CN102732499B

CN102732499B - Artemisia annua amorpha-4,11-diene synthase mutant with improved enzyme activity and application thereof

Info

Publication number: CN102732499B
Application number: CN201110285012.XA
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: 2014-07-16
Anticipated expiration: 2031-09-22
Also published as: CN102732499A

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 Scientists a kind of sesquiterpene lactones containing peroxide bridge of isolation identification from sweet wormwood (Artemisia annua) first, is the medicine of the most effectively treating encephalic malaria and anti-Chloroquine-resistant Falciparum Malaria disease at present.The World Health Organization and Hesperian confirmation and recommendation have been subject to taking Artemisinin as basic composite treatment.Content of artemisinin in sweet wormwood low (0.01-1% dry weight), and it is large affected by kind and region plantation, resource scarcity.

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

The research of Artemisinin biosynthetic pathway obtains many progress in recent years, has cloned AMORPHADIENE synthase (ADS), arteannuinic acid synthetic enzyme (CYP71AV1) and sweet wormwood aldehyde reductase Dbr2.Scientists utilizes metabolic engineering technology to attempt by the microorganism synthetic artemisinin precursor arteannuinic acid that ferments, then by 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 arteannuinic acid.Think at present, utilizing microorganisms producing arteannuinic 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, to improving the content of meta-bolites.

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

Therefore the enzymic activity that, how effectively to improve ADS is that this area need to 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 a first aspect of the present invention, a kind of saltant type 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) there is the albumen of the aminoacid sequence shown in SEQ ID NO:3 (saltant type); Or

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

In another aspect of this invention, provide a kind of polynucleotide of separation, these polynucleotide are selected from lower group:

(i) polynucleotide of the saltant type AMORPHADIENE synthase protein described in coding; Or

(ii) polynucleotide of the polynucleotide complementation and (i).

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

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

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

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, provide a kind of method of producing saltant type AMORPHADIENE synthase, comprise step:

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

(2) segregation mutant AMORPHADIENE synthase from culture.

In another aspect of this invention, the purposes of the saltant type AMORPHADIENE synthase described in providing, for the production of AMORPHADIENE and/or arteannuinic acid.

In another aspect of this invention, a kind of method of producing AMORPHADIENE is provided, described method comprises: utilize described saltant type AMORPHADIENE synthase catalysis method Thessaloniki tetra-sodium (farnesyl pyrophosphate, FPP) cyclisation, thereby obtain AMORPHADIENE.

In another preference, described saltant type AMORPHADIENE synthase catalysis FPP cyclisation in born of the same parents or born of the same parents carry out outward.

In another preference, described saltant type AMORPHADIENE synthase catalysis method Thessaloniki tetra-sodium cyclisation is carried out in born of the same parents, comprise step: the encoding gene of described saltant type AMORPHADIENE synthase is transformed into host cell, cultivates this cell, thus production AMORPHADIENE.

In another preference, described saltant type AMORPHADIENE synthase catalysis method Thessaloniki tetra-sodium cyclisation is carried out in born of the same parents, comprises step: the encoding gene of described saltant type AMORPHADIENE synthase and farnesyl tetra-sodium are produced to the enzyme (enzyme in 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 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, farnesyl diphosphate synthase, DXP synthase, HMG-CoA reductase enzyme, geranyl transferring enzyme etc.) encoding gene transformed host cell, cultivate this cell, thereby production AMORPHADIENE.

In another aspect of this invention, a kind of method of producing arteannuinic acid is provided, described method comprises: by the encoding gene of described saltant type AMORPHADIENE synthase, farnesyl tetra-sodium produces enzyme (enzyme in MVA/MEP approach) encoding gene, arteannuinic acid synthetase-coding gene, cytochrome P450 reductase encoding gene is transformed into host cell; Cultivate this cell, thereby produce arteannuinic acid.

In another preference, also comprise: by obtain arteannuinic acid by organic semi-synthetic production Artemisinin.

In another preference, at 25-45 DEG C, cultivate described cell.

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

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

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

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Brief description of the drawings

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

Fig. 3, temperature (upper figure), the impact of pH (figure below) on wild-type and mutant ADS (T399S) vigor.

Embodiment

The inventor, through long-term research, has unexpectedly obtained a kind of saltant type AMORPHADIENE synthase, and described saltant type AMORPHADIENE synthase is in the situation that keeping catalysate constant, and enzymic activity is much higher than wild-type.Complete the present invention based on this.

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

The present invention also comprises fragment, derivative and the analogue of saltant type AMORPHADIENE synthase.As used herein, term " fragment ", " derivative " refer to and substantially keep biological function or the active polypeptide that saltant type AMORPHADIENE synthase of the present invention is identical with " analogue ".Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferably conservative amino acid residue), and the amino-acid residue of such replacement can not be also to be encoded by genetic code, or (ii) in one or more amino-acid residues, there is the polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as extending the compound of polypeptide transformation period, for example polyoxyethylene glycol) merge the polypeptide that forms, or (iv) additional aminoacid sequence be fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying, or fusion rotein).Belong to the known scope of those skilled in the art according to these fragments of definition, derivative and analogue herein.

In the present invention, term " saltant type AMORPHADIENE synthase " or " saltant type AMORPHADIENE synthase protein " refer to the polypeptide of the SEQ ID NO:3 sequence with saltant type AMORPHADIENE synthase activity.This term also comprises having and variant form saltant type 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 C-terminal and/or N-terminal interpolation or disappearance one or several (being generally in 20, being preferably in 10, is more preferably in 5) amino acid.For example, in the art, while replacement with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, add or one of disappearance or several amino acid also can not change the function of protein conventionally at C-terminal and/or N-terminal.This term also comprises active fragments and the reactive derivative of saltant type 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, albumen that can be coded with the DNA that described saltant type AMORPHADIENE synthase DNA is hybridized under high or low stringency condition and polypeptide or the albumen that utilizes the antiserum(antisera) of anti-mutation type AMORPHADIENE synthase to obtain.The present invention also provides other polypeptide, as the fusion rotein that comprises saltant type AMORPHADIENE synthase or its fragment.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of saltant type AMORPHADIENE synthase protein.Conventionally, this fragment have saltant type AMORPHADIENE synthase sequence at least about 20 continuous amino acids, conventionally 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 saltant type AMORPHADIENE synthase or polypeptide.The difference of these analogues and described saltant type AMORPHADIENE synthase can be the difference on aminoacid sequence, can be also the difference not affecting on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant natural or induction.Induce variation body can obtain by various technology, as by 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-(as D-amino acid), and has the analogue of non-natural amino acid (as β, gamma-amino acid) that exist or synthetic.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide exemplifying.But, in these polypeptide analogs, be Serine corresponding to the amino acid of the 399th of SEQ ID NO:2 aminoacid sequence.

(conventionally the not changing primary structure) form of modification comprises: in body or the chemically derived form of external polypeptide as acetylize or carboxylated.Modify and also comprise glycosylation.Modified forms also comprises the have phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, the conservative property of the saltant type AMORPHADIENE synthase " variation polypeptide " refers to compared with the aminoacid sequence with 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 are replaced by the similar or close amino acid of character and form polypeptide at the most best.These conservative property variation polypeptide preferably carry out amino acid substitution according to table 1 and produce.

Table 1

Initial residue	Representational replacement	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 saltant type 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 SEQ ID NO:3 or the varient of degeneracy.As used herein, " varient of degeneracy " refers to that coding has the protein of SEQ ID NO:3 in the present invention, but with the differentiated nucleotide sequence of coding region sequence shown in 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 (with optional additional code sequence) and the non-coding sequence of mature polypeptide.

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and can be also 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 the fragment of polypeptide, analogue and derivative with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.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 may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of polypeptide of its coding.

The invention still further relates between above-mentioned sequence hybridization and two sequences and have at least 50%, preferably at least 70%, the more preferably polynucleotide of at least 80% (as 85%, 90%, 95%, 99%) homogeny.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " refers to: (1) at the hybridization compared with under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, and 0.1%SDS, 60 DEG C; Or (2) hybridization time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) only at the homogeny between 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 SEQID NO:3.

The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment ", containing 15 Nucleotide, is at least better 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 (as PCR) of nucleic acid to determine 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 obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For 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.In the time that sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment amplifying for each time is stitched together by proper order.

Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated and obtains relevant sequence from the host cell propagation by ordinary method.

In addition, also can synthesize relevant sequence by the method for synthetic, especially fragment length more in short-term.Conventionally, by first synthetic multiple small segments, and then connect and can obtain the fragment that sequence is very long.

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

The present invention also relates to the carrier that comprises polynucleotide of the present invention, and the host cell producing through genetically engineered with carrier of the present invention or saltant type AMORPHADIENE synthase coding sequence, and produce the method for polypeptide of the present invention through recombinant technology.

By conventional recombinant DNA technology (Science, 1984; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to express or the saltant type AMORPHADIENE synthase of Restruction.In general there are following steps:

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

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

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

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

Method well-known to those having ordinary skill in the art can be used for building containing saltant type AMORPHADIENE synthase DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.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 the host cell transforming, as eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of use, or for colibacillary kantlex or amicillin resistance.

Comprise above-mentioned suitable DNA sequence dna and the suitable carrier of promotor or control sequence, can be for transforming suitable host cell, with can marking protein.

Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as 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 insert enhancer sequence in carrier time.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs, act on promotor transcribing with enhancing gene conventionally.

Persons skilled in the art are all known the suitable carrier of How to choose, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaCl ₂method processing, step used is well-known in this area.Another kind method is to use MgCl ₂.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packaging etc.

The transformant obtaining can be cultivated by ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional mediums.Under the condition that is suitable for host cell growth, cultivate.When host cell grows into after suitable cell density, the promotor of selecting with suitable method (as temperature transition or chemical induction) induction, cultivates cell for some time again.

Extracellular can be expressed or be secreted into recombinant polypeptide in the above methods in cell or on cytolemma.If needed, can utilize its physics, the separating by 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 processing, with the combination of protein precipitant processing (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The polynucleotide of saltant type AMORPHADIENE synthase of the present invention (or its fragment, variant form or derivative) are being transformed into after host cell, can be directly used in production AMORPHADIENE, the output that increases AMORPHADIENE can promote the synthetic of arteannuinic acid.

Measure in acquisition after the information of saltant type AMORPHADIENE synthase of the present invention, those skilled in the art are clear how for follow-up preparation AMORPHADIENE or arteannuinic acid.Described saltant type AMORPHADIENE synthase catalysis FPP cyclisation, thus AMORPHADIENE obtained.Preparation method in various born of the same parents or outside born of the same parents all comprises in the present invention, or can be applied in the present invention.

Described saltant type AMORPHADIENE synthase catalysis FPP cyclisation can be in born of the same parents or born of the same parents carry out outward.As a kind of optimal way of the present invention, provide the method for biosynthesizing AMORPHADIENE in a kind of born of the same parents: the encoding gene of described saltant type AMORPHADIENE synthase is transformed into host cell, cultivates this cell, thereby produce arteannuinic acid.

As optimal way of the present invention, provide a kind of in born of the same parents the method for direct production arteannuinic acid, described method comprises: the encoding gene of described saltant type AMORPHADIENE synthase and FPP are produced to enzyme (that is: the enzyme in MVA/MEP approach, be selected from but be not limited to: acetyl-CoA thiolase, HMG-CoA synthetic enzyme, Mevalonic kinase, Phosphomevalonic kinase, tetra-sodium 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, farnesyl diphosphate synthase, DXP synthase, HMG-CoA reductase enzyme, geranyl transferring enzyme etc.) encoding gene transformed host cell, cultivate this cell, thereby production AMORPHADIENE.It is known in the art that FPP produces enzyme and encoding gene thereof, those skilled in the art know that how to obtain these enzymes and how by it transformant.Enzyme in above MVA/MEP approach is also 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 arteannuinic acid, described method comprises: by the encoding gene of described saltant type AMORPHADIENE synthase, FPP produces the enzyme (encoding gene of the enzyme in MVA/MEP approach, and arteannuinic acid synthetic enzyme CYP71AV1 (encoding gene is referring to GenBank:DQ268763) and CPR (encoding gene is referring to GenBank:DQ318192) are transformed into host cell; Cultivate this cell, thereby produce arteannuinic acid.Arteannuinic acid synthetic enzyme and encoding gene thereof are known in the art, those skilled in the art know that how to obtain these enzymes and how by it transformant.The arteannuinic acid obtaining semi-syntheticly can be produced Artemisinin by organic.Compared with traditional method, the method efficiency of this production Artemisinin is high and cost is low.

The inventor has also studied the optimal temperature of described saltant type AMORPHADIENE synthase.Therefore,, as optimal way of the present invention, at 25-45 DEG C, cultivate described cell.More preferably, at 35-43 DEG C, cultivate described cell; More preferably, at 38-42 DEG C, cultivate described cell.Or, at 25-45 DEG C, store described saltant type AMORPHADIENE synthase.More preferably, at 35-43 DEG C, store described saltant type AMORPHADIENE synthase; More preferably, at 38-42 DEG C, store described saltant type AMORPHADIENE synthase.Thereby retain preferably the enzymic activity of described saltant type AMORPHADIENE synthase.

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

Unless otherwise defined, the same meaning that all specialties that use in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.

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

The extraction of a, the total RNA of sweet wormwood

Get sweet wormwood material (about 100mg) fully grinds in liquid nitrogen.Be transferred in 1.5ml centrifuge tube, add 1mL Trizol (Invitrogen, Cat.15596-018), mix, room temperature is placed 5min.The centrifugal 10min of 12,000rpm, abandons or adopts precipitation.In supernatant, add 200 μ L trichloromethanes, mix, the centrifugal 10min of 12,000rpm.Get supernatant, add 500 μ L isopropanol precipitating RNA.The centrifugal 10min of 12,000rpm, precipitation is used 70% washing with alcohol, and vacuum-drying, is dissolved in 20-50 μ L H ₂o (RNase free).10mM Tris-HCl (pH7.5) for RNA is done to suitable dilution, 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 the first chain reverse transcription adopts RNA PCR system (TaKaRa, Cat.DRR019A).Reaction system is as follows:

42 DEG C of reaction 30min.After boiling water boiling 5min, be placed on ice.Reverse transcription product (or after diluting 10 times) can be directly used in 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)。

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

C, rite-directed mutagenesis

Use overlap extension pcr, successfully construct multiple simple point mutation bodies, double-mutant, Trimutant and multimutation body.DNA sequence dna to these mutant is measured.

Primer sequence is as follows:

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 (SEQ IDNO:7) of the corresponding site codon that suddenlys change:

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)。

Protein mutation sequencing result is shown in 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 mutant fragment (ADS-T399S, ADS-T399A, ADS-T399N, ADS-T399D, ADS-T399R), cut and be connected into pET-32a carrier (Amp through NcoI/BamHI enzyme ^r, Novagen, America), obtain recombinant vectors.

B, competent cell preparation

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

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

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

C, conversion

(the above recombinant vectors obtaining, in BL21 (DE3) the E.coli cell that 0.1-0.5 μ g) melts in 50 μ L, mixes, and places 25min on ice to add DNA sample; 42 DEG C of thermal treatment 90s, place 3min on ice; Add 100 μ L liquid LB substratum, 30min is cultivated in 37 DEG C of recoveries; Be applied to and select flat board, cultivate 12-16h.Then choose single bacterium colony and carry out PCR qualification.

The enzyme of DNA agarose gel electrophoresis, fragment is cut, purifying and connection be 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) ₂₀for primer, 10 μ L reaction systems, require to carry out reverse transcription according to reverse transcription test kit (TOYOBO, Osaka, Japan).42 DEG C of reaction 30min.After boiling water boiling 5min, be placed on ice.Reverse transcription product (or after diluting 10 times) can be directly used in PCR and detect.

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

Embodiment 4, prokaryotic expression and enzyme activity determination

A: prokaryotic expression

BL21 cell, containing 37 DEG C of grow overnight on the LB flat board of 50 μ g/mL Ampicillin, was cultivated liquid in the positive single bacterium colony liquid medium within of PCR qualification picking, got 500 μ L culture enlarged culturing to 50mL, until OD ₆₀₀it is 0.5mmol/L to final concentration that ≈ 0.6 adds IPTG, continues at 20 DEG C of inducing culture spend the night (20h).Get 6mL bacterium liquid 12000rpm centrifugal 5 minutes, precipitation be suspended in precooling 3mL Buffer (25mM Mopso, pH 7.0,5mM DTT, and 10%[v/v] glycerine, 5mM MgCl ₂) in, ultrasonic disruption (3S opens, and 7S closes, and processes 3min, 25%power) cell, centrifugal, get supernatant and carry out the qualification of SDS-PAGE electrophoresis or do vitro enzyme biopsy and survey.Or according to Ni-NTA Spin Kit handbook (Qiagen, Valencia, CA), purifying is with the recombinant protein (ADS enzyme or its each mutant) of His-Tag, and electrophoresis qualification.

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, in the above-mentioned albumen of 500 μ L, add FPP (Farnesyl pyrophosphate, Sigma-Aldrich, F6892) to final concentration 40 μ mol/L, above reaction system, cover 400 μ L normal hexanes, 37 DEG C are reacted 1 hour, get 4 μ L and carry 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 setting is set to 220 DEG C.While analysis, heating schedule be 80 DEG C initial, 10 DEG C/min is raised to 250 DEG C, then 20 DEG C/min is raised to 280 DEG C, keep 2.5min.Mass spectrum adopts EI source, and sweep limit is 30-500m/z, and ion source and level Four bar temperature are respectively 250 DEG C and 150 DEG C, and sweep rate is 5 times/s.The structure of compound and title determine jointly by NIST (National Institute of Standards and Technology) and two databases of Wiley libraries.

The GC-MS figure of ADS and mutant thereof is shown in Fig. 2.The albumen (total bacterial protein or if purifying protein has the label of some expression as his-tag, s-tag) separating after BL-21 expression in escherichia coli empty carrier (BL-21), does not have special product and produces; Express wild-type ADS, vitro enzyme biopsy is surveyed and is 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) mutant, can cause enzyme deactivation, the peak of the special AMORPHADIENE in place about 9.5 minutes disappears; Express 399 neutral amino acids residue mutant (except S, as T399N, T399A), can cause enzyme product specific variations, the peak of the special AMORPHADIENE in place about 9.5 minutes becomes bimodal, has also produced 2 kinds of new product α-Gurjunene and Zingiberene except AMORPHADIENE.Express 399 neutral amino acids residue S mutant clons, product does not change.

Embodiment 5, dynamic analysis and optimum temperuture and optimal pH are measured

Water-bath heating, temperature is selected 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C 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 use Tris-hydrochloride buffers (0.05M), pH9.20,9.80,10.10,10.50 and 10.70 use glycine-sodium hydrate buffer solutions (0.05M).Additional 5mM DTT and 5mMMgCl in damping fluid ₂.When measuring, optimal pH with the purifying protein of 3 μ g, reacts 5min at 30 DEG C.

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

Result is as Fig. 3.Show: wild-type and mutant ADS optimum temperuture be all 40 DEG C of left and right, and 40 DEG C of following mutant all improve much than wild-type enzyme is active, mutant deactivation rate is greater than wild-type (the upper figure of Fig. 3) on 40 DEG C.Optimal pH wild-type is in 10.5 left and right, and mutant is in 9.8 left and right, and before pH10, mutant is all than active improve a lot (Fig. 3 figure below) of wild-type enzyme.

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

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

K _mvalue equals enzymatic reaction speed and reaches the corresponding 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 _mthe avidity size of the reaction enzyme-to-substrate that value can be similar to: K _mvalue is large, shows that avidity is little; K _mbe worth littlely, show that avidity is large.

K _catfor catalytic constant, be called again turnover number (TN value).Its unit is s ^-1, k _catbe worth greatlyr, represent that the catalytic rate of enzyme is higher.

K _cat/ K _msize for the catalytic efficiency of the different substrates of more different enzymes or the same enzyme catalysis.

While measuring different concentration of substrate, the speed of response recording and corresponding concentration of substrate are carried out to 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 _cat; Wherein, V _maxfor maximum reaction velocity, at calculating K _mwhen value, try to achieve; [E] is enzyme concn.

Result shows: with amino acid S allelic replacement after the T in original enzyme, can make the ADS after sudden change there is higher catalytic efficiency to substrate FPP, catalytic efficiency improves 71.7%.Its turnover number (K simultaneously _cat) and maximum reaction velocity (V _max) all improve approximately 83%.

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

Substratum: LB; High pressure steam sterilization: 121 DEG C.

Plasmid construction: pET-32a, packs the encoding gene (as previous embodiment 2 " a ") of ADS or its mutant into.

By recombinant plasmid transformed (as previous embodiment 2 " b " and " c ") competent BL21 (DE3) the E.coli cell obtaining above, obtain the cell of restructuring.

Fermenting process:

1. seed culture

From the flat board of cotransformation, picking is containing recombinant plasmid pET-32a (containing the encoding gene of ADS or its mutant enzyme) BL21 (DE3) E.coli bacterium colony, be inoculated in 15 × 150mm test tube that 2ml LB (100mg/L Amp) substratum is housed, with 200rpm, 37 DEG C of overnight incubation, then one-level culture is transferred to by 1% inoculum size in the shaking flask of the 250ml that 30ml LB (100mg/L Amp) is housed, cultivate after about 5h, be transferred in fermention medium as secondary seed.

2. fermentation

With 1% inoculum size, secondary seed is inoculated in the 250ml shaking flask that 20ml LB is housed and (all contains 100mg/L Amp), 280rpm, 37 DEG C while being cultured to OD600=0.4-0.8, with the IPTG abduction delivering of 0.1mM, add the n-dodecane of 4ml to carry out extractive fermentation, 280rpm, 30 DEG C of cultivations simultaneously.Every 24h sampling, centrifugal collection organic phase ,-20 DEG C of preservations.Whole fermenting process maintains 3-5 days.

3. sample preparation

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

Further, in order to improve the fermentation yield of AMORPHADIENE, enzyme in the upstream MVA/MEP approach of ADS (is comprised to isopentenyl diphosphate Δ-isomerase (encoding gene is referring to GenBank:EU896069), 2C-methyl tetrahydroxybutane-2, 4-pyrophosphate synthase (encoding gene is referring to GenBank:EU896619), 4-phosphoric acid-2C-methyl tetrahydroxybutane 4-cytidine phosphates synthase (encoding gene is referring to GenBank:EU896613.1), DXP synthase (encoding gene is referring to GenBank:EU905209), geranyl transferring enzyme (encoding gene is referring to GenBank:EU905203)) encoding gene pack the multiple clone site of above-mentioned recombinant vectors into.The expression of these enzymes can improve the generation of FPP in born of the same parents effectively, thereby improves the amount of ADS catalysis FPP cyclisation formation AMORPHADIENE.

Further, arteannuinic acid synthetic enzyme CYP71AV1 (encoding gene is referring to GenBank:DQ268763) and cytochrome P450 reductase CPR (encoding gene is referring to GenBank:DQ318192) are packed together into the multiple clone site of above-mentioned recombinant vectors, as above transformant and cultivation transformant, thus arteannuinic acid (being present in organic phase) produced efficiently.

All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. a saltant type AMORPHADIENE synthase, it is the albumen of aminoacid sequence as shown in SEQ ID NO:3.

2. polynucleotide for separation, is characterized in that, these polynucleotide are polynucleotide of coding saltant type AMORPHADIENE synthase claimed in claim 1.

3. a carrier, is characterized in that, it contains polynucleotide claimed in claim 2.

4. a genetically engineered host cell, is characterized in that, it contains carrier claimed in claim 3, or in genome, is integrated with polynucleotide claimed in claim 2.

5. a method of producing saltant type AMORPHADIENE synthase, is characterized in that, comprises step:

(1) cultivate host cell claimed in claim 4, obtain culture; With

(2) segregation mutant AMORPHADIENE synthase from culture.

6. the purposes of saltant type AMORPHADIENE synthase claimed in claim 1, for the production of AMORPHADIENE and/or arteannuinic acid.

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

8. method as claimed in claim 7, it is characterized in that, described saltant type AMORPHADIENE synthase catalysis method Thessaloniki tetra-sodium cyclisation is carried out in born of the same parents, comprise step: by the encoding gene transformed host cell of saltant type AMORPHADIENE synthase claimed in claim 1, cultivate this cell, thus production AMORPHADIENE.

9. method as claimed in claim 8, is characterized in that, also in described host cell, transforms the encoding gene of farnesyl tetra-sodium generation enzyme.

10. produce the method for arteannuinic acid for one kind, it is characterized in that, described method comprises: by the encoding gene of saltant type AMORPHADIENE synthase claimed in claim 1, farnesyl tetra-sodium produces the encoding gene of enzyme, arteannuinic acid synthetase-coding gene, cytochrome P450 reductase encoding gene is transformed into host cell; Cultivate this cell, thereby produce arteannuinic acid.

11. methods as described in as arbitrary in claim 8-10, is characterized in that, cultivate described cell at 25-45 DEG C.

12. methods as described in as arbitrary in claim 8-10, is characterized in that, cultivate described cell under pH6.0-10.5.