CN100577805C

CN100577805C - Method of modifying gene and obtained recombinant gene and encoded protin

Info

Publication number: CN100577805C
Application number: CN200510046033A
Authority: CN
Inventors: 吴文芳; 安迎锋; 吕安国
Original assignee: Institute of Applied Ecology of CAS
Current assignee: Institute of Applied Ecology of CAS
Priority date: 2005-03-16
Filing date: 2005-03-16
Publication date: 2010-01-06
Anticipated expiration: 2025-03-16
Also published as: CN1834248A

Abstract

This invention relates to a method for gene directed evolution, specifically, a method for gene recombination and application. The method comprises the steps of: dividing the reaction into two groups, performing recombination, mixing the two reactants, and performing a series of PCR reactions to realize gene recombination. The sequencing results show that the method is better than previous gene recombination methods, and is especially suitable for recombination of genes with low sequence identity. The effectivity of the method is testified by using adenosylmethionine synthetase genes from Escherichia coli and yeast as the starting genes, and can obtain high activity recombined enzyme.

Description

A kind of method and recombination that is obtained and proteins encoded of gene reorganization

Technical field

The present invention relates to the method that gene instruct to be evolved, a kind of specifically gene reorganization method and application thereof are a kind of methods of reorganizing between the lower gene of homology of being specially adapted to.

Background technology

Instructing evolves is exactly the special evolution conditions of artificial creation in the laboratory, simulating nature evolutionary mechanism (as sudden change, reorganization), and orthoselection goes out the protein molecular of required character and does not need to understand proteinic space structure and function.This mechanism has been widely used in pharmaceutical grade protein, agricultural, chemical industry, fields such as biotechnology (Patten, P.A., Howard, R.J.﹠amp at present; Stemmer, W.P.C.Applications of DNA shuffling to pharmaceuticals and vaccines.Curr Opin Biotechnol.1997,8:724-733) particularly be applied to improving the activity (Crameri of enzyme, A., Raillard, S-A., Bermudez, E.﹠amp; Stemmer, W.P.C.DNAshuffling of a family of genes from diverse species acceleratesdirected evolution.Nature.1998,391,288-291.) stability (Oh, K.H., Nam, S.H.﹠amp; Kim, H.S.Improvement of Oxidative and Thermostabilityof N-Carbamyl-D-Amino Acid Amidohydrolase by Directed Evolution.Protein Eng.2002,15,689-695.), and expression level (Bulter, T.et.al.Functional expression of a fungal laccase in Saccharomycescerevisiae by directed evolution.Appl.Environ.Microbiol.2003,69,987-995.).Along with the appearance of various novel mutation technology and novel detection system, for instructing the research of evolving to develop in depth.Say that to a certain extent the elementary tactics of orthogenesis is with evolution is quite similar naturally, two critical step are exactly the mutant that produces molecular diversity and be optimized by directed screening or selection by random mutagenesis and vitro recombination.At present there has been the outer reorganization method of a variety of genosomes to be suggested and successful application, wherein first also be the most classical be DNAshuffling technology (Stemmer, W.P.C.Rapid evolution of a protein in vitroby DNA shuffling.Nature 1994,370,389-391.), this technology is proposed in 1994 by U.S. Stemmer, it is that 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) is reorganized, and creates the method for the gene that makes new advances.The diverse libraries that this method 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.But when homology was lower than 70% sequence and carries out gene reorganization with this method, product often seriously tended to form the chain that regroups of parent's chain.Although more after this depend on recombination method (Ostermeier, M., Shim, the J.H.﹠amp of sequence homology; Benkovic, S.J.A combinatorial approach to hybridenzymes independent of DNAhomology.Nat.Biotechnol.1999,17,1205-1209.; Zhao, H., Giver, L., Shao, Z.﹠amp; Arnold, F.H.Molecular evolution by staggeredextension process (StEP) in vitro recombination.Nat.Biotechnol.1998,16,258-261.; Jon, E.et al.Synthetic shuffling expandsfunctional protein diversity by allowing amino acids to recombineindependently.Nat.Biotechnol.2002,20,1251-1255.) be suggested, but all can not fundamentally address this problem.

Summary of the invention

The objective of the invention is to propose a kind of new gene reorganization method, promptly separation-hybrid system (separating-mixing is called for short SeMi) has effectively reduced the dependence of reorganization process to sequence homology; And provide this method of utilization to produce reorganization gene and the active reorganization albumen that improves.

For achieving the above object, the technical solution used in the present invention is: the present invention has utilized the fragment of cutting through enzyme from the single stranded DNA of the gene homology by its 3 ' end and another gene and the feature of 5 ' end and the identity of autogene, earlier reaction is divided into two groups, each group is earlier through pre-reorganization, again two group reaction things are mixed, through a series of PCR reactions steps, take turns reaction through one and realize intergenic reorganization;

Be specially,

1) chooses two kinds of Linear Double chain gene A and B, the end of gene has respectively can produce the restriction endonuclease recognition sequences (as BglII and PstI etc.) that otch is different viscosity protrusion ends, produces opposite two groups of single stranded DNAs---A ' and the B ' of direction with ExoIII digestion respectively;

2) after digestion, get wherein part respectively, be digested to small segment A with DNaseI " and B ", and carry out purifying;

3) with A ' and B " and A " and B ' mix respectively, form two reaction system I and II, carry out unidirectional PCR then; Subsequently, in the I system, add corresponding A ' the reverse primer of 5 ' end, in the II system, add the reverse primer of the 5 ' end of corresponding B ', oppositely amplification respectively;

4) above-mentioned two individual system are mixed, go out full length fragment through the intussusception pcr amplification; Select the recombination of purpose size, screen and express.

The present invention is a template with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and the total DNA of intestinal bacteria (Escherichia coli) at first respectively, amplification has metk (see figure 1) and the sam2 (see figure 2) gene of EcoRI and NotI respectively, is cloned into the corresponding restriction enzyme site of pSE380 respectively with two.With recombinant plasmid position template, sam2 and metk gene that amplification has BglII and PstI restriction enzyme site respectively and has one section carrier sequence, be above-mentioned two kinds of Linear Double chain gene A and B, it has nucleotide sequence among the sequence table SEQ ID No:1 and the nucleotide sequence among the sequence table SEQ ID No:2 respectively;

With above-mentioned two genes is initial gene, realizes intergenic reorganization through following steps:

1) above two genes are carried out BglII and PstI double digestion, and the fragment after enzyme cut is carried out purifying;

2) with ExoIII digestion (unidirectional enzyme is cut) above-mentioned fragment, to obtain single stranded DNA (ssDNA) and purifying in addition;

3) the single stranded DNA gene of getting 50% total amount of two genes respectively carries out enzyme with DNaseI and cuts, and obtains small segment 80% ethanol sedimentation, reclaims;

4) reaction is divided into two groups, in each group, will mix with total length single stranded DNA from the single stranded DNA fragment of a gene from another gene, and be template with the total length single stranded DNA, by the sequence homology of fragment 3 ' end, the unidirectional template 5 ' end that is expanded under the effect of taq archaeal dna polymerase;

5) add unidirectional primer,, in this process, realize staggered extension by of short duration renaturation/extension by the segmental complementary strand of asymmetric PCR amplification heterozygosis;

6) two group reaction things are mixed, the complimentary piece that direction is opposite is intersegmental will to form the total length two strands in the mode of staggered extension, and realizes amplification in the PCR process;

7) recombination is connected into expression vector and change yeast saccharomyces cerevisiae over to,, obtains the engineering strain of anti-ethionine,, separate obtaining bacterial strain that content improves and the recombination that is comprised thereof by the ademetionine Determination on content by screening.Recombination that is obtained and proteins encoded thereof have nucleotide sequence among the sequence table SEQ ID No:3 and the aminoacid sequence among the sequence table SEQ ID No:4.

The present invention has the following advantages:

1. the present invention provides a new gene reorganization method by the grouping reaction process of uniqueness, and this method is specially adapted to the lower intergenic reorganization of homology;

2. use the present invention to carry out gene reorganization, only take turns reaction and can obtain to reorganize gene by one;

3. the reorganization gene that uses the present invention to obtain is a kind of new recombination, and nucleotide sequence derives from two original genes, and has multiple spot reorganization feature, and this gene can efficiently express in intestinal bacteria;

4. use the recombinant protein that the present invention obtained (energy catalysis ademetionine) to derive from two kinds of initial enzymes respectively; And have two amino acid mutation: 133Ala → Thr, 354Glu → Asp has the adenomethionine synthase activity, and vigor is better than initial enzyme, has higher activity;

5. quote the present invention, can further provide to be directly used in the engineering strain of producing recombinase.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of amplification metk; Lane M:DNA marker DL2000; Lane1: bacillus coli gene group DNA; Lane2:metk.

Fig. 2 is the agarose gel electrophoresis figure of amplification sam2; Lane M:DNA marker DL2000; Lane1: genes of brewing yeast group DNA; Lane2:sam2.

Fig. 3 is the synoptic diagram of reaction process of the present invention; New gene reorganization method: separation-hybrid system; Reaction process is: 1. the DNA of two kinds of different sourcess (the bar shaped post of white is represented metk, and the bar shaped post of black is represented sam2) uses BglII and PstI double digestion respectively; 2. above-mentioned fragment is cut the acquisition single stranded DNA with the unidirectional enzyme of ExoIII respectively; 3. the part single stranded DNA digests with DNaseI; 4. by the unidirectional amplification of no primer PCR; 5. by there being primer PCR to obtain the complementary single stranded DNA; 6. mix the two group reaction products and the full length DNA that increases; 7. recombination is cloned into carrier go forward side by side row filter and selection.

Fig. 4 is the agarose gel electrophoresis figure of reorganization process; Lane M:DNA marker DL2000; Lane1:metk; Lane2:sam2; Lane3: the metk of strand; Lane4: the sam2 of strand; Lane5: through the strand metk of DNaseI digestion; Lane6: through the strand sam2 of DNaseI digestion; Lane7: reorganization DNA; Lane8: specificity is extended the reorganization DNA of purpose size.

Fig. 5 is reorganization gene Ks09 expressed proteins electrophorogram in yeast saccharomyces cerevisiae; M is middle molecular weight protein matter Marker; Lane1-4 represents the abduction delivering albumen of the yeast saccharomyces cerevisiae that includes different plasmids respectively: Lane1 includes pYES2; Lane2 includes pYES2+metk; Lane3 includes pYES2+sam2; Lane4 includes pYES2+Ks09 (reorganization gene).

Fig. 6 is for detecting recombinase abduction delivering bacterial strain ademetionine synthetic HPLC figure (HPLC detects the intracellular accumulation amount of ademetionine); A, b, c, d represent the product of yeast saccharomyces cerevisiae after abduction delivering and extracting that includes different plasmids respectively, and wherein a includes pYES2, and b includes pYES2+metk, and c includes pYES2+sam2, and d includes pYES2+Ks09 (reorganization gene); E is the ademetionine standard.

Fig. 7 is the order-checking collection of illustrative plates of reorganization gene Ks09.

Embodiment

Embodiment 1

A kind of gene metk that derives from the coding adenomethionine synthase (2.5.1.6) of e. coli jm109 bacterial strain has the base sequence among the sequence table SEQ ID No:1.

SEQ?ID?No：1：

ATGGCAAAACACCTTTTTACGTCCGAGTCCGTCTCTGAAGGGCATCCTGACAA

AATTGCTGACCAAATTTCTGATGCCGTTTTAGACGCGATCCTCGAACAGGATC

CGAAAGCACGCGTTGCTTGCGAAACCTACGTAAAAACCGGCATGGTTTTAGTT

GGCGGCGAAATCACCACCAGCGCCTGGGTAGACATCGAAGAGATCACCCGTAA

CACCGTTCGCGAAATTGGCTATGTGCATTCCGACATGGGCTTTGACGCTAACT

CCTGTGCGGTTCTGAGCGCTATCGGCAAACAGTCTCCTGACATCAACCAGGGC

GTTGACCGTGCCGATCCGCTGGAACAGGGCGCGGGTGACCAGGGTCTGATGTT

TGGCTACGCAACTAATGAAACCGACGTGCTGATGCCAGCACCTATCACCTATG

CACACCGTCTGGTACAGCGTCAGGCTGAAGTGCGTAAAAACGGCACTCTGCCG

TGGCTGCGCCCGGACGCGAAAAGCCAGGTGACTTTTCAGTATGACGACGGCAA

AATCGTTGGTATCGATGCTGTCGTGCTTTCCACTCAGCACTCTGAAGAGATCG

ACCAGAAATCGCTGCAAGAAGCGGTAATGGAAGAGATCATCAAGCCAATTCTG

CCCGCTGAATGGCTGACTTCTGCCACCAAATTCTTCATCAACCCGACCGGTCG

TTTCGTTATCGGTGGCCCAATGGGTGACTGCGGTCTGACTGGTCGTAAAATTA

TCGTTGATACCTACGGCGGCATGGCGCGTCACGGTGGCGGTGCATTCTCTGGT

AAAGATCCATCAAAAGTGGACCGTTCCGCAGCCTACGCAGCACGTTATGTCGC

GAAAAACATCGTTGCTGCTGGCCTGGCCGATCGTTGTGAAATTCAGGTTTCCT

ACGCAATCGGCGTGGCTGAACCGACCTCCATCATGGTAGAAACTTTCGGTACT

GAGAAAGTGCCTTCTGAACAACTGACCCTGCTGGTACGTGAGTTCTTCGACCT

GCGCCCATACGGTCTGATTCAGATGCTGGATCTGCTGCACCCGATCTACAAAG

AAACCGCAGCATACGGTCACTTTGGTCGTGAACATTTCCCGTGGGAAAAAACC

GACAAAGCGCAGCTGCTGCGCGATGCTGCCGGTCTGAAGTAA

(1) information of SEQ ID No:1 (referring to sequence table)

(a) sequence signature

* length: 1155 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: linearity

(b) molecule type: DNA

(c) suppose: not

(d) antisense: not

(e) initial source: the coding adenomethionine synthase of intestinal bacteria (E.coli.) JM109 bacterial strain

Metk gene (2.5.1.6)

(2) derive from the coding adenomethionine synthase of intestinal bacteria (E.coli.) JM109 bacterial strain

The preparation of metk gene (2.5.1.6):

Forward primer: 5 '-ACC GAATTCATGGCAAAACACCTTTTTACGTCC-3 ';

Reverse primer: 5 '-TAC GCGGCCGCTTACTTCAGACCGGCAGCATCG-3 ';

The underscore sequence is respectively EcoRI and NotI restriction enzyme site.

Amplification condition: with the total DNA of e. coli jm109 bacterial strain is template, increases with the pfu archaeal dna polymerase.

The pfu archaeal dna polymerase of reaction system: 2.5U, 1 * pfu buffer, 200 μ mol L ^-1DNTP, 1mmol L ^-1The dna profiling of each primer, 100ng, total reaction volume 100 μ l;

Reaction conditions: 94 ℃ of sex change 2min; 94 ℃ of 30s of 30 round-robin, 64.2 ℃ of 30s, 72 ℃ of 3min; 72 ℃ are extended 10min.The PCR product reclaims: by Shanghai China Shun test kit working instructions.The purpose product is seen agarose gel electrophoresis (Fig. 1).Goal gene is cut the corresponding restriction enzyme site that rear clone is gone into the pSE380 carrier through EcoRI and NotI enzyme, make up the pSE380-metk recombinant plasmid.

Embodiment 2

A kind of gene sam2 that derives from the coding adenomethionine synthase (2.5.1.6) of yeast saccharomyces cerevisiae INVScI bacterial strain has base sequence among the sequence table SEQ ID No:2.

SEQ?ID?No：2：

ATGTCCAAGAGCAAAACTTTCTTATTTACCTCTGAATCCGTCGGTGAAGGTCA

CCCAGACAAGATTTGTGACCAAGTTTCTGATGCTATTTTGGACGCTTGTTTAG

AACAAGATCCATTCTCCAAGGTTGCCTGTGAAACAGCTGCCAAAACTGGTATG

ATTATGGTTTTCGGTGAAATTACCACCAAAGCTAGACTTGACTACCAACAAAT

AGTAAGAGATACCATCAAGAAGATTGGTTATGACGATTCTGCCAAGGGTTTCG

ACTACAAGACATGTAATGTTTTAGTAGCTATCGAACAACAATCTCCAGATATC

GCTCAAGGTCTGCACTATGAAAAGAGCTTAGAAGACTTAGGTGCTGGTGACCA

AGGTATAATGTTTGGTTACGCTACAGACGAAACTCCAGAAGGGTTACCATTGA

CCATTCTTTTGGCTCACAAATTGAACATGGCTATGGCAGATGCTAGAAGAGAT

GGTTCTCTCCCATGGTTGAGACCAGACACAAAGACTCAAGTCACTGTCGAATA

CGAAGACGACAATGGTAGATGGGTTCCAAAGAGGATAGATACCGTTGTTATTT

CTGCTCAACATGCTGATGAAATTTCCACCGCTGACTTGAGAACTCAACTTCAA

AAAGATATTGTTGAAAAGGTCATACCAAAGGATATGTTAGACGAAAATACCAA

ATATTTCATCCAACCATCCGGTAGATTCGTCATCGGTGGTCCTCAAGGTGACG

CTGGTTTGACCGGTAGAAAGATTATTGTCGACGCTTACGGTGGTGCCTCATCC

GTCGGTGGTGGTGCCTTCTCCGGTAAGGACTATTCCAAGGTCGATCGTTCCGC

TGCTTACGCTGCTAGATGGGTTGCCAAGTCTCTAGTTGCCGCTGGTTTGTGTA

AGAGAGTCCAAGTCCAATTTTCATATGCTATTGGTATTGCTGAACCATTGTCT

TTACATGTGGACACCTATGGTACAGCTACAAAATCAGATGACGAAATCATTGA

AATTATTAAGAAGAACTTCGACTTGAGACCAGGTGTGTTAGTAAAGGAATTAG

ATTTGGCTAGACCAATTTACTTACCAACCGCTTCTTATGGTCACTTCACTAAT

CAAGAGTACTCATGGGAAAAACCAAAGAAATTGGAATTTTAA

(1) information of SEQ ID No:2 (referring to sequence table)

(A) sequence signature

* length: 1155 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: linearity

(b) molecule type: DNA

(c) suppose: not

(d) antisense: not

(e) initial source: the gene sam2 of the coding adenomethionine synthase (2.5.1.6) of yeast saccharomyces cerevisiae INVScI bacterial strain.

(2) derive from the preparation of gene sam2 of the coding adenomethionine synthase (2.5.1.6) of yeast saccharomyces cerevisiae INVScI bacterial strain:

Forward primer; 5 '-TCA GAATTCATGTCCAAGAGCAAAACT-3 ';

Reverse primer: 5 '-CAA GCGGCCGCTTAAAATTCCAATTTCTTTGG-3 ';

The underscore sequence is respectively EcoRI and NotI restriction enzyme site.

Amplification condition: total DNA is a template with yeast saccharomyces cerevisiae INVScI bacterial strain, increases with the Pfu archaeal dna polymerase.

The pfuDNA polysaccharase of reaction system: 2.5U, 1 * pfubuffer, 200 μ mol L ^-1DNTP, 1mmol L ^-1The dna profiling of each primer, 100ng, total reaction volume 100 μ l;

Reaction conditions: 94 ℃ of sex change 2min; 94 ℃ of 30s of 30 round-robin, 59 ℃ of 30s, 72 ℃ of 3min; 72 ℃ are extended 10min.The PCR product reclaims: by Shanghai China Shun test kit working instructions.The purpose product is seen agarose gel electrophoresis (Fig. 2).Goal gene is cut the corresponding restriction enzyme site that rear clone is gone into the pSE380 carrier through EcoRI and NotI enzyme, make up the pSE380-sam2 recombinant plasmid.

Embodiment 3

Adopt new gene reorganization method that metk and sam2 are reorganized, obtain the heterozygous genes of the high recombinase of coding thermostability, have the base sequence of sequence table SEQ ID No:3.

SEQ?ID?No：3：

ATGTCCAAGAGCAAAACTTTCTTATTTACCTCTGAATCCGTCGGTGAAGGTCA

CCCAGACAAGATTTGTGACCAAGTTTCTGATGCTATTTTGGACGCTTGTTTAG

AACAAGATCCATTCTCCAAGGTTGCCTGTGAAACAGCTGCCAAAACTGGTATG

ATTATGGTTTTCGGTGAAATTACCACCAAAGCTAGACTTGACTACCAACAAAT

AGTAAGAGATACCATCAAGAAGATTGGTTATGACGATTCTGCCAAGGGTTTCG

ACTACAAGACATGTAATGTTTTAGTAGCTATCGAACAACAATCTCCAGATATC

GCTCAAGGTCTGCACTATGAAAAGAGCTTAGAAGACTTAGGTGCTGGTGACCA

AGGTATAATGTTTGGTTACGCTACAAACGAAACTCCAGAAGGGTTACCATTGA

CCATTCTTTTGGCTCACAAATTGAACATGGCTATGGCAGATGCTAGAAGAGAT

GGTTCTCTCCCATGGTTGAGACCAGACACAAAGACTCAAGTCACTGTCGAATA

CGAAGACGACAATGGTAGATGGGTTCCAAAGAGGATAGATACCGTTGTTATTT

CTGCTCAACATGCTGATGAAATTTCCACCGCTGACTTGAGAACTCAACTTCAA

AAAGATATTGTTGAAAAGGTCATACCAAAGGATATGTTAGACGAAAATACCAA

ATTCTTCATCAACCCGACCGGTCGTTTCGTTATCGGTGGCCCAATGGGTGACT

GCGGTCTGACTGGTCGTAAAATTATCGTTGATACCTACGGCGGCATGGCGCGT

CACGGTGGCGGTGCATTCTCTGGTAAAGATCCATCAAAAGTGGACCGTTCCGC

AGCCTACGCAGCACGTTATGTCGCGAAAAACATCGTTGCTGCTGGCCTGGCCG

ATCGTTGTGAAATTCAGGTTTCCTACGCAATCGGCGTGGCTGAACCGACCTCC

ATCATGGTAGAAACTTTCGGTACTGAGAAAGTGCCTTCTGAACAACTGACCCT

GCTGGTACGTGAGTTCTTCGACCTGCGCCCATACGGTCTGATTCAGATGCTGG

ATCTGCTGCACCCGATCTACAAAGAATCCGCAGCATACGGTCACTTTGGTCGT

GAACATTTCCCGTGGGAAAAACCAAAGAAATTGGAATTTTAA

(1) information of SEQ ID No:3 (referring to sequence table)

(A) sequence signature

* length: 1155 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: linearity

(b) molecule type: DNA

(C) suppose: not

(d) antisense: not

(2) adopt new comprehensive gene reorganization mechanism that metk and sam2 are reorganized.

Above-mentioned two adenomethionine synthase gene metk and purified PCR product and the pSE380 carrier of sam2 are digested (with reference to TaKaRa company product description) with EcoRI and NotI respectively; then with magnificent Shun's test kit (Shanghai China Shun Bioisystech Co., Ltd; carry out segmental purifying down together); and respectively the PCR fragment is connected with carrier segments with T4DNA ligase enzyme (TaKaRa), be built into recombinant plasmid pSE380-metk and pSE380-sam2.Two plasmids are applied to respectively increasing and have the goal gene metk ' and the sam2 ' of the terminal homologous fragment of common carrier, and its end is introduced BglII and PstI restriction enzyme site (underscore sequence) respectively.The primer that is used for pcr amplification metk ' is as follows:

f1：5’-CAG AGATCTAATCACTGCATAATTCGTG-3’；

r1：5’-CTG CTGCAGTCGTTTTATTTGATGCCTG-3’；

The primer that is used for pcr amplification sam2 ' is as follows:

f2：5’-CAG CTGCAGAATCACTGCATAATTCGTG-3’；

r2：5’-CTG AGATCTTCGTTTTATTTGATGCCTG-3’；

Above-mentioned two PCR carry out under the following conditions: 94 ℃ of sex change 2min, and 94 ℃ of 30s of 30 round-robin subsequently, 53 ℃ of 30s, 72 ℃ of 3min, 72 ℃ are extended 10min then.Reaction system is as follows: the pfuDNA polysaccharase (TaKaRa) of 2.5U, 2ng template, 1 * pfu buffer, 0.2mmol L ^-1The dNTP mixture, 1.0m mol L ^-1MgCl ₂, reaction cumulative volume 100 μ l.Product PCR purification kit purifying, the fragment of purifying carry out enzyme with BglII and PstI (TaKaRa) to be cut, and reaction conditions is with reference to product description.Enzyme is cut product through 0.8% agarose gel electrophoresis, and the DNA band of purpose size reclaims test kit with glue and reclaims.The gained dna fragmentation is cut through the unidirectional enzyme of ExoIII (TaKaRa) and is obtained the total length single stranded DNA.Two kinds of single stranded DNAs getting 0.2 μ g respectively carry out DNaseI digestion, actual conditions reference literature (Zhao, H.﹠amp; Arno1d, F.H.Optimization of DNAshuffling for high fidelity.Nucleic Acids Res.1997,25,1307-1308.).In step subsequently, reaction is divided into two groups to be carried out, and in each group, the single stranded DNA fragment from another DNA of a kind of single stranded DNA of 20ng and equivalent is blended in respectively in the PCR pipe, and reaction system also comprises 1 * taq buffer, 0.2mmol L ^-1The dNTP mixture, 1.5mmol L ^-1MgCl ₂With 1.25U taq archaeal dna polymerase (TaKaRa), the total reaction system is 50 μ l.Reaction conditions is 94 ℃ of sex change 2min; 94 ℃ of 30s of 10 round-robin, 53 ℃ of 2min; 72 ℃ are extended 10min, add the corresponding reverse primer r1 and r2 of 16pmol then respectively, and enter next PCR reaction, and condition is 94 ℃ of sex change 2min; 94 ℃ of 30s of 60 round-robin, 46 ℃ of 10s.Two group reaction products are mixed, add the taq archaeal dna polymerase of 2U and the following primer of 5.3mmol:

f3：5-CATCCGGCTCGTATAATGTGTG-3；

r3：5-CGCCAGGCAAATTCTGTTTTATC-3；

Reaction conditions is as follows: 94 ℃ of sex change 2min; 94 ℃ of 30s of 40 round-robin, 46 ℃ of 10s; 94 ℃ of 30s of 10 round-robin, 66 ℃ of 10s.Product is behind 0.8% agarose gel electrophoresis, and the fragment of purpose size (about 1.3kb) is used for specific amplifying target genes as template after glue reclaims the test kit purifying, and reaction system is 1 * taq buffer, 0.2mmol L ^-1The dNTP mixture, 50ng template DNA, 1.5mmol L ^-1MgCl ₂With 1.25U taq archaeal dna polymerase cumulative volume be 50 μ l.Reaction conditions is 94 ℃ of sex change 2min; 94 ℃ of 30s of 30 round-robin, 52 ℃ of 30s, 72 ℃ of 3min; 72 ℃ are extended 10min.Product is behind PCR purification kit purifying, with EcoRI and NotI (TaKaRa) digestion.Product is connected into through the pSE380 of same double digestion carrier (reaction conditions is with reference to product description) under the effect of T4DNA ligase enzyme after glue reclaims the test kit purifying.Recombinant plasmid changes E.coli JM109 over to and coats and contains 100 μ g ml ^-1Amp and 10mmol L ^-1On the LB flat board of ethionine, cultivate 12h for 37 ℃.From survival strains, extract recombinant plasmid, and digest with EcoRI and NotI respectively, product is behind 0.8% agarose gel electrophoresis, and the fragment of purpose size subclone after glue reclaims the test kit purifying is gone into the expression vector through the pYES2 of same double digestion, and transformed saccharomyces cerevisiae INVScI.In contrast, will be cloned into the expression vector through the pYES2 of same double digestion through the metk and the sam2 of EcoRI and NotI double digestion, and make up pYES2+metk and pYES2+sam2, above-mentioned two plasmids and pYES2 are transformed into yeast saccharomyces cerevisiae INVScI respectively.Concrete reaction conditions is with reference to product description.SC-U substratum (the 6.7g yeast nitrogen that picking genetic engineering bacterium list colony inoculation is revised to 15ml; The 5g methionine(Met); 0.1g VITAMIN B4, arginine, halfcystine, leucine, Methionin, network propylhomoserin, Threonine; 0.05g aspartic acid, Histidine, Isoleucine, phenylalanine, proline(Pro), Serine, tryptophane and Xie Ansuan; Final volume 1L), comprise 2% raffinose.30 ℃ are cultured to OD ₆₀₀=0.4,4 ℃ of following centrifugal 5min of 1500 * g are resuspended in cell 2ml inducing culture (the SC-U substratum of modification comprises 2% semi-lactosi) then then, inoculate in the inducing culture of 50ml, cultivate 6h under 30 ℃ of conditions in the 150r min-1 shaking table.4 ℃ of following centrifugal 5min of 1500 * g outwell supernatant then, precipitation are resuspended in the pure water of 500 μ l.The centrifugal 5min of 1500 * g under 4 ℃ outwells supernatant, and cell is used to detect the SAM accumulation.Add 5ml 1.5mol L in every 1g thalline ^-1Perchloric acid with 120r min ^-1Shake 1h. and carry out extracting, (Perseptive Biosystem Inc.U.S.A.) detects the SAM level of generation with BioCAD 700E type HPLC.Weak cation exchange column Poros20CM is selected in this test for use, and (4.6mm * 100mm), moving phase is 0.5mol L ^-1HCOONH ₄, pH is adjusted into 4.0, and flow velocity is 5ml min ^-1, under the 260nm wavelength, detect product, 22 ℃ of room temperatures.Containing pYES2, the Wine brewing yeast strain abduction delivering product of pYES2+metk and pYES2+sam2 in contrast, and with 10 μ mol L ^-1The SAM standard substance are demarcated the generation of SAM.

It is centrifugal to get the 1ml fermented liquid, and bacterial sediment 1ml distilled water wash is resuspended in after centrifugal in 1 times the sample-loading buffer, and boiling water bath boils 10min, the centrifugal 2min of 12000 * g.Get 10 μ l and carry out the SDS-PAGE electrophoresis, 0.1% coomassie brilliant blue staining detects proteic expression then.

The result:

1. use gene reorganization method of the present invention, take turns the recombinant DNA that reaction promptly obtains the purpose size through one;

2. in the cell whole protein electrophoresis of engineering bacteria an obvious expression band is arranged, inducible protein as shown in Figure 5.Molecular weight is about 4.2K dalton;

3. detect by HPLC, obtain the Wine brewing yeast strain that a strain ademetionine intracellular accumulation amount improves, and its contained plasmid pYES2+Ks09 is carried out sequential analysis, determine that it contains the reorganization gene, and have the reorganization of multidigit point.

Recombination

SEQUENCE?LISTING

＜110〉Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences

＜120〉a kind of method and recombination that is obtained and proteins encoded of gene reorganization

<130>

<160>6

<170>PatentIn?version?3.1

<210>1

<211>1155

<212>DNA

＜213〉intestinal bacteria (E.coli.) JM109 bacterial strain

<220>

<221>CDS

<222>(1)..(1155)

<223>

<400>1

atg?gca?aaa?cac?ctt?ttt?acg?tcc?gag?tcc?gtc?tct?gaa?ggg?cat?cct 48

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

1 5 10 15

gac?aaa?att?gct?gac?caa?att?tct?gat?gcc?gtt?tta?gac?gcg?atc?ctc 96

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

20 25 30

gaa?cag?gat?ccg?aaa?gca?cgc?gtt?gct?tgc?gaa?acc?tac?gta?aaa?acc 144

Glu?Gln?Asp?Pro?Lys?Ala?Arg?Val?Ala?Cys?Glu?Thr?Tyr?Val?Lys?Thr

35 40 45

ggc?atg?gtt?tta?gtt?ggc?ggc?gaa?atc?acc?acc?agc?gcc?tgg?gta?gac 192

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

50 55 60

atc?gaa?gag?atc?acc?cgt?aac?acc?gtt?cgc?gaa?att?ggc?tat?gtg?cat 240

Ile?Glu?Glu?Ile?Thr?Arg?Asn?Thr?Val?Arg?Glu?Ile?Gly?Tyr?Val?His

65 70 75 80

tcc?gac?atg?ggc?ttt?gac?gct?aac?tcc?tgt?gcg?gtt?ctg?agc?gct?atc 288

Ser?Asp?Met?Gly?Phe?Asp?Ala?Asn?Ser?Cys?Ala?Val?Leu?Ser?Ala?Ile

85 90 95

ggc?aaa?cag?tct?cct?gac?atc?aac?cag?ggc?gtt?gac?cgt?gcc?gat?ccg 336

Gly?Lys?Gln?Ser?Pro?Asp?Ile?Asn?Gln?Gly?Val?Asp?Arg?Ala?Asp?Pro

100 105 110

ctg?gaa?cag?ggc?gcg?ggt?gac?cag?ggt?ctg?atg?ttt?ggc?tac?gca?act 384

Leu?Glu?Gln?Gly?Ala?Gly?Asp?Gln?Gly?Leu?Met?Phe?Gly?Tyr?Ala?Thr

115 120 125

aat?gaa?acc?gac?gtg?ctg?atg?cca?gca?cct?atc?acc?tat?gca?cac?cgt 432

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

130 135 140

ctg?gta?cag?cgt?cag?gct?gaa?gtg?cgt?aaa?aac?ggc?act?ctg?ccg?tgg 480

Leu?Val?Gln?Arg?Gln?Ala?Glu?Val?Arg?Lys?Asn?Gly?Thr?Leu?Pro?Trp

145 150 155 160

ctg?cgc?ccg?gac?gcg?aaa?agc?cag?gtg?act?ttt?cag?tat?gac?gac?ggc 528

Leu?Arg?Pro?Asp?Ala?Lys?Ser?Gln?Val?Thr?Phe?Gln?Tyr?Asp?Asp?Gly

165 170 175

aaa?atc?gtt?ggt?atc?gat?gct?gtc?gtg?ctt?tcc?act?cag?cac?tct?gaa 576

Recombination

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

180 185 190

gag?atc?gac?cag?aaa?tcg?ctg?caa?gaa?gcg?gta?atg?gaa?gag?atc?atc 624

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

195 200 205

aag?cca?att?ctg?ccc?gct?gaa?tgg?ctg?act?tct?gcc?acc?aaa?ttc?ttc 672

Lys?Pro?Ile?Leu?Pro?Ala?Glu?Trp?Leu?Thr?Ser?Ala?Thr?Lys?Phe?Phe

210 215 220

atc?aac?ccg?acc?ggt?cgt?ttc?gtt?atc?ggt?ggc?cca?atg?ggt?gac?tgc 720

Ile?Asn?Pro?Thr?Gly?Arg?Phe?Val?Ile?Gly?Gly?Pro?Met?Gly?Asp?Cys

225 230 235 240

ggt?ctg?act?ggt?cgt?aaa?att?atc?gtt?gat?acc?tac?ggc?ggc?atg?gcg 768

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

245 250 255

cgt?cac?ggt?ggc?ggt?gca?ttc?tct?ggt?aaa?gat?cca?tca?aaa?gtg?gac 816

Arg?His?Gly?Gly?Gly?Ala?Phe?Ser?Gly?Lys?Asp?Pro?Ser?Lys?Val?Asp

260 265 270

cgt?tcc?gca?gcc?tac?gca?gca?cgt?tat?gtc?gcg?aaa?aac?atc?gtt?gct 864

Arg?Ser?Ala?Ala?Tyr?Ala?Ala?Arg?Tyr?Val?Ala?Lys?Asn?Ile?Val?Ala

275 280 285

gct?ggc?ctg?gcc?gat?cgt?tgt?gaa?att?cag?gtt?tcc?tac?gca?atc?ggc 912

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

290 295 300

gtg?gct?gaa?ccg?acc?tcc?atc?atg?gta?gaa?act?ttc?ggt?act?gag?aaa 960

Val?Ala?Glu?Pro?Thr?Ser?Ile?Met?Val?Glu?Thr?Phe?Gly?Thr?Glu?Lys

305 310 315 320

gtg?cct?tct?gaa?caa?ctg?acc?ctg?ctg?gta?cgt?gag?ttc?ttc?gac?ctg 1008

Val?Pro?Ser?Glu?Gln?Leu?Thr?Leu?Leu?Val?Arg?Glu?Phe?Phe?Asp?Leu

325 330 335

cgc?cca?tac?ggt?ctg?att?cag?atg?ctg?gat?ctg?ctg?cac?ccg?atc?tac 1056

Arg?Pro?Tyr?Gly?Leu?Ile?Gln?Met?Leu?Asp?Leu?Leu?His?Pro?Ile?Tyr

340 345 350

aaa?gaa?acc?gca?gca?tac?ggt?cac?ttt?ggt?cgt?gaa?cat?ttc?ccg?tgg 1104

Lys?Glu?Thr?Ala?Ala?Tyr?Gly?His?Phe?Gly?Arg?Glu?His?Phe?Pro?Trp

355 360 365

gaa?aaa?acc?gac?aaa?gcg?cag?ctg?ctg?cgc?gat?gct?gcc?ggt?ctg?aag 1152

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

370 375 380

taa 1155

<210>2

<211>384

<212>PRT

＜213〉intestinal bacteria (E.coli.) JM109 bacterial strain

<400>2

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

1 5 10 15

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

20 25 30

Glu?Gln?Asp?Pro?Lys?Ala?Arg?Val?Ala?Cys?Glu?Thr?Tyr?Val?Lys?Thr

35 40 45

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

50 55 60

Ile?Glu?Glu?Ile?Thr?Arg?Asn?Thr?Val?Arg?Glu?Ile?Gly?Tyr?Val?His

65 70 75 80

Ser?Asp?Met?Gly?Phe?Asp?Ala?Asn?Ser?Cys?Ala?Val?Leu?Ser?Ala?Ile

85 90 95

Gly?Lys?Gln?Ser?Pro?Asp?Ile?Asn?Gln?Gly?Val?Asp?Arg?Ala?Asp?Pro

100 105 110

Leu?Glu?Gln?Gly?Ala?Gly?Asp?Gln?Gly?Leu?Met?Phe?Gly?Tyr?Ala?Thr

115 120 125

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

130 135 140

Leu?Val?Gln?Arg?Gln?Ala?Glu?Val?Arg?Lys?Asn?Gly?Thr?Leu?Pro?Trp

145 150 155 160

Leu?Arg?Pro?Asp?Ala?Lys?Ser?Gln?Val?Thr?Phe?Gln?Tyr?Asp?Asp?Gly

165 170 175

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

180 185 190

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

195 200 205

Recombination

Lys?Pro?Ile?Leu?Pro?Ala?Glu?Trp?Leu?Thr?Ser?Ala?Thr?Lys?Phe?Phe

210 215 220

Ile?Asn?Pro?Thr?Gly?Arg?Phe?Val?Ile?Gly?Gly?Pro?Met?Gly?Asp?Cys

225 230 235 240

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

245 250 255

Arg?His?Gly?Gly?Gly?Ala?Phe?Ser?Gly?Lys?Asp?Pro?Ser?Lys?Val?Asp

260 255 270

Arg?Ser?Ala?Ala?Tyr?Ala?Ala?Arg?Tyr?Val?Ala?Lys?Asn?Ile?Val?Ala

275 280 285

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

290 295 300

Val?Ala?Glu?Pro?Thr?Ser?Ile?Met?Val?Glu?Thr?Phe?Gly?Thr?Glu?Lys

305 310 315 320

Val?Pro?Ser?Glu?Gln?Leu?Thr?Leu?Leu?Val?Arg?Glu?Phe?Phe?Asp?Leu

325 330 335

Arg?Pro?Tyr?Gly?Leu?Ile?Gln?Met?Leu?Asp?Leu?Leu?His?Pro?Ile?Tyr

340 345 350

Lys?Glu?Thr?Ala?Ala?Tyr?Gly?His?Phe?Gly?Arg?Glu?His?Phe?Pro?Trp

355 360 365

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

370 375 380

<210>3

<211>1155

<212>DNA

＜213〉yeast saccharomyces cerevisiae INVScI bacterial strain

<220>

<221>CDS

<222>(1)..(1155)

<223>

<400>3

atg?tcc?aag?agc?aaa?act?ttc?tta?ttt?acc?tct?gaa?tcc?gtc?ggt?gaa 48

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

1 5 10 15

ggt?cac?cca?gac?aag?att?tgt?gac?caa?gtt?tct?gat?gct?att?ttg?gac 96

Gly?His?Pro?Asp?Lys?Ile?Cys?Asp?Gln?Val?Ser?Asp?Ala?Ile?Leu?Asp

20 25 30

gct?tgt?tta?gaa?caa?gat?cca?ttc?tcc?aag?gtt?gcc?tgt?gaa?aca?gct 144

Ala?Cys?Leu?Glu?Gln?Asp?Pro?Phe?Ser?Lys?Val?Ala?Cys?Glu?Thr?Ala

35 40 45

gcc?aaa?act?ggt?atg?att?atg?gtt?ttc?ggt?gaa?att?acc?acc?aaa?gct 192

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

50 55 60

aga?ctt?gac?tac?caa?caa?ata?gta?aga?gat?acc?atc?aag?aag?att?ggt 240

Arg?Leu?Asp?Tyr?Gln?Gln?Ile?Val?Arg?Asp?Thr?Ile?Lys?Lys?Ile?Gly

65 70 75 80

tat?gac?gat?tct?gcc?aag?ggt?ttc?gac?tac?aag?aca?tgt?aat?gtt?tta 288

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

85 90 95

gta?gct?atc?gaa?caa?caa?tct?cca?gat?atc?gct?caa?ggt?ctg?cac?tat 336

Val?Ala?Ile?Glu?Gln?Gln?Ser?Pro?Asp?Ile?Ala?Gln?Gly?Leu?His?Tyr

100 105 110

gaa?aag?agc?tta?gaa?gac?tta?ggt?gct?ggt?gac?caa?ggt?ata?atg?ttt 384

Glu?Lys?Ser?Leu?Glu?Asp?Leu?Gly?Ala?Gly?Asp?Gln?Gly?Ile?Met?Phe

115 120 125

ggt?tac?gct?aca?gac?gaa?act?cca?gaa?ggg?tta?cca?ttg?acc?att?ctt 432

Gly?Tyr?Ala?Thr?Asp?Glu?Thr?Pro?Glu?Gly?Leu?Pro?Leu?Thr?Ile?Leu

130 135 140

ttg?gct?cac?aaa?ttg?aac?atg?gct?atg?gca?gat?gct?aga?aga?gat?ggt 480

Leu?Ala?His?Lys?Leu?Asn?Met?Ala?Met?Ala?Asp?Ala?Arg?Arg?Asp?Gly

145 150 155 160

tct?ctc?cca?tgg?ttg?aga?cca?gac?aca?aag?act?caa?gtc?act?gtc?gaa 528

Ser?Leu?Pro?Trp?Leu?Arg?Pro?Asp?Thr?Lys?Thr?Gln?Val?Thr?Val?Glu

165 170 175

tac?gaa?gac?gac?aat?ggt?aga?tgg?gtt?cca?aag?agg?ata?gat?acc?gtt 576

Tyr?Glu?Asp?Asp?Asn?Gly?Arg?Trp?Val?Pro?Lys?Arg?Ile?Asp?Thr?Val

Recombination

180 185 190

gtt?att?tct?gct?caa?cat?gct?gat?gaa?att?tcc?acc?gct?gac?ttg?aga 624

Val?Ile?Ser?Ala?Gln?His?Ala?Asp?Glu?Ile?Ser?Thr?Ala?Asp?Leu?Arg

195 200 205

act?caa?ctt?caa?aaa?gat?att?gtt?gaa?aag?gtc?ata?cca?aag?gat?atg 672

Thr?Gln?Leu?Gln?Lys?Asp?Ile?Val?Glu?Lys?Val?Ile?Pro?Lys?Asp?Met

210 215 220

tta?gac?gaa?aat?acc?aaa?tat?ttc?atc?caa?cca?tcc?ggt?aga?ttc?gtc 720

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

225 230 235 240

atc?ggt?ggt?cct?caa?ggt?gac?gct?ggt?ttg?acc?ggt?aga?aag?att?att 768

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

245 250 255

gtc?gac?gct?tac?ggt?ggt?gcc?tca?tcc?gtc?ggt?ggt?ggt?gcc?ttc?tcc 816

Val?Asp?Ala?Tyr?Gly?Gly?Ala?Ser?Ser?Val?Gly?Gly?Gly?Ala?Phe?Ser

260 265 270

ggt?aag?gac?tat?tcc?aag?gtc?gat?cgt?tcc?gct?gct?tac?gct?gct?aga 864

Gly?Lys?Asp?Tyr?Ser?Lys?Val?Asp?Arg?Ser?Ala?Ala?Tyr?Ala?Ala?Arg

275 280 285

tgg?gtt?gcc?aag?tct?cta?gtt?gcc?gct?ggt?ttg?tgt?aag?aga?gtc?caa 912

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

290 295 300

gtc?caa?ttt?tca?tat?gct?att?ggt?att?gct?gaa?cca?ttg?tct?tta?cat 960

Val?Gln?Phe?Ser?Tyr?Ala?Ile?Gly?Ile?Ala?Glu?Pro?Leu?Ser?Leu?His

305 310 315 320

gtg?gac?acc?tat?ggt?aca?gct?aca?aaa?tca?gat?gac?gaa?atc?att?gaa 1008

Val?Asp?Thr?Tyr?Gly?Thr?Ala?Thr?Lys?Ser?Asp?Asp?Glu?Ile?Ile?Glu

325 330 335

att?att?aag?aag?aac?ttc?gac?ttg?aga?cca?ggt?gtg?tta?gta?aag?gaa 1056

Ile?Ile?Lys?Lys?Asn?Phe?Asp?Leu?Arg?Pro?Gly?Val?Leu?Val?Lys?Glu

340 345 350

tta?gat?ttg?gct?aga?cca?att?tac?tta?cca?acc?gct?tct?tat?ggt?cac 1104

Leu?Asp?Leu?Ala?Arg?Pro?Ile?Tyr?Leu?Pro?Thr?Ala?Ser?Tyr?Gly?His

355 360 365

ttc?act?aat?caa?gag?tac?tca?tgg?gaa?aaa?cca?aag?aaa?ttg?gaa?ttt 1152

Phe?Thr?Asn?Gln?Glu?Tyr?Ser?Trp?Glu?Lys?Pro?Lys?Lys?Leu?Glu?Phe

370 375 380

taa 1155

<210>4

<211>384

<212>PRT

＜213〉yeast saccharomyces cerevisiae INVScI bacterial strain

<400>4

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

1 5 10 15

Gly?His?Pro?Asp?Lys?Ile?Cys?Asp?Gln?Val?Ser?Asp?Ala?Ile?Leu?Asp

20 25 30

Ala?Cys?Leu?Glu?Gln?Asp?Pro?Phe?Ser?Lys?Val?Ala?Cys?Glu?Thr?Ala

35 40 45

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

50 55 60

Arg?Leu?Asp?Tyr?Gln?Gln?Ile?Val?Arg?Asp?Thr?Ile?Lys?Lys?Ile?Gly

65 70 75 80

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

85 90 95

Val?Ala?Ile?Glu?Gln?Gln?Ser?Pro?Asp?Ile?Ala?Gln?Gly?Leu?His?Tyr

100 105 110

Glu?Lys?Ser?Leu?Glu?Asp?Leu?Gly?Ala?Gly?Asp?Gln?Gly?Ile?Met?Phe

115 120 125

Gly?Tyr?Ala?Thr?Asp?Glu?Thr?Pro?Glu?Gly?Leu?Pro?Leu?Thr?Ile?Leu

130 135 140

Leu?Ala?His?Lys?Leu?Asn?Met?Ala?Met?Ala?Asp?Ala?Arg?Arg?Asp?Gly

145 150 155 160

Ser?Leu?Pro?Trp?Leu?Arg?Pro?Asp?Thr?Lys?Thr?Gln?Val?Thr?Val?Glu

165 170 175

Tyr?Glu?Asp?Asp?Asn?Gly?Arg?Trp?Val?Pro?Lys?Arg?Ile?Asp?Thr?Val

180 185 190

Val?Ile?Ser?Ala?Gln?His?Ala?Asp?Glu?Ile?Ser?Thr?Ala?Asp?Leu?Arg

195 200 205

Thr?Gln?Leu?Gln?Lys?Asp?Ile?Val?Glu?Lys?Val?Ile?Pro?Lys?Asp?Met

Recombination

210 215 220

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

225 230 235 240

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

245 250 255

Val?Asp?Ala?Tyr?Gly?Gly?Ala?Ser?Ser?Val?Gly?Gly?Gly?Ala?Phe?Ser

260 265 270

Gly?Lys?Asp?Tyr?Ser?Lys?Val?Asp?Arg?Ser?Ala?Ala?Tyr?Ala?Ala?Arg

275 280 285

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

290 295 300

Val?Gln?Phe?Ser?Tyr?Ala?Ile?Gly?Ile?Ala?Glu?Pro?Leu?Ser?Leu?His

305 310 315 320

Val?Asp?Thr?Tyr?Gly?Thr?Ala?Thr?Lys?Ser?Asp?Asp?Glu?Ile?Ile?Glu

325 330 335

Ile?Ile?Lys?Lys?Asn?Phe?Asp?Leu?Arg?Pro?Gly?Val?Leu?Val?Lys?Glu

340 345 350

Leu?Asp?Leu?Ala?Arg?Pro?Ile?Tyr?Leu?Pro?Thr?Ala?Ser?Tyr?Gly?His

355 360 365

Phe?Thr?Asn?Gln?Glu?Tyr?Ser?Trp?Glu?Lys?Pro?Lys?Lys?Leu?Glu?Phe

370 375 380

<210>5

<211>1155

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)..(1155)

<223>

<400>5

atg?tcc?aag?agc?aaa?act?ttc?tta?ttt?acc?tct?gaa?tcc?gtc?ggt?gaa 48

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

1 5 10 15

ggt?cac?cca?gac?aag?att?tgt?gac?caa?gtt?tct?gat?gct?att?ttg?gac 96

Gly?His?Pro?Asp?Lys?Ile?Cys?Asp?Gln?Val?Ser?Asp?Ala?Ile?Leu?Asp

20 25 30

gct?tgt?tta?gaa?caa?gat?cca?ttc?tcc?aag?gtt?gcc?tgt?gaa?aca?gct 144

Ala?Cys?Leu?Glu?Gln?Asp?Pro?Phe?Ser?Lys?Val?Ala?Cys?Glu?Thr?Ala

35 40 45

gcc?aaa?act?ggt?atg?att?atg?gtt?ttc?ggt?gaa?att?acc?acc?aaa?gct 192

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

50 55 60

aga?ctt?gac?tac?caa?caa?ata?gta?aga?gat?acc?atc?aag?aag?att?ggt 240

Arg?Leu?Asp?Tyr?Gln?Gln?Ile?Val?Arg?Asp?Thr?Ile?Lys?Lys?Ile?Gly

65 70 75 80

tat?gac?gat?tct?gcc?aag?ggt?ttc?gac?tac?aag?aca?tgt?aat?gtt?tta 288

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

85 90 95

gta?gct?atc?gaa?caa?caa?tct?cca?gat?atc?gct?caa?ggt?ctg?cac?tat 336

Val?Ala?Ile?Glu?Gln?Gln?Ser?Pro?Asp?Ile?Ala?Gln?Gly?Leu?His?Tyr

100 105 110

gaa?aag?agc?tta?gaa?gac?tta?ggt?gct?ggt?gac?caa?ggt?ata?atg?ttt 384

Glu?Lys?Ser?Leu?Glu?Asp?Leu?Gly?Ala?Gly?Asp?Gln?Gly?Ile?Met?Phe

115 120 125

ggt?tac?gct?aca?aac?gaa?act?cca?gaa?ggg?tta?cca?ttg?acc?att?ctt 432

Glv?Tyr?Ala?Thr?Asn?Glu?Thr?Pro?Glu?Gly?Leu?Pro?Leu?Thr?Ile?Leu

130 135 140

ttg?gct?cac?aaa?ttg?aac?atg?gct?atg?gca?gat?gct?aga?aga?gat?ggt 480

Leu?Ala?His?Lys?Leu?Asn?Met?Ala?Met?Ala?Asp?Ala?Arg?Arg?Asp?Gly

145 150 155 160

tct?ctc?cca?tgg?ttg?aga?cca?gac?aca?aag?act?caa?gtc?act?gtc?gaa 528

Ser?Leu?Pro?Trp?Leu?Arg?Pro?Asp?Thr?Lys?Thr?Gln?Val?Thr?Val?Glu

165 170 175

tac?gaa?gac?gac?aat?ggt?aga?tgg?gtt?cca?aag?agg?ata?gat?acc?gtt 576

Tyr?Glu?Asp?Asp?Asn?Gly?Arg?Trp?Val?Pro?Lys?Arg?Ile?Asp?Thr?Val

180 185 190

Recombination

gtt?att?tct?gct?caa?cat?gct?gat?gaa?att?tcc?acc?gct?gac?ttg?aga 624

Val?Ile?Ser?Ala?Gln?His?Ala?Asp?Glu?Ile?Ser?Thr?Ala?Asp?Leu?Arg

195 200 205

act?caa?cttc?aa?aaa?gat?att?gtt?gaa?aag?gtc?ata?cca?aag?gat?atg 672

Thr?Gln?Leu?Gln?Lys?Asp?Ile?Val?Glu?Lys?Val?Ile?Pro?Lys?Asp?Met

210 215 220

tta?gac?gaa?aat?acc?aaa?ttc?ttc?atc?aac?ccg?acc?ggt?cgt?ttc?gtt 720

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

225 230 235 240

atc?ggt?ggc?cca?atg?ggt?gac?tgc?ggt?ctg?act?ggt?cgt?aaa?att?atc 768

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

245 250 255

gtt?gat?acc?tac?ggc?ggc?atg?gcg?cgt?cac?ggt?ggc?ggt?gca?ttc?tct 816

Val?Asp?Thr?Tyr?Gly?Gly?Met?Ala?Arg?His?Gly?Gly?Gly?Ala?Phe?Ser

260 265 270

ggt?aaa?gat?cca?tca?aaa?gtg?gac?cgt?tcc?gca?gcc?tac?gca?gca?cgt 864

Gly?Lys?Asp?Pro?Ser?Lys?Val?Asp?Arg?Ser?Ala?Ala?Tyr?Ala?Ala?Arg

275 280 285

tat?gtc?gcg?aaa?aac?atc?gtt?gct?gct?ggc?ctg?gcc?gat?cgt?tgt?gaa 912

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

290 295 300

att?cag?gtt?tcc?tac?gca?atc?ggc?gtg?gct?gaa?ccg?acc?tcc?atc?atg 960

Ile?Gln?Val?Ser?Tyr?Ala?Ile?Gly?Val?Ala?Glu?Pro?Thr?Ser?Ile?Met

305 310 315 320

gta?gaa?act?ttc?ggt?act?gag?aaa?gtg?cct?tct?gaa?caa?ctg?acc?ctg 1008

Val?Glu?Thr?Phe?Gly?Thr?Glu?Lys?Val?Pro?Ser?Glu?Gln?Leu?Thr?Leu

325 330 335

ctg?gta?cgt?gag?ttc?ttc?gac?ctg?cgc?cca?tac?ggt?ctg?att?cag?atg 1056

Leu?Val?Arg?Glu?Phe?Phe?Asp?Leu?Arg?Pro?Tyr?Gly?Leu?Ile?Gln?Met

340 345 350

ctg?gat?ctg?ctg?cac?ccg?atc?tac?aaa?gaa?tcc?gca?gca?tac?ggt?cac 1104

Leu?Asp?Leu?Leu?His?Pro?Ile?Tyr?Lys?Glu?Ser?Ala?Ala?Tyr?Gly?His

355 360 365

ttt?ggt?cgt?gaa?cat?ttc?ccg?tgg?gaa?aaa?cca?aag?aaa?ttg?gaa?ttt 1152

Phe?Gly?Arg?Glu?His?Phe?Pro?Trp?Glu?Lys?Pro?Lys?Lys?Leu?Glu?Phe

370 375 380

taa 1155

<210>6

<211>384

<212>PRT

＜213〉artificial sequence

<400>6

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

1 5 10 15

Gly?His?Pro?Asp?Lys?Ile?Cys?Asp?Gln?Val?Ser?Asp?Ala?Ile?Leu?Asp

20 25 30

Ala?Cys?Leu?Glu?Gln?Asp?Pro?Phe?Ser?Lys?Val?Ala?Cys?Glu?Thr?Ala

35 40 45

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

50 55 60

Arg?Leu?Asp?Tyr?Gln?Gln?Ile?Val?Arg?Asp?Thr?Ile?Lys?Lys?Ile?Gly

65 70 75 80

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

85 90 95

Val?Ala?Ile?Glu?Gln?Gln?Ser?Pro?Asp?Ile?Ala?Gln?Gly?Leu?His?Tyr

100 105 110

Glu?Lys?Ser?Leu?Glu?Asp?Leu?Gly?Ala?Gly?Asp?Gln?Gly?Ile?Met?Phe

115 120 125

Gly?Tyr?Ala?Thr?Asn?Glu?Thr?Pro?Glu?Gly?Leu?Pro?Leu?Thr?Ile?Leu

130 135 140

Leu?Ala?His?Lys?Leu?Asn?Met?Ala?Met?Ala?Asp?Ala?Arg?Arg?Asp?Gly

145 150 155 160

Ser?Leu?Pro?Trp?Leu?Arg?Pro?Asp?Thr?Lys?Thr?Gln?Val?Thr?Val?Glu

165 170 175

Tyr?Glu?Asp?Asp?Asn?Gly?Arg?Trp?Val?Pro?Lys?Arg?Ile?Asp?Thr?Val

180 185 190

Val?Ile?Ser?Ala?Gln?His?Ala?Asp?Glu?Ile?Ser?Thr?Ala?Asp?Leu?Arg

195 200 205

Thr?Gln?Leu?Gln?Lys?Asp?Ile?Val?Glu?Lys?Val?Ile?Pro?Lys?Asp?Met

210 215 220

Recombination

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

225 230 235 240

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

245 250 255

Val?Asp?Thr?Tyr?Gly?Gly?Met?Ala?Arg?His?Gly?Gly?Gly?Ala?Phe?Ser

260 265 270

Gly?Lys?Asp?Pro?Ser?Lys?Val?Asp?Arg?Ser?Ala?Ala?Tyr?Ala?Ala?Arg

275 280 285

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

290 295 300

Ile?Gln?Val?Ser?Tyr?Ala?Ile?Gly?Val?Ala?Glu?Pro?Thr?Ser?Ile?Met

305 310 315 320

Val?Glu?Thr?Phe?Gly?Thr?Glu?Lys?Val?Pro?Ser?Glu?Gln?Leu?Thr?Leu

325 330 335

Leu?Val?Arg?Glu?Phe?Phe?Asp?Leu?Arg?Pro?Tyr?Gly?Leu?Ile?Gln?Met

340 345 350

Leu?Asp?Leu?Leu?His?Pro?Ile?Tyr?Lys?Glu?Ser?Ala?Ala?Tyr?Gly?His

355 360 365

Phe?Gly?Arg?Glu?His?Phe?Pro?Trp?Glu?Lys?Pro?Lys?Lys?Leu?Glu?Phe

370 375 380

Claims

1. the method for gene reorganization is characterized in that:

1) choose two kinds of Linear Double chain gene A and B, the end of gene has respectively can produce the restriction endonuclease recognition sequences that otch is different viscosity protrusion ends, produces opposite two groups of single stranded DNAs---A ' and the B ' of direction with ExoIII digestion respectively;

2. according to the method for the described gene reorganization of claim 1, it is characterized in that:

The sequence set of described two kinds of Linear Double chain gene A and B becomes to be respectively nucleotide sequence among the sequence table SEQ ID No:1 and the nucleotide sequence among the sequence table SEQ ID No:2, is initial gene with two genes, realizes intergenic reorganization through following steps:

2) digest above-mentioned fragment with ExoIII, to obtain single stranded DNA and purifying in addition;

7) recombination is connected into expression vector and change yeast saccharomyces cerevisiae over to,, obtains the engineering strain of anti-ethionine,, separate obtaining bacterial strain that content improves and the recombination that is comprised thereof by the ademetionine Determination on content by screening.

3. the recombination that obtains of the described method of a claim 1, it is characterized in that: its sequence set becomes the nucleotide sequence among the sequence table SEQ ID No:3.

4. the described method of a claim 1 obtains the proteins encoded of recombination, and it is characterized in that: its sequence set becomes the aminoacid sequence among the sequence table SEQ ID No:4.