CN114058608B

CN114058608B - Engineering bacterium and method for producing putrescine

Info

Publication number: CN114058608B
Application number: CN202010757654.4A
Authority: CN
Inventors: 蔡宇杰; 惠红杰; 梁鑫鑫; 丁彦蕊; 白亚军; 郑晓晖
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2023-08-25
Anticipated expiration: 2040-07-31
Also published as: CN114058608A

Abstract

The invention discloses engineering bacteria and a method for producing putrescine, and belongs to the technical field of bioengineering. According to the invention, two mutants capable of relieving feedback inhibition and improving enzyme activity are obtained by modifying arginine decarboxylase and agmatine urea hydrolase related to a putrescine synthesis pathway, and the mutants are utilized to construct genetic engineering bacteria for producing putrescine, and under the condition of whole cell transformation, arginine is used as a substrate, so that the putrescine yield can reach 120g/L or more. The enzyme conversion method is carried out by utilizing mutants of the two enzymes, arginine is taken as a substrate, and the yield of the putrescine can reach 160g/L. The method for producing the putrescine has the advantages of simple process, easily obtained raw materials and good industrial application prospect.

Description

Engineering bacterium and method for producing putrescine

Technical Field

The invention relates to engineering bacteria and a method for producing putrescine, and belongs to the technical field of bioengineering.

Background

Putrescine (also known as 1,4 butanediamine) has a linear molecular formula of NH ₂ (CH ₂ ) ₄ NH ₂ Is widely distributed in microorganisms, plants and animals, and is an important physiologically active substance. Has wide application prospect in the fields of industry, agriculture and medicine.

In the current report, there are two major synthetic pathways for putrescine in organisms, as shown in fig. 1:

(1) L-arginine is decarboxylated by arginine decarboxylase to agmatine whileCO release ₂ The method comprises the steps of carrying out a first treatment on the surface of the Then, agmatine is subjected to the action of agmatine urea hydrolase to remove one molecule of urea, so that the product putrescine is generated. This pathway is the most common pathway for putrescine synthesis in animals, plants and microorganisms. In Escherichia coli, arginine decarboxylases fall into two categories, one being an inducible arginine decarboxylase (encoded by the AidA gene) and the other being a biosynthetic arginine decarboxylase (encoded by the speA gene). The former functions under acidic conditions, and one molecule of proton is consumed in the decarboxylation process, so that the proton is prevented from being aggregated in cells, and the cell survival under low pH is ensured. At neutral pH, putrescine is synthesized mainly by the arginine decarboxylase encoded by speA. The arginine decarboxylase encoded by speA was chosen for catalysis herein.

(2) Hydrolysis of L-arginine by arginase to ornithine, which under the action of Ornithine Decarboxylase (ODC) produces putrescine, while releasing CO ₂ . Research shows that when L-arginine is exogenously added, E.coli can select to synthesize putrescine by using an ADC (analog-to-digital converter) path without passing through an ornithine path.

Currently, ADCs and AUHs in the synthetic pathway are tightly regulated in strains. For ADC, studies by David R.Morris et al show that Mg ²⁺ Arginine and putrescine have a competitive inhibition relationship to ADC. The data show that low concentrations of Mg ²⁺ The catalytic activity of ADC is greatly reduced, and the inhibition of the putrescine product is also aggravated. When the arginine concentration is higher than a certain concentration, the yield of putrescine is rather decreased. Arginine also produces competitive inhibition of AUH (ki=9×10 ^-3 M). As the double enzymes receive a certain degree of feedback inhibition in the bacterial bodies, the amount of the putrescine synthesized by the method is very low.

Disclosure of Invention

The invention aims to solve the technical problem of efficiently synthesizing putrescine by taking L-arginine as a substrate, in particular to constructing double-gene co-expression engineering bacteria, and the engineering bacteria are utilized to catalyze the L-arginine to synthesize putrescine so as to realize the efficient production of putrescine.

The invention provides a method for synthesizing putrescine by using cheap L-arginine as a substrate. L-arginine is stripped by arginineDecarboxylation of carboxylase to agmatine and CO ₂ . Agmatine is then catalyzed by agmatine urea hydrolase to produce putrescine. The wild-type Arginine Decarboxylase (ADC) selected by the invention is derived from Escherichia coli BL (DE 3), the coding gene of the wild-type arginine decarboxylase is speA, the nucleotide sequence of the wild-type arginine decarboxylase is shown as SEQ ID NO.5, and the corresponding amino acid sequence is shown as GenBank number QJZ 13350.1. The wild Agmatine Urea Hydrolase (AUH) is Escherichia coli BL (DE 3), the coding gene is speB, the nucleotide sequence is shown as SEQ ID NO.6, and the corresponding amino acid sequence is shown as Genbank number QJZ 13349.1.

The invention provides mutants of putrescine synthesis related enzymes, which are arginine decarboxylase and agmatine urea hydrolase;

in one embodiment, the amino acid sequence of the mutant arginine decarboxylase is set forth in SEQ ID NO. 3;

in one embodiment, the amino acid sequence of the mutant agmatine urea hydrolase is shown in SEQ ID NO. 4.

The invention provides a genetically engineered bacterium, which expresses an arginine decarboxylase gene with a nucleotide sequence shown as SEQ ID NO.1 and an agmatine urea hydrolase gene with a nucleotide sequence shown as SEQ ID NO. 2.

In one embodiment, E.coli is used as a host.

In one embodiment, pACYCDuet, pRSFDuet, pCDFDuet or pcoladat is used as an expression vector.

In one embodiment, pACYCDuet, pRSFDuet or pcdfduret is used as an expression vector.

In one embodiment, the arginine decarboxylase gene is linked to the rear of the agmatine urea hydrolase gene, or the agmatine urea hydrolase gene is linked to the rear of the arginine decarboxylase gene.

The invention provides a method for producing putrescine by enzyme conversion, which is characterized in that arginine is converted by utilizing an arginine decarboxylase mutant and an agmatine urea hydrolase mutant together to produce putrescine.

In one embodiment, the reaction is carried out at 40 to 50℃for 70 to 75 hours.

The invention provides a method for producing putrescine, which utilizes whole cell transformation of engineering bacteria to produce putrescine.

In one embodiment, arginine is used as a substrate, and the concentration of arginine is not less than 300g/L.

In one embodiment, the reaction conditions are pH 9.0 to 10.0, 40 to 50℃and 100 to 200rpm for 20 to 30 hours.

The invention also protects the mutant, the engineering bacteria or the application of the method in the production of putrescine or putrescine-containing products or substances taking putrescine as precursors.

The invention has the beneficial effects that:

according to the invention, two mutants capable of relieving feedback inhibition and improving enzyme activity are obtained by modifying arginine decarboxylase and agmatine urea hydrolase related to a putrescine synthesis pathway, and the mutants are utilized to construct genetic engineering bacteria for producing putrescine, and under the condition of whole cell transformation, arginine is used as a substrate, so that the putrescine yield can reach 120g/L or more. The enzyme conversion method is carried out by utilizing mutants of the two enzymes, arginine is taken as a substrate, and the yield of the putrescine can reach 160g/L. The method for producing the putrescine has the advantages of simple process, easily obtained raw materials and good industrial application prospect.

Drawings

FIG. 1 is a schematic diagram of two existing pathways for the synthesis of putrescine.

Detailed Description

1. The strain and plasmid related to the invention

Plasmids such as pCOLADuet-1, pACYCDuet-1, pCDFDuet-1, pETDuet-1, pRSFDuet-1 and strains Escherichia coli JM and Escherichia coli BL (DE 3) were all purchased from Novagen.

2. Construction of polygene coexpression system and cell culture

At present, a plurality of methods for the polygene coexpression of the escherichia coli are adopted, the method is adopted for constructing the escherichia coli by adopting Liu Xianglei (the escherichia coli is modified to produce shikimic acid and resveratrol by adopting a synthetic biological technology, 2016, shanghai medical industry institute, doctor paper), and each gene comprises a T7 promoter and an RBS binding point. Each plasmid contains two genes, the constructed plasmids are thermally transduced into competent cells of the escherichia coli, and are coated on a monoclonal antibody solid flat plate, and positive transformants are obtained by screening, so that the recombinant escherichia coli is obtained.

Culturing cells: according to classical recombinant E.coli culture and induction expression scheme, transferring recombinant E.coli into LB fermentation medium (peptone 10g/L, yeast powder 5g/L, naCl g/L) according to volume ratio of 2%, culturing at 37deg.C and 200rpm to cell OD ₆₀₀ After reaching 0.4-0.6, IPTG was added at a final concentration of 0.4mM, and the expression culture was induced at 15℃for 24 hours. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 10min at 4 ℃.

The concentrations of the corresponding antibiotics were respectively: ampicillin was 100. Mu.g/mL, kanamycin was 40. Mu.g/mL, chloramphenicol was 20. Mu.g/mL, and streptomycin was 40. Mu.g/mL.

3. Selection of related enzymes

(1) Arginine Decarboxylase (ADC)

A gene speA derived from Escherichia coli BL (DE 3) is selected, the nucleotide sequence of the gene speA is shown as SEQ ID NO.5, and the corresponding amino acid sequence is shown as GenBank No. QJZ 13350.1.

(2) Agmatine Urea Hydrolase (AUH)

A gene speB derived from Escherichia coli BL (DE 3) is selected, the nucleotide sequence of the gene speB is shown as SEQ ID NO.6, and the corresponding amino acid sequence is shown as Genbank number QJZ 13349.1.

4. Detection analysis of samples

L-arginine, agmatine and putrescine were measured by methods referenced and optimized in the literature (Evaluation of biogenic amines in organic and non-organic wines by HPLC OPA derivatization, food technology. Biotechnol.45 (1) (2007) 62-68).

Mobile phase a contained 96% 50M acetate buffer, 4% tetrahydrofuran, and mobile phase B was methanol.

The gradient elution ratio is as follows:

solvent A：100％(0min)，100％(17min)，66.7％(22min)，0％(30min)，0％(35min)；

solvent B：0％(0min)，0％(17min)，33.3％(22min)，100％(30min)，100％(35min)。

the column was a Waters Sunfire C18 column, (4.6X250 mm,5 μm).

The liquid chromatography is as follows: (1260Infinity,Agilent Technologies,Santa Clara,CA).

OPA reagent was purchased from Agilent.

5. Construction of iterative combinatorial mutant libraries

According to the result of molecular docking, D502, D531, D535 and H151, D153, H163 and D232 of AUH of ADC were selected for the first round of saturation mutagenesis. Single point mutants with significant ADC viability enhancement are summarized in table 3. Based on the single-point mutants, two mutants with the most obvious vitality improvement were selected for double-point mutation at each site, and the results are shown in Table 3. Finally, three-site combinatorial mutation was performed based on the double-site mutation results, and the results are shown in Table 4. Therefore, the optimal mutant strain for ADC is D502K/D531C/D535P.

The results of screening the single and double mutant libraries of AUH according to the ADC engineering concept are shown in Table 5. The three-site and four-site mutation results are shown in Table 6.

6. Construction and evaluation of mutants

The saturation mutation site primer designs for ADC and AUH are shown in table 1. The combined mutant was constructed by full plasmid PCR using the plasmid from the previous round as template. The mutation primers used for the combination mutation are shown in Table 2.

All mutants in this study were obtained by whole plasmid PCR. The PCR enzyme was 2X FastPfu PCR Supermix from Mioenzyme. The amplification system is as follows: 2X FastPfu PCR Supermix. Mu.L, plasmid template 100-200ng, 1. Mu.L (10. Mu.M) of each of the front/rear primers, ddH2O up to 50. Mu.L. The PCR amplification procedure was: pre-denaturation at 94℃for 2min, denaturation at 98℃for 10sec, annealing at 57℃for 10sec, extension at 72℃for 2min, circulation for 30 times, final extension at 72℃for 10min, and incubation at 10 ℃.

7. Protein purification:

the crude enzyme solution obtained was filtered through a 0.22 μm cellulose filter membrane and AKTA was usedThe avant protein purification system performs purification. The purification comprises the following specific steps: cleaning and exhausting the needed pipeline by using water and corresponding buffer solution respectively; connecting a 5mL Ni-Histrap column to the instrument, flushing at least 10 column volumes with water and binding solution, respectively, at a flow rate of 5mL/min; loading the crude enzyme sample onto a nickel column, and performing linear elution by using an eluent; protein samples at each peak were collected separately for enzyme activity assays. The buffer used had the following composition: 20mM Na ₂ HPO ₄ -NaH ₂ PO ₄ A buffer; the binding liquid comprises: 20mM phosphate buffer, 50mM NaCl and 5mM imidazole; the eluent comprises: 20mM phosphate buffer, 50mM NaCl and 500mM imidazole.

8. Enzyme activity determination:

the arginine decarboxylase ADC reaction system is as follows: 50mM Tris-HCl (pH 7.5), 4mM MgSO ₄ Pyridoxal phosphate (PLP) at 1mM, L-arginine at 0.1mM and 10 mM. The reaction time was 15min, the temperature was 40℃and the reaction volume was 2mL, and finally 400. Mu.L of 40% trichloroacetic acid was added to terminate the reaction.

The reaction system of agmatine urea hydrolase AUH is as follows: 50mM Tris-HCl (pH 7.5), 0.1mM dithiothreitol and 10mM agmatine. The reaction time was 15min, the temperature was 40℃and the reaction volume was 2mL, and finally 400. Mu.L of 40% trichloroacetic acid was added to terminate the reaction.

The content of agmatine and putrescine as reaction products was detected by HPLC.

One enzyme activity unit is defined as: the amount of enzyme required to convert 1. Mu. Mol of substrate per minute.

TABLE 1 design of ADC and AUH Single-site saturation mutagenesis primer

TABLE 2 design of mutant primers for ADC and AUH combinations

Example 1: mutation and screening of arginine decarboxylase

The arginine decarboxylase gene speA was cloned from Escherichia coliBL (DE 3). The nucleotide sequence is shown as SEQ ID NO. 5.

Connecting the cloned gene to a pETDuet-1 vector to obtain a recombinant vector pETDuet-1-speA containing a wild arginine decarboxylase gene, and respectively carrying out full-plasmid PCR by using a primer pair D502-F/R, D531-F/R, D535-F/R in the table 1 with pETDuet-1-speA as a template, and verifying the size of a target fragment by agarose gel electrophoresis after the PCR is completed. After verification is successful, the concentration is measured by using a micro spectrophotometer, and then the Quick Cut Dpn I is utilized to digest the female parent, wherein the digestion reaction system is as follows: 5 μL 10 XQucikcut Buffer, 1 μL Dpn I, 4 μL DNA, 40 μL ddH ₂ O, after being gently mixed, is instantly centrifuged, and is water-bath for 30min at 37 ℃.

Subsequently, 15. Mu.L of the digested PCR product was added to 100. Mu.LE. Coll JM 1009 competence, mixed gently and ice-cooled for 30min. Placing into a preheated 42 ℃ water bath for heat shock treatment for 90s, adding 1mL of LB culture medium after 2min in an ice bath, and culturing for 1h at 37 ℃. Finally, the bacterial cells are evenly coated on an LB plate containing 100 mug/mL ampicillin, and are cultured for 12 hours until single colony is grown. And (3) selecting a single colony for sequencing verification, taking 500 mu L of bacterial liquid for strain preservation from the plasmid with successful mutation after sequencing, and completing the construction of a mutant library.

The plasmid with successful mutation was heat-transferred into E.coli BL21 (DE 3) for protein expression and screened according to the protein expression level.

E.coli BL21 (DE 3) transformed with the recombinant plasmid containing the mutant is inoculated into a 96-deep well plate for culture, wherein each well contains 450 mu L of LB medium and 100 mu g/mL of ampicillin. After 2h of culture, IPTG with a final concentration of 0.4mM was added for induction of expression, and after 5h of culture at 15℃cells were collected by centrifugation. 1000U of lysozyme is added to carry out wall breaking treatment at 37 ℃ for 1 hour. Then, the deep well plate was centrifuged at 4℃and 4000r/min for 15min, and 200. Mu.L of the supernatant was collected and purified to determine the enzyme activity.

The enzyme activity was determined by HPLC: as shown in table 3, the single mutant D502C, D502K, D531Y, D531C, D535M, D535P had higher enzyme activity than the wild type, and therefore these sites were selected, combined and double mutated (the primers used are shown in table 2), specifically:

(1) Mutations were made to D531Y, D531C, D535M, D535P, respectively, on the basis of the single mutant D502C;

(2) Mutations were made to D531Y, D531C, D535M, D535P, respectively, on the basis of the single mutant D502K;

(3) Mutating D535M, D535P on the basis of single mutant 531Y, respectively;

(4) D535M, D535P was mutated separately on the basis of the single mutant D531C.

The results of the double mutants obtained by the same screening and enzyme activity determination method as single mutation are shown in Table 3, the specific enzyme activity of the double mutants of D502K/D531Y, D502K/D531C, D K/D535P, D531C/D535P is higher, and the double mutants are selected for further mutation (the primers used are shown in Table 2):

(1) D535M, D P was mutated separately on the basis of double mutant D502K/D531Y;

(2) D535M, D P was mutated separately on the basis of double mutant D502K/D531C.

The specific enzyme activities of the three mutants were determined using the same screening and enzyme activity determination methods as the single mutation, and the results are shown in Table 4.

The mutation with highest activity is obtained through multiple rounds of mutation, namely D502K/D531C/D535M, and the sequence is shown as SEQ ID NO. 1.

TABLE 3 Single and double mutant library screening results for ADCs

TABLE 4 screening results of ADC triple mutant libraries

Example 2: mutation and screening expression of agmatine urea hydrolase

The agmatine urea hydrolase gene speB is obtained by cloning from Escherichia coliBL (DE 3), and the nucleotide sequence is shown in SEQ ID NO. 6. The expression product is used for catalyzing agmatine to produce putrescine.

The cloned gene is connected to a pETDuet-1 vector to obtain a recombinant vector pETDuet-1-speA containing a wild arginine decarboxylase gene, then the pETDuet-1-speA is used as a template, the primer pairs H151-F/R, H153-F/R, H163-F/R, D232-F/R in the table 1 are respectively used for carrying out full-plasmid PCR, and the size of a target fragment is verified by agarose gel electrophoresis after the PCR is completed. After verification is successful, the concentration is measured by using a micro spectrophotometer, and then the Quick Cut Dpn I is utilized to digest the female parent, wherein the digestion reaction system is as follows: 5 μL 10 XQucikcut Buffer, 1 μL Dpn I, 4 μL DNA, 40 μL ddH ₂ O, after being gently mixed, is instantly centrifuged, and is water-bath for 30min at 37 ℃.

Subsequently, 15. Mu.L of the digested PCR product was added to 100. Mu.LE. Coll JM 109 competence, mixed gently and ice-cooled for 30min. Placing into a preheated 42 ℃ water bath for heat shock treatment for 90s, adding 1mL of LB culture medium after 2min in an ice bath, and culturing for 1h at 37 ℃. And finally, uniformly coating the thalli on an LB plate containing the corresponding antibiotics, and culturing for 12 hours until single colonies grow. And (3) selecting a single colony for sequencing verification, taking 500 mu L of bacterial liquid for strain preservation from the plasmid with successful mutation after sequencing, and completing the construction of a mutant library.

E.coli BL21 (DE 3) with the mutant sequence transferred thereto was inoculated into 96-well plates, each having 450. Mu.L of LB medium containing 100. Mu.g/mL ampicillin, and cultured. After 2h of culture, IPTG with a final concentration of 0.4mM was added for induction of expression, and after 5h of culture at 15℃cells were collected by centrifugation. 1000U of lysozyme is added to carry out wall breaking treatment at 37 ℃ for 1 hour. The deep well plate was centrifuged at 4000r/min at 4℃for 15min, and 200. Mu.L of the supernatant was used for measuring the enzyme activity.

The activity of the enzyme is measured by HPLC with agmatine as a substrate and PLP as a coenzyme: as a result, as shown in Table 5, the wild-type agmatine urea hydrolase had a specific enzyme activity of 0.4U/mg, and the single mutant H151L, D153Y, H163K, H163V, H163A, D232I, D K, and thus, these sites were selected, combined and double mutated (the primers used are shown in Table 2), specifically:

(1) Mutating D153Y, H163V, H163A, D I, D232K based on single mutant H151L respectively;

(2) Mutating H163V, H163A, D232I, D232K based on single mutant 153Y respectively;

(3) D232I, D232K was mutated separately on the basis of the single mutant H163V;

(4) D232I, D K was mutated separately on the basis of the single mutant H163A.

The specific enzyme activities of the double mutants were determined using the same screening and enzyme activity determination methods as the single mutation, and the results are shown in Table 5.

TABLE 5 screening results of AUH single and double mutant libraries

Based on the specific enzyme activities of the double mutants, H151L/D153Y/H163A, H L/H163A/D232I, H L/H163A/D232K, D153Y/H163A/D232I, D153Y/H163A/D232K (the primers used are shown in Table 2) were constructed, and the specific enzyme activities of the three mutants were determined by the same screening and enzyme activity measurement methods described above, and the results are shown in Table 6.

D153Y/H163A/D232I, H L/H163A/D232K with higher specific enzyme activity was selected, on the basis of which H151L was mutated on D153Y/H163A/D232I, and D153Y was mutated on the basis of H151L/H163A/D232K, respectively (the primers used are shown in Table 2), and the results are shown in Table 6.

The mutation with highest activity is obtained through multiple rounds of mutation, namely H151L/D153Y/H163A/D232K, and the sequence is shown as SEQ ID NO. 2. The specific enzyme activities of the enzymes expressed after the combination mutation are as follows: 57U/mg. And by combining experimental results and three-dimensional structural analysis, feedback inhibition of putrescine, spermidine and the like on the mutant strain is relieved.

TABLE 6 results of screening AUH three-and four-mutant libraries

Example 3: construction of recombinant E.coli simultaneously expressing 2 enzymes

Recombinant E.coli construction: the mutated speA and speB genes (nucleotide sequences shown in SEQ ID No.1 and 2, respectively) were selected and ligated into pCOLADuet-1 (kana resistance), pACYCDuet-1 (chloramphenicol resistance), pCDFduret-1 (streptomycin resistance), pETDuet-1 (ampicillin resistance) and pRSFDuet-1 (kana resistance) plasmids, each with 2 genes expressed thereon, each with a T7 promoter and RBS binding site in front of the gene, with a T7 terminator behind the gene.

The plasmid was transferred into E.coli Escherichia coli BL, and positive transformants were obtained by screening with corresponding antibiotic plates, thus obtaining recombinant E.coli capable of enhanced expression of the double genes, as shown in Table 7.

Whole cell transformation to produce putrescine:

in the initial reaction, the wet weight of cells in the reaction system is 20g/L, L-arginine is 300g/L, PLP and the L-arginine is 5g/L, mgSO ₄ 9.6g/L, DTT 0.02g/L, pH 9.5.5, 150rpm, at 45℃for 24 hours. The optimal combination was pACYCDuet-speB-speA. The whole cell catalysis results are shown in Table 7.

TABLE 7 transformation results of different strains

Example 4: in vitro synthesis of putrescine using two enzymes

Ligation of the double genes speA and speB and their mutants to pETDuet, respectivelyOn the vector-1, arginine decarboxylase and agmatine urea hydrolase were obtained by expression and purification in the same manner as in example 1. Then adding the two pure enzymes, namely, arginine decarboxylase and agmatine urea hydrolase, with the addition amounts of 6.3mg and 4mg respectively into a 50mL reaction system, wherein the reaction system contains 300g/L L-arginine and 5g/LPLP of 9.6g/L MgSO ₄ 0.02g/L DTT, pH 9.5, 150rpm, at 45℃for 72 hours. The yield of putrescine was 160g/L as measured by final liquid chromatography.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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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atgagcgaaa agctgcaggc gcacgccgag aagctgctgg ctgccggtaa gcgtatgctc 360

tctttcggtg gtgaccactt tgttacgctg ccgctgctgc gtgctcatgc gaagcatttc 420

ggcaaaatgg cgctggtaca ctttgacgcc ctaacctaca cctatgcgaa cggttgtgaa 480

tttgacgcag gcactatgtt ctataccgcg ccgaaagaag gtctgatcga cccgaatcat 540

tccgtgcaga ttggtattcg taccgagttt gataaagaca acggctttac cgtgctggac 600

gcctgccagg tgaacgatcg cagcgtggat gacgttatcg cccaagtgaa acagattgtg 660

ggtgatatgc cggtttacct gacttttgat atcaaatgcc tggatcctgc ttttgcacca 720

ggcaccggta cgccagtgat tggcggcctg acctccgatc gcgctattaa actggtacgc 780

ggcctgaaag atctcaacat tgttgggatg gacgtagtgg aagtggctcc ggcatacgat 840

cagtcggaaa tcactgctct ggcagcggca acgctggcgc tggaaatgct gtatattcag 900

gcggcgaaaa agggcgagta a 921

<210> 3

<211> 658

<212> PRT

<213> artificial sequence

<400> 3

Met Ser Asp Asp Met Ser Met Gly Leu Pro Ser Ser Ala Gly Glu His

1 5 10 15

Gly Val Leu Arg Ser Met Gln Glu Val Ala Met Ser Ser Gln Glu Ala

20 25 30

Ser Lys Met Leu Arg Thr Tyr Asn Ile Ala Trp Trp Gly Asn Asn Tyr

35 40 45

Tyr Asp Val Asn Glu Leu Gly His Ile Ser Val Cys Pro Asp Pro Asp

50 55 60

Val Pro Glu Ala Arg Val Asp Leu Ala Gln Leu Val Lys Thr Arg Glu

65 70 75 80

Ala Gln Gly Gln Arg Leu Pro Ala Leu Phe Cys Phe Pro Gln Ile Leu

85 90 95

Gln His Arg Leu Arg Ser Ile Asn Ala Ala Phe Lys Arg Ala Arg Glu

100 105 110

Ser Tyr Gly Tyr Asn Gly Asp Tyr Phe Leu Val Tyr Pro Ile Lys Val

115 120 125

Asn Gln His Arg Arg Val Ile Glu Ser Leu Ile His Ser Gly Glu Pro

130 135 140

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

145 150 155 160

His Ala Gly Met Thr Arg Ser Val Ile Val Cys Asn Gly Tyr Lys Asp

165 170 175

Arg Glu Tyr Ile Arg Leu Ala Leu Ile Gly Glu Lys Met Gly His Lys

180 185 190

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

195 200 205

Glu Ala Glu Arg Leu Asn Val Val Pro Arg Leu Gly Val Arg Ala Arg

210 215 220

Leu Ala Ser Gln Gly Ser Gly Lys Trp Gln Ser Ser Gly Gly Glu Lys

225 230 235 240

Ser Lys Phe Gly Leu Ala Ala Thr Gln Val Leu Gln Leu Val Glu Thr

245 250 255

Leu Arg Glu Ala Gly Arg Leu Asp Ser Leu Gln Leu Leu His Phe His

260 265 270

Leu Gly Ser Gln Met Ala Asn Ile Arg Asp Ile Ala Thr Gly Val Arg

275 280 285

Glu Ser Ala Arg Phe Tyr Val Glu Leu His Lys Leu Gly Val Asn Ile

290 295 300

Gln Cys Phe Asp Val Gly Gly Gly Leu Gly Val Asp Tyr Glu Gly Thr

305 310 315 320

Arg Ser Gln Ser Asp Cys Ser Val Asn Tyr Gly Leu Asn Glu Tyr Ala

325 330 335

Asn Asn Ile Ile Trp Ala Ile Gly Asp Ala Cys Glu Glu Asn Gly Leu

340 345 350

Pro His Pro Thr Val Ile Thr Glu Ser Gly Arg Ala Val Thr Ala His

355 360 365

His Thr Val Leu Val Ser Asn Ile Ile Gly Val Glu Arg Asn Glu Tyr

370 375 380

Thr Val Pro Thr Ala Pro Ala Glu Asp Ala Pro Arg Ala Leu Gln Ser

385 390 395 400

Met Trp Glu Thr Trp Gln Glu Met His Glu Pro Gly Thr Arg Arg Ser

405 410 415

Leu Arg Glu Trp Leu His Asp Ser Gln Met Asp Leu His Asp Ile His

420 425 430

Ile Gly Tyr Ser Ser Gly Ile Phe Ser Leu Gln Glu Arg Ala Trp Ala

435 440 445

Glu Gln Leu Tyr Leu Ser Met Cys His Glu Val Gln Lys Gln Leu Asp

450 455 460

Pro Gln Asn Arg Ala His Arg Pro Ile Ile Asp Glu Leu Gln Glu Arg

465 470 475 480

Met Ala Asp Lys Met Tyr Val Asn Phe Ser Leu Phe Gln Ser Met Pro

485 490 495

Asp Ala Trp Gly Ile Lys Gln Leu Phe Pro Val Leu Pro Leu Glu Gly

500 505 510

Leu Asp Gln Val Pro Glu Arg Arg Ala Val Leu Leu Asp Ile Thr Cys

515 520 525

Asp Ser Cys Gly Ala Ile Met His Tyr Ile Asp Gly Asp Gly Ile Ala

530 535 540

Thr Thr Met Pro Met Pro Glu Tyr Asp Pro Glu Asn Pro Pro Met Leu

545 550 555 560

Gly Phe Phe Met Val Gly Ala Tyr Gln Glu Ile Leu Gly Asn Met His

565 570 575

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

580 585 590

Gly Ser Val Glu Val Glu Leu Ser Asp Glu Gly Asp Thr Val Ala Asp

595 600 605

Met Leu Gln Tyr Val Gln Leu Asp Pro Lys Thr Leu Leu Thr Gln Phe

610 615 620

Arg Asp Gln Val Lys Lys Thr Asp Leu Asp Ala Glu Leu Gln Gln Gln

625 630 635 640

Phe Leu Glu Glu Phe Glu Ala Gly Leu Tyr Gly Tyr Thr Tyr Leu Glu

645 650 655

Asp Glu

<210> 4

<211> 306

<212> PRT

<213> artificial sequence

<400> 4

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

1 5 10 15

Phe Gly Phe Leu Arg Leu Pro Met Asn Phe Gln Pro Tyr Asp Ser Asp

20 25 30

Ala Asp Trp Val Ile Thr Gly Val Pro Phe Asp Met Ala Thr Ser Gly

35 40 45

Arg Ala Gly Gly Arg His Gly Pro Ala Ala Ile Arg Gln Val Ser Thr

50 55 60

Asn Leu Ala Trp Glu His Asn Arg Phe Pro Trp Asn Phe Asp Met Arg

65 70 75 80

Glu Arg Leu Asn Val Val Asp Cys Gly Asp Leu Val Tyr Ala Phe Gly

85 90 95

Asp Ala Arg Glu Met Ser Glu Lys Leu Gln Ala His Ala Glu Lys Leu

100 105 110

Leu Ala Ala Gly Lys Arg Met Leu Ser Phe Gly Gly Asp His Phe Val

115 120 125

Thr Leu Pro Leu Leu Arg Ala His Ala Lys His Phe Gly Lys Met Ala

130 135 140

Leu Val His Phe Asp Ala Leu Thr Tyr Thr Tyr Ala Asn Gly Cys Glu

145 150 155 160

Phe Asp Ala Gly Thr Met Phe Tyr Thr Ala Pro Lys Glu Gly Leu Ile

165 170 175

Asp Pro Asn His Ser Val Gln Ile Gly Ile Arg Thr Glu Phe Asp Lys

180 185 190

Asp Asn Gly Phe Thr Val Leu Asp Ala Cys Gln Val Asn Asp Arg Ser

195 200 205

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

210 215 220

Val Tyr Leu Thr Phe Asp Ile Lys Cys Leu Asp Pro Ala Phe Ala Pro

225 230 235 240

Gly Thr Gly Thr Pro Val Ile Gly Gly Leu Thr Ser Asp Arg Ala Ile

245 250 255

Lys Leu Val Arg Gly Leu Lys Asp Leu Asn Ile Val Gly Met Asp Val

260 265 270

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

275 280 285

Ala Ala Thr Leu Ala Leu Glu Met Leu Tyr Ile Gln Ala Ala Lys Lys

290 295 300

Gly Glu

305

<210> 5

<211> 1977

<212> DNA

<213> artificial sequence

<400> 5

atgtctgacg acatgtctat gggtttgcct tcgtcagcgg gcgaacacgg tgtactacgc 60

tccatgcagg aggttgcaat gagctcccag gaagccagca agatgctgcg tacttacaat 120

attgcctggt ggggcaataa ctactatgac gttaacgagc tgggccacat tagcgtgtgc 180

ccggacccgg acgtcccgga agctcgcgtc gatctcgcgc agttagtgaa aactcgtgaa 240

gcacagggcc agcgtctgcc tgcactgttc tgtttcccac agatcctgca gcaccgtttg 300

cgttccatta acgccgcgtt caaacgtgcg agggaatcct acggctataa cggcgattac 360

ttccttgttt atccgatcaa agttaaccag caccgccgcg tgattgagtc cctgattcat 420

tcgggcgaac cgctgggtct ggaagccggt tccaaagccg agttgatggc agtactggca 480

catgctggca tgacccgtag cgtcatcgtc tgcaacggtt ataaagaccg cgaatatatc 540

cgcctggcat taattggcga gaagatgggg cacaaggtct atctggtcat tgagaagatg 600

tcagaaatcg ccattgtgct ggatgaagca gaacgtctga atgtcgttcc tcgtctgggc 660

gtgcgtgcac gtctggcttc gcagggttcg ggtaaatggc agtcctccgg cggggaaaaa 720

tcgaagttcg gcctggctgc gactcaggta ctgcaactgg ttgaaaccct gcgtgaagcc 780

gggcgtctcg acagcctgca actactgcac ttccacctcg gttcgcagat ggcgaatatt 840

cgcgatatcg cgacaggcgt tcgtgaatcc gcgcgtttct atgtggaact gcacaagctg 900

ggcgtcaata ttcagtgctt cgacgtcggc ggcggtctgg gcgtggatta tgaaggtact 960

cgttcgcagt ccgactgttc ggtgaactac ggcctcaatg aatacgccaa caacattatc 1020

tgggcgattg gcgatgcgtg tgaagaaaac ggtctgccgc atccgacggt aatcaccgaa 1080

tcgggtcgtg cggtgactgc gcatcacacc gtgctggtgt ctaatatcat cggcgtggaa 1140

cgtaacgaat acacggtgcc gaccgcgcct gcagaagatg cgccgcgcgc gctgcaaagc 1200

atgtgggaaa cctggcagga gatgcacgaa ccgggaactc gccgttctct gcgtgaatgg 1260

ttacacgaca gtcagatgga tctgcacgac attcatatcg gctactcttc cggcatcttt 1320

agcctgcaag aacgtgcatg ggctgagcag ctttatttga gcatgtgcca tgaagtgcaa 1380

aagcagctgg atccgcaaaa ccgtgctcat cgtccgatta tcgacgagct gcaggaacgt 1440

atggcggaca aaatgtacgt caacttctcg ctgttccagt cgatgccgga cgcatggggg 1500

atcgaccagt tgttcccggt tctgccgctg gaagggctgg atcaagtgcc ggaacgtcgc 1560

gctgtgctgc tggatattac ctgtgactct gacggtgcta tcgaccacta tattgatggt 1620

gacggtattg ccacgacaat gccaatgccg gagtacgatc cagagaatcc gccgatgctc 1680

ggtttcttta tggtcggcgc atatcaggag atcctcggca acatgcacaa cctgttcggt 1740

gataccgaag cggttgacgt gttcgtcttc cctgacggta gcgtagaagt agaactgtct 1800

gacgaaggcg ataccgtggc ggacatgctg caatatgtac agctcgatcc gaaaacgctg 1860

ttaacccagt tccgcgatca agtgaagaaa accgatcttg atgctgaact gcaacaacag 1920

ttccttgaag agttcgaggc aggtttgtac ggttatactt atcttgaaga tgagtaa 1977

<210> 6

<211> 921

<212> DNA

<213> artificial sequence

<400> 6

atgagcacct taggtcatca atacgataac tcactggttt ccaatgcctt tggtttttta 60

cgcctgccga tgaacttcca gccgtatgac agcgatgcag actgggtgat tactggcgtg 120

ccgttcgata tggccacttc tggtcgtgcg ggtggtcgcc acggtccggc agcgatccgt 180

caggtttcga cgaatctggc ctgggaacac aaccgcttcc cgtggaattt cgacatgcgt 240

gagcgtctga acgtcgtgga ctgcggcgat ctggtatatg cctttggcga tgcccgtgag 300

atgagcgaaa agctgcaggc gcacgccgag aagctgctgg ctgccggtaa gcgtatgctc 360

tctttcggtg gtgaccactt tgttacgctg ccgctgctgc gtgctcatgc gaagcatttc 420

ggcaaaatgg cgctggtaca ctttgacgcc cacaccgata cctatgcgaa cggttgtgaa 480

tttgaccacg gcactatgtt ctataccgcg ccgaaagaag gtctgatcga cccgaatcat 540

tccgtgcaga ttggtattcg taccgagttt gataaagaca acggctttac cgtgctggac 600

gcctgccagg tgaacgatcg cagcgtggat gacgttatcg cccaagtgaa acagattgtg 660

ggtgatatgc cggtttacct gacttttgat atcgactgcc tggatcctgc ttttgcacca 720

ggcaccggta cgccagtgat tggcggcctg acctccgatc gcgctattaa actggtacgc 780

ggcctgaaag atctcaacat tgttgggatg gacgtagtgg aagtggctcc ggcatacgat 840

cagtcggaaa tcactgctct ggcagcggca acgctggcgc tggaaatgct gtatattcag 900

gcggcgaaaa agggcgagta a 921

Claims

1. Mutants of enzymes associated with putrescine synthesis, characterized in that the enzymes are arginine decarboxylase and agmatine urea hydrolase;

the amino acid sequence of the mutant of the arginine decarboxylase is shown as SEQ ID NO. 3;

the amino acid sequence of the mutant of agmatine urea hydrolase is shown as SEQ ID NO. 4.

2. A genetically engineered bacterium is characterized by taking escherichia coli as a host and expressing arginine decarboxylase with a nucleotide sequence shown as SEQ ID NO.1 and agmatine urea hydrolase gene with a nucleotide sequence shown as SEQ ID NO. 2.

3. The engineering bacterium according to claim 2, wherein pACYCDuet, pRSFDuet, pCDFDuet or pcoladat is used as an expression vector.

4. A method for producing putrescine by enzymatic conversion, characterized in that arginine is converted to putrescine by using the mutant of claim 1.

5. The method of claim 4, wherein the reaction is carried out at 40-50 ℃ for 70-75 hours.

6. A method for producing putrescine, which is characterized in that arginine is used as a substrate, and the putrescine is produced by whole cell transformation of the genetically engineered bacterium of claim 2 or 3.

7. The method of claim 6, wherein the concentration of arginine is not less than 300g/L.

8. The method according to claim 6, wherein the reaction conditions are pH 9.0 to 10.0, 40 to 50 ℃, 100 to 200rpm, and the reaction is carried out for 20 to 30 hours.

9. Use of a mutant according to claim 1, or a genetically engineered bacterium according to any one of claims 2 to 3, or a method according to any one of claims 4 to 8, for the production of putrescine or a putrescine-containing product or a putrescine-precursor-containing substance.