Method for preparing 1, 5-pentanediamine

Abstract

The invention discloses a method for preparing 1, 5-pentanediamine. The recombinant strain containing the L-lysine decarboxylase coding gene is used for preparing the 1, 5-pentanediamine, so that yield reduction caused by poisoning of host cells by the 1, 5-pentanediamine is greatly reduced, and the yield of the 1, 5-pentanediamine produced by fermentation of the recombinant strain is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for preparing 1,5-pentanediamine. Background Art

[0002] 1,5-pentanediamine (also known as 1,5-diaminopentane, cadaverine) is an important five-carbon compound in the chemical industry. It has a wide range of uses and can be used to manufacture important chemical raw materials such as polyamide, polyurethane, isocyanate, pyridine, piperidine, etc. At present, the microbial production of 1,5-pentanediamine mainly adopts the following two methods: microbial fermentation production and microbial in vitro enzyme catalysis production. Lysine decarboxylase (L-lysine decarboxylase, referred to as LDC, EC 4.1.1.18) used in enzyme catalysis production is widely present in microorganisms, animals and higher plants. It can remove a carboxyl group from L-lysine to generate 1,5-pentanediamine and CO2.

[0003] In the process of lysine decarboxylase catalyzing lysine to produce 1,5-pentanediamine, free lysine decarboxylase or lysine decarboxylase cells are usually used, or a strain that can produce lysine and lysine decarboxylase at the same time is used to ferment and produce 1,5-pentanediamine. However, these production methods result in low efficiency of enzyme or cell reuse, difficulty in product recovery, and high production cost, which is not conducive to the industrial production of 1,5-pentanediamine. In addition, since the concentration of 1,5-pentanediamine that the bacteria itself can tolerate is limited, if lysine decarboxylase is expressed in the early stage of the fermentation system, resulting in excessive conversion of 1,5-pentanediamine, it will cause toxicity to the bacteria, thereby inhibiting the growth of the bacteria and the process of producing L-lysine using glucose (Qian, et al., Biotechnol. Bioeng. 2011; 108: 93–103). Therefore, a new technology for obtaining a higher yield of 1,5-pentanediamine is needed. Summary of the invention

[0004] The purpose of the present invention is to provide a strain for producing 1,5-pentanediamine by fermentation and a method for preparing 1,5-pentanediamine.

[0005] The present invention introduces a gene for expressing L-lysine decarboxylase into Corynebacterium glutamicum CATH2403, and obtains an engineered bacterium that can produce 1,5-pentanediamine in a one-step method (i.e., no additional addition of L-lysine decarboxylase is required, and lysine and L-lysine decarboxylase are produced directly in the cell, and then 1,5-pentanediamine is directly obtained by fermentation). In addition, the present invention uses a stable phase-specific promoter to regulate the expression of L-lysine decarboxylase in the stable phase, which greatly reduces the reduction in production caused by the poisoning of host cells by 1,5-pentanediamine.

[0006] In the first aspect, the present invention provides a nucleic acid molecule comprising an L-lysine decarboxylase encoding gene and a stationary phase-specific promoter capable of initiating expression of the encoding gene, or consisting of the two (e.g., the stationary phase-specific promoter and the L-lysine decarboxylase encoding gene are directly linked in the 5' to 3' direction), wherein the stationary phase-specific promoter is from Corynebacterium glutamicum.

[0007] Herein, L-lysine decarboxylase (abbreviated as LDC, EC 4.1.1.18) can be derived from cells of microorganisms, animals or plants, including but not limited to Escherichia coli, Bacillus subtilis, Bacillus halodurans, Streptomyces coelicolor, Hafnia alvei, Corynebacterium glutamicum or Klebsiella oxytoca, etc. In some embodiments, L-lysine decarboxylase is derived from CadA or LdcC of Escherichia coli.

[0008] In some embodiments, in the above nucleic acid molecule, the L-lysine decarboxylase is from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO:27.

[0009] In some embodiments, in any of the above-mentioned nucleic acid molecules, the L-lysine decarboxylase encoding gene is the cadA gene or ldcC gene from Escherichia coli, and the L-lysine decarboxylase encoding gene is preferably as shown in SEQ ID NO:26.

[0010] Researchers have found that there are some natural promoters in prokaryotes that will start downstream gene expression only after the bacteria grow to the stable phase, which are called stable phase-induced promoters, or stable phase-specific promoters. In order to achieve one-step production of 1,5-pentanediamine, the promoter of the L-lysine decarboxylase encoding gene introduced into Corynebacterium glutamicum can be changed to a stable phase-specific promoter. There are also some stable phase-specific promoters in Corynebacterium glutamicum, such as Pgap, PsigB, Peftu, Psod, PgpmA, etc.

[0011] In some embodiments, in any of the above-mentioned nucleic acid molecules, the stationary phase-specific promoter is selected from any one of the stationary phase-specific promoters of Corynebacterium glutamicum.

[0012] In some embodiments, in any of the above-mentioned nucleic acid molecules, the stable phase-specific promoter is selected from any one of the promoter PgpmA shown in SEQ ID NO:28, the promoter Pgap shown in SEQ ID NO:29, the promoter Psod shown in SEQ ID NO:30, the promoter PsigB shown in SEQ ID NO:31 or the promoter Peftu shown in SEQ ID NO:32.

[0013] In some embodiments, any of the above-described nucleic acid molecules is PgpmA-cadA, Pgap-cadA, Psod-cadA, PsigB-cadA or Peftu-cadA.

[0014] The nucleic acid molecules provided by the present invention can usually be obtained by amplification using a PCR instrument or artificial synthesis.

[0015] In a second aspect, the present invention provides a recombinant vector comprising any of the above-mentioned nucleic acid molecules.

[0016] In some embodiments, in the above-mentioned recombinant vector, the recombinant vector is an expression plasmid or an integration plasmid, the expression plasmid exists in a free form in the host cell and expresses the L-lysine decarboxylase encoding gene it carries during the stable period, and the integration plasmid can integrate any of the above-mentioned nucleic acid molecules into the genome of the host cell by homologous recombination.

[0017] In some embodiments, the expression plasmid vector comprises pEC-XK99E, pXMJ19 or pREST-B.

[0018] In some embodiments, the expression plasmid is pEC-XK99E-PgpmA-cadA, pEC-XK99E-Pgap-cadA, pEC-XK99E-Psod-cadA, pEC-XK99E-PsigB-cadA, pEC-XK99E-Peftu-cadA;

[0019] The construction of these expression plasmids includes the following steps:

[0020] (1) replacing the sequence between the EcoRI and XbaI sites of the plasmid pEC-XK99E with the DNA molecule shown in SEQ ID NO: 26, while leaving the rest of the sequence unchanged, to obtain the expression plasmid pEC-XK99E-ptac-cadA;

[0021] (2) using the stationary phase specific promoter PgpmA sequence shown in SEQ ID NO: 28 as a template and using PgpmA-F and PgpmA-R as primers to perform PCR amplification to obtain a fragment containing the stationary phase specific promoter PgpmA; using the pEC-XK99E-ptac-cadA plasmid as a template, PCR linearizing the pEC-XK99E-ptac-cadA plasmid using primers pEC-F and pEC-R to obtain a linearized plasmid; assembling the fragment containing the stationary phase specific promoter PgpmA and the linearized plasmid by homologous recombination to obtain the expression plasmid pEC-XK99E-PgpmA-cadA;

[0022] Using the stationary phase specific promoter Pgap sequence shown in SEQ ID NO:29 as a template, using Pgap-F and Pgap-R as primers to perform PCR amplification to obtain a fragment containing the stationary phase specific promoter Pgap; using the pEC-XK99E-ptac-cadA plasmid as a template, using primers pEC-F and pEC-R to linearize the pEC-XK99E-ptac-cadA plasmid PCR to obtain a linearized plasmid; assembling the fragment containing the stationary phase specific promoter Pgap and the linearized plasmid by homologous recombination to obtain an expression plasmid pEC-XK99E-Pgap-cadA;

[0023] Using the stationary phase specific promoter Psod sequence shown in SEQ ID NO:30 as a template, using Psod-F and Psod-R as primers to perform PCR amplification to obtain a fragment containing the stationary phase specific promoter Psod; using the pEC-XK99E-ptac-cadA plasmid as a template, using primers pEC-F and pEC-R to linearize the pEC-XK99E-ptac-cadA plasmid PCR to obtain a linearized plasmid; assembling the fragment containing the stationary phase specific promoter Psod and the linearized plasmid by homologous recombination to obtain an expression plasmid pEC-XK99E-Psod-cadA;

[0024] Using the stable phase specific promoter PsigB sequence shown in SEQ ID NO:31 as a template, using PsigB-F and PsigB-R as primers to perform PCR amplification to obtain a fragment containing the stable phase specific promoter PsigB; using the pEC-XK99E-ptac-cadA plasmid as a template, using primers pEC-F and pEC-R to linearize the pEC-XK99E-ptac-cadA plasmid PCR to obtain a linearized plasmid; assembling the fragment containing the stable phase specific promoter PsigB and the linearized plasmid by homologous recombination to obtain an expression plasmid pEC-XK99E-PsigB-cadA;

[0025] The stable phase specific promoter Peftu sequence shown in SEQ ID NO:32 is used as a template, and Peftu-F and Peftu-R are used as primers for PCR amplification to obtain a fragment containing the stable phase specific promoter Peftu; the pEC-XK99E-ptac-cadA plasmid is used as a template, and the pEC-XK99E-ptac-cadA plasmid is PCR linearized by primers pEC-F and pEC-R to obtain a linearized plasmid; the fragment containing the stable phase specific promoter Peftu and the linearized plasmid are assembled by homologous recombination to obtain an expression plasmid pEC-XK99E-Peftu-cadA.

[0026] In some embodiments, the method of secondary homologous recombination of suicide plasmid is used to integrate any of the above-mentioned nucleic acid molecules into the genome of the host cell through the above-mentioned integration plasmid. The vector of the suicide plasmid is preferably plasmid pK18mobsacB or pK18mobrpsL.

[0027] In some embodiments, the homologous fragment of the L-lysine decarboxylase gene expression cassette (i.e., any of the above-mentioned nucleic acid molecules) is first connected to a vector of a suicide plasmid to obtain a recombinant suicide plasmid, and then the recombinant suicide plasmid is electroporated into a competent state of a starting Corynebacterium glutamicum to complete the first homologous recombination; the sucrose lethality principle is then used to complete the second homologous recombination, the suicide plasmid backbone is removed, and the recombinant Corynebacterium glutamicum is obtained, whose genome is integrated with the L-lysine decarboxylase gene expression cassette.

[0028] In some embodiments, in the starting Corynebacterium glutamicum, the gene knockout and replacement are performed by double exchange homologous recombination method: first, the homologous fragment of the L-lysine decarboxylase gene expression frame is connected to the plasmid vector pK18mobsacB, and then the plasmid is electrotransferred into the starting Corynebacterium glutamicum competent state to complete the first homologous recombination; then a secondary homologous screening is performed, and the secondary homologous recombination is completed using the sucrose lethal principle to obtain the target edited strain. The role of the integration plasmid is to modify, knock out or insert new exogenous genes into the genes on the strain's own genome.

[0029] In some embodiments, the integration plasmid used to integrate the L-lysine decarboxylase gene into the genome of Corynebacterium glutamicum is preferably plasmid pK18mobsacB or pK18mobrpsL.

[0030] In some embodiments, Corynebacterium glutamicum CATH2403 is used as the starting strain, and an L-lysine decarboxylase gene expression cassette, such as peftu-cadA, is inserted into a suitable genomic site to construct a one-step Corynebacterium glutamicum expressing L-lysine decarboxylase through genome integration.

[0031] In some embodiments, the suitable genomic loci include: cg1890, cg1895, or cg2037.

[0032] In some embodiments, the integration plasmid is pK18-cg1890::Peftu-cadA, pK18-cg1890::PgpmA-cadA, pK18-cg1890::Pgap-cadA, pK18-cg1890::Psod-cadA, pK18-cg1890::PsigB-cadA;

[0033] The construction of these integration plasmids involves the following steps:

[0034] The genomic DNA of Corynebacterium glutamicum CATH2403 was used as a template, and UP-F and UP-R were used as primers to perform PCR amplification to obtain the upstream fragment cg1890-up; the genomic DNA of Corynebacterium glutamicum CATH2403 was used as a template, and Down-F and Down-R were used as primers to perform PCR amplification to obtain the downstream fragment cg1890-down; and the recombinant expression plasmid pEC-XK99E-Peftu-cadA was used as a template, and peftu-F2 and CadA-R2 were used as primers to perform PCR amplification to obtain the peftu-cadA fragment. Then, the three fragments were fused together in the order of cg1890-up, peftu-cadA, and cg1890-down by overlapping extension PCR to obtain the fusion fragment cg1890::peftu-cadA; pK18mobsacB plasmid was used as a template and pK18-F and pK18-R were used as primers to perform reverse PCR to obtain a linearized fragment, which was recombined and ligated with the fusion fragment cg1890::Peftu-cadA to construct the integration plasmid pK18-cg1890::Peftu-cadA;

[0035] pK18-cg1890::PgpmA-cadA, pK18-cg1890::Pgap-cadA, pK18-cg1890::Psod-cadA, and pK18-cg1890::PsigB-cadA can be obtained using methods similar to those described above.

[0036] In a third aspect, the present invention provides a recombinant Corynebacterium glutamicum comprising any of the above-described nucleic acid molecules and / or any of the above-described recombinant vectors.

[0037] In some embodiments, in the above-mentioned recombinant Corynebacterium glutamicum, any of the above-mentioned nucleic acid molecules exists in the recombinant Corynebacterium glutamicum in the form of a plasmid; in some embodiments, the recombinant Corynebacterium glutamicum is pEC-PgpmA-CadA / CATH-2, pEC-Pgap-CadA / CATH-3, pEC-Psod-CadA / CATH-4, pEC-PsigB-CadA / CATH-5, or pEC-Peftu-CadA / CATH-6.

[0038] In some embodiments, in any of the above-mentioned recombinant Corynebacterium glutamicum, any of the above-mentioned nucleic acid molecules are integrated at some sites in the genome of Corynebacterium glutamicum, such as: cg1890, cg1895, cg2037 genomic sites, etc., preferably integrated at the cg1890 site of Corynebacterium glutamicum; in some embodiments, the recombinant Corynebacterium glutamicum is cg1890::Peftu-CadA / CATH-7, cg1890::PgpmA-CadA / CATH-8, cg1890::Pgap-CadA / CATH-9, cg1890::Psod-CadA / CATH-10, cg1890::PsigB-CadA / CATH-11.

[0039] In a fourth aspect, the present invention provides a method for constructing any of the above-mentioned recombinant Corynebacterium glutamicum, comprising the steps of transforming any of the above-mentioned expression plasmids into the starting Corynebacterium glutamicum; or

[0040] The step of transforming any of the above-mentioned integration plasmids into the starting Corynebacterium glutamicum and carrying out homologous recombination with the genome of the starting Corynebacterium glutamicum.

[0041] In some embodiments, in the above method, the starting Corynebacterium glutamicum is Corynebacterium glutamicum CATH2403, which has been deposited in the China Center for Type Culture Collection (CCTCC) on July 12, 2024, with a deposit number of CCTCC NO: M 20241539.

[0042] In some embodiments, in the above method, for the recombinant Corynebacterium glutamicum pEC-PgpmA-CadA / CATH-2, pEC-Pgap-CadA / CATH-3, pEC-Psod-CadA / CATH-4, pEC-PsigB-CadA / CATH-5, and pEC-Peftu-CadA / CATH-6, the recombinant expression plasmids pEC-XK99E-PgpmA-cadA, pEC-XK99E-Pgap-cadA, pEC-XK99E-Psod-cadA, pEC-XK99E-PsigB-cadA, and pEC-XK99E-Peftu-cadA are respectively transformed into Corynebacterium glutamicum CATH2403.

[0043] In some embodiments, in the above method, for the recombinant Corynebacterium glutamicum cg1890::Peftu-CadA / CATH-7, cg1890::PgpmA-CadA / CATH-8, cg1890::Pgap-CadA / CATH-9, cg1890::Psod-CadA / CATH-10, and cg1890::PsigB-CadA / CATH-11, respectively, the integration plasmids pK18-cg1890::Peftu-cadA, pK18-cg1890::PgpmA-cadA, pK18-cg1890::Pgap-cadA, pK18-cg1890::Psod-cadA, and pK18-cg1890::PsigB-cadA are used, and the recombinant Corynebacterium glutamicum cg1890::Peftu-CadA / CATH-7, cg1890::PgpmA-CadA / CATH-8, cg1890::Pgap-CadA / CATH-9, cg1890::Psod-CadA / CATH-10, and cg1890::PsigB-CadA are respectively The method of secondary homologous recombination of a killing plasmid is to integrate any of the above-mentioned nucleic acid molecules into Corynebacterium glutamicum CATH2403. Specifically: the method comprises transferring any of the above-mentioned integrated plasmids into Corynebacterium glutamicum CATH2403 by electroporation transformation, picking a single colony with kanamycin resistance to perform a recombination verification by colony PCR; then picking a colony with correct recombination verification to culture overnight in LBH medium, so that the strain undergoes secondary recombination and loses the plasmid during the culture process; then spreading a moderately diluted bacterial solution on a plate added with sucrose, selecting a single colony that can grow on the sucrose plate but does not have kanamycin resistance to perform colony PCR verification, and the successfully verified clones are further verified by sequencing, and the clones with correct sequencing verification are one-step Corynebacterium glutamicum with genome-integrated expression of L-lysine decarboxylase.

[0044] In this article, the recombinant Corynebacterium glutamicum contains an L-lysine decarboxylase gene under the control of a stable phase-specific promoter. Thus, by using a stable phase-specific promoter to regulate the expression of L-lysine decarboxylase in the stable phase, the energy consumption during the 1,5-pentanediamine tolerance process is reduced, the production of L-lysine is promoted, and the yield of 1,5-pentanediamine is increased.

[0045] In a fifth aspect, the present invention provides a method for preparing 1,5-pentanediamine, comprising the steps of fermenting and culturing any of the above-mentioned recombinant Corynebacterium glutamicum, and extracting and / or purifying pentamethylenediamine from the fermentation broth.

[0046] In some embodiments, the culture medium of the fermentation culture comprises 50-70 g / L glucose or sucrose, 5-7 g / L CaCO3, 0.8-1.2 g / L KH2PO4, 4.5-5.5 g / L K2HPO4, 0.8-1.2 g / L MgSO4, 20-40 g / L (NH4)2SO4, 5-10 g / L yeast powder or yeast extract, and the balance is water.

[0047] In some embodiments, the temperature of the fermentation culture is 29-32°C, such as 30°C.

[0048] In a sixth aspect, the present invention provides use of any of the above-mentioned nucleic acid molecules, any of the above-mentioned recombinant vectors, and any of the above-mentioned recombinant Corynebacterium glutamicum in the preparation of 1,5-pentanediamine.

[0049] The present invention uses a stable phase-specific promoter to regulate the expression of L-lysine decarboxylase in the stable phase, greatly reduces the production reduction caused by the poisoning of host cells by 1,5-pentanediamine, significantly improves the yield of 1,5-pentanediamine produced by fermentation of the recombinant strain, and significantly reduces the residual L-lysine, thereby simplifying the subsequent process of extracting 1,5-pentanediamine.

[0050] Collection Instructions

[0051] Bacterial species name: Corynebacterium glutamicum CATH2403

[0052] Latin name: Corynebacterium glutamicum CATH2403

[0053] Collection institution: China Center for Type Culture Collection

[0054] Abbreviation of depository institution: CCTCC

[0055] Address: Wuhan University, Wuhan, China

[0056] Deposit date: July 12, 2024

[0057] CCTCC No.: M 20241539 DETAILED DESCRIPTION

[0058] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0059] The specific steps, condition parameters, etc. of PCR amplification, purification, plasmid extraction, enzyme digestion, enzyme digestion product connection, transformation, etc. involved in the following examples are all carried out according to the conditions recommended in the instructions of the purchased relevant enzymes and reagents. The DNA polymerases used for PCR amplification were purchased from Shanghai Baisai Biotechnology Co., Ltd., trademark Takara. Plasmid extraction kits, DNA gel recovery kits, and PCR purification kits were all purchased from Corning Life Sciences (Wujiang) Co., Ltd., trademark Axygen; restriction endonucleases used for enzyme digestion, ligases used for enzyme digestion product connection, and homologous recombination enzymes were purchased from Thermo Fisher Scientific (China) Co., Ltd., trademark Thermo Scientific.

[0060] The expression plasmid pEC-XK99E is a shuttle plasmid.

[0061] Chemical heat shock transformation method (heat shock method): Add the ligation product to 100 μL of E. coli TOP10 competent cells, place on ice for 25 minutes, and heat shock at 42°C for 90 seconds. After incubation on ice for 3 minutes, add 800 μL of LB medium, culture at 37°C and 200 rpm for 1 hour, centrifuge at 4000 rpm for 5 minutes, discard about 600 μL of supernatant medium, gently mix the bacteria at the bottom of the centrifuge tube, and apply it to the LB plate with the corresponding resistance.

[0062] Electroporation transformation method: 500 ng of plasmid was taken and added to the competent cells of Corynebacterium glutamicum CATH2403, mixed evenly, and ice-bathed for 5-10 min; the competent mixture after ice bath was transferred to a pre-cooled electroporation cup, electroporated twice at 1.8 kV and 5 ms, and transferred to 1 mL of LBH medium (containing 5 g / L peptone, 2.5 g / L yeast powder, 10 g / L sodium chloride, 18.5 g / L brain heart extract, 91 g / L sorbitol, and the balance was water) preheated at 46°C in advance, and heat-shocked at 46°C for 6 min; after 2 h of recovery culture at 30°C and 200 rpm, centrifuged at 4000 rpm for 5 min to collect the bacteria; the supernatant was aspirated to resuspend the bacteria when 100-200 μL of culture medium was left, mixed and coated on an LBH plate containing 10 μg / mL kanamycin, and placed in a 30°C constant temperature incubator for culture, and then the correct clone was verified by colony PCR.

[0063] In the present invention, the amounts of L-lysine and 1,5-pentanediamine in the culture medium are detected by a nuclear magnetic resonance method.

[0064] The primers used in the following examples are shown in Table 1.

[0065] Table 1 Primers

[0066]

[0067]

[0068] Note: Underline indicates restriction enzyme cutting site.

[0069] Example 1 Cloning of L-lysine decarboxylase gene cadA in Corynebacterium glutamicum CATH2403

[0070] 1. Using genomic DNA of Escherichia coli str.K-12substr.MG1655 (purchased from Beijing Tianenze Biotechnology Co., Ltd.) as a template and cadA-SacI-F and cadA-XbaI-R as primers, PCR amplification was performed to obtain a fragment containing the L-lysine decarboxylase encoding gene; the fragment containing the L-lysine decarboxylase encoding gene was double-digested with SacI and XbaI to obtain a gene fragment; pUC18 plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) was double-digested with SacI and XbaI to obtain a vector fragment; the gene fragment and the vector fragment were connected to obtain a recombinant plasmid pUC18-cadA.

[0071] The competent cells were prepared using the CaCl2 method, and the recombinant plasmid pUC18-cadA was transformed into Escherichia coli TOP10 (purchased from Takara Biotechnology (Dalian) Co., Ltd.) cells using the heat shock method. Kanamycin was added to LB medium for screening. After cloning PCR and sequencing verification, the plasmid pUC18-cadA was extracted.

[0072] 2. The cadA gene sequence (SEQ ID NO:25) obtained by sequencing was optimized for amino acid codons using the Corynebacterium glutamicum CATH2403 host, so that cadA can be smoothly translated into protein in Corynebacterium glutamicum CATH2403, and the codon-optimized cadA gene sequence (SEQ ID NO:26) (the CadA amino acid sequence encoded by it is shown in SEQID NO:27) was obtained by gene synthesis method, and PCR amplification was performed using it as a template and cadA-F and cadA-R as primers to obtain a fragment containing the cadA gene; EcoRI and XbaI double-digested the fragment containing the cadA gene to obtain a gene fragment; EcoRI and XbaI double-digested the Corynebacterium glutamicum expression plasmid pEC-XK99E (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) to obtain a vector fragment; the gene fragment and the vector fragment were connected to obtain a recombinant expression plasmid pEC-XK99E-Ptac-cadA, wherein the Ptac-cadA sequence is shown in SEQID NO:33. Ptac is not a stationary phase specific promoter.

[0073] The heat shock method was also used to transform into E. coli TOP10. After sequencing verification, the plasmid pEC-XK99E-ptac-cadA was extracted.

[0074] 3. Take 500 ng of pEC-XK99E-Ptac-cadA plasmid and transform it into Corynebacterium glutamicum CATH2403 by electroporation to obtain the recombinant expression strain pEC-Ptac-CadA / CATH-1.

[0075] Example 2 Construction of a strain for inducing recombinant expression of L-lysine decarboxylase using a stable phase specific promoter

[0076] 1. Taking the stable phase specific promoter PgpmA as an example, construct the stable phase specific promoter-induced L-lysine decarboxylase recombinant expression of Corynebacterium glutamicum CATH2403. First, the nucleotide sequence of PgpmA (as shown in SEQ ID NO: 28) was obtained by direct gene synthesis; using it as a template, PCR amplification was performed with PgpmA-F and PgpmA-R as primers to obtain a fragment containing the stable phase specific promoter PgpmA; using the pEC-XK99E-Ptac-cadA plasmid as a template, the pEC-XK99E-Ptac-cadA plasmid was PCR linearized by primers pEC-F and pEC-R to obtain a linearized plasmid; the fragment containing the stable phase specific promoter PgpmA and the linearized plasmid were amplified by homologous recombination kit GeneArt TM The fragments were assembled with Gibson AssemblyHiFi Master Mix to obtain the recombinant expression plasmid pEC-XK99E-PgpmA-cadA, which was then transformed into Corynebacterium glutamicum CATH2403 by electroporation to obtain the recombinant expression strain pEC-PgpmA-CadA / CATH-2.

[0077] In pEC-XK99E-PgpmA-cadA, the PgpmA-cadA sequence is formed by directly connecting the PgpmA sequence and the cadA sequence (SEQ ID NO: 26) in the 5' to 3' direction.

[0078] 2. Similarly, the promoter sequences of Pgap (as shown in SEQ ID NO: 29), Psod (as shown in SEQ ID NO: 30), PsigB (as shown in SEQ ID NO: 31), and Peftu (as shown in SEQ ID NO: 32) were directly obtained by gene synthesis, and PCR amplification was performed with the corresponding primers Pgap-F and Pgap-R, Psod-F and Psod-R, PsigB-F and PsigB-R, Peftu-F and Peftu-R, respectively, to obtain fragments containing the stationary phase specific promoters Pgap, Psod, PsigB, and Peftu, respectively. The pEC-XK99E-Ptac-cadA plasmid was PCR linearized with primers pEC-F and pEC-R to obtain a linearized plasmid; the fragment containing the stationary phase specific promoters Pgap, Psod, PsigB, and Peftu and the linearized plasmid were amplified with the homologous recombination kit GeneArt TMThe fragments assembled by Gibson Assembly HiFi Master Mix were used to obtain recombinant expression plasmids pEC-XK99E-Pgap-cadA, pEC-XK99E-Psod-cadA, pEC-XK99E-PsigB-cadA, and pEC-XK99E-Peftu-cadA, which were then transformed into Corynebacterium glutamicum CATH2403 by electroporation to obtain recombinant expression strains pEC-Pgap-CadA / CATH-3, pEC-Psod-CadA / CATH-4, pEC-PsigB-CadA / CATH-5, and pEC-Peftu-CadA / CATH-6, respectively.

[0079] In pEC-XK99E-Pgap-cadA, the Pgap-cadA sequence is formed by directly connecting the Pgap sequence and the cadA sequence (SEQ ID NO: 26) in the 5' to 3' direction.

[0080] In pEC-XK99E-Psod-cadA, the Psod-cadA sequence is formed by directly connecting the Psod sequence and the cadA sequence (SEQ ID NO: 26) in the 5' to 3' direction.

[0081] In pEC-XK99E-PsigB-cadA, the PsigB-cadA sequence is formed by directly connecting the PsigB sequence and the cadA sequence (SEQ ID NO: 26) in the 5' to 3' direction.

[0082] In pEC-XK99E-Peftu-cadA, the Peftu-cadA sequence is formed by directly connecting the Peftu sequence and the cadA sequence (SEQ ID NO: 26) in the 5' to 3' direction.

[0083] The recombinant expression plasmids and recombinant expression strains for inducing the expression of L-lysine decarboxylase by the non-stable phase specific promoter and different stable phase specific promoters constructed by the present invention are shown in Table 2.

[0084] Table 2

[0085] Promoter Recombinant expression plasmid Recombinant expression strain Ptac pEC-XK99E-Ptac-cadA pEC-Ptac-CadA / CATH-1 PgP pEC-XK99E-PgpmA-cadA pEC-PgpmA-CadA / CATH-2 Pgap pEC-XK99E-Pgap-cadA pEC-Pgap-CadA / CATH-3 Psod pEC-XK99E-Psod-cadA pEC-Psod-CadA / CATH-4 PsigB pEC-XK99E-PsigB-cadA pEC-PsigB-CadA / CATH-5 Peftu pEC-XK99E-Peftu-cadA pEC-Peftu-CadA / CATH-6

[0086] Example 3 Comparison of the lysine and 1,5-pentanediamine yields of recombinant L-lysine decarboxylase expression strains induced by different promoters in Corynebacterium glutamicum CATH2403

[0087] Taking Corynebacterium glutamicum CATH2403 which can produce L-lysine as a control, the recombinant expression strains pEC-Ptac-CadA / CATH-1, pEC-PgpmA-CadA / CATH-2, pEC-Pgap-CadA / CATH-3, pEC-Psod-CadA / CATH-4, pEC-PsigB-CadA / CATH-5, and pEC-Peftu-CadA / CATH-6 were picked and added to 5 mL seed liquid culture medium (containing 40 g / L sucrose, 2 g / L yeast extract, 10 g / L peptone, 1 g / L KH2PO4, 5 g / LK2HPO4, 1 g / L MgSO4, 16 g / L (NH4)2SO4, and 10 μg / mL kanamycin, the balance being water), and cultured at 30°C overnight. The next day, each strain was transferred to 100 mL of fermentation medium (containing 50 g / L glucose, 7 g / L CaCO3, 1 g / L KH2PO4, 5 g / L K2HPO4, 1 g / L MgSO4, 20 g / L (NH4)2SO4, 5 g / L yeast powder, and 10 μg / mL kanamycin, with the remainder being water) at a 2% (v / v) inoculum, and the culture was continued at 30°C with stirring for 24 h. After the culture was completed, samples were taken, and the contents of L-lysine and 1,5-pentanediamine in each culture medium were detected and calculated using nuclear magnetic resonance, and the results are shown in Table 3.

[0088] Table 3

[0089]

[0090] Note: / means not contained, nd means not detected.

[0091] It can be seen from Table 3 that after 24 hours of fermentation, compared with the recombinant expression strain pEC-Ptac-CadA / CATH-1 using a common promoter, the recombinant expression strains using different stable phase specific promoters increased the yield of 1,5-pentanediamine converted at the fermentation endpoint and reduced the unconverted L-lysine residue.

[0092] Example 4: Integrating the L-lysine decarboxylase expression element into the genomic locus of Corynebacterium glutamicum by gene editing to construct a one-step method for expressing L-lysine decarboxylase in the genome of Corynebacterium glutamicum

[0093] Using Corynebacterium glutamicum CATH2403 as the starting strain, the L-lysine decarboxylase gene expression frame peftu-cadA was inserted into the appropriate genomic site to construct a one-step Corynebacterium glutamicum expressing L-lysine decarboxylase by genome integration.

[0094] 1. According to the sequence of about 800-1000 bp in length upstream and about 800-1000 bp in length downstream of the genomic insertion site cg1890 of Corynebacterium glutamicum CATH2403, primers UP-F and UP-R, Down-F and Down-R were designed and synthesized; using the genomic DNA of Corynebacterium glutamicum CATH2403 as a template and UP-F and UP-R as primers, PCR amplification was performed to obtain the upstream fragment cg1890-up; using the genomic DNA of Corynebacterium glutamicum CATH2403 as a template and Down-F and Down-R as primers, PCR amplification was performed to obtain the downstream fragment cg1890-down; and then using the recombinant expression plasmid pEC-XK99E-Peftu-cadA as a template , PCR amplification was performed using peftu-F2 and CadA-R2 as primers to obtain the peftu-cadA fragment; then, the three fragments were fused together in the order of cg1890-up, peftu-cadA, and cg1890-down by overlap extension PCR to obtain the fusion fragment cg1890::peftu-cadA; pK18mobsacB plasmid (purchased from Youbao Bio, product catalog number VT1837) was used as a template and pK18-F and pK18-R were used as primers to perform reverse PCR to obtain a linearized fragment, which was recombined and ligated with the fusion fragment cg1890::Peftu-cadA to construct the integration plasmid pK18-cg1890::Peftu-cadA.

[0095] 2. The integrated plasmid pK18-cg1890::Peftu-cadA was transferred into Corynebacterium glutamicum CATH2403 by electroporation transformation, and a single colony grown on an LBH plate containing 10 μg / mL of kanamycin was selected for a recombination verification by colony PCR. Then a colony with correct recombination verification was selected for overnight culture in LBH medium, so that the strain was recombined and lost plasmid during the culture process. Subsequently, the appropriately diluted bacterial solution was coated on an LBH plate with 100 g / L sucrose added, and a single colony that could grow on a sucrose plate but did not have kanamycin resistance was selected for colony PCR verification, and the successful clone was further verified by sequencing, and the correct clone verified by sequencing was the one-step method Corynebacterium glutamicum cg1890::Peftu-CadA / CATH-7 with genome integration expression of L-lysine decarboxylase.

[0096] Example 5 Verification of Lysine and 1,5-pentanediamine Yields of One-Step Method for Genome-Integrated Expression of L-Lysine Decarboxylase Corynebacterium glutamicum cg1890::Peftu-CadA / CATH-7

[0097] The one-step method for genome-integrated expression of L-lysine decarboxylase in Corynebacterium glutamicum cg1890::Peftu-CadA / CATH-7 does not contain a plasmid and does not require the addition of antibiotics. It uses a stable phase promoter to express L-lysine decarboxylase. The transcription of the downstream gene cadA is initiated only after the bacteria enters the stable phase. Compared with the use of a common promoter, this can further reduce the accumulation of 1,5-pentanediamine during the lysine production process.

[0098] Using L-lysine-producing Corynebacterium glutamicum CATH2403 as a control, the contents of L-lysine and 1,5-pentanediamine after 24 h of fermentation of pEC-Peftu-CadA / CATH-6 and cg1890::Peftu-CadA / CATH-7 were detected. The fermentation conditions were the same as those in Example 3, but the seed culture medium and the fermentation medium did not contain 10 μg / mL kanamycin.

[0099] Table 4

[0100]

[0101] Note: / means not contained, nd means not detected.

[0102] The present invention has found through testing that, when fermented for 24 hours, compared with the control CATH2403 strain, the strain with L-lysine decarboxylase integrated into the genome can convert and generate a certain concentration of 1,5-pentanediamine, thereby realizing the one-step production of L-lysine and 1,5-pentanediamine.

[0103] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0104] sequence

[0105] SEQ ID NO:25, cadA nucleotide sequence

[0106] ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGAACCCATCCG

[0107] TGAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTGTTTACCCGAACGACCGT

[0108] GACGACTTATTAAAACTGATCGAAAACAATGCGCGTCTGTGCGGCGTTATTTTTGACT

[0109] GGGATAAATATAATCTCGAGCTGTGCGAAGAAATTAGCAAAATGAACGAGAACCTGCC

[0110] GTTGTACGCGTTCGCTAATACGTATTCCACTCTCGATGTAAGCCTGAATGACCTGCGTT

[0111] TACAGATTAGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCTAATAAGATC

[0112] AAGCAGACCACTGACGAATATATCAACACTATTCTGCCTCCGCTGACTAAAGCACTGT

[0113] TTAAATATGTTCGTGAAGGTAAATATACTTTCTGTACTCCTGGTCACATGGGCGGTACT

[0114] GCATTCCAGAAAAGCCCGGTAGGTAGCCTGTTCTATGATTTCTTTGGTCCGAATACCAT

[0115] GAAATCTGATATTTCCATTTCAGTATCTGAACTGGGTTCTCTGCTGGATCACAGTGGTC

[0116] CACACAAAGAAGCAGAACAGTATATCGCTCGCGTCTTTAACGCAGACCGCAGCTACAT

[0117] GGTGACCAACGGTACTTCCACTGCGAACAAAATTGTTGGTATGTACTCTGCTCCAGCA

[0118] GGCAGCACCATTCTGATTGACCGTAACTGCCACAAATCGCTGACCCACCTGATGATGA

[0119] TGAGCGATGTTACGCCAATCTATTTCCGCCCGACCCGTAACGCTTACGGTATTCTTGGT

[0120] GGTATCCCACAGAGTGAATTCCAGCACGCTACCATTGCTAAGCGCGTGAAAGAAACA

[0121] CCAAACGCAACCTGGCCGGTACATGCTGTAATTACCAACTCTACCTATGATGGTCTGCT

[0122] GTACAACACCGACTTCATCAAGAAAACACTGGATGTGAAATCCATCCACTTTGACTCC

[0123] GCGTGGGTGCCTTACACCAACTTCTCACCGATTTACGAAGGTAAATGCGGTATGAGCG

[0124] GTGGCCGTGTAGAAGGGAAAGTGATTTACGAAACCCAGTCCACTCACAAACTGCTGG

[0125] CGGCGTTCTCTCAGGCTTCCATGATCCACGTTAAAGGTGACGTAAACGAAGAAACCTT

[0126] TAACGAAGCCTACATGATGCACACCACCACTTCTCCGCACTACGGTATCGTGGCGTCC

[0127] ACTGAAACCGCTGCGGCGATGATGAAAGGCAATGCAGGTAAGCGTCTGATCAACGGT

[0128] TCTATTGAACGTGCGATCAAATTCCGTAAAGAGATCAAACGTCTGAGAACGGAATCTG

[0129] ATGGCTGGTTCTTTGATGTATGGCAGCCGGATCATATCGATACGACTGAATGCTGGCCG

[0130] CTGCGTTCTGACAGCACCTGGCACGGCTTCAAAAACATCGATAACGAGCACATGTATC

[0131] TTGACCCGATCAAAGTCACCCTGCTGACTCCGGGGATGGAAAAAGACGGCACCATGA

[0132] GCGACTTTGGTATTCCGGCCAGCATCGTGGCGAAATACCTCGACGAACATGGCATCGT

[0133] TGTTGAGAAAACCGGTCCGTATAACCTGCTGTTCCTGTTCAGCATCGGTATCGATAAG

[0134] ACCAAAGCACTGAGCCTGCTGCGTGCTCTGACTGACTTTAAACGTGCGTTCGACCTG

[0135] AACCTGCGTGTGAAAAACATGCTGCCGTCTCTGTATCGTGAAGATCCTGAATTCTATG

[0136] AAAACATGCGTATTCAGGAACTGGCTCAGAATATCCACAAACTGATTGTTCACCACAA

[0137] TCTGCCGGATCTGATGTATCGCGCATTTGAAGTGCTGCCGACGATGGTAATGACTCCGT

[0138] ATGCTGCATTCCAGAAAGAGCTGCACGGTATGACCGAAGAAGTTTACCTCGACGAAA

[0139] TGGTAGGTCGTATTAACGCCAATATGATCCTTCCGTACCCGCCGGGAGTTCCTCTGGTA

[0140] ATGCCGGGTGAAATGATCACCGAAGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATG

[0141] CTGTGTGAAATCGGCGCTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACC

[0142] GTCAGGCTGATGGCCGCTATACCGTTAAGGTATTGAAAGAAGAAAGCAAAAAA

[0143] SEQ ID NO:26, cadA nucleotide sequence

[0144] ATGAATGTAATTGCTATCCTGAATCATATGGGCGTATATTTTAAGGAAGAACCTATCCGC

[0145] GAATTGCACCGTGCTTTGGAACGGCTAAATTTTCAAATTGTGTACCCCAATGACCGGG

[0146] ACGACCTCCTCAAGTTAATTGAGAATAACGCCCGCCTCTGTGGGGTGATTTTCGATTG

[0147] GGATAAGTATAACTTGGAGTTGTGTGAAGAGATTAGTAAAATGAATGAAAACTTACCC

[0148] TTGTATGCCTTTGCGAATACCTACTCCACTCTCGATGTGAGTTTAAATGATTTACGGCTA

[0149] CAAATCTCCTTCTTTGAATATGCGCTGGGTGCCGCAGAAGATATTGCTAACAAAATCAA

[0150] ACAGACCACTGACGAGTATATTAACACCATTTTACCTCCTCTGACCAAGGCCTTATTTA

[0151] AATACGTACGGGAAGGTAAATACACCTTTTGTACCCCGGGGCATATGGGCGGTACCGC

[0152] TTTTCAAAAAAGTCCTGTGGGGAGTTTATTTTATGATTTCTTTGGTCCCAACACTATGA

[0153] AATCTGATATCTCCATTAGTGTGTCCGAATTAGGTAGCTTATTGGACCATAGCGGTCCG

[0154] CATAAGGAAGCAGAACAATACATCGCTCGCGTCTTTAATGCTGATCGTAGTTATATGGT

[0155] TACAAATGGTACGAGTACTGCCAACAAAATTGTGGGTATGTATTCCGCGCCCGCGGGA

[0156] AGTACCATTTTGATTGATCGCAATTGCCACAAGAGTTTGACACATCTAATGATGATGTC

[0157] TGACGTGACACCCATCTATTTCCGGCCTACTCGCAACGCCTATGGGATTTTGGGTGGCA

[0158] TCCCTCAAAGCGAATTTCAACACGCAACGATTGCAAAACGAGTTAAAGAAACTCCCA

[0159] ATGCGACCTGGCCCGTGCACGCGGTCATCACAAATAGCACTTACGATGGCCTCCTGTA

[0160] TAATACCGATTTTATCAAGAAAACCCTGGATGTCAAAAGTATCCATTTTGACTCCGCCT

[0161] GGGTACCCTATACTAACTTTTCCCCAATTTATGAAGGTAAATGTGGTATGTCTGGGGGA

[0162] CGTGTGGAAGGAAAAGTGATTTATGAAACCCAATCCACGCATAAACTGTTAGCAGCTT

[0163] TTAGCCAAGCCTCTATGATTCATGTTAAAGGTGATGTTAATGAAGAAACCTTCAATGAG

[0164] GCGTACATGATGCATACTACGACCAGCCCTCATTACGGGATTGTGGCGAGTACGGAAA

[0165] CAGCAGCCGCTATGATGAAGGGTAATGCTGGAAAACGGTTGATTAATGGGTCTATTGA

[0166] ACGGGCTATCAAATTTCGCAAAGAAATTAAGCGTTTGCGCACTGAAAGCGATGGCTG

[0167] GTTTTTTGATGTTTGGCAACCCGACCATATTGATACAACGGAGTGTTGGCCTCTACGTA

[0168] GTGATAGTACCTGGCATGGGTTTAAAAATATCGATAATGAGCACATGTACCTAGACCCG

[0169] ATTAAGGTAACCTTATTAACACCTGGCATGGAAAAAGATGGCACGATGTCCGACTTTG

[0170] GCATTCCGGCGAGCATCGTGGCAAAATACTTAGATGAGCACGGGATTGTGGTTGAAAA

[0171] GACAGGGCCATATAACTTGCTCTTTTTATTCAGTATCGGCATCGATAAAACTAAGGCGT

[0172] TGAGCCTGCTACGGGCCCTGACTGACTTTAAGCGGGCCTTTGATTTGAATTTGCGTGTT

[0173] AAAAATATGCTCCCTTCTTTATACCGAGAAGATCCCGAATTTTATGAAAATATGCGGAT

[0174] CCAAGAATTGGCGCAGAATATCCATAAGTTAATCGTTCACCATAACCTACCAGATCTAA

[0175] TGTACCGCGCCTTTGAAGTGCTGCCCACCATGGTAATGACCCCATATGCAGCCTTTCAA

[0176] AAAGAGTTACATGGGATGACAGAGGAGGTCTATTTAGACGAAATGGTTGGCCGCATCA

[0177] ATGCAAATATGATTCTGCCCTATCCTCCAGGAGTTCCGTTAGTAATGCCTGGCGAAATG

[0178] ATCACTGAAGAGAGTCGCCCCGTTCTGGAGTTTCTACAAATGCTATGCGAAATTGGTG

[0179] CTCATTATCCCGGCTTCGAAACCGATATCCATGGCGCTTATCGGCAGGCAGATGGACGC

[0180] TATACAGTGAAAGTCTTGAAGGAAGAATCCAAAAAA

[0181] SEQ ID NO:27, CadA amino acid sequence

[0182] MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWD

[0183] KYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTT

[0184] DEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISI

[0185] SVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDR

[0186] NCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVI

[0187] TNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQ

[0188] STHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAG

[0189] KRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNE

[0190] HMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDK

[0191] TKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLP

[0192] DLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMP

[0193] GEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKEESKK

[0194] SEQ ID NO:28, Stable-phase specific promoter PgpmA sequence

[0195] TGTTGGCGGTGAACACTCAGGAGGAGATGCAGGATTTGATGCATCACTACGATGCGGA

[0196] TCCGGATCGGATTTCTGTGGTGTCACCGGGTGCGGACGTGGAACTTTATAGCCCTGGA

[0197] AATGATCGCGCGACGGAACGTTCCCGTCGTGAGCTGGGCATTCCGCTGCACACAAAG

[0198] GTGGTGGCTTTTGTAGGTCGGTTGCAGCCGTTTAAGGGCCCGCAGGTGCTGATCAAG

[0199] GCGGTTGCGGCGTTGTTTGATCGCGATCCGGACCGAAATCTGCGCGTCATTATTTGTG

[0200] GCGGCCCTTCTGGTCCGAATGCGACACCGGATACCTATAGGCATATGGCAGAGGAACT

[0201] GGGCGTCGAAAAGCGAATTCGCTTTTTGGACCCGCGCCCGCCGAGCGAGCTAGTGGC

[0202] CGTGTATCGGGCGGCGGATATCGTGGCTGTGCCAAGTTTTAATGAGTCCTTTGGACTC

[0203] GTCGCCATGGAGGCGCAGGCCAGCGGCACACCGGTTATCGCGGCCCGGGTTGGCGGC

[0204] CTGCCCATCGCAGTCGCGGAAGGGGAGACGGGATTGCTTGTCGACGGCCACTCCCCG

[0205] CATGCCTGGGCCGACGCCATAGCCACACTCTTGGACGATGACGAAACGCGCATCAGG

[0206] ATGGGCGAAGATGCCGTCGAACACGCCAGAACATTCTCCTGGGCGGCCACTGCCGCA

[0207] CAGCTATCGTCGCTGTACAACGATGCTATTGCCAACGAAAACGTCGACGGTGAAACGC

[0208] ATCACGGCTAAATAAACGCGCGTCATGGAACATAAAGTGGCAAACTAGTACCT

[0209] SEQ ID NO:29, Stationary-phase specific promoter Pgap sequence

[0210] ACTTCGACGATGCCAATCTGCGCAGGTCAGCCCGAGCAGCAGTTGCCGCTGCAGCGA

[0211] GGGTAGAACGCGCCATGAAGATTCTTGGTGATGATGTTCCTGAGCATTTGGCTGAGGC

[0212] TGGACAGCTGCGTGTGCAGCACCGTCAGGCATCGTTGGAGGAGTTGGGCCGGTTGGC

[0213] TGATCCTCAAATGACCAAGGATGCTGTGGCCGGTCGTATTCGTCGTCTTTTGACGATG

[0214] GCAGATAAGCGCGCCGAAGATCTGAAGATTCCTGATACAAATTCTGTTGTGACGGAAG

[0215] ATTTGTTGGAAGAAATTTAGATGATTGAAGCCTAAAAACGACCGAGCCTATTGGGATT

[0216] ACCATTGAAGCCAGTGTGAGTTGCATCACATTGGCTTCAAATCTGAGACTTTAATTTGT

[0217] GGATTCACGGGGGTGTAATGTAGTTCATAATTAACCCCATTCGGGGGAGCAGATCGTA

[0218] GTGCGAACGATTTCAGGTTCGTTCCCTGCAAAAACTATTTAGCGCAAGTGTTGGAAAT

[0219] GCCCCCGTTTGGGGTCAATGTCCATTTTTGAATGTGTCTGTATGATTTTGCATCTGCTGC

[0220] GAAATCTTTGTTTCCCCGCTAAAGTTGAGGACAGGTTGACACGGAGTTGACTCGACG

[0221] AATTATCCAATGTGAGTAGGTTTGGTGCGTGAGTTGGAAAAATTCGCCATACTCGCCCT

[0222] TGGGTTCTGTCAGCTCAAGAATTCTTGAGTGACCGATGCTCTGATTGACCTAACTGCT

[0223] TGACACATTGCATTTCCTACAATCTTTAGAGGAGACACAAC

[0224] SEQ ID NO:30, Stable-phase specific promoter Psod sequence

[0225] TACTCAGACGGGCTGATGACACCGATTTCCGTGGTGATTTCACCGAAACCGTGCTGTT

[0226] TCAGCGTCTCACCATAGGCATCGACCACGGCTTTTACGCGCGCAGCGTCCTCTTCGTT

[0227] CTCGGTGTAATAAGCAGAGCGGTACTGCGTTCCCACATCGTTACCCTGGCGGAAACCC

[0228] TGAGTCGGATCGTGTGCTTCCAGGCCGCGGGCAACAAGCTGCTCGAGGGAAATCTTC

[0229] GAAGGATCGTAAACAACCTCCACGATTTCAGTGTGGCCGGTGCGCCCAGAACACACC

[0230] TCTCGGTAGGTAGGGTTAGGGGTGAAACCGCCGGCGTAACCAACCGAAGTTCCCTCA

[0231] ACGCCATCCATCTGCCAATACATTTGCTCCACACCCCAGAAACAACCAAGGCCAATCC

[0232] AAATTCGCTGCTGGCCTTCTTTCCACGGACCAGTCACAGGAGTTCCCAACACAGTGT

[0233] GCGGCGCAGGATTTTCTAAAACAGGATGCCTGCCACCTTTAAGCGCCTCATCAGCGGT

[0234] AACCATCACGGGTTCGGGTGCGAAAAACCATGCCATAACAGGAATGTTCCTTTCGAA

[0235] AATTGAGGAAGCCTTATGCCCTTCAACCCTACTTAGCTGCCAATTATTCCGGGCTTGTG

[0236] ACCCACTACCCGATAAATAGGTCGGCTGAAAAATTTCGTTGCAATATCAACAAAAAGG

[0237] CCTATCATTGGGAAGTGTCGCACCAAGTACTTTTGCGAAGCGCCATCTGACGGATTTT

[0238] CAAAAGATGTATATGCTCGGTGCGGAAACCTACGAAAGGATTTTTTACCC

[0239] SEQ ID NO:31, Stable-phase specific promoter PsigB sequence

[0240] GCTCACCCGCACCGATGGTCCTCGTTGGTCCCATGATGTTGATGAGCATGTGGCTTCCA

[0241] TCGTCGCAGGACTTAACCCACATGGACTCCCCTTTGAAGAAATCCTGGAAATGTACGC

[0242] GATGGCTCAAGGTATCGAGGGAGAATCCCTGCACAACGGCGCCATTGCGGCGTTGGT

[0243] GGATCTCATCCGCCACGGATTGGTGTTGCCCGCTGATCTTCTCGATTCTTAAATAAGGA

[0244] CTGATTGTGAAAGCCGTTTTAACCCGTGTGAGTTCCGCCAGCGTCAGCGTGGATGATG

[0245] AAATTGTTGGAGCCATCGATTGCCCCGACACCGGAGGCATTTTGGCGCTGGTTGGAGT

[0246] CGGCGCTGCTGATAGCGACGACGCCTGGGAAACCATGGTGCGAAAAATTGCTGAGCT

[0247] GCGCATCTTGGATGGCGAACAATCCGTCAGTGATGTCAATGCTCCCGTACTGCTTGTTA

[0248] GCCAATTCACCCTGCATGGTCGCACCGCAAAAGGCCGGCGCCCATCGTGGTCTGATGC

[0249] AGCACCTGGTGAGGTGGCTGAGCCGGTGATTGAAAAGATTGCACAAGGTTTACGTGA

[0250] GCGCGGAATCACCGTGGAACAAGGACGATTCGGCGCAATGATGAAGGTCACATCGGT

[0251] TAACGAAGGCCCCTTCACCGTTTTGGTCGAGTGCTAGCCAGTCAATCCTAAGAGCTTG

[0252] AAACGCCCCAATGTGGGGGTGTTAAGAACTCCATAAAAGCGCTTGGGAACTTTTTGT

[0253] GGAAGCAGTCCGTTGAACCTCTTGAACCGCGAATTTAGGAGGCCAGTT

[0254] SEQ ID NO:32, Stable-phase specific promoter Peftu sequence

[0255] TACGGCTTCCTGGCTGGCTACCCACTGGTTAACGTCAAGGCAACCCTTGAAGATGGCG

[0256] CTTACCACGACGTTGACTCCTCTGAAATGGCCTTCAAGCTCGCCGGTTCCCAGGCGTT

[0257] CAAGGAAGCTGTTGCAAAGGCAAAGCCAGTCCTCCTCGAGCCAATCATGTCCGTTGA

[0258] AATCACCACTCCTGAGGAGTACATGGGTGAAGTCATCGGTGACGTGAACTCCCGCCG

[0259] TGGCCAGATCGCTTCCATGGATGACCGTGCAGGCGCCAAGCTGGTTAAGGCTAAGGTT

[0260] CCACTGTCTCAGATGTTCGGTTACGTCGGTGACCTTCGCTCTAAGACCCAGGGTCGTG

[0261] CAAACTACTCCATGGTCTTCGATTCCTACGCTGAGGTCCCAGCCAACGTTGCCGCAGA

[0262] TGTTATTGCTGAGCGCAACGGCACCGCTTCCTAAAGATCGTTTAGATCCGAAGGAAAA

[0263] CGTCGAAAAGCAATTTGCTTTTCGACGCCCCACCCCGCGCGTTTTAGCGTGTCAGTAG

[0264] GCGCGTAGGGTAAGTGGGGTAGCGGCTTGTTAGATATCTTGAAATCGGCTTTCAACAG

[0265] CATTGATTTCGATGTATTTAGCTGGCCGTTACCCTGCGAATGTCCACAGGGTAGCTGGT

[0266] AGTTTGAAAATCAACGCCGTTGCCCTTAGGATTCAGTAACTGGCACATTTTGTAATGC

[0267] GCTAGATCTGTGTGCTCAGTCTTCCAGGCTGCTTATCACAGTGAAAGCAAAACCAATT

[0268] CGTGGCTGCGAAAGTCGTAGCCACCACGAAGTCCAGGAGGACATACA

[0269] SEQ ID NO:33, Ptac-cadA sequence (Ptac sequence is underlined)

[0270] ggcaaatattctgaaatgagctgttgacaattaatcatcggctcgtataatgtgtggaa ATGAATGTAATTGCTATCCTGAA

[0271] TCATATGGGCGTATATTTTAAGGAAGAACCTATCCGCGAATTGCACCGTGCTTTGGAAC

[0272] GGCTAAATTTTCAAATTGTGTACCCCAATGACCGGGACGACCTCCTCAAGTTAATTGA

[0273] GAATAACGCCCGCCTCTGTGGGGTGATTTTCGATTGGGATAAGTATAACTTGGAGTTGT

[0274] GTGAAGAGATTAGTAAAATGAATGAAAACTTACCCTTGTATGCCTTTGCGAATACCTAC

[0275] TCCACTCTCGATGTGAGTTTAAATGATTTACGGCTACAAATCTCCTTCTTTGAATATGCG

[0276] CTGGGTGCCGCAGAAGATATTGCTAACAAAATCAAACAGACCACTGACGAGTATATTA

[0277] ACACCATTTTACCTCCTCTGACCAAGGCCTTATTTAAATACGTACGGGAAGGTAAATAC

[0278] ACCTTTTGTACCCCGGGGCATATGGGCGGTACCGCTTTTCAAAAAAGTCCTGTGGGGA

[0279] GTTTATTTTATGATTTCTTTGGTCCCAACACTATGAAATCTGATATCTCCATTAGTGTGTC

[0280] CGAATTAGGTAGCTTATTGGACCATAGCGGTCCGCATAAGGAAGCAGAACAATACATC

[0281] GCTCGCGTCTTTAATGCTGATCGTAGTTATATGGTTACAAATGGTACGAGTACTGCCAA

[0282] CAAAATTGTGGGTATGTATTCCGCGCCCGCGGGAAGTACCATTTTGATTGATCGCAATT

[0283] GCCACAAGAGTTTGACACATCTAATGATGATGTCTGACGTGACACCCATCTATTTCCGG

[0284] CCTACTCGCAACGCCTATGGGATTTTGGGTGGCATCCCTCAAAGCGAATTTCAACACG

[0285] CAACGATTGCAAAACGAGTTAAAGAAACTCCCAATGCGACCTGGCCCGTGCACGCGG

[0286] TCATCACAAATAGCACTTACGATGGCCTCCTGTATAATACCGATTTTATCAAGAAAACC

[0287] CTGGATGTCAAAAGTATCCATTTTGACTCCGCCTGGGTACCCTATACTAACTTTTCCCC

[0288] AATTTATGAAGGTAAATGTGGTATGTCTGGGGGACGTGTGGAAGGAAAAGTGATTTAT

[0289] GAAACCCAATCCACGCATAAACTGTTAGCAGCTTTTAGCCAAGCCTCTATGATTCATGT

[0290] TAAAGGTGATGTTAATGAAGAAACCTTCAATGAGGCGTACATGATGCATACTACGACC

[0291] AGCCCTCATTACGGGATTGTGGCGAGTACGGAAACAGCAGCCGCTATGATGAAGGGT

[0292] AATGCTGGAAAACGGTTGATTAATGGGTCTATTGAACGGGCTATCAAATTTCGCAAAG

[0293] AAATTAAGCGTTTGCGCACTGAAAGCGATGGCTGGTTTTTTGATGTTTGGCAACCCGA

[0294] CCATATTGATACAACGGAGTGTTGGCCTCTACGTAGTGATAGTACCTGGCATGGGTTTA

[0295] AAAATATCGATAATGAGCACATGTACCTAGACCCGATTAAGGTAACCTTATTAACACCT

[0296] GGCATGGAAAAAGATGGCACGATGTCCGACTTTGGCATTCCGGCGAGCATCGTGGCA

[0297] AAATACTTAGATGAGCACGGGATTGTGGTTGAAAAGACAGGGCCATATAACTTGCTCT

[0298] TTTTATTCAGTATCGGCATCGATAAAACTAAGGCGTTGAGCCTGCTACGGGCCCTGACT

[0299] GACTTTAAGCGGGCCTTTGATTTGAATTTGCGTGTTAAAAATATGCTCCCTTCTTTATAC

[0300] CGAGAAGATCCCGAATTTTATGAAAATATGCGGATCCAAGAATTGGCGCAGAATATCCA

[0301] TAAGTTAATCGTTCACCATAACCTACCAGATCTAATGTACCGCGCCTTTGAAGTGCTGC

[0302] CCACCATGGTAATGACCCCATATGCAGCCTTTCAAAAAGAGTTACATGGGATGACAGA

[0303] GGAGGTCTATTTAGACGAAATGGTTGGCCGCATCAATGCAAATATGATTCTGCCCTATC

[0304] CTCCAGGAGTTCCGTTAGTAATGCCTGGCGAAATGATCACTGAAGAGAGTCGCCCCGT

[0305] TCTGGAGTTTCTACAAATGCTATGCGAAATTGGTGCTCATTATCCCGGCTTCGAAACCG

[0306] ATATCCATGGCGCTTATCGGCAGGCAGATGGACGCTATACAGTGAAAGTCTTGAAGGA

[0307] AGAATCCAAAAAA。

Claims

1. A nucleic acid molecule comprising a L-lysine decarboxylase encoding gene and a stationary phase-specific promoter capable of starting the expression of the encoding gene, wherein the stationary phase-specific promoter is from Corynebacterium glutamicum.

2. The nucleic acid molecule according to claim 1, characterized in that: The L-lysine decarboxylase is CadA or LdcC from Escherichia coli, and the amino acid sequence of the L-lysine decarboxylase is preferably as shown in SEQ ID NO:

27.

3. The nucleic acid molecule according to claim 1 or 2, characterized in that: The L-lysine decarboxylase encoding gene is a cadA gene or a ldcC gene from Escherichia coli, and the L-lysine decarboxylase encoding gene is preferably as shown in SEQ ID NO: 26; and / or; The stationary phase-specific promoter is selected from any one of the promoter PgpmA shown in SEQ ID NO:28, the promoter Pgap shown in SEQ ID NO:29, the promoter Psod shown in SEQ ID NO:30, the promoter PsigB shown in SEQ ID NO:31 or the promoter Peftu shown in SEQ ID NO:

32. A recombinant vector comprising the nucleic acid molecule according to any one of claims 1 to 3.

5. The recombinant vector according to claim 4, characterized in that: The recombinant vector is an expression plasmid or an integration plasmid.

6. A recombinant Corynebacterium glutamicum, comprising the nucleic acid molecule according to any one of claims 1 to 3 and / or the recombinant vector according to claim 4 or 5.

7. The recombinant Corynebacterium glutamicum according to claim 6, characterized in that: The nucleic acid molecule according to any one of claims 1 to 3 is present in the recombinant Corynebacterium glutamicum in the form of a plasmid; or The nucleic acid molecule according to any one of claims 1 to 3 is integrated into the genome of Corynebacterium glutamicum, preferably integrated into the cg1890, cg1895 or cg2037 genomic site of Corynebacterium glutamicum.

8. A method for constructing the recombinant Corynebacterium glutamicum according to claim 6 or 7, comprising the step of transforming the expression plasmid according to claim 5 into the starting Corynebacterium glutamicum; or The step of transforming the integration plasmid according to claim 5 into the starting Corynebacterium glutamicum and performing homologous recombination with the genome of the starting Corynebacterium glutamicum; Preferably, the starting Corynebacterium glutamicum is Corynebacterium glutamicum CATH2403.

9. A method for preparing 1,5-pentanediamine, comprising the steps of fermenting and culturing the recombinant Corynebacterium glutamicum according to claim 6 or 7, and extracting and / or purifying pentamethylenediamine from the fermentation broth.

10. Use of the nucleic acid molecule according to any one of claims 1 to 3, the recombinant vector according to claim 4 or 5, or the recombinant Corynebacterium glutamicum according to claim 6 or 7 in the preparation of 1,5-pentanediamine.

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