CN102732499A

CN102732499A - Artemisia annua amorpha-4,11-diene synthase mutant with improved enzyme activity and application thereof

Info

Publication number: CN102732499A
Application number: CN201110285012XA
Authority: CN
Inventors: 陈晓亚; 李建戌; 王凌健; 洪高洁; 胡文利; 阮菊新; 杨长青; 毛颖波
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2011-04-02
Filing date: 2011-09-22
Publication date: 2012-10-17
Anticipated expiration: 2031-09-22
Also published as: CN102732499B

Abstract

The invention relates to an Artemisia annua amorpha-4,11-diene synthase mutant with improved enzyme activity and application thereof. The amorpha-4,11-diene synthase mutant has enzyme activity substantially higher than that of wild type amorpha-4,11-diene synthase under the condition that catalysate maintains unchanged. Compared to a traditional method, a method of applying the Artemisia annua amorpha-4,11-diene synthase mutant provided by the invention in enzymatic production of artemisinin has the advantages of high efficiency and low cost.

Description

Sweet wormwood AMORPHADIENE synthase mutant and application thereof that a kind of enzymic activity improves

Technical field

The invention belongs to biological technical field; More specifically, the present invention relates to sweet wormwood AMORPHADIENE synthase mutant and the application thereof that a kind of enzymic activity improves.

Background technology

Artemisinin is Chinese science man a kind of sesquiterpene lactones that contains peroxo bridge of isolation identification from sweet wormwood (Artemisia annua) first, is the medicine of at present efficacious therapy encephalic malaria and anti-chloroquine pernicious malaria.With the Artemisinin is that basic composite treatment has received the World Health Organization and Hesperian affirmation and recommendation.Artemislnin content low (0.01-1% dry weight) in the sweet wormwood, and it is big influenced by kind and region plantation, resource scarcity.

In the Artemisinin biosynthesizing, and sesquiterpene composition AMORPHADIENE (Amorphadiene, Amorpha-4 is 11-diene) by the AMORPHADIENE synthase in the sweet wormwood (amorpha-4,11-diene synthase; ADS) catalysis FPP cyclisation forms, and it is the biosynthetic common precursor of beta-Artelinic acid and Artemisinin.The output that increases AMORPHADIENE can promote the synthetic and accumulation of beta-Artelinic acid and Artemisinin.Artificial complete synthesis Artemisinin complex process, cost height.Therefore, at present in the world the Artemisinin raw material mainly rely on from wild and cultivation sweet wormwood and directly extract.

The research of Artemisinin biosynthetic pathway obtains many progress in recent years, has cloned AMORPHADIENE synthase (ADS), beta-Artelinic acid synthetic enzyme (CYP71AV1) and sweet wormwood aldehyde reductase Dbr2.Scientists utilizes the metabolic engineering technology to attempt through microbial fermentation synthetic artemisinin precursor beta-Artelinic acid, then through organic semi-synthetic production Artemisinin, to reduce the production cost of Artemisinin.2006, Jay Keasling carried out genetically engineered to yeast, make it to produce high-caliber beta-Artelinic acid.Think that at present utilizing the microorganisms producing beta-Artelinic acid is a practicable method, can reduce the production cost of Artemisinin.In addition, also can pass through the biosynthetic genes involved of transgenic plant overexpression Artemisinin, in the hope of improving the content of meta-bolites.

The genetic modification of fermentation engineering bacterial classification need be incorporated into Gene A DS in the beta-Artelinic acid biosynthetic pathway and CYP701AV1 in engineering yeast or the engineering bacterial chromosome.And the enzymic activity of ADS is directly connected to the biosynthetic output of beta-Artelinic acid.

Therefore, the enzymic activity that how to improve ADS effectively is that this area need be furtherd investigate.

Summary of the invention

The sweet wormwood AMORPHADIENE synthase mutant and the application thereof that the object of the present invention is to provide a kind of enzymic activity to improve.

In first aspect of the present invention, a kind of mutant AMORPHADIENE synthase is provided, it sports Serine corresponding to the 399th of SEQ IDNO:2 (wild-type) aminoacid sequence by Threonine.

In another preference, this albumen is:

(a) has the albumen of the aminoacid sequence shown in the SEQ ID NO:3 (mutant); Or

(b) replace, lack or add one or several (as 1-20 by the process of the aminoacid sequence shown in (a); Preferably 1-10; More preferably 1-5; 1-3 more preferably) amino acid and reservation (a) protein-active by (a) deutero-albumen; And the amino acid corresponding to the 399th position of SEQ ID NO:3 in this proteic aminoacid sequence is Serine.

In another aspect of this invention, a kind of isolating polynucleotide are provided, these polynucleotide are selected from down group:

(i) polynucleotide of the described mutant AMORPHADIENE synthase protein of coding; Or

(ii) with (i) in polynucleotide complementary polynucleotide.

In another preference, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:3.

In another aspect of this invention, a kind of carrier is provided, it contains described polynucleotide.

In another aspect of this invention, a kind of genetically engineered host cell is provided, it contains described carrier, or is integrated with described polynucleotide in the genome.

In another preference, described host cell is yeast, bacterium or vegetable cell.More preferably, described host cell is intestinal bacteria.

In another aspect of this invention, a kind of method of production mutant AMORPHADIENE synthase is provided, comprises step:

(1) cultivates described host cell, obtain culture; With

(2) segregation mutant AMORPHADIENE synthase from culture.

In another aspect of this invention, the purposes of described mutant AMORPHADIENE synthase is provided, is used to produce AMORPHADIENE and/or beta-Artelinic acid.

In another aspect of this invention; A kind of method of producing AMORPHADIENE is provided; Described method comprises: utilize described mutant AMORPHADIENE synthase catalysis method Thessaloniki tetra-sodium (farnesyl pyrophosphate, FPP) cyclisation, thereby acquisition AMORPHADIENE.

In another preference, described mutant AMORPHADIENE synthase catalysis FPP cyclisation is carried out in born of the same parents or outside the born of the same parents.

In another preference; The cyclisation of described mutant AMORPHADIENE synthase catalysis method Thessaloniki tetra-sodium is carried out in born of the same parents; Comprise step: the encoding sox of described mutant AMORPHADIENE synthase is transformed into host cell, cultivates this cell, thereby produce AMORPHADIENE.

In another preference; The cyclisation of described mutant AMORPHADIENE synthase catalysis method Thessaloniki tetra-sodium is carried out in born of the same parents, comprises step: the encoding sox and the farnesyl tetra-sodium of described mutant AMORPHADIENE synthase are produced the enzyme (enzyme in the MVA/MEP approach; Be selected from but be not limited to: acetyl-CoA thiolase, HMG-CoA synthetic enzyme, HMG-CoA reductase enzyme, Mevalonic kinase, Phosphomevalonic kinase, tetra-sodium RS-Mevalonic acid decarboxylase, IPP isomerase, 5-phosphoric acid-D-1-deoxy-D-xylulose sugar synthetic enzyme 5-phosphoric acid-D-1-deoxy-D-xylulose sugar reduction isomerase, isopentenyl diphosphate isomerase, 5-phosphoric acid-D-1-deoxy-D-xylulose sugar synthetic enzyme, 5-phosphoric acid-D-1-deoxy-D-xylulose sugar reduction isomerase, 4-phosphoric acid-2C-methyl tetrahydroxybutane 4-cytidine phosphates synthase, 2C-methyl tetrahydroxybutane 4-cytidine phosphates kinases, 2C-methyl tetrahydroxybutane-2; 4-pyrophosphate synthase, 1-hydroxy-2-methyl-2-butylene-4-pyrophosphate synthase, 1-hydroxy-2-methyl-2-butylene-4-tetra-sodium reductase enzyme, FPP synthetic enzyme, 1-deoxy-D-xylulose 5-phosphate synthase, HMG-CoA reductase enzyme, geranyl transferring enzyme etc.) the encoding sox transformed host cell; Cultivate this cell, thereby produce AMORPHADIENE.

In another aspect of this invention; A kind of method of producing beta-Artelinic acid is provided; Described method comprises: with the encoding sox of described mutant AMORPHADIENE synthase; The farnesyl tetra-sodium produces enzyme (enzyme in the MVA/MEP approach) encoding sox, the beta-Artelinic acid synthetase-coding gene, and the cytochrome P450 reductase encoding sox is transformed into host cell; Cultivate this cell, thereby produce beta-Artelinic acid.

In another preference, also comprise: with the beta-Artelinic acid that obtains through organic semi-synthetic production Artemisinin.

In another preference, cultivate described cell down at 25-45 ℃.

In another preference, cultivate described cell down at 35-43 ℃; More preferably, cultivate described cell down at 38-42 ℃.

In another preference, under PH, cultivate described cell under the 6.0-10.5.

In another preference, under PH, cultivate described host cell under the 8.5-10; More preferably, under PH, cultivate described host cell under the 9-10; Be PH9.8 best.

Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.

Description of drawings

The sequence comparing result of Fig. 1, ADS and mutant clon.Can find out that from the result 399 amino acids residue T have been mutated into neutral amino acids residue S, A, N and charged amino-acid residue R, D.

The GC-MS figure of Fig. 2, ADS and two mutants thereof.A-F represents wild-type and different mutants GC-MS figure respectively; G and H represent the enlarged view of E figure and F figure respectively.I has shown the structure iron of α-Gurjunene (1) and Zingiberene (2).Wherein AMO represents AMORPHADIENE.

Fig. 3, temperature (last figure), pH (figure below) are to the influence of wild-type and two mutants ADS (T399S) vigor.

Embodiment

The inventor has all of a sudden obtained a kind of mutant AMORPHADIENE synthase through long term studies, and described mutant AMORPHADIENE synthase is keeping under the constant situation of catalysate, and enzymic activity is much higher than wild-type.Accomplished the present invention based on this.

Polypeptide of the present invention (mutant AMORPHADIENE synthase) can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, verivate and the analogue of mutant AMORPHADIENE synthase.As used herein, term " fragment ", " verivate " are meant biological function or the active polypeptide that keeps mutant AMORPHADIENE synthase of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, verivate or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue); And so substituted amino-acid residue can be also can not encoded by genetic code; Or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical; Or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period; Polyoxyethylene glycol for example) merges formed polypeptide; Or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (like leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying or fusion rotein).These fragments of definition, verivate and analogue according to this paper belong to the known scope of those skilled in the art.

In the present invention, term " mutant AMORPHADIENE synthase " or " mutant AMORPHADIENE synthase protein " refer to have the SEQ ID NO:3 polypeptide of sequence of mutant AMORPHADIENE synthase activity.This term also comprises having and variant form mutant AMORPHADIENE synthase identical function, SEQ ID NO:3 sequence.These variant forms comprise (but being not limited to): several (are generally 1-20; 1-10 best; Also better for 1-8,1-5,1-3 or 1-2) amino acid whose disappearance, insertion and/or replacement; And add or lack one or several (being generally in 20, preferably is in 10, more preferably is in 5) amino acid at C-terminal and/or N-terminal.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add or lack one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of mutant AMORPHADIENE synthase.But, in these variant forms, be Serine corresponding to the amino acid of the 399th of SEQ ID NO:2 aminoacid sequence.

The variant form of polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of described mutant AMORPHADIENE synthase DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of mutation type AMORPHADIENE synthase to obtain.The present invention also provides other polypeptide, as comprises mutant AMORPHADIENE synthase or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of mutant AMORPHADIENE synthase protein.Usually; This fragment have mutant AMORPHADIENE synthase sequence at least about 20 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids; More preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of mutant AMORPHADIENE synthase or polypeptide.The difference of these analogues and described mutant AMORPHADIENE synthase can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain through various technology, as through radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to above-mentioned representational polypeptide of giving an example.But, in these polypeptide analogs, be Serine corresponding to the amino acid of the 399th of SEQ ID NO:2 aminoacid sequence.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modify and also comprise glycosylation.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the polypeptide that has improved its anti-proteolyze performance or optimized solubility property.

In the present invention; " the conservative property variation polypeptide of mutant AMORPHADIENE synthase " refers to compare with the aminoacid sequence of SEQ ID NO:3; There are 20 at the most, preferably at the most 10, preferably at the most 8; More preferably at the most 5,3 (as 1,2 or 3) amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also provides the polynucleotide sequence of code book invention mutant AMORPHADIENE synthase or its conservative property variation polypeptide.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:3 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:3, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:3.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:3 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the verivate of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it possibly be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, polynucleotide of at least 80% (as 85%, 90%, 95%, 99%) homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide according to the invention.In the present invention, " stringent condition " is meant: (1) than hybridization under LIS and the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, like 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQID NO:3.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (like PCR) of nucleic acid to confirm and/or to separate the polynucleotide of encoding mutant type AMORPHADIENE synthase.

The Nucleotide full length sequence of encoding mutant type AMORPHADIENE synthase of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.For the pcr amplification method; Can be disclosed according to the present invention about nucleotide sequence; Especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need carries out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, through first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) through chemosynthesis.Can this dna sequence dna be introduced in various existing dna moleculars as known in the art (or like carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention through chemosynthesis.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or mutant AMORPHADIENE synthase coding sequence, and produce the method for polypeptide according to the invention through recombinant technology.

Recombinant DNA technology (Science, 1984 through routine; 224:1431), the mutant AMORPHADIENE synthase of reorganization is expressed or produced to polymerized nucleoside acid sequence of the present invention capable of using.In general following steps are arranged:

(1). with the polynucleotide of encoding mutant type AMORPHADIENE synthase of the present invention (or varient), or with recombinant expression vector conversion that contains these polynucleotide or transduction proper host cell;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, mutant AMORPHADIENE synthase polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carriers.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains mutant AMORPHADIENE synthase DNA sequences encoding and suitable transcribing/the translate expression vector of wave.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness like eukaryotic cell, or be used for colibacillary kantlex or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like vegetable cell.Representative example has: intestinal bacteria, yeast, vegetable cell etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (like temperature transition or chemically induced), cell is cultivated for some time again.

The extracellular can expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.

The polynucleotide of mutant AMORPHADIENE synthase of the present invention (or its fragment, variant form or verivate) can directly be used to produce AMORPHADIENE after being transformed into host cell, the output that increases AMORPHADIENE can promote the synthetic of beta-Artelinic acid.

Measure the information of mutant AMORPHADIENE synthase of the present invention in acquisition after, clear subsequent preparation AMORPHADIENE or the beta-Artelinic acid of how being used for of those skilled in the art.Described mutant AMORPHADIENE synthase catalysis FPP cyclisation, thus AMORPHADIENE obtained.Various born of the same parents preparation method interior or that born of the same parents are outer all comprises in the present invention, or can be applied among the present invention.

Described mutant AMORPHADIENE synthase catalysis FPP cyclisation can be carried out in born of the same parents or outside the born of the same parents.As a kind of optimal way of the present invention, the method for biosynthesizing AMORPHADIENE in a kind of born of the same parents is provided: the encoding sox of described mutant AMORPHADIENE synthase is transformed into host cell, cultivates this cell, thereby produce beta-Artelinic acid.

As optimal way of the present invention, provide a kind of in born of the same parents the method for direct production beta-Artelinic acid, described method comprises: the encoding sox of described mutant AMORPHADIENE synthase and FPP are produced enzyme (that is: the enzyme in the MVA/MEP approach; Be selected from but be not limited to: acetyl-CoA thiolase, HMG-CoA synthetic enzyme, Mevalonic kinase, Phosphomevalonic kinase, tetra-sodium RS-Mevalonic acid decarboxylase, IPP isomerase, 5-phosphoric acid-D-1-deoxy-D-xylulose sugar synthetic enzyme 5-phosphoric acid-D-1-deoxy-D-xylulose sugar reduction isomerase, isopentenyl diphosphate isomerase, 5-phosphoric acid-D-1-deoxy-D-xylulose sugar synthetic enzyme, 5-phosphoric acid-D-1-deoxy-D-xylulose sugar reduction isomerase, 4-phosphoric acid-2C-methyl tetrahydroxybutane 4-cytidine phosphates synthase, 2C-methyl tetrahydroxybutane 4-cytidine phosphates kinases, 2C-methyl tetrahydroxybutane-2; 4-pyrophosphate synthase, 1-hydroxy-2-methyl-2-butylene-4-pyrophosphate synthase, 1-hydroxy-2-methyl-2-butylene-4-tetra-sodium reductase enzyme, FPP synthetic enzyme, 1-deoxy-D-xylulose 5-phosphate synthase, HMG-CoA reductase enzyme, geranyl transferring enzyme etc.) the encoding sox transformed host cell; Cultivate this cell, thereby produce AMORPHADIENE.FPP produces enzyme and encoding sox is known in the art, those skilled in the art know that how to obtain these enzymes and how with it transformant.Enzyme in the above MVA/MEP approach also is well known to those skilled in the art.

As optimal way of the present invention; Provide a kind of in born of the same parents the method for direct production beta-Artelinic acid; Described method comprises: with the encoding sox of described mutant AMORPHADIENE synthase; FPP produces the enzyme (encoding sox of the enzyme in the MVA/MEP approach, and beta-Artelinic acid synthetic enzyme CYP71AV1 (encoding sox is referring to GenBank:DQ268763) and CPR (encoding sox is referring to GenBank:DQ318192) are transformed into host cell; Cultivate this cell, thereby produce beta-Artelinic acid.Beta-Artelinic acid synthetic enzyme and encoding sox thereof are known in the art, those skilled in the art know that how to obtain these enzymes and how with it transformant.The beta-Artelinic acid that is obtained semi-syntheticly can be produced Artemisinin through organic.Compare with traditional method, the method efficient height and the cost of this production Artemisinin are low.

The inventor has also studied the optimal temperature of described mutant AMORPHADIENE synthase.Therefore, as optimal way of the present invention, cultivate described cell down at 25-45 ℃.More preferably, cultivate described cell down at 35-43 ℃; More preferably, cultivate described cell down at 38-42 ℃.Perhaps, store described mutant AMORPHADIENE synthase down at 25-45 ℃.More preferably, store described mutant AMORPHADIENE synthase down at 35-43 ℃; More preferably, store described mutant AMORPHADIENE synthase down at 38-42 ℃.Thereby the enzymic activity that keeps described mutant AMORPHADIENE synthase preferably.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition such as Sambrook and Russell (2001) .Molecular Cloning:A Laboratory Manual (3rd ed.); Condition described in the Cold Spring Harbor Laboratory Press, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.

Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.

The extraction of embodiment 1, the total RNA of sweet wormwood, pcr amplification goal gene ADS and rite-directed mutagenesis

The extraction of a, the total RNA of sweet wormwood

Getting sweet wormwood material (about 100mg) fully grinds in liquid nitrogen.Be transferred in the 1.5ml centrifuge tube, and adding 1mL Trizol (Invitrogen, Cat.15596-018), mixing, room temperature is placed 5min.12, the centrifugal 10min of 000rpm abandons or adopts deposition.Add 200 μ L trichloromethanes, mixing, 12, the centrifugal 10min of 000rpm in the supernatant.Get supernatant, add 500 μ L isopropanol precipitating RNA.12, the centrifugal 10min of 000rpm, deposition is used 70% washing with alcohol, and vacuum-drying is dissolved in 20-50 μ L H ₂O (RNase free).RNA is done suitable dilution with 10mM Tris-HCl (pH7.5), measure the UV absorption value of wavelength between 200-300nm.RNA concentration=40 μ g/mL * A ₂₆₀* extension rate.A ₂₆₀/ A ₂₈₀Should be between 1.9 to 2.1.PolyA mRNA first chain reverse transcription employing RNA PCR system (TaKaRa, Cat.DRR019A).Reaction system is following:

42 ℃ of reaction 30min.After boiling water boils 5min, place on ice.Reverse transcription product (or after diluting 10 times) can directly be used for the pcr amplification goal gene.

B, pcr amplification goal gene ADS

With high-fidelity enzyme KOD-plus archaeal dna polymerase (ToYoBo) amplification ADS full-length gene order (1641bp), primer sequence:

ADS-S：5’-TTT CCATGGCTATGTCTCTTACAGAAGAAAAACCTA-3’(SEQ?ID?NO：14)；

ADS-AS：5’-TTT GGATCCTCATATACTCATAGGATAAACGAGT-3’(SEOID?NO：15)。

The PCR reaction conditions is: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of renaturation 30s, 68 ℃ are extended 120s, 30 to 35 circulations of increasing; 68 ℃ of insulation 10min.4 ℃ of insulations.

C, rite-directed mutagenesis

Use overlap extension pcr, make up successfully a plurality of simple point mutation bodies, double-mutant, Trimutant and multimutation body.Dna sequence dna to these two mutants is measured.

Primer sequence is following:

T → S (ADS-T399S) that ADS is the 399th, the primer of the corresponding site codon that suddenlys change:

ADS-T399S-S：5’-GTTGTAATCATTAGTGGCGGTGCTA-3’(SEQ?ID?NO：4)，

ADS-T399S-AS：5’-TAGCACCGCCACTAATGATTACAAC-3’(SEQ?IDNO：5)；

T → A (ADS-T399A) that ADS is the 399th, the primer of the corresponding site codon that suddenlys change:

ADS-T399A-S：5’-GTTGTAATCATTGCTGGCGGTGCTA-3’(SEQ?ID?NO：6)，

ADS-T399A-AS：5’-TAGCACCGCCAGCAATGATTACAAC-3’；

T → N (ADS-T399N) that ADS is the 399th, the primer of the corresponding site codon that suddenlys change (SEQ IDNO:7):

ADS-T399N-S：5’-GTTGTAATCATTAATGGCGGTGCTA-3’(SEQ?ID?NO：8)，

ADS-T399N-AS：5’-TAGCACCGCCATTAATGATTACAAC-3’(SEQ?IDNO：9)；

T → D (ADS-T399D) that ADS is the 399th, the primer of the corresponding site codon that suddenlys change:

ADS-T399D-S：5’-GTTGTAATCATTGATGGCGGTGCTA-3’(SEQ?ID?NO：?10)，

ADS-T399D-AS：5’-TAGCACCGCCATCAATGATTACAAC-3’(SEQ?IDNO：11)；

T → R (ADS-T399R) that ADS is the 399th, the primer of the corresponding site codon that suddenlys change:

ADS-T399R-S：5’-GTTGTAATCATTCGTGGCGGTGCTA-3’(SEQ?ID?NO：12)，

ADS-T399R-AS：5’-TAGCACCGCCACGAATGATTACAAC-3’(SEQ?IDNO：13)。

The protein mutation sequencing result is seen Fig. 1.

Embodiment 2, vector construction and intestinal bacteria transform

A, vector construction

KOD-plus archaeal dna polymerase amplification ADS coding region fragment (ADS) and two mutants fragment (ADS-T399S, ADS-T399A, ADS-T399N, ADS-T399D ADS-T399R), cuts through the NcoI/BamHI enzyme and to be connected into pET-32a carrier (Amp ^r, Novagen America), obtains recombinant vectors.

B, competent cell preparation

The bacillus coli DH 5 alpha of-70 ℃ of storages or BL-21 rule on solid LB flat board, 37 ℃ of overnight cultures; Picking list bacterium colony in 5mL liquid LB substratum, the 250rpm overnight cultures.Second day, the ratio amplification in 1/50 was inoculated in the 500mL liquid LB substratum, and 18-22 ℃ of cultivation is to OD ₆₀₀≈ 0.5 (about 5-6h), cooled on ice 10min.4 ℃ 2, the centrifugal 10min of 500g, it is resuspended that thalline transforms damping fluid with 160mL, and it is resuspended that the centrifugal supernatant of abandoning, thalline transform damping fluid with 40mL at last, adds 3mL DMSO, mixing.Packing, every pipe 50 μ L, liquid nitrogen flash freezer ,-70 ℃ of preservations.

Transform damping fluid: 55mM MnCl ₂, 15mM CaCl ₂, 250mM KCl, 10mM PIPES (pH6.7), fresh, precooling on ice.

LB substratum (1L): 10g NaCl, the 5g yeast extract, the 10g peptone, pH 7.0.Solid LB substratum adds the 15g/L agar powder.

C, conversion

Add DNA sample (recombinant vectors of above acquisition, 0.1-0.5 μ g) in BL21 (DE3) the E.coli cell that 50 μ L melt, mixing is placed 25min on ice; 42 ℃ of thermal treatment 90s place 3 min on ice; Add 100 μ L liquid LB substratum, 30min is cultivated in 37 ℃ of recoveries; Be applied to and select flat board, cultivate 12-16h.Choose single bacterium colony then and carry out the PCR evaluation.

DNA agarose gel electrophoresis, segmental enzyme are cut, purifying be connected with reference to Sambrook and Russell (Sambrook and Russell (2001) .Molecular Cloning:A Laboratory Manual (3rd ed.); Cold Spring Harbor Laboratory Press).

Embodiment 3, RT-PCR

The total RNA of 1 μ g, Oligo (dt) ₂₀Be primer, 10 μ L reaction systems, (TOYOBO, Osaka Japan) require to carry out reverse transcription according to the reverse transcription test kit.42 ℃ of reaction 30min.After boiling water boils 5min, place on ice.Reverse transcription product (or after diluting 10 times) can directly be used for PCR and detect.

According to known ADS sequence, synthesized the PCR primer, when analyzing the ADS expression, as internal reference, proofread and correct the template amount of RT-PCR reaction with sweet wormwood Actin1 (GenBank:EU531837).The PCR condition is: the renaturation temperature of reaction and extension time are by primer and expanding fragment length decision.General reaction conditions is: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 55-60 ℃ of renaturation 30s, 72 ℃ are extended 30s, 25 to 35 circulations of increasing; 72 ℃ of insulation 10min.4 ℃ of insulations.

Embodiment 4, prokaryotic expression and enzyme activity determination

A: prokaryotic expression

The BL21 cell is 37 ℃ of grow overnight on the LB flat board that contains 50 μ g/mL Ampicillin, and PCR identifies in the positive single bacterium colony liquid medium within of picking and cultivated liquid, gets 500 μ L culture enlarged culturing to 50mL, up to OD ₆₀₀It is 0.5mmol/L to final concentration that ≈ 0.6 adds IPTG, and continuation is at 20 ℃ of inducing culture spend the night (20h).Got 6mL bacterium liquid 12000rpm centrifugal 5 minutes, (25mM Mopso, pH 7.0,5mM DTT and 10% [v/v] glycerine, 5mM MgCl to precipitate the 3mL Buffer that is suspended in precooling ₂) in, (3S opens ultrasonic disruption, and 7S closes, and handles 3min, and 25%power) cell is centrifugal, gets supernatant and carries out the evaluation of SDS-PAGE electrophoresis or do vitro enzyme biopsy survey.Perhaps (CA), purifying has the recombinant protein (ADS enzyme or its each two mutants) of His-Tag for Qiagen, Valencia, and electrophoresis is identified according to Ni-NTA Spin Kit handbook.

B: enzyme activity determination

The ADS enzyme of above-mentioned acquisition or its each mutant protein carry out enzyme activity determination in 1.5mL EP pipe; Add FPP (Farnesyl pyrophosphate in the above-mentioned albumen of 500 μ L; Sigma-Aldrich F6892) to final concentration 40 μ mol/L, covers 400 μ L normal hexanes above reaction system; 37 ℃ were reacted 1 hour, got 4 μ L and carried out GC-MS analysis (Agilent 6890/5973GC-MSD gas chromatography-mass spectrum detector).

C: instrument and chromatographic condition

Agilent 6890/5973GC-MSD gas chromatography-mass spectrum detector adopts HP5-MS quartz capillary column (30m * 0.25mm * 0.25 μ m, Agilent).High-purity helium is as carrier gas, and flow rate of carrier gas is 1ml/min, and temperature is set to 220 ℃.When analyzing, heating schedule be 80 ℃ initial, 10 ℃/min is raised to 250 ℃, 20 ℃/min is raised to 280 ℃ then, keeps 2.5min.Mass spectrum adopts the EI source, and sweep limit is 30-500m/z, and ion source and level Four bar temperature are respectively 250 ℃ and 150 ℃, and sweep rate is 5 times/s.The structure of compound and title determine through NIST (National Institute of Standards and Technology) and two DBs of Wiley libraries jointly.

The GC-MS figure of ADS and two mutants thereof sees Fig. 2.The isolating albumen in BL-21 expression in escherichia coli empty carrier (BL-21) back (total bacterial protein or if purifying protein has label such as his-tag, the s-tag of some expression) does not have special product and produces; Express wild-type ADS, the vitro enzyme biopsy is surveyed and found: the place about 9.5 minutes has produced the peak of a more special AMORPHADIENE; Express 399 bit strip electric charge amino-acid residue (T399D, T399R) two mutants, can cause enzyme deactivation, the peak of the local special AMORPHADIENE about 9.5 minutes disappears; Express 399 neutral amino acids residue two mutants (except that S; Like T399N, T399A); Can cause enzyme product specific variations, the peak of the local special AMORPHADIENE about 9.5 minutes becomes bimodal, except that AMORPHADIENE, has also produced 2 kinds of new product α-Gurjunene and Zingiberene.Express 399 neutral amino acids residue S mutant clons, product does not change.

Embodiment 5, dynamic analysis and optimum temperuture and ph optimum are measured

The water-bath heating, temperature is selected 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ six gradients.PH has selected eight gradients such as 7.50,8.10,8.60,9.20,9.80,10.10,10.50 and 10.70; Wherein pH7.50,8.10 and 8.60 usefulness Tris-hydrochloride buffers (0.05M), pH9.20,9.80,10.10,10.50 and 10.70 usefulness glycocoll-sodium hydrate buffer solutions (0.05M).Additional 5mM DTT and 5mMMgCl in the damping fluid ₂When measuring, ph optimum, reacts 5min at 30 ℃ with the purifying protein of 3 μ g.

Under above-mentioned each condition; Add in wild-type and two mutants ADS to the 1.5mL EP pipe of aforementioned acquisition and carry out enzyme activity determination, add FPP in the 500 μ L damping fluids, above reaction system, cover 400 μ L normal hexanes to final concentration 40 μ mol/L; 37 ℃ were reacted 1 hour, got 4 μ L and carried out the GC-MS analysis.

Result such as Fig. 3.Show: wild-type and two mutants ADS optimum temperuture are all about 40 ℃, and two mutants below 40 ℃ all improves much than wild-type enzyme is active, and the two mutants deactivation rate is greater than wild-type (the last figure of Fig. 3) on 40 ℃.The ph optimum wild-type is about 10.5, and two mutants is about 9.8, and two mutants is all than active improve a lot (Fig. 3 figure below) of wild-type enzyme before pH10.

The purifying protein, 5mM DTT and the 5mM MgCl that comprise 3 μ g during dynamic analysis with 0.025M HEPES damping fluid ₂, at 25 ℃ of reaction 5-10min, the concentration of using substrate FPP from 3 μ m to 100 μ m.K _mUse the Lineweaver-Burk curve calculation.Result such as table 2.

The enzyme of table 2, ADS and ADS mutant clon parameter detecting alive

K _mValue equals enzymatic reaction speed and reaches the pairing concentration of substrate of maximum reaction velocity one half, is one of characteristic constant of enzyme.Different enzyme K _mValue is different, the same enzyme and different substrate reactions K _mValue is also different, K _mValue can proximate reaction enzyme-to-substrate the avidity size: K _mValue is big, shows that avidity is little; K _mBe worth for a short time, show that avidity is big.

k _CatBe catalytic constant, be called turnover number (TN value) again.Its unit is s ^-1, k _CatBe worth greatly more, the catalytic rate of expressed enzyme is high more.

k _Cat/ K _mSize be used for the catalytic efficiency (of different enzymes of comparison or the different substrates of the same enzyme catalysis.

When measuring different concentration of substrate, speed of response and the corresponding concentration of substrate that records carried out regression analysis, try to achieve K _mValue.Calculation formula is Michaelis-Menton equation (Michaelis-Menten equation): v=V _Max* [S]/(Km+ [S]) (formula 1), v represents initial velocity of reaction, V _MaxRepresent maximum reaction velocity, [S] represents concentration of substrate.This computing can obtain Vmax simultaneously.According to formula V _Max=k _Cat* [E] (formula 2) calculates k _CatWherein, V _MaxBe maximum reaction velocity, at calculating K _mTry to achieve during value; [E] is enzyme concn.

The result shows: with amino acid S allelic replacement after the T in the original enzyme, can make the ADS after the sudden change that substrate FPP is had higher catalytic efficiency (, catalytic efficiency (improves 71.7%.While its turnover number (K _Cat) and maximum reaction velocity (V _Max) all improved about 83%.

Embodiment 6, utilization transform the microorganisms producing AMORPHADIENE or the beta-Artelinic acid of the encoding sequence of mutain of the present invention

Substratum: LB; High pressure steam sterilization: 121 ℃.

Plasmid construction: pET-32a, the encoding sox of pack into ADS or its two mutants (like previous embodiment 2 " a ").

With recombinant plasmid transformed (like previous embodiment 2 " b " and " c ") competent BL21 (DE3) the E.coli cell of above acquisition, obtain the cell of reorganization.

Fermenting process:

1. seed culture

Picking contains recombinant plasmid pET-32a (encoding sox that contains ADS or its mutant enzyme) BL21 (DE3) E.coli bacterium colony from the flat board of cotransformation; Be inoculated in 15 * 150mm test tube that 2ml LB (100mg/L Amp) substratum is housed; With 200rpm, 37 ℃ of overnight cultures are transferred to shaking in the bottle of 250ml that 30ml LB (100mg/L Amp) is housed with the one-level culture then by 1% inoculum size; After cultivating about 5h, be transferred in the fermention medium as secondary seed.

2. fermentation

Inoculum size with 1%; Secondary seed is inoculated into the 250ml that 20ml LB is housed shakes in the bottle and (all contain 100mg/L Amp), 280rpm, 37 ℃ when being cultured to OD600=0.4-0.8; IPTG abduction delivering with 0.1mM; The n-dodecane that adds 4ml simultaneously carries out extractive fermentation, 280rpm, 30 ℃ of cultivations.Every 24h sampling, centrifugal collection organic phase ,-20 ℃ of preservations.Whole fermentation process was kept 3-5 days.

3. sample preparation

The organic phase of centrifugal collection comprises AMORPHADIENE.In organic phase: it is miscible that the ratio of ETHYLE ACETATE=1: 700 adds ETHYLE ACETATE, directly detects AMORPHADIENE with GC-MS.The AMORPHADIENE output that comprises the reconstitution cell of ADS mutant code gene is significantly higher than wild-type.

Further; In order to improve the fermentation yield of AMORPHADIENE; With pack into the MCS of above-mentioned recombinant vectors of the encoding sox of the enzyme in the upper reaches MVA/MEP approach of ADS (comprising isopentenyl diphosphate Δ-isomerase (encoding sox is referring to GenBank:EU896069), 2C-methyl tetrahydroxybutane-2,4-pyrophosphate synthase (encoding sox is referring to GenBank:EU896619), 4-phosphoric acid-2C-methyl tetrahydroxybutane 4-cytidine phosphates synthase (encoding sox is referring to GenBank:EU896613.1), 1-deoxy-D-xylulose 5-phosphate synthase (encoding sox is referring to GenBank:EU905209), geranyl transferring enzyme (encoding sox is referring to GenBank:EU905203)).The expression of these enzymes can improve the generation of FPP in the born of the same parents effectively, thereby improves the amount of ADS catalysis FPP cyclisation formation AMORPHADIENE.

Further; With pack into the together MCS of above-mentioned recombinant vectors of beta-Artelinic acid synthetic enzyme CYP71AV1 (encoding sox is referring to GenBank:DQ268763) and cytochrome P450 reductase CPR (encoding sox is referring to GenBank:DQ318192); As above transformant and culture transformation are sub, thereby produce beta-Artelinic acid (being present in the organic phase) efficiently.

All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims

1. mutant AMORPHADIENE synthase, it sports Serine corresponding to the 399th of SEQ ID NO:2 aminoacid sequence by Threonine.

2. albumen as claimed in claim 1 is characterized in that, this albumen is:

(a) has the albumen of the aminoacid sequence shown in the SEQ ID NO:3; Or

(b) by the aminoacid sequence shown in (a) through replacement, lack or add one or several amino acid and reservation (a) protein-active by (a) deutero-albumen; And be Serine corresponding to the amino acid on the 399th of the SEQ ID NO:3 in this proteic aminoacid sequence.

3. isolating polynucleotide is characterized in that, these polynucleotide are selected from down group:

(i) polynucleotide of coding claim 1 or 2 described mutant AMORPHADIENE synthase proteins; Or

(ii) with (i) in polynucleotide complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQID NO:3.

5. a carrier is characterized in that, it contains claim 3 or 4 described polynucleotide.

6. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 5, or is integrated with claim 3 or 4 described polynucleotide in the genome.

7. a method of producing mutant AMORPHADIENE synthase is characterized in that, comprises step:

(1) cultivates the described host cell of claim 6, obtain culture; With

(2) segregation mutant AMORPHADIENE synthase from culture.

8. the purposes of the described mutant AMORPHADIENE of claim 1 synthase is used to produce AMORPHADIENE and/or beta-Artelinic acid.

9. a method of producing AMORPHADIENE is characterized in that, described method comprises: utilize the cyclisation of the described mutant AMORPHADIENE of claim 1 synthase catalysis method Thessaloniki tetra-sodium, thereby obtain AMORPHADIENE.

10. method as claimed in claim 9; It is characterized in that; The cyclisation of described mutant AMORPHADIENE synthase catalysis method Thessaloniki tetra-sodium is carried out in born of the same parents; Comprise step: with the encoding sox transformed host cell of the described mutant AMORPHADIENE of claim 1 synthase, cultivate this cell, thereby produce AMORPHADIENE.

11. method as claimed in claim 10 is characterized in that, also in described host cell, transforms the encoding sox that the farnesyl tetra-sodium produces enzyme.

12. method of producing beta-Artelinic acid; It is characterized in that; Described method comprises: with the encoding sox of the described mutant AMORPHADIENE of claim 1 synthase; The farnesyl tetra-sodium produces the encoding sox of enzyme, the beta-Artelinic acid synthetase-coding gene, and the cytochrome P450 reductase encoding sox is transformed into host cell; Cultivate this cell, thereby produce beta-Artelinic acid.

13. like claim 7, the arbitrary described method of 9-12, it is characterized in that, cultivate described cell down at 25-45 ℃.

14. like claim 7, the arbitrary described method of 9-12, it is characterized in that, under PH, cultivate described cell under the 6.0-10.5.