Thr?Glu?Glu?Asn?Glu?Arg?Asp?Ile?Lys?Leu?Gln?Gln

165 170 175

Leu?Ala?Tyr?Gly?Val?Phe?Ala?Leu?Gly?Asn?Arg?Gln?Tyr?Glu?His?Phe

180 185 190

Asn?Lys?Ile?Gly?Ile?Val?Leu?Asp?Glu?Glu?Leu?Cys?Lys?Lys?Gly?Ala

195 200 205

Lys?Arg?Leu?Ile?Glu?Val?Gly?Leu?Gly?Asp?Asp?Asp?Gln?Ser?Ile?Glu

210 215 220

Asp?Asp?Phe?Asn?Ala?Trp?Lys?Glu?Ser?Leu?Trp?Ser?Glu?Leu?Asp?Lys

225 230 235 240

Leu?Leu?Lys?Asp?Glu?Asp?Asp?Lys?Ser?Val?Ala?Thr?Pro?Tyr?Thr?Ala

245 250 255

Val?Ile?Pro?Glu?Tyr?Arg?Val?Val?Thr?His?Asp?Pro?Arg?Phe?Thr?Thr

260 265 270

Gln?Lys?Ser?Met?Glu?Ser?Asn?Val?Ala?Asn?Gly?Asn?Thr?Thr?Ile?Asp

275 280 285

Ile?His?His?Pro?Cys?Arg?Val?Asp?Val?Ala?Val?Gln?Lys?Glu?Leu?His

290 295 300

Thr?His?Glu?Ser?Asp?Arg?Ser?Cys?Ile?His?Leu?Glu?Phe?Asp?Ile?Ser

305 310 315 320

Arg?Thr?Gly?Ile?Thr?Tyr?Glu?Thr?Gly?Asp?His?Val?Gly?Val?Tyr?Ala

325 330 335

Glu?Asn?His?Val?Glu?Ile?Val?Glu?Glu?Ala?Gly?Lys?Leu?Leu?Gly?His

340 345 350

Ser?Leu?Asp?Leu?Val?Phe?Ser?Ile?His?Ala?Asp?Lys?Glu?Asp?Gly?Ser

355 360 365

Pro?Leu?Glu?Ser?Ala?Val?Pro?Pro?Pro?Phe?Pro?Gly?Pro?Cys?Thr?Leu

370 375 380

Gly?Thr?Gly?Leu?Ala?Arg?Tyr?Ala?Asp?Leu?Leu?Asn?Pro?Pro?Arg?Lys

385 390 395 400

Ser?Ala?Leu?Val?Ala?Leu?Ala?Ala?Tyr?Ala?Thr?Glu?Pro?Ser?Glu?Ala

405 410 415

Glu?Lys?Leu?Lys?His?Leu?Thr?Ser?Pro?Asp?Gly?Lys?Asp?Glu?Tyr?Ser

420 425 430

Gln?Trp?Ile?Val?Ala?Ser?Gln?Arg?Ser?Leu?Leu?Glu?Val?Met?Ala?Ala

435 440 445

Phe?Pro?Ser?Ala?Lys?Pro?Pro?Leu?Gly?Val?Phe?Phe?Ala?Ala?Ile?Ala

450 455 460

Pro?Arg?Leu?Gln?Pro?Arg?Tyr?Tyr?Ser?Ile?Ser?Ser?Cys?Gln?Asp?Trp

465 470 475 480

Ala?Pro?Ser?Arg?Val?His?Val?Thr?Ser?Ala?Leu?Val?Tyr?Gly?Pro?Thr

485 490 495

Pro?Thr?Gly?Arg?Ile?His?Lys?Gly?Val?Cys?Ser?Thr?Trp?Met?Lys?Asn

500 505 510

Ala?Val?Pro?Ala?Glu?Lys?Ser?His?Glu?Cys?Ser?Gly?Ala?Pro?Ile?Phe

515 520 525

Ile?Arg?Ala?Ser?Asn?Phe?Lys?Leu?Pro?Ser?Asn?Pro?Ser?Thr?Pro?Ile

530 535 540

Val?Met?Val?Gly?Pro?Gly?Thr?Gly?Leu?Ala?Pro?Phe?Arg?Gly?Phe?Leu

545 550 555 560

Gln?Glu?Arg?Met?Ala?Leu?Lys?Glu?Asp?Gly?Glu?Glu?Leu?Gly?Ser?Ser

565 570 575

Leu?Leu?Phe?Phe?Gly?Cys?Arg?Asn?Arg?Gln?Met?Asp?Phe?Ile?Tyr?Glu

580 585 590

Asp?Glu?Leu?Asn?Asn?Phe?Val?Asp?Gln?Gly?Val?Ile?Ser?Glu?Leu?Ile

595 600 605

Met?Ala?Phe?Ser?Arg?Glu?Gly?Ala?Gln?Lys?Glu?Tyr?Val?Gln?His?Lys

610 615 620

Met?Met?Glu?Lys?Ala?Ala?Gln?Val?Trp?Asp?Leu?Ile?Lys?Glu?Glu?Gly

625 630 635 640

Tyr?Leu?Tyr?Val?Cys?Gly?Asp?Ala?Lys?Gly?Met?Ala?Arg?Asp?Val?His

645 650 655

Arg?Thr?Leu?His?Thr?Ile?Val?Gln?Glu?Gln?Glu?Gly?Val?Ser?Ser?Ser

660 665 670

Glu?Ala?Glu?Ala?Ile?Val?Lys?Lys?Leu?Gln?Thr?Glu?Gly?Arg?Tyr?Leu

675 680 685

Arg?Asp?Val?Trp

690

<210>39

<211>712

<212>PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400>39

Met?Ser?Ser?Ser?Ser?Ser?Ser?Ser?Thr?Ser?Met?Ile?Asp?Leu?Met?Ala

1 5 10 15

Ala?Ile?Ile?Lys?Gly?Glu?Pro?Val?Ile?Val?Ser?Asp?Pro?Ala?Asn?Ala

20 25 30

Ser?Ala?Tyr?Glu?Ser?Val?Ala?Ala?Glu?Leu?Ser?Ser?Met?Leu?Ile?Glu

35 40 45

Asn?Arg?Gln?Phe?Ala?Met?Ile?Val?Thr?Thr?Ser?Ile?Ala?Val?Leu?Ile

50 55 60

Gly?Cys?Ile?Val?Met?Leu?Val?Trp?Arg?Arg?Ser?Gly?Ser?Gly?Asn?Ser

65 70 75 80

Lys?Arg?Val?Glu?Pro?Leu?Lys?Pro?Leu?Val?Ile?Lys?Pro?Arg?Glu?Glu

85 90 95

Glu?Ile?Asp?Asp?Gly?Arg?Lys?Lys?Val?Thr?Ile?Phe?Phe?Gly?Thr?Gln

100 105 110

Thr?Gly?Thr?Ala?Glu?Gly?Phe?Ala?Lys?Ala?Leu?Gly?Glu?Glu?Ala?Lys

115 120 125

Ala?Arg?Tyr?Glu?Lys?Thr?Arg?Phe?Lys?Ile?Val?Asp?Leu?Asp?Asp?Tyr

130 135 140

Ala?Ala?Asp?Asp?Asp?Glu?Tyr?Glu?Glu?Lys?Leu?Lys?Lys?Glu?Asp?Val

145 150 155 160

Ala?Phe?Phe?Phe?Leu?Ala?Thr?Tyr?Gly?Asp?Gly?Glu?Pro?Thr?Asp?Asn

165 170 175

Ala?Ala?Arg?Phe?Tyr?Lys?Trp?Phe?Thr?Glu?Gly?Asn?Asp?Arg?Gly?Glu

180 185 190

Trp?Leu?Lys?Asn?Leu?Lys?Tyr?Gly?Val?Phe?Gly?Leu?Gly?Asn?Arg?Gln

195 200 205

Tyr?Glu?His?Phe?Asn?Lys?Val?Ala?Lys?Val?Val?Asp?Asp?Ile?Leu?Val

210 215 220

Glu?Gln?Gly?Ala?Gln?Arg?Leu?Val?Gln?Val?Gly?Leu?Gly?Asp?Asp?Asp

225 230 235 240

Gln?Cys?Ile?Glu?Asp?Asp?Phe?Thr?Ala?Trp?Arg?Glu?Ala?Leu?Trp?Pro

245 250 255

Glu?Leu?Asp?Thr?Ile?Leu?Arg?Glu?Glu?Gly?Asp?Thr?Ala?Val?Ala?Thr

260 265 270

Pro?Tyr?Thr?Ala?Ala?Val?Leu?Glu?Tyr?Arg?Val?Ser?Ile?His?Asp?Ser

275 280 285

Glu?Asp?Ala?Lys?Phe?Asn?Asp?Ile?Thr?Leu?Ala?Asn?Gly?Asn?Gly?Tyr

290 295 300

Thr?Val?Phe?Asp?Ala?Gln?His?Pro?Tyr?Lys?Ala?Asn?Val?Ala?Val?Lys

305 310 315 320

Arg?Glu?Leu?His?Thr?Pro?Glu?Ser?Asp?Arg?Ser?Cys?Ile?His?Leu?Glu

325 330 335

Phe?Asp?Ile?Ala?Gly?Ser?Gly?Leu?Thr?Met?Lys?Leu?Gly?Asp?His?Val

340 345 350

Gly?Val?Leu?Cys?Asp?Asn?Leu?Ser?Glu?Thr?Val?Asp?Glu?Ala?Leu?Arg

355 360 365

Leu?Leu?Asp?Met?Ser?Pro?Asp?Thr?Tyr?Phe?Ser?Leu?His?Ala?Glu?Lys

370 375 380

Glu?Asp?Gly?Thr?Pro?Ile?Ser?Ser?Ser?Leu?Pro?Pro?Pro?Phe?Pro?Pro

385 390 395 400

Cys?Asn?Leu?Arg?Thr?Ala?Leu?Thr?Arg?Tyr?Ala?Cys?Leu?Leu?Ser?Ser

405 410 415

Pro?Lys?Lys?Ser?Ala?Leu?Val?Ala?Leu?Ala?Ala?His?Ala?Ser?Asp?Pro

420 425 430

Thr?Glu?Ala?Glu?Arg?Leu?Lys?His?Leu?Ala?Ser?Pro?Ala?Gly?Lys?Asp

435 440 445

Glu?Tyr?Ser?Lys?Trp?Val?Val?Glu?Ser?Gln?Arg?Ser?Leu?Leu?Glu?Val

450 455 460

Met?Ala?Glu?Phe?Pro?Ser?Ala?Lys?Pro?Pro?Leu?Gly?Val?Phe?Phe?Ala

465 470 475 480

Gly?Val?Ala?Pro?Arg?Leu?Gln?Pro?Arg?Phe?Tyr?Ser?Ile?Ser?Ser?Ser

485 490 495

Pro?Lys?Ile?Ala?Glu?Thr?Arg?Ile?His?Val?Thr?Cys?Ala?Leu?Val?Tyr

500 505 510

Glu?Lys?Met?Pro?Thr?Gly?Arg?Ile?His?Lys?Gly?Val?Cys?Ser?Thr?Trp

515 520 525

Met?Lys?Asn?Ala?Val?Pro?Tyr?Glu?Lys?Ser?Glu?Lys?Leu?Phe?Leu?Gly

530 535 540

Arg?Pro?Ile?Phe?Val?Arg?Gln?Ser?Asn?Phe?Lys?Leu?Pro?Ser?Asp?Ser

545 550 555 560

Lys?Val?Pro?Ile?Ile?Met?Ile?Gly?Pro?Gly?Thr?Gly?Leu?Ala?Pro?Phe

565 570 575

Arg?Gly?Phe?Leu?Gln?Glu?Arg?Leu?Ala?Leu?Val?Glu?Ser?Gly?Val?Glu

580 585 590

Leu?Gly?Pro?Ser?Val?Leu?Phe?Phe?Gly?Cys?Arg?Asn?Arg?Arg?Met?Asp

595 600 605

Phe?Ile?Tyr?Glu?Glu?Glu?Leu?Gln?Arg?Phe?Val?Glu?Ser?Gly?Ala?Leu

610 615 620

Ala?Glu?Leu?Ser?Val?Ala?Phe?Ser?Arg?Glu?Gly?Pro?Thr?Lys?Glu?Tyr

625 630 635 640

Val?Gln?His?Lys?Met?Met?Asp?Lys?Ala?Ser?Asp?Ile?Trp?Asn?Met?Ile

645 650 655

Ser?Gln?Gly?Ala?Tyr?Leu?Tyr?Val?Cys?Gly?Asp?Ala?Lys?Gly?Met?Ala

660 665 670

Arg?Asp?Val?His?Arg?Ser?Leu?His?Thr?Ile?Ala?Gln?Glu?Gln?Gly?Ser

675 680 685

Met?Asp?Ser?Thr?Lys?Ala?Glu?Gly?Phe?Val?Lys?Asn?Leu?Gln?Thr?Ser

690 695 700

Gly?Arg?Tyr?Leu?Arg?Asp?Val?Trp

705 710

<210>40

<211>667

<212>PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400>40

Met?Leu?Ile?Glu?Asn?Arg?Gln?Phe?Ala?Met?Ile?Val?Thr?Thr?Ser?Ile

1 5 10 15

Ala?Val?Leu?Ile?Gly?Cys?Ile?Val?Met?Leu?Val?Trp?Arg?Arg?Ser?Gly

20 25 30

Ser?Gly?Asn?Ser?Lys?Arg?Val?Glu?Pro?Leu?Lys?Pro?Leu?Val?Ile?Lys

35 40 45

Pro?Arg?Glu?Glu?Glu?Ile?Asp?Asp?Gly?Arg?Lys?Lys?Val?Thr?Ile?Phe

50 55 60

Phe?Gly?Thr?Gln?Thr?Gly?Thr?Ala?Glu?Gly?Phe?Ala?Lys?Ala?Leu?Gly

65 70 75 80

Glu?Glu?Ala?Lys?Ala?Arg?Tyr?Glu?Lys?Thr?Arg?Phe?Lys?Ile?Val?Asp

85 90 95

Leu?Asp?Asp?Tyr?Ala?Ala?Asp?Asp?Asp?Glu?Tyr?Glu?Glu?Lys?Leu?Lys

100 105 110

Lys?Glu?Asp?Val?Ala?Phe?Phe?Phe?Leu?Ala?Thr?Tyr?Gly?Asp?Gly?Glu

115 120 125

Pro?Thr?Asp?Asn?Ala?Ala?Arg?Phe?Tyr?Lys?Trp?Phe?Thr?Glu?Gly?Asn

130 135 140

Asp?Arg?Gly?Glu?Trp?Leu?Lys?Asn?Leu?Lys?Tyr?Gly?Val?Phe?Gly?Leu

145 150 155 160

Gly?Asn?Arg?Gln?Tyr?Glu?His?Phe?Asn?Lys?Val?Ala?Lys?Val?Val?Asp

165 170 175

Asp?Ile?Leu?Val?Glu?Gln?Gly?Ala?Gln?Arg?Leu?Val?Gln?Val?Gly?Leu

180 185 190

Gly?Asp?Asp?Asp?Gln?Cys?Ile?Glu?Asp?Asp?Phe?Thr?Ala?Trp?Arg?Glu

195 200 205

Ala?Leu?Trp?Pro?Glu?Leu?Asp?Thr?Ile?Leu?Arg?Glu?Glu?Gly?Asp?Thr

210 215 220

Ala?Val?Ala?Thr?Pro?Tyr?Thr?Ala?Ala?Val?Leu?Glu?Tyr?Arg?Val?Ser

225 230 235 240

Ile?His?Asp?Ser?Glu?Asp?Ala?Lys?Phe?Asn?Asp?Ile?Asn?Met?Ala?Asn

245 250 255

Gly?Asn?Gly?Tyr?Thr?Val?Phe?Asp?Ala?Gln?His?Pro?Tyr?Lys?Ala?Asn

260 265 270

Val?Ala?Val?Lys?Arg?Glu?Leu?His?Thr?Pro?Glu?Ser?Asp?Arg?Ser?Cys

275 280 285

Ile?His?Leu?Glu?Phe?Asp?Ile?Ala?Gly?Ser?Gly?Leu?Thr?Tyr?Glu?Thr

290 295 300

Gly?Asp?His?Val?Gly?Val?Leu?Cys?Asp?Asn?Leu?Ser?Glu?Thr?Val?Asp

305 310 315 320

Glu?Ala?Leu?Arg?Leu?Leu?Asp?Met?Ser?Pro?Asp?Thr?Tyr?Phe?Ser?Leu

325 330 335

His?Ala?Glu?Lys?Glu?Asp?Gly?Thr?Pro?Ile?Ser?Ser?Ser?Leu?Pro?Pro

340 345 350

Pro?Phe?Pro?Pro?Cys?Asn?Leu?Arg?Thr?Ala?Leu?Thr?Arg?Tyr?Ala?Cys

355 360 365

Leu?Leu?Ser?Ser?Pro?Lys?Lys?Ser?Ala?Leu?Val?Ala?Leu?Ala?Ala?His

370 375 380

Ala?Ser?Asp?Pro?Thr?Glu?Ala?Glu?Arg?Leu?Lys?His?Leu?Ala?Ser?Pro

385 390 395 400

Ala?Gly?Lys?Asp?Glu?Tyr?Ser?Lys?Trp?Val?Val?Glu?Ser?Gln?Arg?Ser

405 410 415

Leu?Leu?Glu?Val?Met?Ala?Glu?Phe?Pro?Ser?Ala?Lys?Pro?Pro?Leu?Gly

420 425 430

Val?Phe?Phe?Ala?Gly?Val?Ala?Pro?Arg?Leu?Gln?Pro?Arg?Phe?Tyr?Ser

435 440 445

Ile?Ser?Ser?Ser?Pro?Lys?Ile?Ala?Glu?Thr?Arg?Ile?His?Val?Thr?Cys

450 455 460

Ala?Leu?Val?Tyr?Glu?Lys?Met?Pro?Thr?Gly?Arg?Ile?His?Lys?Gly?Val

465 470 475 480

Cys?Ser?Thr?Trp?Met?Lys?Asn?Ala?Val?Pro?Tyr?Glu?Lys?Ser?Glu?Asn

485 490 495

Cys?Ser?Ser?Ala?Pro?Ile?Phe?Val?Arg?Gln?Ser?Asn?Phe?Lys?Leu?Pro

500 505 510

Ser?Asp?Ser?Lys?Val?Pro?Ile?Ile?Met?Ile?Gly?Pro?Gly?Thr?Gly?Leu

515 520 525

Ala?Pro?Phe?Arg?Gly?Phe?Leu?Gln?Glu?Arg?Leu?Ala?Leu?Val?Glu?Ser

530 535 540

Gly?Val?Glu?Leu?Gly?Pro?Ser?Val?Leu?Phe?Phe?Gly?Cys?Arg?Asn?Arg

545 550 555 560

Arg?Met?Asp?Phe?Ile?Tyr?Glu?Glu?Glu?Leu?Gln?Arg?Phe?Val?Glu?Ser

565 570 575

Gly?Ala?Leu?Ala?Glu?Leu?Ser?Val?Ala?Phe?Ser?Arg?Glu?Gly?Pro?Thr

580 585 590

Lys?Glu?Tyr?Val?Gln?His?Lys?Met?Met?Asp?Lys?Ala?Ser?Asp?Ile?Trp

595 600 605

Asn?Met?Ile?Ser?Gln?Gly?Ala?Tyr?Leu?Tyr?Val?Cys?Gly?Asp?Ala?Lys

610 615 620

Gly?Met?Ala?Arg?Asp?Val?His?Arg?Ser?Leu?His?Thr?Ile?Ala?Gln?Glu

625 630 635 640

Gln?Gly?Ser?Met?Asp?Ser?Thr?Lys?Ala?Glu?Gly?Phe?Val?Lys?Asn?Leu

645 650 655

Gln?Thr?Ser?Gly?Arg?Tyr?Leu?Arg?Asp?Val?Trp

660 665

<210>41

<211>560

<212>PRT

＜213〉Eschscholtzia californica (Eschscholzia stolonifera)

<400>41

Met?Glu?Lys?Pro?Ile?Leu?Leu?Gln?Leu?Gln?Ala?Gly?Ile?Leu?Gly?Leu

1 5 10 15

Leu?Ala?Leu?Ile?Cys?Phe?Leu?Tyr?Tyr?Val?Ile?Lys?Val?Ser?Leu?Ser

20 25 30

Thr?Arg?Asn?Cys?Asn?Gln?Leu?Val?Lys?His?Pro?Pro?Glu?Ala?Ala?Gly

35 40 45

Ser?Trp?Pro?Ile?Val?Gly?His?Leu?Pro?Gln?Leu?Val?Gly?Ser?Gly?Lys

50 55 60

Pro?Leu?Phe?Arg?Val?Leu?Gly?Asp?Met?Ala?Asp?Lys?Phe?Gly?Pro?Ile

65 70 75 80

Phe?Met?Val?Arg?Phe?Gly?Val?Tyr?Pro?Thr?Leu?Val?Val?Ser?Thr?Trp

85 90 95

Glu?Met?Ala?Lys?Glu?Cys?Phe?Thr?Ser?Asn?Asp?Lys?Phe?Leu?Ala?Ser

100 105 110

Arg?Pro?Pro?Ser?Ala?Ala?Ser?Ser?Tyr?Met?Thr?Tyr?Asp?His?Ala?Met

115 120 125

Phe?Gly?Phe?Ser?Phe?Tyr?Gly?Pro?Tyr?Trp?Arg?Glu?Ile?Arg?Lys?Ile

130 135 140

Ser?Thr?Leu?His?Leu?Leu?Ser?His?Arg?Arg?Leu?Glu?Leu?Leu?Lys?His

145 150 155 160

Val?Pro?His?Thr?Glu?Ile?His?Asn?Phe?Ile?Lys?Gly?Leu?Phe?Gly?Ile

165 170 175

Trp?Lys?Asp?His?Gln?Lys?Gln?Gln?Gln?Pro?Thr?Gly?Arg?Glu?Asp?Arg

180 185 190

Asp?Ser?Val?Met?Leu?Glu?Met?Ser?Gln?Leu?Phe?Gly?Tyr?Leu?Thr?Leu

195 200 205

Asn?Val?Val?Leu?Ser?Leu?Val?Val?Gly?Lys?Arg?Val?Cys?Asn?Tyr?His

210 215 220

Ala?Asp?Gly?His?Leu?Asp?Asp?Gly?Glu?Glu?Ala?Gly?Gln?Gly?Gln?Lys

225 230 235 240

Leu?His?Gln?Thr?Ile?Thr?Asp?Phe?Phe?Lys?Leu?Ser?Gly?Val?Ser?Val

245 250 255

Ala?Ser?Asp?Ala?Leu?Pro?Leu?Leu?Gly?Leu?Phe?Asp?Leu?Gly?Gly?Lys

260 265 270

Lys?Glu?Ser?Met?Lys?Arg?Val?Ala?Lys?Glu?Met?Asp?Phe?Phe?Ala?Glu

275 280 285

Arg?Trp?Leu?Gln?Asp?Lys?Lys?Leu?Ser?Leu?Ser?Leu?Ser?Ser?Glu?Thr

290 295 300

Asn?Asn?Lys?Gln?Asn?Asp?Ala?Gly?Glu?Gly?Asp?Gly?Asp?Asp?Phe?Met

305 310 315 320

Asp?Val?Leu?Met?Ser?Ile?Leu?Pro?Asp?Asp?Asp?Asp?Ser?Leu?Phe?Thr

325 330 335

Lys?Tyr?Ser?Arg?Asp?Thr?Val?Ile?Lys?Ala?Thr?Ser?Leu?Ser?Met?Val

340 345 350

Val?Ala?Ala?Ser?Asp?Thr?Thr?Ser?Val?Ser?Leu?Thr?Trp?Ala?Leu?Ser

355 360 365

Leu?Leu?Leu?Asn?Asn?Ile?Gln?Val?Leu?Arg?Lys?Ala?Gln?Asp?Glu?Leu

370 375 380

Asp?Thr?Lys?Val?Gly?Arg?Asp?Arg?His?Val?Glu?Glu?Lys?Asp?Ile?Asp

385 390 395 400

Asn?Leu?Val?Tyr?Leu?Gln?Ala?Ile?Val?Lys?Glu?Thr?Leu?Arg?Met?Tyr

405 410 415

Pro?Ala?Gly?Pro?Leu?Ser?Val?Pro?His?Glu?Ala?Ile?Glu?Asp?Cys?Asn

420 425 430

Val?Gly?Gly?Tyr?His?Ile?Lys?Thr?Gly?Thr?Arg?Leu?Leu?Val?Asn?Ile

435 440 445

Trp?Lys?Leu?Gln?Arg?Asp?Pro?Arg?Val?Trp?Ser?Asn?Pro?Ser?Glu?Phe

450 455 460

Arg?Pro?Glu?Arg?Phe?Leu?Asp?Asn?Gln?Ser?Asn?Gly?Thr?Leu?Leu?Asp

465 470 475 480

Phe?Arg?Gly?Gln?His?Phe?Glu?Tyr?Ile?Pro?Phe?Gly?Ser?Gly?Arg?Arg

485 490 495

Met?Cys?Pro?Gly?Val?Asn?Phe?Ala?Thr?Leu?Ile?Leu?His?Met?Thr?Leu

500 505 510

Ala?Arg?Leu?Leu?Gln?Ala?Phe?Asp?Leu?Ser?Thr?Pro?Ser?Ser?Ser?Pro

515 520 525

Val?Asp?Met?Thr?Glu?Gly?Ser?Gly?Leu?Thr?Met?Pro?Lys?Val?Thr?Pro

530 535 540

Leu?Lys?Val?Leu?Leu?Thr?Pro?Arg?Leu?Pro?Leu?Pro?Leu?Tyr?Asp?Tyr

545 550 555 560

<210>42

<211>493

<212>PRT

＜213〉Vinca (Catharanthus roseus)

<400>42

Met?Asp?Tyr?Leu?Thr?Ile?Ile?Leu?Thr?Leu?Leu?Phe?Ala?Leu?Thr?Leu

1 5 10 15

Tyr?Glu?Ala?Phe?Ser?Tyr?Leu?Ser?Arg?Arg?Thr?Lys?Asn?Leu?Pro?Pro

20 25 30

Gly?Pro?Ser?Pro?Leu?Pro?Phe?Ile?Gly?Ser?Leu?His?Leu?Leu?Gly?Asp

35 40 45

Gln?Pro?His?Lys?Ser?Leu?Ala?Lys?Leu?Ser?Lys?Lys?His?Gly?Pro?Ile

50 55 60

Met?Ser?Leu?Lys?Leu?Gly?Gln?Ile?Thr?Thr?Ile?Val?Ile?Ser?Ser?Ser

65 70 75 80

Thr?Met?Ala?Lys?Glu?Val?Leu?Gln?Lys?Gln?Asp?Leu?Ala?Phe?Ser?Ser

85 90 95

Arg?Ser?Val?Pro?Asn?Ala?Leu?His?Ala?His?Asn?Gln?Phe?Lys?Phe?Ser

100 105 110

Val?Val?Trp?Leu?Pro?Val?Ala?Ser?Arg?Trp?Arg?Ser?Leu?Arg?Lys?Val

115 120 125

Leu?Asn?Ser?Asn?Ile?Phe?Ser?Gly?Asn?Arg?Leu?Asp?Ala?Asn?Gln?His

130 135 140

Leu?Arg?Thr?Arg?Lys?Val?Gln?Glu?Leu?Ile?Ala?Tyr?Cys?Arg?Lys?Asn

145 150 155 160

Ser?Gln?Ser?Gly?Glu?Ala?Val?Asp?Val?Gly?Arg?Ala?Ala?Phe?Arg?Thr

165 170 175

Ser?Leu?Asn?Leu?Leu?Ser?Asn?Leu?Ile?Phe?Ser?Lys?Asp?Leu?Thr?Asp

180 185 190

Pro?Tyr?Ser?Asp?Ser?Ala?Lys?Glu?Phe?Lys?Asp?Leu?Val?Trp?Asn?Ile

195 200 205

Met?Val?Glu?Ala?Gly?Lys?Pro?Asn?Leu?Val?Asp?Phe?Phe?Pro?Leu?Leu

210 215 220

Glu?Lys?Val?Asp?Pro?Gln?Gly?Ile?Arg?His?Arg?Met?Thr?Ile?His?Phe

225 230 235 240

Gly?Glu?Val?Leu?Lys?Leu?Phe?Gly?Gly?Leu?Val?Asn?Glu?Arg?Leu?Glu

245 250 255

Gln?Arg?Arg?Ser?Lys?Gly?Glu?Lys?Asn?Asp?Val?Leu?Asp?Val?Leu?Leu

260 265 270

Thr?Thr?Ser?Gln?Glu?Ser?Pro?Glu?Glu?Ile?Asp?Arg?Thr?His?Ile?Glu

275 280 285

Arg?Met?Cys?Leu?Asp?Leu?Phe?Val?Ala?Gly?Thr?Asp?Thr?Thr?Ser?Ser

290 295 300

Thr?Leu?Glu?Trp?Ala?Met?Ser?Glu?Met?Leu?Lys?Asn?Pro?Asp?Lys?Met

305 310 315 320

Lys?Lys?Thr?Gln?Asp?Glu?Leu?Ala?Gln?Val?Ile?Gly?Arg?Gly?Lys?Thr

325 330 335

Ile?Glu?Glu?Ser?Asp?Ile?Asn?Arg?Leu?Pro?Tyr?Leu?Arg?Cys?Val?Met

340 345 350

Lys?Glu?Thr?Leu?Arg?Ile?His?Pro?Pro?Val?Pro?Phe?Leu?Ile?Pro?Arg

355 360 365

Lys?Val?Glu?Gln?Ser?Val?Glu?Val?Cys?Gly?Tyr?Asn?Val?Pro?Lys?Gly

370 375 380

Ser?Gln?Val?Leu?Val?Asn?Ala?Trp?Ala?Ile?Gly?Arg?Asp?Glu?Thr?Val

385 390 395 400

Trp?Asp?Asp?Ala?Leu?Ala?Phe?Lys?Pro?Glu?Arg?Phe?Met?Glu?Ser?Glu

405 410 415

Leu?Asp?Ile?Arg?Gly?Arg?Asp?Phe?Glu?Leu?Ile?Pro?Phe?Gly?Ala?Gly

420 425 430

Arg?Arg?Ile?Cys?Pro?Gly?Leu?Pro?Leu?Ala?Leu?Arg?Thr?Val?Pro?Leu

435 440 445

Met?Leu?Gly?Ser?Leu?Leu?Asn?Ser?Phe?Asn?Trp?Lys?Leu?Glu?Gly?Gly

450 455 460

Met?Ala?Pro?Lys?Asp?Leu?Asp?Met?Glu?Glu?Lys?Phe?Gly?Ile?Thr?Leu

465 470 475 480

Gln?Lys?Ala?His?Pro?Leu?Arg?Ala?Val?Pro?Ser?Thr?Leu

485 490

<210>43

<211>496

<212>PRT

＜213〉Vinca (Catharanthus roseus)

<400>43

Met?Leu?Leu?Phe?Cys?Phe?Ile?Leu?Ser?Lys?Thr?Thr?Lys?Lys?Phe?Gly

1 5 10 15

Gln?Asn?Ser?Gln?Tyr?Ser?Asn?His?Asp?Glu?Leu?Pro?Pro?Gly?Pro?Pro

20 25 30

Gln?Ile?Pro?Ile?Leu?Gly?Asn?Ala?His?Gln?Leu?Ser?Gly?Gly?His?Thr

35 40 45

His?His?Ile?Leu?Arg?Asp?Leu?Ala?Lys?Lys?Tyr?Gly?Pro?Leu?Met?His

50 55 60

Leu?Lys?Ile?Gly?Glu?Val?Ser?Thr?Ile?Val?Ala?Ser?Ser?Pro?Gln?Ile

65 70 75 80

Ala?Glu?Glu?Ile?Phe?Arg?Thr?His?Asp?Ile?Leu?Phe?Ala?Asp?Arg?Pro

85 90 95

Ser?Asn?Leu?Glu?Ser?Phe?Lys?Ile?Val?Ser?Tyr?Asp?Phe?Ser?Asp?Met

100 105 110

Val?Val?Ser?Pro?Tyr?Gly?Asn?Tyr?Trp?Arg?Gln?Leu?Arg?Lys?Ile?Ser

115 120 125

Met?Met?Glu?Leu?Leu?Ser?Gln?Lys?Ser?Val?Gln?Ser?Phe?Arg?Ser?Ile

130 135 140

Arg?Glu?Glu?Glu?Val?Leu?Asn?Phe?Ile?Lys?Ser?Ile?Gly?Ser?Lys?Glu

145 150 155 160

Gly?Thr?Arg?Ile?Asn?Leu?Ser?Lys?Glu?Ile?Ser?Leu?Leu?Ile?Tyr?Gly

165 170 175

Ile?Thr?Thr?Arg?Ala?Ala?Phe?Gly?Glu?Lys?Asn?Lys?Asn?Thr?Glu?Glu

180 185 190

Phe?Ile?Arg?Leu?Leu?Asp?Gln?Leu?Thr?Lys?Ala?Val?Ala?Glu?Pro?Asn

195 200 205

Ile?Ala?Asp?Met?Phe?Pro?Ser?Leu?Lys?Phe?Leu?Gln?Leu?Ile?Ser?Thr

210 215 220

Ser?Lys?Tyr?Lys?Ile?Glu?Lys?Ile?His?Lys?Gln?Phe?Asp?Val?Ile?Val

225 230 235 240

Glu?Thr?Ile?Leu?Lys?Gly?His?Lys?Glu?Lys?Ile?Asn?Lys?Pro?Leu?Ser

245 250 255

Gln?Glu?Asn?Gly?Glu?Lys?Lys?Glu?Asp?Leu?Val?Asp?Val?Leu?Leu?Asn

260 265 270

Ile?Gln?Arg?Arg?Asn?Asp?Phe?Glu?Ala?Pro?Leu?Gly?Asp?Lys?Asn?Ile

275 280 285

Lys?Ala?Ile?Ile?Phe?Asn?Ile?Phe?Ser?Ala?Gly?Thr?Glu?Thr?Ser?Ser

290 295 300

Thr?Thr?Val?Asp?Trp?Ala?Met?Cys?Glu?Met?Ile?Lys?Asn?Pro?Thr?Val

305 310 315 320

Met?Lys?Lys?Ala?Gln?Glu?Glu?Val?Arg?Lys?Val?Phe?Asn?Glu?Glu?Gly

325 330 335

Asn?Val?Asp?Glu?Thr?Lys?Leu?His?Gln?Leu?Lys?Tyr?Leu?Gln?Ala?Val

340 345 350

Ile?Lys?Glu?Thr?Leu?Arg?Leu?His?Pro?Pro?Val?Pro?Leu?Leu?Leu?Pro

355 360 365

Arg?Glu?Cys?Arg?Glu?Gln?Cys?Lys?Ile?Lys?Gly?Tyr?Thr?Ile?Pro?Ser

370 375 380

Lys?Ser?Arg?Val?Ile?Val?Asn?Ala?Trp?Ala?Ile?Gly?Arg?Asp?Pro?Asn

385 390 395 400

Tyr?Trp?Ile?Glu?Pro?Glu?Lys?Phe?Asn?Pro?Asp?Arg?Phe?Leu?Glu?Ser

405 410 415

Lys?Val?Asp?Phe?Lys?Gly?Asn?Ser?Phe?Glu?Tyr?Leu?Pro?Phe?Gly?Gly

420 425 430

Gly?Arg?Arg?Ile?Cys?Pro?Gly?Ile?Thr?Phe?Ala?Leu?Ala?Asn?Ile?Glu

435 440 445

Leu?Pro?Leu?Ala?Gln?Leu?Leu?Phe?His?Phe?Asp?Trp?Gln?Ser?Asn?Thr

450 455 460

Glu?Lys?Leu?Asn?Met?Lys?Glu?Ser?Arg?Gly?Val?Thr?Val?Arg?Arg?Glu

465 470 475 480

Asp?Asp?Leu?Tyr?Leu?Thr?Pro?Val?Asn?Phe?Ser?Ser?Ser?Ser?Pro?Ala

485 490 495

<210>44

<211>505

<212>PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400>44

Met?Asp?Leu?Leu?Leu?Leu?Glu?Lys?Ser?Leu?Ile?Ala?Val?Phe?Val?Ala

1 5 10 15

Val?Ile?Leu?Ala?Thr?Val?Ile?Ser?Lys?Leu?Arg?Gly?Lys?Lys?Leu?Lys

20 25 30

Leu?Pro?Pro?Gly?Pro?Ile?Pro?Ile?Pro?Ile?Phe?Gly?Asn?Trp?Leu?Gln

35 40 45

Val?Gly?Asp?Asp?Leu?Asn?His?Arg?Asn?Leu?Val?Asp?Tyr?Ala?Lys?Lys

50 55 60

Phe?Gly?Asp?Leu?Phe?Leu?Leu?Arg?Met?Gly?Gln?Arg?Asn?Leu?Val?Val

65 70 75 80

Val?Ser?Ser?Pro?Asp?Leu?Thr?Lys?Glu?Val?Leu?Leu?Thr?Gln?Gly?Val

85 90 95

Glu?Phe?Gly?Ser?Arg?Thr?Arg?Asn?Val?Val?Phe?Asp?Ile?Phe?Thr?Gly

100 105 110

Lys?Gly?Gln?Asp?Met?Val?Phe?Thr?Val?Tyr?Gly?Glu?His?Trp?Arg?Lys

115 120 125

Met?Arg?Arg?Ile?Met?Thr?Val?Pro?Phe?Phe?Thr?Asn?Lys?Val?Val?Gln

130 135 140

Gln?Asn?Arg?Glu?Gly?Trp?Glu?Phe?Glu?Ala?Ala?Ser?Val?Val?Glu?Asp

145 150 155 160

Val?Lys?Lys?Asn?Pro?Asp?Ser?Ala?Thr?Lys?Gly?Ile?Val?Leu?Arg?Lys

165 170 175

Arg?Leu?Gln?Leu?Met?Met?Tyr?Asn?Asn?Met?Phe?Arg?Ile?Met?Phe?Asp

180 185 190

Arg?Arg?Phe?Glu?Ser?Glu?Asp?Asp?Pro?Leu?Phe?Leu?Arg?Leu?Lys?Ala

195 200 205

Leu?Asn?Gly?Glu?Arg?Ser?Arg?Leu?Ala?Gln?Ser?Phe?Glu?Tyr?Asn?Tyr

210 215 220

Gly?Asp?Phe?Ile?Pro?Ile?Leu?Arg?Pro?Phe?Leu?Arg?Gly?Tyr?Leu?Lys

225 230 235 240

Ile?Cys?Gln?Asp?Val?Lys?Asp?Arg?Arg?Ile?Ala?Leu?Phe?Lys?Lys?Tyr

245 250 255

Phe?Val?Asp?Glu?Arg?Lys?Gln?Ile?Ala?Ser?Ser?Lys?Pro?Thr?Gly?Ser

260 265 270

Glu?Gly?Leu?Lys?Cys?Ala?Ile?Asp?His?Ile?Leu?Glu?Ala?Glu?Gln?Lys

275 280 285

Gly?Glu?Ile?Asn?Glu?Asp?Asn?Val?Leu?Tyr?Ile?Val?Glu?Asn?Ile?Asn

290 295 300

Val?Ala?Ala?Ile?Glu?Thr?Thr?Leu?Trp?Ser?Ile?Glu?Trp?Gly?Ile?Ala

305 310 315 320

Glu?Leu?Val?Asn?His?Pro?Glu?Ile?Gln?Ser?Lys?Leu?Arg?Asn?Glu?Leu

325 330 335

Asp?Thr?Val?Leu?Gly?Pro?Gly?Val?Gln?Val?Thr?Glu?Pro?Asp?Leu?His

340 345 350

Lys?Leu?Pro?Tyr?Leu?Gln?Ala?Val?Val?Lys?Glu?Thr?Leu?Arg?Leu?Arg

355 360 365

Met?Ala?Ile?Pro?Leu?Leu?Val?Pro?His?Met?Asn?Leu?His?Asp?Ala?Lys

370 375 380

Leu?Ala?Gly?Tyr?Asp?Ile?Pro?Ala?Glu?Ser?Lys?Ile?Leu?Val?Asn?Ala

385 390 395 400

Trp?Trp?Leu?Ala?Asn?Asn?Pro?Asn?Ser?Trp?Lys?Lys?Pro?Glu?Glu?Phe

405 410 415

Arg?Pro?Glu?Arg?Phe?Phe?Glu?Glu?Glu?Ser?His?Val?Glu?Ala?Asn?Gly

420 425 430

Asn?Asp?Phe?Arg?Tyr?Val?Pro?Phe?Gly?Val?Gly?Arg?Arg?Ser?Cys?Pro

435 440 445

Gly?Ile?Ile?Leu?Ala?Leu?Pro?Ile?Leu?Gly?Ile?Thr?Ile?Gly?Arg?Met

450 455 460

Val?Gln?Asn?Phe?Glu?Leu?Leu?Pro?Pro?Pro?Gly?Gln?Ser?Lys?Val?Asp

465 470 475 480

Thr?Ser?Glu?Lys?Gly?Gly?Gln?Phe?Ser?Leu?His?Ile?Leu?Asn?His?Ser

485 490 495

Ile?Ile?Val?Met?Lys?Pro?Arg?Asn?Cys

500 505

<210>45

<211>512

<212>PRT

＜213〉sweet basil (Ocimum basilicum)

<400>45

Met?Ala?Ala?Leu?Leu?Leu?Leu?Leu?Leu?Leu?Pro?Leu?Leu?Leu?Pro?Ala

1 5 10 15

Ile?Phe?Leu?Leu?His?His?Leu?Tyr?Tyr?Arg?Leu?Arg?Phe?Arg?Leu?Pro

20 25 30

Pro?Gly?Pro?Arg?Pro?Leu?Pro?Ile?Val?Gly?Asn?Leu?Tyr?Asp?Val?Lys

35 40 45

Pro?Val?Arg?Phe?Arg?Cys?Phe?Ala?Asp?Trp?Ala?Gln?Ser?Tyr?Gly?Pro

50 55 60

Ile?Ile?Ser?Val?Trp?Phe?Gly?Ser?Thr?Leu?Asn?Val?Ile?Val?Ser?Asn

65 70 75 80

Thr?Glu?Leu?Ala?Lys?Glu?Val?Leu?Lys?Glu?Lys?Asp?Gln?Gln?Leu?Ala

85 90 95

Asp?Arg?His?Arg?Ser?Arg?Ser?Ala?Ala?Lys?Phe?Ser?Arg?Asp?Gly?Gln

100 105 110

Asp?Leu?Ile?Trp?Ala?Asp?Tyr?Gly?Pro?His?Tyr?Val?Lys?Val?Arg?Lys

115 120 125

Val?Cys?Thr?Leu?Glu?Leu?Phe?Ser?Pro?Lys?Arg?Leu?Glu?Ala?Leu?Arg

130 135 140

Pro?Ile?Arg?Glu?Asp?Glu?Val?Thr?Ala?Met?Val?Glu?Ser?Ile?Tyr?His

145 150 155 160

Asp?Cys?Thr?Ala?Pro?Asp?Asn?Ala?Gly?Lys?Ser?Leu?Leu?Val?Lys?Lys

165 170 175

Tyr?Leu?Gly?Ala?Val?Ala?Phe?Asn?Asn?Ile?Thr?Arg?Leu?Ala?Phe?Gly

180 185 190

Lys?Arg?Phe?Val?Asn?Ser?Glu?Gly?Ile?Ile?Asp?Lys?Gln?Gly?Leu?Glu

195 200 205

Phe?Lys?Ala?Ile?Val?Ser?Asn?Gly?Leu?Lys?Leu?Gly?Ala?Ser?Leu?Ala

210 215 220

Met?Ala?Glu?His?Ile?Pro?Ser?Leu?Arg?Trp?Met?Phe?Pro?Leu?Asp?Glu

225 230 235 240

Asp?Ala?Phe?Ala?Lys?His?Gly?Ala?Arg?Arg?Asp?Gln?Leu?Thr?Arg?Glu

245 250 255

Ile?Met?Glu?Glu?His?Thr?Arg?Ala?Arg?Glu?Glu?Ser?Gly?Gly?Ala?Lys

260 265 270

Gln?His?Phe?Phe?Asp?Ala?Leu?Leu?Thr?Leu?Lys?Asp?Lys?Tyr?Asp?Leu

275 280 285

Ser?Glu?Asp?Thr?Ile?Ile?Gly?Leu?Leu?Trp?Asp?Met?Ile?Thr?Ala?Gly

290 295 300

Met?Asp?Thr?Thr?Ala?Ile?Ser?Val?Glu?Trp?Ala?Met?Ala?Glu?Leu?Ile

305 310 315 320

Lys?Asn?Pro?Arg?Val?Gln?Gln?Lys?Ala?Gln?Glu?Glu?Leu?Asp?Arg?Val

325 330 335

Ile?Gly?Tyr?Glu?Arg?Val?Met?Thr?Glu?Leu?Asp?Phe?Ser?Asn?Leu?Pro

340 345 350

Tyr?Leu?Gln?Cys?Val?Ala?Lys?Glu?Ala?Leu?Arg?Leu?His?Pro?Pro?Thr

355 360 365

Pro?Leu?Met?Leu?Pro?His?Arg?Ser?Asn?Ser?Asn?Val?Lys?Ile?Gly?Gly

370 375 380

Tyr?Asp?Ile?Pro?Lys?Gly?Ser?Asn?Val?His?Val?Asn?Val?Trp?Ala?Val

385 390 395 400

Ala?Arg?Asp?Pro?Ala?Val?Trp?Lys?Asn?Pro?Cys?Glu?Phe?Arg?Pro?Glu

405 410 415

Arg?Phe?Leu?Glu?Glu?Asp?Val?Asp?Met?Lys?Gly?His?Asp?Phe?Arg?Leu

420 425 430

Leu?Pro?Phe?Gly?Ala?Gly?Arg?Arg?Val?Cys?Pro?Gly?Ala?Gln?Leu?Gly

435 440 445

Ile?Asn?Leu?Val?Thr?Ser?Met?Ile?Gly?His?Leu?Leu?His?His?Phe?Asn

450 455 460

Trp?Ala?Pro?Pro?Ser?Gly?Val?Ser?Ser?Asp?Glu?Leu?Asp?Met?Gly?Glu

465 470 475 480

Asn?Pro?Gly?Leu?Val?Thr?Tyr?Met?Arg?Thr?Pro?Leu?Glu?Ala?Val?Pro

485 490 495

Thr?Pro?Arg?Leu?Pro?Ser?Asp?Leu?Tyr?Lys?Arg?Ile?Ala?Val?Asp?Leu

500 505 510

<210>46

<211>521

<212>PRT

＜213〉the cultivated soybean (Glycine max)

<400>46

Met?Leu?Leu?Glu?Leu?Ala?Leu?Gly?Leu?Phe?Val?Leu?Ala?Leu?Phe?Leu

1 5 10 15

His?Leu?Arg?Pro?Thr?Pro?Ser?Ala?Lys?Ser?Lys?Ala?Leu?Arg?His?Leu

20 25 30

Pro?Asn?Pro?Pro?Ser?Pro?Lys?Pro?Arg?Leu?Pro?Phe?Ile?Gly?His?Leu

35 40 45

His?Leu?Leu?Lys?Asp?Lys?Leu?Leu?His?Tyr?Ala?Leu?Ile?Asp?Leu?Ser

50 55 60

Lys?Lys?His?Gly?Pro?Leu?Phe?Ser?Leu?Ser?Phe?Gly?Ser?Met?Pro?Thr

65 70 75 80

Val?Val?Ala?Ser?Thr?Pro?Glu?Leu?Phe?Lys?Leu?Phe?Leu?Gln?Thr?His

85 90 95

Glu?Ala?Thr?Ser?Phe?Asn?Thr?Arg?Phe?Gln?Thr?Ser?Ala?Ile?Arg?Arg

100 105 110

Leu?Thr?Tyr?Asp?Asn?Ser?Val?Ala?Met?Val?Pro?Phe?Gly?Pro?Tyr?Trp

115 120 125

Lys?Phe?Val?Arg?Lys?Leu?Ile?Met?Asn?Asp?Leu?Leu?Asn?Ala?Thr?Thr

130 135 140

Val?Asn?Lys?Leu?Arg?Pro?Leu?Arg?Thr?Gln?Gln?Ile?Arg?Lys?Phe?Leu

145 150 155 160

Arg?Val?Met?Ala?Gln?Ser?Ala?Glu?Ala?Gln?Lys?Pro?Leu?Asp?Val?Thr

165 170 175

Glu?Glu?Leu?Leu?Lys?Trp?Thr?Asn?Ser?Thr?Ile?Ser?Met?Met?Met?Leu

180 185 190

Gly?Glu?Ala?Glu?Glu?Ile?Arg?Asp?Ile?Ala?Arg?Glu?Val?Leu?Lys?Ile

195 200 205

Phe?Gly?Glu?Tyr?Ser?Leu?Thr?Asp?Phe?Ile?Trp?Pro?Leu?Lys?Tyr?Leu

210 215 220

Lys?Val?Gly?Lys?Tyr?Glu?Lys?Arg?Ile?Asp?Asp?Ile?Leu?Asn?Lys?Phe

225 230 235 240

Asp?Pro?Val?Val?Glu?Arg?Val?Ile?Lys?Lys?Arg?Arg?Glu?Ile?Val?Arg

245 250 255

Arg?Arg?Lys?Asn?Gly?Glu?Val?Val?Glu?Gly?Glu?Ala?Ser?Gly?Val?Phe

260 265 270

Leu?Asp?Thr?Leu?Leu?Glu?Phe?Ala?Glu?Asp?Glu?Thr?Met?Glu?Ile?Lys

275 280 285

Ile?Thr?Lys?Glu?Gln?Ile?Lys?Gly?Leu?Val?Val?Asp?Phe?Phe?Ser?Ala

290 295 300

Gly?Thr?Asp?Ser?Thr?Ala?Val?Ala?Thr?Glu?Trp?Ala?Leu?Ala?Glu?Leu

305 310 315 320

Ile?Asn?Asn?Pro?Arg?Val?Leu?Gln?Lys?Ala?Arg?Glu?Glu?Val?Tyr?Ser

325 330 335

Val?Val?Gly?Lys?Asp?Arg?Leu?Val?Asp?Glu?Val?Asp?Thr?Gln?Asn?Leu

340 345 350

Pro?Tyr?Ile?Arg?Ala?Ile?Val?Lys?Glu?Thr?Phe?Arg?Met?His?Pro?Pro

355 360 365

Leu?Pro?Val?Val?Lys?Arg?Lys?Cys?Thr?Glu?Glu?Cys?Glu?Ile?Asn?Gly

370 375 380

Tyr?Val?Ile?Pro?Glu?Gly?Ala?Leu?Val?Leu?Phe?Asn?Val?Trp?Gln?Val

385 390 395 400

Gly?Arg?Asp?Pro?Lys?Tyr?Trp?Asp?Arg?Pro?Ser?Glu?Phe?Arg?Pro?Glu

405 410 415

Arg?Phe?Leu?Glu?Thr?Gly?Ala?Glu?Gly?Glu?Ala?Gly?Pro?Leu?Asp?Leu

420 425 430

Arg?Gly?Gln?His?Phe?Gln?Leu?Leu?Pro?Phe?Gly?Ser?Gly?Arg?Arg?Met

435 440 445

Cys?Pro?Gly?Val?Asn?Leu?Ala?Thr?Ser?Gly?Met?Ala?Thr?Leu?Leu?Ala

450 455 460

Ser?Leu?Ile?Gln?Cys?Phe?Asp?Leu?Gln?Val?Leu?Gly?Pro?Gln?Gly?Gln

465 470 475 480

Ile?Leu?Lys?Gly?Asp?Asp?Ala?Lys?Val?Ser?Met?Glu?Glu?Arg?Ala?Gly

485 490 495

Leu?Thr?Val?Pro?Arg?Ala?His?Ser?Leu?Val?Cys?Val?Pro?Leu?Ala?Arg

500 505 510

Ile?Gly?Val?Ala?Ser?Lys?Leu?Leu?Ser

515 520

<210>47

<211>456

<212>PRT

＜213〉Avid kyowamycin (Streptomyces avermitilis)

<400>47

Met?Met?Ser?Gln?Ser?Thr?Ser?Ser?Ile?Pro?Glu?Ala?Pro?Gly?Ala?Trp

1 5 10 15

Pro?Val?Val?Gly?His?Val?Pro?Pro?Leu?Met?Arg?Gln?Pro?Leu?Glu?Phe

20 25 30

Leu?Arg?Ser?Ala?Ala?Asp?His?Gly?Asp?Leu?Leu?Lys?Leu?Arg?Leu?Gly

35 40 45

Pro?Lys?Thr?Ala?Tyr?Leu?Ala?Thr?His?Pro?Asp?Leu?Val?Arg?Thr?Met

50 55 60

Leu?Val?Ser?Ser?Gly?Ser?Gly?Asp?Phe?Thr?Arg?Ser?Lys?Gly?Ala?Gln

65 70 75 80

Gly?Ala?Ser?Arg?Phe?Ile?Gly?Pro?Ile?Leu?Val?Ala?Val?Ser?Gly?Glu

85 90 95

Thr?His?Arg?Arg?Gln?Arg?Arg?Arg?Met?Gln?Pro?Gly?Phe?His?Arg?Gln

100 105 110

Arg?Leu?Glu?Ser?Tyr?Val?Ala?Thr?Met?Ala?Ala?Ala?Ala?Gln?Glu?Thr

115 120 125

Ala?Asp?Ser?Trp?Ser?Ala?Gly?Gln?Val?Val?Asp?Val?Glu?Gln?Ala?Ala

130 135 140

Cys?Asp?Leu?Ser?Leu?Ala?Met?Ile?Thr?Lys?Thr?Leu?Phe?Phe?Ser?Asp

145 150 155 160

Leu?Gly?Ala?Lys?Ala?Glu?Ala?Ala?Leu?Arg?Lys?Thr?Gly?His?Asp?Ile

165 170 175

Leu?Lys?Val?Ala?Arg?Leu?Ser?Ala?Leu?Ala?Pro?Thr?Leu?Tyr?Glu?Val

180 185 190

Leu?Pro?Thr?Ala?Gly?Lys?Arg?Ser?Val?Gly?Arg?Thr?Ser?Ala?Thr?Ile

195 200 205

Arg?Glu?Ala?Ile?Thr?Ala?Tyr?Arg?Ala?Asp?Gly?Arg?Asp?His?Gly?Asp

210 215 220

Leu?Leu?Ser?Thr?Met?Leu?Arg?Ala?Thr?Asp?Ala?Glu?Gly?Ala?Ser?Met

225 230 235 240

Thr?Asp?Gln?Glu?Val?His?Asp?Glu?Val?Met?Gly?Ile?Ala?Val?Ala?Gly

245 250 255

Ile?Gly?Gly?Pro?Ala?Ala?Ile?Thr?Ala?Trp?Ile?Phe?His?Glu?Leu?Gly

260 265 270

Gln?Asn?Ala?Glu?Ile?Glu?Ser?Arg?Leu?His?Ala?Glu?Leu?Asp?Thr?Val

275 280 285

Leu?Gly?Gly?Arg?Leu?Pro?Thr?His?Glu?Asp?Leu?Pro?Arg?Leu?Pro?Tyr

290 295 300

Thr?Gln?Asn?Leu?Val?Lys?Glu?Ala?Leu?Arg?Lys?Tyr?Pro?Gly?Trp?Val

305 310 315 320

Gly?Ser?Arg?Arg?Thr?Val?Arg?Pro?Val?Arg?Leu?Gly?Gly?His?Asp?Leu

325 330 335

Pro?Ala?Asp?Val?Glu?Val?Met?Tyr?Ser?Ala?Tyr?Ala?Ile?Gln?Arg?Asp

340 345 350

Pro?Arg?Trp?Tyr?Pro?Glu?Pro?Glu?Arg?Leu?Asp?Pro?Gly?Arg?Trp?Glu

355 360 365

Thr?Lys?Gly?Ser?Ser?Arg?Gly?Val?Pro?Lys?Gly?Ala?Trp?Val?Pro?Phe

370 375 380

Ala?Leu?Gly?Thr?Tyr?Lys?Cys?Ile?Gly?Asp?Asn?Phe?Ala?Leu?Leu?Glu

385 390 395 400

Thr?Ala?Val?Thr?Val?Ala?Val?Val?Ala?Ser?His?Trp?Arg?Leu?His?Ala

405 410 415

Leu?Pro?Gly?Asp?Glu?Val?Arg?Pro?Lys?Thr?Lys?Ala?Thr?His?Val?Phe

420 425 430

Pro?Asn?Arg?Leu?Arg?Met?Ile?Ala?Glu?Pro?Arg?Ser?Val?Val?Arg?Leu

435 440 445

Glu?Glu?Pro?Ala?Ala?Met?Gly?Ala

450 455

<210>48

<21l>401

<212>PRT

＜213〉than basic streptomycete (Streptomyces bikiniensis)

<400>48

Met?Gly?Leu?Pro?Leu?Thr?Ser?Thr?Lys?Thr?Ala?Pro?Val?Ser?Tyr?Pro

1 5 10 15

Phe?Gly?Arg?Pro?Glu?Gly?Leu?Asp?Leu?Asp?Glu?Ala?Tyr?Glu?Gln?Ala

20 25 30

Arg?Lys?Ser?Glu?Gly?Leu?Leu?Trp?Val?His?Met?Pro?Tyr?Gly?Glu?Pro

35 40 45

Gly?Trp?Leu?Val?Ser?Arg?Tyr?Asp?Asp?Ala?Arg?Phe?Val?Leu?Gly?Asp

50 55 60

Arg?Arg?Phe?Ser?His?Ala?Ala?Glu?Ala?Glu?Asn?Asp?Ala?Pro?Arg?Met

65 70 75 80

Arg?Glu?Leu?Arg?Thr?Pro?Asn?Gly?Ile?Ile?Gly?Met?Asp?Ala?Pro?Asp

85 90 95

His?Thr?Arg?Leu?Arg?Gly?Leu?Val?Thr?Lys?Ala?Phe?Thr?Pro?Arg?Arg

100 105 110

Val?Glu?Ala?Met?Arg?Pro?His?Val?Arg?Arg?Met?Thr?Ala?Ser?Leu?Leu

115 120 125

Arg?Asp?Met?Thr?Ala?Leu?Gly?Ser?Pro?Val?Asp?Leu?Val?Asp?His?Tyr

130 135 140

Ala?Val?Pro?Leu?Pro?Val?Ala?Val?Ile?Cys?Gly?Leu?Leu?Gly?Val?Pro

145 150 155 160

Glu?Glu?Asp?Arg?Asp?Leu?Phe?Arg?Gly?Trp?Cys?Glu?Ile?Ala?Met?Ser

165 170 175

Thr?Ser?Ser?Leu?Thr?Ala?Glu?Asp?His?Val?Arg?Leu?Ala?Gly?Glu?Leu

180 185 190

Thr?Gly?Tyr?Leu?Ala?Asp?Leu?Ile?Thr?Ala?Arg?Arg?Ala?Ala?Pro?Arg

195 200 205

Asp?Asp?Leu?Val?Ser?Ala?Leu?Val?Glu?Ala?Arg?Asp?Ala?Gln?Gly?Arg

210 215 220

Leu?Ser?Gln?Glu?Glu?Leu?Val?Asp?Leu?Ile?Val?Phe?Leu?Leu?Phe?Ala

225 230 235 240

Gly?His?Glu?Thr?Thr?Ala?Ser?Gln?Ile?Ser?Asn?Phe?Val?Leu?Val?Leu

245 250 255

Leu?Glu?Gln?Pro?Asp?Gln?Leu?Ala?Leu?Leu?Arg?Asp?Arg?Pro?Asp?Leu

260 265 270

Leu?Asp?Asn?Ala?Val?Glu?Glu?Leu?Thr?Arg?Phe?Val?Pro?Leu?Gly?Ser

275 280 285

Gln?Ala?Gly?Phe?Pro?Arg?Tyr?Ala?Thr?Glu?Asp?Val?Glu?Val?Gly?Gly

290 295 300

Thr?Leu?Val?Arg?Ala?Gly?Asp?Pro?Val?Leu?Val?Gln?Met?Asn?Ala?Ala

305 310 315 320

Asn?Arg?Asp?Ala?Leu?Arg?Phe?Arg?Ser?Pro?Gly?Val?Leu?Asp?Ile?Thr

325 330 335

Arg?Asp?Asp?Ala?Gly?Arg?His?Leu?Gly?Tyr?Gly?His?Gly?Pro?His?His

340 345 350

Cys?Leu?Gly?Ala?Ser?Leu?Ala?Arg?Leu?Glu?Leu?Gln?Glu?Ala?Leu?Arg

355 360 365

Thr?Leu?Leu?Asp?Glu?Leu?Pro?Gly?Leu?His?Leu?Ala?Gln?Pro?Val?Glu

370 375 380

Trp?Lys?Thr?Glu?Met?Val?Val?Arg?Gly?Pro?Arg?Thr?Met?Leu?Val?Gly

385 390 395 400

Trp

<210>49

<211>1497

<212>DNA

＜213〉Herba Artemisiae annuae (Artemisia annua)

<400>49

catatgaagt?ctattctgaa?agcaatggct?ctgtctctga?ccactagcat?cgccctggcg?60

actatcctgc?tgtttgtgta?caaattcgcg?acccgttcta?aaagcactaa?gaaatctctg?120

ccggaaccgt?ggcgtctgcc?aatcatcggt?cacatgcacc?acctgatcgg?caccaccccg?180

caccgtggcg?tacgcgacct?ggcgcgtaag?tacggctctc?tgatgcatct?gcagctgggc?240

gaggtaccta?ctatcgtcgt?ttcctccccg?aagtgggcca?aagaaatcct?gactacctat?300

gacatcactt?tcgccaaccg?cccggaaacg?ctgaccggcg?aaattgtcct?gtaccataac?360

acggatgtgg?ttctggcccc?gtacggtgag?tactggcgcc?agctgcgcaa?aatttgtact?420

ctggaactgc?tgagcgttaa?aaaggttaaa?tccttccaga?gcctgcgtga?agaggaatgc?480

tggaacctgg?tgcaggagat?taaagcgtct?ggcagcggtc?gtccagttaa?cctgtctgag?540

aatgttttta?aactgatcgc?tactatcctg?tctcgcgcgg?cattcggtaa?aggtatcaaa?600

gatcagaaag?aactgaccga?aatcgttaag?gaaatcctgc?gccagactgg?tggcttcgac?660

gttgcggaca?tcttcccgtc?caaaaagttc?ctgcaccatc?tgtctggcaa?acgcgctcgt?720

ctgacctccc?tgcgtaagaa?aattgataac?ctgattgaca?acctggtcgc?tgagcacact?780

gtgaacacct?cttctaaaac?caacgaaacc?ctgctggacg?tactgctgcg?cctgaaggac?840

tctgccgaat?ttccactgac?tagcgacaat?atcaaagcaa?tcatcctgga?catgttcggc?900

gccggtaccg?atacgtcctc?ttccacgatt?gagtgggcta?tttccgaact?gatcaaatgc?960

ccgaaggcga?tggaaaaagt?gcaggcggaa?ctgcgtaaag?cgctgaacgg?taaagagaaa?1020

attcatgaag?aggacatcca?ggaactgtcc?tacctgaata?tggtaatcaa?agaaactctg?1080

cgtctgcatc?cgccgctgcc?actggttctg?ccgcgtgaat?gccgtcagcc?ggttaacctg?1140

gccggctaca?acattccgaa?caaaacgaag?ctgatcgtca?acgttttcgc?gatcaaccgc?1200

gatcctgaat?actggaaaga?cgcggaagcg?ttcattccgg?aacgctttga?gaactcctct?1260

gccaccgtta?tgggcgctga?atacgagtac?ctgccgttcg?gtgcgggtcg?ccgtatgtgc?1320

ccgggtgctg?cactgggcct?ggcgaacgtt?caactgccac?tggcgaacat?cctgtaccac?1380

ttcaactgga?aactgcctaa?cggcgtatct?tatgatcaaa?tcgacatgac?cgaaagctcc?1440

ggcgcgacca?tgcagcgtaa?aaccgaactg?ctgctggttc?cgtcctttta?acctagg 1497

<210>50

<211>1497

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase nucleic acid of Xiu Shiing

<400>50

catatgaagt?ctattctgaa?agcaatggct?ctgtctctga?ccactagcat?cgccctggcg?60

actatcctgc?tgtttgtgta?caaattcgcg?acccgttcta?aaagcactaa?gaaatctctg?120

ccggaaccgt?ggcgtctgcc?aatcatcggt?cacatgcacc?acctgatcgg?caccaccccg?180

caccgtggcg?tacgcgacct?ggcgcgtaag?tacggctctc?tgatgcatct?gcagctgggc?240

gaggtaccta?ctatcgtcgt?ttcctccccg?aagtgggcca?aagaaatcct?gactacctat?300

gacatcactt?tcgccaaccg?cccggaaacg?ctgaccggcg?aaattgtcct?gtaccataac?360

acggatgtgg?ttctggcccc?gtacggtgag?tactggcgcc?agctgcgcaa?aatttgtact?420

ctggaactgc?tgagcgttaa?aaaggttaaa?tccttccaga?gcctgcgtga?agaggaatgc?480

tggaacctgg?tgcaggagat?taaagcgtct?ggcagcggtc?gtccagttaa?cctgtctgag?540

aatgttttta?aactgatcgc?tactatcctg?tctcgcgcgg?cattcggtaa?aggtatcaaa?600

gatcagaaag?aactgaccga?aatcgttaag?gaaatcctgc?gccagactgg?tggcttcgac?660

gttgcggaca?tcttcccgtc?caaaaagttc?ctgcaccatc?tgtctggcaa?acgcgctcgt?720

ctgacctccc?tgcgtaagaa?aattgataac?ctgattgaca?acctggtcgc?tgagcacact?780

gtgaacacct?cttctaaaac?caacgaaacc?ctgctggacg?tactgctgcg?cctgaaggac?840

tctgccgaat?ttccactgac?tagcgacaat?atcaaagcaa?tcatcctgga?catgttcggc?900

gccggtaccg?atacgtcctc?ttccacgatt?gagtgggcta?tttccgaact?gatcaaatgc?960

ccgaaggcga?tggaaaaagt?gcaggcggaa?ctgcgtaaag?cgctgaacgg?taaagagaaa?1020

attcatgaag?aggacatcca?ggaactgtcc?tacctgaata?tggtaatcaa?agaaactctg?1080

cgtctgcatc?cgccgctgcc?actggttctg?ccgcgtgaat?gccgtcagcc?ggttaacctg?1140

gccggctaca?acattccgaa?caaaacgaag?ctgatcgtca?acgttttcgc?gatcaaccgc?1200

gatcctgaat?actggaaaga?cgcggaagcg?ttcattccgg?aacgctttga?gaactcctct?1260

gccaccgtta?tgggcgctga?atacgagtac?ctgccgttcg?gtgcgggtcg?ccgtatgtgc?1320

ccgggtgctg?cactgggcct?ggcgaacgtt?caactgccac?tggcgaacat?cctgtaccac?1380

ttcaactgga?aactgcctaa?cggcgtatct?tatgatcaaa?tcgacatgac?cgaaagctcc?1440

ggcgcgacca?tgcagcgtaa?aaccgaactg?ctgctggttc?cgtcctttta?acctagg 1497

<210>51

<211>1488

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase nucleic acid of Xiu Shiing

<400>51

atgaagtcta?ttctgaaagc?aatggctctg?tctctgacca?ctagcatcgc?cctggcgact?60

atcctgctgt?ttgtgtacaa?attcgcgacc?cgttctaaaa?gcactaagaa?atctctgccg?120

gaaccgtggc?gtctgccaat?catcggtcac?atgcaccacc?tgatcggcac?caccccgcac?180

cgtggcgtac?gcgacctggc?gcgtaagtac?ggctctctga?tgcatctgca?gctgggcgag?240

gtacctacta?tcgtcgtttc?ctccccgaag?tgggccaaag?aaatcctgac?tacctatgac?300

atcactttcg?ccaaccgccc?ggaaacgctg?accggcgaaa?ttgtcctgta?ccataacacg?360

gatgtggttc?tggccccgta?cggtgagtac?tggcgccagc?tgcgcaaaat?ttgtactctg?420

gaactgctga?gcgttaaaaa?ggttaaatcc?ttccagagcc?tgcgtgaaga?ggaatgctgg?480

aacctggtgc?aggagattaa?agcgtctggc?agcggtcgtc?cagttaacct?gtctgagaat?540

gtttttaaac?tgatcgctac?tatcctgtct?cgcgcggcat?tcggtaaagg?tatcaaagat?600

cagaaagaac?tgaccgaaat?cgttaaggaa?atcctgcgcc?agactggtgg?cttcgacgtt?660

gcggacatct?tcccgtccaa?aaagttcctg?caccatctgt?ctggcaaacg?cgctcgtctg?720

acctccctgc?gtaagaaaat?tgataacctg?attgacaacc?tggtcgctga?gcacactgtg?780

aacacctctt?ctaaaaccaa?cgaaaccctg?ctggacgtac?tgctgcgcct?gaaggactct?840

gccgaatttc?cactgactag?cgacaatatc?aaagcaatca?tcctggacat?gttcggcgcc?900

ggtaccgata?cgtcctcttc?cacgattgag?tgggctattt?ccgaactgat?caaatgcccg?960

aaggcgatgg?aaaaagtgca?ggcggaactg?cgtaaagcgc?tgaacggtaa?agagaaaatt?1020

catgaagagg?acatccagga?actgtcctac?ctgaatatgg?taatcaaaga?aactctgcgt?1080

ctgcatccgc?cgctgccact?ggttctgccg?cgtgaatgcc?gtcagccggt?taacctggcc?1140

ggctacaaca?ttccgaacaa?aacgaagctg?atcgtcaacg?ttttcgcgat?caaccgcgat?1200

cctgaatact?ggaaagacgc?ggaagcgttc?attccggaac?gctttgagaa?ctcctctgcc?1260

accgttatgg?gcgctgaata?cgagtacctg?ccgttcggtg?cgggtcgccg?tatgtgcccg?1320

ggtgctgcac?tgggcctggc?gaacgttcaa?ctgccactgg?cgaacatcct?gtaccacttc?1380

aactggaaac?tgcctaacgg?cgtatcttat?gatcaaatcg?acatgaccga?aagctccggc?1440

gcgaccatgc?agcgtaaaac?cgaactgctg?ctggttccgt?ccttttaa 1488

<210>52

<211>495

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase of Xiu Shiing

<400>52

Met?Lys?Ser?Ile?Leu?Lys?Ala?Met?Ala?Leu?Ser?Leu?Thr?Thr?Ser?Ile

1 5 10 15

Ala?Leu?AIa?Thr?Ile?Leu?Leu?Phe?Val?Tyr?Lys?Phe?Ala?Thr?Arg?Ser

20 25 30

Lys?Ser?Thr?Lys?Lys?Ser?Leu?Pro?Glu?Pro?Trp?Arg?Leu?Pro?Ile?Ile

35 40 45

Gly?His?Met?His?His?Leu?Ile?Gly?Thr?Thr?Pro?His?Arg?Gly?Val?Arg

50 55 60

Asp?Leu?Ala?Arg?Lys?Tyr?Gly?Ser?Leu?Met?His?Leu?Gln?Leu?Gly?Glu

65 70 75 80

Val?Pro?Thr?Ile?Val?Val?Ser?Ser?Pro?Lys?Trp?Ala?Lys?Glu?Ile?Leu

85 90 95

Thr?Thr?Tyr?Asp?Ile?Thr?Phe?Ala?Asn?Arg?Pro?Glu?Thr?Leu?Thr?Gly

100 105 110

Glu?Ile?Val?Leu?Tyr?His?Asn?Thr?Asp?Val?Val?Leu?Ala?Pro?Tyr?Gly

115 120 125

Glu?Tyr?Trp?Arg?Gln?Leu?Arg?Lys?Ile?Cys?Thr?Leu?Glu?Leu?Leu?Ser

130 135 140

Val?Lys?Lys?Val?Lys?Ser?Phe?Gln?Ser?Leu?Arg?Glu?Glu?Glu?Cys?Trp

145 150 155 160

Asn?Leu?Val?Gln?Glu?Ile?Lys?Ala?Ser?Gly?Ser?Gly?Arg?Pro?Val?Asn

165 170 175

Leu?Ser?Glu?Asn?Val?Phe?Lys?Leu?Ile?Ala?Thr?Ile?Leu?Ser?Arg?Ala

180 185 190

Ala?Phe?Gly?Lys?Gly?Ile?Lys?Asp?Gln?Lys?Glu?Leu?Thr?Glu?Ile?Val

195 200 205

Lys?Glu?Ile?Leu?Arg?Gln?Thr?Gly?Gly?Phe?Asp?Val?Ala?Asp?Ile?Phe

210 215 220

Pro?Ser?Lys?Lys?Phe?Leu?His?His?Leu?Ser?Gly?Lys?Arg?Ala?Arg?Leu

225 230 235 240

Thr?Ser?Leu?Arg?Lys?Lys?Ile?Asp?Asn?Leu?Ile?Asp?Asn?Leu?Val?Ala

245 250 255

Glu?His?Thr?Val?Asn?Thr?Ser?Ser?Lys?Thr?Asn?Glu?Thr?Leu?Leu?Asp

260 265 270

Val?Leu?Leu?Arg?Leu?Lys?Asp?Ser?Ala?Glu?Phe?Pro?Leu?Thr?Ser?Asp

275 280 285

Asn?Ile?Lys?Ala?Ile?Ile?Leu?Asp?Met?Phe?Gly?Ala?Gly?Thr?Asp?Thr

290 295 300

Ser?Ser?Ser?Thr?Ile?Glu?Trp?Ala?Ile?Ser?Glu?Leu?Ile?Lys?Cys?Pro

305 310 315 320

Lys?Ala?Met?Glu?Lys?Val?Gln?Ala?Glu?Leu?Arg?Lys?Ala?Leu?Asn?Gly

325 330 335

Lys?Glu?Lys?Ile?His?Glu?Glu?Asp?Ile?Gln?Glu?Leu?Ser?Tyr?Leu?Asn

340 345 350

Met?Val?Ile?Lys?Glu?Thr?Leu?Arg?Leu?His?Pro?Pro?Leu?Pro?Leu?Val

355 360 365

Leu?Pro?Arg?Glu?Cys?Arg?Gln?Pro?Val?Asn?Leu?Ala?Gly?Tyr?Asn?Ile

370 375 380

Pro?Asn?Lys?Thr?Lys?Leu?Ile?Val?Asn?Val?Phe?Ala?Ile?Asn?Arg?Asp

385 390 395 400

Pro?Glu?Tyr?Trp?Lys?Asp?Ala?Glu?Ala?Phe?Ile?Pro?Glu?Arg?Phe?Glu

405 410 415

Asn?Ser?Ser?Ala?Thr?Val?Met?Gly?Ala?Glu?Tyr?Glu?Tyr?Leu?Pro?Phe

420 425 430

Gly?Ala?Gly?Arg?Arg?Met?Cys?Pro?Gly?Ala?Ala?Leu?Gly?Leu?Ala?Asn

435 440 445

Val?Gln?Leu?Pro?Leu?Ala?Asn?Ile?Leu?Tyr?His?Phe?Asn?Trp?Lys?Leu

450 455 460

Pro?Asn?Gly?Val?Ser?Tyr?Asp?Gln?Ile?Asp?Met?Thr?Glu?Ser?Ser?Gly

465 470 475 480

Ala?Thr?Met?Gln?Arg?Lys?Thr?Glu?Leu?Leu?Leu?Val?Pro?Ser?Phe

485 490 495

<210>53

<211>3018

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase nucleic acid of Xiu Shiing

<400>53

catatgaccg?tacacgacat?catcgcaacg?tacttcacta?aatggtacgt?aattgtgccg?60

ctggcactga?ttgcgtatcg?cgtgctggat?tatttctacg?cgacccgttc?taaaagcact?120

aagaaatctc?tgccggaacc?gtggcgtctg?ccaatcatcg?gtcacatgca?ccacctgatc?180

ggcaccaccc?cgcaccgtgg?cgtacgcgac?ctggcgcgta?agtacggctc?tctgatgcat?240

ctgcagctgg?gcgaggtacc?tactatcgtc?gtttcctccc?cgaagtgggc?caaagaaatc?300

ctgactacct?atgacatcac?tttcgccaac?cgcccggaaa?cgctgaccgg?cgaaattgtc?360

ctgtaccata?acacggatgt?ggttctggcc?ccgtacggtg?agtactggcg?ccagctgcgc?420

aaaatttgta?ctctggaact?gctgagcgtt?aaaaaggtta?aatccttcca?gagcctgcgt?480

gaagaggaat?gctggaacct?ggtgcaggag?attaaagcgt?ctggcagcgg?tcgtccagtt?540

aacctgtctg?agaatgtttt?taaactgatc?gctactatcc?tgtctcgcgc?ggcattcggt?600

aaaggtatca?aagatcagaa?agaactgacc?gaaatcgtta?aggaaatcct?gcgccagact?660

ggtggcttcg?acgttgcgga?catcttcccg?tccaaaaagt?tcctgcacca?tctgtctggc?720

aaacgcgctc?gtctgacctc?cctgcgtaag?aaaattgata?acctgattga?caacctggtc?780

gctgagcaca?ctgtgaacac?ctcttctaaa?accaacgaaa?ccctgctgga?cgtactgctg?840

cgcctgaagg?actctgccga?atttccactg?actagcgaca?atatcaaagc?aatcatcctg?900

gacatgttcg?gcgccggtac?cgatacgtcc?tcttccacga?ttgagtgggc?tatttccgaa?960

ctgatcaaat?gcccgaaggc?gatggaaaaa?gtgcaggcgg?aactgcgtaa?agcgctgaac?1020

ggtaaagaga?aaattcatga?agaggacatc?caggaactgt?cctacctgaa?tatggtaatc?1080

aaagaaactc?tgcgtctgca?tccgccgctg?ccactggttc?tgccgcgtga?atgccgtcag?1140

ccggttaacc?tggccggcta?caacattccg?aacaaaacga?agctgatcgt?caacgttttc?1200

gcgatcaacc?gcgatcctga?atactggaaa?gacgcggaag?cgttcattcc?ggaacgcttt?1260

gagaactcct?ctgccaccgt?tatgggcgct?gaatacgagt?acctgccgtt?cggtgcgggt?1320

cgccgtatgt?gcccgggtgc?tgcactgggc?ctggcgaacg?ttcaactgcc?actggcgaac?1380

atcctgtacc?acttcaactg?gaaactgcct?aacggcgtat?cttatgatca?aatcgacatg?1440

accgaaagct?ccggcgcgac?catgcagcgt?aaaaccgaac?tgctgctggt?tccgtccttt?1500

taacctaggc?atatgaccgt?acacgacatc?atcgcaacgt?acttcactaa?atggtacgta?1560

attgtgccgc?tggcactgat?tgcgtatcgc?gtgctggatt?atttctacgc?gacccgttct?1620

aaaagcacta?agaaatctct?gccggaaccg?tggcgtctgc?caatcatcgg?tcacatgcac?1680

cacctgatcg?gcaccacccc?gcaccgtggc?gtacgcgacc?tggcgcgtaa?gtacggctct?1740

ctgatgcatc?tgcagctggg?cgaggtacct?actatcgtcg?tttcctcccc?gaagtgggcc?1800

aaagaaatcc?tgactaccta?tgacatcact?ttcgccaacc?gcccggaaac?gctgaccggc?1860

gaaattgtcc?tgtaccataa?cacggatgtg?gttctggccc?cgtacggtga?gtactggcgc?1920

cagctgcgca?aaatttgtac?tctggaactg?ctgagcgtta?aaaaggttaa?atccttccag?1980

agcctgcgtg?aagaggaatg?ctggaacctg?gtgcaggaga?ttaaagcgtc?tggcagcggt?2040

cgtccagtta?acctgtctga?gaatgttttt?aaactgatcg?ctactatcct?gtctcgcgcg?2100

gcattcggta?aaggtatcaa?agatcagaaa?gaactgaccg?aaatcgttaa?ggaaatcctg?2160

cgccagactg?gtggcttcga?cgttgcggac?atcttcccgt?ccaaaaagtt?cctgcaccat?2220

ctgtctggca?aacgcgctcg?tctgacctcc?ctgcgtaaga?aaattgataa?cctgattgac?2280

aacctggtcg?ctgagcacac?tgtgaacacc?tcttctaaaa?ccaacgaaac?cctgctggac?2340

gtactgctgc?gcctgaagga?ctctgccgaa?tttccactga?ctagcgacaa?tatcaaagca?2400

atcatcctgg?acatgttcgg?cgccggtacc?gatacgtcct?cttccacgat?tgagtgggct?2460

atttccgaac?tgatcaaatg?cccgaaggcg?atggaaaaag?tgcaggcgga?actgcgtaaa?2520

gcgctgaacg?gtaaagagaa?aattcatgaa?gaggacatcc?aggaactgtc?ctacctgaat?2580

atggtaatca?aagaaactct?gcgtctgcat?ccgccgctgc?cactggttct?gccgcgtgaa?2640

tgccgtcagc?cggttaacct?ggccggctac?aacattccga?acaaaacgaa?gctgatcgtc?2700

aacgttttcg?cgatcaaccg?cgatcctgaa?tactggaaag?acgcggaagc?gttcattccg?2760

gaacgctttg?agaactcctc?tgccaccgtt?atgggcgctg?aatacgagta?cctgccgttc?2820

ggtgcgggtc?gccgtatgtg?cccgggtgct?gcactgggcc?tggcgaacgt?tcaactgcca?2880

ctggcgaaca?tcctgtacca?cttcaactgg?aaactgccta?acggcgtatc?ttatgatcaa?2940

atcgacatga?ccgaaagctc?cggcgcgacc?atgcagcgta?aaaccgaact?gctgctggtt?3000

ccgtcctttt?aacctagg 3018

<210>54

<211>1500

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase nucleic acid of Xiu Shiing

<400>54

atgaccgtac?acgacatcat?cgcaacgtac?ttcactaaat?ggtacgtaat?tgtgccgctg?60

gcactgattg?cgtatcgcgt?gctggattat?ttctacgcga?cccgttctaa?aagcactaag?120

aaatctctgc?cggaaccgtg?gcgtctgcca?atcatcggtc?acatgcacca?cctgatcggc?180

accaccccgc?accgtggcgt?acgcgacctg?gcgcgtaagt?acggctctct?gatgcatctg?240

cagctgggcg?aggtacctac?tatcgtcgtt?tcctccccga?agtgggccaa?agaaatcctg?300

actacctatg?acatcacttt?cgccaaccgc?ccggaaacgc?tgaccggcga?aattgtcctg?360

taccataaca?cggatgtggt?tctggccccg?tacggtgagt?actggcgcca?gctgcgcaaa?420

atttgtactc?tggaactgct?gagcgttaaa?aaggttaaat?ccttccagag?cctgcgtgaa?480

gaggaatgct?ggaacctggt?gcaggagatt?aaagcgtctg?gcagcggtcg?tccagttaac?540

ctgtctgaga?atgtttttaa?actgatcgct?actatcctgt?ctcgcgcggc?attcggtaaa?600

ggtatcaaag?atcagaaaga?actgaccgaa?atcgttaagg?aaatcctgcg?ccagactggt?660

ggcttcgacg?ttgcggacat?cttcccgtcc?aaaaagttcc?tgcaccatct?gtctggcaaa?720

cgcgctcgtc?tgacctccct?gcgtaagaaa?attgataacc?tgattgacaa?cctggtcgct?780

gagcacactg?tgaacacctc?ttctaaaacc?aacgaaaccc?tgctggacgt?actgctgcgc?840

ctgaaggact?ctgccgaatt?tccactgact?agcgacaata?tcaaagcaat?catcctggac?900

atgttcggcg?ccggtaccga?tacgtcctct?tccacgattg?agtgggctat?ttccgaactg?960

atcaaatgcc?cgaaggcgat?ggaaaaagtg?caggcggaac?tgcgtaaagc?gctgaacggt?1020

aaagagaaaa?ttcatgaaga?ggacatccag?gaactgtcct?acctgaatat?ggtaatcaaa?1080

gaaactctgc?gtctgcatcc?gccgctgcca?ctggttctgc?cgcgtgaatg?ccgtcagccg?1140

gttaacctgg?ccggctacaa?cattccgaac?aaaacgaagc?tgatcgtcaa?cgttttcgcg?1200

atcaaccgcg?atcctgaata?ctggaaagac?gcggaagcgt?tcattccgga?acgctttgag?1260

aactcctctg?ccaccgttat?gggcgctgaa?tacgagtacc?tgccgttcgg?tgcgggtcgc?1320

cgtatgtgcc?cgggtgctgc?actgggcctg?gcgaacgttc?aactgccact?ggcgaacatc?1380

ctgtaccact?tcaactggaa?actgcctaac?ggcgtatctt?atgatcaaat?cgacatgacc?1440

gaaagctccg?gcgcgaccat?gcagcgtaaa?accgaactgc?tgctggttcc?gtccttttaa?1500

<210>55

<211>499

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase of Xiu Shiing

<400>55

Met?Thr?Val?His?Asp?Ile?Ile?Ala?Thr?Tyr?Phe?Thr?Lys?Trp?Tyr?Val

1 5 10 15

Ile?Val?Pro?Leu?Ala?Leu?Ile?Ala?Tyr?Arg?Val?Leu?Asp?Tyr?Phe?Tyr

20 25 30

Ala?Thr?Arg?Ser?Lys?Ser?Thr?Lys?Lys?Ser?Leu?Pro?Glu?Pro?Trp?Arg

35 40 45

Leu?Pro?Ile?Ile?Gly?His?Met?His?His?Leu?Ile?Gly?Thr?Thr?Pro?His

50 55 60

Arg?Gly?Val?Arg?Asp?Leu?Ala?Arg?Lys?Tyr?Gly?Ser?Leu?Met?His?Leu

65 70 75 80

Gln?Leu?Gly?Glu?Val?Pro?Thr?Ile?Val?Val?Ser?Ser?Pro?Lys?Trp?Ala

85 90 95

Lys?Glu?Ile?Leu?Thr?Thr?Tyr?Asp?Ile?Thr?Phe?Ala?Asn?Arg?Pro?Glu

100 105 110

Thr?Leu?Thr?Gly?Glu?Ile?Val?Leu?Tyr?His?Asn?Thr?Asp?Val?Val?Leu

115 120 125

Ala?Pro?Tyr?Gly?Glu?Tyr?Trp?Arg?Gln?Leu?Arg?Lys?Ile?Cys?Thr?Leu

130 135 140

Glu?Leu?Leu?Ser?Val?Lys?Lys?Val?Lys?Ser?Phe?Gln?Ser?Leu?Arg?Glu

145 150 155 160

Glu?Glu?Cys?Trp?Asn?Leu?Val?Gln?Glu?Ile?Lys?Ala?Ser?Gly?Ser?Gly

165 170 175

Arg?Pro?Val?Asn?Leu?Ser?Glu?Asn?Val?Phe?Lys?Leu?Ile?Ala?Thr?Ile

180 185 190

Leu?Ser?Arg?Ala?Ala?Phe?Gly?Lys?Gly?Ile?Lys?Asp?Gln?Lys?Glu?Leu

195 200 205

Thr?Glu?Ile?Val?Lys?Glu?Ile?Leu?Arg?Gln?Thr?Gly?Gly?Phe?Asp?Val

210 215 220

Ala?Asp?Ile?Phe?Pro?Ser?Lys?Lys?Phe?Leu?His?His?Leu?Ser?Gly?Lys

225 230 235 240

Arg?Ala?Arg?Leu?Thr?Ser?Leu?Arg?Lys?Lys?Ile?Asp?Asn?Leu?Ile?Asp

245 250 255

Asn?Leu?Val?Ala?Glu?His?Thr?Val?Asn?Thr?Ser?Ser?Lys?Thr?Asn?Glu

260 265 270

Thr?Leu?Leu?Asp?Val?Leu?Leu?Arg?Leu?Lys?Asp?Ser?Ala?Glu?Phe?Pro

275 280 285

Leu?Thr?Ser?Asp?Asn?Ile?Lys?Ala?Ile?Ile?Leu?Asp?Met?Phe?Gly?Ala

290 295 300

Gly?Thr?Asp?Thr?Ser?Ser?Ser?Thr?Ile?Glu?Trp?Ala?Ile?Ser?Glu?Leu

305 310 315 320

Ile?Lys?Cys?Pro?Lys?Ala?Met?Glu?Lys?Val?Gln?Ala?Glu?Leu?Arg?Lys

325 330 335

Ala?Leu?Asn?Gly?Lys?Glu?Lys?Ile?His?Glu?Glu?Asp?Ile?Gln?Glu?Leu

340 345 350

Ser?Tyr?Leu?Asn?Met?Val?Ile?Lys?Glu?Thr?Leu?Arg?Leu?His?Pro?Pro

355 360 365

Leu?Pro?Leu?Val?Leu?Pro?Arg?Glu?Cys?Arg?Gln?Pro?Val?Asn?Leu?Ala

370 375 380

Gly?Tyr?Asn?Ile?Pro?Asn?Lys?Thr?Lys?Leu?Ile?Val?Asn?Val?Phe?Ala

385 390 395 400

Ile?Asn?Arg?Asp?Pro?Glu?Tyr?Trp?Lys?Asp?Ala?Glu?Ala?Phe?Ile?Pro

405 410 415

Glu?Arg?Phe?Glu?Asn?Ser?Ser?Ala?Thr?Val?Met?Gly?Ala?Glu?Tyr?Glu

420 425 430

Tyr?Leu?Pro?Phe?Gly?Ala?Gly?Arg?Arg?Met?Cys?Pro?Gly?Ala?Ala?Leu

435 440 445

Gly?Leu?Ala?Asn?Val?Gln?Leu?Pro?Leu?Ala?Asn?Ile?Leu?Tyr?His?Phe

450 455 460

Asn?Trp?Lys?Leu?Pro?Asn?Gly?Val?Ser?Tyr?Asp?Gln?Ile?Asp?Met?Thr

465 470 475 480

Glu?Ser?Ser?Gly?Ala?Thr?Met?Gln?Arg?Lys?Thr?Glu?Leu?Leu?Leu?Val

485 490 495

Pro?Ser?Phe

<210>56

<211>2988

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase of Xiu Shiing

<400>56

catatgatcg?aacaactgct?ggaatactgg?tacgtggttg?tgcctgttct?gtatattatc?60

aaacagctgc?tggcgtacac?taaagcgacc?cgttctaaaa?gcactaagaa?atctctgccg?120

gaaccgtggc?gtctgccaat?catcggtcac?atgcaccacc?tgatcggcac?caccccgcac?180

cgtggcgtac?gcgacctggc?gcgtaagtac?ggctctctga?tgcatctgca?gctgggcgag?240

gtacctacta?tcgtcgtttc?ctccccgaag?tgggccaaag?aaatcctgac?tacctatgac?300

atcactttcg?ccaaccgccc?ggaaacgctg?accggcgaaa?ttgtcctgta?ccataacacg?360

gatgtggttc?tggccccgta?cggtgagtac?tggcgccagc?tgcgcaaaat?ttgtactctg?420

gaactgctga?gcgttaaaaa?ggttaaatcc?ttccagagcc?tgcgtgaaga?ggaatgctgg?480

aacctggtgc?aggagattaa?agcgtctggc?agcggtcgtc?cagttaacct?gtctgagaat?540

gtttttaaac?tgatcgctac?tatcctgtct?cgcgcggcat?tcggtaaagg?tatcaaagat?600

cagaaagaac?tgaccgaaat?cgttaaggaa?atcctgcgcc?agactggtgg?cttcgacgtt?660

gcggacatct?tcccgtccaa?aaagttcctg?caccatctgt?ctggcaaacg?cgctcgtctg?720

acctccctgc?gtaagaaaat?tgataacctg?attgacaacc?tggtcgctga?gcacactgtg?780

aacacctctt?ctaaaaccaa?cgaaaccctg?ctggacgtac?tgctgcgcct?gaaggactct?840

gccgaatttc?cactgactag?cgacaatatc?aaagcaatca?tcctggacat?gttcggcgcc?900

ggtaccgata?cgtcctcttc?cacgattgag?tgggctattt?ccgaactgat?caaatgcccg?960

aaggcgatgg?aaaaagtgca?ggcggaactg?cgtaaagcgc?tgaacggtaa?agagaaaatt?1020

catgaagagg?acatccagga?actgtcctac?ctgaatatgg?taatcaaaga?aactctgcgt?1080

ctgcatccgc?cgctgccact?ggttctgccg?cgtgaatgcc?gtcagccggt?taacctggcc?1140

ggctacaaca?ttccgaacaa?aacgaagctg?atcgtcaacg?ttttcgcgat?caaccgcgat?1200

cctgaatact?ggaaagacgc?ggaagcgttc?attccggaac?gctttgagaa?ctcctctgcc?1260

accgttatgg?gcgctgaata?cgagtacctg?ccgttcggtg?cgggtcgccg?tatgtgcccg?1320

ggtgctgcac?tgggcctggc?gaacgttcaa?ctgccactgg?cgaacatcct?gtaccacttc?1380

aactggaaac?tgcctaacgg?cgtatcttat?gatcaaatcg?acatgaccga?aagctccggc?1440

gcgaccatgc?agcgtaaaac?cgaactgctg?ctggttccgt?ccttttaacc?taggcatatg?1500

atcgaacaac?tgctggaata?ctggtacgtg?gttgtgcctg?ttctgtatat?tatcaaacag?1560

ctgctggcgt?acactaaagc?gacccgttct?aaaagcacta?agaaatctct?gccggaaccg?1620

tggcgtctgc?caatcatcgg?tcacatgcac?cacctgatcg?gcaccacccc?gcaccgtggc?1680

gtacgcgacc?tggcgcgtaa?gtacggctct?ctgatgcatc?tgcagctggg?cgaggtacct?1740

actatcgtcg?tttcctcccc?gaagtgggcc?aaagaaatcc?tgactaccta?tgacatcact?1800

ttcgccaacc?gcccggaaac?gctgaccggc?gaaattgtcc?tgtaccataa?cacggatgtg?1860

gttctggccc?cgtacggtga?gtactggcgc?cagctgcgca?aaatttgtac?tctggaactg?1920

ctgagcgtta?aaaaggttaa?atccttccag?agcctgcgtg?aagaggaatg?ctggaacctg?1980

gtgcaggaga?ttaaagcgtc?tggcagcggt?cgtccagtta?acctgtctga?gaatgttttt?2040

aaactgatcg?ctactatcct?gtctcgcgcg?gcattcggta?aaggtatcaa?agatcagaaa?2100

gaactgaccg?aaatcgttaa?ggaaatcctg?cgccagactg?gtggcttcga?cgttgcggac?2160

atcttcccgt?ccaaaaagtt?cctgcaccat?ctgtctggca?aacgcgctcg?tctgacctcc?2220

ctgcgtaaga?aaattgataa?cctgattgac?aacctggtcg?ctgagcacac?tgtgaacacc?2280

tcttctaaaa?ccaacgaaac?cctgctggac?gtactgctgc?gcctgaagga?ctctgccgaa?2340

tttccactga?ctagcgacaa?tatcaaagca?atcatcctgg?acatgttcgg?cgccggtacc?2400

gatacgtcct?cttccacgat?tgagtgggct?atttccgaac?tgatcaaatg?cccgaaggcg?2460

atggaaaaag?tgcaggcgga?actgcgtaaa?gcgctgaacg?gtaaagagaa?aattcatgaa?2520

gaggacatcc?aggaactgtc?ctacctgaat?atggtaatca?aagaaactct?gcgtctgcat?2580

ccgccgctgc?cactggttct?gccgcgtgaa?tgccgtcagc?cggttaacct?ggccggctac?2640

aacattccga?acaaaacgaa?gctgatcgtc?aacgttttcg?cgatcaaccg?cgatcctgaa?2700

tactggaaag?acgcggaagc?gttcattccg?gaacgctttg?agaactcctc?tgccaccgtt?2760

atgggcgctg?aatacgagta?cctgccgttc?ggtgcgggtc?gccgtatgtg?cccgggtgct?2820

gcactgggcc?tggcgaacgt?tcaactgcca?ctggcgaaca?tcctgtacca?cttcaactgg?2880

aaactgccta?acggcgtatc?ttatgatcaa?atcgacatga?ccgaaagctc?cggcgcgacc?2940

atgcagcgta?aaaccgaact?gctgctggtt?ccgtcctttt?aacctagg 2988

<210>57

<211>1485

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase nucleic acid of Xiu Shiing

<400>57

atgatcgaac?aactgctgga?atactggtac?gtggttgtgc?ctgttctgta?tattatcaaa?60

cagctgctgg?cgtacactaa?agcgacccgt?tctaaaagca?ctaagaaatc?tctgccggaa?120

ccgtggcgtc?tgccaatcat?cggtcacatg?caccacctga?tcggcaccac?cccgcaccgt?180

ggcgtacgcg?acctggcgcg?taagtacggc?tctctgatgc?atctgcagct?gggcgaggta?240

cctactatcg?tcgtttcctc?cccgaagtgg?gccaaagaaa?tcctgactac?ctatgacatc?300

actttcgcca?accgcccgga?aacgctgacc?ggcgaaattg?tcctgtacca?taacacggat?360

gtggttctgg?ccccgtacgg?tgagtactgg?cgccagctgc?gcaaaatttg?tactctggaa?420

ctgctgagcg?ttaaaaaggt?taaatccttc?cagagcctgc?gtgaagagga?atgctggaac?480

ctggtgcagg?agattaaagc?gtctggcagc?ggtcgtccag?ttaacctgtc?tgagaatgtt?540

tttaaactga?tcgctactat?cctgtctcgc?gcggcattcg?gtaaaggtat?caaagatcag?600

aaagaactga?ccgaaatcgt?taaggaaatc?ctgcgccaga?ctggtggctt?cgacgttgcg?660

gacatcttcc?cgtccaaaaa?gttcctgcac?catctgtctg?gcaaacgcgc?tcgtctgacc?720

tccctgcgta?agaaaattga?taacctgatt?gacaacctgg?tcgctgagca?cactgtgaac?780

acctcttcta?aaaccaacga?aaccctgctg?gacgtactgc?tgcgcctgaa?ggactctgcc?840

gaatttccac?tgactagcga?caatatcaaa?gcaatcatcc?tggacatgtt?cggcgccggt?900

accgatacgt?cctcttccac?gattgagtgg?gctatttccg?aactgatcaa?atgcccgaag?960

gcgatggaaa?aagtgcaggc?ggaactgcgt?aaagcgctga?acggtaaaga?gaaaattcat?1020

gaagaggaca?tccaggaact?gtcctacctg?aatatggtaa?tcaaagaaac?tctgcgtctg?1080

catccgccgc?tgccactggt?tctgccgcgt?gaatgccgtc?agccggttaa?cctggccggc?1140

tacaacattc?cgaacaaaac?gaagctgatc?gtcaacgttt?tcgcgatcaa?ccgcgatcct?1200

gaatactgga?aagacgcgga?agcgttcatt?ccggaacgct?ttgagaactc?ctctgccacc?1260

gttatgggcg?ctgaatacga?gtacctgccg?ttcggtgcgg?gtcgccgtat?gtgcccgggt?1320

gctgcactgg?gcctggcgaa?cgttcaactg?ccactggcga?acatcctgta?ccacttcaac?1380

tggaaactgc?ctaacggcgt?atcttatgat?caaatcgaca?tgaccgaaag?ctccggcgcg?1440

accatgcagc?gtaaaaccga?actgctgctg?gttccgtcct?tttaa 1485

<210>58

<211>494

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase of Xiu Shiing

<400>58

Met?Ile?Glu?Gln?Leu?Leu?Glu?Tyr?Trp?Tyr?Val?Val?Val?Pro?Val?Leu

1 5 10 15

Tyr?Ile?Ile?Lys?Gln?Leu?Leu?Ala?Tyr?Thr?Lys?Ala?Thr?Arg?Ser?Lys

20 25 30

Ser?Thr?Lys?Lys?Ser?Leu?Pro?Glu?Pro?Trp?Arg?Leu?Pro?Ile?Ile?Gly

35 40 45

His?Met?His?His?Leu?Ile?Gly?Thr?Thr?Pro?His?Arg?Gly?Val?Arg?Asp

50 55 60

Leu?Ala?Arg?Lys?Tyr?Gly?Ser?Leu?Met?His?Leu?Gln?Leu?Gly?Glu?Val

65 70 75 80

Pro?Thr?Ile?Val?Val?Ser?Ser?Pro?Lys?Trp?Ala?Lys?Glu?Ile?Leu?Thr

85 90 95

Thr?Tyr?Asp?Ile?Thr?Phe?Ala?Asn?Arg?Pro?Glu?Thr?Leu?Thr?Gly?Glu

100 105 110

I1e?Val?Leu?Tyr?His?Asn?Thr?Asp?Val?Val?Leu?Ala?Pro?Tyr?Gly?Glu

115 120 125

Tyr?Trp?Arg?Gln?Leu?Arg?Lys?Ile?Cys?Thr?Leu?Glu?Leu?Leu?Ser?Val

130 135 140

Lys?Lys?Val?Lys?Ser?Phe?Gln?Ser?Leu?Arg?Glu?Glu?Glu?Cys?Trp?Asn

145 150 155 160

Leu?Val?Gln?Glu?Ile?Lys?Ala?Ser?Gly?Ser?Gly?Arg?Pro?Val?Asn?Leu

165 170 175

Ser?Glu?Asn?Val?Phe?Lys?Leu?Ile?Ala?Thr?Ile?Leu?Ser?Arg?Ala?Ala

180 185 190

Phe?Gly?Lys?Gly?Ile?Lys?Asp?Gln?Lys?Glu?Leu?Thr?Glu?Ile?Val?Lys

195 200 205

Glu?Ile?Leu?Arg?Gln?Thr?Gly?Gly?Phe?Asp?Val?Ala?Asp?Ile?Phe?Pro

210 215 220

Ser?Lys?Lys?Phe?Leu?His?His?Leu?Ser?Gly?Lys?Arg?Ala?Arg?Leu?Thr

225 230 235 240

Ser?Leu?Arg?Lys?Lys?Ile?Asp?Asn?Leu?Ile?Asp?Asn?Leu?Val?Ala?Glu

245 250 255

His?Thr?Val?Asn?Thr?Ser?Ser?Lys?Thr?Asn?Glu?Thr?Leu?Leu?Asp?Val

260 265 270

Leu?Leu?Arg?Leu?Lys?Asp?Ser?Ala?Glu?Phe?Pro?Leu?Thr?Ser?Asp?Asn

275 280 285

Ile?Lys?Ala?Ile?Ile?Leu?Asp?Met?Phe?Gly?Ala?Gly?Thr?Asp?Thr?Ser

290 295 300

Ser?Ser?Thr?Ile?Glu?Trp?Ala?Ile?Ser?Glu?Leu?Ile?Lys?Cys?Pro?Lys

305 310 315 320

Ala?Met?Glu?Lys?Val?Gln?Ala?Glu?Leu?Arg?Lys?Ala?Leu?Asn?Gly?Lys

325 330 335

Glu?Lys?Ile?His?Glu?Glu?Asp?Ile?Gln?Glu?Leu?Ser?Tyr?Leu?Asn?Met

340 345 350

Val?Ile?Lys?Glu?Thr?Leu?Arg?Leu?His?Pro?Pro?Leu?Pro?Leu?Val?Leu

355 360 365

Pro?Arg?Glu?Cys?Arg?Gln?Pro?Val?Asn?Leu?Ala?Gly?Tyr?Asn?Ile?Pro

370 375 380

Asn?Lys?Thr?Lys?Leu?Ile?Val?Asn?Val?Phe?Ala?Ile?Asn?Arg?Asp?Pro

385 390 395 400

Glu?Tyr?Trp?Lys?Asp?Ala?Glu?Ala?Phe?Ile?Pro?Glu?Arg?Phe?Glu?Asn

405 410 415

Ser?Ser?Ala?Thr?Val?Met?Gly?Ala?Glu?Tyr?Glu?Tyr?Leu?Pro?Phe?Gly

420 425 430

Ala?Gly?Arg?Arg?Met?Cys?Pro?Gly?Ala?Ala?Leu?Gly?Leu?Ala?Asn?Val

435 440 445

Gln?Leu?Pro?Leu?Ala?Asn?Ile?Leu?Tyr?His?Phe?Asn?Trp?Lys?Leu?Pro

450 455 460

Asn?Gly?Val?Ser?Tyr?Asp?Gln?Ile?Asp?Met?Thr?Glu?Ser?Ser?Gly?Ala

465 470 475 480

Thr?Met?Gln?Arg?Lys?Thr?Glu?Leu?Leu?Leu?Val?Pro?Ser?Phe

485 490

<210>59

<211>2946

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase nucleic acid of Xiu Shiing

<400>59

catatggctc?tgctgctggc?tgtcttcctg?ggtctgtcct?gcctgctgct?gctgtccctg?60

tgggcgaccc?gttctaaaag?cactaagaaa?tctctgccgg?aaccgtggcg?tctgccaatc?120

atcggtcaca?tgcaccacct?gatcggcacc?accccgcacc?gtggcgtacg?cgacctggcg?180

cgtaagtacg?gctctctgat?gcatctgcag?ctgggcgagg?tacctactat?cgtcgtttcc?240

tccccgaagt?gggccaaaga?aatcctgact?acctatgaca?tcactttcgc?caaccgcccg?300

gaaacgctga?ccggcgaaat?tgtcctgtac?cataacacgg?atgtggttct?ggccccgtac?360

ggtgagtact?ggcgccagct?gcgcaaaatt?tgtactctgg?aactgctgag?cgttaaaaag?420

gttaaatcct?tccagagcct?gcgtgaagag?gaatgctgga?acctggtgca?ggagattaaa?480

gcgtctggca?gcggtcgtcc?agttaacctg?tctgagaatg?tttttaaact?gatcgctact?540

atcctgtctc?gcgcggcatt?cggtaaaggt?atcaaagatc?agaaagaact?gaccgaaatc?600

gttaaggaaa?tcctgcgcca?gactggtggc?ttcgacgttg?cggacatctt?cccgtccaaa?660

aagttcctgc?accatctgtc?tggcaaacgc?gctcgtctga?cctccctgcg?taagaaaatt?720

gataacctga?ttgacaacct?ggtcgctgag?cacactgtga?acacctcttc?taaaaccaac?780

gaaaccctgc?tggacgtact?gctgcgcctg?aaggactctg?ccgaatttcc?actgactagc?840

gacaatatca?aagcaatcat?cctggacatg?ttcggcgccg?gtaccgatac?gtcctcttcc?900

acgattgagt?gggctatttc?cgaactgatc?aaatgcccga?aggcgatgga?aaaagtgcag?960

gcggaactgc?gtaaagcgct?gaacggtaaa?gagaaaattc?atgaagagga?catccaggaa?1020

ctgtcctacc?tgaatatggt?aatcaaagaa?actctgcgtc?tgcatccgcc?gctgccactg?1080

gttctgccgc?gtgaatgccg?tcagccggtt?aacctggccg?gctacaacat?tccgaacaaa?1140

acgaagctga?tcgtcaacgt?tttcgcgatc?aaccgcgatc?ctgaatactg?gaaagacgcg?1200

gaagcgttca?ttccggaacg?ctttgagaac?tcctctgcca?ccgttatggg?cgctgaatac?1260

gagtacctgc?cgttcggtgc?gggtcgccgt?atgtgcccgg?gtgctgcact?gggcctggcg?1320

aacgttcaac?tgccactggc?gaacatcctg?taccacttca?actggaaact?gcctaacggc?1380

gtatcttatg?atcaaatcga?catgaccgaa?agctccggcg?cgaccatgca?gcgtaaaacc?1440

gaactgctgc?tggttccgtc?cttttgacct?aggcatatgg?ctctgctgct?ggctgtcttc?1500

ctgggtctgt?cctgcctgct?gctgctgtcc?ctgtgggcga?cccgttctaa?aagcactaag?1560

aaatctctgc?cggaaccgtg?gcgtctgcca?atcatcggtc?acatgcacca?cctgatcggc?1620

accaccccgc?accgtggcgt?acgcgacctg?gcgcgtaagt?acggctctct?gatgcatctg?1680

cagctgggcg?aggtacctac?tatcgtcgtt?tcctccccga?agtgggccaa?agaaatcctg?1740

actacctatg?acatcacttt?cgccaaccgc?ccggaaacgc?tgaccggcga?aattgtcctg?1800

taccataaca?cggatgtggt?tctggccccg?tacggtgagt?actggcgcca?gctgcgcaaa?1860

atttgtactc?tggaactgct?gagcgttaaa?aaggttaaat?ccttccagag?cctgcgtgaa?1920

gaggaatgct?ggaacctggt?gcaggagatt?aaagcgtctg?gcagcggtcg?tccagttaac?1980

ctgtctgaga?atgtttttaa?actgatcgct?actatcctgt?ctcgcgcggc?attcggtaaa?2040

ggtatcaaag?atcagaaaga?actgaccgaa?atcgttaagg?aaatcctgcg?ccagactggt?2100

ggcttcgacg?ttgcggacat?cttcccgtcc?aaaaagttcc?tgcaccatct?gtctggcaaa?2160

cgcgctcgtc?tgacctccct?gcgtaagaaa?attgataacc?tgattgacaa?cctggtcgct?2220

gagcacactg?tgaacacctc?ttctaaaacc?aacgaaaccc?tgctggacgt?actgctgcgc?2280

ctgaaggact?ctgccgaatt?tccactgact?agcgacaata?tcaaagcaat?catcctggac?2340

atgttcggcg?ccggtaccga?tacgtcctct?tccacgattg?agtgggctat?ttccgaactg?2400

atcaaatgcc?cgaaggcgat?ggaaaaagtg?caggcggaac?tgcgtaaagc?gctgaacggt?2460

aaagagaaaa?ttcatgaaga?ggacatccag?gaactgtcct?acctgaatat?ggtaatcaaa?2520

gaaactctgc?gtctgcatcc?gccgctgcca?ctggttctgc?cgcgtgaatg?ccgtcagccg?2580

gttaacctgg?ccggctacaa?cattccgaac?aaaacgaagc?tgatcgtcaa?cgttttcgcg?2640

atcaaccgcg?atcctgaata?ctggaaagac?gcggaagcgt?tcattccgga?acgctttgag?2700

aactcctctg?ccaccgttat?gggcgctgaa?tacgagtacc?tgccgttcgg?tgcgggtcgc?2760

cgtatgtgcc?cgggtgctgc?actgggcctg?gcgaacgttc?aactgccact?ggcgaacatc?2820

ctgtaccact?tcaactggaa?actgcctaac?ggcgtatctt?atgatcaaat?cgacatgacc?2880

gaaagctccg?gcgcgaccat?gcagcgtaaa?accgaactgc?tgctggttcc?gtccttttaa?2940

cctagg 2946

<210>60

<211>1464

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase nucleic acid of Xiu Shiing

<400>60

atggctctgc?tgctggctgt?cttcctgggt?ctgtcctgcc?tgctgctgct?gtccctgtgg?60

gcgacccgtt?ctaaaagcac?taagaaatct?ctgccggaac?cgtggcgtct?gccaatcatc?120

ggtcacatgc?accacctgat?cggcaccacc?ccgcaccgtg?gcgtacgcga?cctggcgcgt?180

aagtacggct?ctctgatgca?tctgcagctg?ggcgaggtac?ctactatcgt?cgtttcctcc?240

ccgaagtggg?ccaaagaaat?cctgactacc?tatgacatca?ctttcgccaa?ccgcccggaa?300

acgctgaccg?gcgaaattgt?cctgtaccat?aacacggatg?tggttctggc?cccgtacggt?360

gagtactggc?gccagctgcg?caaaatttgt?actctggaac?tgctgagcgt?taaaaaggtt?420

aaatccttcc?agagcctgcg?tgaagaggaa?tgctggaacc?tggtgcagga?gattaaagcg?480

tctggcagcg?gtcgtccagt?taacctgtct?gagaatgttt?ttaaactgat?cgctactatc?540

ctgtctcgcg?cggcattcgg?taaaggtatc?aaagatcaga?aagaactgac?cgaaatcgtt?600

aaggaaatcc?tgcgccagac?tggtggcttc?gacgttgcgg?acatcttccc?gtccaaaaag?660

ttcctgcacc?atctgtctgg?caaacgcgct?cgtctgacct?ccctgcgtaa?gaaaattgat?720

aacctgattg?acaacctggt?cgctgagcac?actgtgaaca?cctcttctaa?aaccaacgaa?780

accctgctgg?acgtactgct?gcgcctgaag?gactctgccg?aatttccact?gactagcgac?840

aatatcaaag?caatcatcct?ggacatgttc?ggcgccggta?ccgatacgtc?ctcttccacg?900

attgagtggg?ctatttccga?actgatcaaa?tgcccgaagg?cgatggaaaa?agtgcaggcg?960

gaactgcgta?aagcgctgaa?cggtaaagag?aaaattcatg?aagaggacat?ccaggaactg?1020

tcctacctga?atatggtaat?caaagaaact?ctgcgtctgc?atccgccgct?gccactggtt?1080

ctgccgcgtg?aatgccgtca?gccggttaac?ctggccggct?acaacattcc?gaacaaaacg?1140

aagctgatcg?tcaacgtttt?cgcgatcaac?cgcgatcctg?aatactggaa?agacgcggaa?1200

gcgttcattc?cggaacgctt?tgagaactcc?tctgccaccg?ttatgggcgc?tgaatacgag?1260

tacctgccgt?tcggtgcggg?tcgccgtatg?tgcccgggtg?ctgcactggg?cctggcgaac?1320

gttcaactgc?cactggcgaa?catcctgtac?cacttcaact?ggaaactgcc?taacggcgta?1380

tcttatgatc?aaatcgacat?gaccgaaagc?tccggcgcga?ccatgcagcg?taaaaccgaa?1440

ctgctgctgg?ttccgtcctt?ttaa 1464

<210>61

<211>487

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the AMORPHADIENE oxydase of Xiu Shiing

<400>61

Met?Ala?Leu?Leu?Leu?Ala?Val?Phe?Leu?Gly?Leu?Ser?Cys?Leu?Leu?Leu

1 5 10 15

Leu?Ser?Leu?Trp?Ala?Thr?Arg?Ser?Lys?Ser?Thr?Lys?Lys?Ser?Leu?Pro

20 25 30

Glu?Pro?Trp?Arg?Leu?Pro?Ile?Ile?Gly?His?Met?His?His?Leu?Ile?Gly

35 40 45

Thr?Thr?Pro?His?Arg?Gly?Val?Arg?Asp?Leu?Ala?Arg?Lys?Tyr?Gly?Ser

50 55 60

Leu?Met?His?Leu?Gln?Leu?Gly?Glu?Val?Pro?Thr?Ile?Val?Val?Ser?Ser

65 70 75 80

Pro?Lys?Trp?Ala?Lys?Glu?Ile?Leu?Thr?Thr?Tyr?Asp?Ile?Thr?Phe?Ala

85 90 95

Asn?Arg?Pro?Glu?Thr?Leu?Thr?Gly?Glu?Ile?Val?Leu?Tyr?His?Asn?Thr

100 105 110

Asp?Val?Val?Leu?Ala?Pro?Tyr?Gly?Glu?Tyr?Trp?Arg?Gln?Leu?Arg?Lys

115 120 125

Ile?Cys?Thr?Leu?Glu?Leu?Leu?Ser?Val?Lys?Lys?Val?Lys?Ser?Phe?Gln

130 135 140

Ser?Leu?Arg?Glu?Glu?Glu?Cys?Trp?Asn?Leu?Val?Gln?Glu?Ile?Lys?Ala

145 150 155 160

Ser?Gly?Ser?Gly?Arg?Pro?Val?Asn?Leu?Ser?Glu?Asn?Val?Phe?Lys?Leu

165 170 175

Ile?Ala?Thr?Ile?Leu?Ser?Arg?Ala?Ala?Phe?Gly?Lys?Gly?Ile?Lys?Asp

180 185 190

Gln?Lys?Glu?Leu?Thr?Glu?Ile?Val?Lys?Glu?Ile?Leu?Arg?Gln?Thr?Gly

195 200 205

Gly?Phe?Asp?Val?Ala?Asp?Ile?Phe?Pro?Ser?Lys?Lys?Phe?Leu?His?His

210 215 220

Leu?Ser?Gly?Lys?Arg?Ala?Arg?Leu?Thr?Ser?Leu?Arg?Lys?Lys?Ile?Asp

225 230 235 240

Asn?Leu?Ile?Asp?Asn?Leu?Val?Ala?Glu?His?Thr?Val?Asn?Thr?Ser?Ser

245 250 255

Lys?Thr?Asn?Glu?Thr?Leu?Leu?Asp?Val?Leu?Leu?Arg?Leu?Lys?Asp?Ser

260 265 270

Ala?Glu?Phe?Pro?Leu?Thr?Ser?Asp?Asn?Ile?Lys?Ala?Ile?Ile?Leu?Asp

275 280 285

Met?Phe?Gly?Ala?Gly?Thr?Asp?Thr?Ser?Ser?Ser?Thr?Ile?Glu?Trp?Ala

290 295 300

Ile?Ser?Glu?Leu?Ile?Lys?Cys?Pro?Lys?Ala?Met?Glu?Lys?Val?Gln?Ala

305 310 315 320

Glu?Leu?Arg?Lys?Ala?Leu?Asn?Gly?Lys?Glu?Lys?Ile?His?Glu?Glu?Asp

325 330 335

Ile?Gln?Glu?Leu?Ser?Tyr?Leu?Asn?Met?Val?Ile?Lys?Glu?Thr?Leu?Arg

340 345 350

Leu?His?Pro?Pro?Leu?Pro?Leu?Val?Leu?Pro?Arg?Glu?Cys?Arg?Gln?Pro

355 360 365

Val?Asn?Leu?Ala?Gly?Tyr?Asn?Ile?Pro?Asn?Lys?Thr?Lys?Leu?Ile?Val

370 375 380

Asn?Val?Phe?Ala?Ile?Asn?Arg?Asp?Pro?Glu?Tyr?Trp?Lys?Asp?Ala?Glu

385 390 395 400

Ala?Phe?Ile?Pro?Glu?Arg?Phe?Glu?Asn?Ser?Ser?Ala?Thr?Val?Met?Gly

405 410 415

Ala?Glu?Tyr?Glu?Tyr?Leu?Pro?Phe?Gly?Ala?Gly?Arg?Arg?Met?Cys?Pro

420 425 430

Gly?Ala?Ala?Leu?Gly?Leu?Ala?Asn?Val?Gln?Leu?Pro?Leu?Ala?Asn?Ile

435 440 445

Leu?Tyr?His?Phe?Asn?Trp?Lys?Leu?Pro?Asn?Gly?Val?Ser?Tyr?Asp?Gln

450 455 460

Ile?Asp?Met?Thr?Glu?Ser?Ser?Gly?Ala?Thr?Met?Gln?Arg?Lys?Thr?Glu

465 470 475 480

Leu?Leu?Leu?Val?Pro?Ser?Phe

485

<210>62

<211>10633

<212>DNA

＜213〉artificial sequence

<220>

＜223〉recombinant plasmid

<400>62

accttcggga?gcgcctgaag?cccgttctgg?acgccctggg?gccgttgaat?cgggatatgc?60

aggccaaggc?cgccgcgatc?atcaaggccg?tgggcgaaaa?gctgctgacg?gaacagcggg?120

aagtccagcg?ccagaaacag?gcccagcgcc?agcaggaacg?cgggcgcgca?catttccccg?180

aaaagtgcca?cctgacgtct?aagaaaccat?tattatcatg?acattaacct?ataaaaatag?240

gcgtatcacg?aggccctttc?gtcttcaaga?attctcatgt?ttgacagctt?atcatcgata?300

agctttaatg?cggtagttta?tcacagttaa?attgctaacg?cagtcaggca?ccgtgtatga?360

aatctaacaa?tgcgctcatc?gtcatcctcg?gcaccgtcac?cctggatgct?gtaggcatag?420

gcttggttat?gccggtactg?ccgggcctct?tgcgggatat?cgtccattcc?gacagcatcg?480

ccagtcacta?tggcgtgctg?ctagcgctat?atgcgttgat?gcaatttcta?tgcgcacccg?540

ttctcggagc?actgtccgac?cgctttggcc?gccgcccagt?cctgctcgct?tcgctacttg?600

gagccactat?cgactacgcg?atcatggcga?ccacacccgt?cctgtggatc?ctctacgccg?660

gacgcatcgt?ggccggcatc?accggcgcca?caggtgcggt?tgctggcgcc?tatatcgccg?720

acatcaccga?tggggaagat?cgggctcgcc?acttcgggct?catgagcgct?tgtttcggcg?780

tgggtatggt?ggcaggcccc?gtggccgggg?gactgttggg?cgccatctcc?ttgcatgcac?840

cattccttgc?ggcggcggtg?ctcaacggcc?tcaacctact?actgggctgc?ttcctaatgc?900

aggagtcgca?taagggagag?cgtcgaccga?tgcccttgag?agccttcaac?ccagtcagct?960

ccttccggtg?ggcgcggggc?atgactatcg?tcgccgcact?tatgactgtc?ttctttatca?1020

tgcaactcgt?aggacaggtg?ccggcagcgc?tctgggtcat?tttcggcgag?gaccgctttc?1080

gctggagcgc?gacgatgatc?ggcctgtcgc?ttgcggtatt?cggaatcttg?cacgccctcg?1140

ctcaagcctt?cgtcactggt?cccgccacca?aacgtttcgg?cgagaagcag?gccattatcg?1200

ccggcatggc?ggccgacgcg?ctgggctacg?tcttgctggc?gttcgcgacg?cgaggctgga?1260

tggccttccc?cattatgatt?cttctcgctt?ccggcggcat?cgggatgccc?gcgttgcagg?1320

ccatgctgtc?caggcaggta?gatgacgacc?atcagggaca?gcttcaagga?tcgctcgcgg?1380

ctcttaccag?cctaacttcg?atcactggac?cgctgatcgt?cacggcgatt?tatgccgcct?1440

cggcgagcac?atggaacggg?ttggcatgga?ttgtaggcgc?cgccctatac?cttgtctgcc?1500

tccccgcgtt?gcgtcgcggt?gcatggagcc?gggccacctc?gacctgaatg?gaagccggcg?1560

gcacctcgct?aacggattca?ccactccaag?aattggagcc?aatcaattct?tgcggagaac?1620

tgtgaatgcg?caaatgcgcc?caatacgcaa?accgcctctc?cccgcgcgtt?ggccgattca?1680

ttaatgcagc?tggcacgaca?ggtttcccga?ctggaaagcg?ggcagtgagc?gcaacgcaat?1740

taatgtgagt?tagctcactc?attaggcacc?ccaggcttta?cactttatgc?ttccggctcg?1800

tatgttgtgt?ggaattgtga?gcggataaca?atttcacaca?ggaaacagct?atgaccatga?1860

ttacgccaag?cgcgcaatta?accctcacta?aagggaacaa?aagctgggta?ccgggccccc?1920

cctcgaggtc?gacggtatcg?ataagcttga?tatcgaattc?ctgcagtagg?aggaattaac?1980

catgtcatta?ccgttcttaa?cttctgcacc?gggaaaggtt?attatttttg?gtgaacactc?2040

tgctgtgtac?aacaagcctg?ccgtcgctgc?tagtgtgtct?gcgttgagaa?cctacctgct?2100

aataagcgag?tcatctgcac?cagatactat?tgaattggac?ttcccggaca?ttagctttaa?2160

tcataagtgg?tccatcaatg?atttcaatgc?catcaccgag?gatcaagtaa?actcccaaaa?2220

attggccaag?gctcaacaag?ccaccgatgg?cttgtctcag?gaactcgtta?gtcttttgga?2280

tccgttgtta?gctcaactat?ccgaatcctt?ccactaccat?gcagcgtttt?gtttcctgta?2340

tatgtttgtt?tgcctatgcc?cccatgccaa?gaatattaag?ttttctttaa?agtctacttt?2400

acccatcggt?gctgggttgg?gctcaagcgc?ctctatttct?gtatcactgg?ccttagctat?2460

ggcctacttg?ggggggttaa?taggatctaa?tgacttggaa?aagctgtcag?aaaacgataa?2520

gcatatagtg?aatcaatggg?ccttcatagg?tgaaaagtgt?attcacggta?ccccttcagg?2580

aatagataac?gctgtggcca?cttatggtaa?tgccctgcta?tttgaaaaag?actcacataa?2640

tggaacaata?aacacaaaca?attttaagtt?cttagatgat?ttcccagcca?ttccaatgat?2700

cctaacctat?actagaattc?caaggtctac?aaaagatctt?gttgctcgcg?ttcgtgtgtt?2760

ggtcaccgag?aaatttcctg?aagttatgaa?gccaattcta?gatgccatgg?gtgaatgtgc?2820

cctacaaggc?ttagagatca?tgactaagtt?aagtaaatgt?aaaggcaccg?atgacgaggc?2880

tgtagaaact?aataatgaac?tgtatgaaca?actattggaa?ttgataagaa?taaatcatgg?2940

actgcttgtc?tcaatcggtg?tttctcatcc?tggattagaa?cttattaaaa?atctgagcga?3000

tgatttgaga?attggctcca?caaaacttac?cggtgctggt?ggcggcggtt?gctctttgac?3060

tttgttacga?agagacatta?ctcaagagca?aattgacagc?ttcaaaaaga?aattgcaaga?3120

tgattttagt?tacgagacat?ttgaaacaga?cttgggtggg?actggctgct?gtttgttaag?3180

cgcaaaaaat?ttgaataaag?atcttaaaat?caaatcccta?gtattccaat?tatttgaaaa?3240

taaaactacc?acaaagcaac?aaattgacga?tctattattg?ccaggaaaca?cgaatttacc?3300

atggacttca?taggaggcag?atcaaatgtc?agagttgaga?gccttcagtg?ccccagggaa?3360

agcgttacta?gctggtggat?atttagtttt?agatacaaaa?tatgaagcat?ttgtagtcgg?3420

attatcggca?agaatgcatg?ctgtagccca?tccttacggt?tcattgcaag?ggtctgataa?3480

gtttgaagtg?cgtgtgaaaa?gtaaacaatt?taaagatggg?gagtggctgt?accatataag?3540

tcctaaaagt?ggcttcattc?ctgtttcgat?aggcggatct?aagaaccctt?tcattgaaaa?3600

agttatcgct?aacgtattta?gctactttaa?acctaacatg?gacgactact?gcaatagaaa?3660

cttgttcgtt?attgatattt?tctctgatga?tgcctaccat?tctcaggagg?atagcgttac?3720

cgaacatcgt?ggcaacagaa?gattgagttt?tcattcgcac?agaattgaag?aagttcccaa?3780

aacagggctg?ggctcctcgg?caggtttagt?cacagtttta?actacagctt?tggcctcctt?3840

ttttgtatcg?gacctggaaa?ataatgtaga?caaatataga?gaagttattc?ataatttagc?3900

acaagttgct?cattgtcaag?ctcagggtaa?aattggaagc?gggtttgatg?tagcggcggc?3960

agcatatgga?tctatcagat?atagaagatt?cccacccgca?ttaatctcta?atttgccaga?4020

tattggaagt?gctacttacg?gcagtaaact?ggcgcatttg?gttgatgaag?aagactggaa?4080

tattacgatt?aaaagtaacc?atttaccttc?gggattaact?ttatggatgg?gcgatattaa?4140

gaatggttca?gaaacagtaa?aactggtcca?gaaggtaaaa?aattggtatg?attcgcatat?4200

gccagaaagc?ttgaaaatat?atacagaact?cgatcatgca?aattctagat?ttatggatgg?4260

actatctaaa?ctagatcgct?tacacgagac?tcatgacgat?tacagcgatc?agatatttga?4320

gtctcttgag?aggaatgact?gtacctgtca?aaagtatcct?gaaatcacag?aagttagaga?4380

tgcagttgcc?acaattagac?gttcctttag?aaaaataact?aaagaatctg?gtgccgatat?4440

cgaacctccc?gtacaaacta?gcttattgga?tgattgccag?accttaaaag?gagttcttac?4500

ttgcttaata?cctggtgctg?gtggttatga?cgccattgca?gtgattacta?agcaagatgt?4560

tgatcttagg?gctcaaaccg?ctaatgacaa?aagattttct?aaggttcaat?ggctggatgt?4620

aactcaggct?gactggggtg?ttaggaaaga?aaaagatccg?gaaacttatc?ttgataaata?4680

ggaggtaata?ctcatgaccg?tttacacagc?atccgttacc?gcacccgtca?acatcgcaac?4740

ccttaagtat?tgggggaaaa?gggacacgaa?gttgaatctg?cccaccaatt?cgtccatatc?4800

agtgacttta?tcgcaagatg?acctcagaac?gttgacctct?gcggctactg?cacctgagtt?4860

tgaacgcgac?actttgtggt?taaatggaga?accacacagc?atcgacaatg?aaagaactca?4920

aaattgtctg?cgcgacctac?gccaattaag?aaaggaaatg?gaatcgaagg?acgcctcatt?4980

gcccacatta?tctcaatgga?aactccacat?tgtctccgaa?aataactttc?ctacagcagc?5040

tggtttagct?tcctccgctg?ctggctttgc?tgcattggtc?tctgcaattg?ctaagttata?5100

ccaattacca?cagtcaactt?cagaaatatc?tagaatagca?agaaaggggt?ctggttcagc?5160

ttgtagatcg?ttgtttggcg?gatacgtggc?ctgggaaatg?ggaaaagctg?aagatggtca?5220

tgattccatg?gcagtacaaa?tcgcagacag?ctctgactgg?cctcagatga?aagcttgtgt?5280

cctagttgtc?agcgatatta?aaaaggatgt?gagttccact?cagggtatgc?aattgaccgt?5340

ggcaacctcc?gaactattta?aagaaagaat?tgaacatgtc?gtaccaaaga?gatttgaagt?5400

catgcgtaaa?gccattgttg?aaaaagattt?cgccaccttt?gcaaaggaaa?caatgatgga?5460

ttccaactct?ttccatgcca?catgtttgga?ctctttccct?ccaatattct?acatgaatga?5520

cacttccaag?cgtatcatca?gttggtgcca?caccattaat?cagttttacg?gagaaacaat?5580

cgttgcatac?acgtttgatg?caggtccaaa?tgctgtgttg?tactacttag?ctgaaaatga?5640

gtcgaaactc?tttgcattta?tctataaatt?gtttggctct?gttcctggat?gggacaagaa?5700

atttactact?gagcagcttg?aggctttcaa?ccatcaattt?gaatcatcta?actttactgc?5760

acgtgaattg?gatcttgagt?tgcaaaagga?tgttgccaga?gtgattttaa?ctcaagtcgg?5820

ttcaggccca?caagaaacaa?acgaatcttt?gattgacgca?aagactggtc?taccaaagga?5880

ataactgcag?cccgggagga?ggattactat?atgcaaacgg?aacacgtcat?tttattgaat?5940

gcacagggag?ttcccacggg?tacgctggaa?aagtatgccg?cacacacggc?agacacccgc?6000

ttacatctcg?cgttctccag?ttggctgttt?aatgccaaag?gacaattatt?agttacccgc?6060

cgcgcactga?gcaaaaaagc?atggcctggc?gtgtggacta?actcggtttg?tgggcaccca?6120

caactgggag?aaagcaacga?agacgcagtg?atccgccgtt?gccgttatga?gcttggcgtg?6180

gaaattacgc?ctcctgaatc?tatctatcct?gactttcgct?accgcgccac?cgatccgagt?6240

ggcattgtgg?aaaatgaagt?gtgtccggta?tttgccgcac?gcaccactag?tgcgttacag?6300

atcaatgatg?atgaagtgat?ggattatcaa?tggtgtgatt?tagcagatgt?attacacggt?6360

attgatgcca?cgccgtgggc?gttcagtccg?tggatggtga?tgcaggcgac?aaatcgcgaa?6420

gccagaaaac?gattatctgc?atttacccag?cttaaataac?ccgggggatc?cactagttct?6480

agagcggccg?ccaccgcgga?ggaggaatga?gtaatggact?ttccgcagca?actcgaagcc?6540

tgcgttaagc?aggccaacca?ggcgctgagc?cgttttatcg?ccccactgcc?ctttcagaac?6600

actcccgtgg?tcgaaaccat?gcagtatggc?gcattattag?gtggtaagcg?cctgcgacct?6660

ttcctggttt?atgccaccgg?tcatatgttc?ggcgttagca?caaacacgct?ggacgcaccc?6720

gctgccgccg?ttgagtgtat?ccacgcttac?tcattaattc?atgatgattt?accggcaatg?6780

gatgatgacg?atctgcgtcg?cggtttgcca?acctgccatg?tgaagtttgg?cgaagcaaac?6840

gcgattctcg?ctggcgacgc?tttacaaacg?ctggcgttct?cgattttaag?cgatgccgat?6900

atgccggaag?tgtcggaccg?cgacagaatt?tcgatgattt?ctgaactggc?gagcgccagt?6960

ggtattgccg?gaatgtgcgg?tggtcaggca?ttagatttag?acgcggaagg?caaacacgta?7020

cctctggacg?cgcttgagcg?tattcatcgt?cataaaaccg?gcgcattgat?tcgcgccgcc?7080

gttcgccttg?gtgcattaag?cgccggagat?aaaggacgtc?gtgctctgcc?ggtactcgac?7140

aagtatgcag?agagcatcgg?ccttgccttc?caggttcagg?atgacatcct?ggatgtggtg?7200

ggagatactg?caacgttggg?aaaacgccag?ggtgccgacc?agcaacttgg?taaaagtacc?7260

taccctgcac?ttctgggtct?tgagcaagcc?cggaagaaag?cccgggatct?gatcgacgat?7320

gcccgtcagt?cgctgaaaca?actggctgaa?cagtcactcg?atacctcggc?actggaagcg?7380

ctagcggact?acatcatcca?gcgtaataaa?taagagctcc?aattcgccct?atagtgagtc?7440

gtattacgcg?cgctcactgg?ccgtcgtttt?acaacgtcgt?gactgggaaa?accctggcgt?7500

tacccaactt?aatcgccttg?cagcacatcc?ccctttcgcc?agctggcgta?atagcgaaga?7560

ggcccgcacc?gatcgccctt?cccaacagtt?gcgcagcctg?aatggcgaat?ggaaattgta?7620

agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc?attttttaac?7680

caataggccg?actgcgatga?gtggcagggc?ggggcgtaat?ttttttaagg?cagttattgg?7740

tgcccttaaa?cgcctggtgc?tacgcctgaa?taagtgataa?taagcggatg?aatggcagaa?7800

attcgaaagc?aaattcgacc?cggtcgtcgg?ttcagggcag?ggtcgttaaa?tagccgctta?7860

tgtctattgc?tggtttaccg?gtttattgac?taccggaagc?agtgtgaccg?tgtgcttctc?7920

aaatgcctga?ggccagtttg?ctcaggctct?ccccgtggag?gtaataattg?acgatatgat?7980

catttattct?gcctcccaga?gcctgataaa?aacggtgaat?ccgttagcga?ggtgccgccg?8040

gcttccattc?aggtcgaggt?ggcccggctc?catgcaccgc?gacgcaacgc?ggggaggcag?8100

acaaggtata?gggcggcgag?gcggctacag?ccgatagtct?ggaacagcgc?acttacgggt?8160

tgctgcgcaa?cccaagtgct?accggcgcgg?cagcgtgacc?cgtgtcggcg?gctccaacgg?8220

ctcgccatcg?tccagaaaac?acggctcatc?gggcatcggc?aggcgctgct?gcccgcgccg?8280

ttcccattcc?tccgtttcgg?tcaaggctgg?caggtctggt?tccatgcccg?gaatgccggg?8340

ctggctgggc?ggctcctcgc?cggggccggt?cggtagttgc?tgctcgcccg?gatacagggt?8400

cgggatgcgg?cgcaggtcgc?catgccccaa?cagcgattcg?tcctggtcgt?cgtgatcaac?8460

caccacggcg?gcactgaaca?ccgacaggcg?caactggtcg?cggggctggc?cccacgccac?8520

gcggtcattg?accacgtagg?ccgacacggt?gccggggccg?ttgagcttca?cgacggagat?8580

ccagcgctcg?gccaccaagt?ccttgactgc?gtattggacc?gtccgcaaag?aacgtccgat?8640

gagcttggaa?agtgtcttct?ggctgaccac?cacggcgttc?tggtggccca?tctgcgccac?8700

gaggtgatgc?agcagcattg?ccgccgtggg?tttcctcgca?ataagcccgg?cccacgcctc?8760

atgcgctttg?cgttccgttt?gcacccagtg?accgggcttg?ttcttggctt?gaatgccgat?8820

ttctctggac?tgcgtggcca?tgcttatctc?catgcggtag?ggtgccgcac?ggttgcggca?8880

ccatgcgcaa?tcagctgcaa?cttttcggca?gcgcgacaac?aattatgcgt?tgcgtaaaag?8940

tggcagtcaa?ttacagattt?tctttaacct?acgcaatgag?ctattgcggg?gggtgccgca?9000

atgagctgtt?gcgtaccccc?cttttttaag?ttgttgattt?ttaagtcttt?cgcatttcgc?9060

cctatatcta?gttctttggt?gcccaaagaa?gggcacccct?gcggggttcc?cccacgcctt?9120

cggcgcggct?ccccctccgg?caaaaagtgg?cccctccggg?gcttgttgat?cgactgcgcg?9180

gccttcggcc?ttgcccaagg?tggcgctgcc?cccttggaac?ccccgcactc?gccgccgtga?9240

ggctcggggg?gcaggcgggc?gggcttcgcc?ttcgactgcc?cccactcgca?taggcttggg?9300

tcgttccagg?cgcgtcaagg?ccaagccgct?gcgcggtcgc?tgcgcgagcc?ttgacccgcc?9360

ttccacttgg?tgtccaaccg?gcaagcgaag?cgcgcaggcc?gcaggccgga?ggcttttccc?9420

cagagaaaat?taaaaaaatt?gatggggcaa?ggccgcaggc?cgcgcagttg?gagccggtgg?9480

gtatgtggtc?gaaggctggg?tagccggtgg?gcaatccctg?tggtcaagct?cgtgggcagg?9540

cgcagcctgt?ccatcagctt?gtccagcagg?gttgtccacg?ggccgagcga?agcgagccag?9600

ccggtggccg?ctcgcggcca?tcgtccacat?atccacgggc?tggcaaggga?gcgcagcgac?9660

cgcgcagggc?gaagcccgga?gagcaagccc?gtagggcgcc?gcagccgccg?taggcggtca?9720

cgactttgcg?aagcaaagtc?tagtgagtat?actcaagcat?tgagtggccc?gccggaggca?9780

ccgccttgcg?ctgcccccgt?cgagccggtt?ggacaccaaa?agggaggggc?aggcatggcg?9840

gcatacgcga?tcatgcgatg?caagaagctg?gcgaaaatgg?gcaacgtggc?ggccagtctc?9900

aagcacgcct?accgcgagcg?cgagacgccc?aacgctgacg?ccagcaggac?gccagagaac?9960

gagcactggg?cggccagcag?caccgatgaa?gcgatgggcc?gactgcgcga?gttgctgcca?10020

gagaagcggc?gcaaggacgc?tgtgttggcg?gtcgagtacg?tcatgacggc?cagcccggaa?10080

tggtggaagt?cggccagcca?agaacagcag?gcggcgttct?tcgagaaggc?gcacaagtgg?10140

ctggcggaca?agtacggggc?ggatcgcatc?gtgacggcca?gcatccaccg?tgacgaaacc?10200

agcccgcaca?tgaccgcgtt?cgtggtgccg?ctgacgcagg?acggcaggct?gtcggccaag?10260

gagttcatcg?gcaacaaagc?gcagatgacc?cgcgaccaga?ccacgtttgc?ggccgctgtg?10320

gccgatctag?ggctgcaacg?gggcatcgag?ggcagcaagg?cacgtcacac?gcgcattcag?10380

gcgttctacg?aggccctgga?gcggccacca?gtgggccacg?tcaccatcag?cccgcaagcg?10440

gtcgagccac?gcgcctatgc?accgcaggga?ttggccgaaa?agctgggaat?ctcaaagcgc?10500

gttgagacgc?cggaagccgt?ggccgaccgg?ctgacaaaag?cggttcggca?ggggtatgag?10560

cctgccctac?aggccgccgc?aggagcgcgt?gagatgcgca?agaaggccga?tcaagcccaa?10620

gagacggccc?gag 10633

<210>63

<211>4263

<212>DNA

＜213〉artificial sequence

<220>

＜223〉recombination of polynucleotide

<400>63

cttgatatcg?aattcctgca?gcccggggat?cctctagagt?cgactaggag?gaatataaaa?60

tgaaaaattg?tgtcatcgtc?agtgcggtac?gtactgctat?cggtagtttt?aacggttcac?120

tcgcttccac?cagcgccatc?gacctggggg?cgacagtaat?taaagccgcc?attgaacgtg?180

caaaaatcga?ttcacaacac?gttgatgaag?tgattatggg?taacgtgtta?caagccgggc?240

tggggcaaaa?tccggcgcgt?caggcactgt?taaaaagcgg?gctggcagaa?acggtgtgcg?300

gattcacggt?caataaagta?tgtggttcgg?gtcttaaaag?tgtggcgctt?gccgcccagg?360

ccattcaggc?aggtcaggcg?cagagcattg?tggcgggggg?tatggaaaat?atgagtttag?420

ccccctactt?actcgatgca?aaagcacgct?ctggttatcg?tcttggagac?ggacaggttt?480

atgacgtaat?cctgcgcgat?ggcctgatgt?gcgccaccca?tggttatcat?atggggatta?540

ccgccgaaaa?cgtggctaaa?gagtacggaa?ttacccgtga?aatgcaggat?gaactggcgc?600

tacattcaca?gcgtaaagcg?gcagccgcaa?ttgagtccgg?tgcttttaca?gccgaaatcg?660

tcccggtaaa?tgttgtcact?cgaaagaaaa?ccttcgtctt?cagtcaagac?gaattcccga?720

aagcgaattc?aacggctgaa?gcgttaggtg?cattgcgccc?ggccttcgat?aaagcaggaa?780

cagtcaccgc?tgggaacgcg?tctggtatta?acgacggtgc?tgccgctctg?gtgattatgg?840

aagaatctgc?ggcgctggca?gcaggcctta?cccccctggc?tcgcattaaa?agttatgcca?900

gcggtggcgt?gccccccgca?ttgatgggta?tggggccagt?acctgccacg?caaaaagcgt?960

tacaactggc?ggggctgcaa?ctggcggata?ttgatctcat?tgaggctaat?gaagcatttg?1020

ctgcacagtt?ccttgccgtt?gggaaaaacc?tgggctttga?ttctgagaaa?gtgaatgtca?1080

acggcggggc?catcgcgctc?gggcatccta?tcggtgccag?tggtgctcgt?attctggtca?1140

cactattaca?tgccatgcag?gcacgcgata?aaacgctggg?gctggcaaca?ctgtgcattg?1200

gcggcggtca?gggaattgcg?atggtgattg?aacggttgaa?ttaaggagga?cagctaaatg?1260

aaactctcaa?ctaaactttg?ttggtgtggt?attaaaggaa?gacttaggcc?gcaaaagcaa?1320

caacaattac?acaatacaaa?cttgcaaatg?actgaactaa?aaaaacaaaa?gaccgctgaa?1380

caaaaaacca?gacctcaaaa?tgtcggtatt?aaaggtatcc?aaatttacat?cccaactcaa?1440

tgtgtcaacc?aatctgagct?agagaaattt?gatggcgttt?ctcaaggtaa?atacacaatt?1500

ggtctgggcc?aaaccaacat?gtcttttgtc?aatgacagag?aagatatcta?ctcgatgtcc?1560

ctaactgttt?tgtctaagtt?gatcaagagt?tacaacatcg?acaccaacaa?aattggtaga?1620

ttagaagtcg?gtactgaaac?tctgattgac?aagtccaagt?ctgtcaagtc?tgtcttgatg?1680

caattgtttg?gtgaaaacac?tgacgtcgaa?ggtattgaca?cgcttaatgc?ctgttacggt?1740

ggtaccaacg?cgttgttcaa?ctctttgaac?tggattgaat?ctaacgcatg?ggatggtaga?1800

gacgccattg?tagtttgcgg?tgatattgcc?atctacgata?agggtgccgc?aagaccaacc?1860

ggtggtgccg?gtactgttgc?tatgtggatc?ggtcctgatg?ctccaattgt?atttgactct?1920

gtaagagctt?cttacatgga?acacgcctac?gatttttaca?agccagattt?caccagcgaa?1980

tatccttacg?tcgatggtca?tttttcatta?acttgttacg?tcaaggctct?tgatcaagtt?2040

tacaagagtt?attccaagaa?ggctatttct?aaagggttgg?ttagcgatcc?cgctggttcg?2100

gatgctttga?acgttttgaa?atatttcgac?tacaacgttt?tccatgttcc?aacctgtaaa?2160

ttggtcacaa?aatcatacgg?tagattacta?tataacgatt?tcagagccaa?tcctcaattg?2220

ttcccagaag?ttgacgccga?attagctact?cgcgattatg?acgaatcttt?aaccgataag?2280

aacattgaaa?aaacttttgt?taatgttgct?aagccattcc?acaaagagag?agttgcccaa?2340

tctttgattg?ttccaacaaa?cacaggtaac?atgtacaccg?catctgttta?tgccgccttt?2400

gcatctctat?taaactatgt?tggatctgac?gacttacaag?gcaagcgtgt?tggtttattt?2460

tcttacggtt?ccggtttagc?tgcatctcta?tattcttgca?aaattgttgg?tgacgtccaa?2520

catattatca?aggaattaga?tattactaac?aaattagcca?agagaatcac?cgaaactcca?2580

aaggattacg?aagctgccat?cgaattgaga?gaaaatgccc?atttgaagaa?gaacttcaaa?2640

cctcaaggtt?ccattgagca?tttgcaaagt?ggtgtttact?acttgaccaa?catcgatgac?2700

aaatttagaa?gatcttacga?tgttaaaaaa?taaggaggat?tacactatgg?ttttaaccaa?2760

taaaacagtc?atttctggat?cgaaagtcaa?aagtttatca?tctgcgcaat?cgagctcatc?2820

aggaccttca?tcatctagtg?aggaagatga?ttcccgcgat?attgaaagct?tggataagaa?2880

aatacgtcct?ttagaagaat?tagaagcatt?attaagtagt?ggaaatacaa?aacaattgaa?2940

gaacaaagag?gtcgctgcct?tggttattca?cggtaagtta?cctttgtacg?ctttggagaa?3000

aaaattaggt?gatactacga?gagcggttgc?ggtacgtagg?aaggctcttt?caattttggc?3060

agaagctcct?gtattagcat?ctgatcgttt?accatataaa?aattatgact?acgaccgcgt?3120

atttggcgct?tgttgtgaaa?atgttatagg?ttacatgcct?ttgcccgttg?gtgttatagg?3180

ccccttggtt?atcgatggta?catcttatca?tataccaatg?gcaactacag?agggttgttt?3240

ggtagcttct?gccatgcgtg?gctgtaaggc?aatcaatgct?ggcggtggtg?caacaactgt?3300

tttaactaag?gatggtatga?caagaggccc?agtagtccgt?ttcccaactt?tgaaaagatc?3360

tggtgcctgt?aagatatggt?tagactcaga?agagggacaa?aacgcaatta?aaaaagcttt?3420

taactctaca?tcaagatttg?cacgtctgca?acatattcaa?acttgtctag?caggagattt?3480

actcttcatg?agatttagaa?caactactgg?tgacgcaatg?ggtatgaata?tgatttctaa?3540

aggtgtcgaa?tactcattaa?agcaaatggt?agaagagtat?ggctgggaag?atatggaggt?3600

tgtctccgtt?tctggtaact?actgtaccga?caaaaaacca?gctgccatca?actggatcga?3660

aggtcgtggt?aagagtgtcg?tcgcagaagc?tactattcct?ggtgatgttg?tcagaaaagt?3720

gttaaaaagt?gatgtttccg?cattggttga?gttgaacatt?gctaagaatt?tggttggatc?3780

tgcaatggct?gggtctgttg?gtggatttaa?cgcacatgca?gctaatttag?tgacagctgt?3840

tttcttggca?ttaggacaag?atcctgcaca?aaatgttgaa?agttccaact?gtataacatt?3900

gatgaaagaa?gtggacggtg?atttgagaat?ttccgtatcc?atgccatcca?tcgaagtagg?3960

taccatcggt?ggtggtactg?ttctagaacc?acaaggtgcc?atgttggact?tattaggtgt?4020

aagaggcccg?catgctaccg?ctcctggtac?caacgcacgt?caattagcaa?gaatagttgc?4080

ctgtgccgtc?ttggcaggtg?aattatcctt?atgtgctgcc?ctagcagccg?gccatttggt?4140

tcaaagtcat?atgacccaca?acaggaaacc?tgctgaacca?acaaaaccta?acaatttgga?4200

cgccactgat?ataaatcgtt?tgaaagatgg?gtccgtcacc?tgcattaaat?cctaagtcga?4260

cct 4263

Claims

1. nucleic acid, order with 5 ' to 3 ', described nucleic acid comprises the nucleotide sequence of the coding structural domain that operability connects and the nucleotide sequence of Codocyte cytochrome p 450 enzyme, described structural domain is selected from membrane spaning domain, secretory structure territory, solubilising structural domain or film and inserts albumen, wherein said structural domain and described cytochrome P 450 enzymes allos.

2. nucleic acid as claimed in claim 1 is characterized in that described membrane spaning domain has function in prokaryotic host cell.

3. nucleic acid as claimed in claim 1 is characterized in that, the nucleotide sequence of described Codocyte cytochrome p 450 enzyme has accepted to adapt to express in the prokaryotic host cell codon optimized.

4. nucleic acid as claimed in claim 1 is characterized in that, described cytochrome P 450 enzymes is the isoprenoid precursor modifying enzyme that the isoprenoid precursor is modified in catalysis.

5. nucleic acid as claimed in claim 4 is characterized in that described modification is selected from oxidation, hydroxylation or epoxidation.

6. nucleic acid as claimed in claim 1 also contains the nucleotide sequence of Codocyte cytochrome p 450 reductase enzyme.

7. one kind contains the described expression of nucleic acids carrier of claim 1.

8. host cell that contains the described expression vector of claim 7.

9. host cell as claimed in claim 8 is characterized in that, described host cell is the cell that does not produce tetra-sodium isoamyl-1-alkene ester usually by mevalonate pathway.

10. host cell as claimed in claim 9 is characterized in that described host cell is a prokaryotic cell prokaryocyte.

11. host cell as claimed in claim 8 is characterized in that, described host cell also comprises the nucleic acid of the nucleotide sequence that contains coding allos diterpene synthase.

12. host cell as claimed in claim 8 is characterized in that, described host cell also comprises the nucleic acid of the nucleotide sequence that contains Codocyte cytochrome p 450 reductase enzyme.

13. host cell as claimed in claim 9 is characterized in that, described host cell is to cross with one or more nucleic acid genetic modifications of the nucleotide sequence that contains two or more mevalonate pathway enzymes of encoding.

14. a method that produces the biosynthetic pathway product in host cell, described method comprises:

The host cell of cultivating genetic modification in suitable culture medium has the cytochrome P 450 enzymes of the modification of enzymic activity with generation, wherein use the described host cell of nucleic acid genetic modification of the nucleotide sequence that contains Codocyte cytochrome p 450 enzyme, described cytochrome P 450 enzymes operability is connected in and is selected from membrane-spanning domain, secretory structure territory, solubilising structural domain and film and inserts proteic structural domain

Wherein, in the presence of the biosynthetic pathway intermediate, produce described modified cytochrome P 450 enzymes and cause zymetology to modify described biosynthetic pathway intermediate and the described biosynthetic pathway product of generation.

15. method as claimed in claim 14, it is characterized in that, described cytochrome P 450 enzymes is an isoprenoid precursor modifying enzyme, wherein, in the presence of the isoprenoid precursor compound, produce described isoprenoid precursor modifying enzyme and cause zymetology to modify described isoprenoid precursor and the described isoprenoid compound of generation.

16. method as claimed in claim 14 is characterized in that, described host cell is an eukaryotic host cell.

17. method as claimed in claim 16 is characterized in that, described host cell is a yeast cell.

18. method as claimed in claim 16 is characterized in that, described host cell is a vegetable cell.

19. method as claimed in claim 14 is characterized in that, described host cell is a prokaryotic cell prokaryocyte.

20. method as claimed in claim 15, it is characterized in that, also use the described host cell of nucleic acid genetic modification of the nucleotide sequence that contains coding allos diterpene synthase, wherein said cultivation can produce described diterpene synthase, described diterpene synthase can be modified many isopentene of tetra-sodium ester, to produce the substrate of described isoprenoid modifying enzymes.

21. method as claimed in claim 20 is characterized in that, described many isopentene of tetra-sodium ester is selected from farnesyl pyrophosphate, tetra-sodium Mang ox ester or tetra-sodium geranyl Mang ox ester.

22. method as claimed in claim 14 is characterized in that, also uses the described host cell of nucleic acid genetic modification of the nucleotide sequence that contains Codocyte cytochrome p 450 reductase enzyme (CPR).

23. method as claimed in claim 15, it is characterized in that, described host cell is the cell that does not synthesize tetra-sodium isoamyl-1-alkene ester (IPP) usually by mevalonate pathway, wherein with containing two or more enzymes in the coding mevalonate pathway, the IPP isomerase, the described host cell of one or more nucleic acid genetic modifications of the nucleotide sequence of prenyltransferase and diterpene synthase, described cultivation can produce the mevalonate pathway enzyme, wherein produces described two or more mevalonate pathway enzymes, the IPP isomerase, prenyltransferase, diterpene synthase and isoprenoid precursor modifying enzyme cause producing the isoprenoid compound.

24. method as claimed in claim 23, it is characterized in that, described two or more mevalonate pathway enzymes comprise Mevalonic kinase, Phosphomevalonic kinase and tetra-sodium mevalonate decarboxylase, wherein cultivate described host cell in the presence of mevalonic acid.

25. method as claimed in claim 23, it is characterized in that described two or more mevalonate pathway enzymes comprise Acetoacetyl-CoA thiolase, Hydroxymethylglutaryl-CoA synthase, hydroxymethyl glutaryl-CoA reductase enzyme, Mevalonic kinase, Phosphomevalonic kinase and tetra-sodium mevalonate decarboxylase.

26. method as claimed in claim 14 is characterized in that, the nucleotide sequence operability of described Codocyte cytochrome p 450 enzyme is connected in inducible promoter.

27. method as claimed in claim 15 is characterized in that, the output of described isoprenoid compound is at least about 10 mg/litre.

28. method as claimed in claim 14 also comprises and separates described biosynthetic pathway product.