CN118406625A - Recombinant halomonas for producing 1, 3-propanediamine and construction method and application thereof - Google Patents

Abstract

The present invention provides a recombinant salt mononas for producing 1,3-propylenediamine, a construction method thereof and an application thereof. The recombinant salt mononas expresses 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase from Acinetobacter baumannii. The present invention introduces a 1,3-DAP heterologous synthesis pathway from Acinetobacter baumannii, which increases the yield of 1,3-DAP, improves the efficiency of industrial production, and reduces production costs. The present invention utilizes fermentation to produce 1,3-DAP, has a short time cycle, a simple production process, and a green and pollution-free environment. At the same time, no additional antibiotics and inducers need to be added, which is conducive to industrial large-scale production.

Description

A recombinant halomonas bacteria for producing 1,3-propylenediamine and a construction method and application thereof

Technical Field

The present invention relates to the field of biotechnology, and in particular to a recombinant halomonas bacteria for producing 1,3-propylenediamine, and a construction method and application thereof.

Background technique

1,3-Diaminopropane (1,3-DAP) is a tricarbon diamine that can be used as an important chemical with wide application potential. 1,3-DAP can be used as a crosslinker for epoxy resins, which are precursors to 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 through 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, the use of synthetic biology methods to construct a 1,3-DAP cell factory is in line with the current green development trend, and it is of great significance to promote the development of bio-based chemicals, reduce dependence on limited resources, and reduce environmental impacts.

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 that converts spermidine to 1,3-DAP. However, spermidine synthase encoded by the speE gene requires S-adenosyl-3-methylthiopropylamine as a cofactor, which leads to low efficiency of the C5 pathway. The existing microbial fermentation method for preparing 1,3-DAP has a long production cycle (80 to 100 hours), which leads to low production intensity. The chassis strains all require a strict aseptic production environment. The existing microbial production technologies all rely on plasmid induction expression systems. Expensive antibiotics and inducers need to be added during the production process, which greatly increases the production cost and is not conducive to large-scale industrial production. Therefore, there is an urgent need for a method for 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

In view of the defects in the prior art, the present invention provides a recombinant Halomonas for producing 1,3-propanediamine and a construction method and application thereof.

The invention discloses a recombinant halomonas bacteria for producing 1,3-propanediamine. The recombinant halomonas bacteria express 2-oxoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase derived from Acinetobacter.

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

Furthermore, the recombinant Halomonas also expresses the aspC gene from Corynebacterium glutamicum AT1302 and the rocG gene from Bacillus subtilis 168.

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

Furthermore, the expression of the 2-oxoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase used the pSEVA321 vector.

Furthermore, the pSEVA321 vector comprises a promoter, and the promoter is porin68.

Furthermore, the Halomonas includes Halomonas sp., preferably Halomonas sp. TD01.

The present invention also provides application of the recombinant Halomonas in producing 1,3-propylenediamine.

The present invention also provides a method for constructing the recombinant Halomonas, which comprises introducing any one of the following groups into the recombinant Halomonas:

(1) a nucleotide sequence encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase; or

(2) a nucleotide sequence encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase and an asd-lysC nucleotide sequence; or

(3) a nucleotide sequence encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase, an asd-lysC nucleotide sequence, and an aspC-rocG nucleotide sequence; or

(4) nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase, asd-lysC nucleotide sequence, aspC-rocG nucleotide sequence, and ppc-pyc nucleotide sequence;

The nucleotide sequence encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase is 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; and the ppc-pyc nucleotide sequence is shown in SEQ ID No.64.

The present invention also provides a method for producing 1,3-propylenediamine 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;

Preferably, the fermentation temperature is 35-37°C;

Preferably, the pH of the fermentation is 8.0-9.0;

Preferably, the fermentation time is 36 to 48 hours.

In summary, compared with the prior art, the present invention achieves the following technical effects:

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

(2) The present invention has a short production cycle for 1,3-DAP, a simple production process, and a green and pollution-free environment. In addition, no antibiotics or inducers need to be added, which is conducive to industrial large-scale production.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

FIG1 is a 1,3-DAP synthesis pathway diagram of the present invention;

FIG2 is a map of the pSEVA321 _Mmp1-dat-ddc plasmid used in Example 1 of the present invention;

FIG3 is an electrophoresis diagram of the dat-ddc target fragment in Example 1 of the present invention;

FIG4 shows the yield of 1,3-DAP under different inducers in Example 2 of the present invention;

FIG5 is a map of the pSEVA321 _{porin68-dat-ddc} plasmid used in Example 3 of the present invention;

FIG6 is a map of the pRE112 _{G4-porin68-dat-ddc} plasmid used in Example 3 of the present invention;

FIG7 is an electrophoresis diagram of the dat-ddc target fragment in Example 3 of the present invention;

FIG8 is a map of the pSEVA321 _{porin58-asd-lysC} plasmid used in Example 4 of the present invention;

FIG9 is a map of the pRE112 _{G7-porin58-asd-lysC} plasmid used in Example 4 of the present invention;

Figure 10 is an electrophoretic diagram of the asd-lysC target fragment in Example 4 of the present invention;

Figure 11 is a map of the pSEVA321 _{porin140-aspC-rocG} plasmid used in Example 5 of the present invention;

Figure 12 is a map of the pRE112 _{G43-porin140-aspC-rocG} plasmid used in Example 5 of the present invention;

Figure 13 is an electrophoresis diagram of the aspC-rocG target fragment in Example 5 of the present invention;

Figure 14 is a plasmid map of pSEVA321 _{porin226-ppc-pyc} used in Example 6 of the present invention;

Figure 15 is a map of the pSEVA321 _{G49-porin226-ppc-pyc} plasmid used in Example 6 of the present invention;

Figure 16 is an electrophoresis diagram of the ppc-pyc target fragment in the TD01-W4 strain in Example 7 of the present invention.

Detailed ways

In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Halomonas sp.TD01 itself does not have the ability to synthesize 1,3-DAP, so in order to achieve the synthesis of 1,3-DAP, Halomonas sp.TD01 needs to introduce an additional exogenous synthesis pathway. The present invention introduces a 1,3-DAP heterologous synthesis pathway from Acinetobacter baumannii, namely, 2-oxoglutarate 4-aminotransferase and L-2,4-diaminobutyrate decarboxylase encoded by dat and ddc genes, and both genes are codon-optimized to adapt to Halomonas sp.TD01. The heterologous introduction pathway is first induced to express by a plasmid, achieving a breakthrough in the production of 1,3-DAP by Halomonas, and then the heterologous gene is integrated into the genome, and then the genes on the pathway are strengthened, so that Halomonas can produce 1,3-DAP in high yield without relying on antibiotics and inducers.

The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials and reagents used can be obtained from commercial sources unless otherwise specified. Halomonas sp. TD01 was obtained from Tsinghua University.

Example 1 Construction of 1,3-DAP Synthesis Pathway

Plasmid construction: The dat, ddc and plasmid pSEVA321 _Mmp1 backbone amplified by PCR were recombined with Gibson ligase to form a new plasmid named pSEVA321 _{Mmp1 -dat-ddc} . The dat-ddc fragment was amplified by PCR using pSEVA321 _Mmp1-dat-ddc as a template. Part of the product was sent to a biological company for sequencing. The plasmid information is shown in Figure 2.

(1) The primer sequences (5'-3') for PCR amplification of dat, ddc and the pSEVA321 _Mmp1 backbone are as follows:

dat-F: see SEQ ID No.1;

dat-R: see SEQ ID No.2;

ddc-F: see SEQ ID No. 3;

ddc-R: see SEQ ID No.4;

pSEVA321 _Mmp1-dat-ddc -F: see SEQ ID No.5;

pSEVA321 _Mmp1-dat-ddc -R: See SEQ ID No. 6.

The amplification system and amplification procedure are shown in Table 1 and Table 2:

Table 1 Amplification system

Table 2 Amplification program

After the PCR reaction is completed, prepare agarose gel of corresponding concentration and perform electrophoresis to observe the size of DNA bands. Place the gel under ultraviolet light and quickly cut out the gel of the target DNA fragment, cutting off as much excess gel as possible.

(2) Gibson Assembly method connection

The concentration of the recovered DNA was tested, and the proportion of DNA added was calculated according to the length and concentration of the target fragment and the backbone, and the Gibson mixed enzyme was used for connection. The Gibson Assembly connection system and procedure are shown in Tables 3 and 4.

Table 3 Gibson Assembly connection system

Table 4 Gibson Assembly connection procedure

(3) S17-1 Escherichia coli transformation

Step 1: Take out the prepared S17-1 E. coli competent cells from -80℃, thaw them on ice, and wait for the bacteria to melt after 5 minutes;

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

Step 3: Place on ice for 30 minutes, heat shock at 42°C for 2 minutes, and immediately place on ice for 2 minutes. Note: shaking will reduce transformation efficiency.

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

Step 5: Centrifuge at 5000 rpm for 5 min to collect the bacteria, discard 350 μL of the supernatant, keep 100 μL of the suspension, gently blow to resuspend the bacteria, and spread it on LB medium containing the corresponding antibiotics;

Step 6: Invert the culture medium into a 37°C incubator and culture for 12 to 16 hours.

(4) Monoclonal colony positive verification

Colonies were picked out on the corresponding resistance LB plates and verified by colony PCR. PCR products with the correct band size were sent to a biological company for sequencing.

(5) Select a single colony with the correct sequence for expansion. After 12 to 16 hours, combine it with Halomonas sp.TD01 in a 20LB plate. After 8 hours, pick a small amount of the combined bacteria and spread it on a 60LB plate with corresponding resistance. After 36 to 48 hours, perform another single clone colony verification.

(6) Product identification

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

Example 2 Recombinant bacteria shake flask fermentation test

The strain constructed in Example 1 was used for fermentation culture.

(1) Seed solution preparation

① Bacteria activation

The recombinant strain was streaked on a 60LB plate and activated at 37°C for 24 h until a single clone grew out.

②First-level seed cultivation:

A single colony was picked and inoculated into a shaking tube containing 5 mL of seed culture medium (60LB+Cm), and cultured in a shaking incubator at 37°C and 220 rpm for 12 h.

③Secondary seed cultivation:

The primary bacteria were aspirated and inoculated into a 150 mL conical flask containing 20 mL seed culture medium (60 LB + Cm) at a 1% inoculation rate, and then placed in a shaker at 37°C and 220 rpm for 12 h.

(2) Shake flask fermentation to produce 1,3-DAP

(a) Culture medium preparation: 50MM culture medium system:

Base material: NaCl 50g/L, yeast powder 1g/L;

Component I: MgSO ₄ 20 g/L, CO(NH ₂ ) ₂ 150 g/L;

Component II: KH ₂ PO ₄ 175 g/L;

Component III: 5 g/L Fe(III)-NH ₄ -Citrate, 2 g/L CaCl ₂ ·2H ₂ O and 41.7 mL (12 mol/L) concentrated hydrochloric acid were thoroughly mixed and the volume was fixed to 1 L;

Component IV: ZnSO ₄ ·7H ₂ O 0.1g/L, MnCl ₂ ·4H ₂ O 0.03g/L, H ₃ BO ₃ 0.3g/L, CoCl ₂ ·6H ₂ O0.2g/L, CuSO ₄ ·5H ₂ O 0.01g/L, NiCl ₂ ·6H ₂ O 0.02g/L and NaMoO ₄ ·2H ₂ O 0.03g/L;

Component III & IV: Take 100 mL of component III and 10 mL of component IV, add 90 mL of deionized water and mix, and finally adjust the pH value to 4.5-5.5 with 5M NaOH;

Carbon source (g/L): glucose 30 g/L.

(b) 60LB seed liquid culture medium

Sodium chloride 60g/L, yeast powder 5g/L, tryptone 10g/L, sodium chloride 60g/L, pH 8.5;

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

(3) After the fermentation is completed, the cells are collected for testing

(a) _OD600 determination:

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

(b) Detection of 1,3-DAP

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

(4) Fermentation results

As shown in Figure 4, when the IPTG addition amount was 2 mg/L, the recombinant bacteria TD01 could produce 0.93 g/L of 1,3-DAP.

Example 3 Replacement of constitutive promoter

1. Promoter replacement plasmid construction

The plasmid pSEVA321 _{Mmp1-dat-ddc was} used as a template for amplification to obtain the fragment dat-ddc sequence. The plasmid pSEVA321 _porin68-GFP was used as a template for amplification to obtain the backbone pSEVA321 _porin68 , and the plasmid pSEVA321 _{porin68-dat-ddc} was constructed according to the method of Example 1. The plasmid information is shown in Figure 5.

The primer sequences (5'-3') for PCR amplification of the dat-ddc gene sequence and the backbone pSEVA321 _porin68 are as follows:

dat-ddc-F: see SEQ ID No.7;

dat-ddc-R: see SEQ ID No.8;

pSEVA321 _porin68 -F: see SEQ ID No. 9;

pSEVA321 _porin68 -R: see SEQ ID No.10.

2. Construction of Insertion Plasmid

Referring to the plasmid construction method of Example 1, _{porin68-dat-ddc} was amplified using plasmid pSEVA321 porin68-dat-ddc as a template. Backbone pRE112 was amplified using plasmid pRE112-Backbone as a template, and homology arms G4L and G4R were amplified using Halomonas sp. TD01 genome as a template to construct plasmid pRE112 _{G4-porin68-dat-ddc} , as shown in FIG6 .

The primer sequences (5'-3') for PCR amplification of the porin68-dat-ddc gene sequence and the backbone pRE112 are as follows:

porin68-dat-ddc-F: see SEQ ID No.11;

porin68-dat-ddc-R: see SEQ ID No.12;

G4L-F: see SEQ ID No. 13;

G4L-R: see SEQ ID No. 14;

G4R-F: see SEQ ID No.15;

G4R-R: see SEQ ID No. 16;

pRE112-F: see SEQ ID No. 17;

pRE112-R: see SEQ ID No.18.

3. Gene integration

(1) The integrated plasmid constructed above was cultured and conjugated with Halomonas sp.TD01 in a 20LB plate after 12 to 16 hours. After 8 hours, a small amount of conjugated bacteria was selected and spread on a 60LB plate with corresponding resistance. After 36 to 48 hours, single clone colony verification was performed again.

(2) After a single colony grows on the plate, resistance verification can be performed.

(3) The colonies that successfully de-antigenized were selected for double verification (universal F1/R and specific F2/R). The bands were verified by PCR cloning. The results are shown in Figure 7. The length of the fragment amplified by the universal primer band was 5600 bp, and there was no specific primer band. The PCR reaction solution that successfully verified the double verification was selected for testing. After successful sequencing, the strain obtained was named TD01-W1.

Example 4 Construction of Aspartate to Aspartate Semialdehyde Pathway

1. Construction of asd and lysC gene expression plasmids

Halomonas sp.TD01 was used as a template for amplification to obtain the asd fragment; Corynebacterium glutamicum AT1302 was used as a template for amplification to obtain the lysC target gene fragment; plasmid pSEVA321 _porin58-GFP was used as a template for amplification to obtain the backbone pSEVA321 _porin58 . The specific implementation operation of the plasmid was the same as that in Example 1 to obtain the plasmid pSEVA321 _{porin58-asd-lysC} . The plasmid information is shown in Figure 8.

The primer sequences (5'-3') for PCR amplification of asd, lysC gene sequences and backbone pSEVA321 _porin58 are as follows:

asd-F: see SEQ ID No.19;

asd-R: see SEQ ID No.20;

lysC-F: see SEQ ID No.21;

lysC-R: see SEQ ID No.22;

pSEVA321 _porin68 -F: see SEQ ID No. 23;

pSEVA321 _porin68 -R: see SEQ ID No.24.

2. Construction of asd and lysC gene integration plasmid

Using the pSEVA321 _{porin58-asd-lysC} plasmid as a template, PCR amplification was performed to obtain the porin58-asd-lysC gene fragment; using pRE112-Backbone as a template, PCR amplification of the pRE112 backbone was performed; homology arms G7L and G7R were PCR amplified using the Halomonas p.TD01 genome as a template to obtain the plasmid pRE112 _{G7-porin58-asd-lysC} plasmid map, see Figure 9.

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

porin58-asd-lysC-F: see SEQ ID No.25;

porin58-asd-lysC-R: see SEQ ID No.26;

G7L-F: see SEQ ID No. 27;

G7L-R: see SEQ ID No. 28;

G7R-F: see SEQ ID No. 29;

G7R-R: see SEQ ID No.30;

pRE112-G7-F: see SEQ ID No.31;

pRE112-G7-R: see SEQ ID No.32.

3. lysC and asd gene integration

Specific steps refer to Example 2, the asd-lysC gene sequence is inserted into the genome of the TD01-W1 strain, and the PCR cloning verification band is correct, indicating that the asd-lysC gene sequence is successfully inserted into the genome of the TD01-W1 strain, as shown in Figure 10, the target fragment is 5100bp, which meets the expected results, and the obtained strain is named TD01-W2, and the asd-lysC gene sequence is shown in SEQ ID No.62 (wherein 183-1295bp is the asd gene sequence, and 1292-2587bp is the lysC gene).

Example 5 Construction of the oxaloacetate to aspartate pathway

1. Construction of aspC and rocG gene expression plasmids

Using Corynebacterium glutamicum AT1302 as a template, PCR amplification was performed to obtain the aspC gene fragment; using Bacillus subtilis 168 as a template, PCR amplification was performed to obtain the rocG gene fragment; using plasmid pSEVA321 _porin140-GFP as a template, PCR amplification of the backbone pSEVA321 was performed to obtain the plasmid pSEVA321 _{porin140-aspC-rocG} . The plasmid information is shown in FIG. 11 .

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

aspC-F: see SEQ ID No.33;

aspC-R: see SEQ ID No.34;

PDC-F: see SEQ ID No.35;

PDCG-R: see SEQ ID No.36;

pSEVA321 _porin140-GFP -F: see SEQ ID No. 37;

pSEVA321 _porin140-GFP -R: see SEQ ID No.38.

2. Construction of aspC and rocG gene integration plasmids

Using the pSEVA321 _{porin140-aspC-rocG} plasmid as a template, PCR amplification was performed to obtain the porin140-aspC-rocG gene fragment; using pRE112-Backbone as a template, PCR amplification of the pRE112 backbone was performed; homology arms G43L and G43R were PCR amplified using the Halomonassp.TD01 genome as a template to construct the pRE112 _{G43-porin140-aspC-rocG} plasmid. The plasmid information is shown in Figure 12.

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

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

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

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

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

G43L-F: see SEQ ID No.43;

G43L-R: see SEQ ID No.44;

G43R-F: see SEQ ID No.45;

G43R-R: see SEQ ID No.46.

3. aspC and rocG gene integration

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

Example 6 Strengthening the construction of phosphoenolpyruvate to aspartate and pyruvate to aspartate pathways

1. Construction of ppc and pyc gene expression plasmids

Using Corynebacterium glutamicum AT1302 as a template, PCR amplification was performed to obtain ppc and pyc gene fragments; using plasmid pSEVA321 _porin226 as a template, PCR amplification of the backbone pSEVA321 _porin226 was performed. The plasmid construction steps were referred to Example 2 to obtain plasmid pSEVA321 _{porin226-ppc-pyc} . The plasmid information is shown in Figure 14.

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

ppc-F: see SEQ ID No.47;

ppc-R: see SEQ ID No.48;

pyc-F: see SEQ ID No.49;

pyc-R: see SEQ ID No.50;

pSEVA321 _porin226 -F: see SEQ ID No.51;

pSEVA321 _porin226 -R: see SEQ ID No.52.

2. Construction of ppc and pyc gene integration plasmids

Using the pSEVA321 _{porin226-ppc-pyc} plasmid as a template, PCR amplification was performed to obtain the porin226-ppc-pyc gene fragment; using pRE112-Backbone as a template, PCR amplification of the pRE112 backbone was performed; homology arms G49L and G49R were PCR amplified using the Halomonas p.TD01 genome as a template to obtain the plasmid pSEVA321 _{G49-porin226-ppc-pyc} . The plasmid information is shown in Figure 15.

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

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

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

pRE112-G49-F: see SEQ ID No.55;

pRE112-G49-R: see SEQ ID No.56;

G49L-F: see SEQ ID No.57;

G49L-R: see SEQ ID No.58;

G49R-F: see SEQ ID No.59;

G49R-R: see SEQ ID No.60.

3. ppc, pyc gene integration

The specific steps refer to Example 2, and the ppc-pyc gene sequence is inserted into the genome of the TD01-W3 strain. The PCR cloning verification band is correct, indicating that the ppc and pyc genes are successfully inserted into the genome of the TD01-W3 strain. As shown in Figure 16, the target fragment is 7500bp, which is in line with the expected results. The obtained strain is named TD01-W4, and the ppc-pyc gene sequence is shown in SEQ ID No. 64 (wherein 177 to 2936bp is the ppc gene sequence, and 2962 to 6384bp is the pyc gene sequence).

Example 7 Fermentation production of 1,3-DAP by four modified bacteria

The five modified bacteria constructed in Examples 3 to 5 were used for fermentation culture. No inducer was added to the fermentation medium. The fermentation production adopted a 50MM culture system. The seed liquid was inoculated at 5% in a 150mL conical flask (base material 18mL, component I 0.4mL, component II 0.4mL, component III & IV 0.4mL, 500g / L glucose 1.2mL, 200g / L urea 0.4mL). For specific fermentation and detection steps, see Example 2.

The fermentation results are shown in Table 5.

Table 5 Fermentation results of four recombinant strains

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 several module exogenous genes at the chromosome level can effectively increase 1,3-DAP production, among which TD01-W4 strain produced a higher yield of 1,3-DAP, up to 4.1 g/L.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A recombinant halomonas for producing 1, 3-propanediamine, wherein said recombinant halomonas expresses a 2-ketoglutarate 4-aminotransferase and a L-2, 4-diaminobutyrate decarboxylase from acinetobacter.

2. The recombinant Halomonas according to claim 1, wherein said recombinant Halomonas further expresses the asd gene from Halomonas sp.td01 and the lysC gene from corynebacterium glutamicum Corynebacterium glutamicum AT 1302.

3. The recombinant halomonas according to claim 2, wherein said recombinant halomonas further expresses an aspC gene derived from corynebacterium glutamicum Corynebacterium glutamicum AT 1302; rocG gene derived from bacillus subtilis Bacillus subtilis.

4. A recombinant halomonas according to claim 3, wherein said recombinant halomonas also expresses ppc and pyc genes derived from corynebacterium glutamicum AT 1302.

5. The recombinant halomonas according to claim 1, wherein the expression of 2-ketoglutarate 4-aminotransferase and L-2, 4-diaminobutyrate decarboxylase uses a pSEVA321 vector.

6. The recombinant halomonas of claim 5, wherein said pSEVA321 vector comprises a promoter, said promoter being porin.

7. The recombinant Halomonas according to any of claims 1-6, wherein said Halomonas comprises Halomonas sp.

8. Use of the recombinant halomonas of any of claims 1-6 for the production of 1, 3-propanediamine.

9. A method of constructing a recombinant halomonas according to any one of claims 1 to 6, comprising introducing into the recombinant halomonas any one of the group:

(1) Nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2, 4-diaminobutyrate decarboxylase; or (b)

(2) Nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2, 4-diaminobutyrate decarboxylase and asd-lysC nucleotide sequences; or (b)

(3) Nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2, 4-diaminobutyrate decarboxylase, asd-lysC nucleotide sequence and aspC-rocG nucleotide sequence; or (b)

(4) Nucleotide sequences encoding 2-ketoglutarate 4-aminotransferase and L-2, 4-diaminobutyrate decarboxylase, asd-lysC nucleotide sequence, aspC-rocG nucleotide sequence and ppc-pyc nucleotide sequence;

The nucleotide sequences of the coded 2-ketoglutarate 4-aminotransferase and the coded L-2, 4-diaminobutyric acid decarboxylase are shown as SEQ ID No. 61; the asd-lysC nucleotide sequence is shown as SEQ ID No. 62; the aspC-rocG nucleotide sequence is shown as SEQ ID No. 63; the ppc-pyc nucleotide sequence is shown as SEQ ID No. 64.

10. A method for producing 1, 3-propanediamine using the recombinant halomonas of any one of claims 1-6, comprising the steps of: activating the strain, culturing shaking bottle seeds, and then fermenting and culturing in a fermentation medium;

preferably, the temperature of the fermentation is 35-37 ℃;

preferably, the pH of the fermentation is 8.0 to 9.0;

Preferably, the fermentation time is 36 to 48 hours.