CN110229773B

CN110229773B - Method for collecting and preserving wet cells of genetically engineered bacteria

Info

Publication number: CN110229773B
Application number: CN201910486085.1A
Authority: CN
Inventors: 张君轩; 付妍; 付雪蓉; 解雨晨; 任泓宇; 刘立明; 刘佳; 陈修来
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-06-05
Filing date: 2019-06-05
Publication date: 2021-08-24
Anticipated expiration: 2039-06-05
Also published as: CN110229773A

Abstract

The invention discloses a method for collecting and storing wet cells of genetically engineered bacteria, belonging to the technical field of biological engineering. The method is used for collecting and storing wet cells of escherichia coli which co-expresses threonine deaminase, formate dehydrogenase and leucine dehydrogenase, the thawed wet cells are converted to produce L-2-aminobutyric acid after the wet cells are stored for 2 months, the conversion rate of a substrate L-threonine reaches 96.8%, and the enzyme activities of the three enzymes relative to fresh wet cells can reach more than 90.0%.

Description

Method for collecting and preserving wet cells of genetically engineered bacteria

Technical Field

The invention relates to a method for collecting and storing wet cells of a genetic engineering bacterium, belonging to the technical field of biological engineering.

Background

L-2-aminobutyric acid (L-ABA) is an unnatural amino acid, is used as an important chemical raw material and a chiral medical intermediate, and is widely applied to the field of synthesis of antibacterial and antitubercular drugs of ethambutol hydrochloride, novel antiepileptic drugs of levetiracetam, brivaracetam and the like.

At present, reported methods for synthesizing L-ABA include chemical methods and biological methods. The chemical methods mainly comprise a ketobutyric acid reduction method, a desulfurization reaction method and the like, the chemical methods are easy to form racemes, are not beneficial to the synthesis of chiral compounds, have severe reaction conditions, high reagent cost and serious environmental pollution, and are not easy to carry out industrial production.

The biological method is widely concerned due to the characteristics of mild reaction conditions, high stereoselectivity, green environmental protection and the like. The enzyme method mainly utilizes threonine deaminase (L-TD), Leucine Dehydrogenase (LDH), coupled Formate Dehydrogenase (FDH) or Glucose Dehydrogenase (GDH), synthesizes L-ABA by taking L-threonine as a substrate, and simultaneously realizes the circulation of coenzyme NADH by FDH or GDH. The work of producing L-ABA by using amino acid dehydrogenase conversion was earlier carried out abroad. In 1997 Galkin et al used 2-ketobutyrate as a substrate, L-ABA of 36g/L was finally obtained by coupling Leucine Dehydrogenase (LDH) derived from T.intermedia and Formate Dehydrogenase (FDH) which is an NADH recycling enzyme, and the product yield was 88%, but the substrate 2-ketobutyrate was unstable and high in price. Tao et al expressed L-TD, LDH and FDH in E.coli respectively in 2014, and converted 30mol of L-threonine into 29.2mol of L-ABA in a 30L conversion system, the yield was 97.3%, and the space-time yield reached 6.37 g/(L.h). Zhang Caij in 2018 constructs two recombinant escherichia coli which respectively express TD and co-express LDH/GDH, on the basis, a whole-cell transformation system which takes L-threonine and D-glucose as substrates and coproduces L-ABA and D-gluconic acid is constructed, a fed-batch strategy is adopted, and the L-ABA at 141.6g/L and the D-gluconic acid at 269.4g/L are finally transformed, so that the transformation effect of the method is good, but two thalli and two substrates need to be added, the two products are separated, and the complexity of the process is increased.

The inventor previously uses the recombinant strain of double plasmids to co-express L-TD, LDH and FDH, realizes the production of L-ABA (patent publication No.: CN109266595A) by adding single strain and transforming L-threonine by whole cells, and simplifies the production process. In the practical application process, the bacterial sludge collected by fermentation needs to be preserved and then is unfrozen for a conversion experiment, and the enzyme activity is influenced by the collection and storage modes, so that the conversion effect is influenced. At lower temperatures, for example, FDH is deactivated by oxidation of the free thiol groups. Therefore, the method for collecting and preserving the whole cells, which has the advantages of high efficiency, low cost, easy amplification and capability of reducing enzyme activity loss in the preservation process, is provided, and has important application value for realizing the enzyme method industrial production.

Disclosure of Invention

The invention aims to provide a method for collecting and preserving wet cells of a genetic engineering strain, which comprises the steps of cooling fermentation liquor of the genetic engineering strain to 4-15 ℃, adding 50-100 mmol/L cysteine and/or 5-20 mu mol/L coenzyme pyridoxal phosphate, uniformly mixing, and centrifuging at 6000-8000 rpm for 5-10 min to obtain wet cells; the genetic engineering strain takes escherichia coli as a host to co-express threonine deaminase, formate dehydrogenase and leucine dehydrogenase.

In one embodiment of the present invention, the centrifugation temperature is 4 to 15 ℃.

In one embodiment of the invention, the moisture content of the collected wet cell bacterial sludge is 75% to 80%.

In one embodiment of the invention, the engineered strain is stored frozen at-10 to-20 ℃.

In one embodiment of the invention, the genetically engineered strain is stored in batches and is not subjected to repeated freeze thawing.

In one embodiment of the invention, when the engineering strain is used for a transformation experiment, 3.5-4.5 times of deionized water with the volume of 15-25 ℃ is added, and the engineering strain is naturally thawed or is stirred and thawed at a low speed of 25-50 rpm.

In one embodiment of the invention, the threonine deaminase is derived from Escherichia coli W3110, and has a gene sequence shown in SEQ ID NO.1 and an amino acid sequence shown in SEQ ID NO. 4.

In one embodiment of the invention, the leucine dehydrogenase is derived from Bacillus thuringiensis serovar kurstaki YBT-1520, the gene sequence is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 5.

In one embodiment of the present invention, the formate dehydrogenase is derived from Candida, and has a gene sequence shown in SEQ ID NO.3 and an amino acid sequence shown in SEQ ID NO. 6.

In one embodiment of the invention, the pETDuet-1 plasmid is used to express threonine deaminase, formate dehydrogenase and the pRSFDuet-1 plasmid is used to express leucine dehydrogenase.

In one embodiment of the present invention, the escherichia coli is escherichia coli BL 21.

The invention also aims to provide the application of the method in producing the L-2-aminobutyric acid, wherein the L-threonine is used as a substrate, and wet cells of the genetic engineering strain are used as a whole-cell catalyst.

In one embodiment of the invention, the conversion conditions are: in a 5L fermentation tank, stirring at 400r/min and 37 deg.C, aerating at 3vvm, dissolving L-threonine in purified water at 80g/L, ammonium formate at 40g/L, and NAD⁺30mg/L of wet cells, 20g/L of wet cells, pH 8.0 controlled with 4mol/L NaOH solution, and conversion volume 2L.

The invention has the beneficial effects that:

(1) according to the method, the treatment processes of freeze drying, addition of a large amount of protective agents and the like are not needed, only 50-100 mmol/L cysteine and 5-20 mu mol/L coenzyme pyridoxal phosphate (PLP) are added into the fermentation liquor after the fermentation liquor is cooled, and then the thalli are collected through low-temperature centrifugation, so that the cost is low, and the operation is simple and convenient.

(2) After the thalli are collected according to the method, the thalli are stored in a refrigeration house at the temperature of-10 to-20 ℃ for 2 months, the enzyme activity is maintained above 90 percent, the conversion rate can still reach 96.8 percent, and the method has industrial application value.

Drawings

FIG. 1: influence of the addition of the protective agent on the activity of the FDH preserved enzyme (a) an L-TD enzyme activity change curve; (b) LDH enzyme activity change curve; (c) FDH enzyme activity change curve

FIG. 2: production of L-2-aminobutyric acid by frozen genetic engineering strain transformation

Detailed Description

The following examples were all carried out by a conventional experimental method, the experimental materials were commercially available, and the double plasmid coexpression L-TD, LDH and FDH gene engineering strains were constructed in the early stage from the subject group (patent publication No.: CN 109266595A).

Method for measuring content of (I) 2-aminobutyric acid

Sample pretreatment: centrifuging the transformation solution at 12000rpm for 10min, collecting supernatant, and preparing standard solution with alpha-ketobutyric acid or 2-aminobutyric acid as standard. Filtering the supernatant and the standard solution after appropriate dilution with 0.22 μm microporous membrane, and measuring 2-aminobutyric acid by high performance liquid chromatography.

Determination of 2-aminobutyric acid content: high performance liquid chromatography with ortho-phthalaldehyde (OPA) as derivatization reagent, ZO RBAX SB-C18 as chromatographic column, and 10mmol/L KH as mobile phase A₂PO₄(4mol/L KOH pH 5.3), mobile phase B acetonitrile: methanol: and (3) carrying out gradient elution on the phase A (5: 3:1 (pH 5.3 adjusted by glacial acetic acid), wherein the flow rate is 1mL/min, and the detection wavelength is 330, 460nm and the column temperature is 30 ℃.

(II) definition and detection method of enzyme activities of L-TD, LDH and FDH

And (3) detecting the enzymatic activity of threonine deaminase: 200. mu.L of appropriately diluted BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD bacterial solution was taken, 1800. mu.L of a substrate (substrate system: 50mmol/L of threonine dissolved in potassium dihydrogenphosphate-dipotassium hydrogenphosphate buffer solution of pH 7.5) was added, and the mixture was reacted in a thermostatic water bath at 37 ℃ for 15 minutes, followed by boiling to terminate the reaction. Samples were diluted 10-fold and threonine reduction was measured using HPLC-OPA pre-column derivatization. The enzyme activity unit U is defined as the amount of enzyme required for a 1. mu. mol reduction of threonine in 1 min.

And (3) detecting the activity of leucine dehydrogenase: 200. mu.L of appropriately diluted BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD bacterial solution was taken, and 1800. mu.L of substrate (substrate system: 30mg/L NAD was dissolved using 50mmol/L pH 8.0 monobasic sodium phosphate-dibasic sodium phosphate buffer solution ⁺50 mmol/L2-ketobutyric acid, 40mmol/L ammonium formate), reacting in a thermostatic water bath at 37 ℃ for 15min, and then boiling to terminate the reaction. The sample was diluted 10-fold and the amount of 2-aminobutyric acid produced was measured by HPLC-OPA pre-column derivatization. The enzyme activity unit U is defined as the amount of enzyme required for 1. mu. mol increase of 2-aminobutyric acid within 1 min.

Detecting the activity of the formate dehydrogenase: 200. mu.L of appropriately diluted BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD bacterial solution was taken, and 1600. mu.L of substrate (substrate system: 0.167mmol/L ammonium formate dissolved in 50mmol/L pH 8.0 monobasic sodium phosphate-dibasic sodium phosphate buffer, 1.67mmol/L NAD⁺) The reaction was carried out at 37 ℃ and absorbance of NADH at 340nm was measured every 30 seconds by a microplate reader and plotted. Different concentrations are configuredAbsorbance of NADH standard solution (0-0.6mmol/L) was measured at 340nm, a standard curve was plotted, and a regression equation was fitted. The enzyme activity unit U is defined as the amount of enzyme required to produce 1. mu. mol NADH within 1 min.

Definition of 100% enzyme activity: and (3) detecting the enzyme activity when fresh wet cells are collected after fermentation is finished.

(III) culture Medium

The seed culture medium formula comprises: LB culture medium, yeast powder 5g/L, tryptone 10g/L, NaCl 10 g/L.

The fermentation medium formula comprises: 8g/L of glycerol, 8g/L of yeast powder, 10g/L of soybean peptone and K₂HPO₄·12H₂O 6.0g/L，KH₂PO₄ 10.0g/L。

Composition of the feed medium: glycerol 500g/L, MgSO₄·7H₂O 10g/L。

Example 1 Effect of protective agent addition on enzyme Activity

Inoculating BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD into a fermentation culture medium according to the inoculation amount of 5%, wherein the ventilation amount is 2.0vvm, the temperature is 37 ℃, the stirring speed is 500rpm, the fermentation is carried out for 5-6 h, when the dissolved oxygen suddenly rises, the dissolved oxygen-related feeding is started, the dissolved oxygen is controlled to be 30-40%, and the culture is carried out until the OD is OD₆₀₀Reducing the temperature to 25 ℃ and adding 10g/L lactose to induce the expression of target proteins L-TD, LDH and FDH, and performing fermentation culture for 22-24 h and OD₆₀₀At 70-75 ℃, ending fermentation, closing ventilation, reducing the stirring speed to 50rpm, and cooling the fermentation tank to 10-15 ℃.

And respectively adding 50mmol/L Cys and 10 mu mol/L PLP into the fermentation liquor, simultaneously adding 50mmol/L Cys and 10 mu mol/L PLP, uniformly mixing, centrifuging at the temperature of 4-15 ℃, centrifuging at 6000rpm, and collecting wet cells for 5 min. The wet cells are frozen at the temperature of minus 20 ℃, and are sampled at intervals of different days, and the detection of the change condition of the enzyme activity of the FDH is taken as an index because the FDH storage enzyme activity is unstable. The experimental results are shown in FIG. 1 (a-c): along with the prolonging of the freezing preservation time, the enzyme activities of L-TD, LDH and FDH are continuously reduced; when no protective agent is added, the enzyme activities of L-TD, LDH and FDH are reduced to 88.2%, 70.2% and 72.6% of the original enzyme activities, and the freezing enzyme activity stability is L-TD > FDH > LDH; when the three enzymes are stored for 30 days, and 50mmol/L Cys and 10 mu mol/L PLP are added simultaneously, the enzyme activities of the three enzymes are all maintained at more than 95.0 percent.

Example 2 Effect of different centrifugation methods on enzyme Activity

BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD was fermented in the culture mode of example 1, 50mmol/L Cys and 10. mu. mol/L PLP were added to the fermentation broth, the centrifugation temperature was 4-25 ℃, the centrifugation speed was 4000-8000 rpm, and wet cells were collected for 5 min. Freezing wet cells at-20 ℃, freezing for 30 days to detect relative enzyme activities of L-TD, LDH and FDH, wherein experimental results are shown in table 1, and the influence of centrifugal rotating speed and time on the enzyme activity is respectively considered in experiment 1, experiment 2 and experiment 3 groups and experiment 2, experiment 4 and experiment 5 groups, when the centrifugal rotating speed is low, the time is short, the water content of the wet cells is high, the protein is damaged due to the formation of ice crystals in the freezing process, and after freezing for 30 days, only the L-TD enzyme activity with the best stability can be maintained at about 90%; 2. groups 6, 7 and 8 investigate the influence of different centrifugal temperatures on enzyme activity, when the centrifugal temperatures are 4 ℃ and 15 ℃, the enzyme activities of the three enzymes can be maintained to be more than 94.9 percent, when the centrifugal temperatures are increased to be 25 ℃ and 30 ℃, partial protein denaturation is inactivated by high temperature, and the enzyme activities of LDH and FDH are reduced to be less than 90 percent.

TABLE 1 Effect of different centrifugation methods on enzyme Activity

Example 3 Effect of repeated Freeze/thaw mode on enzyme Activity

A system for producing L-2-aminobutyric acid by converting L-threonine in a 5L fermentation tank: stirring at 400r/min and 37 deg.C, aerating at 3vvm, dissolving L-threonine in purified water at 80g/L and ammonium formate at 40g/L, and dissolving NAD in water⁺30mg/L of wet cells, 20g/L of wet cells, pH 8.0 controlled with 4mol/L NaOH solution, and conversion volume 2L.

BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD were fermented in the same manner as in example 1, 50mmol/L Cys and 10. mu. mol/L PLP were added to the fermentation broth, the centrifugation temperature was 10 ℃, the centrifugation speed was set at 8000rpm, wet cells were collected for 5min, and the wet cells were frozen at-20 ℃ for 30 days and then used for transformation experiments. Thawing frozen wet cells, then freezing, namely freezing and thawing once, naturally thawing or stirring thalli by using a magnetic stirrer for thawing, and investigating the influence of repeated freezing and thawing modes on the transformation production of the L-2-aminobutyric acid by the wet cells, wherein the results are shown in Table 2, the thawed thalli in the

experiment groups

1, 2 and 3 are directly used for transformation, the yield of the L-2-aminobutyric acid is 68.1g/L, and the yield of the freezing and thawing once and twice is respectively reduced to 63.2g/L and 56.9 g/L;

experiments

1, 4, 5 and 6 investigate the influence of different stirring rates on enzyme activity, the thalli which are naturally thawed and are stirred and thawed at a substrate of 50rpm are used for a conversion experiment, the conversion rate of L-threonine is more than 98.0 percent, the stirring rotating speed is increased, and the conversion rate is obviously reduced; the cell membrane is incomplete due to freeze thawing or rapid stirring and thawing of the thalli, and the heterogeneously expressed intracellular enzyme has no stable protection system, so that the enzyme activity is reduced, and the conversion period is prolonged.

TABLE 2 Effect of repeated freezing and thawing mode on enzyme Activity

EXAMPLE 4 production of L-2-aminobutyric acid by transformation of frozen genetically engineered Strain

BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD were fermented in the same manner as in example 1, 50mmol/L Cys and 10. mu. mol/L PLP were added to the fermentation broth, 8000rpm was applied, and the mixture was centrifuged at 15 ℃ for 5min to collect wet cells. The wet cells are frozen at the temperature of minus 20 ℃, the enzyme activities of L-TD, LDH and FDH are detected after the wet cells are frozen for 30, 60 and 90 days, the wet cells are naturally unfrozen and then put into a transformation system for transformation experiment, the transformation mode is the same as that of example 3, and the experimental results are shown in figure 2: the wet thalli is preserved according to the method of the invention, and is frozen for 60 days, and the enzyme activity of three enzymes in the genetic engineering strain can reach more than 90 percent of that of the fresh thalli; freezing for 60 days, wherein the yield of L-2-aminobutyric acid used for converting and producing the target product is 67.0g/L, and the conversion rate of L-threonine is 96.8%; after the mixture is frozen for 90 days, the relative enzyme activities of L-TD, LDH and FDH are respectively reduced to 93.2 percent, 85.3 percent and 89.3 percent, and the yield of the L-2-aminobutyric acid only can reach 62.1 g/L.

Comparative example 1

BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD were fermented in the same manner as in example 1, 50 mmol/L. beta. -mercaptoethanol and 10. mu. mol/L PLP were added to the fermentation broth, the centrifugation temperature was 10 ℃ and the centrifugation speed was set at 8000rpm, and wet cells were collected for 5 min. After 30 days of storage, the relative enzyme activities of L-TD, LDH and FDH were 84.8%, 81.9% and 80.8%, respectively.

Comparative example 2

BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD were fermented in the same manner as in example 1, 50mmol/L glutathione and 10. mu. mol/L PLP were added to the fermentation broth, the centrifugation temperature was 10 ℃ and the centrifugation speed was set at 8000rpm, and wet cells were collected for 5 min. After 30 days of storage, the relative enzyme activities of L-TD, LDH and FDH were 85.6%, 82.3% and 80.3%, respectively.

Comparative example 3

BL21/pRSFDuet-BtLeuDH + pETduet-CbFDH-EcTD were fermented in the same manner as in example 1, 50mmol/L dithiothreitol and 10. mu. mol/L PLP were added to the fermentation broth, the centrifugation temperature was 10 ℃ and the centrifugation speed was set at 8000rpm, and wet cells were collected for 5 min. After 30 days of storage, the relative enzyme activities of L-TD, LDH and FDH were 82.6%, 83.7% and 87.6%, respectively.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr

1 5 10 15

Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr

20 25 30

Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile

35 40 45

Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg

50 55 60

Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His

65 70 75 80

Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe

85 90 95

Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala

100 105 110

Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val

115 120 125

Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu

130 135 140

Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro

145 150 155 160

Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln

165 170 175

Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu

180 185 190

Val Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys

195 200 205

Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu

210 215 220

Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu

225 230 235 240

Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln

245 250 255

Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala

260 265 270

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

275 280 285

Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn

290 295 300

Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn

305 310 315 320

Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln

325 330 335

Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe

340 345 350

Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn

355 360 365

Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg

370 375 380

Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn

385 390 395 400

Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys

405 410 415

Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln

420 425 430

Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu

435 440 445

Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His

450 455 460

Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu

465 470 475 480

Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly

485 490 495

Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu

500 505 510

Ala Gly

<210> 5

<211> 366

<212> PRT

<213> Artificial Synthesis

<400> 5

Met Thr Leu Glu Ile Phe Glu Tyr Leu Glu Lys Tyr Asp Tyr Glu Gln

1 5 10 15

Val Val Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala

20 25 30

Ile His Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp

35 40 45

Thr Tyr Asp Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala

50 55 60

Lys Gly Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly

65 70 75 80

Ala Lys Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Ser Glu Ala

85 90 95

Met Phe Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr

100 105 110

Ile Thr Ala Glu Asp Val Gly Thr Thr Val Asp Asp Met Asp Ile Ile

115 120 125

His Glu Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ser Phe Gly Ser

130 135 140

Ser Gly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met

145 150 155 160

Lys Ala Ala Ala Lys Glu Ala Phe Gly Thr Asp Asn Leu Glu Gly Lys

165 170 175

Val Ile Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Lys

180 185 190

His Leu His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys

195 200 205

Glu Ala Val Gln Arg Ala Val Glu Glu Phe Gly Ala Ser Ala Val Glu

210 215 220

Pro Asn Glu Ile Tyr Gly Val Glu Cys Asp Ile Tyr Ala Pro Cys Ala

225 230 235 240

Leu Gly Ala Thr Val Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys

245 250 255

Val Ile Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Asn Arg His Gly

260 265 270

Asp Ile Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile

275 280 285

Asn Ala Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn

290 295 300

Arg Glu Arg Ala Leu Lys Arg Val Glu Ser Ile Tyr Asp Thr Ile Ala

305 310 315 320

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

325 330 335

Ala Asp Arg Leu Ala Glu Glu Arg Ile Ala Ser Leu Lys Asn Ser Arg

340 345 350

Ser Thr Tyr Leu Arg Asn Gly His Asp Ile Ile Ser Arg Arg

355 360 365

<210> 6

<211> 364

<212> PRT

<213> Artificial Synthesis

<400> 6

Met Lys Ile Val Leu Val Leu Tyr Asp Ala Gly Lys His Ala Ala Asp

1 5 10 15

Glu Glu Lys Leu Tyr Gly Cys Thr Glu Asn Lys Leu Gly Ile Ala Asn

20 25 30

Trp Leu Lys Asp Gln Gly His Glu Leu Ile Thr Thr Ser Asp Lys Glu

35 40 45

Gly Gly Asn Ser Val Leu Asp Gln His Ile Pro Asp Ala Asp Ile Ile

50 55 60

Ile Thr Thr Pro Phe His Pro Ala Tyr Ile Thr Lys Glu Arg Ile Asp

65 70 75 80

Lys Ala Lys Lys Leu Lys Leu Val Val Val Ala Gly Val Gly Ser Asp

85 90 95

His Ile Asp Leu Asp Tyr Ile Asn Gln Thr Gly Lys Lys Ile Ser Val

100 105 110

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

115 120 125

Met Thr Met Leu Val Leu Val Arg Asn Phe Val Pro Ala His Glu Gln

130 135 140

Ile Ile Asn His Asp Trp Glu Val Ala Ala Ile Ala Lys Asp Ala Tyr

145 150 155 160

Asp Ile Glu Gly Lys Thr Ile Ala Thr Ile Gly Ala Gly Arg Ile Gly

165 170 175

Tyr Arg Val Leu Glu Arg Leu Val Pro Phe Asn Pro Lys Glu Leu Leu

180 185 190

Tyr Tyr Asp Tyr Gln Ala Leu Pro Lys Asp Ala Glu Glu Lys Val Gly

195 200 205

Ala Arg Arg Val Glu Asn Ile Glu Glu Leu Val Ala Gln Ala Asp Ile

210 215 220

Val Thr Val Asn Ala Pro Leu His Ala Gly Thr Lys Gly Leu Ile Asn

225 230 235 240

Lys Glu Leu Leu Ser Lys Phe Lys Lys Gly Ala Trp Leu Val Asn Thr

245 250 255

Ala Arg Gly Ala Ile Cys Val Ala Glu Asp Val Ala Ala Ala Leu Glu

260 265 270

Ser Gly Gln Leu Arg Gly Tyr Gly Gly Asp Val Trp Phe Pro Gln Pro

275 280 285

Ala Pro Lys Asp His Pro Trp Arg Asp Met Arg Asn Lys Tyr Gly Ala

290 295 300

Gly Asn Ala Met Thr Pro His Tyr Ser Gly Thr Thr Leu Asp Ala Gln

305 310 315 320

Thr Arg Tyr Ala Gln Gly Thr Lys Asn Ile Leu Glu Ser Phe Phe Thr

325 330 335

Gly Lys Phe Asp Tyr Arg Pro Gln Asp Ile Ile Leu Leu Asn Gly Glu

340 345 350

Tyr Val Thr Lys Ala Tyr Gly Lys His Asp Lys Lys

355 360

Claims

1. A method for collecting and storing wet cells of a genetic engineering strain is characterized in that fermentation liquor of the genetic engineering strain is cooled to 4-15 ℃, 50-100 mmol/L cysteine and 5-20 mu mol/L coenzyme pyridoxal phosphate are added, the mixture is uniformly mixed and then centrifuged at 6000-8000 rpm for 5-10 min to obtain wet cells, and the wet cells are frozen and stored at-10-20 ℃; the genetic engineering strain takes escherichia coli as a host to co-express threonine deaminase, formate dehydrogenase and leucine dehydrogenase; the centrifugation temperature is 4-15 ℃.

2. The collection and preservation method according to claim 1, wherein the moisture content of the collected wet cell bacterial sludge is 75% to 80%.

3. The collection and storage method according to claim 1, wherein the amino acid sequence of threonine deaminase is represented by SEQ ID No.4, the amino acid sequence of leucine dehydrogenase is represented by SEQ ID No.5, and the amino acid sequence of formate dehydrogenase is represented by SEQ ID No. 6.

4. The collection and storage method according to claim 1, wherein the genetically engineered strain expresses threonine deaminase and formate dehydrogenase using pETDuet-1 plasmid and leucine dehydrogenase using pRSFDuet-1 plasmid.

5. The collection and storage method according to claim 1, wherein the Escherichia coli is Escherichia coli (Escherichia coli) BL 21.

6. Use of a process according to any one of claims 1 to 5 for the production of L-2-aminobutyric acid.

7. The application of claim 6, wherein the genetically engineered strain is prepared by co-expressing three enzymes of threonine deaminase, formate dehydrogenase and leucine dehydrogenase with Escherichia coli as a host, cooling fermentation liquor of the genetically engineered strain to 4-15 ℃, adding 50-100 mmol/L cysteine and 5-20 μmol/L coenzyme pyridoxal phosphate, mixing uniformly, centrifuging at 6000-8000 rpm for 5-10 min to obtain wet cells, and producing the L-2-aminobutyric acid by taking the obtained wet cells of the genetically engineered strain as a whole-cell catalyst with L-threonine as a substrate.

8. The use of claim 7, wherein when the genetically engineered strain is used in transformation experiments, 3.5-4.5 times of deionized water with a volume of 15-25 ℃ is added, and the strain is naturally thawed or is thawed by stirring at a low speed of 25-50 rpm.