CN109943548A - A method of it improving Corynebacterium crenatum and synthesizes L-arginine yield - Google Patents

Abstract

The invention discloses a kind of methods of raising Corynebacterium crenatum synthesis L-arginine yield; specifically disclose it is a kind of shift/go by the difunctional uridine acyl of integration mutation transformation dezymotize GlnD; it is acylated to modify pII signal transducer GlnK uridine; the method for improving L-arginine yield, belongs to technical field of bioengineering.The present invention, which is successfully realized, shifts/goes the integration for dezymotizing GlnD mutation transformation to difunctional uridine acyl, and overexpression pII signal transducer GlnK, the uridine for enhancing GlnK are acylated on this basis.Using 5-L fermentor batch fermentation strategy, optimization of fermentation conditions, final recombinant corynebacterium crematum Cc-HD_AA/ pXMJ19-glnK ferments to 96h, and the L-arginine yield of recombinant bacterium reaches 52.2g/L, and production intensity reaches 0.544g/Lh, realizes the high yield of L-arginine.

Description

A method of it improving Corynebacterium crenatum and synthesizes L-arginine yield

Technical field

The present invention relates to a kind of methods of raising Corynebacterium crenatum synthesis L-arginine yield, and in particular to one kind passes through whole The difunctional uridine acyl of conjunction mutation transformation, which is shifted/gone, dezymotizes GlnD, and modification pII signal transducer GlnK uridine is acylated, raising L- The method of arginine yield, belongs to technical field of bioengineering.

Background technique

Nitrogen is the material for constituting organism macromolecular nucleic acid, protein and other nitrogen compounds, is all kinds of micro- lifes Most basic one of the growth factor of object.By long-term evolution, microorganism forms very delicate global nitrogen regulated and control network, with Cope with the survival pressure under varying environment.NH₄ ⁺It is the ultimate constituent of culture medium, is that most microorganism overrides utilize Nitrogen source.

Corynebacterium glutamicum is widely used in the production of amino acid, in Corynebacterium glutamicum as gram-positive bacteria In, regulation of the nitrogen metabolism by global nitrogen transcription regulatory factor AmtR.As extracellular NH₄ ⁺When less, pII signal transducer GlnK It is shifted/is gone by difunctional uridine acyl to dezymotize and form GlnK-UMP after GlnD (UTase/UR, glnD) uridine is acylated, GlnK, which is in, to be lost State living, GlnK-UMP and nitrogen transcription regulatory factor AmtR reciprocation, the inhibition of AmtR regulator are eliminated, transcription intracellular It operates normally.Meanwhile the GlnK-UMP that uridine is acylated cannot form compound in conjunction with the ammonium transporter AmtB on film, therefore Ammonium is activated to transport channel, NH₄ ⁺Channel can be transported by AmtB ammonium to enter into the cell.On the contrary, working as NH intracellular₄ ⁺When sufficient, GlnD egg White to go uridine to be acylated for GlnK in GlnK-UMP albumen, GlnK is active at this time, the GlnK meeting being activated and cell membrane On target protein AmtB be combined into compound AmtB-GlnK, hinder transhipment of the AmtB to ammonium, uridine acyl enzyme quilt gone due to GlnD Activation, not in conjunction with nitrogen transcription regulatory factor AmtR, AmtR is integrated on promoter DNA GlnK-UMP, to hinder intracellular The generation of other fixed nitrogen related gene responsive transcriptions, endocellular metabolism activity weaken.

Corynebacterium crenatum (Corynebacterium crenatum) be a kind of isolated cognate shape of China researcher, Nonspore-bearing gram-positive bacteria is widely used in the amino acids production of mutant strain at home, but to its genetic background Research is also in space state.Corynebacterium crenatum SYPA5-5 is the high yield arginine mutant strain (bacterium that this laboratory screening obtains Strain deposit number is CGMCC NO.0890, is disclosed in the patent of Publication No. CN1441055A).

L-arginine (C₆H₁₂N₄O₂) it is used as semi-dispensable amino acid, there are different physiological roles, be a kind of important industrial ammonia Base acid, is widely used in the industries such as medicine, food, chemical industry, is one of the hot spot of amino acids industry research and development.In L- essence ammonia In sour forming process, a large amount of nitrogen source is needed to supply, in the environment of high nitrogen source, nitrogen transcription regulatory factor AmtR inhibits transcription intracellular Progress.

Therefore it provides a kind of method of new production L-arginine, has important answer for industrially prepared L-arginine With value.

Summary of the invention

Shift the first purpose of the invention is to provide a kind of difunctional uridine acyl/enzyme mutant is removed, ID containing SEQ Amino acid sequence shown in NO.1.

A second object of the present invention is to provide the gene for encoding above-mentioned difunctional uridine acyl and shift/remove enzyme mutant, It encodes the difunctional uridine acyl and shifts/go the nucleotide sequence for dezymotizing GlnD as shown in SEQ ID NO.2.

Third object of the present invention is to provide a kind of genetic engineering bacteriums, express above-mentioned difunctional uridine acyl and shift/go Except enzyme mutant.

Further, the genetic engineering bacterium is host with Corynebacterium crenatum.

Further, the genetic engineering bacterium is host with Corynebacterium crenatum SYPA5-5.

Fourth object of the present invention is to provide a kind of method for improving L-arginine yield, is to apply recombination cognate stick Bacillus Cc-HD_AAIt ferments, the recombinant corynebacterium crematum Cc-HD_AAIt is to express above-mentioned difunctional uridine acyl transfer/removal The Corynebacterium crenatum of enzyme mutant is host, expresses pII signal transducer GlnK.

Further, the amino acid sequence of pII signal transducer GlnK encodes the pII as shown in SEQ ID NO.3 The nucleotide sequence of signal transducer GlnK is as shown in SEQ ID NO.4.

Further, using pXMJ19 as carrier.

Further, it the described method comprises the following steps:

(1) the gene glnK for encoding pII signal transducer GlnK is connect with fabric shuttle-type carrier pXMJ19, is recombinated Plasmid pXMJ19-glnK；

(2) recombinant plasmid pXMJ19-glnK electricity is transferred to Corynebacterium crenatum strain Cc-HD_AA, obtain recombinant bacterium Cc-HD_AA/ pXMJ19-glnK；

(3) ferment recombinant bacterium Cc-HD_AA/pXMJ19-glnK。

Further, the fermentation condition in step (3) are as follows: seed liquor is inoculated in fermentation medium by 6% and is sent out Ferment culture, fermentation temperature are 30 DEG C, and speed of agitator is 500~600r/min, and ferment 90-110h.In fermentation process, temperature with stir It mixes revolving speed to be automatically controlled by fermentor, pH maintains to stablize by adding 50% ammonium hydroxide.

The fermentation medium components: glucose 150g/L, ammonium sulfate 40g/L, yeast extract 15g/L, seven hydration sulphur Sour magnesium 0.5g/L, potassium chloride 1g/L, potassium dihydrogen phosphate 1.5g/L, green vitriol 0.02g/L, Manganous sulfate monohydrate 0.02g/L, 0.05mM L-arginine, deionized water configuration, pH 7.0.50% ammonium hydroxide of above-mentioned fermentation medium is adjusted Its pH to 7.0, the high-temperature sterilization 30min at 121 DEG C, grape sugar disappear.

The preparation method of the seed liquor is that the LBG culture containing kanamycins is inoculated in from picking single colonie on activation plate In base, for 24 hours, then all switching is in seed culture medium, and 30 DEG C of cultures are for 24 hours for 30 DEG C of shaken cultivations.Seed culture medium composition: Portugal Grape sugar 50g/L, ammonium sulfate 25g/L, yeast extract 15g/L, bitter salt 0.5g/L, potassium dihydrogen phosphate 1.5g/L are gone Ionized water configuration, pH 7.0.

Fifth object of the present invention is to provide above-mentioned enzyme mutant pharmacy, food or chemical field application.

Beneficial effects of the present invention:

The present invention, which is successfully realized, shifts/goes the integration for dezymotizing GlnD mutation to difunctional uridine acyl, and on this basis Overexpression pII signal transducer GlnK proves successfully to reduce the vigor that uridine acyl goes to dezymotize by detecting GlnK-UMP, The GlnK-UMP residual quantity of original strain drops to 15 μm from 80 μM before being transformed, Cc-HD after transformation_AAThe GlnK-UMP residual quantity of bacterial strain 75 μM are dropped to from 80 μM, illustrates that uridine acyl goes the vigor dezymotized to be reduced.Using 5-L fermentor batch fermentation strategy, optimization Fermentation condition, final recombinant corynebacterium crematum Cc-HD_AA/ pXMJ19-glnK ferments to 96h, and L-arginine yield reaches 52.2g/ L compares starting strain, the output increased of L-arginine 25.3%.Meanwhile recombinant corynebacterium crematum Cc-HD_AA/pXMJ19- GlnK also adds NH₄ ⁺Utilization, illustrate difunctional uridine acyl is shifted/gone the transformation for dezymotizing GlnD success.

Specific embodiment

(1) culture medium and buffer

LBG culture medium: peptone 10g/L, yeast extract 5g/L, the glucose of sodium chloride 10g/L, 5-10%.

Seed culture medium: glucose 50g/L, ammonium sulfate 25g/L, yeast extract 15g/L, bitter salt 0.5g/ L, potassium dihydrogen phosphate 1.5g/L, deionized water configuration, pH 7.0.

PBS buffer solution: NaCl 8g/L, KCL 0.2g/L, Na₂HPO₄1.42g/L KH₂PO₄0.27g/L, NaOH are adjusted PH is 7.4.

(2) definition and detection method of enzyme activity

The definition of enzyme activity and detection method reference literature Zhang Y, Pohlmann E L, Serate J, et al.Mutagenesis and Functional Characterization of the Four Domains of GlnD,a Bifunctional Nitrogen Sensor Protein[J].Journal of Bacteriology,2010,192(11): 2711-2721, the residual quantitative response uridine acyl by detecting GlnK-UMP go the enzyme activity dezymotized.

Embodiment 1: the difunctional uridine acyl of integration mutation transformation, which is shifted/gone, dezymotizes GlnD

Using the genomic DNA of Corynebacterium crenatum SYPA5-5 as template, with F1 (sequence is as shown in SEQ ID NO.5) and Rm (sequence is as shown in SEQ ID NO.6), Fm (sequence is as shown in SEQ ID NO.7) and R1 (sequence is as shown in SEQ ID NO.8) It for primer, carries out PCR amplification and goes out two sections of homology arms of upstream and downstream, carry out fusion extension PCR, obtaining size is 2137bp gene piece Section, is connected to pK18mobsacB carrier and obtains pk18-glnD_AA, by pk18-glnD_AASequencing carries out sequence ratio by DNAMAN It is right, the results showed that mutant gene sequence is errorless.By recombinant plasmid pk18-glnD_AAElectricity is gone in Corynebacterium crenatum SYPA5-5, The single colonie grown is expanded culture, is forwarded on the plate containing sucrose and is screened, verified using bacterium colony PCR, Screening obtains glnD_AAMutant strain in the genome is integrated in success, is named as Corynebacterium crenatum Cc-HD_AA。

Embodiment 2:pII signal transducer GlnK is in Corynebacterium crenatum Cc-HD_AAIn Corynebacterium crenatum SYPA5-5 Clonal expression

Using the genomic DNA of Corynebacterium crenatum SYPA5-5 as template, PCR amplification is carried out with primer glnKF1/R1, is obtained The genetic fragment of 339bp.After HindIII and EcoRI double digestion, glnK segment is recycled, is linearized with same enzyme is utilized PXMJ19 is ligated and transformed into e. coli bl21, and picking positive transformant extracts plasmid, is template using plasmid, is carried out PCR verifying, the results showed that plasmid pXMJ19-glnK is constructed successfully.By recombinant plasmid pXMJ19-glnK electrotransformation to cognate rod Bacterial strain Cc-HD_AAIn SYPA5-5, recombinant bacterium Cc-HD is obtained_AA/ pXMJ19-glnK and Cc/pXMJ19-glnK.

Two plants of recombinant bacteriums are cultivated into 16-20h in the 50mL container equipped with 10mL LBG culture medium respectively, then press 1%- 2% inoculum concentration, which is transferred in the 250mL container equipped with 50mL LBG culture medium, cultivates 3-6h, is added the 238mg/mL's of 25 μ L IPTG is induced, and entire incubation carries out in 30 DEG C, the reciprocal shaker of 180rpm.It is received after Fiber differentiation 10-12h Collect cell, cell is crushed using Ultrasonic Cell Disruptor, supernatant is collected after centrifugation, verifies glnK in Corynebacterium crenatum Cc- HD_AAWith successful expression in Cc/SYPA5-5, and Cc-HD_AAThe acylated band of/pXMJ19-glnK uridine is remarkably reinforced, and illustrates GlnD Protein integration is mutated successfully decrease uridine acyl and goes the activity dezymotized, and successfully reduces urine after proving transformation by detection GlnK-UMP Glycosides acyl removes the vigor dezymotized, and the GlnK-UMP residual quantity of original strain drops to 15 μm from 80 μM before being transformed, Cc-HD after transformation_AABacterium The GlnK-UMP residual quantity of strain drops to 75 μM from 80 μM, illustrates that uridine acyl goes the vigor dezymotized to be reduced.

Embodiment 3: recombinant bacterium Cc-HD_AAThe fermentation of/pXMJ19-glnK

(1) seed culture

From picking recombinant corynebacterium crematum Cc-HD on activation plate_AAThe single colonie of/pXMJ19-glnK is inoculated in 10mL In LBG culture medium (chloramphenicol containing 100mg/mL), 30 DEG C of shaken cultivations for 24 hours, are then all transferred in 150mL seed culture medium In, 30 DEG C of cultures are for 24 hours.

(2) fermented and cultured

Preliminary fermentation volume of culture is 2.5L, and the fermentation medium components of use are as follows:

Fermentation medium components: glucose 150g/L, ammonium sulfate 40g/L, yeast extract 15g/L, bitter salt 0.5g/L, potassium chloride 1g/L, potassium dihydrogen phosphate 1.5g/L, green vitriol 0.02g/L, Manganous sulfate monohydrate 0.02g/L, Deionized water configuration, pH 7.0.Above-mentioned fermentation medium is adjusted into its pH to 7.0 with 50% ammonium hydroxide, high temperature goes out at 121 DEG C Bacterium 30min.

Fermentation condition: above-mentioned cultured seed liquor is inoculated in fermentation medium by 6% inoculum concentration and carries out fermentation training It supports, fermentation temperature is 30 DEG C, pH 7.0, speed of agitator 600r/min, fermentation time 96h.

In fermentation process, temperature and speed of agitator are automatically controlled by fermentor, and pH maintains to stablize by adding 50% ammonium hydroxide. From for 24 hours, every 12h sampling is primary, cell OD value is measured at 562nm using spectrophotometer, with bio-sensing analyzer The content of (SBA-50, Shandong Province academy sciences Biology Research Institute) glucose is measured, and using HPLC measurement L-arginine content (peace Prompt human relations 1100, the U.S.).The result shows that recombinant bacterium growing state is more preferable compared to starting strain, sugar consumption faster, is fermented to 96h, Recombinant bacterium Cc-HD_AAThe L-arginine yield of/pXMJ19-glnK reaches 52.2g/L, improves compared with starting strain SYPA5-5 25.3%, production intensity reaches 0.544g/Lh, realizes the high yield of L-arginine.

Embodiment 4: detection recombinant bacterium Cc-HD_AAIt is intracellular outer in/pXMJ19-glnK and control strain SYPA5-5 fermentation process NH₄ ⁺Concentration

Using the outer NH intracellular of sulfate by ion chromatography recombinant bacterium and wild type₄ ⁺Concentration, ferment in Example 3 36h When culture solution, 10000rpm be centrifuged 10min, remove supernatant, collect cell；Cell is washed with the PBS buffer solution that pH is 7.2 Three times, the smudge cells on ultrasonic cell instrument, 10000rpm are centrifuged 20min, cell residue are removed, by the clasmatosis liquid of collection It is diluted to deionized water less than 0.1ppm, the clasmatosis liquid after dilution is boiled into 10-20min, is centrifuged deproteinized, then use Membrane filtration, upper machine carry out ion chromatography detection.Using ammonium sulfate as standard specimen.

Ion chromatography testing conditions: analytical column: IonPac CS12A splitter (4 × 250mm) and IonPac CG12A are protected Guard post (4 × 50mm), mobile phase: methane sulfonic acid 20mM, flow velocity: 1.00mL/min, the time: 15min, post case temperature: 30 DEG C, into Sample volume: 25 μ L, detection mode: suppressive conductance, 500 4mm of CERS, instrument: Thermo ICS 5000+.

Pass through outer NH intracellular in contrasting detection recombinant bacterium and control strain SYPA5-5 fermentation process₄ ⁺The variation of concentration, hence it is evident that It was found that recombinant bacterium increases NH₄ ⁺Utilization.In initial NH₄ ⁺Concentration be 300mM under conditions of, wild type when fermentation ends The extracellular remaining NH of SYPA5-5₄ ⁺Concentration is 92 ± 0.3mM, and the extracellular remaining NH of recombinant bacterium₄ ⁺Concentration is 45.0 ± 0.5mM.

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

SEQUENCE LISTING

<110>Southern Yangtze University

<120>a kind of method for improving Corynebacterium crenatum synthesis L-arginine yield

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 692

<212> PRT

<213>artificial sequence

<400> 1

Met Asn Asp Pro Ala Gln Leu Arg His Gly Ala Glu Gln Asp Ala Leu

1 5 10 15

Ala Leu Leu Gly Ser Leu Ala Leu Pro Ala Gly Thr Thr Leu Ala Ala

20 25 30

Thr Gly Ser Leu Ala Arg Ser Glu Leu Thr Pro Tyr Ser Asp Leu Asp

35 40 45

Leu Ile Leu Ile His Ser Pro Gly Gln Thr Pro Glu Gly Val Glu Asp

50 55 60

Leu Trp Tyr Pro Ile Trp Asp Ala Lys Lys Arg Leu Asp Tyr Ser Val

65 70 75 80

Arg Thr Pro Ala Glu Cys Val Glu Met Ile Ser Ala Asp Ser Thr Ala

85 90 95

Ala Leu Ala Met Leu Asp Ile Arg Phe Ile Ala Gly Glu Glu Asp Leu

100 105 110

Phe Ala Thr Thr Arg Arg Lys Ile Leu Asp Lys Trp Arg Gln Glu Leu

115 120 125

Asn Lys Asn Phe Asp Ala Val Val Gln Thr Ala Ile Ala Arg Trp Arg

130 135 140

Arg Ser Gly Pro Val Val Ala Met Thr Arg Pro Asp Leu Lys His Gly

145 150 155 160

Arg Gly Gly Leu Arg Asp Phe Glu Leu Ile Asn Ala Leu Ala Leu Gly

165 170 175

His Leu Cys Asp Val Pro Lys Leu Asp Ser Gln His Arg Leu Leu Val

180 185 190

Asp Val Arg Thr Leu Leu His Val His Ala Arg Arg Ser Arg Asp Val

195 200 205

Leu Asp Pro Glu Phe Ala Val Asp Ile Ala Leu Asp Leu Gly Phe Val

210 215 220

Asp Arg Tyr His Leu Gly Arg Glu Ile Ala Asp Ala Ala Arg Ala Ile

225 230 235 240

Asp Asp Ala Leu Thr Ala Ala Leu Ala Thr Ala Arg Gly Leu Leu Pro

245 250 255

Arg Arg Gly Gly Phe Ala Phe Arg Asn Thr Val Arg Arg Pro Leu Asp

260 265 270

Val Asp Val Val Asp Ala Asn Gly Thr Ile Ala Leu Ser Lys Lys Pro

275 280 285

Asp Leu Thr Asp Pro Ala Leu Pro Leu Arg Val Ala Ala Ala Ala Ala

290 295 300

Lys Thr Gly Leu Pro Val Ser Asp Ser Thr Trp Ala Arg Leu Ala Glu

305 310 315 320

Cys Pro Glu Leu Pro Glu Pro Trp Pro Pro Ala Ala Ser Ala Asp Phe

325 330 335

Phe Arg Ile Leu Ser Ser Pro Glu Asn Thr Arg Arg Val Val Lys Gln

340 345 350

Met Asp Arg His His Leu Trp Glu Arg Tyr Val Pro Glu Trp Asp Arg

355 360 365

Ile Arg Gly Leu Met Pro Arg Glu Pro Ser His Val Ser Thr Ile Asp

370 375 380

Glu His Ser Leu Asn Thr Val Ala Gly Cys Ala Leu Glu Thr Val Thr

385 390 395 400

Val Ala Arg Pro Asp Leu Leu Val Leu Gly Ala Leu Tyr His Asp Ile

405 410 415

Gly Lys Gly Thr Asp Arg Pro His Glu Gln Val Gly Ala Glu Met Val

420 425 430

Ala Lys Ala Ala Ser Arg Met Gly Leu Asn Leu Arg Asp Arg Ala Ser

435 440 445

Val Gln Thr Leu Val Ala Glu His Thr Thr Leu Ala Lys Ile Ala Ser

450 455 460

Arg Leu Asp Pro Arg Ser Glu Glu Ala Val Glu Lys Leu Leu Asp Ala

465 470 475 480

Val His Tyr Asp Leu Val Thr Leu Asn Leu Leu Glu Val Leu Thr Glu

485 490 495

Ala Asp Ala Glu Ala Thr Gly Pro Gly Val Trp Ser Thr Arg Leu Glu

500 505 510

Gln Ala Leu Arg Ile Val Cys Gln Cys Ala Arg Ser Arg Leu Ile Asp

515 520 525

Ile Arg Pro Val Ala Pro Met Leu Val His Arg Gly Asp Ile Ala Leu

530 535 540

Val Glu Lys Asp Gly Thr Asn Thr Val Arg Trp His Gly Asp Asp Leu

545 550 555 560

Arg Lys Ile Phe Ser Val Met Ala Ala Lys Gly Trp Thr Val Asn Ala

565 570 575

Ala Arg Met Val Ala Asn Gly Glu Trp Thr Gly Glu Phe Asp Val Arg

580 585 590

Ala Asn Gly Pro Gln Asp Phe Asp Pro Gln Leu Phe Leu Gln Ser Tyr

595 600 605

Gln Ser Gly Ile Tyr Ser Asp Val Pro Ala Pro Ala Pro Gly Leu Thr

610 615 620

Ala Thr Phe Trp Tyr Gly Asn Ile Leu Glu Val Arg Thr Glu Leu Arg

625 630 635 640

Ile Gly Ala Ile Tyr Ala Leu Leu Arg Thr Leu Pro Ser Ala Leu Trp

645 650 655

Ile Asn Ala Ile Thr Arg Gly Ala Thr Leu Ile Val Gln Ala Ala Leu

660 665 670

Ala Pro Gly Phe Asp Arg Val Ala Val Glu Arg Asp Val Met Lys Val

675 680 685

Leu Ala Ala Ser

690

<210> 2

<211> 2079

<212> DNA

<213>artificial sequence

<400> 2

atgaatgacc cagcccagct ccgccatggc gccgaacaag atgccctggc gttgctgggc 60

tctttggcgc ttccagcagg aacaaccctt gctgccaccg gttcactagc tcgctcagaa 120

ctaacgccct attcggatct tgatctaatt ttgatccact caccagggca gacccccgag 180

ggcgtcgaag atctctggta ccccatttgg gacgctaaga aacgcctcga ctattcggtt 240

cggacacccg cggaatgcgt ggaaatgatt tccgcagact ccacggcagc gcttgcaatg 300

ctcgatattc gatttattgc tggcgaggaa gatctctttg ccaccacgcg ccgcaagatt 360

ttggacaagt ggcgccaaga acttaataaa aactttgatg cggttgtcca aaccgcgatt 420

gcgcgctggc gacgctccgg ccctgtcgtc gctatgaccc gccccgacct taaacatggc 480

aggggagggc tccgcgattt tgaacttatc aacgcgctag cgttgggcca cctctgcgat 540

gtgcccaagc ttgattcgca acacagattg cttgtcgacg ttcgcacgct gcttcacgtt 600

cacgcccgac gctcacgcga cgtgctggac ccagagtttg ctgtcgacat cgctcttgac 660

cttggtttcg ttgaccgcta tcacttaggt cgcgaaatcg ccgacgccgc ccgagccatt 720

gatgacgcgc ttactgcggc acttgcgaca gcccgtggac tgttgcctcg ccgcggtggc 780

tttgccttcc gcaataccgt gaggcgacca ctcgatgttg acgtggtgga tgccaacggc 840

actatcgcgt tgtcgaaaaa accagatctc accgatcccg cactgccgtt gcgtgtagct 900

gcggctgctg cgaaaactgg tttgcctgtc tctgacagca catgggcacg tcttgctgag 960

tgccctgagc taccagaacc ttggccacct gcagcttcag ccgacttttt ccggattctc 1020

tccagcccag aaaataccag gcgtgtagtc aaacaaatgg atcgtcacca cctgtgggaa 1080

cgctacgttc cagagtggga tcgcatcaga ggactcatgc cgcgtgaacc cagccacgtg 1140

tccaccatcg atgagcacag tctgaacact gtggcaggct gcgcattaga aaccgtcacc 1200

gtggctcgcc ctgacctatt ggtgctgggc gcgttgtacc acgacattgg taaaggaact 1260

gatcgtccgc atgagcaagt cggtgcggag atggtggcaa aagccgcaag tcggatggga 1320

ctaaatctgc gtgaccgcgc cagcgtgcaa acgctggtcg cggaacacac aacgctggcc 1380

aaaattgcca gccgccttga tccgcgctcg gaggaggccg tcgaaaagct cttagatgct 1440

gtgcactatg acttggtcac gctcaatctt ctcgaagtgt tgacagaggc agacgcagag 1500

gcaactggcc caggcgtgtg gtcaacgcga cttgagcagg cattgcggat tgtgtgtcag 1560

tgcgcccgtt cgaggcttat cgatattcgg cctgttgcac ccatgcttgt gcatcgaggc 1620

gacatcgcgt tggtggaaaa agacggcacc aacacggtgc gatggcatgg cgatgacctg 1680

cgcaaaatct ttagtgtcat ggctgctaaa ggttggacag tcaacgcggc gcgcatggtg 1740

gcgaatgggg agtggacagg cgaatttgat gtccgcgcca acgggccgca ggattttgat 1800

ccgcagttat tcctccagtc ttatcaatct ggaatctatt cagatgttcc ggcaccggcc 1860

ccgggtctga ccgcgacatt ttggtacggc aacattttgg aagtacgcac cgaactgcgc 1920

attggtgcga tctatgcgtt gcttaggacg ctgccatccg ccctctggat caacgccatc 1980

acacgtggcg ccaccttaat tgttcaagcg gcccttgccc caggatttga tcgcgtagcc 2040

gtcgagcgag acgtgatgaa ggtcttagcc gcaagctga 2079

<210> 3

<211> 109

<212> PRT

<213>artificial sequence

<400> 3

Met Lys Leu Ile Thr Ala Ile Val Lys Pro Phe Thr Leu Thr Asp Ile

1 5 10 15

Lys Asp Ala Leu Glu Gln Ala Val Cys Arg Ala Leu Ser Pro Lys Pro

20 25 30

Lys Ala Leu Ala Ser Arg Lys Ala Thr Pro Arg Cys Arg Gly Ala Glu

35 40 45

Tyr Ala Val Asp Phe Val Pro Lys Val Lys Ile Glu Val Ile Ile Ser

50 55 60

Asp Ala Gln Val Arg Lys Ser Ser Thr Leu Ser Ser Arg Pro His Ala

65 70 75 80

Pro Ala Lys Ser Ala Thr Ala Lys Cys Gly Thr Asn Ile Glu Glu Leu

85 90 95

Val Arg Val Arg Thr Gly Glu Arg Gly Glu Ala Ala Leu

100 105

<210> 4

<211> 333

<212> DNA

<213>artificial sequence

<400> 4

atgaaactca tcaccgcaat tgtcaagccg tttaccctca ccgacattaa agatgctctc 60

gagcaggcgg tgtgcagggc atgactgtca ccgaaaccca aggctttggc cagcagaaag 120

gccacaccga ggtgtcgtgg tgctgaatac gctgtcgatt ttgtgcctaa ggtcaagatt 180

gaagttatta tctccgatgc tcaggtgagg aagtcatcaa cattatcgtc gagaccgcac 240

gcaccggcaa agtcggcgac ggcaaagtgt gggactaaca tcgaagagct ggttcgtgtt 300

cgtaccggtg agcgcggcga agcagccctt taa 333

<210> 5

<211> 27

<212> DNA

<213>artificial sequence

<400> 5

cggaattcat gaatgaccca gcccagc 27

<210> 6

<211> 38

<212> DNA

<213>artificial sequence

<400> 6

aatgtccggg tacaacgcgc ccagcaccaa taggtcag 38

<210> 7

<211> 36

<212> DNA

<213>artificial sequence

<400> 7

ctgggcgcgt tgtacgccgc cattggtaaa ggaact 36

<210> 8

<211> 27

<212> DNA

<213>artificial sequence

<400> 8

gctctagagg tcttagccgc aagctga 27

<210> 9

<211> 40

<212> DNA

<213>artificial sequence

<400> 9

gatttataac gcccgccaag gttttagagc tagaaatagc 40

<210> 10

<211> 40

<212> DNA

<213>artificial sequence

<400> 10

cttggcgggc gttataaatc actagtatta tacctaggac 40

Claims (10)

1. a kind of difunctional uridine acyl shifts/remove enzyme mutant, which is characterized in that amino acid shown in the NO.1 of ID containing SEQ Sequence.

2. the gene that difunctional uridine acyl described in coding claim 1 shifted/removed enzyme mutant.

3. a kind of genetic engineering bacterium, which is characterized in that express difunctional uridine acyl described in claim 1 shift/go dezymotize it is prominent Variant.

4. genetic engineering bacterium as claimed in claim 3, which is characterized in that with Corynebacterium crenatum be host.

5. a kind of method for improving L-arginine yield, which is characterized in that apply recombinant corynebacterium crematum Cc-HD_AAIt is sent out Ferment, the recombinant corynebacterium crematum Cc-HD_AAIt is with genetic engineering bacterium described in claim 3 or 4 for host, expresses pII signal Transducin GlnK.

6. method as claimed in claim 5, which is characterized in that the amino acid sequence such as SEQ of pII signal transducer GlnK Shown in ID NO.3.

7. method as claimed in claim 5, which is characterized in that recombinant corynebacterium crematum Cc-HD_AAUsing pXMJ19 as carrier.

8. the method for claim 7, which is characterized in that the method specifically includes the following steps:

(1) the gene glnK for encoding pII signal transducer GlnK is connect with carrier pXMJ19, obtains recombinant plasmid pXMJ19-glnK；

(2) recombinant plasmid pXMJ19-glnK electricity is transferred to Corynebacterium crenatum strain Cc-HD_AA, obtain recombinant bacterium Cc-HD_AA/pXMJ19- glnK；

(3) ferment recombinant bacterium Cc-HD_AA/pXMJ19-glnK。

9. method according to claim 8, which is characterized in that the fermentation condition in step (3) are as follows: seed liquor is pressed 5-10% It is inoculated in fermentation medium and carries out fermented and cultured, fermentation temperature is 28-30 DEG C, revolving speed 500-600r/min, and ferment 90- 110h。

10. enzyme mutant described in claim 1 is in the application of pharmacy, food or chemical field.