CN1823162A - Variant serine acetyltransferase and process for producing l-cysteine - Google Patents

Variant serine acetyltransferase and process for producing l-cysteine Download PDF

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CN1823162A
CN1823162A CNA2004800202648A CN200480020264A CN1823162A CN 1823162 A CN1823162 A CN 1823162A CN A2004800202648 A CNA2004800202648 A CN A2004800202648A CN 200480020264 A CN200480020264 A CN 200480020264A CN 1823162 A CN1823162 A CN 1823162A
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sat
halfcystine
ala
dna
gly
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CN100471949C (en
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柏木立己
开裕子
石川弘纪
铃木荣一郎
高木博史
M·K·齐野蒂洛夫
E·I·雷德基纳
M·M·谷斯野蒂纳
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Ajinomoto Co Inc
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Abstract

Culturing bacteria of Escherichia can produce O-acetyl serine, L-cysteine and derived sulfurous compounds. The bacteria have acetyl transferase variant maintained, and the variant is wild serine acetyl transferase with its amino acid sequence of 89-96 sites substituted with any one of the amino acids shown in SEQ ID Nos. 4-9 and with desensitization on the feedback inhibition L-cysteine generates.

Description

Variant serine acetyltransferase and the method for producing the L-halfcystine
Invention field
The present invention relates to microbiological industry and more specifically relate to the amino acid whose method of producing.More specifically, the present invention relates to participate in the purposes of the biosynthetic new anti-feedback enzyme of halfcystine.More specifically, the present invention relates to new anti-feedback variant serine acetyltransferase, intestinal bacteria (E.coli) bacterial strain that carries this enzyme and this bacterial strain of use method by fermentative production L-halfcystine.
Description of Related Art
Usually, utilized microorganism strains that obtains from natural origin or the described bacterial strain mutant of having been modified to improve L-amino acid throughput to come industrial production L-amino acid by fermentation process.
The a lot of technology that improve L-amino acid throughput are disclosed, for example by transform microorganism (seeing for example U.S. Patent No. 4,278,765) with recombinant DNA.These technology participate in the active of amino acid bio synthetic enzyme and/or make target enzyme (see for example day disclosure application No.56-18596 (1981), WO 95/16042 or U.S. Patent No. 5 to the feedback inhibition desensitization that is produced by the L-amino acid that produces based on increasing, 661,012 and 6,040,160).
In intestinal bacteria by L-Serine biosynthesizing L-halfcystine be serine acetyltransferase, cysK and cysM genes encoding by the cysE genes encoding called after-A and-O-acetylserine (mercaptan) the lyase isozyme of B carries out.Serine acetyltransferase (is also referred to as " SAT "; EC 2.3.1.30) catalysis forms O-acetyl-L-Serine by acetyl-CoA and L-Serine and play regulating effect (Escherichia coli and Salmonella in the halfcystine biosynthesizing by L-halfcystine feedback inhibition, Second Edition, Editor in Chief:F.C.Neidhardt, ASM Press, Washington D.C., 1996).
Denk D. separated with Bock A. from colibacillary to the L-halfcystine do not have the feedback SAT mutant of susceptibility and wild-type SAT (J.Gen.Microbiol., 1987,133 (Pt3), 515-25).The cysE gene mutation body shows the 767th single sequence change, causes the 256th methionine(Met) to be replaced into Isoleucine.This mutant secretion L-halfcystine.
Replace wild-type Met-256 with 19 other amino-acid residues among the intestinal bacteria SAT of cysE genes encoding, or the introducing terminator codon in most of the cases causes anti-feedback (fbr) phenotype with brachymemma 256-273C end region.Yet the SAT mutant protein does not keep the activity level (1998,64,5,1607-1611, WO 97/15673 for people such as Nakamori S., Appl.Environ.Microbiol.) of wild-type SAT.The bacterial strain of plasmid that carries the cysE gene of these changes produces the halfcystine be up to 200mg/l, comprises Gelucystine.
Obtain by PCR by random mutagenesis from the insensitive SAT mutant of colibacillary a lot of feedbacks.Identify sudden change along the whole aminoacid sequences of SAT, but all SAT mutant all show significantly reduced specific activity level (Takagi, people such as H., FEBS Lett., 1999,452,323-327).
People such as Mino K. (Biosci.Biotechnol.Biochem.1999,63,1, the 168-179) vital role of the C-terminal district that also shows SAT in the feedback inhibition that the L-halfcystine produces.The SAT of brachymemma, it cuts wild-type SAT between Ser 253 and Met 254, cause the disappearance of 20 amino-acid residues of C-terminal, and is more much smaller than wild-type SAT to the susceptibility of feedback inhibition.
Reported that also the bacterial strain that belongs to Escherichia (Escherichia) can produce the L-halfcystine, it is characterized in that the L-halfcystine decomposing system that is suppressed, for example the cysteine desulfhydrase activity is lower, the reservation of the SAT that the feedback inhibition that produces with the L-halfcystine reduces, for example the 256th methionine(Met) of the 256th of wild-type SAT is by the SAT mutant (JP11155571A2) of another radical amino acid replacement.
Have the recombinant serine Transacetylase that suddenlys change or have disappearance in amino acid 237-240,245-259 and 267-269C end sequence district in amino acid 97-100,164-169,237,239-240,245-259 and 267-269 sequence area and compare the susceptibility reduction that demonstrates the L-halfcystine with wild-type enzyme, and at United States Patent (USP) 6, open in 218,168.The following sudden change of being encoded by double mutant cysEXIV allelotrope among the SAT shows the good resistance (K to halfcystine i＞1000 μ M), have high relatively activity (0.453 μ M/ minute * mg): become G at the 721st A and cause Thr167 to become Ala167 and become TAG causing producing terminator codon rather than Met256 at 988-990 position ATG.The JM15 bacterial strain that transforms with cysEXIV allelotrope produces 2.3g/l L-halfcystine in charging batch fermentation method after 48 hours.Yet the optimum yield of L-halfcystine (3.9g/l) is to use the cysEDe1_255 mutant allele to obtain, thereby 18 amino acid in C-terminal district are blocked (United States Patent (USP) 6,218,168).
In general, the fbr phenotype of enzyme be with other amino-acid residue in the protein sequence replace one or more amino-acid residues and produce and these replace to reduce enzymic activitys.
Therefore the shortcoming of the enzyme mutant body that is obtained by method as mentioned above is that the activity of comparing the enzyme mutant body with wild-type enzyme reduces.Very clear, the activity of fbr phenotype enzyme mutant body need be kept in this area.
Summary of the invention
The purpose of this invention is to provide a kind of one or several sudden change that in wild-type serine acetyltransferase the 89th to 96 amino acids sequence, has, and the variant serine acetyltransferase of the feedback inhibition desensitization that the L-halfcystine is produced.
Another purpose of the present invention provides aforesaid variant serine acetyltransferase, and the aminoacid sequence that wherein is equivalent to the 89th to 96 of wild-type serine acetyltransferase is selected from SEQ ID NO:4,5,6,7,8 and 9 aminoacid sequence replaces.
Another purpose of the present invention provides aforesaid variant serine acetyltransferase, and wherein the wild-type serine acetyltransferase stems from intestinal bacteria;
Another purpose of the present invention provides aforesaid variant serine acetyltransferase, described mutant is included in one or more topagnosises, displacement, the insertion except that the 89th to 96 or adds one or several amino acid, wherein get rid of the 51st Asn and be replaced into Lys, the 91st Arg is replaced into His and the 233rd His is replaced into Tyr.
Another purpose of the present invention provides the DNA of the aforesaid variant serine acetyltransferase of coding.
Another purpose of the present invention provides the bacterium that belongs to Escherichia, and described bacterium transforms and have the ability of generation L-halfcystine with aforesaid DNA.
Another purpose of the present invention provides a kind of method of the L-of production halfcystine, and described method is included in cultivates aforesaid bacterium and collect the L-halfcystine from this substratum in the substratum.
SAT with any as mentioned above fbr sudden change can be called as " SAT mutant ", and the DNA of coding SAT mutant can be called as " cysE gene mutation body ", and the SAT that does not have a sudden change can be called " wild-type SAT ".
The accompanying drawing summary
Fig. 1 has shown the structure of primer SEQ ID NO:18 and 19.
Fig. 2 has shown the oligomerization structure of SAT.
Fig. 3 has shown the three-dimensional structure of SAT subunit.
Fig. 4 has shown the constructing plan in cysE gene mutation body storehouse.
Fig. 5 has shown the catalytic performance of SAT mutant.
The preferred embodiment of invention
The present invention relates to the structure of anti-feedback high active enzyme mutant, this enzyme plays a crucial role in intestinal bacteria biosynthesizing halfcystine.The invention still further relates to the completely random of using the cysE gene fragment and be combined to the method for a big group cysE gene mutation body.
Have that simultaneous radical amino acid replacement can produce protein mutant in the protein fragments of fbr sudden change, because the correct assessment of enzyme three-dimensional structure, described protein mutant has the activity level that approaches natural horizontal.
To be described in further detail the present invention now:
＜1〉SAT mutant and cysE gene mutation body
According to the SAT three-dimensional structure that the inventor obtains, disclosed and interact with the L-halfcystine and be responsible for the new SAT zone (see embodiment 1) of SAT L-halfcystine susceptibility necessity.
SAT mutant of the present invention and cysE gene mutation body obtain by random fragment-directed mutagenesis.A lot of sudden changes are that the randomization of 24 nucleotide fragments by the cysE gene obtains in the cysE gene.In this fragment coding SAT protein sequence from the zone of 96 l-asparagines of the 89th arginine to the.So each two adjacent amino acids residues of randomization are the randomization in 4 experimentations subsequently of proteic whole 89-96 zone.
The fbr variant of selecting and screen SAT mutant that allow to select to have the different biological activity level subsequently with recombinant clone of the cysE gene mutation body that is cloned into expression vector is up to and comprises the activity level of wild-type (wt) SAT that disinthibites.As wild-type SAT, comprise SAT (SEQ ID NO:2) from intestinal bacteria (EC-2.3.1.30).
The present invention includes the aminoacid sequence of SAT mutant with fbr phenotype.The SAT mutant can be introduced the acquisition of wild-type cysE gene by using usual way to suddenly change.Wild-type cysE gene can be by for example utilizing the PCR (polymerase chain reaction based on the primer of the nucleotide sequence of this gene preparation; With reference to White, people such as T.J., Trends Genet., 5,185 (1989)) obtain.As wild-type cysE gene, comprise colibacillary cysE gene (GenBank registration number NC_000913.1; Nucleotide in the sequence of gi:16127994 numbers 3779368 to 3780189).Encoding wild type SAT or can obtain in a similar manner from the gene of the SAT mutant of other microorganism.The introducing wild-type of will suddenling change cysE gene can be undertaken by for example site-directed mutagenesis.
The the 89th to 96 amino acids sequence contains one or several sudden change in the SAT mutant of the present invention.Preferably, the 89th to 96 amino acids sequence is any one sequence shown in the SEQ IDNO:4 to 9 in the SAT mutant of the present invention.Table 1 has been illustrated the corresponding aminoacid sequence of SAT mutant of the present invention, and wherein Val-95 and Asp-96 are by Arg-95 and Pro-96, Gly-95 and Gly-96 or Leu-95 and Pro-96 replacement; Ala-94 is replaced by Thr-94; Arg-89 is replaced by Pro-89; Arg-89 and Thr-90 are replaced by Ser-89 and Leu-90; With intestinal bacteria wild-type SAT.Table 1 has also shown the example of the nucleotide sequence of these aminoacid sequences of encoding.
Table 1
The allelotrope of cysE gene The sequence (89 → 96 amino acid) in SAT albumen randomization zone SEQ ID NO： The segmental dna sequence dna of the randomization of cysE gene (5 ' → 3 ') SEQ ID NO：
Wt Arg Thr Arg Asp Pro Ala Val Asp 3 cgt acc cgc gac ccg gca gtc gat 10
CysE5 Arg Thr Arg Asp Pro Ala Arg Pro 4 cgt acc cgc gac ccg gca AGA CCC 11
CysE12 Arg Thr Arg Asp Pro Ala Gly Gly 5 cgt acc cgc gac ccg gca GGT GGT 12
CysE15 Arg Thr Arg Asp Pro Ala Leu Pro 6 cgt acc cgc gac ccg gca CTA CCA 13
CysE1 Pro Thr Arg Asp Pro Ala Val Asp 7 CCC acc cgc gac ccg gca gtc gat 14
CysE102 Arg Thr Arg Asp Pro Thr Val Asp 8 cgt acc cgc gac cct ACA gtc gat 15
CysE142 Ser Leu Arg Asp Pro Ala Val Asp 9 AGT CTA cgc gac ccg gca gtc gat 16
The SAT mutant can be included in one or more topagnosises, displacement, the insertion except the 89th to 96 or add one or several amino acid, and prerequisite is that the SAT activity does not reduce.Term " SAT activity " meaning is the catalysis ethanoyl is transferred to the L-Serine from acetyl-CoA a reactive activity.Can use for example Kredich, N.M. and Tomkins, G.M. (J.Biol.Chem.1966,241,21,4955-4965) method of describing is measured the SAT activity.Get rid of and to have the 51st Asn and be replaced into Lys, the 91st Arg and be replaced into the triple mutant body that His and the 233rd His are replaced into Tyr, because Takagi, people such as H. (FEBS Lett., 1999,452, described mutant had been described before 323-327).
The position that " several " amino acid whose number depends on amino-acid residue in the protein three-dimensional structure is with the amino acid type and different.This is because the following reasons.That is to say that some amino acid have high homology each other, and this amino acid whose difference can not influence proteic three-dimensional structure greatly.Therefore, SAT mutant of the present invention can be to have with whole amino-acid residues of forming SAT to be not less than 30 to 50%, preferably is not less than 50 to 70%, more preferably is not less than 70 to 90%, most preferably be not less than 95% homology, and have the active mutant of fbr SAT.Perhaps, the number of " several " amino-acid residue specifically can be 2 to 20 referred in this, and is preferred 2 to 10, more preferably 2 to 15.
In the present invention, " aminoacid sequence that is equivalent to the 89th to 96 bit sequence " meaning is the aminoacid sequence that is equivalent to the 89th to 96 amino acids sequence in the aminoacid sequence of intestinal bacteria wild-type SAT.The position of amino-acid residue can change.For example, if an amino-acid residue inserts the N-terminal portions, the so intrinsic amino-acid residue that is positioned at 89 becomes 90.In this case, the 90th amino-acid residue is appointed as and is equivalent to original the 89th amino-acid residue of the present invention.
Therefore, the change of aforesaid those SAT is generally guarded, thereby keeps the SAT activity.Displacement changes the change comprise that at least one residue has been removed and has inserted in its position different residues in the aminoacid sequence wherein.The examples of amino acids that can replace original amino acid in the SAT albumen and be considered to conservative substitution comprises: replace Ala with ser or thr; Replace arg with gln, his or lys; Replace asn with glu, gln, lys, his, asp; Replace asp with asn, glu or gln; Replace cys with ser or ala; Replace gln with asn, glu, lys, his, asp or arg; Replace glu with asn, gln, lys or asp; Replace gly with pro; Replace his with asn, lys, gln, arg, tyr; Replace ile with leu, met, val, phe; Replace leu with ile, met, val, phe; Replace lys with asn, glu, gln, his, arg; Replace met with ile, leu, val, phe; Replace phe with trp, tyr, met, ile or leu; Replace ser with thr, ala; Replace thr with ser or ala; Replace trp with phe, tyr; Replace tyr and replace val with his, phe or trp with met, ile, leu.
Identical with the aforesaid SAT mutant basically proteic DNA of coding can obtain by the following method, for example pass through to modify this nucleotide sequence, thereby for example make one or more amino-acid residues in concrete site be lacked, replace, insert or add by site-directed mutagenesis method.The DNA of Xiu Shiing can handle by common known sudden change and obtain as mentioned above.This sudden change is handled and is comprised the method for handling the DNA that contains external cysE gene mutation body, for example use azanol, with the processing method of microorganism, for example handle the bacterium that belongs to Escherichia of carrying the cysE gene mutation body, N-methyl-N '-nitro-N-nitrosoguanidine (NTG) and the nitrous acid of described mutagens as being generally used for this processing with uviolizing or mutagens.
The displacement of aforesaid Nucleotide, disappearance, insertion or add the sudden change (mutant or variant) that also comprises natural generation, for example random mutation, the species of bacterium that carry SAT or the difference of genus.
Identical with the SAT mutant basically proteic DNA that encodes can obtain by following method, hybridize with known cysE gene order (SEQ ID NO:1) or its part under stringent condition from the cellular segregation of carrying the SAT mutant, and coding has the active proteic DNA of SAT as probe.
Term " stringent condition " comprises the condition that forms so-called specific hybrid and do not form non-specific hybridization.Be difficult to use any numerical value accurately to express this condition.Yet for example stringent condition comprises following condition, has high homology under this condition each other, for example has the DNAs hybridization that is not less than 50% homology, and have each other than above-mentioned homology more the DNAs of low homology do not hybridize.Perhaps, stringent condition is included in the condition that DNA is hybridized each other with the salt concn under the usual terms that is equivalent to washing in the southern blotting technique hybridization under this condition, promptly 60 ℃, 1 * SSC, 0.1%SDS, preferred 0.1 * SSC, 0.1%SDS.
The gene that to hybridize under condition as mentioned above comprises gene with the terminator codon that produces in the gene coding region and owing to the active centre sudden change does not have active gene.Yet, connect this gene with obtainable expression vector on the market, and the SAT activity of institute's expressing protein can easily be eliminated this inconvenience.
＜2〉bacterium that belongs to Escherichia of the present invention.
Bacterium of the present invention is the bacterium that belongs to Escherichia of wherein introducing aforesaid cysE gene mutation body.Term " bacterium that the belongs to Escherichia " meaning is the bacterium that is classified as Escherichia according to the known classification of microbiological technique personnel.The bacterium example that belongs to Escherichia comprises intestinal bacteria.
" bacterium with the ability that produces the L-halfcystine " meaning is a kind of bacterium, and when cultivating bacterium of the present invention in substratum, described bacterium has the ability of accumulation L-halfcystine in substratum.Can give or improve the ability that produces the L-halfcystine by breeding.Term " bacterium with the ability that the produces the L-halfcystine " meaning also is a kind of bacterium as used herein, and it can produce in substratum and accumulate than wild-type or the more substantial L-halfcystine of parent strain.The example of wild-type e. coli bacterial strain comprises coli strain MG1655 (ATCC47076, ATCC700926, VKPM B-6195), K-12 or the like.The MG1655 bacterial strain can obtain from American type culture collection (VA 20110, the U.S. for 10801 University Boulevard, Manassas).
Can introduce the cysE gene mutation body by for example transforming the bacterium that belongs to Escherichia with the recombinant DNA that is included in carrier that function is arranged in the bacterium that belongs to Escherichia and cysE gene mutation body.Also can introduce the cysE gene mutation body by on karyomit(e), replacing the cysE gene with the cysE gene mutation body.
The suitable carrier example of introducing the cysE gene mutation body comprises plasmid vector such as pBR322, pMW118, pUC19 or the like, phage vector as 11059,1BF101, M13mp9 or the like and transposon such as Mu, Tn10, Tn5 or the like.
DNA is introduced the bacterium that belongs to Escherichia can be by for example D.A.Morrison (Methods in Enzymology, 68,326 (1979)) method or handle the bacterial receptor cell to increase the infiltrative method (Mandel of DNA with calcium chloride, M. and Higa, A., J.Mol.Biol., 53,159, (1970)) or the like carry out.
By the cysE gene mutation body is introduced the L-halfcystine amount that the bacterium that belongs to Escherichia that produces the L-halfcystine as mentioned above can increase generation.The bacterium of having introduced the cysE gene mutation body equally, in advance can be endowed the ability that produces the L-halfcystine.
Have the bacterium example that belongs to Escherichia that produces the L-cysteine activity and comprise coli strain JM15, it is encoded and resists the different cysE allelotrope of feedback serine acetyltransferase to transform (United States Patent (USP) 6,218,168); The coli strain W3110 (United States Patent (USP) 5,972,663) of the proteic gene that having encodes is suitable for secretory cell toxicant, overexpression; Has active coli strain W3110 that increases of positive transcriptional regulatory agent (PCT application WO 0127307A1) of the halfcystine regulon of low active coli strain of cysteine desulfhydrase (JP11155571A2) and cys B genes encoding or the like.
＜3〉method of the present invention.
Method of the present invention comprises a kind of method of the L-of production halfcystine, and it may further comprise the steps, and cultivates bacterium of the present invention in substratum, allow to produce the L-halfcystine and collect the L-halfcystine in the cumulative L-halfcystine from substratum.
In the present invention, can carry out with conventional fermentation process, wherein use microorganisms amino acid from culture medium culturing, collection and purifying L-cysteine or the like.
The substratum that uses among the present invention can be synthetic medium or natural medium, carbon source, nitrogenous source, sulphur source and mineral substance and if necessary comprises the appropriate amount nutrition that microorganism growth is required as long as this substratum comprises.
Carbon source can comprise various carbohydrate such as dextrose plus saccharose and various organic acid.According to the assimilation pattern of use microorganism, can use alcohol, comprise ethanol and glycerine.
As nitrogenous source, can use the organism of fermentation of various ammonium salts such as ammonia and ammonium salt, other nitrogen compound such as amine, natural nitrogenous source such as peptone, soya hydrolysate and digestion.As the sulphur source, can use vitriol and thiosulphate.
As mineral substance, can use a potassiumphosphate, sodium-chlor, calcium chloride, magnesium salts, ferrous salt, manganese salt or the like.
Can in substratum, add other nutrition if necessary.For example, if microorganism growth needs methionine(Met) (methionine(Met) auxotroph), then can in substratum, add the capacity methionine(Met).
Preferably under aerobic conditions cultivate, shake and the stir culture thing preferred 37 to 40 ℃ as under 20 to 42 ℃ temperature, ventilating.The pH of culture normally 5 to 9, preferred 6.5 to 7.2.The pH of culture can use ammonia, lime carbonate, various acid, various alkali and damping fluid to regulate.Usually, cultivation in 1 to 5 day causes cumulative target L-amino acid in the liquid nutrient medium.
After the cultivation, can from liquid nutrient medium, remove solid such as cell, can collect target L-amino acid then and use ion-exchange, concentrated and crystallization method purifying with centrifugal or membrane filtration.
Embodiment
Will be with reference to the present invention of following non-limiting examples specific explanations.
The three-dimensional structure of embodiment 1:SAT
Following mensuration of carrying out the SAT three-dimensional structure.
＜1〉preparation of seleno methionyl SAT and purifying
In order in intestinal bacteria, to produce reorganization SAT, use carrier pQE* to make up the cysE expression of gene plasmid of coding SAT.This carrier produces by modifying coli expression carrier pQE30 (Qiagen).
Use pQE30 to implement PCR as template with Oligonucleolide primers SEQ ID NO:17 and 18.Nucleotide sequence based on carrier pQE30 (www.qiagen.com) makes up primer.Primer SEQ ID NO:18 contains SphI recognition site and the mispairing that Fig. 1 describes.The unique amplified band that digests 150bp with restriction enzyme XhoI and SphI is to reclaim the fragment of 80bp.This fragment is connected with the big fragment of pQE30 that has digested with the same restrictions restriction endonuclease in advance.The plasmid pQE* that generates contains the SphI cleavage site at the initiator codon place, rather than the sequence of six the continuous His amino-acid residues of encoding and BamHI site.
With the genomic dna of bacillus coli DH 5 alpha and the Oligonucleolide primers SEQ ID NO:19 and 20 that makes up according to cysE gene nucleotide series (GenBank registration number M15745), prepare the dna fragmentation of cysE gene by PCR.Primer SEQ ID NO:19 contains SphI recognition site and the mispairing that Fig. 1 describes.Primer SEQ ID NO:20 contains the HindIII recognition site at 5 ' end.With unique amplified band of restriction enzyme SphI and HindIII digestion 840bp, be connected with the big fragment of pQE* that has digested with the same restrictions restriction endonuclease in advance then and make up plasmid pQE-SAT.As a result, the proline(Pro) that the natural serine residue of the 2nd TCG triplet coding is encoded by the CCG triplet among the reorganization SAT replaces.
Cultivating the intestinal bacteria B834/DE3 cell (Novagen Co., the U.S.) that carries SAT expression plasmid pQE-SAT at 37 ℃ with the LB substratum that contains penbritin (5 μ g/ml) [20g/l bacto-tryptone, 10g/l bacterium with yeast extract, 20g/l NaCl] spends the night.Subsequently, this inoculum is transferred to [15.1g/l Na in the M9 substratum that contains selenomethionine (40 μ g/ml) and penbritin (50 μ g/ml) 2HPO 4.12H 2O, 3g/lKH 2PO 4, 0.5g/l NaCl, 1g/l NH 4Cl, 4g/l glucose, 2ml 1M MgSO 4, 1ml 1M CaCl 2, 1ml FeCl 3(3.5mg/ml), 2.4ml VitB1 (1mg/ml)].Continue to cultivate at 37 ℃.When the 560nm optical density(OD) reaches 0.6, add 1mM sec.-propyl-1-β-D-galactopyranoside and induce seleno methionyl SAT to express.Centrifugal results cultured cells.
Cell precipitation is suspended in 50mM Tris-HCl (hydrochloric acid Tutofusin tris), 2mM (±)-dithiothreitol (DTT), 5mM EDTA-Na 2Carry out fragmentation in (edta disodium dihydrate) solution and by ultrasonic.Supernatant liquor is 60 ℃ of incubations 10 minutes and cooled on ice 10 minutes.The centrifugal precipitation of removing.With ammonium sulfate precipitation purifying dissolved seleno methionyl SAT solution and by 50mM Tris-HCl, pH7.5,2mM (±) dithiothreitol (DTT) and 5mMEDTA-Na 2The solution dialysis.Then it is applied to use 50mM Tris-HCl, pH7.5,2mM (±) dithiothreitol (DTT) and 5mM EDTA-Na 2Equilibrated anion-exchange column (ResourceQ 6ml, Amersham Pharmacia Biotech, Tokyo, Japan).After washing this post with the same buffer of 1 column volume, with 0 to 1M linear gradient sodium-chlor in 10 column volumes, with 6ml/ minute flow velocity wash-out seleno methionyl SAT.Collect the fraction of wash-out.After adding ammonium sulfate, this solution is applied to 0.75M ammonium sulfate, 50mM Tris-HCl, pH7.5,2mM (±) dithiothreitol (DTT) and 5mM EDTA-Na 2Equilibrated hydrophobic interaction chromatography post (HiPrep16/10Butyl; Amersham Pharmacia Biotech, Tokyo, Japan).After washing this post with the same buffer of 1 column volume, with 0.75 to 0M linear gradient ammonium sulfate in 10 column volumes, with 5ml/ minute flow velocity wash-out seleno methionyl SAT.For buffer exchange, use 50mM Tris-HCl, pH7.5,2mM (±) dithiothreitol (DTT), 5mM EDTA-Na 2The fraction that the solution dialysis merges.
＜2〉crystallization
Carry out the crystallization of seleno methionyl SAT.Obtain growing to size (0.2 * 0.2 * 0.2mm) the cubic system that is enough in about 5 days by X-ray diffraction.
＜3〉data gathering and processing
Use is at Photon Factory of the National Laboratory for HighEnergy Physics, Tsukuba, the Quantam 4R CCD detector (ADSC) of the last installation of beamline (beam line) 6B of Japan is collected seleno methionyl SAT crystalline multi-wavelength anomalous dispersion (MAD) data.Collect before the X ray diffracting data record X-ray fluorescence spectra and the optimal wavelength that is used to select to carry out the MAD data gathering.During measured X ray fluorescence spectra and data gathering, at containing 35v/v%2-methyl-2, behind the freezing solvent balance of 4-pentanediol, 0.1M2-(N-morpholino) ethyl sulfonic acid-NaOH (pH6.2) and 1mM L-halfcystine, seleno methionyl SAT crystal dodges cooling (flash-cool) with 95K.With 0.9791 (flex point of fluorescence spectrum, f ' minimum), 0.9789 (f " maximum), 0.9500 (remote high energy wavelength) and 1.0500 (remote low energy wavelength) collection data.Whole four groups of data are collected with crystal-detector distance and 1.0 ° of amplitudes of each image of use of 220mm from the phase allomeric.Service routine DPS/MOSFLM processing diffraction data (Rossman, M.G. and vanBeek, C.G. (1999) Acta Crystallogr.Sect.D55,1631-1640).The crystalline diffraction of seleno methionyl SAT is up to the resolving power of 2.7 .It belongs to spacer R3, unit cell dimension a=101.2 , c=223.2 .Each asymmetric cell of crystalline contains four seleno methionyl SAT molecules (molecule A, B, C and D), and it has 35.3% solvent.
＜4〉MAD becomes phase (Phasing) and improves mutually
(760-763) program SCALEIT measures the MAD data for Beiley, S. (1994) Acta Crystallogr.Sect.D50 with CCP4.Have in 36 Se sites of expecting in the asymmetric cell 19 service routine SOLVE determine (Terwilliger, T.C. and Berendzen, J. (1996) Acta Crystallogr.Sect.D52,743-748).Calculate initial MAD phase with program MLPHARE among the CCP4.The final numeral of eigenwert becomes 0.541 (40.0-2.7 resolving power).
Program DM improves the MAD phase among the use CCP4.At first, under the condition of 30% solvent, keep on solvent delustring (flattening) step, wherein use 40.0-2.7 resolution data.Although electron density map shows albumen-solvent border relatively clearly, it of poor quality and be difficult to explain.Go up the explanation that service routine QUANTA (Accelrys Inc.) carries out electron density map and modelling at Octane graphics workstation (Silicon Graphics Inc.).
Next step further improves phase by the average step of molecule.Noncrystal symmetry (NCS) parameter is tentatively determined in position by stack Se atom.Molecule A and B and molecule C and D connect by noncrystal 2 solid axles respectively.Molecule A and C and molecule B and D connect by 1/2 translation along the c axle respectively.Refining NCS parameter in the average step process of molecule of under 40.0-2.7 resolving power, carrying out.Complete successfully the average step of molecule, thereby produce the average characteristics numeral (0.644) of combination phase and the improvement value of relation conefficient (0.617 → 0.868).Figure is enhanced and shows many secondary structures.On this figure, make up the three-dimensional structure of seleno methionyl SAT.
＜5〉three-dimensional structure of SAT
Fig. 2 has shown the oligomerization structure of SAT.As shown in Figure 1, SAT forms and has 32 point-symmetric six poly structures.Three relevant dimers of noncrystal 2 solid axles connect by crystallography 3 solid axles.
Fig. 3 has shown the three-dimensional structure of SAT subunit.The SAT subunit is formed by two structural domains.The N-terminal structural domain contains many alpha-helixs.N-terminal structural domain spiral closely interacts each other in the SAT sexamer, and plays an important role in sexamer stable.The structure of C-terminal structural domain is the parallel β spirane structure of left hand territory (L β H).As if L β H structure is folded into the major coil prism, and volume is the left hand helix around prismatic surface.The face of L β H structure is formed by three plane parallel beta sheets.In SAT sexamer structure, between the adjacent L β H structural domain big breach is arranged, described breach connects by crystallography 3 solid axles.(Takagi, people such as H. (1999) FEBS Lett.452 323-327) extensively is distributed in this breach responsible many residues that feedback inhibition is desensitized.Therefore, this breach may be the essential zone of feedback inhibition.Therefore the zone of containing residue 89-96 also is present in this breach, and is chosen as and is used to carry out mutagenesis.
Embodiment 2: the SATs mutant that obtains to have the zone that contains randomization residue 89-96
＜1〉randomization fragment-site-directed mutagenesis
At first, obtain two plasmid pMW-P OmpCAnd pMW-P NlpD
Plasmid pMW-P OmpCBe to obtain by the PaeI-SalI site that the 0.3kb dna fragmentation that will contain the ompC gene promoter region is cloned into plasmid pMW118.The dna fragmentation that contains the ompC promotor is to use primer P4 (SEQ ID NO:21) and P6 (SEQ ID NO:22) and uses chromosomal DNA from coli strain MG1655 to obtain by PCR as template.The ompC promoter region is as the hybridization target of primer 4.Arbitrary sequence can replace the ompC promotor, as long as primer P4 is designed to and this sequence hybridization.
Plasmid pMW-P NlpDBe to obtain by the PaeI-SalI site that the 0.3kb dna fragmentation that will contain the nlpD gene promoter region is cloned into plasmid pMW118.The dna fragmentation that contains the nlpD promotor is to use primer P5 (SEQ ID NO:23) and P 7 (SEQ ID NO:24) and uses chromosomal DNA from coli strain MG1655 to obtain by PCR as template.The nlpD promoter region not with above-mentioned primer hybridization.Arbitrary sequence can replace the nlpD promotor, if primer P4 not with this sequence hybridization.
Complete wild-type cysE gene is with primer P1 (SEQ ID NO:25) and P2 (SEQ IDNO:26), and use from the chromosomal DNA of coli strain MG 1655 as template by the PCR acquisition.The dna fragmentation (0.83kb) that obtains is cloned into plasmid pMW-P OmpCAnd pMW-P NlpDThe SalI-XbaI site, produce plasmid pMW-P respectively OmpC-cysE and pMW-P NlpD-cysE.
The Pyrobest that is used for pcr amplification TMArchaeal dna polymerase obtains from Takara Shuzo Co. (Japan), and uses under the condition that supplier recommends.
For structure has the cysE gene mutation body storehouse of 285 to 291 of randomizations, at first, by the 1st cysE gene fragment of pcr amplification coding SAT to the sequence of 102 amino acids residues.Carry out PCR, wherein use plasmid pMW-P OmpC-cysE as template and contain 6 randomization Nucleotide primer P3 (SEQ ID NO:27) and with the primer P4 (SEQ ID NO:21) (referring to Fig. 1) of ompC gene promoter region sequence homology.Primer P3 fixed 19-Nucleotide 3 ' end sequence and cysE gene A sp-96 codon downstream sequence homology, and fixed 20-Nucleotide 5 ' terminal and cysE gene Val-95 upstream sequence homology.Equally, primer P3 contains 6 random nucleotides describing with letter " n " among the SEQ ID NO:27.In the PCR solution (50 μ l) of each (10pmol) that contain two kinds of primers, add 20ng plasmid pMW-P OmpC-cysE is as template.Carry out 25 PCR circulation (96 ℃ 0.4 minute, 60 ℃ 0.4 minute, 72 ℃ 1 minute) with 2400 type DNA thermal cyclers (Perkin-ElmerCo., Foster City, California, the U.S.).Obtain the 0.3kbDNA fragment in 25 working cyclees of PCR.
Second step, by the agarose gel electrophoresis purifying 0.3kbp dna fragmentation that obtains of step and used as " primer " in the primer extension step in front, obtain the full sequence of cysE gene with increase by 10 round-robin (96 ℃ 1 minute, 40 ℃ 1 minute, 72 ℃ 0.5 minute).With plasmid pMW-P NlpD-cysE is used as template to prevent the amplification at PCR wild-type cysE gene in the 3rd step.
The 3rd step, in the fresh reactant mixture (40 μ l) of the primer P4 that contains 50pmol and ompC gene promoter region sequence homology (SEQ ID NO:21) (referring to Fig. 4) and primer P2 (SEQ ID NO:26), add 10 μ l aliquots containig reaction mixtures, and carry out other 15 circulations (94 ℃ 0.5 minute, 57 ℃ 0.5 minute, 72 ℃ 2 minutes).
By the 0.83kbpDNA fragment in agarose gel electrophoresis purifying coding cysE gene mutation body storehouse, with SalI and XbaI digestion, then with the pMW-P that digests with the same restrictions restriction endonuclease OmpCCarrier connects.
With the about 100ng pMW-P of gained OmpC-cysE (at random) is used for the conversion of intestinal bacteria recipient cell.
＜2〉separation of new cysE gene mutation body
With coli strain LE392 cysE ∷ Km RCarry out further experiment as F-strain.This bacterial strain is to be obtained by destroying the cysE gene by coli strain LE392 people such as (, MolecularCloning, 1989) J.Sambrook.Use bacterial strain JC7623 to carry out the destruction of cysE gene by introducing kalamycin resistance gene, as Kushner, S.R., H.Nagaishi and A.J.Clark. (Proc.Natl.Acad.Sci.USA, 1972,69:1366-1370) described.
Use plasmid pMW-P OmpC-cysE (at random) storehouse transformed into escherichia coli F-strain LE392cysE ∷ Km ROn the M9 agar plate that replenishes 0.5% glucose and 50mg/l methionine(Met), by coli strain LE392 cysE ∷ Km RThe complementation of middle karyomit(e) cysE sudden change selects coding that the cysE gene mutation body of active SAT is arranged.
The clone who tests all acquisitions supplies the ability of halfcystine auxotroph, and chooses about 15 variants.Purifying is also used the dna sequence dna of double deoxidating chain termination measuring cysE gene structure part from these clones' plasmid.For measuring the SAT activity, transform halfcystine auxotroph, bacterial strain LE392 cysE ∷ Km again with these plasmids R
Obtain the 767th cysE gene mutation body (cysE256) by the standard side-directed mutagenesis with single sequence change, thereby cause replacing methionine(Met) 256 (DenkD. and Bock A., J.Gen.Microbiol., 1987 with Isoleucine, 133 (Pt 3), 515-25).
Embodiment 3: amino-acid substitution is to the influence of SAT catalytic performance
Use Kredich, N.M. and Tomkins, G.M. (J.Biol.Chem., 1966,241,21,4955-4965) method of describing is measured the catalytic performance of SATs mutant, and this method has been done slight change.The acetyl-CoA that uses and other chemical reagent are from Sigma ChemicalCo. (St.Louis, MO, the U.S.).
For measuring the activity of SAT mutant, the Bacillus coli cells LE392 cysE ∷ Km of recombinant plasmid will be carried RGrowth, with 0.14M NaCl solution washing and was resuspended in the 2ml damping fluid (pH7.5,50mM potassiumphosphate and 100mM KCl) until late period exponential phase of growth in 5ml M9 substratum.The supersound process cell also precipitates with the 13000rpm centrifugation.With 5 times of saturated (NH of volume 4) 2SO 4Precipitate the protein fractions that contains SAT in the gained supernatant liquor, and precipitation is dissolved in the 2ml damping fluid (pH7.5,50mM potassiumphosphate and 100mM KCl).The solution that obtains is added into 0.1ml reaction mixture (500mM Tris-HCl (pH8.5), 5mM L-Serine, 0.1mM acetyl-CoA) and 37 ℃ of incubations 10 minutes.By adding 0.3ml ethanol termination reaction, centrifugal with 13000rpm subsequently.0.95ml 0.24mM DTNB (5,5-dithio-two-2-nitrobenzoate) solution is added in the supernatant liquor, and with mixture incubation 15 minutes.Measure the SAT activity by the absorbancy of measuring 412nm.Table 2 and Fig. 5 provide the data that obtain.
Table 2
CysE gene on the plasmid Specific activity, relative unit Residual activity in the presence of 25 μ M halfcystines, % 50% suppresses (I 50) semicystinol concentration, μ M
Wild type cysE cysE256 cysE5 cysE12 cysE15 cysE1 cysE102 cysE142 2300 1400 900 600 480 400 650 2600 0 88 100 95 100 93 85 38 5 120 ＞700 ＞700 ＞700 ＞700 130 15
As the data visible that provides from table 2 and Fig. 5, cysE gene mutation body, especially cysE5, cysE12, cysE15 and the cysE1 of the coding SAT mutant of acquisition show that the feedback inhibition that the L-halfcystine is produced does not have susceptibility.This SATs mutant of the feedback inhibition complete resistance that the L-halfcystine is produced can be used for using the bacterium that produces the L-halfcystine to prepare the L-halfcystine.
Embodiment 4:cysE gene mutation body is expressed and is improved the influence that the L-halfcystine is produced
With the parent strain of coli strain MG1655 (ATCC47076, ATCC700926) as the L-halfcystine production impact evaluation of assessment cysE gene mutation body expression raising.
With plasmid pMW-P OmpC-cysEX (contain the cysEX gene mutation body of coding SAT mutant, wherein Thr167 is replaced by Ala, and it is at United States Patent (USP) 6,218,168 in describe) and pMW-P OmpC-cysE5 imports coli strain MG1655.The bacterial strain MG1655/pMW-P that is produced OmpC-cysEX and MG1655/pMW-P OmpC-cysE5 spends the night in 34 ℃ of shaking culture in the 2ml nutrient liquid substratum that replenishes the 100mg/l penbritin.The culture that 0.2ml is obtained is inoculated in the 2ml fermention medium that contains penbritin (100mg/l) in 20 * 200mm test tube, and with rotary shaker with 250rpm in 34 ℃ of cultivations 42 hours.Fermention medium composed as follows: 15.0g/l (NH 4) 2SO 4, 1.5g/l KH 2PO 4, 1.0g/l MgSO 4, 20.0g/l CaCO 3, 0.1mg/l VitB1,1%LB, 4% glucose, 300mg/l L-methionine(Met) and 0.5g/l Na 2S 2O 3
After the cultivation, use Gaitonde, the method that M.K. (Biochem.J., 104:2,627-33 (1967)) describes is measured the amount of cumulative L-halfcystine in the substratum.Table 3 provides the data that obtain.
Table 3
Bacterial strain The amount of L-halfcystine, g/l
MG1655/pMW-P ompC-cysEX MG1655/pMW-P ompC-cysE5 0.27 0.37
As can be seen from Table 3, the overexpression of cysE5 gene mutation body has improved the halfcystine throughput of this bacterial strain among the coli strain MG1655.
Industrial applicibility
According to the present invention, provide the variant serine acetyltransferase of the feedback inhibition desensitization that Cys is produced. In addition, according to the present invention, can increase the Cys production capacity of the bacterium that belongs to Escherichia.
Sequence table
<110>Ajinomoto Co.，Inc.
＜120〉method of variant serine acetyltransferase and production L-halfcystine
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aaa tac tca acc ccg ttg tta tac ctg aag ggt ttt cat gcc ttg cag 336
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Claims (7)

1. variant serine acetyltransferase, the aminoacid sequence that wherein is equivalent in the wild-type serine acetyltransferase the 89th to 96 contains one or several sudden change, and the feedback inhibition desensitization that the L-halfcystine is produced of wherein said variant serine acetyltransferase.
2. according to the variant serine acetyltransferase of claim 1, the aminoacid sequence that wherein is equivalent to the 89th to 96 of wild-type serine acetyltransferase is selected from SEQ ID NO:4,5,6,7,8 and 9 aminoacid sequence replaces.
3. according to the variant serine acetyltransferase of claim 1, wherein the wild-type serine acetyltransferase stems from intestinal bacteria.
4. according to the variant serine acetyltransferase of claim 1, it is included in one or more topagnosises, displacement, the insertion except that the 89th to 96 or adds one or several amino acid, wherein gets rid of the 51st Asn and is replaced into that Lys, the 91st Arg are replaced into His and the 233rd His is replaced into Tyr.
5. the DNA of the variant serine acetyltransferase of the claim 1 of encoding.
6. bacterium that belongs to Escherichia, described bacterium transforms with the DNA of claim 5 and wherein said bacterium has the ability that produces the L-halfcystine.
7. method of producing the L-halfcystine, it is included in the bacterium of cultivating claim 6 in the substratum, and collects the L-halfcystine from this substratum.
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JP2009118740A (en) 2006-03-03 2009-06-04 Ajinomoto Co Inc Method for producing l-amino acid
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