CN104630300A - Production method of L-amino acid - Google Patents
Production method of L-amino acid Download PDFInfo
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- CN104630300A CN104630300A CN201310566206.6A CN201310566206A CN104630300A CN 104630300 A CN104630300 A CN 104630300A CN 201310566206 A CN201310566206 A CN 201310566206A CN 104630300 A CN104630300 A CN 104630300A
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Abstract
The invention relates to a production method of L-amino acid. The invention discloses a method for increasing expression or activity of glutaminase YbaS in L-amino acid producing bacteria to improve the yield of L-amino (such as L-lysine, L-threonine and L-tryptophan) for the first time.
Description
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to a kind of L-amino acids production method.
Background technology
Kind more than 20 can be had in the world at present with the amino acid of fermentative Production.Amino acid alpha-carbon atom connects hydrogen atom, carboxyl and amino and side chain with covalent linkage respectively.Side chain is different, amino acid whose different in kind.Amino acid is widely used in food, feed, medicine industry, comprising: nutrient fortified food freshener (tryptophane is in wheat for Methionin, Threonine); Msg powder type and aspartic acid, phenylalanine and aspartic acid can be used for manufacturing dipeptide sweetener low in calories (α-aspartame); The indispensable amino acids such as methionine(Met) can be used for manufacturing animal-feed; Multiple mixed amino acid is by infusion treatment nutrition or Metabolic disorder.
Amino acid whose production method has multiple, comprises direct fermentation, enzyme process (utilizing the enzyme of microorganism cells or microorganism generation to manufacture amino acid), extraction method (proteolysis extracts from hydrolyzed solution) etc.Traditional extraction method, enzyme process and chemical synthesis due to the cost of precursor high, complex process, is difficult to the object reaching suitability for industrialized production.Along with the structure of carrier, receptor system and the day by day perfect of outer-gene recombinant technology, amino acid bio engineering bacteria be built with further development.
Summary of the invention
The object of the present invention is to provide a kind of L-amino acids production method.
In a first aspect of the present invention, provide a kind of method improving the L-amino acid production of L-amino acids production bacterium, described method comprises: in L-amino acids production bacterium, improve L-Glutamine deaminase YbaS expression or activity; Such as, process LAN (external source) L-Glutamine deaminase YbaS in L-amino acids production bacterium; Cultivate this bacterial strain, thus produce L-amino acid.
In a preference, described L-amino acid includes, but is not limited to: 1B, L-Trp, L-threonine.
In another preference, the aminoacid sequence of described L-Glutamine deaminase YbaS is: (a) GenBank accession number: NP_415018.1; Or its congenerous variant, as by (a) polypeptide through one or more (as 1-20; Preferably 1-10; Preferably 1-4; More preferably 1-3; More preferably 1-2) replacement of amino-acid residue or interpolation formed, and with the polypeptide of (a) polypeptide congenerous.
In another preference, described method comprises:
A () provides a kind of recombinant plasmid, comprise a recombinant expression cassettes in described recombinant plasmid, and this recombinant expression cassettes contains: L-Glutamine deaminase YbaS encoding gene; With
B () by the recombinant plasmid transformed L-amino acids production bacterium of (a), thus improves expression or the activity of L-Glutamine deaminase YbaS;
C () cultivates (b) bacterial strain, thus produce L-amino acid.
In another preference, described L-amino acid is 1B, and it is the intestinal bacteria comprising pDCtet plasmid and pDCkan plasmid that 1B produces bacterium; Or
Described L-amino acid is L-Trp, and it is CIBTS2 that L-Trp produces bacterium; Or
Described L-amino acid is L-threonine, and it is comprise (external source) ppc, aspA, pntAB, thrA that L-threonine produces bacterium
*the intestinal bacteria (being preferably MG1655) of gene.
In another preference, described L-amino acids production bacterium is MG1655.
In another aspect of this invention, provide the purposes of L-Glutamine deaminase YbaS, for improving the L-amino acid production of L-amino acids production bacterium.
In a preference, described L-amino acid includes, but is not limited to: 1B, L-Trp, L-threonine.
In another aspect of this invention, provide a kind of L-amino acids production bacterium of restructuring, described L-amino acids production bacterium GLN enzyme YbaS high expression level or high reactivity.
In a preference, in the genome of described L-amino acids production bacterium, be integrated with the L-Glutamine deaminase YbaS encoding gene of external source.
In another aspect of this invention, provide a kind of for the production of the amino acid whose test kit of L-, described test kit comprises the L-amino acids production bacterium of described restructuring.
In a preference, also comprise in described test kit:
The substratum of L-amino acids production bacterium; Or
Induced expression agent (as IPTG).
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, pTrc-ybaS restriction enzyme mapping.1:pTrc-ybaS utilizes EcoRV enzyme to cut, and obtains about 1.3kb/4.1kb fragment; M:marker.
Fig. 2, pTrc-yabS plasmid map.
Fig. 3, pThr plasmids Plasmids collection of illustrative plates.
Embodiment
The present inventor, through deep research, disclosing a kind of expression or activity by increasing L-Glutamine deaminase YbaS in L-amino acids production bacterium first, improving the method for L-amino acid (as 1B, L-threonine, L-Trp) output.Described method can significantly improve the amino acid whose output of L-.
As used herein, " external source " or " allos " refers to for cell, and described L-Glutamine deaminase YbaS encoding gene not comes from itself, but builds insertion by recombination method.
Described " L-amino acids production bacterium " refers under suitable culture condition, can the amino acid whose bacterial strain of recombinant expressed production L-, and preferably described bacterial strain is intestinal bacteria.Constructed some L-amino acid preparation strains in prior art, these bacterial strains all can be applicable in the present invention.Further, those skilled in the art all understand conventional L-amino acid as 1B, L-Trp, and L-threonine produces the construction process of bacterial strain.
For the production path of L-amino acid in intestinal bacteria, those skilled in the art have the understanding of certain level.Therefore, people know how to select " L-amino acids production bacterium ", such as, in EP1253195, EP1477564, report the structure of some L-amino acids production bacterium.
As optimal way of the present invention, described L-amino acids production bacterium is intestinal bacteria, and preferably following in described intestinal bacteria one or more genes are weakened or express reduction: the adhE gene of a. coding ethanol dehydrogenase; B. the ackA gene of encoding acetate kinase; C. to encode the pta gene of phosphate acetyltransferase; D. the ldhA gene of encoding lactate dehydrogenase; E. to encode the focA gene of formate transporter; F. the pflB gene of encode pyruvate formate lyase; G. the oxidasic poxB gene of encode pyruvate; H. the thrA gene of codes for aspartate kinase I/ homoserine dehydrogenase I bifunctional enzyme; I. the thrB gene of encoded homoserine kinase; J. the ldcC gene of encodes lysine decarboxylase; Or the cadA gene of h. encodes lysine decarboxylase.
As optimal way of the present invention, be selected from following one or more genes in described 1B production bacterium (intestinal bacteria) and be enhanced or process LAN: the dapA gene of the dihydrodi pyridine synthetic enzyme that a. Coded Discharge lysine feedback suppresses; B. the dapB gene of coding dihydrodi pyridine dicarboxylic acid reductase enzyme; C. to encode the ddh gene of diaminopimelate dehydrogenase; D. encode the dapD of tetrahydropyridine dicarboxylic acid succinyl enzyme and the dapE of coding succinyl-diaminopimelic acid deacylase; E. the asd gene of codes for aspartate-semialdehyde dehydrogenase; F. the ppc gene of Orynebacterium carboxylase; Or the pntAB gene of g. coding niacinamide usp adenine dinucleotide transhydrogenase.
As the preferred embodiment of the present invention, it is the intestinal bacteria comprising pDCtet plasmid and pDCkan plasmid that described 1B produces bacterium.When producing L-Trp, it is CIBTS2 that described L-Trp produces bacterium.When producing L-threonine, it is ppc, aspA, pntAB, thrA of comprising external source that described L-threonine produces bacterium
*the intestinal bacteria (being preferably MG1655) of gene (polypeptide of coding SEQ ID NO:24).Ppc, aspA, pntAB, thrA
*genes such as (thrA rite-directed mutagenesis G433R) is all genes well known in the art.
The sequence that NCBI discloses " L-Glutamine deaminase YbaS ", such as its sequence can see shown in GenBank accession number NP_415018.1.
The Nucleotide full length sequence of " encoding gene of L-Glutamine deaminase YbaS " of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.
In order to increase the L-amino acid production of L-amino acids production bacterium, the present inventor has done to study widely, have found the gene being suitable for carrying out improveing, and constructs corresponding construction.
Therefore, the invention provides a kind of construction, described construction comprises: the expression cassette of the encoding gene of L-Glutamine deaminase YbaS.Described expression cassette possesses all elements (comprising promotor, coding DNA and terminator etc.) needed for genetic expression, thus can intactly give expression to corresponding albumen.
Usually, described construction is positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described construction.Described expression vector is usually also containing replication orgin and/or marker gene etc.Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, synthesizes to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.
In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as penbritin, apramycin, kalamycin resistance that prokaryotic cell prokaryocyte is cultivated.
Comprise the carrier of above-mentioned suitable polynucleotide sequence and suitable promotor or control sequence, may be used for transforming suitable host.In the method for the invention, described host is L-amino acids production bacterium.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell, such as calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.As the preferred mode of one, can electricity consumption transform method carry out.
The invention still further relates to for the production of the amino acid whose test kit of L-, described test kit comprises: the L-amino acids production bacterium of restructuring, wherein L-Glutamine deaminase YbaS high expression level or high reactivity.Described reconstitution cell or cell are placed in suitable container.
As optimal way of the present invention, also comprise other chemical composition of producing gsh for subsequent chemical reaction in described test kit, include but not limited to: halfcystine, glycine, L-glutamic acid or its salt (as sodium salt), inorganic salt and ATP; Preferably, described inorganic salt comprise: seven aqueous magnesium chlorides, acetylphosphate dilithium salt.
As optimal way of the present invention, in described test kit, also comprise working instructions, the concentration of various chemical reagent or expression vector or cell or product of cell lysis, usage and dosage are described.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Embodiment 1, structure pTrc-ybaS plasmid
(1) take ybaS-F/ybaS-R as primer, MG1655 (CGSC6300) (purchased from Coli Genetic Stock Center) genome is template, pcr amplification ybaS ORF fragment, about 1kb;
ybaS-F：5’GCGGTCTCCATGTTAGATGCAAACAAATTACAG3’(SEQ ID NO:1)；
ybaS-R：5’GCCTCGAGTCAGCCCTTAAACACGTTATAGC3’(SEQ ID NO:2)；
(2) ybaS ORF fragment utilizes BsaI/XhoI enzyme to cut, and be inserted into the NcoI/XhoI site of pTrcHis2B carrier (available from Shanghai plant physiological ecology institute of the Chinese Academy of Sciences), the plasmid called after pTrc-yabS of structure, plasmid map is as Fig. 2.The pTrc-ybaS fragment built can utilize EcoRV digestion verification, the results are shown in Figure 1.
Embodiment 2, pTrc-ybaS proceed to 1B and produce bacterial strain (MG1655/pDCkan/pDCtet) test
(1) MG1655/pDCkan/pDCtet builds as follows: be transferred to by pDCtet (US20120107882A1) and pDCkan (US20120107882A1) in MG1655 (purchased from Coli Genetic Stock Center).Between, pTrc-ybaS plasmid is transferred in MG1655/pDCkan/pDCtet competent cell, obtains MG1655/pDCkan/pDCtet/pTrc-ybaS;
(2) respectively from picking list bacterium colony MG1655/pDCkan/pDCtet (contrast), MG1655/pDCkan/pDCtet/pTrc-ybaS flat board, be inoculated in 4ml containing tetracycline (15 μ g/ml) and/or card receive mycin (50 μ g/ml) and or ammonia benzyl mycin (100 μ g/ml) LB test tube in, 37 DEG C, 200rpm cultivates about 10h;
(3) transfer with 10% inoculum size in 50ml fermention medium by bacterium liquid, add final concentration is that the IPTG of 0.1mM induces simultaneously; After shake flask fermentation 48h, to the dense (OD of bacterium
600) measure, centrifugal 10min under fermented liquid 12000rpm condition, carries out HPLC mensuration to the lysine content in supernatant, the results are shown in Table 2 simultaneously.
Fermentative medium formula is as table 1.
Table 1
| Yeast extract | 5g/L |
| (NH 4) 2SO 4 | 16g/L |
| KPH 2PO 4 | 1g/L |
| MgSO 4·7H 2O | 1g/L |
| FeSO 4·7H 2O | 0.01g/L |
| MnSO 4·5H 2O | 0.01g/L |
| L-Met | 0.5g/L |
| L-Thr | 0.1g/L |
| L-Ile | 0.05g/L |
| CaCO 3 | 20g/L |
| Glucose | 40g/L |
HPLC detection method is as follows:
Chromatographic condition: Agilent XDB-C8 (150mm) neutral pillar, determined wavelength 334nm, column temperature: 35 DEG C.
Derivating agent configures:
Get 0.3430g o-phthalaldehyde(OPA)+5ml dehydrated alcohol+0.1472gN-acetyl-L halfcystine, fixed molten to 50ml with 0.05mol/L borate buffer (PH=9.5), lucifuge is for subsequent use.
Mobile phase A: take a certain amount of sodium-acetate, is made into the damping fluid 1000ml of 0.05mol/L.
Mobile phase B: methyl alcohol.
Derivative program:
Draw0.0ul from Vial91；
Draw6.0ul from Vial92；
Draw0.0ul from Vial91；
Draw3.0ul from Vial94；
Draw0.0ul from Vial92；
Mix9.0ul in air rept2times in200ul/min；
Draw4.0ul from Vial93；
Draw0.0ul from Vial91；
Draw2.0ul from Sample；
Draw0.0ul from Vial91；
Mix15.0ul in air rept6times in200ul/min；
Wait2.0min；
Inject；
Wherein No. 91 is distilled water, and No. 92 is borate buffer (ph=9.5), and No. 93 is derivating agent, and No. 94 is distilled water.
Gradient elution program:
Data acquisition time 18min.
Table 2, process LAN ybaS affect thalline 1B bacterial strain MG1655/pDCkan/pDCtet
| Strain name | OD 600 | 1B (g/L) |
| MG1655/pDCkan/pDCtet | 6.77 | 0.2 |
| MG1655/pDCkan/pDCtet/pTrc-ybaS | 8.72 | 2.28 |
From table 2, the output of the thalline production 1B of process LAN ybaS significantly increases, and increases by more than 10 times.The growing multiplication of process LAN ybaS to thalline has promoter action.
Embodiment 3, pTrc-yabS proceeded in tryptophan-producing Strain CIBTS2 and tests
(1) pTrc-ybaS plasmid is transferred in tryptophan-producing Strain CIBTS2 (see CN 201010598350.4) competent cell, obtains CIBTS2/pTrc-ybaS;
(2) respectively from picking list bacterium colony CIBTS2 (contrast), CIBTS2/ybaS flat board, being inoculated in 4ml contains in the test tube of tetracycline (15 μ g/ml) and/or ammonia benzyl mycin (100 μ g/ml) LB substratum, 37 DEG C, 200rpm incubated overnight;
(3) be then seeded in 25ml shake-flask seed substratum by the inoculum size of 1%, 37 DEG C, 220rpm cultivates 24 hours, is forwarded in 25ml Medium of shaking flask fermentation subsequently in the ratio of 20%, add final concentration is that the IPTG of 0.1mM carries out induction 37 DEG C simultaneously, and 220rpm cultivates 28 hours.
Fermentation ends, by fermented liquid centrifugal 10min under 12000rpm condition, carry out HPLC mensuration (the results are shown in Table 5) to the tryptophane in supernatant, the condition determination of HPLC is as follows:
HPLC (Agilent technologies, 1100), pillar: ZORBAX SB-C18 (4.6 × 250mm), moving phase forms, water: acetonitrile=92:8 (v/v), flow velocity is 1ml/min, in view of tryptophane has maximum absorption at 226nm place, therefore determined wavelength 226nm.
Shake-flask seed culture medium prescription is as table 3.Fermention medium is as table 4.
Table 3
| Title | Consumption (g/L) |
| Glucose | 28 |
| KH 2PO 4 | 10 |
| K 2HPO 4 | 24 |
| MgSO 4·7H 2O | 1.0 |
| (NH4) 2SO 4 | 5 |
Table 4
| Title | Consumption (g/L) |
| Glucose | 35 |
| KH 2PO 4 | 7.5 |
| MgSO 4·7H 2O | 0.02 |
| Citric acid | 2.0 |
| (NH 4) 2SO 4 | 25 |
| Na 2SO 4 | 1 |
| MnSO 4 | 0.2 |
| FeSO 4·7H 2O | 0.16 |
| ZnCl 2 | 0.002 |
| CoCl 2·6H 2O | 0.002 |
| CuSO 4·5H 2O | 0.00003 |
| CaCO 3 | 20 |
Table 5, process LAN ybaS affect thalline L-Trp bacterial strain
| Strain name | OD 600 | L-Trp (g/L) |
| CIBTS2 | 9.07 | 1.083 |
| CIBTS2/pTrc-ybaS | 9.32 | 1.212 |
From table 5, the L-Trp of process LAN ybaS produces the output increase that bacterium produces L-Trp, increases about 1.12 times.The growing multiplication of process LAN ybaS to thalline has certain promotion.
Embodiment 4, pTrc-yabS proceeded in threonine producing strain and tests
1, L-threonine-producing strain builds
(1) pMWK builds
With pPIC3.5K (Invitrogen) carrier for template, Kandn/Kanup is primer, pcr amplification Kan fragment, about 0.9kb; PMW118 carrier (purchased from Nippon Gene) utilizes ApaLI/EcoO1109I double digestion, reclaims 2.7kb fragment and fills; Above-mentioned fragment connects, and coat containing kantlex (50ug/ml) LB dull and stereotyped, transformant utilizes KanF/M13F-47 to verify, can obtain 500bp fragment person, be and be correctly connected into direction, obtains pMWK plasmid.
Kandn：5’CCAACCAATTAACCAATTCTGATTAG3’(SEQ ID NO:3)；
Kanup：5’CCTGCAGGGGGGGGGGGAAAGCCACGTTGTGTC3’(SEQ ID NO:4)；
kanF：5’TCGGAATCGCAGACCGATAC3’(SEQ ID NO:5)；
M13F-47：5’CGCCAGGGTTTTCCCAGTCACGAC3’(SEQ ID NO:6)。
(2) pMW-ppc builds
With MG1655 genome for template, ppcdn/ppcup is primer, pcr amplification ppc fragment, and about 3kb, ppc fragment utilizes AflII enzyme to cut to fill; PBR322 carrier (purchased from TAKARA) utilizes EcoO109I enzyme to cut and fills and dephosphorization; Two fragments connect, and coat containing on penbritin (100ug/ml) LB flat board, transformant utilizes ampR/ppcF to verify, obtains 500bp fragment then for be correctly connected into direction, called after pBR-ppc.
PBR-ppc utilizes SmaI/ScaI enzyme to cut, and reclaims 3.6kb fragment; Be inserted into the SmaI site of pMW118 carrier, transformant utilizes ppcF/M13F-47 to verify, obtains pMW-ppc plasmid.
ppcdn：5’GGAATTCCTTAAGGATATCTGAAGGTATATTCAGAATTTG3’(SEQ ID NO:7)；
ppcup：5’AGGAAGCTTAAGCCCGGGTCGACCGGTCGACCGGCGATTTTTTAACATTTCCATAAGT3’(SEQ ID NO:8)；
ampR：5’CGTGCACCCAACTGATCTTC3’(SEQ ID NO:9)；
ppcF：5’ATCTGCCGTGGATTGCAGAG3’(SEQ ID NO:10)。
(3) pMW-aspA builds
With MG1655 genome for template, aspAup/aspAdn is primer, pcr amplification aspA fragment, about 2.1kb; AspA fragment is inserted into the SmaI site of pMW118 carrier, and coat containing on ammonia benzyl mycin (100ug/ml) LB flat board, transformant utilizes aspAR/M13R-48 primer to verify, amplifies about 500bp fragment, is and strives for, obtains pMW-aspA plasmid.
aspAup：5′-TGATCAGCGAAACACTTTTA-3′(SEQ ID NO:11)；
aspAdn：5′-CAGCAAAACTATGATGAGTTCTAC-3′(SEQ ID NO:12)；
aspAR：5′-CATCAGCTGGAACTTCCCTGG-3′(SEQ ID NO:13)。
(4) pMW-pntAB builds
With MG1655 genome for template, pntABdn/pntABup is primer, pcr amplification pntAB fragment, about 3.2kb, pntAB fragment utilizes HindIII/BamHI to be inserted into the HindIII/BamHI site of pMW118 carrier, and coat containing ammonia benzyl mycin (100ug/ml) LB plate screening, transformant utilizes pntB-F/M13F-47 to verify, about 400bp fragment can be amplified be correctly, obtain pMW-pntAB plasmid.
pntABdn：5’AGAAGCTTTCTCAATAAAGAGTGACGG3’(SEQ ID NO:14)；
pntABup：5’AACCCGGGATCCAGATCACAGGCATAATTTTCAG3’(SEQ ID NO:15)；
pntB-F：5’GAACGTATTGCTGGCTGAAG3’(SEQ ID NO:16)。
(5) pMW-ppc-aspA builds
PMW118-aspA plasmid first utilizes SacI enzyme cut and fill, then cuts with HindIII enzyme, obtains 2kb fragment; PMW118-ppc plasmid first utilizes XbaI enzyme cutting and fills, then cuts with HindIII enzyme, obtains 6kb fragment; Two fragments connect, and coat containing on ammonia benzyl mycin (100ug/ml) LB flat board, transformant utilizes aspAR/ppcF to verify, can amplify 1.5kb fragment, namely obtains pMW-ppc-aspA plasmid.
(6) pMW-ppc-aspA-pntAB builds
PMW118-ppc-aspA plasmid first utilizes XbaI enzyme cutting and fills, and recycling HindIII enzyme is cut, and reclaims about 9kb fragment; PMW-pntAB plasmid utilizes HindIII/SmaI enzyme to cut, and reclaims about 3.2kb fragment; Two fragments connect, and coat containing on ammonia benzyl mycin (100ug/ml) LB flat board, obtain pMW-ppc-aspA-pntAB plasmid.
(7) pMWK-ppc-aspA-pntAB builds
PMW-ppc-aspA-pntAB plasmid utilizes HindIII/SmaI enzyme to cut, and reclaims about 9kb fragment and fills; PMWK plasmid utilize EcoRI enzyme to cut and fill, dephosphorization; Two fragments connect, and coat containing on kantlex (50ug/ml) LB flat board, transformant utilizes pntB-F/M13F-47 to verify, can amplify about 400bp fragment, namely obtain pMWK-ppc-aspA-pntAB plasmid.
pntB-F：5’GAACGTATTGCTGGCTGAAG3’(SEQ ID NO:17)。
(8) pMW-Pthr builds
With MG1655 genome for template, Pthr-F/Pthr-R is primer, pcr amplification Pthr fragment, and about 300bp, Pthr fragment utilizes XbaI/SacI enzyme to cut, and is inserted into the XbaI/SacI site of pMW118, obtains pMW-Pthr plasmid.
Pthr-F：5’GGGAGCTCTACGCGAACGAGCCATGACATTG3’(SEQ ID NO:18)；
Pthr-R：5’GGACTCTAGAGTCTTTATCTGTCTGTGCGCTATGCC3’(SEQ ID NO:19)。
(9) pMW-Pthr-thrA
*build
With MG1655 genome for template, thrA-F/thrA-R2 is primer, pcr amplification thrA-1 fragment, about 1.3kb; Simultaneously with MG1655 genome for template, thrA-F2/thrA-R is primer, pcr amplification thrA-2 fragment, about 1.2kb; With thrA-1/thrA-2 fragment for template, fusion pcr amplification thrA
*fragment, about 2.5kb, namely obtain G433R mutant fragments thrA
*("
*" represent and undergo mutation), thrA
*fragment utilizes XbaI enzyme cutting; PMW-Pthr plasmid is first cut with HindII enzyme and is filled, then uses XbaI enzyme cutting; Two fragments connect, and obtain pMW-Pthr-thrA
*plasmid;
thrA-F：5’GACTCTAGAGTCCGACCAAAGGTAACGAGGTAACAAC3’(SEQ ID NO:20)；
thrA-R2：5’GTTCAGAAGATCTCTGAGCAATGG3’(SEQ ID NO:21)；
thrA-F2：5’CCATTGCTCAGAGATCTTCTGAAC3’(SEQ ID NO:22)；
thrA-R：5’CCTCTAGAACTAGTCCTAGGTTTAAACGCGGCCGCTTAGACTCCTAACTTCCATGAGA3’(SEQ ID NO:23)。
(10) pThr builds
PMW-Pthr-thrA
*plasmid utilizes SacI/SpeI enzyme to cut, and glue reclaims 2.9kb fragment, and be inserted into the SacI/XbaI site of pMWK-ppc-aspA-pntAB, obtain pThr plasmid, plasmid map is as Fig. 3.
2, L-threonine-producing strain fermentation
(1) pThr and/or pTrc-ybaS plasmid is transferred in MG1655 competent cell, obtain MG1655/pThr (contrast), MG1655/pThr/pTrc-ybaS bacterial strain, picking list bacterium colony from flat board, being inoculated in 4ml contains in the LB test tube of kantlex (50 μ g/ml) and/or ammonia benzyl mycin (100 μ g/ml), 37 DEG C of incubated overnight;
(2) incubated overnight bacterium liquid is got respectively 1ml to transfer in 50ml containing in LB kantlex (50 μ g/ml) and/or ammonia benzyl mycin (100 μ g/ml)+20g/L (final concentration) glucose, add 0.1mM IPTG induction simultaneously, under 37 DEG C of conditions, 200r/m cultivates 48h, it is dense that fermentation ends measures bacterium, get bacterium liquid centrifuging and taking supernatant simultaneously and HPLC mensuration is carried out to threonine content, the results are shown in Table 7.
HPLC detection method is as follows:
Chromatographic condition: Agilent XDB-C8 (150mm) neutral pillar, determined wavelength 334nm, column temperature: 35 DEG C.
Derivating agent configures: get 0.3430g o-phthalaldehyde(OPA)+5ml dehydrated alcohol+0.1472gN-acetyl-L halfcystine, and fixed molten to 50ml with 0.05mol/L borate buffer (PH=9.5), lucifuge is for subsequent use.
Mobile phase A: take a certain amount of sodium-acetate, is made into the damping fluid 1000ml of 0.05mol/L.
Mobile phase B: methyl alcohol
Derivative program:
Draw0.0ul from Vial91；
Draw6.0ul from Vial92；
Draw0.0ul from Vial91；
Draw3.0ul from Vial94；
Draw0.0ul from Vial92；
Mix9.0ul in air rept2times in200ul/min；
Draw4.0ul from Vial93；
Draw0.0ul from Vial91；
Draw2.0ul from Sample；
Draw0.0ul from Vial91；
Mix15.0ul in air rept6times in200ul/min；
Wait2.0min；
Inject；
Wherein No. 91 is distilled water, and No. 92 is borate buffer (ph=9.5), and No. 93 is derivating agent, and No. 94 is distilled water.
Gradient elution program:
Data acquisition time 18min.
Shake-flask seed substratum is as table 6.
Table 6
| Title | Consumption (g/L) |
| Glucose | 20 |
| Peptone (OXOID) | 10 |
| Yeast extract (OXOID) | 5 |
| Sodium-chlor | 10 |
| pH | 7.0 |
Table 7, process LAN ybaS affect thalline L-threonine-producing strain
| Strain name | OD 600 | L-threonine (g/L) |
| MG1655/pThr | 3.42 | 0.046 |
| MG1655/pThr/pTrc-ybaS | 4.14 | 0.052 |
From table 7, the L-threonine of process LAN ybaS produces the output increase that bacterium produces L-threonine, increases about 1.13 times.The growing multiplication of process LAN ybaS to thalline has certain promotion.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. produce a method for L-amino acid production, it is characterized in that, described method comprises: in L-amino acids production bacterium, improve L-Glutamine deaminase YbaS expression or activity; Cultivate this bacterial strain, thus produce L-amino acid.
2. the method for claim 1, is characterized in that, described L-amino acid comprises: 1B, L-Trp, L-threonine.
3. the method for claim 1, is characterized in that, described method comprises:
A () provides a kind of recombinant plasmid, comprise a recombinant expression cassettes in described recombinant plasmid, and this recombinant expression cassettes contains: L-Glutamine deaminase YbaS encoding gene; With
B () by the recombinant plasmid transformed L-amino acids production bacterium of (a), thus improves expression or the activity of L-Glutamine deaminase YbaS;
C () cultivates (b) bacterial strain, thus produce L-amino acid.
4. method as claimed in claim 3, it is characterized in that, described L-amino acid is 1B, and it is intestinal bacteria that 1B produces bacterium; Preferably, it is the intestinal bacteria comprising pDCtet plasmid and pDCkan plasmid; Or
Described L-amino acid is L-Trp, and it is CIBTS2 that L-Trp produces bacterium; Or
Described L-amino acid is L-threonine, and it is comprise ppc, aspA, pntAB, Pthr, thrA that L-threonine produces bacterium
*the intestinal bacteria of gene.
5. the method as described in as arbitrary in claim 1-4, it is characterized in that, described L-amino acids production bacterium is MG1655.
6. the purposes of L-Glutamine deaminase YbaS, is characterized in that, for the production of L-amino acid.
7. purposes as claimed in claim 6, it is characterized in that, described L-amino acid comprises: 1B, L-Trp, L-threonine.
8. a L-amino acids production bacterium for restructuring, is characterized in that, described L-amino acids production bacterium GLN enzyme YbaS high expression level or high reactivity.
9. for the production of the amino acid whose test kit of L-, it is characterized in that, described test kit comprises the L-amino acids production bacterium of restructuring according to claim 8.
10. test kit as claimed in claim 9, is characterized in that, also comprise in described test kit:
The substratum of L-amino acids production bacterium; Or
Induced expression agent.
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