CN114058608B - A kind of engineering bacteria and method for producing putrescine - Google Patents

Abstract

The invention discloses an engineering bacterium and a method for producing putrescine, belonging to the technical field of bioengineering. The present invention obtains two mutants capable of releasing feedback inhibition and improving enzyme activity by transforming the related arginine decarboxylase and agmatine urea hydrolase in the putrescine synthesis pathway, and using the mutants to construct Genetically engineered bacteria are used for the production of putrescine. Under the condition of whole cell transformation, with arginine as the substrate, the output of putrescine can reach 120g/L and above. The mutants of the two enzymes are used for enzymatic conversion, and the putrescine output can reach 160 g/L with arginine as the substrate. The method for producing putrescine provided by the invention is simple in process and easy to obtain raw materials, and has good industrial application prospect.

Description

A kind of engineering bacteria and method for producing putrescine

technical field

The invention relates to an engineering bacterium and a method for producing putrescine, belonging to the technical field of bioengineering.

Background technique

Putrescine, also known as 1,4-butylene diamine, has a linear molecular formula of NH ₂ (CH ₂ ) ₄ NH ₂ . It is widely distributed in microorganisms, plants and animals, and is an important physiologically active substance. It has broad application prospects in the fields of industry, agriculture and medicine.

In the current report, there are two main synthetic pathways for putrescine in organisms, as shown in Figure 1:

(1) L-arginine is decarboxylated by arginine decarboxylase to generate agmatine, and CO ₂ is released at the same time; then, agmatine is removed under the action of agmatine urea hydrolase Urea, producing putrescine as a product. This pathway is the most common pathway for putrescine synthesis in animals, plants and microorganisms. In Escherichia coli, arginine decarboxylase is divided into two types, one is inducible arginine decarboxylase (encoded by AidA gene), and the other is biosynthetic arginine decarboxylase (encoded by speA gene ). The former functions under acidic conditions, and a molecule of proton will be consumed during the decarboxylation process, thereby preventing the accumulation of protons in the cell and ensuring the survival of bacteria at low pH. At neutral pH, putrescine is mainly synthesized by arginine decarboxylase encoded by speA. In this paper, arginine decarboxylase encoded by speA was selected for catalysis.

(2) L-arginine is hydrolyzed by arginase to generate ornithine, and ornithine generates putrescine under the action of ornithine decarboxylase (ODC), and releases CO ₂ at the same time. Studies have shown that when L-arginine is added exogenously, E.coli will choose to use the ADC pathway to synthesize putrescine instead of the ornithine pathway.

Currently, ADC and AUH in the synthetic pathway are strictly regulated in the strain. For ADC, research by DavidR.Morris.et al. shows that Mg ²⁺ , arginine and putrescine have a competitive inhibitory relationship on ADC. The data showed that low concentration of Mg ²⁺ would greatly reduce the catalytic activity of ADC and also aggravate the product inhibition of putrescine. When the concentration of arginine was higher than a certain concentration, the production of putrescine would decrease instead. Arginine also competitively inhibits AUH (Ki=9×10 ^-3 M). Since both enzymes are subject to a certain degree of feedback inhibition in the bacteria, the amount of putrescine synthesized by this pathway is very low.

Contents of the invention

The technical problem to be solved in the present invention is to efficiently synthesize putrescine with L-arginine as a substrate, especially to construct a double-gene co-expression engineering bacterium, and use the engineering bacterium to catalyze L-arginine to synthesize putrescine to realize the production of putrescine. Efficient production.

The invention provides a method for synthesizing putrescine by using cheap L-arginine as a substrate. L-arginine is decarboxylated by arginine decarboxylase to generate agmatine and CO ₂ . Subsequently, agmatine is catalyzed by agmatine urea hydrolase to produce putrescine. The wild-type arginine decarboxylase (ADC) selected in the present invention is derived from Escherichia coli BL21 (DE3), its coding gene is speA, its nucleotide sequence is shown in SEQ ID NO.5, and the corresponding amino acid sequence is shown in GenBank No. Shown in QJZ13350.1. The selected wild-type agmatine urea hydrolase (AUH) is derived from Escherichia coli BL21 (DE3), its coding gene is speB, its nucleotide sequence is shown in SEQ ID NO.6, and the corresponding amino acid sequence is shown in Genbank No. It is shown in QJZ13349.1.

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

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

In one embodiment, the amino acid sequence of the mutant of 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 as shown in SEQ ID NO.1 and a guanidine with a nucleotide sequence as shown in SEQ ID NO.2 butylaminourea hydrolase gene.

In one embodiment, Escherichia coli is used as the host.

In one embodiment, pACYCDuet, pRSFDuet, pCDFDuet or pCOLADuet is used as the expression vector.

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

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

The invention provides a method for producing putrescine by enzymatic conversion, which is characterized in that the mutant of arginine decarboxylase and the mutant of arginine urea hydrolase are used to convert arginine together to generate putrescine.

In one embodiment, the reaction is carried out at 40-50° C. for 70-75 hours.

The invention provides a method for producing putrescine, which utilizes whole cells of the engineering bacteria to transform and produce putrescine.

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

In one embodiment, the reaction conditions are pH 9.0-10.0, 40-50° C., 100-200 rpm, and react for 20-30 hours.

The present invention also protects the mutant, or the engineering bacterium, or the application of the method in the production of putrescine or a product containing putrescine or a substance with putrescine as a precursor.

Beneficial effects of the present invention:

The present invention obtains two mutants capable of releasing feedback inhibition and improving enzyme activity by transforming the related arginine decarboxylase and agmatine urea hydrolase in the putrescine synthesis pathway, and using the mutants to construct Genetically engineered bacteria are used for the production of putrescine. Under the condition of whole cell transformation, with arginine as the substrate, the output of putrescine can reach 120g/L and above. The mutants of the two enzymes are used for enzymatic conversion, and the putrescine production can reach 160 g/L with arginine as the substrate. The method for producing putrescine provided by the invention is simple in process and easy to obtain raw materials, and has good industrial application prospect.

Description of drawings

Figure 1 is a schematic diagram of two existing synthetic putrescine pathways.

Detailed ways

1. Bacterial strains and plasmids involved in the present invention

Plasmids such as pCOLADuet-1, pACYCDuet-1, pCDFDuet-1, pETDuet-1, pRSFDuet-1 and strains Escherichia coli JM 109 and Escherichia coli BL21 (DE3) were purchased from Novagen.

2. Construction of multi-gene co-expression system and cell culture

At present, there are many methods for the co-expression of multiple genes in Escherichia coli. The present invention adopts the method described by Liu Xianglei (Synthetic biology technology transforms E. coli to produce shikimic acid and resveratrol, 2016, Shanghai Pharmaceutical Industry Research Institute, doctoral dissertation). All genes contain T7 promoter and RBS junction. Each plasmid contains two genes, and the constructed plasmid is thermally transfected into E. coli competent cells, and coated on a monoclonal antibody solid plate, and positive transformants are obtained by screening, that is, recombinant E. coli is obtained.

Cell culture: According to the classical recombinant Escherichia coli culture and induced expression scheme, the recombinant Escherichia coli was transferred to LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/ In L), culture at 37°C and 200rpm until the OD ₆₀₀ of the cells reaches 0.4-0.6, then add IPTG with a final concentration of 0.4mM, induce expression and culture at 15°C for 24h. After induction of expression, the cells were collected by centrifugation at 4°C, 8000 rpm, and 10 min.

The concentrations of the corresponding antibiotics were: 100 μg/mL for ampicillin, 40 μg/mL for kanamycin, 20 μg/mL for chloramphenicol, and 40 μg/mL for streptomycin.

3. Selection of relevant enzymes

(1) Arginine decarboxylase (ADC)

The gene speA derived from Escherichia coli BL21 (DE3) was selected, its nucleotide sequence is shown in SEQ ID NO.5, and the corresponding amino acid sequence is shown in GenBank No. QJZ13350.1.

(2) Agmatine urea hydrolase (AUH)

The gene speB derived from Escherichia coli BL21 (DE3) was selected, its nucleotide sequence is shown in SEQ ID NO.6, and the corresponding amino acid sequence is shown in Genbank No. QJZ13349.1.

4. Detection and analysis of samples

L-arginine, agmatine and putrescine were determined with reference to and optimized methods in relevant literature (Evaluation of biological amines in organic and non-organic wines by HPLC OPA derivatization, Food Technol.Biotechnol.45(1)(2007) 62-68).

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

The ratio of gradient elution 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 chromatographic column is Waters Sunfire C18 column, (4.6×250mm, 5μm).

Liquid chromatography was: (1260 Infinity, Agilent Technologies, Santa Clara, CA).

OPA reagent was purchased from Agilent.

5. Construction of iterative combinatorial mutant library

According to the results of molecular docking, D502, D531, D535 of ADC and H151, D153, H163 and D232 of AUH were selected for the first round of saturation mutation. The single point mutants with significantly improved ADC activity are summarized in Table 3. According to the single-point mutants, two mutants with the most obvious improvement in activity were selected for each site for double-site mutation, and the results are shown in Table 3. Finally, based on the results of double-site mutations, three-site combined mutations were performed, and the results are shown in Table 4. Therefore, the optimal mutant strain of ADC is D502K/D531C/D535P.

According to the transformation idea of ADC, the screening results of single mutation and double mutation library of AUH are shown in Table 5. The results of three-position and four-position mutations are shown in Table 6.

6. Construction and evaluation of mutants

The design of primers for saturation mutation sites of ADC and AUH is shown in Table 1. The construction of combinatorial mutants was carried out by PCR of the whole plasmid with the plasmid of the previous round as a template. The mutation primers used for combinatorial mutation are shown in Table 2.

All mutants in this study were obtained by whole-plasmid PCR. The PCR enzyme was 2×FastPfu PCR Supermix from Mioenzyme Company. The amplification system is: 2×FastPfu PCR Supermix 25μL, plasmid template 100-200ng, front/back primer 1μL (10μM), ddH2O supplemented to 50μL. The PCR amplification program was as follows: pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 sec, annealing at 57°C for 10 sec, extension at 72°C for 2 min, 30 cycles, final extension at 72°C for 10 min, and incubation at 10°C.

7. Protein purification:

The obtained crude enzyme solution was filtered through a 0.22 μm cellulose filter membrane, and purified using the AKTA avant protein purification system. The specific steps of purification are: clean and exhaust the required pipelines with water and corresponding buffer solution respectively; connect 5mL Ni-HistrapTM column to the instrument, wash at least 10 column volumes with water and binding solution respectively, and the flow rate is 5mL/min ; The crude enzyme sample is loaded on the nickel column, and the eluent is used for linear elution; the protein samples at each peak are collected separately for the determination of enzyme activity. The buffer components used are as follows: 20mM Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer; the binding solution contains: 20mM phosphate buffer, 50mM NaCl and 5mM imidazole; the eluent contains: 20mM phosphate buffer, 50mM NaCl and 500mM imidazole.

8. Enzyme activity assay:

The reaction system of arginine decarboxylase ADC is: 50mM Tris-HCl (pH 7.5), 4mM MgSO ₄ , 1mM pyridoxal phosphate (PLP), 0.1mM and 10mM L-arginine. The reaction time was 15 min, the temperature was 40° C., and the reaction volume was 2 mL. Finally, 400 μL of 40% trichloroacetic acid was added to terminate the reaction.

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

The contents of the reaction products agmatine and putrescine were detected by HPLC.

A unit of enzyme activity is defined as the amount of enzyme required to convert 1 μmol of substrate per minute.

Table 1 Design of primers for single point saturation mutation of ADC and AUH

Table 2 Design of primers for combined mutation of ADC and AUH

Example 1: Mutation and screening of arginine decarboxylase

The arginine decarboxylase gene speA was cloned from Escherichia coli BL21 (DE3). The nucleotide sequence is shown in SEQ ID NO.5.

The cloned gene was connected to the pETDuet-1 vector to obtain the recombinant vector pETDuet-1-speA containing the wild-type arginine decarboxylase gene, and then using pETDuet-1-speA as a template, using the primer pairs in Table 1 D502-F/R, D531-F/R, and D535-F/R primers were used for PCR of the whole plasmid. After the PCR was completed, the size of the target fragment was verified by agarose gel electrophoresis. After the verification is successful, use a micro-spectrophotometer to measure the concentration, and then use Quick Cut Dpn I to digest the parent. The digestion reaction system is: 5 μL 10×Qucikcut Buffer, 1 μL Dpn I, 4 μL DNA, 40 μL ddH ₂ O, and mix gently Afterwards, it was centrifuged briefly and placed in a water bath at 37°C for 30 minutes.

Then take 15 μL of the digested PCR product and add it to 100 μL of LE.coli JM 1009 competent medium, mix gently, and ice-bath for 30 minutes. Place in a preheated 42°C water bath for heat shock treatment for 90s, ice-bath for 2min, add 1mL LB medium and incubate at 37°C for 1h. Finally, the cells were evenly spread on LB plates containing 100 μg/mL ampicillin, and cultured for 12 hours until a single colony grew. Pick a single colony for sequencing verification, and take 500 μL of the mutated plasmid after sequencing for strain preservation to complete the construction of the mutant library.

The successfully mutated plasmid was thermally transferred into E.coli BL21(DE3) for protein expression and screened according to the protein expression level.

The E.coli BL21(DE3) transformed with the recombinant plasmid containing the mutant was inserted into a 96-deep well plate for culture, each well contained 450 μL of LB medium, and 100 μg/mL of ampicillin. After culturing for 2 hours, IPTG was added at a final concentration of 0.4 mM to induce expression, and cells were collected by centrifugation after culturing at 15°C for 5 hours. Add 1000 U of lysozyme at 37°C for 1 hour to break the wall. Afterwards, the deep-well plate was centrifuged at 4°C and 4000r/min for 15min, and 200μL of the supernatant was taken for purification and determination of enzyme activity.

The activity of the enzyme was determined by HPLC: as shown in Table 3, the single mutants D502C, D502K, D531Y, D531C, D535M, and D535P all had higher enzyme activities than the wild type. Therefore, these sites were selected, combined and double-mutated (The primers used are shown in Table 2), specifically:

(1) D531Y, D531C, D535M, and D535P were mutated on the basis of the single mutant D502C;

(2) On the basis of single mutant D502K, mutate D531Y, D531C, D535M, and D535P respectively;

(3) On the basis of single mutant 531Y, mutate D535M and D535P respectively;

(4) On the basis of the single mutant D531C, the D535M and D535P were mutated respectively.

Using the same screening and enzyme activity assay methods as the single mutation, the results of the double mutants obtained are shown in Table 3. The specific enzyme activities of the D502K/D531Y, D502K/D531C, D502K/D535P, and D531C/D535P double mutants are higher. These double mutants were selected for further mutation (primers used are shown in Table 2):

(1) On the basis of the double mutant D502K/D531Y, mutate D535M and D535P respectively;

(2) On the basis of double mutant D502K/D531C, D535M and D535P were mutated respectively.

The specific enzymatic activity of the triple mutant was measured using the same screening and enzymatic activity assay methods as the single mutant, and the results are shown in Table 4.

After multiple rounds of mutation, the mutation with the highest activity was D502K/D531C/D535M, the sequence of which is shown in SEQ ID NO.1.

Table 3 ADC single mutation and double mutation library screening results

Table 4 ADC triple mutation library screening results

Example 2: Mutation and screening expression of agmatine urea hydrolase

The arginidine urea hydrolase gene speB was cloned from Escherichia coliBL21 (DE3), and the nucleotide sequence is shown in SEQ ID NO.6. The expressed product is used to catalyze the production of putrescine from agmatine.

The cloned gene was connected to the pETDuet-1 vector to obtain the recombinant vector pETDuet-1-speA containing the wild-type arginine decarboxylase gene, and then using pETDuet-1-speA as a template, using the primer pairs in Table 1 For H151-F/R, H153-F/R, H163-F/R, and D232-F/R, carry out the whole plasmid PCR. After the PCR is completed, verify the size of the target fragment by agarose gel electrophoresis. After the verification is successful, use a micro-spectrophotometer to measure the concentration, and then use Quick Cut Dpn I to digest the parent. The digestion reaction system is: 5 μL 10×Qucikcut Buffer, 1 μL Dpn I, 4 μL DNA, 40 μL ddH ₂ O, and mix gently Afterwards, it was centrifuged briefly and placed in a water bath at 37°C for 30 minutes.

Then take 15 μL of the digested PCR product and add it to 100 μL E.coli JM 109 competent medium, mix gently, and ice-bath for 30 minutes. Place in a preheated 42°C water bath for heat shock treatment for 90s, ice-bath for 2min, add 1mL LB medium and incubate at 37°C for 1h. Finally, the bacteria were evenly spread on the LB plate containing the corresponding antibiotics, and cultured for 12 hours until a single colony grew. Pick a single colony for sequencing verification, and take 500 μL of the mutated plasmid after sequencing for strain preservation to complete the construction of the mutant library.

The successfully mutated plasmid was thermally transferred into E.coli BL21(DE3) for protein expression and screened according to the protein expression level.

The E.coli BL21 (DE3) transformed with the mutant sequence was cultured in a 96-deep well plate, and each well contained 450 μL of LB medium containing 100 μg/mL ampicillin. After culturing for 2 hours, IPTG was added at a final concentration of 0.4 mM to induce expression, and cells were collected by centrifugation after culturing at 15°C for 5 hours. Add 1000 U of lysozyme at 37°C for 1 hour to break the wall. Afterwards, the deep-well plate was centrifuged at 4°C and 4000 r/min for 15 min, and 200 μL of the supernatant was taken to measure the enzyme activity.

With agmatine as the substrate, PLP as the coenzyme, the activity of the enzyme was measured by HPLC: the results are shown in Table 5, the specific enzyme activity of the agmatine urea hydrolase wild type is 0.4U/mg, single mutant H151L, D153Y, H163K, H163V, H163A, D232I, D232K, therefore, select these sites, combine and carry out double mutation (primers used are shown in Table 2), specifically:

(1) On the basis of single mutant H151L, mutate D153Y, H163V, H163A, D232I, and D232K respectively;

(2) On the basis of the single mutant 153Y, mutate H163V, H163A, D232I, and D232K respectively;

(3) On the basis of the single mutant H163V, mutate D232I and D232K respectively;

(4) On the basis of single mutant H163A, D232I and D232K were mutated respectively.

The specific enzymatic activity of the double mutants was measured using the same screening and enzymatic activity assay methods as the single mutants, and the results are shown in Table 5.

Table 5 AUH single mutation and double mutation library screening results

According to the specific enzyme activity of double mutants, on the basis of double mutants, construct H151L/D153Y/H163A, H151L/H163A/D232I, H151L/H163A/D232K, D153Y/H163A/D232I, D153Y/H163A/D232K (primers used See Table 2), using the above-mentioned same screening and enzyme activity determination method, the specific enzyme activity of the triple mutant was determined, and the results are shown in Table 6.

Select D153Y/H163A/D232I and H151L/H163A/D232K with higher specific enzyme activity, and on the basis of them, mutate H151L on D153Y/H163A/D232I, and mutate D153Y on the basis of H151L/H163A/D232K (used Primers are shown in Table 2), and the results are shown in Table 6.

After multiple rounds of mutation, the mutation with the highest activity was H151L/D153Y/H163A/D232K, the sequence of which is shown in SEQ ID NO.2. The specific enzyme activity of the expressed enzyme after combined mutation is: 57U/mg. Combined with the experimental results and three-dimensional structure analysis, the feedback inhibition of putrescine and spermidine in the mutant strain has been relieved.

Table 6 AUH triple mutation and quadruple mutation library screening results

Example 3: Construction of recombinant Escherichia coli expressing two enzymes simultaneously

Recombinant Escherichia coli construction: select mutated speA and speB genes (nucleotide sequences shown in SEQ ID NO.1 and 2 respectively), and connect to pCOLADuet-1 (Kana resistance), pACYCDuet-1 (chloramphenicol resistance), pCDFduet-1 (streptomycin resistance), pETDuet-1 (ampicillin resistance) and pRSFDuet-1 (canna resistance) plasmids, each expressing 2 genes, each gene before Both contain T7 promoter and RBS junction, with T7 terminator behind the gene.

The plasmid was transformed into E. coli Escherichia coli BL21, and positive transformants were obtained by screening with corresponding antibiotic plates, that is, recombinant E. coli capable of enhanced expression of double genes was obtained, as shown in Table 7.

Whole-cell transformation to produce putrescine:

During the initial reaction, the wet weight of cells in the reaction system was 20g/L, L-arginine was 300g/L, PLP was 5g/L, _MgSO4 was 9.6g/L, DTT was 0.02g/L, pH 9.5, 150rpm, React at 45°C for 24 hours. The most suitable combination was obtained as pACYCDuet-speB-speA. The whole-cell catalytic results are shown in Table 7.

Transformation results of different bacterial strains in table 7

Embodiment 4: Application of two kinds of enzymes to synthesize putrescine in vitro

The double genes speA and speB and their mutants were respectively connected to the pETDuet-1 vector, expressed and purified by the same method as in Example 1 to obtain arginine decarboxylase and agmatine urea hydrolase. Then add these two pure enzymes in 50mL reaction system, the addition amount of arginine decarboxylase and agmatine urea hydrolase is respectively 6.3mg and 4mg, contains 300g/L L-arginine in the reaction system, 5g /LPLP is, 9.6g/L MgSO ₄ , 0.02g/L DTT, pH 9.5, 150rpm, reaction at 45°C, conversion for 72 hours. The output of putrescine measured by final liquid chromatography was 160g/L.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> A kind of engineering bacteria and method for producing putrescine

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 1977

<212>DNA

<213> Artificial sequence

<400> 1

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

atcaaacagt tgttcccggt tctgccgctg gaagggctgg atcaagtgcc ggaacgtcgc 1560

gctgtgctgc tggatattac ctgtgactct tgcggtgcta tcatgcacta 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> 2

<211> 921

<212>DNA

<213> Artificial sequence

<400> 2

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 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 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 (9)

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.