CN100537754C - A kind of S-adenosylmethionine is produced the structure and the large scale fermentation of bacterial strain - Google Patents
A kind of S-adenosylmethionine is produced the structure and the large scale fermentation of bacterial strain Download PDFInfo
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- CN100537754C CN100537754C CNB2006101178280A CN200610117828A CN100537754C CN 100537754 C CN100537754 C CN 100537754C CN B2006101178280 A CNB2006101178280 A CN B2006101178280A CN 200610117828 A CN200610117828 A CN 200610117828A CN 100537754 C CN100537754 C CN 100537754C
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Abstract
The invention discloses a kind of new S-adenosylmethionine synthetase albumen and encoding gene thereof.The invention also discloses the carrier that contains described encoding gene.The invention also discloses and can be used for producing the bacterial strain of S-adenosylmethionine and the method for producing S-adenosylmethionine.The S-adenosylmethionine output that the S-adenosylmethionine that the present invention makes up is produced bacterial strain can reach more than the 9g/L, and the L-methionine(Met) can reach 40% to the transformation efficiency of S-adenosylmethionine.
Description
Technical field
The invention belongs to biotechnology engineering field, relate to a kind of new S-adenosylmethionine synthetic enzyme and application thereof.
Background technology
S-adenosylmethionine (S-Adenosyl-L-Methionine), breviary commonly used are SAM or AdoMet.External medical trade(brand)name has S-Amet (Castejon factory, Spain), Samyr (Bioresearch, Italy).Molecular formula: C
15H
22N
6O
5S.SAM was found by Cantoni in nineteen fifty-three.No matter be prokaryotic cell prokaryocyte or eukaryotic cell, it all is very important intermediary metabolism substance, mainly plays the transmethylase effect in vivo, changes the aminopropyl effect and changes the sulfenyl effect.
As far back as the end of the seventies, S-adenosylmethionine is used in Europe clinically, at first as the anti-inflammation analgesic of treatment of arthritis, after be applied to anti-depression drug and treat various hepatopathys.Therefore as far back as the eighties in last century, the pharmaceutical use of S-adenosylmethionine just admitted widely in Europe, entered American market in 1999 after, sales volume reaches nearly 1,000,000,000 dollars very soon.China does not have like product production at present, only depends on import.Along with the raising of people's quality of life, the renewal of health concept, prediction will get more and more to the demand of S-adenosylmethionine.
The preparation method of S-adenosylmethionine mainly contains 3 kinds of chemical synthesis, fermentation method and Enzymatic transformation methods.Chemical synthesis adopts S-adenosyl homocysteine and methyl donor (CH
3I) synthetic, S-adenosylmethionine has (+) and (-) two kinds of configurations, has only (-) configuration that biological activity is just arranged, but contains a small amount of (+) type SAM in the synthetic product, and is difficult to separate.
Traditional fermentation method adopts and add the L-methionine(Met) in substratum, with yeast fermentation production (-) type S-adenosylmethionine, is the main method of producing S-adenosylmethionine the sixties in last century to the eighties.Schlenk etc. find Candida utilis, several common yeast such as Saccharomyces cerevisiae, and under a large amount of relatively L-methionine(Met) existence conditions, yeast cell is grown, not growing all can accumulate S-adenosylmethionine.Shiozaki etc. screen from multiple microorganism and obtain a strain Saccharomycesake, and culture can accumulate S-adenosylmethionine.The SAM output that this bacterium is cultivated after 7 days in the 10L fermentor tank is higher.But calculate with the L-methionine(Met), its maximum conversion rate of producing SAM has only about 20%, even lower.
The Enzymatic transformation method is compared with chemical synthesis, fermentation method, has end product accumulation volume height, separate to purify easily, reaction time is short and advantage such as pollution-free, thereby be comparatively effective industrialized preparing process.Enzyme transforming process mainly utilizes the S-adenosylmethionine synthetic enzyme, catalytic substrate L-methionine(Met) and ATP generate (-) type S-adenosylmethionine, and the S-adenosylmethionine synthetic enzyme has the triphosphatase activity, makes triphosphoric acid be decomposed into tetra-sodium and phosphoric acid, and enzymatic reaction is as follows:
The S-adenosylmethionine synthetic enzyme extensively is present in animal and plant and the microbe.Cantoli uses the S-adenosylmethionine synthetic enzyme that extracts first from rat liver, catalytic substrate L-methionine(Met) and ATP synthesize S-adenosylmethionine.There is the people from intestinal bacteria and yeast, to separate the S-adenosylmethionine synthetic enzyme subsequently again and is used for the S-adenosylmethionine preparation.It is not high that but the S-adenosylmethionine synthetic enzyme is few at animal and plant and microbe intensive amount, enzyme is lived, and the separation and purification difficulty, and for example 400g yeast stem cell can only separation and purification obtain 8U S-adenosylmethionine synthetic enzyme, is 0.05U/mg than vigor.Therefore, be subjected to S-adenosylmethionine synthetic enzyme vigor and enzyme quantitative limitation with Enzymatic transformation method mass preparation S-adenosylmethionine.
Utilizing genetic engineering means, will have high enzyme S-adenosylmethionine synthase gene alive and change in the suitable expression system, make up the genetic engineering bacterium with S-adenosylmethionine synthetic enzyme, is following main direction of producing S-adenosylmethionine.Utilize the reorganization bacterium to produce S-adenosylmethionine and mainly contain dual mode, a kind of is directly to utilize the reorganization bacterium to come fermentative production SAM.Also having a kind of is to extract the S-adenosylmethionine synthetic enzyme, at the synthetic S-adenosylmethionine of external use enzymatic method.A kind of method biggest advantage in back is that separation and purification is simple, pollutes and lacks, but because the ATP price that external source is added is higher, make cost rise.So from industrialized angle, preceding a kind of method is more suitable for large-scale production.
Abroad since the mid-90 just the research of recombination engineering bacterium expression S-adenosylmethionine synthetic enzyme.Pajares has made up the recombination bacillus coli of expressing rat liver S-adenosylmethionine synthetic enzyme.They are cloned into pSSRL with the cDNA of the coding rat liver S-adenosylmethionine synthetic enzyme of one section 1.2Kb, are inserted in the T7-7 expression vector that contains the T7 promotor, change host bacterium E.coli BL21 (DE3) over to.After IPTG induced 3h, the output of endobacillary S-adenosylmethionine rate ratio E.coli BL21 (DE3) itself increased, yet still can not reach the required level of producing.
It is in recent years, first-class among Yuan that (patent No. CN 1357630A CN1385537A) is cloned into pPIC3.5K and pGAPZ α A respectively with the sam2 gene in yeast saccharomyces cerevisiae source respectively, transforms pichia spp GS115, and the output of optimizing back SAM is respectively 1.58 and 2.49g/L.
Yet this area also needs further to improve the activity and the S-adenosylmethionine synthesis capability of S-adenosylmethionine synthetic enzyme, in the hope of increasing substantially the output of S-adenosylmethionine, advances S-adenosylmethionine large-scale industrial production process.
Summary of the invention
The object of the present invention is to provide a kind of new S-adenosylmethionine synthetic enzyme and application thereof.
In a first aspect of the present invention, improve a kind of S-adenosylmethionine synthetase albumen, its enzymic activity surpasses 25U/mg (as 25-40U/mg), and forming the needed enzyme amount of 1 μ mol S-adenosylmethionine with 37 ℃ of following reactions catalysis in 60 minutes is that 1U calculates.
In another preference of the present invention, the L-methionine(Met) of described albumen in host cell to the transformation efficiency of S-adenosylmethionine meets or exceeds 35% (as 35-60%, preferred as 40-50%) in the L-methionine(Met).
In another preference of the present invention, the output that contains S-adenosylmethionine under the bacterial strain that is integrated with this protein coding gene in the bacterial strain of the carrier that carries described protein coding gene or the genome adds the L-methionine(Met) in external source the condition surpasses 9g/L (as 9-20g/L).
In another preference of the present invention, the aminoacid sequence of described S-adenosylmethionine synthetase albumen sports V corresponding to the 337th of SEQ ID NO:2 aminoacid sequence by I.
In another preference of the present invention, also comprise: the aminoacid sequence of described S-adenosylmethionine synthetase albumen sports E corresponding to the 5th of SEQ ID NO:2 aminoacid sequence by K.
In another preference of the present invention, described albumen is:
(a) has the albumen of the aminoacid sequence shown in the SEQ ID NO:4; Or
(b) by the aminoacid sequence shown in (a) through replacement, lack or add one or several amino acid and S-adenosylmethionine synthase activity surpass 25U/mg by (a) deutero-albumen.
In a second aspect of the present invention, a kind of isolating polynucleotide are provided, these polynucleotide are selected from down group:
(i) polynucleotide of the described S-adenosylmethionine synthetase albumen of coding; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
In another preference of the present invention, described polynucleotide obtain by DNA Shuffl ing technology.
In another preference of the present invention, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:4.
In another preference of the present invention, these polynucleotide have the nucleotide sequence shown in the SEQ ID NO:3.
In a third aspect of the present invention, a kind of carrier is provided, it contains described polynucleotide.
In another preference of the present invention, described carrier is a methyl alcohol nutritional type recombinant yeast expression vector.
In a fourth aspect of the present 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 of the present invention, contain the described polynucleotide of multiple copied in the described host cell.
In another preference of the present invention, described host cell expression:
The S-adenosylmethionine synthetic enzyme, and/or
S-adenosylmethionine.
In another preference of the present invention, described host cell is a methyl alcohol nutritional type recombination yeast.
Preferably, described host cell is selected from recombinant yeast pichia pastoris (Pichia), reorganization debaryomyces hansenii (Hansenula), reorganization candiyeast (Candida) or reorganization torulopsis (Torulopsis).Preferred, described host cell is recombinant yeast pichia pastoris (Pichia).
In a fifth aspect of the present invention, a kind of method of producing the S-adenosylmethionine synthetic enzyme is provided, comprise step:
(1) cultivates described host cell, obtain culture; With
(2) from culture, separate the S-adenosylmethionine enzyme.
In a sixth aspect of the present invention, a kind of method of producing S-adenosylmethionine is provided, described method comprises step:
(S1) in the presence of the S-adenosylmethionine synthetic enzyme, make L-methionine(Met) and ATP reaction, obtain reaction product;
(S2) from reaction product, isolate S-adenosylmethionine.
In another preference of the present invention, in step (S1), described being reflected in the cell carried out, and cultivates described host cell in the presence of the L-methionine(Met) that is:, makes it to produce S-adenosylmethionine.
In another preference of the present invention, described method comprises step:
Described host cell is added in the fermention medium glycerine OD in the control substratum
600=120-600 added methyl alcohol 2-6g/L/ hour, added the L-methionine(Met) 0.5-2g/L/ days, collected S-adenosylmethionine in 3-6 days (preferred, as to be 4 days) back.
In another preference of the present invention, described substratum is selected from: BSM nutrient solution or BMMY nutrient solution.
In another preference of the present invention, culture temperature is 25-35 ℃.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
That Fig. 1 is shown is the result who utilizes PCR method amplification coding S-adenosylmethionine synthase gene from total DNA from three kinds of different sources microorganisms.Swimming lane 1 is represented DL15000; Swimming lane 2 is represented E.colisam2; Swimming lane 3 is represented Streptomyces spectabilis; Swimming lane 4 is represented Saccharomycescerevisiae sam2.
What Fig. 2 showed is the plasmid map of expression plasmid pPIC3.5K-SAMrm.
That Fig. 3 is shown is the SDS-PAGE of restructured Pichia pastoris in expression S-adenosylmethionine synthetic enzyme.Wherein, swimming lane 1Marker; Swimming lane 2 is not induced; Swimming lane 3 is induced 8hr; Swimming lane 4 is induced 16hr; Swimming lane 5 is induced 24hr; Swimming lane 6 is induced 32hr; Swimming lane 7 is induced 40hr; Swimming lane 8 is induced 48hr; Swimming lane 9 is induced 56hr.
What Fig. 4 was shown is to utilize HPLC to measure the collection of illustrative plates (retention time 26.944min) of S-adenosylmethionine content.Wherein, Fig. 4 A is a SAM mark product 0.6g/L peak area 3800; Fig. 4 B is reorganization bacterium fermented sample (diluting 4 times) 10.5g/L peak area 16700; Fig. 4 C is a GS115 fermented sample 0.11g/L peak area 710.
Embodiment
The inventor is through long term studies, obtained a S-adenosylmethionine synthetic enzyme that can make bacterial strain mass production S-adenosylmethionine under the condition of external source interpolation L-methionine(Met), the enzyme of described S-adenosylmethionine synthetic enzyme is lived and is surpassed the 25U/mg substratum.Can make the output of the S-adenosylmethionine of bacterial strain surpass 9g/L, and L-methionine(Met) to the transformation efficiency of S-adenosylmethionine meet or exceed 35%.Finished the present invention based on this.
As used herein, described " S-adenosylmethionine synthase activity " is that the unit of activity with enzyme defines.Reaction catalysis in 60 minutes formed the needed enzyme amount of 1 μ mol SAM under a unit of activity (1U) of enzyme was defined as 37 ℃.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating S-adenosylmethionine synthetase albumen or polypeptide " is meant that the S-adenosylmethionine synthetase albumen is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying S-adenosylmethionine synthetase albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention 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 to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).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, derivative and the analogue of S-adenosylmethionine synthetase albumen.As used herein, term " fragment ", " derivative " are meant biological function or the active polypeptide that keeps natural S-adenosylmethionine synthetase albumen of the present invention identical basically 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 (preferred conservative amino acid residue), and the amino-acid residue of such replacement 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 (as 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, derivative and analogue according to this paper belong to the known scope of those skilled in the art.
In the present invention, term " S-adenosylmethionine synthetase albumen " refers to have the active SEQ ID of S-adenosylmethionine synthetase albumen NO:4 polypeptide of sequence.This term also comprises having and variant form S-adenosylmethionine synthetase albumen identical function, SEQ ID NO:4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 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 at C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is in 10, more preferably is in 5) amino acid.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 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 S-adenosylmethionine synthetase albumen.Best, in the variant form of these SEQ ID NO:4, the 5th amino acid is that E or the 337th amino acids are V.Preferred, the variant form of these SEQ ID NO:4 satisfied the 5th amino acid simultaneously is that E, the 337th amino acids are V.
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 S-adenosylmethionine synthetase albumen DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-S-adenosylmethionine synthetase albumen to obtain.The present invention also provides other polypeptide, as comprises S-adenosylmethionine synthetase albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of S-adenosylmethionine synthetase albumen.Usually, this fragment have S-adenosylmethionine synthetase albumen 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 S-adenosylmethionine synthetase albumen or polypeptide.The difference of these analogues and natural S-adenosylmethionine synthetase albumen 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 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 non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(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 (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, " S-adenosylmethionine synthetase albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ IDNO:4, there are 10 at the most, 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 S-adenosylmethionine synthetase albumen 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:4, 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:4 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 derivative 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 may 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 of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 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:4.
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 (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding S-adenosylmethionine synthetase albumen.
S-adenosylmethionine synthetase albumen Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic 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 needs to carry 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 obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by 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) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by 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 S-adenosylmethionine synthetase albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to express or produce the S-adenosylmethionine synthetase albumen of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding S-adenosylmethionine synthetase albumen of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, S-adenosylmethionine synthetase albumen 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 virus or other carriers.In a word, as long as can duplicate in host 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 S-adenosylmethionine synthetase albumen 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 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 as 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, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell such as 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 (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be 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 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 handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
As an example of the present invention, a kind of S-adenosylmethionine synthase gene is provided, its sequence is as described in the SEQ ID NO:3, and its encoded protein is shown in SEQ ID NO:4.
The inventor goes up the S-adenosylmethionine synthetic enzyme E.coli sam2 (Genbank accession number AE000377) of 3 kinds of different sourcess announcing according to ncbi (http://www.ncbi.nlm.nih.gov/), Streptomyces spectabilis (Genbank accession number AF117274), the dna sequence dna of coding S-adenosylmethionine synthetic enzyme Saccharomycescerevisiae sam2 (Genbank accession number M23368)) has designed primer, from its total DNA, amplify the DNA of required coding S-adenosylmethionine synthetic enzyme, obtain the gene of the coding S-adenosylmethionine synthetic enzyme of a series of reorganization by DNA Shuffling technology, and be cloned on the expression vector, made up the expression plasmid of reorganization S-adenosylmethionine synthase gene.After described expression plasmid linearizing, electricity transforms pichia spp GS115, passes through His
+And the recombinant bacterial strain of G418 screening high yield S-adenosylmethionine.And from superior strain, clone the SAM synthase gene and check order, checking obtains to have high enzymic activity and S-adenosylmethionine synthetic enzyme that can impel bacterial strain high yield S-adenosylmethionine.
DNA Shuffling technology is one group of gene colony (dna sequence dna of being correlated with in the evolution or the improvement in performance sequence that once filtered out) to be recombinated create the method for new gene.Because this method also may be introduced point mutation in the dna fragmentation assembling process, so it also is effective to instruct evolution protein from unique sequence.The diverse libraries that the DNAShuffling technology produces can effectively accumulate useful sudden change, gets rid of detrimental mutation and neutral mutation, also can realize the coevolution of target protein multifrequency nature simultaneously.Comprised the process that DNA re-assemblies just because of this technology, made it the different of matter arranged with in the past induced-mutation technique.
In an example of the present invention, make the superior strain that carries described S-adenosylmethionine synthetic enzyme in the 5L fermentor tank, carry out fermenting experiment, the output of S-adenosylmethionine is (other S-adenosylmethionine output is many about 5g/L) more than the 9g/L, the L-methionine(Met) can meet or exceed 35% to the transformation efficiency of S-adenosylmethionine, existing head and shoulders above level (about 20%).
In a preferred examples of the present invention, utilize widely used methyl alcohol nutritional type expression of recombinant yeast system, structure contains the methyl alcohol nutritional type recombination yeast of reorganization S-adenosylmethionine synthase gene of the present invention, and add substrate L-methionine(Met) by external source and make this reorganization bacterium High-efficient Production S-adenosylmethionine, solve yielding poorly of present S-adenosylmethionine production existence, the cost height, the problem that substrate conversion efficiency is low.
When express producing S-adenosylmethionine, described S-adenosylmethionine gene can be imported to and anyly can add the L-methionine(Met) by external source and produce in the bacterial strain of S-adenosylmethionine by bacterial strain.Preferably, described bacterial strain is for adopting alcohol oxidase promotor PAOX1, PAOX2 methyl alcohol nutritional type yeast.Characteristics such as methyl alcohol nutritional type recombination yeast is a kind of expression system commonly used, has the output height, and is with low cost.
Preferred, described bacterial strain such as pichia spp (Pichia), debaryomyces hansenii (Hansenula), candiyeast (Candida) or torulopsis (Torulopsis) etc., this recombination yeast can adopt different expression vector (as pPIC3.5K etc.) when making up, and carrier can have different existing waies (as additive type, integrated etc.) in yeast, and this bacterium can possess different phenotype (Mut
+, Mut
sDeng).
As another example of the present invention, produce the S-adenosylmethionine synthetic enzyme by genetic engineering means, such as utilizing any suitable genetic engineering bacterium to produce described S-adenosylmethionine synthetic enzyme, separate described S-adenosylmethionine synthetic enzyme.Described S-adenosylmethionine synthetic enzyme is used for external enzymatic method produces S-adenosylmethionine.Because S-adenosylmethionine synthetic enzyme of the present invention has very high enzymic activity, the scale that makes enzymatic method produce S-adenosylmethionine increases greatly, and efficient improves greatly.
Major advantage of the present invention is:
Obtain an adenosine methilanin synthase that can make bacterial strain mass production S-adenosylmethionine under the condition of external source interpolation L-methionine(Met) first, the enzyme of described-adenosine methilanin synthase is lived and is surpassed 25U/mg, and L-methionine(Met) to the transformation efficiency of S-adenosylmethionine meets or exceeds 35%.
S-adenosylmethionine production method of the present invention is compared with existing other modes, (1) output height; (2) raw materials cost is low; (3) technology is easy.Therefore reduced production cost significantly, and be more suitable for producing S-adenosylmethionine in large-scale industrialization.
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 and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1 carries the structure of the expression plasmid of reorganization S-adenosylmethionine synthetic enzyme
Adopt conventional method, extract E.coli JM109 (available from invitrogen company) respectively, Streptomyces spectabilis (available from invitrogen company), the genomic dna of Saccharomycescerevisiae (available from invitrogen company), with described genomic dna is that template is carried out PCR, designed primer such as table 2.
Table 2
PCR obtains being about the dna sequence dna of 1.2Kb, and electrophoresis result is seen shown in Figure 1.After reclaiming with ethanol sedimentation after phenol-chloroform extracting, be cloned in the multiple clone site of pGM-T carrier (is Time Inc. available from the sky) (condition of contact is with reference to this product description) carrier construction pGM-T-SAMsp1, pGM-T-SAMsp2, pGM-T-SAMsp3.
Gene segment with the coding S-adenosylmethionine synthetic enzyme on above-mentioned pGM-T-SAMsp1, pGM-T-SAMsp2, the pGM-T-SAMsp3 under the EcoRI/NotI double digestion, with DNase I it is degraded to small segment, carry out self primer PCR (DNA Shuffling), from self primer PCR product, select the fragment about 1.2Kb, be cloned into once more on the pGM-T carrier, obtain carrier pGM-T-SAMrm.
Then, with EcoR I/Not I double digestion pGM-T-SAMrm carrier, reorganization S-adenosylmethionine gene clone on the pGM-T-SAMrm carrier between the EcoR I and Not I site of Pichia anomala expression plasmid pPic3.5K, is obtained recombinant plasmid pPIC3.5K-SAMrm.Wherein, the plasmid map of the pPic3.5K of reorganization is seen shown in Figure 2.
The structure of embodiment 2 genetically engineered recombinant yeast pichia pastoris
Constructed recombinant plasmid pPIC3.5K-SAMrm is carried out linearizing with Bgl II enzyme, after phenol-chloroform extracting, reclaim the linearization plasmid segment with ethanol sedimentation.
Linearization plasmid segment after purifying reclaims is changeed pichia spp GS115 (available from invitrogen company) according to the electric method for transformation electricity on the Multi-copy PichiaExpression Kit specification sheets of Invitrogen company, and the condition that electricity changes is carried out according to the default Pichiapastors program of the MicroPulser electroporation of Bio-RAD company.
Electricity changes the bacterium liquid that obtains and is coated on MD (1.34%YNB, 4 * 10
-5Vitamin H (biotin), 2% glucose) cultivated screening-gene type His 3-4 days in 30 ℃ on the flat board
+Recon, the recon that obtains obtains the recon GS115/pPIC3.5K-SAMrm that multiple copied (as the 1-3 copy) gene inserts according to the screening of the G418 screening method on the Multi-copy Pichia Expression Kit specification sheets of Invitrogen company again, and the pPIC3.5K plasmid that will not contain foreign gene changes GS115 over to according to identical method, structure contrast bacterium GS115/pPIC3.5K.
Screening and the checking of embodiment 3 S-adenosylmethionine High-efficient Production bacterium
The genetically engineered recombinant yeast pichia pastoris mono-clonal access of the anti-1.0-1.5mg/ml G418 of energy is contained 25mlBMGY substratum (1% yeast extract (Yeast extract), 2% peptone (peptone), 0.2M PBSpH=6.0,1.34%YNB, 4 * 10
-5Vitamin H (biotin), 1% glycerine) 250ml shakes bottle and ferments 30 ℃ of culture temperature, rotating speed 220rpm.
The centrifugal collection thalline of 5000rpm after 24 hours that ferments inserts according to the ratio of 1:1 and to contain 25ml BMMY substratum (1% yeast extract (Yeast extract), 2% peptone (peptone), 0.2M PBS pH=6.0,1.34%YNB, 4*10
-5Vitamin H (biotin), 0.5% methyl alcohol) 250ml shakes and carries out abduction delivering in the bottle, and mends every day into the L-methionine(Met) of 0.5-1g/L and the methyl alcohol of 0.6-1.2%.Induce centrifugal collection thalline after 4 days, supernatant discarded adds the S-adenosylmethionine of isopyknic trichoroacetic acid(TCA) in 4 ℃ of extracting born of the same parents.Measure the content of S-adenosylmethionine after the centrifugal removal of extract precipitates with HPLC, therefrom screening obtains superior strain, and by the expression that SDS-PAGE checks the S-adenosylmethionine synthetic enzyme, the results are shown in shown in Figure 3.
HPLC condition: HPLC instrument: Agilent 1100 series; Separator column: Hypersil SCX 5 μ m 25cm; Moving phase: 0.5mol/L ammonium formiate (transferring to pH4.0) with formic acid; Detect wavelength: 254nm; Retention time: about 27min.The HPLC spectrogram as shown in Figure 4.
Therefrom screening obtains the bacterial strain that a strain S-adenosylmethionine output reaches 1.4g/L (shake a bottle output, calculate by pure SAM content), utilizes conventional pcr clone to go out the SAM synthase gene, has obtained the sequence of new gene by order-checking.This gene is compared with the S-adenosylmethionine synthase gene that derives from Saccharomyces cerevisiae, there are 3 sites that sudden change has taken place, i.e. the 9th G → A, the 13rd A → G, the 1009th A → G, caused encoded protein matter to have 2 places to undergo mutation thus, i.e. the 5th K → E, the 337th I → V.
The S-adenosylmethionine synthetase gene sequence that derives from Saccharomyces cerevisiae is shown in SEQ ID NO:1:
atgtccaaga gcaaaacttt cttatttacc tctgaatccg tcggtgaagg tcacccagac 60
aagatttgtg accaagtttc tgatgctatt ttggacgctt gtttagaaca agatccattc 120
tccaaggttg cctgtgaaac agctgccaaa actggtatga ttatggtttt cggtgaaatt 180
accaccaaag ctagacttga ctaccaacaa atagtaagag ataccatcaa gaagattggt 240
tatgacgatt ctgccaaggg tttcgactac aagacatgta atgttttagt agctatcgaa 300
caacaatctc cagatatcgc tcaaggtctg cactatgaaa agagcttaga agacttaggt 360
gctggtgacc aaggtataat gtttggttac gctacagacg aaactccaga agggttacca 420
ttgaccattc ttttggctca caaattgaac atggctatgg cagatgctag aagagatggt 480
tctctcccat ggttgagacc agacacaaag actcaagtca ctgtcgaata cgaagacgac 540
aatggtagat gggttccaaa gaggatagat accgttgtta tttctgctca acatgctgat 600
gaaatttcca ccgctgactt gagaactcaa cttcaaaaag atattgttga aaaggtcata 660
ccaaaggata tgttagacga aaataccaaa tatttcatcc aaccatccgg tagattcgtc 720
atcggtggtc ctcaaggtga cgctggtttg accggtagaa agattattgt cgacgcttac 780
ggtggtgcct catccgtcgg tggtggtgcc ttctccggta aggactattc caaggtcgat 840
cgttccgctg cttacgctgc tagatgggtt gccaagtctc tagttgccgc tggtttgtgt 900
aagagagtcc aagtccaatt ttcatatgct attggtattg ctgaaccatt gtctttacat 960
gtggacacct atggtacagc tacaaaatca gatgacgaaa tcattgaaat tattaagaag 1020
aacttcgact tgagaccagg tgtgttagta aaggaattag atttggctag accaatttac 1080
ttaccaaccg cttcttatgg tcacttcact aatcaagagt actcatggga aaaaccaaag 1140
aaattggaat tt 1152
Protein sequence (SEQ ID NO:2):
MSKSKTFLFT SESVGEGHPD KICDQVSDAI LDACLEQDPF SKVACETAAK TGMIMVFGEI 60
TTKARLDYQQ IVRDTIKKIG YDDSAKGFDY KTCNVLVAIE QQSPDIAQGL HYEKSLEDLG 120
AGDQGIMFGY ATDETPEGLP LTILLAHKLN MAMADARRDG SLPWLRPDTK TQVTVEYEDD 180
NGRWVPKRID TVVISAQHAD EISTADLRTQ LQKDIVEKVI PKDMLDENTK YFIQPSGRFV 240
IGGPQGDAGL TGRKIIVDAY GGASSVGGGA FSGKDYSKVD RSAAYAARWV AKSLVAAGLC 300
KRVQVQFSYA IGIAEPLSLH VDTYGTATKS DDEIIEIIKK NFDLRPGVLV KELDLARPIY 360
LPTASYGHFT NQEYSWEKPK KLEF 384
S-adenosylmethionine synthetase gene sequence of the present invention is shown in SEQ ID NO:3:
atgtccaaaa gcgaaacttt cttatttacc tctgaatccg tcggtgaagg tcacccagac 60
aagatttgtg accaagtttc tgatgctatt ttggacgctt gtttagaaca agatccattc 120
tccaaggttg cctgtgaaac agctgccaaa actggtatga ttatggtttt cggtgaaatt 180
accaccaaag ctagacttga ctaccaacaa atagtaagag ataccatcaa gaagattggt 240
tatgacgatt ctgccaaggg tttcgactac aagacatgta atgttttagt agctatcgaa 300
caacaatctc cagatatcgc tcaaggtctg cactatgaaa agagcttaga agacttaggt 360
gctggtgacc aaggtataat gtttggttac gctacagacg aaactccaga agggttacca 420
ttgaccattc ttttggctca caaattgaac atggctatgg cagatgctag aagagatggt 480
tctctcccat ggttgagacc agacacaaag actcaagtca ctgtcgaata cgaagacgac 540
aatggtagat gggttccaaa gaggatagat accgttgtta tttctgctca acatgctgat 600
gaaatttcca ccgctgactt gagaactcaa cttcaaaaag atattgttga aaaggtcata 660
ccaaaggata tgttagacga aaataccaaa tatttcatcc aaccatccgg tagattcgtc 720
atcggtggtc ctcaaggtga cgctggtttg accggtagaa agattattgt cgacgcttac 780
ggtggtgcct catccgtcgg tggtggtgcc ttctccggta aggactattc caaggtcgat 840
cgttccgctg cttacgctgc tagatgggtt gccaagtctc tagttgccgc tggtttgtgt 900
aagagagtcc aagtccaatt ttcatatgct attggtattg ctgaaccatt gtctttacat 960
gtggacacct atggtacagc tacaaaatca gatgacgaaa tcattgaagt tattaagaag 1020
aacttcgact tgagaccagg tgtgttagta aaggaattag atttggctag accaatttac 1080
ttaccaaccg cttcttatgg tcacttcact aatcaagagt actcatggga aaaaccaaag 1140
aaattggaat tt 1152
Protein sequence (SEQ ID NO:4):
MSKSETFLFT SESVGEGHPD KICDQVSDAI LDACLEQDPF SKVACETAAK TGMIMVFGEI 60
TTKARLDYQQ IVRDTIKKIG YDDSAKGFDY KTCNVLVAIE QQSPDIAQGL HYEKSLEDLG 120
AGDQGIMFGY ATDETPEGLP LTILLAHKLN MAMADARRDG SLPWLRPDTK TQVTVEYEDD 180
NGRWVPKRID TVVISAQHAD EISTADLRTQ LQKDIVEKVI PKDMLDENTK YFIQPSGRFV 240
IGGPQGDAGL TGRKIIVDAY GGASSVGGGA FSGKDYSKVD RSAAYAARWV AKSLVAAGLC 300
KRVQVQFSYA IGIAEPLSLH VDTYGTATKS DDEIIEVIKK NFDLRPGVLV KELDLARPIY 360
LPTASYGHFT NQEYSWEKPK KLEF 384
The S-adenosylmethionine rate ratio that experiment showed, the superior strain that carries described new gene utilizes the production bacterial strain of 3 kinds of S-adenosylmethionine synthase genes structures that set out to exceed 2-8 doubly.
The mensuration that embodiment 4 enzymes are lived
The S-adenosylmethionine synthetic enzyme that embodiment 3 is obtained carries out enzyme activity determination, and compares with other S-adenosylmethionine synthetic enzyme.
1. cytoclasis
Fermented liquid centrifugal (3000 * 10min, 4 ℃) is collected thalline, and (potassium phosphate buffer that contains 50mMpH7.4,5% glycerine ((v/v), the coloured glaze base ethanol of 5mM and the EDTA of Imm) cleans once with lysis buffer.Add the long-pending pickling glass pearl (diameter 0.1mm) of thalline monoploid and the lysis buffer of 4 times of volumes, handled lysing cell 10 minutes in 0 ℃ with ultrasonic wave (in maximum output, 50% work period).4 ℃ of following 10000g of granulated glass sphere and cell relic removed in centrifugal 15 minutes.Crude extract is after 12000g * 30min is centrifugal, and supernatant liquor is acellular enzyme liquid.
2. the foundation of reaction system
Set the reaction mixture of 1mL, wherein contain the L-methionine(Met) of 20mM, the ATP of 20mM, the reduced glutathion of 8mM (GSH), the MgCL of 20mM
2, the KCL of 100mM, the Tris-HCL of the pH7.4 of 150mM and the cell pyrolysis liquid of 0.5mL were 37 ℃ of incubations 60 minutes.The reaction that does not add the L-methionine(Met) is as blank.After reaction finishes, add the 20% perchloric acid solution stopped reaction of 0.5mL.Centrifugal, remove precipitation, the supernatant of gained is analyzed the content of SAM by HPLC.A unit of activity of enzyme is that reaction catalysis in 60 minutes forms the needed enzyme amount of 1 μ mol SAM under 37 ℃.
Found that the vigor of the S-adenosylmethionine synthetic enzyme of superior strain of the present invention has reached 28.45U/mg, and the vigor of the production bacterial strain of 3 kinds of S-adenosylmethionine synthase gene structures that set out is respectively:
E.coli sam2：10.31U/mg；
Streptomyces spectabilis sam2：8.64U/mg；
Saccharomyces cerevisiae sam2：20.24U/mg。
Therefore as seen, the superior strain Billy that the present invention makes up is significantly increased with the S-adenosylmethionine synthetic enzyme vigor of the production bacterial strain of 3 kinds of S-adenosylmethionine synthase genes structures that set out, and has also improved more than 40% than the enzyme activity of Saccharomyces cerevisiae sam2.
1. adopt the fermentation of GS115 bacterial strain
The high yield restructuring yeast strains of gained among the embodiment 3 inserted by 5% inoculum size contain 2L BSM and (contain 0.93g/L CaSO
4, 18.2g/L K
2SO
4, 4% glycerine (glycerol), 4.13g/L KOH, 26.7ml/L85% H
3PO
4, 14.9g/L MgSO
47H
2O, 12ml/L PTM1,2ml steeps the enemy) carry out the fed-batch high density fermentation in the 5L jar of substratum, at first wait to begin to flow glycerol adding to OD after glycerine exhausts in the basestocks
600=120-600, methanol induction then, benefit rate 2-6g/L/hr, and add 0.5-2g/L L-methionine(Met) every day.
After inducing 4 days, after measured, the output of S-adenosylmethionine reaches more than the 9g/L, and the L-methionine(Met) is 40% to the S-adenosylmethionine transformation efficiency.
2. adopt the fermentation of SMD1163 bacterial strain
Adopt aforementioned identical method, the inventor imports SMD1163 yeast (pichia spp a kind of, protease-deficient with aforementioned S-adenosylmethionine synthase gene; Available from invitrogen company) in.
As a result, the S-adenosylmethionine output of the bacterial strain of acquisition reaches 9.54g/L, and the L-methionine(Met) reaches 35% to the S-adenosylmethionine transformation efficiency.
And under the precursor of the fermentation of adopting the SMD1163 bacterial strain, the enzyme that inventor's employing has been measured the S-adenosylmethionine synthetic enzyme as embodiment 4 described enzyme activity determination methods is lived, and compares with other S-adenosylmethionine synthetic enzyme.
Found that the vigor of the S-adenosylmethionine synthetic enzyme of superior strain of the present invention has reached 27.20U/mg, be significantly higher than the vigor of the production bacterial strain of 3 kinds of S-adenosylmethionine synthase genes structures that set out.
Therefore as seen, the superior strain Billy of the present invention's structure is significantly increased with the S-adenosylmethionine synthetic enzyme vigor of the production bacterial strain of 3 kinds of S-adenosylmethionine synthase genes structures that set out.
The varient of embodiment 6 S-adenosylmethionine synthetic enzyme of the present invention
1. varient 1
In addition, in as embodiment 3 described screenings, the inventor has also obtained the high-yield bacterial strain of a strain S-adenosylmethionine, utilizes conventional pcr clone to go out the SAM synthase gene, has obtained the sequence of another new gene by order-checking.This gene is compared with the sequence shown in the SEQ ID NO:1, at the 1009th A → G sudden change takes place, and has caused its encoded protein matter to have 1 place to undergo mutation thus, i.e. the 337th I → V.
This gene is expressed in GS115, be 23.17U/mg, output 9.21g/L on the 5L jar than living.
2. varient 2
The inventor has carried out rite-directed mutagenesis with the new S-adenosylmethionine synthase gene (SEQ ID NO:3) that embodiment 3 obtains, and causes the 33rd A variation of its amino acids coding to be V.Afterwards, adopt foregoing method, the gene behind this rite-directed mutagenesis is imported among the GS115, measure its enzyme work and L-methionine(Met) transformation efficiency to S-adenosylmethionine.
Found that significant change does not take place for the enzymic activity of the S-adenosylmethionine synthase gene after process makes a variation and transformation efficiency, enzyme about 28U/mg alive, transformation efficiency 40%.
Embodiment 7 enzymatic methods are produced S-adenosylmethionine
Adopt conventional external enzymatic reaction condition, the inventor utilizes described S-adenosylmethionine synthetic enzyme, and catalytic substrate L-methionine(Met) and ATP generate (-) type S-adenosylmethionine, and enzymatic reaction is as follows:
Found that the yield of S-adenosylmethionine is very high.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition 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 institute restricted portion equally.
Sequence table
＜110〉East China University of Science
＜120〉a kind of S-adenosylmethionine is produced the structure and the large scale fermentation of bacterial strain
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Claims (7)
1. a S-adenosylmethionine synthetase albumen is characterized in that, its enzymic activity surpasses 25U/mg, and forming the needed enzyme amount of 1 μ mol S-adenosylmethionine with 37 ℃ of following reactions catalysis in 60 minutes is that 1U calculates; This albumen is:
(a) albumen of the aminoacid sequence shown in SEQ ID NO:4; Or
(b) by the aminoacid sequence shown in (a) through replacement, disappearance or add 1-5 amino acid and the S-adenosylmethionine synthase activity above 25U/mg by (a) deutero-albumen; And the 337th of this proteic aminoacid sequence is V, and the 5th is E.
2. isolating polynucleotide is characterized in that, these polynucleotide are selected from down group:
(i) polynucleotide of the described S-adenosylmethionine synthetase albumen of coding claim 1; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
3. polynucleotide as claimed in claim 2 is characterized in that, the polypeptide of this polynucleotide encoding aminoacid sequence shown in SEQ IDNO:4.
4. a carrier is characterized in that, it contains the described polynucleotide of claim 2.
5. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 4, or is integrated with the described polynucleotide of claim 2 in the genome.
6. a method of producing the S-adenosylmethionine synthetic enzyme is characterized in that, comprises step:
(1) cultivates the described host cell of claim 5, obtain culture; With
(2) from culture, separate the S-adenosylmethionine enzyme.
7. a method of producing S-adenosylmethionine is characterized in that, described method comprises step:
(S1) in the presence of the described S-adenosylmethionine synthetase albumen of claim 1, make L-methionine(Met) and ATP reaction, obtain reaction product;
(S2) from reaction product, isolate S-adenosylmethionine.
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| 酿酒酵母S-腺苷甲硫氨酸合成酶基因的克隆及其在大肠杆菌中的表达. 杨静等.中国药科大学学报，第33卷第3期. 2002 |
| 酿酒酵母S-腺苷甲硫氨酸合成酶基因的克隆及其在大肠杆菌中的表达. 杨静等.中国药科大学学报，第33卷第3期. 2002 * |
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