Method for producing spermidine

Abstract

The present invention belongs to the technical field of synthetic biology. Disclosed is a method for producing spermidine. A genetically engineered strain of Serratia marcescens and a genetically engineered strain of Escherichia coli with a high spermidine yield are respectively obtained by introducing a new exogenous path, relieving the inhibition of various products from the spermidine synthesis route, changing the spermidine transfer system, and enhancing the enzyme activity in a key pathway by using a metabolic engineering method. The concentration of spermidine in the supernatant of the culture of the genetically engineered strain of Serratia marcescens in a shake flask reaches 26.8 g / L, and the concentration of spermidine in the supernatant of the culture of the genetically engineered strain of Escherichia coli in the shake flask reaches 24.7 g / L, both demonstrating promising industrial application prospects.

Description

A kind of production method of spermidine Technical Field

[0001] The invention relates to a method for producing spermidine, and belongs to the technical field of synthetic biology. Background Art

[0002] Spermidine, with the molecular formula NH2(CH2)3NH(CH2)4NH2, is widely present in microorganisms, plants, and animals and is an important physiologically active substance. A multifunctional polyamine, spermidine plays a vital role in numerous biological processes, including cell proliferation, differentiation, DNA stability, and cellular stress response. In industrial production, spermidine is often used as a catalyst for certain plastics and polymers and as a plant growth regulator in agriculture.

[0003] Despite its widespread industrial and agricultural applications, traditional chemical synthesis methods present a range of challenges. These include the use of highly toxic chemical reagents, which pose a threat to operator safety and lead to environmental pollution and waste disposal issues. Furthermore, the synthesis typically requires multiple, complex, and inefficient steps, resulting in low yields.

[0004] Compared with chemical synthesis, the use of microorganisms to synthesize spermidine has some significant advantages. The microbial synthesis process is relatively environmentally friendly and does not require the use of highly toxic reagents, reducing the negative impact on the environment. Secondly, biosynthesis can be carried out under mild conditions, reducing energy consumption and production costs. Most importantly, microorganisms have diverse metabolic pathways that can synthesize spermidine from naturally occurring metabolites. However, existing microbial production methods also have limitations. The stability of the production strain is a key issue, especially during long fermentation processes, when microorganisms may lose the ability to synthesize spermidine. In addition, the yield of spermidine is usually not high enough and needs to be improved through metabolic engineering methods. The fermentation process may also be complex and require careful design and optimization.

[0005] Therefore, one of the methods to solve the current problem of spermidine production by microbial fermentation is to transform microorganisms through genetic engineering to make them produce high spermidine.

[0006] Summary of the Invention

[0007] The present invention finds that Serratia marcescens (Serratia marcescens) is all outstanding in growth rate and metabolic adaptability, can grow under multiple nutrient sources and environmental conditions;And the metabolic network of Serratia marcescens has naturally adapted to the synthesis of polyamines, which provides it with natural production advantages. Compared to traditional Escherichia coli (1.5g / L), Serratia marcescens shows a higher tolerance to spermidine (7g / L), which is conducive to the high concentration production of spermidine. Therefore, the present invention provides a kind of Serratia marcescens genetic engineering bacteria and a kind of Escherichia coli genetic engineering bacteria, respectively, introduces a new way of synthesizing spermidine from exogenous, and uses Serratia marcescens and Escherichia coli as hosts to produce spermidine in high yield.

[0008] The first object of the present invention is to provide a genetically engineered bacterium, wherein the engineered bacterium has introduced an exogenous spermidine synthesis pathway and contains genes for carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase and aminopropylagmatine urea hydrolase.

[0009] In one embodiment, the genetically engineered bacteria is an engineered Serratia marcescens bacteria or an engineered Escherichia coli bacteria.

[0010] Optionally, the genetically engineered Escherichia coli bacteria are constructed using Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli Top10 or Escherichia coli MG1655 as hosts.

[0011] Optionally, the engineered Serratia marcescens bacteria is constructed using Serratia marcescens HBQA7 as a host.

[0012] In one embodiment, the carboxyaminopropylagmatine dehydrogenase can be a carboxyaminopropylagmatine dehydrogenase from any source; alternatively, it can be a carboxyaminopropylagmatine dehydrogenase from Spirochaeta thermophila, Denitrovibrio acetiphilus, Geobacter sulfurreducens, Ilumatobacter coccineus, etc. Further alternatively, it can be from Spirochaeta thermophila DSM 6578, Denitrovibrio acetiphilus DSM 12809, Geobacter sulfurreducens KN400, or Ilumatobacter coccineus YM16-304.

[0013] In one embodiment, the carboxyaminopropylagmatine decarboxylase can be a carboxyaminopropylagmatine dehydrogenase from any source; alternatively, it can be a carboxyaminopropylagmatine dehydrogenase from Spirochaeta thermophila, Denitrovibrio acetiphilus, Geobacter sulfurreducens, Ilumatobacter coccineus, etc. Further alternatively, it can be from Spirochaeta thermophila DSM 6578, Denitrovibrio acetiphilus DSM 12809, Geobacter sulfurreducens KN400, or Ilumatobacter coccineus YM16-304.

[0014] In one embodiment, the aminopropylagmatine urea hydrolase can be a carboxyaminopropylagmatine dehydrogenase from any source; alternatively, it can be a carboxyaminopropylagmatine dehydrogenase from Spirochaeta thermophila, Denitrovibrio acetiphilus, Geobacter sulfurreducens, Ilumatobacter coccineus, etc. Further alternatively, it can be from Spirochaeta thermophila DSM 6578, Denitrovibrio acetiphilus DSM 12809, Geobacter sulfurreducens KN400, or Ilumatobacter coccineus YM16-304.

[0015] In one embodiment, the engineered bacteria further knocks out the gene encoding the repressor protein argR and constructs an argA negative feedback mutant.

[0016] In one embodiment, the argA negative feedback mutant of the engineered bacteria is a mutant in which the 40th base C of the argA gene is mutated to T, and the encoded histidine is changed to tyrosine, thereby significantly increasing the production of arginine.

[0017] In one embodiment, the engineered bacteria further utilizes a strong promoter to replace the natural promoters of the gene encoding aspartate kinase, lysC, asd, argA, N-acetylglutamate synthetase, argH, and speA, respectively. For example, the strong promoter trc is used to replace the natural promoters of lysC, asd, argA, argH, and speA to enhance the expression of aspartate kinase, aspartate semialdehyde dehydrogenase, N-acetylglutamate synthetase, arginine succinate lyase, and arginine decarboxylase. Alternatively, the strong promoter sequence is shown in SEQ ID NO. 187.

[0018] In one embodiment, the engineered bacteria also knocks out the gene encoding the transport protein PotABCD, reducing the transfer of spermidine into the cell; optionally, further, the expression of the transport protein MdtJI that transfers spermidine to the extracellular space is enhanced, for example, by replacing the natural promoter of the MdtJI gene encoding the strong promoter.

[0019] In one embodiment, the engineered bacteria further has the thrA and metL genes encoding homoserine synthase knocked out; or, based on the knockout of the thrA and metL genes, the dapA gene encoding dihydropicolinate synthase is further knocked out; or, based on the knockout of the thrA, metL, and dapA genes, the proC encoding pyrroline 5-carboxylate reductase is further knocked out.

[0020] In one embodiment, the engineered bacteria further has a knockout of the astA gene encoding arginine N-succinyltransferase.

[0021] In one embodiment, the strong promoter is the trc promoter.

[0022] In one embodiment, the principle of homologous recombination is used to integrate and express genes in the genetically engineered bacteria.

[0023] In one embodiment, the knockout gene is knocked out using homologous recombination technology.

[0024] In one embodiment, the gene is knocked out using Crispr / Cas9 gene editing technology.

[0025] The second object of the present invention is to provide a use of the above-mentioned genetically engineered Serratia marcescens in producing spermidine.

[0026] The present invention also provides the use of the above-mentioned genetically engineered Escherichia coli bacteria in producing spermidine. Any of the above-mentioned genetically engineered bacteria is placed in a culture medium containing glucose and fermented to produce spermidine.

[0027] In one embodiment, the fermentation is carried out at pH 7, 37° C., a shaking flask speed of 200 rpm, and fermentation for 24 hours.

[0028] In one embodiment, the genetically engineered bacteria can be used for high-density culture to produce spermidine.

[0029] The present invention also provides a method for increasing the yield of spermidine, wherein any of the above-mentioned genetically engineered bacteria is placed in a culture medium containing glucose and fermented to produce spermidine.

[0030] In one embodiment, the fermentation is carried out at pH 7, 37° C., a shaking flask speed of 200 rpm, and fermentation for 24 hours.

[0031] In one embodiment, the genetically engineered bacteria can be used for high-density culture to produce spermidine.

[0032] The present invention also provides a method for transforming genetically engineered bacteria, comprising: using common primers to amplify and obtain gene fragments of carboxyaminopropylagmatine dehydrogenase and carboxyaminopropylagmatine decarboxylase; using double enzymes HindIII and EcoRV to digest the first multiple cloning site of plasmid pBBR1MCS-2; purifying the digestion product; and connecting the digested plasmid with the gene fragments of carboxyaminopropylagmatine dehydrogenase and carboxyaminopropylagmatine decarboxylase to construct a plasmid connected with the carboxyaminopropylagmatine dehydrogenase and carboxyaminopropylagmatine decarboxylase genes;

[0033] An aminopropylagmatine urea hydrolase gene fragment was amplified using aminopropylagmatine urea hydrolase gene primers, and the second multiple cloning site of the plasmid containing the carboxyaminopropylagmatine dehydrogenase and carboxyaminopropylagmatine decarboxylase genes was digested using the enzymes EcoRI and SpeI. After purification of the digestion product, the digested plasmid was ligated with the aminopropylagmatine urea hydrolase gene fragment to construct a plasmid containing the carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase, and aminopropylagmatine urea hydrolase genes.

[0034] A plasmid containing carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase, and aminopropylagmatine ureahydrolase genes was transformed into JM109 competent cells, and positive clones were selected on a sodium-resistant card plate and further verified by colony PCR. The plasmid was extracted from the JM109 strain and transformed into competent genetically engineered bacteria to construct a spermidine-synthesizing strain.

[0035] Optionally, the competent genetic engineering bacteria include Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli MG1655 or Serratia marcescens HBQA7;

[0036] Alternatively, the carboxyaminopropylagmatine dehydrogenase gene, the carboxyaminopropylagmatine decarboxylase gene and the aminopropylagmatine ureahydrolase gene can be cloned from the genomes of Spirochaeta thermophila DSM 6578, Denitrovibrio acetiphilus DSM 12809, Geobacter sulfurreducens KN400, and Ilumatobacter coccineus YM16-304;

[0037] Optionally, the nucleotide sequence of the common primer is shown as SEQ ID NOs. 1-2; the nucleotide sequence of the aminopropylagmatine urea hydrolase gene primer is shown as SEQ ID NOs. 3-4. Beneficial effects

[0038] Based on synthetic biology methods, this invention constructs a genetically engineered strain of Serratia marcescens and Escherichia coli that can produce high levels of spermidine using glucose as a raw material. The production process is simple, the raw materials are readily available, and the cost is low, showing good prospects for industrial application. The details are as follows:

[0039] (1) The present invention introduces an exogenous pathway to achieve a new pathway for the synthesis of spermidine in Serratia marcescens by introducing exogenous carboxylaminopropyl agmatine dehydrogenase genes, exogenous carboxylaminopropyl agmatine decarboxylase genes, and exogenous aminopropyl agmatine urea hydrolase genes. Compared to traditional production pathways, the genetically engineered Serratia marcescens of the present invention can greatly increase the yield of spermidine. When only the exogenous pathway is introduced, the spermidine concentration reaches 5.9 g / L.

[0040] (2) The present invention relieves the feedback inhibition of arginine in the genetically engineered Serratia marcescens and relieves the inhibition of the pathway by ArgR. After optimization, the production of spermidine reaches 8.6 g / L.

[0041] (3) The present invention enhances the expression of aspartate kinase encoded by the lysC gene and aspartate semialdehyde dehydrogenase encoded by the asd gene in the genetically engineered bacteria of Serratia marcescens, which are key enzymes that catalyze the synthesis of aspartate semialdehyde from aspartate; N-acetylglutamate synthetase and arginine succinate lyase encoded by the argA and argH genes, which are the first and last enzymes in the arginine synthesis pathway, respectively; and arginine decarboxylase encoded by the speA gene, which is a key enzyme that catalyzes the further synthesis of agmatine from arginine. The expression of these five enzymes can increase the yield of spermidine to 16.3 g / L.

[0042] (4) The present invention improves the transport pathway of spermidine in genetically engineered Serratia marcescens. PotABCD and MdtJI proteins are proteins that transport spermidine into and out of cells, respectively. Knocking out PotA and enhancing the expression of MdtJI can increase the production of spermidine to 21.4 g / L.

[0043] (5) The present invention knocks out the genes thrA, metL, dapA, and proC in the genetically engineered bacteria of Serratia marcescens, and the spermidine production reaches 23.3 g / L.

[0044] (6) The present invention further knocks out the ast series genes in the genetically engineered bacteria of Serratia marcescens to increase the production of spermidine to 26.8 g / L.

[0045] (7) The present invention introduces an exogenous pathway, and realizes a new pathway for the synthesis of spermidine in Escherichia coli by introducing exogenous carboxylaminopropyl agmatine dehydrogenase gene, exogenous carboxylaminopropyl agmatine decarboxylase gene, and exogenous aminopropyl agmatine urea hydrolase gene. Escherichia coli almost does not produce spermidine without genetic engineering modification, and the output is extremely low, close to zero. The traditional spermidine synthesis pathway in Escherichia coli synthesizes spermidine through carboxylation of S-adenosylmethionine and putrescine under the action of spermidine synthase. The synthesis pathway of carboxylation of S-adenosylmethionine has a low production efficiency. Compared with the traditional production pathway, the genetically engineered Escherichia coli of the present invention can greatly increase the output of spermidine. When only the exogenous pathway is introduced, the spermidine concentration reaches 6.7 g / L.

[0046] (8) The present invention eliminates feedback inhibition and repression in genetically engineered Escherichia coli. Arginine synthesis is a multi-stage enzyme chain reaction starting from glutamate, and it is necessary to remove the inhibitory effect of arginine on N-acetylglutamate synthase encoded by argA. ArgR is a repressor protein of the arginine (Arg) pathway. The present invention eliminates the feedback inhibition of arginine and the repression of the pathway by ArgR. After optimization, the production of spermidine reaches 7.8g / L.

[0047] (9) The present invention enhances the expression of key enzymes in genetically engineered Escherichia coli. Aspartate kinase encoded by the lysC gene and aspartate semialdehyde dehydrogenase encoded by the asd gene are key enzymes that catalyze the synthesis of aspartate semialdehyde from aspartate. N-acetylglutamate synthetase and arginine succinate lyase encoded by the argA and argH genes are the first and last enzymes in the arginine synthesis pathway, respectively. Arginine decarboxylase encoded by the speA gene is a key enzyme that catalyzes the further synthesis of agmatine from arginine. Enhanced expression of these five enzymes can increase spermidine production to 14.8 g / L.

[0048] (10) The present invention improves the transport pathway of spermidine in genetically engineered Escherichia coli. PotABCD and MdtJI proteins are proteins that transport spermidine into and out of cells, respectively. Knocking out PotA and enhancing the expression of MdtJI can increase the production of spermidine to 18.5 g / L.

[0049] (11) The present invention weakens the competitive pathways of the spermidine process in genetically engineered Escherichia coli. In the spermidine synthesis process, the aspartaldehyde intermediate has multiple competitive pathways for the production of amino acids, such as the lysine pathway and the homoserine pathway; there are also competitive pathways for the glutamate intermediate, such as the synthesis of Pro. By weakening these pathways, the spermidine production reached 21.3 g / L after knocking out the genes thrA, metL, dapA, and proC.

[0050] (12) The present invention knocks out the relevant decomposition pathways in the genetically engineered Escherichia coli. Escherichia coli contains enzymes that utilize and degrade the intermediates and products of spermidine synthesis. For example, the arginine N-succinyltransferase encoded by the astA gene degrades arginine to produce N2-succinylarginine, which is further degraded into L-Glu and succinic acid through the ast series genes, thereby increasing the spermidine yield to 24.7 g / L.

[0051] (13) The genetically engineered Escherichia coli of the present invention can produce a maximum yield of 266 g / L through high-density fermentation.

[0052] Biological deposit materials

[0053] The Serratia marcescens HBQA7 in the present invention is taxonomically named Serratia marcescens HBQA7, which was deposited in the China Center for Type Culture Collection on February 23, 2023, with a deposit number of CCTCC NO: M 2023184, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG1 shows the pathway for spermidine synthesis by Serratia marcescens and Escherichia coli in the present invention. DETAILED DESCRIPTION

[0055] 1. Strains and plasmids involved in the present invention

[0056] pRSFDuet-1, pTrc99a, PKD46, pCP20, T19 vector plasmids, pMD18-T vector plasmid, Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli Top10, and Escherichia coli MG1655 were purchased from Novagen. pBBR1MCS-2 was purchased from the MiaoLing Plasmid Platform, and Serratia marcescens HBQA7 (CCTCC NO: M 2023184) was collected from the laboratory. Illustrative examples of Serratia marcescens include, but are not limited to, Serratia fonticola ATCC 29845, Serratia odorifera ATCC 33077, Serratia plymuthica ATCC 15928, Serratia liquefaciens ATCC 27592, Serratia rubidaea ATCC 19279, Serratia oryzae ATCC 1011, Serratia ureilytica ATCC BAA-2620, Serratia entomophila ATCC 43705, Serratia ficaria ATCC 33105, Serratia marcescens ATCC 13880, and Serratia proteamaculans ATCC 19323, all available from the ATCC culture collection. Serratia symbiotica DSM 23270, Serratia nematodiphila DSM 21420, Serratia quinivorans DSM 4597, and Serratia grimesi DSM 30063 were purchased from the DSMZ strain bank in Germany. Serratia marcescens HBQA7 was deposited in the China Center for Type Culture Collection with the accession number CCTCC NO: M 2023184.

[0057] 2. Fermentation conditions

[0058] The shake flask fermentation medium used in the strain yield verification of the present invention is formulated as follows: per liter, 10 g glucose, 10 g yeast extract, 20 g NaCl, 50% (v / v) R / 2 medium, and 5 mL of trace metal element stock solution; the R / 2 medium comprises per liter 2 g (NH4)2HPO4, 6.75 g KH2PO4, 0.85 g citric acid, and 0.7 g MgSO4·7H2O, with a pH of 6.86; and the trace metal element stock solution comprises per liter 5 M HCl, 10 g FeSO4·7H2O, 2.25 g ZnSO4·7H2O, 1 g CuSO4·5H2O, 0.5 g MnSO4·5H2O, 0.23 g Na2B4O7·10H2O, 2 g CaCl2·2H2O, and 0.1 g (NH4)6Mo7O 24 The shake flask fermentation temperature was controlled at 37°C and the initial pH was 7. The liquid volume was 1 / 5 of the shake flask volume, the rotation speed was controlled at 200 rpm, and the shake flask fermentation time was 24 hours.

[0059] 3. Determination of spermidine content

[0060] Preparation of OPA derivatization reagent: Prepare OPA reagent using the Uren method and Karababa (15). Dissolve 0.20 g of OPA in 9.0 mL of methanol, add 1.0 mL of 0.40 M (pH 9.0) borate buffer and 160 μL of 2-mercaptoethanol (reducing agent). Store OPA reagent at 4°C.

[0061] Spermidine was first derivatized with o-phthalaldehyde (OPA; Sigma, St. Louis, MO). 50 μL of sample and 450 μL of water were added to 400 μL of methanol. After adding 100 μL of OPA reagent, the mixture was filtered through a 0.2-μm filter membrane, and 20.0 μL of the filtrate was immediately injected into the HPLC instrument.

[0062] HPLC detection conditions: Spermidine was detected according to the method in the reference (Qian, ZG, XXXia, and SYLee, Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine. Biotechnol Bioeng, 2009. 104 (4): p. 651-662): A Luna 5-μm C18 (2) 100A column (250 × 4.6 mm; Phenomenex) was used for all sample separations at 25° C. and a mobile phase of 0.8 mL / min. The mobile phase consisted of solvent A (55% methanol in 0.1 M sodium acetate, pH 7.2) and solvent B (methanol). The following gradient was applied: 1-6 min, 100% A; 6-10 min, linear gradient from 0% to 30% B; 10-15 min, linear gradient from 30% to 50% B; 15-19 min, linear gradient from 50% to 100% B; 19-23 min, 100% B; 23-25 ​​min, linear gradient from 100% to 30% B; 25-28 min, linear gradient from 30% to 0% B (all in % by volume).

[0063] 4. Gene integration expression plan

[0064] The principle of homologous recombination is used for gene integration and expression. Specifically,

[0065] The target gene was amplified using primers "KZ-F / R" and integrated into the multiple cloning site of the T19 vector plasmid via homologous recombination. Using the 1000 bp above and below the target gene as a template, the left homology arm (LA) and right homology arm (RA) were amplified and ligated to the ends of the target gene on the T19 vector plasmid to construct the homology arms. The plasmid containing the ligated left and right arms was extracted, and the target fragment (LA-trc-target gene-RA) was amplified using the plasmid T19-LA-trc-target gene-RA as a template using corresponding primers. The PCR product was purified and the template effect was eliminated using the restriction endonuclease DpnI. The digested product was then purified again. The homologous recombination enzyme-expressing PKD46 plasmid was extracted using a plasmid extraction kit and transformed into competent Serratia marcescens HBQA7 cells to construct Serratia marcescens HBQA7-PKD46. After preparing Serratia marcescens HBQA7-PKD46 competent cells, store them at -80°C for later use. The purified product was transferred into the above-mentioned Serratia marcescens competent cells, and positive transformants were screened on resistance plates using the carnamycin resistance marker. To verify whether recombination occurred, PCR was performed using the corresponding primers "target gene-YZ-F" and "target gene-YZ-R". If recombination occurred, a fragment of about 1200 bp could be amplified, otherwise no fragment could be amplified. At the same time, the corresponding PCR method was used on ampicillin and carnamycin double-resistance plates to verify Serratia marcescens HBQA7 PKD46 (kana R ). Then, the resistance to carnamycin was eliminated. First, Serratia marcescens HBQA7 PKD46 (kana R ) competent cells, and the pCP20 plasmid was transformed into Serratia marcescens HBQA7-PKD46 (kana R ) After the cells were competent, resistance-eliminated transformants were screened using non-resistant plates and resistant plates, and transformants were identified by colony PCR to screen out transformants that integrated the target gene and eliminated resistance.

[0066] By using the above-mentioned integration expression method and integration operation steps, the mutated gene and the replaced promoter sequence are integrated into the required position of the genome, and the strain is subjected to corresponding metabolic engineering transformation.

[0067] Enzyme digestion reaction system: 10×Q Cut Buffer 5μL, DNA 20μL, ddH2O 33μL, endonuclease-1 1μL, endonuclease-2 1μL, mix well, and heat in a 37℃ water bath for 0.5-1h.

[0068] Ligation reaction system: 5 μL of Lightning Cloning Enzyme, 1-2 μL of double-enzyme digested plasmid (10-100 ng), 4-5 μL of homologous target fragment, add ddH2O to 10 μL, mix well, and heat in a 50°C water bath for 30 min.

[0069] DpnI template effect elimination reaction system (60 μL): DpnI 3 μL, PCR purified product 20 μL, 10× Fast Digest Buffer 6 μL, ddH2O 31 μL.

[0070] PCR amplification target gene reaction system (50 μL): DNA template 1 μL, 5× PrimeSTAR Buffer 10 μL, dNTP Mixture 4 μL, amplification primer 1 μL, amplification primer 1 μL, PrimeSTAR HS DNA Polymerase 0.5 μL, ddH2O 32.5 μL.

[0071] PCR amplification program: 30 cycles (98°C for 10 s, 55°C for 10 s, 72°C for 1 kb / min), 72°C for 10 min, and storage at 4°C.

[0072] Colony PCR reaction system: 2×Tag Master Mix 10μL, upstream primer (concentration 10μM) 1μL, downstream primer (concentration 10μM) 1μL, ddH2O 8μL, a small amount of colonies or bacterial liquid as template.

[0073] Example 1: Introduction of exogenous spermidine synthesis pathway - construction of genetically engineered Serratia marcescens

[0074] The present invention introduces the CAPADH / CAPADC / APAUH pathway from various microbial strains into Serratia marcescens HBQA7 for heterologous expression. A schematic diagram of the construction is shown in Figure 1. First, carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase, and aminopropylagmatine ureahydrolase from various sources are expressed in Serratia marcescens via plasmids. The enzymes with the highest catalytic efficiency are then selected and exogenously integrated into the CAPADH / CAPADC / APAUH pathway via homologous recombination. The genomic integration site selected in the present invention is upstream of the argA gene of Serratia marcescens HBQA7.

[0075] The specific process is as follows:

[0076] The carboxyaminopropylagmatine dehydrogenase genes stcapadh, dacapadh, gscapadh, and iccapadh were cloned from the genomes of Spirochaeta thermophila DSM 6578, Denitrovibrio acetiphilus DSM 12809, Geobacter sulfurreducens KN400, and Ilumatobacter coccineus YM16-304 using amplification primers. The amino acid sequences of these genes are listed in NCBI accession numbers WP_014623968.1, WP_013011851.1, WP_010943175.1, and WP_015442124.1, respectively; The carboxylaminopropylagmatine decarboxylase genes stcapadc, dacapadc, gscapadc, and iccapadc were cloned from the genome of YM16-304, and their amino acid sequences are numbered WP_014623969.1, WP_013011852.1, WP_004513933.1, and WP_015442123.1 in NCBI, respectively. The aminopropylagmatine urea hydrolase genes stapauh, daapauh, lbapauh, and icapauh were cloned from the YM16-304 genome. Their amino acid sequences are listed in NCBI as WP_014623970.1, WP_013011853.1, WP_015770028.1, and WP_015442122.1, respectively. These three cloned genes were ligated into the same pBBR1MCS-2 plasmid to generate recombinant Serratia marcescens expressing these three genes.

[0077] CAPADH and CAPADC in the above-mentioned microorganisms are located adjacent to each other in a gene cluster. Using stcapadh-HindⅢ-F and stcapadc-EcoRⅤ-R as primers and the genome of Spirochaeta thermophila DSM 6578 as a template, the target fragment stcapadh-stcapadc was amplified. The first multiple cloning site of the plasmid pBBR1MCS-2 was digested with the double enzyme HindⅢ and EcoRⅤ enzymes. After purification of the digestion product, the cut plasmid was ligated with the amplified target fragment stcapadh-stcapadc. The ligation product pBBR1MCS-2-stcapadh-stcapadc was transformed into JM109 competent cells, and positive clones were selected on a resistance plate sodium plate. Colony PCR was further used to verify its correctness. Using stapauh-EcoRⅠ-F and stapauh-SpeⅠ-R as primers, the first multiple cloning site of the plasmid pBBR1MCS-2 was digested with the double enzyme HindⅢ and EcoRⅤ enzymes. The target fragment, stapauh, was amplified using the 6578 genome as a template. The second multiple cloning site of the plasmid pBBR1MCS-2-stcapadh-stcapadc was digested with EcoRI and SpeI. The digestion product was purified and ligated with the amplified target fragment, stapauh. The ligation product, pBBR1MCS-2-stcapadh-stcapadc-stapauh, was transformed into JM109 competent cells. Positive clones were selected on resistant cardiomyocytes plates and verified by colony PCR. The JM109 strain with the correct ligation was selected, and the plasmid pBBR1MCS-2-stcapadh-stcapadc-stapauh was extracted and transformed into competent Serratia marcescens HBQA7 cells to construct the Serratia marcescens HBQA7-stcapadh-stcapadc-stapauh spermidine-biosynthesizing strain. The remaining strains were constructed similarly to the Serratia marcescens HBQA7-stcapadh-stcapadc-stapauh strain. Carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase, and aminopropylagmatine ureahydrolase from different strains were ligated into two multiple cloning sites of the pBBR1MCS-2 plasmid. Other genes were ligated in a similar manner. The primer sequences used are shown in Table 1.

[0078] Table 1 Primer sequences (SEQ ID NO. 1-4)

[0079] The recombinant Serratia marcescens was transferred into a shake flask fermentation medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L) at a volume ratio of 2%. 600 After the pH reached 0.6-0.8, IPTG was added to a final concentration of 0.4 mM and expression was induced at 20°C for 12 hours. After induction, the supernatant was collected by centrifugation at 8000 rpm for 20 minutes at 4°C. The spermidine concentration in the supernatant was determined by HPLC, as shown in Table 2.

[0080] Table 2

[0081] Overexpressing key genes for spermidine synthesis using expression vectors imposes a metabolic burden on the bacteria, affecting their growth. The expression vectors are structurally unstable and easily lost during bacterial reproduction, and their copy number in the cells is unstable. The addition of antibiotics affects bacterial growth and pollutes the environment. Increased costs due to IPTG induction limit the expansion of production. Therefore, the strain Serratia marcescens-stcapadh-stcapadc-stapauh, which has the best effect of inducing spermidine synthesis, was selected. The promoter J23119 and ribosome binding site (RBS) B0034 from Serratia marcescens were ligated to the stcapadh-stcapadc-stapauh gene to construct an exogenous gene expression fragment. Following the aforementioned "4. Gene Integration and Expression Plan," the exogenous gene expression fragment was integrated into the knockout strain Serratia marcescens HBQA7 to construct the Serratia marcescens-stcapadh-stcapadc-stapauh strain, designated S1, which served as the base strain for subsequent studies.

[0082] The production of S1 spermidine was verified by shake flask culture, and the spermidine concentration in the shake flask supernatant was measured to be 5.9 g / L.

[0083] Example 2: Removing feedback inhibition of important enzymes in the Serratia marcescens genetically engineered bacteria pathway and knocking out the repressor protein

[0084] In the process of synthesizing important intermediates such as aspartate semialdehyde and arginine, there is an important repressor protein that inhibits the synthesis of intermediates and final products. The DNA-binding dual transcriptional regulator encoded by the argR gene (amino acid sequence accession number WP_004933572 on NCBI) inhibits the expression of the arg series genes and the excessive synthesis of the required important intermediates ornithine and arginine. Therefore, in order to ensure the stable and high-rate synthesis of each intermediate, the gene encoding this repressor protein was deleted to increase the metabolic flux.

[0085] In the metabolic pathway of arginine synthesis, N-acetylglutamate synthase encoded by argA (amino acid sequence accession number on NCBI is ALE97678.1) is subject to feedback inhibition by the product arginine. To improve the activity of this enzyme, based on the knockout of the argR gene, the gene encoding the enzyme was further mutated to construct a negative feedback mutant, thereby relieving feedback inhibition and obtaining the engineered bacterium Serratia marcescens argA. fbr ΔArgR, named S2.

[0086] The specific process is as follows:

[0087] (1) Knockout of the repressor protein ArgR

[0088] The primer designs are shown in Table 3 below:

[0089] Table 3 Primer sequences (SEQ ID 5-14)

[0090] The homologous recombination enzyme-expressing PKD46 plasmid was extracted using a plasmid extraction kit, purified, and transformed into competent S1 cells to construct S1-PKD46. After preparation, S1-PKD46 competent cells were stored at -80°C until use. The homologous recombination fragment template plasmid T19 vector was extracted using a plasmid extraction kit and purified using a gel tapping recovery kit. Primers ApaI-LA-argR and argR-LA-EcoRI, and KpnI-RA-argR and argR-RA-SalI were designed to amplify the corresponding homology arms LA and RA fragments, which were then purified and recovered using a gel tapping recovery kit. The T19 vector was double-digested with ApaI and EcoRI, and the digestion results were confirmed by electrophoresis. The digestion product was 5041 bp in size and purified. The double-digested T19 vector product was ligated with the left arm LA fragment. The ligated product was transformed into JM109 competent cells and plated onto a Kansas-resistant plate. Positive clones were screened and verified by colony PCR using primers LA-YZ-F and LA-YZ-R. The band size was approximately 1100 bp. Successful strains were sequenced to further confirm successful fragment ligation. The T19 plasmid with the LA fragment ligated was double-digested with KpnI and SalI. The RA fragment was ligated using the same method as above, and the ligated plasmid was verified. The T19 vector plasmid with the LA and RA fragments ligated was extracted and used as a template for PCR with primers ApaI-LA-argR and argR-RA-SalI. The target fragment, argR-LA-FRT-Kan-FRT-RA, was obtained. Electrophoresis confirmed the band size to be approximately 3324 bp. The PCR product was purified and the template effect was eliminated using the restriction endonuclease DpnI. The target fragment argR-LA-FRT-Kan-FRT-RA PCR product after restriction endonuclease DpnI digestion is further purified and transferred into Serratia marcescens HBQA-PKD46 competent cells after purification. Positive transformants are screened on resistance plates using the carnamycin resistance marker. To verify whether the positive transformants screened have indeed undergone recombination, the positive transformants are used as templates and primers argR-YZ-F and argR-YZ-R are used to amplify a fragment of about 1100bp, which can be judged as recombination. Conversely, if no fragment can be amplified, it can be judged as a false positive. While verifying whether recombination has occurred by PCR amplification, the corresponding PCR verification bacteria are spotted on ampicillin sodium plates. The correct transformant S1-PKD46 (kana R ) to eliminate carnamycin resistance, firstly, S1-PKD46 (kana R ) competent cells, the extracted pCP20 plasmid was transformed into S1-PKD46 (kana R) After the cells were competent, resistance-eliminated transformants were screened using non-resistant plates and resistant plates, and transformants were identified by colony PCR. The transformant S1-ΔargR with knockout of the argR gene and elimination of resistance was selected.

[0091] (2)argA fbr Construction of mutants

[0092] The argA gene is subject to feedback inhibition by the target product arginine. In the present invention, the 40th base C of the argA gene is mutated to T. After the encoded histidine is changed to tyrosine, the production of arginine is significantly increased. Therefore, we mutated this site and integrated it into the plasmid pRSFDuet-1 plasmid.

[0093] The specific process is as follows:

[0094] The primer designs are shown in Table 4 below:

[0095] Table 4 Primer sequences (SEQ ID NO. 15-18)

[0096] First, the pRSFDuet-1 (high copy) plasmid was extracted using a plasmid extraction kit and double-digested with BamHI and HindIII. The digestion system (50 μL) was as follows: 5 μL 10×Q Cut Buffer, 20 μL plasmid, 1 μL BamHI, 1 μL HindIII, and 33 μL ddH2O. After digestion, the fragment was purified. Next, the Serratia marcescens HBQA genome was extracted and used as a template to amplify the argA gene fragment. After PCR verification, the product was ligated into the digested pRSFDuet-1 plasmid. E. coli JM109 competent cells were prepared using the CaCl2 method. The pRSFDuet-1 plasmid ligated with the argA fragment was transformed into competent cells. Positive transformants were selected and identified as successfully transformed E. coli JM109-argA strains by colony PCR. The plasmid pRSFDuet1-argA from E. coli JM109-argA was extracted and used as a template for site-directed mutagenesis of argA. The mutant gene was designed and extracted using the Fast Mutangenesis System kit. The extracted pRSFDuet1-argA plasmid was used as a template and the argA mutant primers argA-TB-F / R were used to amplify argA. fbr Fragment, wherein the PCR amplification system (50 μL) is as follows: plasmid template 3 μL, argA-TB-F 1 μL, argA-TB-R 1 μL, Fly PCR SuperMix 25μL, ddH2O 20μL. The PCR amplification program is: 94℃ 2-5min, 20-25 cycles (94℃ 20s, 55℃ 20s, 72℃ 2-6kb / min), 72℃ 10min, and store at 4℃. Add 1-3μL DMT enzyme to the obtained PCR product for digestion, mix well, and incubate at 37℃ for 1h. After purifying the obtained DMT enzyme digestion product using a PCR product purification kit, take 2-5μL and add it to 50μL JM109 competent cells for transformation. Select positive transformants, preliminarily identify them by colony PCR, and screen out the correct transformant JM109-argA after sequencing by the company. fbr .

[0097] Extract transformant JM109-argA fbr Plasmid pRSFDuet1-argA fbr According to the above “4. Gene integration expression plan”, the gene fragment argA fbr Integrate into the knockout strain S1-ΔargR to construct strain S1-argA fbr ΔArgR, named S2, for subsequent studies.

[0098] The production of S2 arginine was verified by shake flask culture, and the arginine concentration in the shake flask supernatant was measured to be 8.6 g / L.

[0099] Example 3: Enhancing the expression of key enzymes in genetically engineered Serratia marcescens

[0100] The present invention utilizes the strong trc promoter to replace the native promoter, enhancing its expression and promoting arginine metabolic flux. The trc promoter (SEQ ID NO. 187: 5'-TTGACAATTAATCATCCGGCTCGTATAATG-3') is derived from the pTrc99a plasmid. Aspartate kinase, encoded by the lysC gene, catalyzes the phosphorylation of aspartate, while aspartate semialdehyde dehydrogenase, encoded by the asd gene, catalyzes the phosphorylated aspartate to produce aspartate semialdehyde. To enhance the enzymatic activity and metabolic flux of this metabolic pathway, the strong trc promoter is used to replace the native promoter, intensifying its expression and accumulating more target product.

[0101] Using S2 as the starting strain, the key enzymes of the synthesis pathway, lysC (amino acid sequence accession number WP_004936686.1), asd (amino acid sequence accession number TEW88852.1), argA (amino acid sequence accession number ALE97678.1), argH (amino acid sequence accession number TEW89265.1) and speA (amino acid sequence accession number TEW85704.1) were further enhanced, and the strong trc promoter was used to replace the natural promoter of the gene to obtain the engineered strain S2-P. trc lysC-P trc asd-P trc argA-P trc argH-P trc speA strain.

[0102] The specific process is as follows:

[0103] Primers were designed using Primer Premier5 based on the lysC fragment of Serratia marcescens HBQA from NCBI (Table 5). The target gene fragment, lysC, was amplified by PCR using the Serratia marcescens HBQA genome as a template and primers lysC-KZ-F / R. EcoRI and HindIII restriction sites were added to the upstream and downstream ends of the fragment, respectively. The PCR fragment was purified. The pTrc99a plasmid was double-digested with EcoRI and HindIII, the digested plasmid was purified, and the purified lysC fragment was ligated and transformed into competent E. coli JM109 cells. Positive transformants were selected and verified by colony PCR and sequencing using the validation primers lysC-99a-YZ-F / R. The successfully transformed plasmid pTrc-lysC was extracted and used as a template. Using primers BamHI-trc-lysC-F and trc-lysC-SacI-R, the target fragment trc-lysC was amplified. The fragment was approximately 1398 bp, verified by gel analysis, and purified. The T19 vector plasmid was double-digested with BamHI and SacI, purified, and ligated with the trc-lysC fragment for transformation. The fragment was verified by colony PCR and company sequencing using the verification primers trc-lysC-T19-YZF / YZR, and the correct positive transformant, T19-trc-lysC, was selected. Using the lysC genome in Serratia marcescens HBQA as a template, lysC-LA-ApaI and EcoRI-lysC-LA were used as primers to amplify the left arm fragment LA, and lysC-RA-KpnI and SalI-lysC-RA were used as primers to amplify the right arm fragment RA. After purification, the fragments were ligated to the left and right arms of the double-enzyme-digested and purified plasmid T19-trc-lysC, respectively. The verification primers lysC-LA-YZ-F and lysC-LA-YA-R, lysC-RA-YZ-F and lysC-RA-YA-R were used to perform preliminary colony PCR verification, and the fragments were sent to the company for sequencing to find the plasmid T19-LA-trc-lysC-RA that correctly connected the left and right arms. The plasmid T19-LA-trc-lysC-RA with the left and right arms connected was extracted and used as a template. LysC-LA-ApaI and SalI-lysC-RA were used as primers to amplify the target fragment LA-trc-lysC-RA with a size of about 3700 bp. After purification, the restriction endonuclease DpnI was used to eliminate the template effect.The digested product was further purified and then transferred into S2-PKD46 (the homologous recombinase expression plasmid PKD46 was extracted using a plasmid extraction kit, purified, and transformed into competent cells S2 to construct S2-PKD46). Positive transformants were screened on resistance plates using the carnamycin resistance marker. To verify recombination, primers lysC-YZ-F and lysC-YZ-R should amplify a fragment of approximately 1000 bp; otherwise, no fragment should be amplified. Simultaneously, the corresponding PCR verification bacteria were spotted on ampicillin sodium plates. The correct transformant, S2-PKD46 (kanamycin), was confirmed. R To eliminate carnamycin resistance, first prepare S2-PKD46 (kana R ) competent cells, and the pCP20 plasmid was transformed into S2-PKD46 (kana R ) competent state, the resistance-eliminating transformants were screened using non-resistant plates and resistant plates, and the transformants were identified by colony PCR. The transformants S2-P that replaced the lysC gene promoter and eliminated the resistance were screened. trc lysC.

[0104] According to the above method, the promoters of genes asd, argA, argH and speA were replaced in sequence. trc lysC-P trc asd-P trc argA-P trc argH-P trc speA, and named it S3. The primers used are shown in Table 5 below:

[0105] Table 5 Primer sequences (SEQ ID NO. 19-108)

[0106] The production of S3 spermidine was verified by shake flask culture, and the spermidine concentration in the shake flask supernatant was measured to be 16.3 g / L.

[0107] Example 4: Enhancing the transport pathway of genetically engineered Serratia marcescens

[0108] PotABCD is the transporter that Serratia marcescens absorbs spermidine into the cell, so it is knocked out; Because PotABCD protein is composed of four subunits encoded by gene cluster potA, potB, potC, potD gene, potA encodes ATP binding subunit, for transshipment provides necessary energy, potB encodes ABC transport membrane subunit PotB, potC encodes ABC transport membrane subunit PotC, potD encodes ABC transporter protein periplasmic binding protein, the four genes of the operon are necessary for the correct expression of the operon function, and the deletion of any one gene will lead to functional inactivation, therefore, the ATP binding subunit potA (amino acid sequence accession number on NCBI is TEW94582.1) is deleted in the present invention, the ability of the protein transporting spermidine is weakened, and the bacterial strain that improves the transport pathway is constructed on the basis of S3. According to the method for Red homologous recombination in Example 2, PotA is knocked out to obtain bacterial strain S3-ΔPotA. MdtJI (amino acid sequence accession numbers TEW81964.1 and TEW81965.1 at NCBI) is a protein that transports spermidine out of the cell of Serratia marcescens. It is encoded by a gene cluster consisting of the mdtJ and mdtI genes. Therefore, its expression was enhanced using the strong trc promoter. According to the method described in Example 2, the promoter was replaced to obtain the strain S3-ΔPotA-P with improved transport pathway. trc MdtJI and named it S4. The primers are shown in Table 6.

[0109] Table 6 Primer sequences (SEQ ID NO. 109-136)

[0110] The production of S4 spermidine was verified by shake flask culture, and the spermidine concentration in the shake flask supernatant was measured to be 21.4 g / L.

[0111] Example 5: Weakening the Competition Pathway of Genetically Engineered Serratia Marcescens

[0112] (1) Homoserine competition pathway:

[0113] The intermediate aspartate semialdehyde is further converted to homoserine by homoserine synthases encoded by the thrA (NCBI amino acid sequence accession number TEW91726.1) and metL (NCBI amino acid sequence accession number ALE98426.1) genes. To eliminate this competing metabolic pathway, strain S4-ΔthrAΔmetL was constructed by deleting the thrA and metL genes using Red homologous recombination, as described in Example 2. The resulting strain was named S5-1.

[0114] (2) Lys competition pathway:

[0115] The dihydrodipicolinate synthase encoded by the dapA gene (NCBI accession number TEW94959.1) converts the intermediate aspartate semialdehyde into dihydrodipicolinate. To mitigate the impact of this branched pathway and redirect more aspartate toward the aspartate semialdehyde synthesis pathway, strain S4-ΔthrAΔmetL was constructed by knocking out the dapA gene using Red homologous recombination, as described in Example 1. The resulting strain was named S5-2.

[0116] (3) There is a competitive pathway for Glu: Glutamate kinase encoded by proB and glutamate semialdehyde dehydrogenase encoded by proA form glutamate semialdehyde, which can be spontaneously converted to pyrroline-5-carboxylic acid, which is converted to proline (Pro) by pyrroline 5-carboxylic acid reductase encoded by proC (amino acid sequence accession number on NCBI is ALE97888.1). Therefore, in order to prevent the metabolic flow of the competitive pathway, we will delete the gene proC and weaken this pathway. According to the method described in Example 2, based on the strain S4ΔthrAΔmetLΔdapA, the expression of the competitive pathway gene proC was deleted using the Red homologous recombination method to construct the strain S4ΔthrAΔmetLΔdapAΔproC. The resulting strain was named S5-3.

[0117] The required primers are shown in Table 7 below.

[0118] Table 7 Primer sequences (SEQ ID NO. 137-176)

[0119] The spermidine production of S5-1, S5-2, and S5-3 was verified by shake flask culture. The results showed that the spermidine production in the supernatant of S5-3 was the highest, with a spermidine concentration of 23.3 g / L.

[0120] Example 6: Knockout of key enzymes in the decomposition pathway of genetically engineered Serratia marcescens

[0121] There are two pathways for the utilization and degradation of Arg (competitive pathways): (1) Arg is degraded by arginine N-succinyltransferase encoded by the astA gene (the amino acid sequence is accession number TEW83071.1 on NCBI) to produce N 2 -succinylarginine, which is further degraded into L-Glu and succinate by the ast series of genes. To reduce the degradation of Arg, we weakened the Arg degradation pathway by deleting the astA gene.

[0122] According to the method described in Example 2, strain S5-3 was used as the starting strain for this metabolic pathway. The astA gene was deleted using Red homologous recombination to prevent further degradation of Arg and direct more metabolic flux toward spermidine synthesis. Strain S5-3ΔastA was obtained and named S6. The required primers are shown in Table 8:

[0123] Table 8 Primer sequences (SEQ ID NO. 177-186)

[0124] The production of S6 spermidine was verified by shake flask culture, and the spermidine concentration in the shake flask supernatant was measured to be 26.8 g / L.

[0125] Example 7: Introduction of exogenous spermidine synthesis pathway - genetically engineered Escherichia coli

[0126] Aspartate semialdehyde is an important intermediate metabolite in cells. It is a direct precursor for the synthesis of methionine and decarboxylated S-adenosylmethionine, and is also a key precursor for spermidine synthesis. The metabolic pathway from aspartate semialdehyde to decarboxylated S-adenosylmethionine is complex and variable, long, and involves the metabolism of multiple amino acid intermediates. Excessive modification of this metabolic pathway can easily affect bacterial growth. For these reasons, in this example, the CAPADH / CAPADC / APAUH pathway from different microbial strains was introduced into E. coli for heterologous expression. The construction schematic is shown in Figure 1.

[0127] The carboxyaminopropylagmatine dehydrogenase encoded by CAPADH directly uses aspartate semialdehyde and agmatine as precursors to synthesize carboxyaminopropylagmatine. This is then decarboxylated to aminopropylagmatine by the carboxyaminopropylagmatine decarboxylase encoded by CAPADC. Finally, spermidine is generated by the aminopropylagmatine ureahydrolase encoded by APAUH. First, carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase, and aminopropylagmatine ureahydrolase from various sources are expressed in Escherichia coli via plasmids. The enzyme with the highest catalytic efficiency is then selected and exogenously integrated into the CAPADH / CAPADC / APAUH pathway via homologous recombination. The genomic integration site selected in the present invention is upstream of the argA gene of E. coli BL21.

[0128] The specific process is as follows:

[0129] The carboxyaminopropylagmatine dehydrogenase genes stcapadh, dacapadh, gscapadh, and iccapadh were cloned from the genomes of Spirochaeta thermophila DSM 6578, Denitrovibrio acetiphilus DSM 12809, Geobacter sulfurreducens KN400, and Ilumatobacter coccineus YM16-304 using amplification primers. The amino acid sequences of these genes are listed in NCBI accession numbers WP_014623968.1, WP_013011851.1, WP_010943175.1, and WP_015442124.1, respectively; The carboxylaminopropylagmatine decarboxylase genes stcapadc, dacapadc, gscapadc, and iccapadc were cloned from the genome of YM16-304, and their amino acid sequences are numbered WP_014623969.1, WP_013011852.1, WP_004513933.1, and WP_015442123.1 in NCBI, respectively. The aminopropylagmatine urea hydrolase genes stapauh, daapauh, lbapauh, and icapauh were cloned from the YM16-304 genome. Their amino acid sequences are listed in NCBI as WP_014623970.1, WP_013011853.1, WP_015770028.1, and WP_015442122.1, respectively. These three cloned genes were ligated into the same pRSFDuet-1 plasmid to generate recombinant E. coli expressing these three genes.

[0130] CAPADH and CAPADC in the above-mentioned microorganisms are located adjacent to each other in a gene cluster. Using stcapadh-BamHI-F and stcapadc-HindⅢ-R as primers and the genome of Spirochaeta thermophila DSM 6578 as a template, the target fragment stcapadh-stcapadc was amplified. The first multiple cloning site of the plasmid pRSFDuet-1 was digested with the double enzymes BamHI and HindⅢ. After purification of the digestion product, the cut plasmid was ligated with the amplified target fragment stcapadh-stcapadc. The ligation product pRSFDuet-1-stcapadh-stcapadc was transformed into JM109 competent cells, and positive clones were selected on a resistant card sodium plate. Colony PCR was used to further verify its correctness. Using stapauh-KpnI-F and stapauh-XhoI-R as primers, the genome of Spirochaeta thermophila DSM The target fragment, stapauh, was amplified using the 6578 genome as a template. The second multiple cloning site of the plasmid pRSFDuet-1-stcapadh-stcapadc was digested with KpnI and XhoI. The digestion product was purified and ligated with the amplified target fragment, stapauh. The ligation product, pRSFDuet-1-stcapadh-stcapadc-stapauh, was transformed into JM109 competent cells. Positive clones were selected on resistant cardiomyocytes plates and further verified by colony PCR. The JM109 strain with the correct ligation was selected, and the plasmid pRSFDuet-1-stcapadh-stcapadc-stapauh was extracted and transformed into competent E. coli BL21 cells to construct the E. coli-stcapadh-stcapadc-stapauh spermidine-biosynthesizing strain. The remaining strains were constructed similarly to the E. coli-stcapadh-stcapadc-stapauh strain. Carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase, and aminopropylagmatine ureahydrolase from different strains were ligated into two multiple cloning sites of the pRSFDuet-1 plasmid. Other genes were ligated in a similar manner. The primer sequences used are shown in Table 9:

[0131] Table 9 Primer sequences (SEQ ID NO. 188-191)

[0132] The recombinant E. coli was transferred into the shake flask fermentation medium at a volume ratio of 2%. 600After the pH reached 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 20°C for 12 hours. After induction, the supernatant was collected by centrifugation at 8000 rpm for 20 minutes at 4°C. The spermidine concentration in the supernatant was determined by HPLC, as shown in Table 10.

[0133] Table 10

[0134] The use of expression vectors to overexpress key genes for spermidine synthesis will impose a metabolic burden on the bacteria and affect their growth. The expression vector is structurally unstable and easily lost during bacterial reproduction, and its copy number in the cell is unstable. The addition of antibiotics affects bacterial growth and pollutes the environment. The increased cost caused by IPTG induction limits the expansion of production scale. Therefore, the strain E. coli-stcapadh-stcapadc-stapauh with the best effect of inducing expression of spermidine synthesis was selected, and the promoter J23119 and ribosome binding site (RBS) B0034 in Escherichia coli were connected to the stcapadh-stcapadc-stapauh gene to form an exogenous gene expression fragment. According to the aforementioned "4. Gene integration and expression plan", the exogenous gene expression fragment was integrated into the knockout strain E. coli BL21 to construct the strain E. coli-stcapadh-stcapadc-stapauh, named E1, and used as the basic strain for subsequent studies.

[0135] The production of E1 spermidine was verified by shake flask culture, and the spermidine concentration in the shake flask supernatant was measured to be 6.7 g / L.

[0136] Example 8: Removing feedback inhibition of important enzymes in the Escherichia coli genetically engineered pathway and knocking out the repressor protein

[0137] In the process of synthesizing important intermediates such as aspartate semialdehyde and arginine, there is an important repressor protein that inhibits the synthesis of intermediates and final products. The DNA-binding dual transcriptional regulator encoded by the argR gene (amino acid sequence accession number QJZ05382.1 on NCBI) inhibits the expression of the arg series genes and the excessive synthesis of the required important intermediates ornithine and arginine. Therefore, in order to ensure the stable and high-rate synthesis of each intermediate, the gene encoding the repressor protein was deleted to increase the metabolic flux.

[0138] In the metabolic pathway of arginine synthesis, N-acetylglutamate synthase encoded by argA (amino acid sequence accession number on NCBI is QJZ04982.1) is subject to feedback inhibition by the product arginine. In order to improve the activity of this enzyme, on the basis of knocking out the argR gene, the gene encoding this enzyme was further mutated to construct a negative feedback mutant, thereby relieving feedback inhibition and obtaining the engineered bacterium E. coli argA fbr ΔArgR, named E2.

[0139] The specific process is as follows:

[0140] (1) Knockout of the repressor protein ArgR

[0141] The primer designs are shown in Table 11 below:

[0142] Table 11 Primer sequences (SEQ ID NO. 192-201)

[0143] The homologous recombination enzyme-expressing PKD46 plasmid was extracted using a plasmid extraction kit, purified, and transformed into competent E1 cells to construct E1-PKD46. E1-PKD46 competent cells were prepared using the CaCl2 method and stored at -80°C until use. The homologous recombination fragment template plasmid T19 vector was extracted using a plasmid extraction kit and purified using a gel tapping recovery kit. Primers ApaI-LA-argR and argR-LA-EcoRI, and KpnI-RA-argR and argR-RA-SalI were designed to amplify the corresponding homology arms LA and RA fragments, which were then purified and recovered using a gel tapping recovery kit. The T19 vector was double-digested with ApaI and EcoRI, and the digestion results were confirmed by electrophoresis. The digestion product was 5041 bp in size and purified. The double-digested T19 vector product was ligated with the left arm LA fragment. The ligated product was transformed into JM109 competent cells and plated onto a Kansas-resistant plate. Positive clones were screened and verified by colony PCR using primers LA-YZ-F and LA-YZ-R. The band size was approximately 1100 bp. Successful strains were sequenced to further confirm successful fragment ligation. The T19 plasmid with the LA fragment ligated was double-digested with KpnI and SalI. The RA fragment was ligated using the same method as above, and the ligated plasmid was verified. The T19 vector plasmid with the LA and RA fragments ligated was extracted and used as a template for PCR with primers ApaI-LA-argR and argR-RA-SalI. The target fragment, argR-LA-FRT-Kan-FRT-RA, was obtained. Electrophoresis confirmed the band size to be approximately 3324 bp. The PCR product was purified and the template effect was eliminated using the restriction endonuclease DpnI. The target fragment argR-LA-FRT-Kan-FRT-RA PCR product after restriction endonuclease DpnI digestion is further purified and transferred into E.coli BL21-PKD46 competent cells after purification. Positive transformants are screened on resistance plates using the carnamycin resistance marker. To verify whether the positive transformants screened have indeed undergone recombination, the positive transformants are used as templates and primers argR-YZ-F and argR-YZ-R are used to amplify a fragment of about 1100bp, which can be judged to be a recombination. Conversely, if no fragment can be amplified, it can be judged to be a false positive. While verifying whether recombination has occurred by PCR amplification, the corresponding PCR verification bacteria are spotted on ampicillin sodium plates. The correct transformant E1-PKD46 (kana R ) to eliminate carnamycin resistance, firstly, E1-PKD46 (kana R ) competent cells, the extracted pCP20 plasmid was transformed into E1-PKD46 (kana R) After the cells were competent, resistance-eliminated transformants were screened using non-resistant plates and resistant plates, and transformants were identified by colony PCR. The transformant E1-ΔargR with knockout of the argR gene and elimination of resistance was selected.

[0144] (2)argA fbr Construction of mutants

[0145] The argA gene is subject to feedback inhibition by the target product arginine. In the present invention, the 43rd base C of the argA gene is mutated to T. After the encoded histidine is changed to tyrosine, the production of arginine is significantly increased. Therefore, we mutated this site and integrated it into the plasmid pRSFDuet-1 plasmid.

[0146] The specific process is as follows:

[0147] The primer designs are shown in Table 12 below:

[0148] Table 12 Primer sequences (SEQ ID NO. 202-205)

[0149] First, the pRSFDuet-1 (high copy) plasmid was extracted using a plasmid extraction kit and double-digested with BamHI and HindIII. The digestion system (50 μL) was as follows: 5 μL 10×Q Cut Buffer, 20 μL plasmid, 1 μL BamHI, 1 μL HindIII, and 33 μL ddH2O. After digestion, the product was purified. Next, the wild-type E. coli BL21 genome was extracted and used as a template to amplify the argA gene fragment. After PCR verification, the product was ligated into the digested pRSFDuet-1 plasmid. E. coli JM109 competent cells were prepared using the CaCl2 method. The pRSFDuet-1 plasmid ligated with the argA fragment was transformed into competent cells. Positive transformants were selected and identified as successfully transformed strains, E. coli JM109-argA, by colony PCR. The plasmid pRSFDuet1-argA from E. coli JM109-argA was extracted and used as a template for site-directed mutagenesis of argA. The mutant gene was designed and extracted using the Fast Mutangenesis System kit. The extracted pRSFDuet1-metA plasmid was used as a template to amplify argA using the argA mutant primers argA-TB-F / R. fbr Fragment, wherein the PCR amplification system (50 μL) is as follows: plasmid template 3 μL, argA-TB-F 1 μL, argA-TB-R 1 μL, Fly PCR SuperMix 25μL, ddH2O 20μL. The PCR amplification program is: 94℃ 2-5min, 20-25 cycles (94℃ 20s, 55℃ 20s, 72℃ 2-6kb / min), 72℃ 10min, and store at 4℃. Add 1-3μL DMT enzyme to the obtained PCR product for digestion, mix well, and incubate at 37℃ for 1h. After purifying the obtained DMT enzyme digestion product using a PCR product purification kit, take 2-5μL and add it to 50μL JM109 competent cells for transformation. Select positive transformants, preliminarily identify them by colony PCR, and screen out the correct transformant JM109-argA after sequencing by the company. fbr .

[0150] Extract transformant JM109-argA fbr Plasmid pRSFDuet1-argA fbr According to the above “4. Gene integration expression plan”, the gene fragment argA fbr Integrate into the knockout strain E1-ΔargR to construct strain E1-argA fbr ΔArgR was named E2 for subsequent studies.

[0151] The production of E2 arginine was verified by shake flask culture, and the arginine concentration in the shake flask supernatant was measured to be 7.8 g / L.

[0152] Example 9: Enhanced expression of key enzymes in the pathway

[0153] In the metabolic process of synthesizing important intermediates such as decarboxylated aspartate semialdehyde and arginine, several genes act as rate-limiting enzymes, strictly regulating the expression of the entire metabolic pathway. The argA gene encodes N-acetylglutamate synthase, the rate-limiting enzyme in the arginine metabolic synthesis pathway. The argH gene encodes argininosuccinate lyase, which catalyzes the synthesis of arginine from argininosuccinate. These two enzymes significantly inhibit excessive arginine synthesis. The speA gene encodes arginine decarboxylase, which catalyzes the conversion of arginine to agmatine, a key precursor for the novel spermidine production pathway. Therefore, we replaced the native promoter with the strong trc promoter to enhance its expression and promote the synthesis of arginine metabolic flux. The trc promoter (SEQ ID NO. 187: 5'-TTGACAATTAATCATCCGGCTCGTATAATG-3') is derived from the pTrc99a plasmid. The aspartate kinase encoded by the lysC gene catalyzes the phosphorylation of aspartate, and the aspartate semialdehyde dehydrogenase encoded by the asd gene catalyzes the phosphorylated aspartate to produce aspartate semialdehyde. In order to enhance the enzyme activity and metabolic flux of this metabolic pathway, the strong promoter trc was used to replace its natural promoter, strengthen its expression, and accumulate more target products.

[0154] Using E2 as the starting strain, the key enzymes of the synthesis pathway, lysC (amino acid sequence accession number on NCBI is QZI64953.1), asd (amino acid sequence accession number on NCBI is QJZ05564.1), argA (amino acid sequence accession number on NCBI is QJZ04982.1), argH (amino acid sequence accession number on NCBI is QJZ06087.1) and speA (amino acid sequence accession number on NCBI is QJZ05082.1) were further enhanced, and the strong trc promoter was used to replace the natural promoter of the gene to obtain the engineered strain E2-P. trc lysC-P trc asd-P trc argA-P trc argH-P trc speA strain.

[0155] The specific process is as follows:

[0156] Based on the lysC fragment of E. coli BL21 from NCBI, primer Premier5 was used to design the corresponding fragment primers (Table 13). Using the E. coli BL21 genome as a template and lysC-KZ-F / R as primers, PCR amplified the target gene fragment lysC. EcoRI and HindIII restriction sites were added to the upstream and downstream ends, respectively, and the PCR fragment was purified. The pTrc99a plasmid was double-digested with EcoRI and HindIII, the digested plasmid was purified, and the purified lysC fragment was ligated and transformed into competent E. coli JM109 cells. Positive transformants were selected and verified by colony PCR and sequencing using the validation primers lysC-99a-YZ-F / R. The successfully transformed plasmid pTrc-lysC was extracted and used as a template. Using primers BamHI-trc-lysC-F and trc-lysC-SacI-R, the target fragment trc-lysC was amplified. The fragment was approximately 1453 bp, verified by gel analysis, and purified. The T19 vector plasmid was double-digested with BamHI and SacI, purified, and ligated with the trc-lysC fragment for transformation. The fragment was verified by colony PCR and company sequencing using the verification primers trc-lysC-T19-YZF / YZR, and the correct positive transformant, T19-trc-lysC, was selected. Using the lysC genome in E. coli BL21 as a template, lysC-LA-ApaI and EcoRI-lysC-LA were used as primers to amplify the left arm fragment LA, and lysC-RA-KpnI and SalI-lysC-RA were used as primers to amplify the right arm fragment RA. After purification, they were ligated to the left and right arms of the double-enzyme-digested and purified plasmid T19-trc-lysC, respectively. The verification primers lysC-LA-YZ-F and lysC-LA-YA-R, lysC-RA-YZ-F and lysC-RA-YA-R were used to perform preliminary colony PCR verification, and the fragments were sent to the company for sequencing to find the plasmid T19-LA-trc-lysC-RA that correctly connected the left and right arms. The plasmid T19-LA-trc-lysC-RA with the left and right arms connected was extracted and used as a template. LysC-LA-ApaI and SalI-lysC-RA were used as primers to amplify the target fragment LA-trc-lysC-RA with a size of about 3700 bp. After purification, the restriction endonuclease DpnI was used to eliminate the template effect.The digested product was further purified and then transferred into E2-PKD46 (the homologous recombinase expression plasmid PKD46 was extracted using a plasmid extraction kit, purified, and transformed into competent E2 cells to construct E2-PKD46). Positive transformants were screened on resistance plates using the carnamycin resistance marker. To verify recombination, primers lysC-YZ-F and lysC-YZ-R should amplify a fragment of approximately 1000 bp; otherwise, no fragment should be amplified. Simultaneously, the corresponding PCR was performed on ampicillin sodium plates to verify the presence of the transformant. The correct transformant, E2-PKD46 (carnamycin), was confirmed. R To eliminate carnamycin resistance, firstly, E2-PKD46 (kana R ) competent cells, and the pCP20 plasmid was transformed into E2-PKD46 (kana R ) After the competent state, the resistance-eliminated transformants were screened using non-resistant plates and resistant plates, and the transformants were identified by colony PCR. The transformants E2-lysCP that replaced the lysC gene promoter and eliminated resistance were screened. trc .

[0157] According to the above method, the promoters of genes asd, argA, argH and speA were replaced in sequence. trc lysC-P trc asd-P trc argA-P trc argH-P trc speA, and named it E3. The primers used are shown in Table 13 below:

[0158] Table 13 Primer sequences (SEQ ID NO. 206-295)

[0159] The production of E3 spermidine was verified by shake flask culture, and the spermidine concentration in the shake flask supernatant was measured to be 14.8 g / L.

[0160] Example 10: Improving transport pathways

[0161] PotABCD is the transporter that Escherichia coli absorbs spermidine into the cell, so it is knocked out; Because PotABCD protein is composed of four subunits encoded by gene cluster potA, potB, potC, and potD genes, potA encodes ATP binding subunit, for transshipment provides necessary energy, potB encodes ABC transport membrane subunit PotB, potC encodes ABC transport membrane subunit PotC, and potD encodes ABC transporter protein periplasmic binding protein. The four genes of the operon are necessary for the correct expression of the operon function, and the deletion of any one gene will lead to functional inactivation. Therefore, the ATP binding subunit potA (amino acid sequence accession number on NCBI is QJZ03606.1) is deleted in the present invention, the ability of the transferred protein to transport spermidine is weakened, and a bacterial strain improving the transport pathway is constructed on the basis of E3. PotA is knocked out according to the method for Red homologous recombination in Example 2 to obtain bacterial strain E3-ΔPotA. MdtJI (amino acid sequence accession number CAQ32076.1 at NCBI) is a protein that transports spermidine out of Escherichia coli. It is encoded by a gene cluster consisting of the mdtJ and mdtI genes. Therefore, its expression was enhanced using the strong trc promoter. According to the method described in Example 2, its promoter was replaced to obtain the strain E3-ΔPotA-P with improved transport pathway. trc MdtJI, and named it E4, the primer sequence is shown in Table 14.

[0162] Table 14 Primer sequences (SEQ ID NO. 296-323)

[0163] The production of E4 spermidine was verified by shake flask culture, and the spermidine concentration in the shake flask supernatant was measured to be 18.5 g / L.

[0164] Example 11: Weakening competition approach

[0165] (1) Homoserine competition pathway:

[0166] The intermediate aspartate semialdehyde is further converted to homoserine by homoserine synthases encoded by the thrA (NCBI amino acid sequence accession number QJZ02606.1) and metL (NCBI amino acid sequence accession number QJZ06068.1) genes. To eliminate this competing metabolic pathway, strain E4-ΔthrAΔmetL was constructed by deleting the thrA and metL genes using Red homologous recombination, as described in Example 1. The resulting strain was named E5-1.

[0167] (2) Lys competition pathway:

[0168] The dihydrodipicolinate synthase encoded by the dapA gene (NCBI accession number QJZ04698.1) converts the intermediate aspartate semialdehyde into dihydrodipicolinate. To mitigate the impact of this branched pathway and redirect more aspartate toward the aspartate semialdehyde synthesis pathway, strain E4-ΔthrAΔmetL was constructed by knocking out the dapA gene using Red homologous recombination, as described in Example 1. The resulting strain was designated E5-2.

[0169] (3) There is a competitive pathway for Glu: Glutamate kinase encoded by proB and glutamate semialdehyde dehydrogenase encoded by proA form glutamate semialdehyde, which can be spontaneously converted to pyrroline-5-carboxylic acid, which is converted to proline (Pro) by pyrroline 5-carboxylic acid reductase encoded by proC (amino acid sequence accession number on NCBI is QJZ02915.1). Therefore, in order to prevent the metabolic flow of the competitive pathway, we will delete the gene proC and weaken this pathway. According to the method described in Example 1, based on the strain E4ΔthrAΔmetLΔdapA, the expression of the competitive pathway gene proC was deleted using the Red homologous recombination method to construct the strain E4ΔthrAΔmetLΔdapAΔproC. The resulting strain was named E5-3.

[0170] The required primers are shown in Table 15 below.

[0171] Table 15 Primer sequences (SEQ ID NO. 324-353)

[0172] The spermidine production of E5-1, E5-2, and E5-3 was verified by shake flask culture, and the spermidine concentrations in the shake flask supernatants were measured to be 19.3 g / L, 19.7 g / L, and 21.3 g / L.

[0173] Example 12: Knockout of key enzymes in related decomposition pathways

[0174] There are two pathways for the utilization and degradation of Arg (competitive pathways): (1) Arg is degraded by arginine N-succinyltransferase encoded by the astA gene (the amino acid sequence is accession number QJZ04124.1 on NCBI) to produce N 2 -succinylarginine, which is further degraded into L-Glu and succinate by the ast series of genes. To reduce the degradation of Arg, we weakened the Arg degradation pathway by deleting the astA gene.

[0175] Following the method described in Example 2, strain E5-3 was used as the starting strain for this metabolic pathway. Red homologous recombination was used to delete the astA gene, preventing further degradation of Arg and directing more metabolic flux toward spermidine synthesis. This resulted in strain E5-3ΔastA, designated E6. The required primers are listed in Table 16:

[0176] Table 16 Primer sequences (SEQ ID NO. 354-363)

[0177] The production of E6 spermidine was verified by shake flask culture, and the spermidine concentration in the shake flask supernatant was measured to be 24.7 g / L.

[0178] Example 13: Comparison of various integrated expression strains and high-density culture

[0179] The same procedures as in Examples 7-12 were further performed in Escherichia coli JM109, DH5α, Top10, and MG1655 to obtain similar strains E.JM, E.DH, E.TO, and E.MG, each harboring a novel integrated spermidine production pathway. These strains were cultured in parallel at high density for 18 hours. The medium composition and fed-batch operation were as described in the literature (Simple fed-batch technique for high cell density cultivation of Escherichia coli. Journal of Biotechnology 1995, 39: 59-65). The bacterial cell count and spermidine production are shown in Table 17.

[0180] E.JM, E.DH, E.TO and E.MG were cultured in shake flasks, and the spermidine production was 19.7, 21.3, 22.7 and 26.2 g / L, respectively.

[0181] Table 17 Results of high-density culture of different strains

[0182] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone 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 based on the definition of the claims.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria express exogenous carboxylaminopropylagmatine dehydrogenase, carboxylaminopropylagmatine decarboxylase and aminopropylagmatine urea hydrolase.

2. The genetically engineered bacterium according to claim 1, characterized in that: The genetically engineered bacteria are genetically engineered bacteria of Serratia marcescens or genetically engineered bacteria of Escherichia coli; Optionally, the genetically engineered Escherichia coli bacteria is constructed using Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli Top10 or Escherichia coli MG1655 as a host.

3. The genetically engineered bacterium according to claim 1, characterized in that The genetically engineered bacteria knock out the gene encoding the repressor protein argR and construct an argA negative feedback mutant.

4. The genetically engineered bacterium according to claim 1, characterized in that: The genetically engineered bacteria utilizes a strong promoter to replace the natural promoters of the gene lysC encoding aspartate kinase, the gene asd encoding aspartate semialdehyde dehydrogenase, the gene argA encoding N-acetylglutamate synthetase, the gene argH encoding arginine succinate lyase, and the gene speA encoding arginine decarboxylase.

5. The genetically engineered bacterium according to claim 3, characterized in that: The genetically engineered bacteria utilizes a strong promoter to replace the natural promoters of the gene lysC encoding aspartate kinase, the gene asd encoding aspartate semialdehyde dehydrogenase, the gene argA encoding N-acetylglutamate synthetase, the gene argH encoding arginine succinate lyase, and the gene speA encoding arginine decarboxylase.

6. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The genetically engineered bacteria knock out the gene encoding the transporter PotA, or on the basis of knocking out the gene encoding the transporter PotA, strengthen the expression of the transporter MdtJI that transfers spermidine to the extracellular space.

7. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The genetically engineered bacteria also have thrA and metL genes encoding homoserine synthetase knocked out; or, based on the knockout of thrA and metL genes, the dapA gene encoding dihydropicolinate synthetase is further knocked out; or, based on the knockout of thrA, metL, dapA genes, the proC encoding pyrroline 5-carboxylate reductase is further knocked out.

8. The genetically engineered bacterium according to any one of claims 1 to 7, characterized in that: The genetically engineered bacteria knock out the astA gene encoding arginine N-succinyltransferase.

9. The genetically engineered bacterium according to any one of claims 3 to 8, characterized in that: The strong promoter is the trc promoter.

10. Use of the genetically engineered bacteria according to any one of claims 1 to 9 in producing spermidine, characterized in that: The genetically engineered bacteria according to any one of claims 1 to 9 are placed in a medium containing glucose and fermented to prepare spermidine.

11. The use according to claim 10, characterized in that: The fermentation was carried out at pH 7, 37°C, with a shaking flask speed of 200 rpm for 24 hours.

12. A method for increasing spermidine production, characterized in that: The genetically engineered bacteria according to any one of claims 1 to 9 are placed in a medium containing glucose and fermented to prepare spermidine.

13. The method according to claim 12, characterized in that The fermentation was carried out at pH 7, 37°C, with a shaking flask speed of 200 rpm for 24 hours.

14. A method for modifying genetically engineered bacteria, characterized in that: A common primer is used to amplify the gene fragments of carboxyaminopropyl agmatine dehydrogenase and carboxyaminopropyl agmatine decarboxylase, the first multiple cloning site of the plasmid pBBR1MCS-2 is treated with double enzymes HindⅢ and EcoRⅤ, the digested product is purified, and then the cut plasmid is connected with the gene fragments of carboxyaminopropyl agmatine dehydrogenase and carboxyaminopropyl agmatine decarboxylase to construct a plasmid connected with the carboxyaminopropyl agmatine dehydrogenase and carboxyaminopropyl agmatine decarboxylase genes; Amplifying an aminopropyl agmatine urea hydrolase gene fragment using aminopropyl agmatine urea hydrolase gene primers, digesting the second multiple cloning site of the plasmid connected with carboxyaminopropyl agmatine dehydrogenase and carboxyaminopropyl agmatine decarboxylase genes using two enzymes EcoRⅠ and SpeⅠ, purifying the digestion product, and connecting the cut plasmid with the aminopropyl agmatine urea hydrolase gene fragment to construct a plasmid connected with carboxyaminopropyl agmatine dehydrogenase, carboxyaminopropyl agmatine decarboxylase and aminopropyl agmatine urea hydrolase genes; The plasmid connected with carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase and aminopropylagmatine urea hydrolase genes was transformed into JM109 competent cells, and positive clones were selected on a resistant plate sodium card plate, and colony PCR was further used to verify its correctness; the plasmid was extracted from the JM109 strain and transformed into competent genetic engineering bacteria to construct a spermidine synthesis strain; Optionally, the competent genetic engineering bacteria include Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli MG1655 or Serratia marcescens HBQA7; Alternatively, the carboxyaminopropylagmatine dehydrogenase gene, the carboxyaminopropylagmatine decarboxylase gene and the aminopropylagmatine ureahydrolase gene can be cloned from the genomes of Spirochaeta thermophila DSM 6578, Denitrovibrio acetiphilus DSM 12809, Geobacter sulfurreducens KN400, and Ilumatobacter coccineus YM16-304; Optionally, the nucleotide sequence of the common primer is shown in SEQ ID NOs. 1-2; the nucleotide sequence of the aminopropylagmatine urea hydrolase gene primer is shown in SEQ ID NOs. 3-4.

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