Spermidine synthetase mutant and application thereof in synthesis of spermidine

Abstract

The invention discloses a spermidine synthetase mutant and application of the spermidine synthetase mutant in synthesis of spermidine, and belongs to the technical field of genetic engineering and metabolic engineering. According to the method, the spermidine synthetase is mutated firstly, the efficiency of the spermidine synthetase is improved, and on the basis, the dissolved oxygen and fed-batch concentration are optimally controlled through fermentation, so that the yield of spermidine is improved. The corynebacterium glutamicum provided by the invention is an engineering bacterium with plasmids and without defects, and after the corynebacterium glutamicum is subjected to fed-batch fermentation in a 5L fermentation tank for 68 hours, the yield of spermidine reaches 25.1 g / L, and the sugar-acid conversion rate is 17.9%.

Description

TECHNICAL FIELD

[0001] The present application relates to a mutant of spermidine synthetase and its application in synthesizing spermidine, and belongs to the technical field of genetic engineering and metabolic engineering. BACKGROUND

[0002] Spermidine, which usually exists in the form of trihydrochloride and contains three positively charged amino groups, is a low-molecular-weight natural polyamine widely present in organisms and is produced through the biosynthesis pathway of putrescine and plays a key regulatory function in numerous life activities. In recent years, the research on spermidine has been gaining momentum, especially in the field of anti-aging. A large number of animal models and some human studies have shown that spermidine supplementation can significantly prolong the lifespan of model organisms and improve problems related to aging, such as cognitive function decline and cardiovascular function degradation, and the core mechanism is believed to be closely related to the strong induction of autophagy. In addition, the potential benefits of spermidine in neuroprotection, heart health, metabolic regulation, and immune regulation have also attracted widespread attention, making it an important target for nutritional health and biomedical research and development.

[0003] Although spermidine has great potential, its production still faces certain challenges. At present, the core technology for large-scale preparation of spermidine still focuses on chemical synthesis, and its industrial production system generally uses 1,4-butanediamine and acrylonitrile as basic raw materials for coupling reaction. Currently, the main approaches for synthesizing spermidine by biological methods are the SAM pathway and the ASA pathway. Compared with chemical synthesis, biological synthesis has the advantages of fewer by-products and environmental friendliness, but still has the disadvantages of low production concentration, high cost, and long production cycle. At present, the deep integration of gene editing technology and systems biology is promoting microbial metabolic engineering into the era of precise regulation, and it is particularly noteworthy that the modular design concept advocated by synthetic biology is promoting the cross-species transplantation of metabolic pathways, which provides the possibility for microorganisms to efficiently'manufacture' secondary metabolites such as spermidine. In Corynebacterium glutamicum, metabolic engineering technology has been successfully applied to enhance precursor supply, eliminate product inhibition, optimize energy metabolism, and restructure transport systems, etc., which has promoted the continuous improvement of the production level of metabolic products.

[0004] Therefore, based on the spermidine synthetase mutant and fermentation optimization engineering to improve the production of spermidine in Corynebacterium glutamicum, there is a high practical value and economic value. SUMMARY

[0005] To solve the above problems, the present application uses Corynebacterium glutamicum ARG12 as the chassis cell, and introduces pEC XK99E-metk-speD, pXMJ19-speC-speE V161Q / L199N As a high-yield arginine-producing strain, Corynebacterium glutamicum has the ability to synthesize spermidine using glucose.

[0006] C. glutamicum ARG12 is described in Zhenqiang Zhao et al. Genomics and transcriptomics-guided metabolic engineering Corynebacterium glutamicum for L-arginine production.

[0007] The present application provides a mutant spermidine synthase, which has one or two amino acid mutations at position 161 or 199 based on the amino acid sequence shown in SEQ ID NO. 2.

[0008] SEQ ID NO. 2:

[0009] LSELWYTEKQTKNFGITLKVKQTLHTEQTEFQHLEMVETEEFGNMLFLDGMVMTSEKDEFVYHEMVAHVPLFTHPNPENVLVVGGGDGGVIREILKHPSVKKATLVDIDGKVIEYSKKFLPSIAGKLDDPRVDVRVDDGFMHIAKSENEYDVIMVDSTEPVGPAVNLFSKGFYAGISKALKEDGIFVAQTDNPWFTPELITNVQRDVKEIFPITRLYTANIPTYPSGLWTFTIGSKKYDPLEVEDSRFFDIDTKYYTKELHKAAFVLPKFVSDLIK

[0010] In one embodiment, the valine at position 161 is mutated to glutamine, designated as speE V161Q ;

[0011] or the leucine at position 199 is mutated to asparagine, designated as speE L199N ;

[0012] or the valine at position 161 is mutated to glutamine and the leucine at position 199 is mutated to asparagine, designated as speE V161Q / L199N .

[0013] In one embodiment, the valine at position 161 is mutated to glutamine and the leucine at position 199 is mutated to asparagine, the mutant spermidine synthase amino acid sequence is shown in SEQ ID NO. 3.

[0014] SEQ ID NO. 3:

[0015] LSELWYTEKQTKNFGITLKVKQTLHTEQTEFQHLEMVETEEFGNMLFLDGMVMTSEKDEFVYHEMVAHVPLFTHPNPENVLVVGGGDGGVIREILKHPSVKKATLVDIDGKVIEYSKKFLPSIAGKLDDPRVDVRVDDGFMHIAKSENEYDVIMVDSTEPQGPAVNLFSKGFYAGISKALKEDGIFVAQTDNPWFTPENITNVQRDVKEIFPITRLYTANIPTYPSGLWTFTIGSKKYDPLEVEDSRFFDIDTKYYTKELHKAAFVLPKFVSDLIK

[0016] The present application also provides a gene encoding the spermidine synthetase mutant.

[0017] The present application also provides a recombinant vector carrying the mutant gene.

[0018] In one embodiment, the nucleotide sequence of the wild-type spermidine synthetase is shown as SEQ ID NO. 1.

[0019] In one embodiment, the vector includes pXMJ19, pECXK99E, etc.

[0020] The present application also provides a recombinant cell expressing the mutant or carrying the gene or the recombinant vector.

[0021] In one embodiment, the recombinant cell is a bacterial or fungal host cell.

[0022] In one embodiment, the cell includes Corynebacterium glutamicum, Escherichia coli.

[0023] The present application also provides a Corynebacterium glutamicum genetically engineered bacterium expressing any of the above-mentioned spermidine synthetase mutants.

[0024] The present application also provides the use of any of the above-mentioned spermidine synthetase mutants or the above-mentioned gene or any of the above-mentioned plasmid vectors or any of the above-mentioned cells or the above-mentioned Corynebacterium glutamicum genetically engineered bacterium in the preparation of spermidine or a product containing spermidine.

[0025] The present application also provides a method for improving the production of spermidine by Corynebacterium glutamicum, overexpressing a spermidine synthetase mutant mutant having one or two amino acid mutations at position 161 or 199 based on the amino acid sequence of SEQ ID NO. 2.

[0026] In an embodiment, the valine at position 161 is mutated to glutamine, corresponding to the amino acid sequence shown in SEQ ID NO. 2; or,

[0027] the leucine at position 199 is mutated to asparagine; or,

[0028] the valine at position 161 is mutated to glutamine, and the leucine at position 199 is mutated to asparagine.

[0029] The present application also provides a recombinant enzyme catalyst containing the mutant spermidine synthetase, which is in any of the following forms:

[0030] (1) culturing a recombinant expression transformant containing the mutant spermidine synthetase, and isolating transformant cells containing the recombinant mutant spermidine synthetase enzyme;

[0031] (2) culturing a recombinant expression transformant containing the mutant spermidine synthetase, and isolating transformant cells containing the recombinant mutant spermidine synthetase enzyme, crushing the transformant cells containing the recombinant mutant spermidine synthetase enzyme to obtain a cell crushing solution;

[0032] (3) culturing a recombinant expression transformant containing the mutant spermidine synthetase, and isolating transformant cells containing the recombinant mutant spermidine synthetase enzyme, crushing the transformant cells containing the recombinant mutant spermidine synthetase enzyme to obtain a cell crushing solution, and freeze-drying the cell crushing solution of the recombinant mutant spermidine synthetase enzyme to obtain a freeze-dried enzyme powder.

[0033] The present application also provides a genetically engineered bacterium, which is Corynebacterium glutamicum as a chassis cell, and expresses the mutant spermidine synthetase described above, and also expresses S-adenosylmethionine synthetase, S-adenosylmethionine decarboxylase, and ornithine decarboxylase.

[0034] In an embodiment, the amino acid sequence of the S-adenosylmethionine synthetase is shown in SEQ ID NO. 5, the amino acid sequence of the S-adenosylmethionine decarboxylase is shown in SEQ ID NO. 6, and the amino acid sequence of the ornithine decarboxylase is shown in SEQ ID NO. 4.

[0035] In an embodiment, the nucleotide sequence encoding the S-adenosylmethionine synthetase is shown in SEQ ID NO. 8, the nucleotide sequence encoding the S-adenosylmethionine decarboxylase is shown in SEQ ID NO. 9, and the nucleotide sequence encoding the ornithine decarboxylase is shown in SEQ ID NO. 7.

[0036] In one embodiment, the genetically engineered bacteria uses C. glutamicum ARG12 as the chassis cell, pECXK99E as the expression vector to express S-adenosylmethionine synthase and S-adenosylmethionine decarboxylase, and pXMJ19 as the expression vector to express wild-type ornithine decarboxylase and spermidine synthase mutants.

[0037] The present invention also provides a method for increasing spermidine production, wherein the method comprises using the above-mentioned genetically engineered bacteria to ferment and prepare spermidine using glucose or methionine as a substrate.

[0038] The present invention also provides a method for preparing spermidine, wherein the method comprises preparing spermidine by fermentation using the above-mentioned genetically engineered bacteria.

[0039] In one embodiment, the method involves inoculating the seed culture of the genetically engineered bacteria into a fermentation medium. The fermentation conditions are: temperature 30–37°C, rotation speed 300–800 rpm, aeration rate 3–4 VVM, pH 6.5–7.5, fermentation time 60–80 h, dissolved oxygen controlled at 0–50%, and residual sugar 10–30 g / L. In another embodiment, after inoculating the seed culture of the genetically engineered bacteria into the fermentation medium, methionine is added to the medium.

[0040] In one embodiment, the amount of methionine added is 0-10 g / L.

[0041] The present invention also provides the use of the above-mentioned spermidine synthase mutant, or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned genetically engineered bacteria, or the above-mentioned method in the preparation of spermidine or products containing spermidine.

[0042] Beneficial effects

[0043] The *Corynebacterium glutamicum* strain provided by this invention is an engineered bacterium with plasmids, resistance, and no defects in its ability to synthesize spermidine from glucose. First, the spermidine synthase was mutated, improving the efficiency of spermidine synthesis. Based on this, further fermentation optimization was performed. The recombinant *Corynebacterium glutamicum* strain constructed in this invention was used in a 5L fed-batch fermenter, achieving a spermidine yield of 25.1 g / L and a sugar-acid conversion rate of 17.9% after 68 hours. Attached Figure Description

[0044] Figure 1 Fermentation of recombinant strain Arg-4. Detailed Implementation

[0045] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0046] The C. glutamicum ARG12 involved in the following examples is described in Zhenqiang Zhao et al. Genomics and transcriptomics-guided metabolic engineering Corynebacterium glutamicum for L-arginine production.

[0047] The raw materials used in the following examples are as follows:

[0048] Escherichia coli BL21 was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0049] Corynebacterium glutamicum competent medium: 10 g / L proteose peptone, 5 g / L yeast extract, 10 g / L NaCl, 5 g / L glucose, 1 g / L Tween-80, 3 g / L glycine.

[0050] Seed medium: 25 g / L glucose, 1.5 g / L K2HPO4·3H2O, 0.6 g / L MgSO4, 30 g / L corn syrup, 2.5 g / L urea, 0.005 g / L FeSO4·7H2O, 0.005 g / L MnSO4·4H2O, pH 7.0.

[0051] Fermentation medium: glucose 140 g / L, potassium phosphate dibasic 1.5 g / L, magnesium sulfate heptahydrate 0.6 g / L, corn syrup 5.0 g / L, ferrous sulfate heptahydrate 0.005 g / L, manganese sulfate monohydrate 0.005 g / L, urea 7.0 g / L, pH 7.0.

[0052] The test methods involved in the following examples are as follows:

[0053] 1. Determination of glucose content and sugar acid conversion rate:

[0054] The Bio-SBA biological analyzer was used to analyze the glucose content in the fermentation broth. 25 μL of standard liquid SBA was taken for calibration. After calibration, 1 mL of fermentation broth was diluted, and 25 μL of diluted fermentation broth was taken for determination. The data was recorded.

[0055] 2. The calculation formula of sugar acid conversion rate is as follows:

[0056]

[0057] Wherein: η: sugar acid conversion rate; CL-glutamic acid: the concentration of L-glutamic acid in the fermenter after fermentation, g / L; Co: initial sugar concentration, g / L; Cglucose: the concentration of glucose in the fermenter after fermentation, g / L; V1: the volume of 80% glucose solution consumed, L; V: the volume of fermentation broth before tanking, L.

[0058] 3. Determination of bacterial concentration:

[0059] The absorbance value at 600 nm was determined using a UV-2000Z ultraviolet visible spectrophotometer.

[0060] 4. Determination of spermidine yield

[0061] The yield of spermidine in the fermentation broth was determined by HPLC using opA pre-column derivatization method.

[0062] The gene sequences involved in the following examples are as follows:

[0063] The nucleotide sequence of the wild-type spermidine synthetase gene speE is shown as SEQ ID NO. 1:

[0064] ttgagcgagctttggtatacggagaagcagacgaagaatttcggcattacattaaaggtgaaacaaacattacatacggaacagacggaatttcagcatctggaaatggtggagacagaagaattcggcaacatgctgttcttagacggaatggtcatgacttctgaaaaagatgaattcgtctatcatgaaatggttgcgcacgtcccgttatttacgcacccgaatccggaaaacgtcctcgttgtcggcggcggagacggcggtgtcatcagagagattctgaagcacccaagcgtaaaaaaagcgacgcttgtcgacattgacggcaaggttattgaatactctaaaaaattccttccgtccattgcgggcaagcttgatgatccgcgcgtcgacgtgagagtcgatgacggtttcatgcacatcgcgaaatcagaaaatgagtatgacgtcatcatggttgattcaacagaaccggtcggaccggccgtcaatctgttctcaaaaggcttctatgccggaatttccaaggcgttaaaagaagacggcatcttcgtcgcgcagacggacaacccgtggtttacaccggagctgattacaaacgtacagcgtgacgtgaaagaaatcttcccgatcacccgtctgtatacggctaatatcccgacgtatccgagcggcctgtggacatttacgatcggttctaaaaagtacgatccgcttgaagtggaggacagccgtttctttgacatcgacacgaaatattacacgaaagagcttcacaaagcggcatttgttcttccgaaatttgtgagcgacttaatcaaataa

[0065] The nucleotide sequence of the wild-type ornithine decarboxylase gene speC is shown in SEQ ID NO. 7:

[0066]

[0067] The nucleotide sequence of the wild-type S-adenosylmethionine decarboxylase gene speD is shown in SEQ ID NO. 9:

[0068] ttgaaaaaactgaaactgcatggctttaataatctgaccaaaagtctgagtttttgtatttacgatatctgctacgccaaaactgccgaagagcgcgacggttatattgcttatatcgatgaactctataatgccaaccgtctgaccgaaatcctgtcagaaacctgttccattatcggggctaatattcttaacatcgcccgccaggattacgaaccacagggtgccagcgtcactattctggtgagtgaagaaccggttgacccgaaactcatcgacaaaacagaacaccccggcccactgccagaaacggtcgttgcccatcttgataaaagtcatatttgcgtacatacctacccggaaagtcatcctgaaggcggtttatgtaccttccgcgccgatattgaagtctctacctgcggcgtgatttctccgctgaaggcgctgaattacctgatccaccagcttgagtccgatatcgtaaccattgattatcgcgtgcgcggttttacccgcgacattaacggtatgaagcactttatcgaccatgagattaattcgattcagaactttatgtctgacgatatgaaggcgctgtatgacatggtggatgtgaacgtctatcaggaaaatatcttccataccaagatgttgcttaaagagttcgaccttaagcactacatgttccacaccaaaccggaagacttaaccgacagcgagcgccaggaaattaccgctgcgctgtggaaagaaatgcgcgagatttattacgggcgcaatatgccagctgtttaa

[0069] The nucleotide sequence of the wild-type S-adenosylmethionine synthetase gene metk is shown in SEQ ID NO. 8:

[0070]

[0071] Example 1: Construction of spermidine synthetase mutant recombinant strain

[0072] The specific steps are as follows:

[0073] 1. Synthesis of genes

[0074] The wild-type spermidine synthetase gene speE with the nucleotide sequence as shown in SEQ ID NO. 1 and the wild-type ornithine decarboxylase gene speC with the nucleotide sequence as shown in SEQ ID NO. 7 were chemically synthesized.

[0075] 2. Construction of recombinant plasmid

[0076] The gene speE and the gene speC were inserted into pXMJ19 between Hind III and EcoR I to construct the recombinant plasmid pXMJ19-speC-speE.

[0077] On the basis of the recombinant plasmid pXMJ19-speC-speE, the valine at position 161 of speE was mutated to glutamine or isoleucine; the leucine at position 199 was mutated to asparagine or valine; the valine at position 161 of speE was mutated to glutamine, and the leucine at position 199 was mutated to asparagine; the primers are shown in Table 1, and the recombinant plasmids pXMJ19-speC-speE V161Q , pXMJ19-speC-speE L199N , pXMJ19-speC-speE V161Q / L199N , pXMJ19-speC-speE V161I , pXMJ19-speC-speE L199V .

[0078] Table 1 Mutant primer sequences

[0079]

[0080]

[0081] 3. Construction of chassis strain C. glutamicum ARG12 / pEC-XK99E-metk-speD

[0082] (1) The S-adenosylmethionine decarboxylase gene speD with the nucleotide sequence as shown in SEQ ID NO. 9 and the S-adenosylmethionine synthetase gene metk with the nucleotide sequence as shown in SEQ ID NO. 8 were chemically synthesized.

[0083] (2) The gene speD and the gene metk prepared in step (1) are respectively inserted into pEC-XK99E between EcoRI and Kpnl to construct the recombinant plasmid pEC-XK99E-metk-speD.

[0084] (3) The pEC-XK99E-metk-speD prepared is electrotransformed into C. glutamicum ARG12 to prepare the recombinant strain C. glutamicum ARG12 / pEC-XK99E-metk-speD.

[0085] 4. Construction of spermidine synthase mutant recombinant strain

[0086] The recombinant plasmids pXMJ19-speC-speE, pXMJ19-speC-speE V161Q , pXMJ19-speC-speE L199N , pXMJ19-speC-speE V161Q / L199N , pXMJ19-speC-speE V161I , pXMJ19-speC-speE L199V respectively constructed in step 2 are electrotransformed into C. glutamicum ARG12 / pEC-XK99E-metk-speD competent cells to obtain the recombinant strains Arg-1 (C. glutamicum ARG12 / pEC-XK99E-metk-speD, pXMJ19-speC-speE), Arg-2 (C. glutamicum ARG12 / pEC-XK99E-metk-speD, pXMJ19-speC-speE V161Q ), Arg-3 (C. glutamicum ARG12 / pEC-XK99E-metk-speD, pXMJ19-speC-speE L199N ), Arg-4 (C. glutamicum ARG12 / pEC-XK99E-metk-speD, pXMJ19-speC-speE V161Q / L199N ), Arg-5 (C. glutamicum ARG12 / pEC-XK99E-metk-speD, pXMJ19-speC-speE V161I ), and Arg-6 (C. glutamicum ARG12 / pEC-XK99E-metk-speD, pXMJ19-speC-speE L199V ).

[0087] Example 2: Determination of spermidine production of spermidine synthetase mutant recombinant strains

[0088] The specific steps are as follows:

[0089] (1) The recombinant strains Arg-1, Arg-2, Arg-3, Arg-4, Arg-5, Arg-6 and the starting strain C. glutamicum ARG12 constructed in Example 1 were streaked on an antibiotic-free BHI plate and incubated at 30°C in an inverted incubator for 18-24 h. Larger single colonies were picked from the plate and inoculated into 500 mL baffle shake flasks containing 30 mL seed medium, and incubated at 30°C and 220 rpm for 24 h to prepare the first-stage seed liquid.

[0090] (2) The second-stage seed liquid was prepared by inoculating 1 L baffle shake flasks containing 200 mL seed medium with the first-stage seed liquid at a 10% (v / v) inoculation amount and incubating at 30°C and 220 rpm for 18 h.

[0091] (3) The second-stage seed liquid was inoculated into 5 L fermentors containing 1.8 L fermentation medium at a 10% (v / v) inoculation amount, and fermented for 68 h. The parameters of the fermentor were controlled as follows: initial rotation speed 300 rpm, temperature 30°C, aeration rate 4 vvm, pH 7.0, and dissolved oxygen controlled at 20%, with residual sugar 20 g / L. The results of the fed-batch fermentation in the 5 L fermentor are shown in Table 2.

[0092] Table 2: Production and yield of recombinant strains containing different mutants

[0093]

[0094] The results show that the spermidine production of C. glutamicum ARG12, Arg-1, Arg-2, Arg-3 and Arg-4 strains after 68 h of fermentation was 0, 7.4, 8.6, 10.1 and 13.4 g / L, respectively, and the sugar acid conversion rate was 0%, 9.7%, 11.5%, 11.8% and 12.2%, respectively. It can be seen that the spermidine production and sugar acid conversion rate of the spermidine synthetase mutant recombinant strains are significantly improved compared with Arg-1.

[0095] Example 3: Fermentation optimization of spermidine synthetase mutant strain Arg-4 (C. glutamicum ARG12 / pEC-XK99E-metk-speD, pXMJ19-speC-speE V161Q / L199N )

[0096] The specific embodiment is the same as that of Example 2, except that the strain is adjusted to be: C. glutamicum ARG12 / pEC-XK99E-metk-speD, pXMJ19-speC-speE V161Q / L199N The fermentation conditions are optimized, and the specific optimization is as follows:

[0097] 1. Increasing the sugar acid conversion rate by controlling the dissolved oxygen

[0098] The specific embodiment is the same as that of Example 2, except that the dissolved oxygen is controlled at 0%, 10%, 20%, and 50% respectively during the fermentation of the Arg-4 strain, and the fermentation culture is carried out according to the method of Example 2, and the results show that:

[0099] It is found that the spermidine yield is 11.1, 15.4, 13.4, and 12.5 g / L respectively when the dissolved oxygen is 0%, 10%, 20%, and 50%, and the sugar acid conversion rate is 11.7%, 12.9%, 10.9%, and 8.4% respectively. The yield and conversion rate are the highest when the dissolved oxygen is 10%.

[0100] 2. Substrate addition

[0101] The exogenous addition of methionine provides precursor substances for the synthesis of SAM, thereby promoting the synthesis of spermidine. In order to investigate the effect of methionine addition amount on the yield of spermidine, the specific embodiment is the same as that of Example 2, except that 0, 2, 5, and 10 g / L of methionine are added to the initial fermentation medium, and the spermidine yield is 12.5, 25.4, 20.1, and 21.5 g / L respectively. The comparative test finds that the yield and conversion rate are the highest when 2 g / L of methionine is contained in the fermentation.

[0102] 3. Fermentation preparation of spermidine under the conditions of 10% dissolved oxygen and 2 g / L methionine

[0103] The specific embodiment is the same as that of Example 2, except that the dissolved oxygen is controlled at 10% during the fermentation of the Arg-4 strain, and 2 g / L of methionine is added to the initial fermentation medium, and the fermentation culture is carried out according to the method of Example 2, and the results show that:

[0104] 5L fermenter fed-batch fermentation and Figure 1 The results show that the spermidine yield of the Arg-4 strain is 25.1 g / L after 68 h of fermentation, and the sugar acid conversion rate is 17.9%.

[0105] It can be seen that the strain with spermidine synthetase double mutant for synthesizing spermidine from glucose created by the application has obvious improvement in spermidine yield and sugar acid conversion rate after fermentation optimization, and has broad industrial application prospect.

[0106] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is not intended to limit the present application thereto, and various modifications and alterations can be made thereto by those skilled in the art without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the appended claims.

Claims

1. A mutant of spermidine synthase characterized in that, the agmatine synthase mutant is that the valine at position 161 of the agmatine synthase with the amino acid sequence as shown in SEQ ID NO. 2 is mutated into glutamine; or the leucine at position 199 of the agmatine synthase with the amino acid sequence as shown in SEQ ID NO. 2 is mutated into asparagine; or the valine at position 161 of the agmatine synthase with the amino acid sequence as shown in SEQ ID NO. 2 is mutated into glutamine, and the leucine at position 199 is mutated into asparagine.

2. A gene encoding the agmatine synthase mutant of claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector, preferably, the recombinant cell is a bacterial or fungal host cell.

4. An enzyme catalyst comprising the mutant spermidine synthase of claim 1, wherein, is any one of the following forms: (1) culturing the recombinant expression transformant containing the agmatine synthase mutant, and isolating the transformant cell containing the agmatine synthase mutant enzyme; (2) culturing the recombinant expression transformant containing the agmatine synthase mutant, isolating the transformant cell containing the agmatine synthase mutant enzyme, and crushing the transformant cell containing the agmatine synthase mutant enzyme to obtain a cell crushing solution; (3) culturing the recombinant expression transformant containing the agmatine synthase mutant, isolating the transformant cell containing the agmatine synthase mutant enzyme, crushing the transformant cell containing the recombinant agmatine synthase mutant enzyme to obtain a cell crushing solution, and freeze-drying the agmatine synthase mutant enzyme cell crushing solution to obtain a freeze-dried enzyme powder.

5. A genetically engineered bacterium, characterized by, The genetically engineered bacteria are Corynebacterium glutamicum as a chassis cell, expressing the agmatine synthase mutant of claim 1, and also expressing S-adenosylmethionine synthetase, S-adenosylmethionine decarboxylase, and ornithine decarboxylase.

6. The genetically engineered bacteria of claim 5, wherein, The amino acid sequence of the S-adenosylmethionine synthetase is as shown in SEQ ID NO. 5, the amino acid sequence of the S-adenosylmethionine decarboxylase is as shown in SEQ ID NO. 6, and the amino acid sequence of the ornithine decarboxylase is as shown in SEQ ID NO. 4; Preferably, the nucleotide sequence encoding the S-adenosylmethionine synthetase is as shown in SEQ ID NO. 8, the nucleotide sequence encoding the S-adenosylmethionine decarboxylase is as shown in SEQ ID NO. 9, and the nucleotide sequence encoding the ornithine decarboxylase is as shown in SEQ ID NO.

7.

7. A method of increasing the production of spermidine in Corynebacterium glutamicum, characterized in that, The method is to use Corynebacterium glutamicum as a chassis cell, express the agmatine synthase mutant of claim 1, and also express S-adenosylmethionine synthetase, S-adenosylmethionine decarboxylase, and ornithine decarboxylase; Preferably, the amino acid sequence of the S-adenosylmethionine synthetase is as shown in SEQ ID NO. 5, the amino acid sequence of the S-adenosylmethionine decarboxylase is as shown in SEQ ID NO. 6, and the amino acid sequence of the ornithine decarboxylase is as shown in SEQ ID NO. 4; Preferably, the nucleotide sequence encoding the S-adenosylmethionine synthetase is shown as SEQ ID NO. 8; the nucleotide sequence encoding the S-adenosylmethionine decarboxylase is shown as SEQ ID NO. 9; and the nucleotide sequence encoding the ornithine decarboxylase is shown as SEQ ID NO.

7.

8. A method of preparing spermidine, characterized by, The method is to add the genetically engineered bacteria of claim 5 or 6 to a fermentation medium to prepare spermidine by fermentation.

9. The method of claim 8, wherein, The fermentation conditions are: temperature 30-37℃, rotation speed 300-800rpm, aeration amount 3-4VVM, pH 6.5-7.5, fermentation time 60-80h, dissolved oxygen controlled at 0-50%, residual sugar 10-30g / L; Preferably, methionine is added to the fermentation medium after inoculation of the genetically engineered bacteria. Preferably, the amount of methionine added is 0-10g / L.

10. Use of the spermidine synthetase mutant of claim 1, the gene or recombinant vector of claim 2, the recombinant cell of claim 3, the enzyme catalyst of claim 4, the genetically engineered bacteria of claim 5 or 6, or the method of any one of claims 7-9 in the preparation of spermidine or a product containing spermidine.

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