A recombinant Halomonas for producing 1,3-propanediamine, and a construction method and application thereof

Abstract

The present invention provides a recombinant Halomonas for producing 1,3-propanediamine, and a construction method and application thereof. The recombinant Halomonas expresses 2-ketoglutaric acid 4-aminotransferase and L-2,4-diaminobutyric acid decarboxylase derived from Acinetobacter. The present invention introduces a heterologous synthesis pathway of 1,3-DAP derived from Acinetobacter baumannii, improves the yield of 1,3-DAP, enhances the efficiency of industrial production, and reduces production costs. The present invention uses fermentation to produce 1,3-DAP, which has a short time cycle, a simple production process, is environmentally friendly and pollution-free, and does not require additional addition of antibiotics and inducers, facilitating large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a recombinant Halomonas for producing 1,3-diaminopropane, a construction method thereof and an application thereof. Background Art

[0002] 1,3-Diaminopropane (1,3-DAP) is a three-carbon diamine and can be used as an important chemical with broad application potential. 1,3-DAP can be used as a cross-linking agent for epoxy resins, and epoxy resins are precursors of pharmaceuticals, agrochemicals and organic chemicals. In addition, it may also be used as a monomer for polyamides. In traditional production processes, 1,3-DAP is obtained by chemical synthesis methods using fossil fuels as raw materials, but this method has high costs, high energy consumption and unsustainable impacts on the environment. Therefore, constructing a 1,3-DAP cell factory using synthetic biology methods is in line with the current trend of green development, and is of great significance for promoting the development of bio-based chemicals, reducing dependence on limited resources and reducing environmental impacts.

[0003] 1,3-DAP can be biosynthesized through the C5 pathway. In the C5 pathway, spermidine dehydrogenase encoded by the spdH gene is the key enzyme for converting spermidine into 1,3-DAP. However, the spermidine synthase encoded by the speE gene requires S-adenosyl-3-methylthiopropylamine as a cofactor, resulting in low efficiency of the C5 pathway. In the existing microbial fermentation method for preparing 1,3-DAP, the production cycle is relatively long (80-100 h), which leads to low production intensity. The chassis strains all require a strict aseptic production environment, and the existing microbial production technologies all rely on plasmid induction expression systems, and expensive antibiotics and inducers need to be added during the production process, which greatly increases the production cost and is extremely unfavorable for large-scale industrial production. Therefore, there is an urgent need for a method for producing high-yield 1,3-DAP that can reduce the production cycle, improve production efficiency, reduce production costs and is environmentally friendly. Summary of the Invention

[0004] Aiming at the defects in the prior art, the present invention provides a recombinant Halomonas for producing 1,3-diaminopropane, a construction method thereof and an application thereof.

[0005] The present invention discloses a recombinant Halomonas for producing 1,3-diaminopropane, and the recombinant Halomonas expresses 2-ketoglutaric acid 4-aminotransferase and L-2,4-diaminobutyric acid decarboxylase derived from Acinetobacter.

[0006] Furthermore, the recombinant Halomonas also expresses the asd gene derived from Halomonas sp. TD01 and the lysC gene derived from Corynebacterium glutamicum AT1302.

[0007] Furthermore, the recombinant Halomonas also expresses the aspC gene derived from Corynebacterium glutamicum AT1302; the rocG gene derived from Bacillus subtilis 168.

[0008] Furthermore, the recombinant Halomonas also expresses the ppc gene and the pyc gene derived from Corynebacterium glutamicum AT1302.

[0009] Furthermore, the expression of the 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase uses the pSEVA321 vector.

[0010] Furthermore, the pSEVA321 vector contains a promoter, and the promoter is porin68.

[0011] Furthermore, the Halomonas includes Halomonas sp., preferably Halomonas sp. TD01.

[0012] The present invention also provides the application of the recombinant Halomonas in the production of 1,3-propanediamine.

[0013] The present invention also provides a construction method of the recombinant Halomonas, and the construction method includes introducing any one of the following groups into the recombinant Halomonas:

[0014] (1) The nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase; or

[0015] (2) The nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase and the asd-lysC nucleotide sequences; or

[0016] (3) The nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase, the asd-lysC nucleotide sequences, and the aspC-rocG nucleotide sequences; or

[0017] (4) The nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase, the asd-lysC nucleotide sequences, the aspC-rocG nucleotide sequences, and the ppc-pyc nucleotide sequences;

[0018] The nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase are shown in SEQ ID No. 61; the asd-lysC nucleotide sequence is shown in SEQ ID No. 62; the aspC-rocG nucleotide sequence is shown in SEQ ID No. 63; the ppc-pyc nucleotide sequence is shown in SEQ ID No. 64.

[0019] The present invention also provides a method for producing 1,3-propanediamine using the recombinant Halomonas, comprising the following steps: activating the strain, performing shake flask seed culture, and then performing fermentation culture in a fermentation medium;

[0020] Preferably, the temperature of the fermentation is 35-37 °C;

[0021] Preferably, the pH of the fermentation is 8.0-9.0;

[0022] Preferably, the fermentation time is 36-48 h.

[0023] In summary, compared with the prior art, the present invention has achieved the following technical effects:

[0024] (1) The engineered bacteria of the present invention can produce 1,3-DAP, with a yield of up to 4.1 g / L, improving the efficiency of industrial production and reducing production costs.

[0025] (2) The time period for producing 1,3-DAP in the present invention is short, the production process is simple, the environment is green and pollution-free, and there is no need to additionally add antibiotics and inducers, which is conducive to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0027] Figure 1 It is a pathway diagram of the 1,3-DAP synthesis pathway of the present invention;

[0028] Figure 2 It is pSEVA321 used in Example 1 of the present invention Mmp1-dat-ddc Plasmid map;

[0029] Figure 3 It is an electrophoresis diagram of the dat-ddc target fragment in Example 1 of the present invention;

[0030] Figure 4 The yield of 1,3-DAP under different inducers in Example 2 of the present invention;

[0031] Figure 5 pSEVA321 used in Example 3 of the present invention porin68-dat-ddc Plasmid map;

[0032] Figure 6 pRE112 used in Example 3 of the present invention G4-porin68-dat-ddc Plasmid map;

[0033] Figure 7 Electrophoresis map of the dat-ddc target fragment in Example 3 of the present invention;

[0034] Figure 8 pSEVA321 used in Example 4 of the present invention porin58-asd-lysC Plasmid map;

[0035] Figure 9 pRE112 used in Example 4 of the present invention G7-porin58-asd-lysC Plasmid map;

[0036] Figure 10 Electrophoresis map of the asd-lysC target fragment in Example 4 of the present invention;

[0037] Figure 11 pSEVA321 used in Example 5 of the present invention porin140-aspC-rocG Plasmid map;

[0038] Figure 12 pRE112 used in Example 5 of the present invention G43-porin140-aspC-rocG Plasmid map;

[0039] Figure 13 Electrophoresis map of the aspC-rocG target fragment in Example 5 of the present invention;

[0040] Figure 14 pSEVA321 used in Example 6 of the present invention porin226-ppc-pyc Plasmid map;

[0041] Figure 15 pSEVA321 used in Example 6 of the present invention G49-porin226-ppc-pyc Plasmid map;

[0042] Figure 16 Electrophoresis map of the ppc-pyc target fragment in the TD01-W4 strain in Example 7 of the present invention. Detailed implementation manners

[0043] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0044] Halomonas sp. TD01 itself does not have the ability to synthesize 1,3-DAP. Therefore, to achieve the synthesis of 1,3-DAP, Halomonas sp. TD01 needs to introduce an exogenous synthesis pathway additionally. The present invention introduces a heterologous synthesis pathway of 1,3-DAP from Acinetobacter baumannii, namely 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyric acid decarboxylase encoded by the dat and ddc genes. The codons of both genes have been optimized to adapt to Halomonas sp. TD01. The heterologously introduced pathway is first induced and expressed by a plasmid, achieving a breakthrough in the production of 1,3-DAP by Halomonas sp. TD01. Subsequently, the heterologous genes are integrated into the genome, and then by strengthening the genes on the pathway, Halomonas sp. TD01 can produce high-yield 1,3-DAP without relying on antibiotics and inducers.

[0045] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified. Halomonas sp. TD01 was obtained from Tsinghua University.

[0046] Example 1 Construction of the 1,3-DAP Synthesis Pathway

[0047] Plasmid construction: The dat, ddc amplified by PCR and the plasmid pSEVA321 Mmp1 skeleton, under the action of Gibson ligase, the dat, ddc fragments and the pSEVA321 Mmp1 skeleton are recombined to form a new plasmid, named pSEVA321 Mmp1-dat-ddc , and the dat-ddc fragment is obtained by PCR amplification using pSEVA321 Mmp1-dat-ddc as a template. Part of the product is sent to a biological company for sequencing, and the plasmid information is as Figure 2 shown.

[0048] (1) The primer sequences (5'-3') for amplifying dat, ddc and the pSEVA321 Mmp1 skeleton by PCR are as follows:

[0049] dat-F: See SEQ ID No.1;

[0050] dat-R: See SEQ ID No. 2;

[0051] ddc-F: See SEQ ID No. 3;

[0052] ddc-R: See SEQ ID No. 4;

[0053] pSEVA321 Mmp1-dat-ddc -F: See SEQ ID No. 5;

[0054] pSEVA321 Mmp1-dat-ddc -R: See SEQ ID No. 6.

[0055] Its amplification system and amplification procedure are shown in Table 1 and Table 2:

[0056] Table 1 Amplification System Table

[0057]

[0058] Table 2 Amplification Procedure Table

[0059]

[0060] After the PCR reaction is completed, prepare an agarose gel with the corresponding concentration, perform electrophoresis to observe the size of the DNA bands, place the gel under an ultraviolet lamp, quickly cut the gel of the target DNA fragment, and cut off as much excess gel as possible.

[0061] (2) Ligation by Gibson Assembly

[0062] Detect the concentration of the recovered DNA, then calculate the addition ratio of DNA according to the lengths and concentrations of the target fragment and the backbone, and use Gibson Mix Enzyme for ligation. The Gibson Assembly ligation system and procedure are shown in Table 3 and Table 4.

[0063] Table 3 Gibson Assembly Ligation System Table

[0064]

[0065] Table 4 Gibson Assembly Ligation Procedure

[0066]

[0067] (3) Transformation of Escherichia coli S17-1

[0068] Step 1: Take out the pre-prepared Escherichia coli S17-1 competent cells from -80 °C, thaw them on ice, and wait for the bacterial mass to melt after 5 minutes;

[0069] Step 2: Add 5 μL of the ligation product to the competent cells, and gently flick the tube wall to mix the reaction solution (do not mix by shaking). Note: The transformation volume of the ligation product should not exceed 1 / 10 of the volume of the competent cells used;

[0070] Step 3: Incubate on ice for 30 min, heat shock in a 42 °C water bath for 2 min, and immediately cool on ice for 2 min. Note: Shaking will reduce the transformation efficiency;

[0071] Step 4: Add 400 μL of LB medium (without antibiotics) to the centrifuge tube, mix well, and place in a 37 °C shaker at 200 rpm for 60 min for resuscitation;

[0072] Step 5: Centrifuge at 5000 rpm for 5 min to collect the bacteria, discard 350 μL of the supernatant, retain 100 μL, gently pipette to resuspend the bacterial pellet, and spread it on LB medium containing the corresponding antibiotic;

[0073] Step 6: Invert the medium and culture it in a 37 °C incubator for 12 - 16 h.

[0074] (4) Positive verification of monoclonal colonies

[0075] Pick colonies on the corresponding resistant LB plate and perform colony PCR verification. Send the PCR products with the correct band size to a biological company for sequencing.

[0076] (5) Select monoclonal colonies with the correct sequence for amplification. After 12 - 16 h, conjugate with Halomonas sp. TD01 on a 20LB plate. After 8 h, pick a small amount of the conjugated bacteria and spread them on a 60LB plate with the corresponding resistance. Perform monoclonal colony verification again after 36 - 48 h.

[0077] (6) Product identification

[0078] The size of the target product was verified by PCR. The results showed that the Halomonas sp. TD01 strain was successfully transformed with the dat - ddc gene sequence. As Figure 3 shown, the target fragment was 3800 bp, which was in line with the expected result. The dat - ddc gene sequence was as shown in SEQ ID No. 61 (where 1 - 1374 bp is the dat gene sequence and 1041 - 2933 bp is the ddc gene sequence).

[0079] Example 2 Recombinant bacteria shake - flask fermentation test

[0080] Use the strain constructed in Example 1 for fermentation culture.

[0081] (1) Seed solution preparation

[0082] ①Activation of bacterial strain

[0083] Streak the recombinant strain on a 60LB plate and activate it at 37°C for 24 h until monoclonal colonies grow out.

[0084] ②Primary seed culture:

[0085] Pick a single colony and inoculate it into a shaking flask containing 5 mL of seed medium (60LB + Cm). Incubate it on a shaker at 37°C and 220 rpm for 12 h.

[0086] ③Secondary seed culture:

[0087] Aspirate the primary bacteria and inoculate them into a 150 mL conical flask containing 20 mL of seed medium (60LB + Cm) at an inoculation amount of 1%. Incubate it on a shaker at 37°C and 220 rpm for 12 h.

[0088] (2) Shake flask fermentation for the production of 1,3 - DAP

[0089] (a) Medium preparation: 50MM medium system:

[0090] Bottom material: NaCl 50 g / L, yeast powder 1 g / L;

[0091] Component I: MgSO4 20 g / L, CO(NH2)2 150 g / L;

[0092] Component II: KH2PO4 175 g / L;

[0093] Component III: Mix 5 g / L Fe(III)-NH4-Citrate, 2 g / L CaCl2·2H2O and 41.7 mL of concentrated hydrochloric acid (12 mol / L) thoroughly and make up the volume to 1 L;

[0094] Component IV: ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H3BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L and NaMoO4·2H2O 0.03 g / L;

[0095] Component III & IV: Take 100 mL of Component III and 10 mL of Component IV, add 90 mL of deionized water and mix. Finally, adjust the pH value to 4.5 - 5.5 with 5M NaOH.

[0096] Carbon source (g / L): Glucose 30 g / L.

[0097] (b) 60LB seed liquid medium

[0098] 60 g / L sodium chloride, 5 g / L yeast powder, 10 g / L tryptone, 60 g / L sodium chloride, pH 8.5;

[0099] For fermentation production, a 50 mL culture system was used. It was inoculated into a 500 mL conical flask containing 50 mL of seed medium (50 mM + Cm). The concentration of inducer IPTG added to the system was changed (0, 2, 10, 20, 200 mg / L respectively), and cultured at 37 °C and 200 rpm for 48 h, then placed in a shaker at 37 °C and cultured for 48 h.

[0100] (3) After fermentation, the cells were collected for detection

[0101] (a) OD 600 Determination:

[0102] Take 1 mL of the fermented bacterial liquid, centrifuge at 12000 r / min for 10 min, discard the supernatant, add an equal volume of deionized water, resuspend the cells, and use a spectrophotometer to detect the absorbance value at a wavelength of 600 nm (if necessary, dilute the bacterial liquid to ensure that the reading of the spectrophotometer at a wavelength of 600 nm is within the range of 0.3 - 0.8).

[0103] (b) Detection of 1,3 - DAP

[0104] Take the appropriately diluted bacterial cells after breaking the cell wall, centrifuge the bacterial liquid at 12000 rpm for 10 min, take the supernatant, perform derivatization treatment, filter through a 0.22 μm microporous filter membrane, and perform HPLC analysis. The liquid phase conditions are as follows: C18 chromatographic column. The mobile phase is acetonitrile (solution A) and 20 mM sodium acetate (solution B), A:B = 70:30; injection volume 10 μL; flow rate 1 mL / min; detection wavelength 235 nm.

[0105] (4) Fermentation results

[0106] As Figure 4 shown, when the IPTG addition amount is 2 mg / L, the recombinant bacterium TD01 can produce 0.93 g / L of 1,3 - DAP.

[0107] Example 3 Replacement of the constitutive promoter

[0108] 1. Construction of the promoter replacement plasmid

[0109] Using plasmid pSEVA321 Mmp1-dat-ddc as a template for amplification to obtain the fragment dat - ddc sequence. Using plasmid pSEVA321 porin68-GFP as a template for amplification to obtain the backbone pSEVA321 porin68 , and constructing it into plasmid pSEVA321 porin68-dat-ddc, the plasmid information is as Figure 5 shown.

[0110] The primer sequences (5'-3') for amplifying the dat-ddc gene sequence and the backbone pSEVA321 porin68 by PCR are as follows:

[0111] dat-ddc-F: See SEQ ID No.7;

[0112] dat-ddc-R: See SEQ ID No.8;

[0113] pSEVA321 porin68 -F: See SEQ ID No.9;

[0114] pSEVA321 porin68 -R: See SEQ ID No.10.

[0115] 2. Construction of the inserted plasmid

[0116] Referring to the plasmid construction method in Example 1, porin68-dat-ddc was amplified using the plasmid pSEVA321 porin68-dat-ddc as a template. The backbone pRE112 was amplified using the plasmid pRE112-Backbone as a template, and the homologous arms G4L and G4R were amplified using the Halomonas sp. TD01 genome as a template to construct the plasmid pRE112 G4-porin68-dat-ddc , as Figure 6 shown.

[0117] The primer sequences (5'-3') for amplifying the porin68-dat-ddc gene sequence and the backbone pRE112 by PCR are as follows:

[0118] porin68-dat-ddc-F: See SEQ ID No.11;

[0119] porin68-dat-ddc-R: See SEQ ID No.12;

[0120] G4L-F: See SEQ ID No.13;

[0121] G4L-R: See SEQ ID No.14;

[0122] G4R-F: See SEQ ID No.15;

[0123] G4R-R: See SEQ ID No.16;

[0124] pRE112-F: See SEQ ID No.17;

[0125] pRE112-R: See SEQ ID No. 18.

[0126] 3. Gene integration

[0127] (1) Amplify the above constructed integration plasmid. After 12 - 16 h, perform conjugation with Halomonas sp. TD01 on a 20LB plate. After 8 h, pick a small amount of conjugated bacteria and spread them on a 60LB plate with the corresponding resistance. After 36 - 48 h, verify the monoclonal colonies again.

[0128] (2) After single colonies grow on the plate, perform drag resistance verification.

[0129] (3) Select the colonies with successful drag resistance for double verification (universal F1 / R and specific F2 / R). After verifying that the bands are correct by PCR cloning, the results are as Figure 7 shown. The length of the fragment amplified by the universal primer band is 5600 bp, and there is no specific primer band. Select the PCR reaction solution with successful double verification for submission for sequencing. After successful sequencing, the strain is named TD01-W1.

[0130] Example 4 Construction of the aspartate to aspartate semialdehyde pathway

[0131] 1. Construction of asd and lysC gene expression plasmids

[0132] Amplify using Halomonas sp. TD01 as a template to obtain the asd fragment; amplify using Corynebacterium glutamicum AT1302 as a template to obtain the lysC target gene fragment; amplify using plasmid pSEVA321 porin58-GFP as a template to obtain the backbone pSEVA321 porin58 . The specific implementation operations of the plasmid are the same as in Example 1 to obtain plasmid pSEVA321 porin58-asd-lysC . The plasmid information is as Figure 8 shown.

[0133] Use PCR to amplify the asd and lysC gene sequences and the backbone pSEVA321 porin58 The primer sequences (5'-3') are as follows:

[0134] asd-F: See SEQ ID No. 19;

[0135] asd-R: See SEQ ID No. 20;

[0136] lysC-F: See SEQ ID No. 21;

[0137] lysC-R: See SEQ ID No.22;

[0138] pSEVA321 porin68 -F: See SEQ ID No.23;

[0139] pSEVA321 porin68 -R: See SEQ ID No.24.

[0140] 2. Construction of asd and lysC gene integration plasmids

[0141] Using pSEVA321 porin58-asd-lysC plasmid as a template, the porin58 - asd - lysC gene fragment was amplified by PCR; using pRE112 - Backbone as a template, the pRE112 backbone was amplified by PCR; the homologous arms G7L and G7R were amplified by PCR using the Halomonassp.TD01 genome as a template to obtain the plasmid pRE112 G7-porin58-asd-lysC Plasmid map, see Figure 9 .

[0142] The primer sequences for PCR amplification are as follows (5'-3'):

[0143] porin58 - asd - lysC - F: See SEQ ID No.25;

[0144] porin58 - asd - lysC - R: See SEQ ID No.26;

[0145] G7L - F: See SEQ ID No.27;

[0146] G7L - R: See SEQ ID No.28;

[0147] G7R - F: See SEQ ID No.29;

[0148] G7R - R: See SEQ ID No.30;

[0149] pRE112 - G7 - F: See SEQ ID No.31;

[0150] pRE112 - G7 - R: See SEQ ID No.32.

[0151] 3. Integration of lysC and asd genes

[0152] The specific steps refer to Example 2. The asd - lysC gene sequence was inserted into the genome of the TD01 - W1 strain. After verification by PCR cloning that the band was correct, it indicated that the asd - lysC gene sequence was successfully inserted into the genome of the TD01 - W1 strain, asFigure 10 As shown in Figure 10 , the target fragment was 5100 bp, which was in line with the expected result. The obtained strain was named TD01-W2, and the asd-lysC gene sequence was as shown in SEQ ID No.62 (where 183-1295 bp was the asd gene sequence and 1292-2587 bp was the lysC gene).

[0153] Example 5 Construction of the Oxaloacetate-to-Aspartate Pathway

[0154] 1. Construction of Expression Plasmids for aspC and rocG Genes

[0155] Using Corynebacterium glutamicum AT1302 as a template, the aspC gene fragment was amplified by PCR; using Bacillus subtilis 168 as a template, the rocG gene fragment was amplified by PCR; using plasmid pSEVA321 porin140-GFP as a template to PCR amplify the backbone pSEVA321, obtaining plasmid pSEVA321 porin140-aspC-rocG , and the plasmid information is as Figure 11 shown.

[0156] The primer sequences (5'-3') for PCR amplification of the aspC and rocG genes are as follows:

[0157] aspC-F: See SEQ ID No.33;

[0158] aspC-R: See SEQ ID No.34;

[0159] rocG-F: See SEQ ID No.35;

[0160] rocG-R: See SEQ ID No.36;

[0161] pSEVA321 porin140-GFP -F: See SEQ ID No.37;

[0162] pSEVA321 porin140-GFP -R: See SEQ ID No.38.

[0163] 2. Construction of Integration Plasmids for aspC and rocG Genes

[0164] Using pSEVA321 porin140-aspC-rocGUsing the plasmid as a template, the porin140-aspC-rocG gene fragment was amplified by PCR; using pRE112-Backbone as a template, the pRE112 backbone was amplified by PCR; the homologous arms G43L and G43R were amplified by PCR using the Halomonassp.TD01 genome as a template to construct the pRE112 G43-porin140-aspC-rocG plasmid, and the plasmid information is as Figure 12 shown.

[0165] The primer sequences (5'-3') used for PCR amplification are as follows:

[0166] porin140-aspC-rocG-F: see SEQ ID No.39;

[0167] porin140-aspC-rocG-R: see SEQ ID No.40;

[0168] pRE112-G43-F: see SEQ ID No.41;

[0169] pRE112-G43-R: see SEQ ID No.42;

[0170] G43L-F: see SEQ ID No.43;

[0171] G43L-R: see SEQ ID No.44;

[0172] G43R-F: see SEQ ID No.45;

[0173] G43R-R: see SEQ ID No.46.

[0174] 3. Integration of aspC and rocG genes

[0175] Specific steps refer to Example 2. The porin140-aspC-rocG sequence was inserted into the genome of the TD01-W2 strain. After PCR cloning verification, the correct band was obtained, indicating that the lysC and asd genes were successfully inserted into the genome of the TD01-W2 strain. As Figure 13 shown, the target fragment was 5700bp, which was in line with the expected results. The obtained strain was named TD01-W3, and the aspC-rocG sequence was as shown in SEQID No.63 (where 177-1457bp is the aspC gene sequence and 1484-2758 is the rocG gene sequence).

[0176] Example 6 Construction of the enhanced phosphoenolpyruvate-to-aspartate and pyruvate-to-aspartate pathways

[0177] 1. Construction of ppc and pyc gene expression plasmids

[0178] Using Corynebacterium glutamicum AT1302 as a template, ppc and pyc gene fragments were amplified by PCR; using plasmid pSEVA321 porin226 as a template, the backbone pSEVA321 was amplified by PCR porin226 , and the plasmid construction steps were referred to Example 2 to obtain plasmid pSEVA321 porin226-ppc-pyc , and the plasmid information is as Figure 14 shown.

[0179] The primer sequences for PCR amplification (5'-3') are as follows:

[0180] ppc-F: See SEQ ID No.47;

[0181] ppc-R: See SEQ ID No.48;

[0182] pyc-F: See SEQ ID No.49;

[0183] pyc-R: See SEQ ID No.50;

[0184] pSEVA321 porin226 -F: See SEQ ID No.51;

[0185] pSEVA321 porin226 -R: See SEQ ID No.52.

[0186] 2. Construction of ppc and pyc gene integration plasmids

[0187] Using plasmid pSEVA321 porin226-ppc-pyc as a template, the porin226-ppc-pyc gene fragment was amplified by PCR; using pRE112-Backbone as a template, the pRE112 backbone was amplified by PCR; the homologous arms G49L and G49R were amplified by PCR using the Halomonassp.TD01 genome as a template, and plasmid pSEVA321 G49-porin226-ppc-pyc was obtained, and the plasmid information is as Figure 15 shown.

[0188] The primer sequences for PCR amplification (5'-3') are as follows:

[0189] porin226-ppc-pyc-F: See SEQ ID No.53;

[0190] porin226-ppc-pyc-R: See SEQ ID No.54;

[0191] pRE112-G49-F: See SEQ ID No.55;

[0192] pRE112-G49-R: See SEQ ID No.56;

[0193] G49L-F: See SEQ ID No.57;

[0194] G49L-R: See SEQ ID No.58;

[0195] G49R-F: See SEQ ID No.59;

[0196] G49R-R: See SEQ ID No.60.

[0197] 3. Integration of ppc and pyc genes

[0198] Refer to Example 2 for the specific steps. Insert the ppc-pyc gene sequence into the genome of TD01-W3 strain. After PCR cloning verification, the correct band indicates that the ppc and pyc genes have been successfully inserted into the genome of TD01-W3 strain. As Figure 16 shown, the target fragment is 7500bp, which is in line with the expected result. The obtained strain is named TD01-W4, and the ppc-pyc gene sequence is as shown in SEQ ID No.64 (where 177 - 2936bp is the ppc gene sequence and 2962 - 6384bp is the pyc gene sequence).

[0199] Example 7 Fermentation of Four Genetically Modified Bacteria to Produce 1,3-DAP

[0200] Use the five genetically modified bacteria constructed in Examples 3 - 5 for fermentation culture. No inducer is added to the fermentation medium. The 50MM culture system is adopted for fermentation production. Inoculate the seed liquid at 5% into a 150 mL conical flask (18 mL of bottom material, 0.4 mL of Component I, 0.4 mL of Component II, 0.4 mL of Component III & IV, 1.2 mL of 500 g / L glucose, 0.4 mL of 200 g / L urea). For the specific fermentation and detection steps, refer to Example 2.

[0201] The fermentation results are shown in Table 5.

[0202] Table 5 Fermentation Results of Four Recombinant Strains

[0203]

[0204] According to the fermentation results, strains TD01-W1, TD01-W2, TD01-W3, and TD01-W4 all successfully produced 1,3-DAP, with yields of 1.2 g / L, 2.4 g / L, 3.2 g / L, and 4.1 g / L respectively. This indicates that overexpression of exogenous genes in several modules at the chromosomal level can effectively increase the yield of 1,3-DAP. Among them, the TD01-W4 strain has a higher fermentation yield of 1,3-DAP, reaching 4.1 g / L.

[0205] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant Halomonas for producing 1,3-propanediamine, characterized in that, The recombinant Halomonas expresses the dat-ddc nucleotide sequence, the asd-lysC nucleotide sequence, and the aspC-rocG nucleotide sequence; The dat-ddc nucleotide sequence is as shown in SEQ ID NO.61, the asd-lysC nucleotide sequence is as shown in SEQ ID NO.62, and the aspC-rocG nucleotide sequence is as shown in SEQ ID NO.

63.

2. The recombinant Halomonas according to claim 1, characterized in that, The recombinant Halomonas also expresses the ppc gene and the pyc gene derived from Corynebacterium glutamicum AT1302.

3. The recombinant Halomonas according to claim 1, wherein The expression of the dat-ddc nucleotide sequence uses the pSEVA321 vector.

4. The recombinant Halomonas according to claim 3, characterized in that, The pSEVA321 vector contains a promoter, and the promoter is porin68.

5. The recombinant Halomonas according to any one of claims 1 to 4, characterized in that, The Halomonas includes Halomonas sp.

6. Use of the recombinant Halomonas according to any one of claims 1 to 5 in the production of 1,3-propanediamine.

7. The construction method of the recombinant Halomonas according to any one of claims 1 to 5, characterized in that, The construction method includes introducing any one of the following groups into the recombinant Halomonas: (1) The dat-ddc nucleotide sequence, the asd-lysC nucleotide sequence, and the aspC-rocG nucleotide sequence; or (2) The dat-ddc nucleotide sequence, the asd-lysC nucleotide sequence, the aspC-rocG nucleotide sequence, and the ppc-pyc nucleotide sequence; The ppc-pyc nucleotide sequence is as shown in SEQ ID No.

64.

8. A method for producing 1,3-propanediamine using the recombinant Halomonas described in any one of claims 1 to 5, characterized in that, It includes the following steps: activating the strain, performing shake flask seed culture, and then performing fermentation culture in a fermentation medium.

9. The method according to claim 8, wherein The temperature of the fermentation is 35 to 37 °C.

10. The method according to claim 8, characterized in that, The pH of the fermentation is 8.0 to 9.

0.

11. The method according to claim 8, wherein The time of the fermentation is 36 to 48 h.

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