Genetically engineered bacterium for synthesizing 1, 5-pentanediamine and method for synthesizing 1, 5-pentanediamine

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a genetically engineered bacterium for synthesizing 1, 5-pentanediamine and a method for synthesizing the 1, 5-pentanediamine. According to the invention, lysine decarboxylase and lysine-cadaverine antiporter are co-expressed in a basic strain of high-yield L-lysine, the obtained genetically engineered bacterium synthesizes L-lysine by using a carbon source, the L-lysine is converted into 1, 5-pentamethylene diamine under the action of the lysine decarboxylase, and the synthesized 1, 5-pentamethylene diamine is converted into 1, 5-pentamethylene diamine through the lysine-cadaverine antiporter. According to the present invention, the 1, 5-pentanediamine is effectively transferred to the outside of the cell, such that the one-step conversion from the carbon source, especially the glucose, to the 1, 5-pentanediamine is achieved, the efficient synthesis of the 1, 5-pentanediamine is achieved, the process is simple, and the production cost is low. Results of embodiments show that the concentration of 1, 5-pentanediamine synthesized by using the genetically engineered bacterium provided by the invention is 3.35-4.90 mM, and the conversion rate of glucose is close to 100%.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a genetic engineering bacterium for synthesizing 1,5-pentanediamine and a method for synthesizing 1,5-pentanediamine. Background Art

[0002] 1,5-Pentanediamine is an important core raw material for the synthesis of bio-based nylon 5X. Moreover, 1,5-Pentanediamine can be directly synthesized from biomass through microbial fermentation, which can not only reduce the nylon industry's dependence on petroleum-based raw material hexamethylenediamine, but also form the basis for the independent development of nylon 5X production technology.

[0003] At present, there are two methods for synthesizing 1,5-pentanediamine: chemical method and biological method. The chemical method has harsh conditions, poor catalyst stability, serious equipment corrosion, and environmental pollution. The biological method for synthesizing 1,5-pentanediamine includes enzyme conversion method and microbial fermentation method. The enzyme conversion method uses L-lysine hydrochloride as raw material, induces the production of lysine decarboxylase in vitro, and further catalyzes the synthesis of 1,5-pentanediamine, but the enzyme conversion process is complicated and the cost is high. The direct fermentation production method of microorganisms has low raw material cost and can directly ferment and synthesize pentamethylenediamine, but the content of 1,5-pentanediamine in the fermentation supernatant is low. Summary of the invention

[0004] The purpose of the present invention is to make up for the deficiencies of the prior art and provide a method for efficiently synthesizing 1,5-pentanediamine, which has a simple process, reduces production costs, and improves the content and conversion rate of 1,5-pentanediamine.

[0005] The present invention provides a genetically engineered bacterium for synthesizing 1,5-pentanediamine, wherein the genetically engineered bacterium comprises a basic strain and a gene expression vector introduced into the basic strain;

[0006] The gene expression vector co-expresses lysine decarboxylase and lysine-cadaverine antiporter;

[0007] The basic strains include Corynebacterium, Micrococcus or Lactobacillus strains.

[0008] Preferably, the basic strain is Corynebacterium glutamicum.

[0009] Preferably, the gene expression vector comprises a gene encoding the lysine decarboxylase and a gene encoding a lysine-cadaverine antiporter;

[0010] The gene encoding lysine decarboxylase is from Hafnia alvei, Bacillus halodurans, Bacillus cereus, Bacterium cadaveris, Burkholderia vietnamensia, Chromobacterium violaceum, Vibrio cholerae, Streptomyces polosus, Selenomonas ruminantium, Edwardsiella tarda, Salmonella typhimurium, Salmonella bongori, Serratia, Bordetella, Vibrio cholerae, unclassified Aeromonas Aeromonas and Klebsiella bacteria and lysine decarboxylase genes of mutants of the bacteria;

[0011] The gene encoding the lysine-cadaverine antiporter is derived from Escherichia coli.

[0012] Preferably, the amino acid sequence of the lysine decarboxylase is shown in SEQ ID NO.1;

[0013] The amino acid sequence of the lysine-cadaverine antiporter is shown in SEQ ID NO.2.

[0014] Preferably, the starting vector of the gene expression vector comprises a Corynebacterium glutamicum / Escherichia coli shuttle vector.

[0015] Preferably, the starting vector is a pJXM19 plasmid with chloramphenicol resistance.

[0016] The present invention also provides the use of the genetically engineered bacteria described in the above technical solution in synthesizing 1,5-pentanediamine.

[0017] The present invention also provides a method for synthesizing 1,5-pentanediamine, comprising the following steps:

[0018] The genetically engineered bacteria were inoculated into the culture medium and cultured until OD 600When the value is 0.6-0.8, the inducer is added, and the solid-liquid separation is performed after induction culture at 20-50°C for 30-48 hours, and the supernatant is collected;

[0019] The culture medium uses one or more of glucose, sucrose, fructose and lactose as carbon sources; the mass concentration of the carbon source in the culture medium is 0.5% to 1%.

[0020] Preferably, the inducing agent comprises IPTG.

[0021] Preferably, the working concentration of the inducer is 0.1-1.0 mmol / L.

[0022] Beneficial effects:

[0023] The present invention introduces lysine decarboxylase and lysine-cadaverine antiporter into a basic strain with high L-lysine production, and the obtained genetically engineered bacteria can co-express lysine decarboxylase and lysine-cadaverine antiporter. The genetically engineered bacteria provided by the present invention utilize carbon sources to synthesize L-lysine, convert L-lysine into 1,5-pentanediamine under the action of lysine decarboxylase, and effectively transport the synthesized 1,5-pentanediamine to the outside of the cell through the lysine-cadaverine antiporter, thereby realizing a one-step conversion of carbon sources, especially glucose, to 1,5-pentanediamine, with a simple process and low production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 The plasmid map of pJXM19-ldcI / cadB constructed in Example 1;

[0026] Figure 2 The results are shown in Table 3 for the detection of lysine and 1,5-pentanediamine contents in Example 3. DETAILED DESCRIPTION

[0027] The present invention provides a genetically engineered bacterium for synthesizing 1,5-pentanediamine, wherein the genetically engineered bacterium comprises a basic strain with high L-lysine production and a gene expression vector introduced into the basic strain;

[0028] The gene expression vector co-expresses lysine decarboxylase and lysine-cadaverine antiporter;

[0029] The basic strains include Corynebacterium, Micrococcus or Lactobacillus strains.

[0030] In the present invention, the basic strain is a basic strain for high-yield L-lysine, preferably Corynebacterium glutamicum, more preferably any one of Corynebacterium glutamicum GDMCC 1.497, Corynebacterium glutamicum CICC 20814, Corynebacterium glutamicum ATCC 13032 and Corynebacterium glutamicum ATCC 14067 and mutant strains thereof for high-yield L-lysine, more preferably Corynebacterium glutamicum ATCC 13032 or Corynebacterium glutamicum CICC 20814. The present invention has no strict requirements on the source of the basic strain, and conventional purchase is sufficient. The C. glutamicum CICC 20814 used in the embodiment of the present invention is purchased from the China Industrial Microbiological Culture Collection Management Center (the purchase link is http: / / www.china-cicc.org / search / ?classtype=0&keyword=CICC20814).

[0031] In the present invention, the gene expression vector comprises a gene encoding the lysine decarboxylase and a gene encoding a lysine-cadaverine antiporter. The gene encoding lysine decarboxylase of the present invention is preferably from Hafnia alvei, Bacillus halodurans, Bacillus cereus, Bacterium cadaveris, Burkholderia vietnamensia, Chromobacterium violaceum, Vibrio cholerae, Streptomyces polosus, Selenomonas ruminantium, Edwardsiella tarda, Salmonella typhimurium, Salmonella bongori, Serratia, Bordetella, Vibrio cholerae. cholerae), unclassified Aeromonas and Klebsiella bacteria and the lysine decarboxylase gene of the mutant of the bacteria, further preferably selected from the lysine decarboxylase gene of Bacterium cadaveris, Hafnia alvei or Klebsiella bacteria, more preferably selected from the lysine decarboxylase gene of Hafnia alvei bacteria. The amino acid sequence of the lysine decarboxylase of the present invention is preferably as shown in SEQ ID NO.1. The amino acid sequence of the lysine decarboxylase defined in the present invention can further convert L-lysine into 1,5-pentanediamine, thereby improving the conversion efficiency of 1,5-pentanediamine.

[0032] In the present invention, the gene encoding the lysine-cadaverine antiporter is preferably derived from Escherichia coli. The amino acid sequence of the lysine-cadaverine antiporter of the present invention is preferably as shown in SEQ ID NO.2.

[0033] The sequences shown in SEQ ID NO.1-2 are as follows:

[0034] SEQ ID NO.1:MNIIAILNHMGVYFKEEPIRELHKALEAQNFQIVYPNDREDLLKLIDNNARLCGVIFDWDTYNLDLCRDISEMNEHLPVYAFANTHSTLDVSLSDLRLNVEFFEYALGAAEDIALKIRQSTDAYVDEILPPLTKALFNYVKEGKYTFCTPGHMGGTAFQKSPVGSLFYDFYGANAMKSDISISVSELGSLLDHTGPHKEAEEYIARTFNAERSYMVTNGTSTANKIVGMYSAPAGSTIMIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPKSEFARETIEERVKNTPNATWPVHAVVTNSTYDGLFYNAEYIKKTLDVKSIHFDSAWVPYTNFSPIYKGLCGMSGDRVEGKVIYETQSTHKLLAAFSQASMIHVKGDINEETFNEAFMMHTSTSPHYGIVASIETAAAMMKGNAGKRLINGSIERAIRFRKEIKRLRSESDGWFFDVWQPEHIDEAKCWNLDPKESWHGFKDIDENHMFLDPIKVTLLTPGMKEDGTMADTGIPASIVAKYLDEHGIIVEKTGPYNLLFLFSIGIDKTKAMSLLRGLTDFKRAYDLNLRVKNMLPSLYREDPEFYENMRIQELAQGIHALIQHHNLPDLMYRAFEVLPTMVMNPHNAFQMELRGQTEEVYLEEMIGKVNANMILPYPPGVPLVMPGEMLTEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKTEQKHHHHHH*;

[0035] SEQ ID NO.2: MSSAKKIGLFACTGVVAGNMMGSGIALLPANLASIGGIAIWGWIISIIGAMSLAYVYARLATKNPQQGGPIAYAGEISPAFGFQTGVLYYHANWIGNLAIGITAVSY LSTFFPVLNDPVPAGIACIAIVWVFTFVNMLGGTWVSRLTTIGLVLVLIPVVMTAIVGWHWFDAATYAANWNTADTTDGHAIIKSILLCLWAFVGVESAAVSTGMVKNPKRTV PLATMLGTGLAGIVYIAATQVLSGMYPSSVMAASGAPFAISASTILGNWAAPLVSAFTAFACLTSLGSWMMLVGQAGVRAANDGNFPKVYGEVDSNGIPKKGLLLAAVKMTAL MILITLMNSAGGKASDLFGELTGIAVLLTMLPYFYSCVDLIRFEGVNIRNFVSLICSVLGCVFCFIALMGASSFELAGTFIVSLIILMFYARKMHERQSHSMDNHTASNAH*.

[0036] In the present invention, the starting vector of the gene expression vector is preferably a Corynebacterium glutamicum / Escherichia coli shuttle vector, and more preferably a pJXM19 plasmid with chloramphenicol resistance. The promoter of the starting vector of the gene expression vector of the present invention is preferably a tac promoter.

[0037] The present invention also preferably provides a method for constructing the genetically engineered bacteria described in the above technical solution, comprising the following steps:

[0038] inserting a lysine decarboxylase encoding gene and a lysine-cadaverine antiporter encoding gene into the coding frame of the starting vector in sequence to obtain a gene expression vector;

[0039] The gene expression vector is introduced into a basic strain with high L-lysine production to obtain a genetically engineered bacterium.

[0040] The present invention sequentially inserts the lysine decarboxylase encoding gene and the lysine-cadaverine antiporter encoding gene into the coding frame of the gene expression vector to obtain the gene expression vector. In the present invention, the starting vector preferably includes a Corynebacterium glutamicum / Escherichia coli shuttle vector, and more preferably a pJXM19 plasmid with chloramphenicol resistance; the promoter of the starting vector of the gene expression vector of the present invention is preferably a tac promoter. The pJXM19 plasmid with chloramphenicol resistance used in the embodiment of the present invention is purchased at http: / / www.miaolingbio.com / plasmid / P1020.html. The present invention has no strict requirements for the method of sequentially inserting the lysine decarboxylase encoding gene and the lysine-cadaverine antiporter encoding gene into the coding frame of the gene expression vector, and the conventional method in the art can be used, using enzyme cutting, connection or Gibson assembly.

[0041] After obtaining the gene expression vector, the present invention introduces the gene expression vector into the basic strain with high yield of L-lysine to obtain a genetically engineered bacterium. The relevant contents of the basic strain with high yield of L-lysine have been recorded above and will not be repeated here. The present invention has no strict requirements on the method of introduction, and the conventional method in the art can be used, such as electrotransformation.

[0042] The present invention co-expresses lysine decarboxylase and lysine-cadaverine antiporter in a basic strain with high L-lysine production, and the obtained genetically engineered bacteria synthesize L-lysine using a carbon source, convert L-lysine into 1,5-pentanediamine under the action of lysine decarboxylase, and effectively transport the synthesized 1,5-pentanediamine to the outside of the cell through the lysine-cadaverine antiporter, thereby realizing the one-step conversion of the carbon source to synthesize 1,5-pentanediamine. Therefore, the application of the genetically engineered bacteria in the synthesis of 1,5-pentanediamine described in the above technical solution also belongs to the protection scope of the present invention.

[0043] The present invention also preferably provides a method for synthesizing 1,5-pentanediamine, comprising the following steps:

[0044] The genetically engineered bacteria were inoculated into the culture medium and cultured until OD 600 When the value is 0.8, the inducer is added, and the solid-liquid separation is performed after induction culture at 20-50°C for 30-48 hours, and the supernatant is collected;

[0045] The culture medium uses one or more of glucose, sucrose, fructose and lactose as carbon source; the mass concentration of the carbon source is 0.5% to 1%.

[0046] In the present invention, the temperature of the induction culture is preferably 20-32°C, more preferably 30°C; the time of the induction culture is preferably 35-45h, more preferably 40h. In the embodiments of the present invention, the induction culture can preferably be carried out in a segmented culture manner, more preferably culturing at 20-30°C for 38h followed by culturing at 30-50°C for 2h, more preferably culturing at 30°C for 38h followed by culturing at 30°C for 2h. The culture temperature before adding the inducer in the present invention is preferably 30°C. The solid-liquid separation method of the present invention is preferably centrifugation at 4°C.

[0047] In the present invention, the culture medium uses one or more of glucose, sucrose, fructose and lactose as a carbon source, more preferably uses glucose and / or sucrose as a carbon source, and more preferably uses glucose as the sole carbon source. The mass concentration of the carbon source in the culture medium of the present invention is 0.5% to 1%, preferably 1%. The culture medium of the present invention preferably includes 10 g / L of peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, 5 to 10 g / L of glucose and the remainder of water.

[0048] In the present invention, the inducer preferably includes IPTG; the working concentration of the IPTG is preferably 0.1-10 mmol / L, and more preferably 0.1 mmol / L.

[0049] The method provided by the present invention can realize the one-step conversion of carbon source, especially glucose, into 1,5-pentanediamine, reduces the production cost, and has potential industrial application value. The results of the embodiment show that when glucose is used as the carbon source, the concentration of the synthesized 1,5-pentanediamine is 3.35-4.90 mM, and the conversion rate of glucose is close to 100%.

[0050] In order to further illustrate the present invention, a genetically engineered bacterium for synthesizing 1,5-pentanediamine and a method for synthesizing 1,5-pentanediamine provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The culture medium and strain culture temperature used in the examples are as follows:

[0052] LB medium (E. coli medium): peptone 10g / L; yeast powder 5g / L; sodium chloride 10g / L, solid medium with 15g / L agar powder. Sterilize at 121℃ for 20min.

[0053] LBG medium (Corynebacterium glutamicum medium): peptone 10 g / L; yeast powder 5 g / L; sodium chloride 10 g / L, glucose 5 g / L, solid culture medium added with 15 g / L agar powder, high temperature sterilization at 115°C for 20 min.

[0054] Epo medium (preparation of electro-transformed competent cells of Corynebacterium glutamicum): yeast extract 5 g / L; peptone 10 g / L; NaCl 10 g / L; glycine 25 g / L; Tween 80 1 g / L; isoniazid 4 g / L, high temperature sterilization at 115°C for 20 min.

[0055] LBHIS medium (electric transformation recovery medium for Corynebacterium glutamicum): brain heart infusion 10g / L; peptone 10g / L; yeast powder 5g / L; sodium chloride 10g / L; sorbitol 91g / L, add 15g / L agar powder to the solid culture medium, and sterilize at 115℃ for 20min.

[0056] The culture conditions of E. coli were: 37°C, 200 r / min, antibiotic concentration: chloramphenicol 50 μg / mL;

[0057] The culture conditions of Corynebacterium glutamicum are: 30°C, 200r / min, antibiotic concentration: chloramphenicol 10μg / mL.

[0058] Example 1

[0059] Construction of expression vector pJXM19-ldcI / cadB

[0060] 1. Cloning of ldcI, cadB genes and pJXM19 vector

[0061] (1) Primer design

[0062] Primers were designed using plasmid pETDuet-ldcI-cadB (disclosed in CN115125229A) as a template to amplify the ldcI and cadB genes; primers were designed using expression vector pJXM19 as a template to amplify the pJXM19 vector. The specific primer sequences designed are shown in Table 1, where the underlined parts are the positions of the homology arms.

[0063] Table 1 Primers used to construct pJXM19-ldcI / cadB expression vector

[0064] Primer name Nucleotide sequence (5'-3') ldcI-F <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> ttgcatgcctgcaggtcgac <h2 style=";text-align:left;direction:ltr"> atgaatattattgccatcctgaatca(SEQ ID NO.3)]]><h2 style=";text-align:left;direction:ltr"> ldcI-R <![CDATA[ gtacccggggatcctctaga ttaatggtgatggtgatgatgtttc(SEQ ID NO.4)]]> cadB-F <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> ccattaatctagaggatcc <h2 style=";text-align:left;direction:ltr"> atgaaatacctgctgccgacc(SEQ ID NO.5)]]><h2 style=";text-align:left;direction:ltr"> cadB-R <![CDATA[ cagccaagctgaattc tattgctcagcggtggca(SEQ ID NO.6)]]> pJXM19-F <![CDATA[ gaattcagcttggctgttttgg (SEQ ID NO.7)]]> pJXM19-R <![CDATA[ gtcgacctgcaggcatgc (SEQ ID NO.8)]]>

[0065] (2) Target gene amplification and recovery

[0066] Using the primers and template of step (1) to perform PCR amplification, respectively amplify the ldcI, cadB genes and the linearized pJXM19 vector;

[0067] Among them, the system of PCR amplification is shown in Table 2:

[0068] Table 2 PCR amplification system

[0069] Components Dosage 2×Phanta Max Master 25μL The upstream primer 2μL The downstream primer 2μL Template 1μL <![CDATA[ddH 2 The]]> To 50μL

[0070] The PCR amplification program is shown in Table 3:

[0071] Table 3 PCR amplification program

[0072]

[0073]

[0074] After PCR amplification, agarose gel electrophoresis was performed to cut out the bright bands of 2195 bp, 1496 bp, and 6606 bp, which were consistent with the ldcI gene, cadB gene, and linear pJXM19 vector, respectively, and recovered and purified according to the gel recovery kit (Tian Gen) method to obtain the target gene fragment.

[0075] (3) Connection of cloning vector

[0076] One-step cloning and ligation were performed using ClonExpress Ultra One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd.) to obtain ligation products;

[0077] The connection system is: 2×ConExpress Mix 5 μL, ldcI gene fragment 1.8 μL, cadB gene fragment 1.8 μL and linear pJXM19 vector fragment 1.4 μL.

[0078] (4) Vector transformation and screening and identification of positive clones

[0079] The ligation product of step (3) was transferred into E. coli Trelief by chemical method. TM 5α competent cells (purchased from Nanjing Novozyme Biotechnology Co., Ltd.), the entire operation was carried out on ice. After recovery, coating and culture. The colonies grown on the chloramphenicol resistance (50 mg / L) plate were picked, and sequencing primers cx-pJXM19-F (5'-gagcggataacaatttcacacagga-3', SEQ ID NO.9) and cx-pJXM19-R (5'-tgcgttctgatttaatctgtatcaggc-3', SEQ ID NO.10) were used for PCR detection and sequencing identification, and the correct recombinant plasmid was named pJXM19-ldcI / cadB, as shown in the following figure. Figure 1 shown.

[0080] Example 2

[0081] Construction of recombinant expression strain

[0082] (1) Preparation of competent cells of Corynebacterium glutamicum

[0083] Take two strains of C. glutamicum ATCC 13032 and C. glutamicum CICC 20814 stored at -80℃, streak them on LBG plate medium, pick a single clone and inoculate it in LBG medium, and rotate at 200r·min -1 , 30℃ overnight culture; transfer the overnight cultured seed solution to 50mL EPO medium, 200r·min -1 , cultured at 30℃ for about 5h, OD 600 After reaching about 0.8; 60r·min -1 , ice bath for 20 min, transfer all culture medium into 50 mL pre-cooled centrifuge tubes, and centrifuge at 4000 r·min -1 , centrifuge at 4℃ for 10min; take 30mL of pre-cooled 10% glycerol, fully suspend the bacteria, and centrifuge at 4000r·min -1 , centrifuge at 4°C for 10 min, repeat 3 times; finally, resuspend the cells with 500 μL pre-cooled 10% glycerol and dispense into centrifuge tubes, 100 μL per tube, to obtain C. glutamicum ATCC 13032 competent cells and C. glutamicum CICC 20814 competent cells.

[0084] All operations related to the bacterial solution in this step are completed in the clean bench.

[0085] (2) Construction of recombinant expression strains C. glutamicum Y-AB and C. glutamicum G-AB

[0086] Extract the recombinant plasmid pJXM19-ldcI / cadB verified correctly in Example 1. For detailed steps, see the plasmid extraction kit (the plasmid extraction kit was purchased from Tiangen). Use an ELISA reader to measure the concentration and OD of the gel recovery product. 260 / 280 , meeting the requirements of subsequent experiments.

[0087] a. Place C.glutamicum ATCC 13032 competent cells and pJXM19-ldcI / cadB plasmid to be transformed in an ice bath for 5 min respectively; take 5 μL of plasmid, with a total amount of about 300 ng, transform C.glutamicum ATCC 13032 competent cells, gently blow and mix, and place in an ice bath for 10 min; transfer to a pre-cooled 0.1 cm electric transfer cup, and shock at 1.8 KV for 5 ms; immediately add to a sterile EP tube containing 800 μL of LBHIS medium (antibiotic-free) preheated at 46°C, mix in a metal bath at 46°C for 6 min, and shake on a shaker at 30°C, 150 r·min -1 Incubate for 3 h; after incubation, 4000 r / min -1Centrifuge for 3 minutes, keep 100 μL of bacterial solution, resuspend and apply it on a plate containing chloramphenicol resistance (10 mg / L), and culture at 30°C for 48 hours to obtain transformants; the transformants were verified to be correct by primers cx-pJXM19-F and cx-pJXM19-R and named C.glutamicumY-AB.

[0088] b. Same as step a, the only difference is that the C. glutamicum ATCC 13032 competent cells are replaced with C. glutamicum CICC 20814 competent cells, and the transformants are verified by primers cx-pJXM19-F and cx-pJXM19-R to be the correct strain named C. glutamicum G-AB.

[0089] Example 3

[0090] 1. Cultivation of recombinant strains

[0091] Single clones of C. glutamicum Y-AB, C. glutamicum ATCC 13032, C. glutamicum G-AB, and C. glutamicum CICC 20814 were picked from the solid culture medium and inoculated into 5 mL of LBG medium (chloramphenicol resistance 10 mg / L) at 30°C and 200 r·min. -1 Overnight culture, 1 mL of seed solution was inoculated into 50 mL of fresh LBG medium, 30 ° C, 200 r / min -1 Culture to OD 600 The value was about 0.8, 50 μL IPTG (working concentration was 0.1 mmol / L) was added to the above bacterial solution, 30°C, 200 r·min -1 , culture for 40h;

[0092] When C.glutamicumY-AB and C.glutamicumG-AB were cultured, chloramphenicol resistance was added to the LBG medium at a concentration of 10 mg / L; when C.glutamicum ATCC 13032 and C.glutamicum CICC 20814 were cultured as monoclonal cultures, no antibiotics were added to the LBG medium.

[0093] 2. Determination of Lysine and 1,5-pentanediamine

[0094] (1) After culturing the four strains in step 1 for 40 h, the OD value of the bacterial solution was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer (METASH). The same amount of bacterial solution was taken from each sample and incubated at 4°C and 6000 r / min. -1Centrifuge for 6 minutes, take the supernatant and mix with the pre-column derivatization system according to Table 4, leave to react at room temperature for 10 minutes, transfer to a 60-80°C metal bath for 2 hours to perform a derivatization reaction, and obtain the system to be tested;

[0095] Table 4 Composition of the derivatization system and the amount of supernatant

[0096]

[0097] (2) Using high performance liquid chromatography SPD diode array, the contents of lysine and 1,5-pentanediamine in the test system were determined by binary high pressure gradient elution. The results are shown in Table 5 and Figure 2 As shown;

[0098] Wherein, mobile phase A is 100% acetonitrile; mobile phase B is 25mM sodium acetate buffer solution at pH 4.8, flow rate is 0.5mL / min; detection column is C18; column detection temperature: 40°C; injection volume: 2-10μL; wavelength: 284nm;

[0099] The elution gradient program was: 0-4 min: 20%-35% mobile phase A, 80%-65% mobile phase B;

[0100] 4-20min: 35%-55% mobile phase A, 65%-45% mobile phase B;

[0101] 20-27min: 55%-20% mobile phase A, 45%-80% mobile phase B;

[0102] 27~27.01min: 20% mobile phase A, 80% mobile phase B.

[0103] Table 5 Lysine and 1,5-pentanediamine content test results

[0104] strain Lysine content (mM) 1,5-Pentanediamine content (mM) C.glutamicumY-AB 0 3.35 C. glutamicum ATCC 13032 4.37 0 C.glutamicumG-AB 0 4.27 C. glutamicum CICC 20814 4.65 0

[0105] According to Table 5 and Figure 2It can be seen that the blank controls C.glutamicum ATCC 13032 and C.glutamicumCICC20814 produced lysine contents of 4.37 mM and 4.65 mM, respectively, without 1,5-pentanediamine production; the constructed strains C.glutamicum Y-AB and C.glutamicum G-AB produced 1,5-pentanediamine contents of 3.35 mM and 4.27 mM, respectively, without any lysine residue, indicating that the introduced ldcI gene (lysine decarboxylase gene) and cadB gene (lysine-cadaverine antiporter gene) were expressed and functioned in the cells, decarboxylating lysine to produce 1,5-pentanediamine and transporting it out of the cells, thus realizing the one-step production of 1,5-pentanediamine in Corynebacterium glutamicum using cheap glucose as a substrate.

[0106] Example 4

[0107] 1. Pick C.glutamicum Y-AB and C.glutamicum G-AB monoclones from the solid culture medium and inoculate them into 5 mL LBG medium (chloramphenicol resistance 10 mg / L) at 30°C and 200 r·min. -1 Overnight culture;

[0108] (1) Treatment 1: 1 mL of seed solution was inoculated into 50 mL of fresh LBG medium (chloramphenicol resistance 10 mg / L), 30°C, 200 r·min -1 Culture to OD 600 The value was about 0.8, 50 μL IPTG (working concentration was 0.1 mmol / L) was added to the above bacterial solution, and the mixture was incubated at 200 r·min at 20°C. -1 After 38 h of induction culture, the cells were transferred to 30 °C for 2 h;

[0109] (2) Treatment 2: Same as treatment 1, except that 50 μL IPTG was added and the mixture was heated at 200 r / min at 20 °C. -1 After 38 h of induction culture, the cells were transferred to 50 °C for 2 h;

[0110] (3) Treatment 3: Same as treatment 1, except that 50 μL IPTG was added and the mixture was incubated at 30 °C for 200 r / min. -1 After 38 h of induction culture, the cells were transferred to 30 °C for 2 h;

[0111] (4) Treatment 4: Same as treatment 1, except that 50 μL IPTG was added and the mixture was heated at 30 °C for 200 r / min. -1 After 38 h of induction culture, the cells were transferred to 50 °C for 2 h;

[0112] 2. After the treatments 1 to 4 in step 1 were completed, the contents of lysine and 1,5-pentanediamine were determined in the manner of step 2 in Example 3. The results are shown in Tables 6 to 7.

[0113] Table 6 Detection results of lysine and 1,5-pentanediamine contents in C. glutamicum Y-AB

[0114] Processing Lysine content (mM) 1,5-Pentanediamine content (mM) Treatment 1 (20℃ for 38h, then 30℃ for 2h) 1.07 1.52 Treatment 2 (20℃ for 38h, then 50℃ for 2h) 1.41 0.98 Treatment 3 (30℃ for 38h followed by 30℃ for 2h) 0 3.14 Treatment 4 (30℃ for 38h, then 50℃ for 2h) 0.98 2.69

[0115] Table 7 Detection results of lysine and 1,5-pentanediamine contents in C.glutamicum G-AB

[0116] Processing Lysine content (mM) 1,5-Pentanediamine content (mM) Treatment 1 (20℃ for 38h, then 30℃ for 2h) 2.05 2.07 Treatment 2 (20℃ for 38h, then 50℃ for 2h) 2.31 1.81 Treatment 3 (30℃ for 38h followed by 30℃ for 2h) 0 4.15 Treatment 4 (30℃ for 38h, then 50℃ for 2h) 1.36 3.58

[0117] According to Tables 6 and 7, it can be seen that compared with the induction temperature of 20°C, strains C. glutamicum Y-AB and C. glutamicum G-AB can produce more pentamethylenediamine when induced at 30°C, which indicates that 30°C is a suitable induction and reaction temperature for strains C. glutamicum Y-AB and C. glutamicum G-AB.

[0118] Example 5

[0119] Effects of different glucose concentrations in culture medium on the production of pentamethylenediamine by C.glutamicum Y-AB and C.glutamicum G-AB

[0120] Single clones of C. glutamicum Y-AB and C. glutamicum G-AB were picked from the solid culture medium and inoculated into culture tubes containing 5 mL of LB culture medium (chloramphenicol resistance 10 mg / L) and the seed solution was shaken at 30°C and 200 r·min. -1 1 mL of seed solution was inoculated into 50 mL LB medium (chloramphenicol resistance 10 mg / L) with glucose concentrations of 0.5%, 1%, 1.5%, 2%, 2.5%, 5%, and 10%, respectively, and incubated at 30°C and 200 r·min -1 Culture for 3-5h until OD 600 =0.8, add 50 μL IPTG (working concentration is 0.1 mmol / L) to the above bacterial solution, 30°C, 200 r·min -1 , culture for 40h.

[0121] After the culture was completed, the OD value of the bacterial liquid was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer (METASH), the contents of lysine and 1,5-pentanediamine were determined according to step 2 in Example 3, and the residual glucose content was detected using a high performance liquid chromatography differential refractive index detector, the mobile phase was acetonitrile: methanol: water = 75:20:5, the flow rate was 1 mL / min; the detection column was VG-504E; the column temperature was 40°C; the injection volume was 2-10 μL, and the results are shown in Tables 8-9.

[0122] Table 8 Results of glucose, lysine and 1,5-pentanediamine content in C.glutamicum Y-AB

[0123]

[0124] Table 9 Results of glucose, lysine and 1,5-pentanediamine content in C.glutamicum G-AB

[0125]

[0126]

[0127] According to Tables 8 and 9, when the glucose concentration is 0.5wt.% to 1wt.%, the production of pentamethylenediamine by C.glutamicum Y-AB and C.glutamicum G-AB is the largest, among which C.glutamicum Y-AB is 3.19 to 3.84mM, and C.glutamicum G-AB is 4.25 to 4.90mM, and there is almost no glucose left, which is consumed. When the glucose concentration exceeds 1wt.%, as the glucose concentration gradually increases, the production of pentamethylenediamine gradually decreases, which indicates that too high a glucose concentration may affect the growth of the strain, thereby affecting the production of pentamethylenediamine.

[0128] It can be seen from the above content that the genetically engineered bacteria provided by the present invention can realize the one-step conversion and synthesis of 1,5-pentanediamine using a carbon source, especially glucose, with a simple process and low production cost.

[0129] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium for synthesizing 1,5-pentanediamine, It is characterized in that The genetically engineered bacteria include a basic strain and a gene expression vector introduced into the basic strain; The gene expression vector co-expresses lysine decarboxylase and lysine-cadaverine antiporter; The basic strains include Corynebacterium, Micrococcus or Lactobacillus strains.

2. The genetically engineered bacteria according to claim 1, It is characterized in that The basic strain is Corynebacterium glutamicum.

3. The genetically engineered bacterium according to claim 1, It is characterized in that The gene expression vector comprises a gene encoding the lysine decarboxylase and a gene encoding a lysine-cadaverine antiporter; The gene encoding the lysine decarboxylase is from Hafnia alvei, Bacillus halodurans, Bacillus cereus, Bacterium cadaveris, Burkholderia vietnamensia, Chromobacterium violaceum, Vibrio cholerae, Streptomyces polosus, Selenomonas ruminantium, Edwardsiella tarda, Salmonella typhimurium, Salmonella bongori, Serratia, Bordetella, Vibrio cholerae, unclassified Aeromonas Aeromonas and Klebsiella bacteria and lysine decarboxylase genes of mutants of the bacteria; The gene encoding the lysine-cadaverine antiporter is derived from Escherichia coli.

4. The genetically engineered bacterium according to claim 1 or 3, It is characterized in that The amino acid sequence of the lysine decarboxylase is shown in SEQ ID NO.1; The amino acid sequence of the lysine-cadaverine antiporter is shown in SEQ ID NO.

2.

5. The genetically engineered bacterium according to claim 1, It is characterized in that The starting vector of the gene expression vector is a Corynebacterium glutamicum / Escherichia coli shuttle vector.

6. The construction method according to claim 5, It is characterized in that The starting vector is a pJXM19 plasmid with chloramphenicol resistance.

7. Use of the genetically engineered bacteria according to any one of claims 1 to 6 in the synthesis of 1,5-pentanediamine.

8. A method for synthesizing 1,5-pentanediamine, It is characterized in that The steps include: The genetically engineered bacteria according to any one of claims 1 to 6 are inoculated into a culture medium and cultured until OD 600 When the value is 0.6-0.8, the inducer is added, and the solid-liquid separation is performed after induction culture at 20-50°C for 30-48 hours, and the supernatant is collected; The culture medium uses one or more of glucose, sucrose, fructose and lactose as carbon sources; the mass concentration of the carbon source in the culture medium is 0.5% to 1%.

9. The method according to claim 8, It is characterized in that The inducing agent includes IPTG.

10. The method according to claim 9, It is characterized in that The working concentration of the inducer is 0.1-1.0 mmol / L.

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