Genetically engineered bacterium for producing 1, 4-butanediamine as well as construction method and application of genetically engineered bacterium

Abstract

The invention belongs to the field of metabolic engineering, and discloses a genetically engineered bacterium for producing 1, 4-butanediamine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes escherichia coli W3110 as an original strain, and is obtained by performing the following gene editing on a genome of the escherichia coli W3110, proteins such as lactose operon repressor protein, ornithine carbamyl transferase 1 and ornithine carbamyl transferase 2 are not expressed; proteins such as ornithine decarboxylase, ornithine synthetic operon, N-acetylglutamate synthetase and the like are overexpressed. The genetically engineered bacterium for producing the 1, 4-butanediamine, which is constructed by the invention, is fermented for 48 hours in a 5L fermentation tank by utilizing a double-stage dissolved oxygen control process, and the yield of the 1, 4-butanediamine can reach 39.2-55.8 g / L; in addition, the invention further provides an efficient extraction method, the total recovery rate of 1, 4-butanediamine in the fermentation liquor reaches 67.1%-78.5%, and the production efficiency and the resource utilization rate are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering and discloses a genetic engineering bacterium for producing 1,4-butanediamine and a construction method and application thereof. Background Art

[0002] 1,4-Butanediamine, also known as putrescine, has a chemical name of 1,4-diaminobutane, a molecular formula of H2N(CH2)4NH2, a molecular weight of 88.15, and is a white crystal with the smell of hexahydropyridine. 1,4-Butanediamine has important value in the production of engineering plastics, drugs, agricultural chemicals and surfactants.

[0003] At present, 1,4-butanediamine is mainly synthesized by chemical methods, but its reaction process requires expensive catalysts. At the same time, the synthesis process is highly toxic and flammable, which will have adverse effects on the human body and the environment. 1,4-Butanediamine can also be prepared by biological methods, mainly including conversion methods and direct fermentation methods. Chen Kequan et al. (CN202410321612.4) heterologously expressed cattle ( Bos taurus ) ARG1 , the gene encoding ornithine decarboxylase endogenous to Escherichia coli speC and pyridoxal kinase encoding gene pdxK The recombinant E. coli constructed uses arginine or arginine salt as substrate to catalyze the synthesis of 1,4-butanediamine. The conversion method requires L-arginine or ornithine as substrate, and L-arginine and ornithine need to be obtained by biological fermentation, so the total fermentation cycle is long and the cost is high.

[0004] Noh et al. (NOH M, YOO SM, KIM WJ, et al.Gene expression knockdown bymodulating synthetic small RNA expression in Escherichia coli [J]. Cell Systems, 2017, 5(4): 418-426.) Using Escherichia coli XQ52 as the starting strain, the glutamine synthetase gene was downregulated. glnA and ornithine carbamoyltransferase gene argFThe expression of can effectively improve the synthesis of 1,4-butanediamine. After 34 hours of fermentation in a 6.6 L fermenter, the yield of 1,4-butanediamine reached 42.3 g / L, which is the highest level of 1,4-butanediamine fermentation using Escherichia coli as the host. Although this achievement has achieved a breakthrough in the direct fermentation production of 1,4-butanediamine, its yield still has room for further improvement. At the same time, the strains of the existing transformation method and direct fermentation method all carry recombinant plasmids, so antibiotics need to be added during the fermentation process to ensure the stable inheritance of the plasmid, and inducers need to be added to transcribe the recombinant gene, which not only increases the production cost, but also inevitably causes antibiotics and inducers to remain in 1,4-butanediamine. Summary of the invention

[0005] In view of these defects, the present invention provides a genetically engineered bacterium for producing 1,4-butanediamine, and a construction method and application thereof. The genetically engineered bacterium for producing 1,4-butanediamine provided by the present invention does not carry a plasmid, does not require the addition of an inducer, and avoids the risk of plasmid loss or instability. The output of 1,4-butanediamine in a 5 L fermenter can reach 55.8 g / L, and no other organic acids are detected during the entire fermentation process. The metabolic pathway is highly specific, and glucose can be efficiently converted into 1,4-butanediamine. At the same time, the present invention also provides 1,4-butanediamine produced by the strain and a method for extracting 1,4-butanediamine from the fermentation broth. By optimizing the fermentation conditions and the extraction process, the yield is further increased and the cost is reduced, providing an efficient, stable and economically feasible solution for the biological industrial production of 1,4-butanediamine, and strongly promoting the industrial application of 1,4-butanediamine biosynthesis technology.

[0006] To achieve the above object, the present invention provides the following technical solutions: In the first aspect, the present invention provides a genetically engineered bacterium for producing 1,4-butanediamine, wherein the genetically engineered bacterium is obtained by using Escherichia coli W3110 as a starting strain and performing the following gene editing on its genome: lactose operon repressor protein, ornithine carbamoyltransferase 1, ornithine carbamoyltransferase 2, arginine repressor protein, acetate kinase, pyruvate dehydrogenase and lactate dehydrogenase are not expressed; ornithine decarboxylase, ornithine synthesis operon, N-acetylglutamate synthetase, phosphoketolase, citrate synthase are overexpressed 、 Isocitrate dehydrogenase, glutamate dehydrogenase, and putrescine transporter.

[0007] The "non-expression" in the present invention means that the amount of the protein or protease is significantly lower than the original level, for example, significantly reduced by at least 50%, 60%, 70%, 80%, 90%, 100%. Gene editing on the genome of the starting strain to make the protein or protease non-expressed can be achieved by conventional means in the art, including but not limited to gene knockout, gene knock-in, point mutation, deletion mutation and their combination.

[0008] The present invention E. coli W3110 was used as the starting strain. By using systemic metabolic engineering methods, a recombinant strain producing 1,4-butanediamine was constructed by increasing the supply of acetyl-CoA and reducing power, strengthening the synthesis flux of 1,4-butanediamine, enhancing its efflux system, and strengthening the TCA cycle. Specifically, the gene encoding phosphoketolase was overexpressed and the gene encoding acetate kinase was knocked out. 、 The genes encoding pyruvate dehydrogenase and lactate dehydrogenase can be used to increase the supply of acetyl-CoA and reducing power; the genes encoding citrate synthase and isocitrate dehydrogenase can be used to strengthen the TCA cycle and increase the accumulation of α-ketoglutarate; the genes encoding glutamate dehydrogenase can be used to increase the supply of acetyl-CoA and reducing power. 、 The coding genes of N-acetylglutamate synthetase, ornithine synthesis operon and ornithine decarboxylase were knocked out, and the coding genes of ornithine carbamoyltransferase 1 and ornithine carbamoyltransferase 2 were knocked out to increase the accumulation of L-glutamate, the precursor of 1,4-butanediamine, strengthen the synthesis pathway of L-glutamate-L-ornithine-1,4-butanediamine, and reduce the branch consumption of L-ornithine; the 1,4-butanediamine transport system was strengthened by overexpressing the coding gene of putrescine transporter, and the transport of 1,4-butanediamine to the extracellular space was promoted; the coding gene of lactose operon repressor protein was knocked out to release the lacI Repressor protein on promoter P trc By knocking out the gene encoding the arginine repressor protein, the argC Gene, argB Gene, argG Genes and carA The gene repression is to avoid affecting the production of 1,4-butanediamine. E. coli The above gene editing was performed on the W3110 genome, and a genetically engineered E. coli strain that produces 1,4-butanediamine was successfully constructed. The experimental results showed that the strain's ability to produce 1,4-butanediamine was significantly improved. After 48 hours of staged fermentation in a 5 L fermenter, the 1,4-butanediamine production reached 55.8 g / L, and no other organic acids were detected during the entire fermentation process.

[0009] In the present invention, the lactose operon repressor protein has Protein ID: BAE76127.1 in NCBI; ornithine carbamoyltransferase 1 has Protein ID: BAE78251.1 in NCBI; ornithine carbamoyltransferase 2 has Protein ID: BAE76057.1 in NCBI; arginine repressor protein has Protein ID: BAE77280.1 in NCBI; acetate kinase has Protein ID: BAA16135.1 in NCBI; pyruvate dehydrogenase has Protein ID: BAA35585.1 in NCBI; and lactate dehydrogenase has Protein ID: BAA14990.1 in NCBI.

[0010] The "overexpression" described in the present invention means that the amount of the protease or transporter is significantly higher than the original level. Gene editing is performed on the genome of the starting strain, and the method of overexpressing the protease or transporter can be achieved by conventional means in the art. It can be achieved by replacing the promoter of the corresponding gene with a strong promoter on the genome of the starting strain, or by inserting the corresponding gene into the genome, increasing the gene copy number, or a combination thereof. When selecting a promoter, a reasonable selection can be made based on the specific expression level requirements and the characteristics of the target gene.

[0011] Preferably, the ornithine decarboxylase, ornithine biosynthesis operon, N-acetylglutamate synthetase, phosphoketolase, citrate synthase 、 At least one of the genes encoding isocitrate dehydrogenase, glutamate dehydrogenase and putrescine transporter uses a promoter of P trc Promoter or P lpp promoter; the P trc The nucleotide sequence of the promoter is shown in the sequence table SEQ ID NO.1. lpp The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.2.

[0012] Preferably, the nucleotide sequence of the ornithine decarboxylase encoding gene is shown as SEQ ID NO.3.

[0013] Ornithine decarboxylase (ODC) is a key rate-limiting enzyme in the synthesis of 1,4-butanediamine. Overexpression of ornithine decarboxylase can increase the activity level of ODC, thereby increasing the yield of 1,4-butanediamine. The ornithine decarboxylase encoding gene can be derived from Enterobacter cloacae ( Enterobacter cloacae ) Ornithine decarboxylase encoding gene from ATCC 13047 speF .

[0014] Preferably, the nucleotide sequence of the gene encoding the ornithine biosynthesis operon is shown in SEQ ID NO.4-6.

[0015] Overexpression of the ornithine synthesis operon can promote the conversion of L-glutamate into L-ornithine. The ornithine synthesis operon encoding gene can be derived from Corynebacterium glutamicum (Corynebacterium glutamicum) Ornithine biosynthesis operon of ATCC13032 argJBD .

[0016] Preferably, the nucleotide sequence of the N-acetylglutamate synthetase encoding gene is shown as SEQ ID NO.7.

[0017] Overexpression of N-acetylglutamate synthetase can promote the conversion of L-glutamate to L-ornithine. The N-acetylglutamate synthetase encoding gene can be derived from the N-acetylglutamate synthetase encoding gene of Escherichia coli W3110. argA .

[0018] Preferably, the nucleotide sequence of the phosphoketolase encoding gene is shown as SEQ ID NO.8.

[0019] Phosphoketolase can catalyze the key step in the non-oxidative glycolysis pathway to increase the accumulation of acetyl-CoA. The phosphoketolase encoding gene can be derived from Bifidobacterium adolescentis (Bifidobacterium adolescentis) The gene encoding phosphoketolase of ATCC15703 fxpK .

[0020] Preferably, the nucleotide sequence of the citrate synthase encoding gene is shown in SEQ ID NO.9.

[0021] Citrate synthase (CS) is the first enzyme in the TCA cycle and participates in the initial reaction of the TCA cycle. Overexpression of CS can enhance the carbon flow into the TCA cycle and promote the synthesis of L-glutamate. The citrate synthase encoding gene can be derived from the citrate synthase encoding gene of Escherichia coli W3110. gltA .

[0022] Preferably, the nucleotide sequence of the isocitrate dehydrogenase encoding gene is shown as SEQ ID NO.10.

[0023] Isocitrate dehydrogenase (ICDH) can catalyze the reversible reaction of isocitrate oxidative decarboxylation to form α-ketoglutarate, promote the TCA cycle by catalyzing the key oxidative decarboxylation reaction, and produce the important energy molecule NADPH. The isocitrate dehydrogenase encoding gene can be derived from Escherichia coli W3110. icd .

[0024] Preferably, the nucleotide sequence of the glutamate dehydrogenase encoding gene is shown as SEQ ID NO.11.

[0025] Glutamate dehydrogenase can convert α-ketoglutarate into L-glutamate. The coding gene of the glutamate dehydrogenase can be derived from Bacillus subtilis. (Bacillus subtilis) 168 Glutamate dehydrogenase encoding gene rocG .

[0026] Preferably, the nucleotide sequence of the putrescine transporter encoding gene is one of the following nucleotide sequences: the nucleotide sequence is shown in SEQ ID NO.12; the nucleotide sequence is shown in SEQ ID NO.13; the nucleotide sequence is shown in SEQ ID NO.14; and the nucleotide sequence is shown in SEQ ID NO.15.

[0027] Putrescine transporter PotE acts as an antiporter that can absorb and excrete putrescine and take in ornithine.

[0028] A putrescine transporter encoding gene from Escherichia coli W3110 can be used potE , whose nucleotide sequence is shown in SEQ ID NO.12; or using the mythical pig host-associated Lactobacillus ( Ligilactobacillus saerimneri Putrescine transporter encoding gene from potE , whose nucleotide sequence is shown in SEQ ID NO.13; or using Pasteurella multocida ( Pasteurella multocida Putrescine transporter encoding gene from potE , whose nucleotide sequence is shown in SEQ ID NO.14; or using Pseudomonas auriculatus ( Pseudomonas plecoglossicida Putrescine transporter encoding gene from potE , whose nucleotide sequence is shown in SEQ ID NO.15.

[0029] More preferably, the nucleotide sequence of the putrescine transporter encoding gene is as shown in SEQ ID NO.12, and the promoter used for overexpression of the putrescine transporter encoding gene is P lpp Promoter.

[0030] In a second aspect, the present invention also provides a method for constructing a genetically engineered bacterium for producing 1,4-butanediamine, comprising the following steps: using Escherichia coli W3110 as a starting strain, knocking out the lactose operon repressor protein encoding gene, ornithine carbamoyltransferase 1 encoding gene, ornithine carbamoyltransferase 2 encoding gene, arginine repressor protein encoding gene, acetate kinase encoding gene, pyruvate dehydrogenase encoding gene and lactate dehydrogenase encoding gene in its genome; integrating the ornithine decarboxylase encoding gene, ornithine synthesis operator gene, N-acetylglutamate synthetase encoding gene, phosphoketolase encoding gene, citrate synthase encoding gene 、 Isocitrate dehydrogenase encoding gene, glutamate dehydrogenase encoding gene and putrescine transporter encoding gene; the order of editing the above genes is not important. After the above gene editing, a genetically engineered bacterium that produces 1,4-butanediamine is finally obtained.

[0031] The present invention utilizes gene editing technology to overexpress and knock out the target gene of the starting strain, thereby constructing a genetically engineered bacterium that produces 1,4-butanediamine. In this process, the specific sequence of gene editing can be flexibly adjusted according to experimental requirements, and different editing sequences can achieve the expected genetic engineering goals without affecting the performance of the genetically engineered bacteria and the yield of 1,4-butanediamine that are finally constructed.

[0032] When integrating the above genes, the gene insertion site can be reasonably selected based on the specific operational requirements and the characteristics of the target gene, taking into account factors such as PAM sequence, genomic environment, functional requirements and off-target risks.

[0033] Preferably, the ornithine decarboxylase encoding gene insertion site is yjiP .

[0034] Preferably, the ornithine synthesis operator insertion site is ycgH .

[0035] Preferably, the N-acetylglutamate synthetase encoding gene insertion site is yciQ .

[0036] Preferably, the phosphoketolase encoding gene insertion site is yghX .

[0037] Preferably, the citrate synthase encoding gene insertion site is yjiT .

[0038] Preferably, the isocitrate dehydrogenase encoding gene insertion site is lfhA .

[0039] Preferably, the glutamate dehydrogenase encoding gene insertion site is ygaY .

[0040] Preferably, the putrescine transporter encoding gene insertion site is yjgX .

[0041] Specifically, a method for constructing a genetically engineered bacterium for producing 1,4-butanediamine is provided, comprising the following steps: (1) Using the Escherichia coli W3110 genome as a template, amplify the gene encoding the lactose operon repressor protein lacI The upstream homology arm and the downstream homology arm of lacI The upstream homology arm and the downstream homology arm are fused to obtain lacI The upstream homology arm and the downstream homology arm overlap fragments; lacI The upstream homology arm and the downstream homology arm overlap fragments and contain lacI -gRNA vector was transformed into the starting strain Escherichia coli W3110 to obtain knockout lacI Recombinant strains E. coli W3110 ∆lacI ; (2) Using the wild-type Escherichia coli W3110 genome as a template, amplify the gene yjiP The upstream homology arm and downstream homology arm of the ornithine decarboxylase were amplified by PCR. speF gene fragment; yjiP The upstream homology arm, the downstream homology arm and the speF Gene fragment fusion, speF The gene knock-in fragment speF Gene knock-in fragments and yjiP -gRNA vector transformation E. coli W3110 ∆lacI strain, gene knocked out yjiP and in yjiP Ornithine decarboxylase gene speF Recombinant strains of (3) Based on the recombinant strain obtained in step (2), the same method as step (1) was used to construct a knockout strain encoding ornithine carbamoyltransferase 1. argI and ornithine carbamoyltransferase 2 encoding gene argF Recombinant strains of (4) Based on the recombinant strain obtained in step (3), the same method as step (2) was used to construct ycgH Ornithine biosynthesis operon argJBD Recombinant strains of (5) Based on the recombinant strain obtained in step (4), the same method as step (1) was used to construct a knockout arginine repressor protein encoding gene argR Recombinant strains of (6) Based on the recombinant strain obtained in step (5), the same method as step (2) is used and repeated, with the recombinant strain obtained in the previous round as the construction basis, and the recombinant strains obtained in step (5) are constructed in sequence. yciQ N-acetylglutamate synthetase encoding gene argA The recombinant strains yghX phosphoketolase encoding gene fxpK The recombinant strains yjiT citrate synthase encoding gene gltA The recombinant strains lfhA Isocitrate dehydrogenase gene icd The recombinant strains and ygaY Glutamate dehydrogenase gene rocG Recombinant strains of (7) In step (6), the ygaY Glutamate dehydrogenase gene rocG Based on the recombinant strain, the same method as step (1) was repeated to construct a knockout acetate kinase encoding gene. ackA , pyruvate dehydrogenase encoding gene poxB and lactate dehydrogenase encoding genes ldhA Recombinant strains of (8) Based on the recombinant strain obtained in step (7), the same method as in step (2) was used to yjgX Putrescine transporter encoding gene potE , a genetically engineered bacterium for producing 1,4-butanediamine was constructed.

[0042] The present invention " lacI -gRNA" indicates that lacI The sequence-designed and synthesized RNA molecule that guides the Cas9 nuclease to recognize the target DNA sequence can be a combination of natural crRNA and tracrRNA, or it can be an engineered single guide RNA (sgRNA); the description of the gRNA of other genes is similar.

[0043] Furthermore, the vector is a pGRB plasmid.

[0044] Preferably, the nucleotide sequence of the ornithine decarboxylase encoding gene is shown in SEQ ID NO.3; the nucleotide sequence of the ornithine synthesis operator is shown in SEQ ID NO.4-6; the nucleotide sequence of the N-acetylglutamate synthetase encoding gene is shown in SEQ ID NO.7; the nucleotide sequence of the phosphoketolase encoding gene is shown in SEQ ID NO.8; the nucleotide sequence of the citrate synthase encoding gene is shown in SEQ ID NO.9; the nucleotide sequence of the isocitrate dehydrogenase encoding gene is shown in SEQ ID NO.10; the nucleotide sequence of the glutamate dehydrogenase encoding gene is shown in SEQ ID NO.11; the nucleotide sequence of the putrescine transporter encoding gene is shown in SEQ ID NO.12, and the promoter used for overexpression of the putrescine transporter encoding gene is P lpp Promoter.

[0045] In a third aspect, the present invention also provides the use of the above-mentioned genetically engineered bacteria for producing 1,4-butanediamine in the fermentation production of 1,4-butanediamine.

[0046] In a fourth aspect, the present invention also provides a method for producing 1,4-butanediamine by fermentation, using the genetically engineered bacteria or the genetically engineered bacteria obtained by the above-mentioned construction method, and fermenting with glucose or a culture medium containing glucose as a substrate.

[0047] Preferably, the method comprises the following steps: (1) inoculating the activated genetically engineered bacteria onto a seed culture medium to perform seed culture to obtain a seed culture; (2) inoculating the seed culture obtained in step (1) onto a fermentation medium at an inoculation rate of 5% to 10%, performing aerobic fermentation, feeding a glucose solution, maintaining the residual sugar concentration in the fermentation broth, and obtaining a fermentation broth containing 1,4-butanediamine; The seed culture medium comprises 30 g / L glucose, 5 ml / L corn steep liquor, 2 g / L peptone, 2 g / L arginine, 2 g / L KH2PO4, 1.5 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, 1.3 mg / L B1 , 0.3 mg / LV H ; pH 7.0-7.2; The fermentation medium comprises: 10 g / L glucose, 3 ml / L corn steep liquor, 1 g / L glutamic acid, 2 g / L arginine, 3 g / L KH2PO4, 1.8 g / L MgSO4·7H2O, 2 g / L sodium citrate dihydrate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, 2 mg / L B1 , 0.2 mg / LV H ; pH is 7.0-7.2.

[0048] By using the genetically engineered bacteria for producing 1,4-butanediamine and the method for producing 1,4-butanediamine by fermentation provided by the present invention, the yield of 1,4-butanediamine reaches 39.2-55.8 g / L after 48 h of fermentation in a 5 L fermenter.

[0049] In the seed fermentation of step (1) and the aerobic fermentation of step (2) of the present invention, the dissolved oxygen level in the fermentation liquid is controlled to be not less than 20%, preferably 20%-45%. The aerobic fermentation process in step (2) can be divided into different fermentation stages according to the production and metabolic process of the recombinant strain, and the fermentation temperature and dissolved oxygen level can be accurately controlled and adjusted according to the specific needs of each stage to optimize the fermentation conditions and improve production efficiency and product quality. The dissolved oxygen level control can be controlled by parameters such as stirring speed, ventilation volume, tank pressure, etc.

[0050] More preferably, in step (1), the stirring speed is 200-800 rpm, and the ventilation volume is 2-4 m 3 / h, culture at 37℃ for 10 h.

[0051] More preferably, the fermentation cycle of the aerobic fermentation in step (2) is 48 h, and the pH of the fermentation liquid is maintained at 7.0-7.5 and the residual sugar concentration is maintained at 0.1%-0.5% during the fermentation process; the aerobic fermentation is divided into two stages, in the first stage, the fermentation temperature is set at 32-37°C, and the dissolved oxygen level in the fermentation liquid is maintained at 20%-30%; in the second stage, the fermentation temperature is set at 32-37°C, and the dissolved oxygen level in the fermentation liquid is maintained at 30%-45%.

[0052] More preferably, in step (2), the seed culture obtained in step (1) is inoculated with 8% of the seed culture into a fermentation tank filled with fermentation medium, the fermentation cycle is 48 hours, and the pH of the fermentation liquid is maintained at 7.0-7.5 and the residual sugar concentration is maintained at 0.1%-0.5% during the fermentation process; the aerobic fermentation is divided into two stages, the first stage is set at a fermentation temperature of 37°C, a stirring speed of 300-900 rpm, and a ventilation volume of 2-4 m 3 / h, the dissolved oxygen level in the fermentation liquid was maintained at 20%-30%, and the fermentation time was 16 h; in the second stage, the fermentation temperature was set at 32°C, the stirring speed was 400-900 rpm, and the ventilation volume was 3-5 m 3 / h, the dissolved oxygen level in the fermentation broth is maintained at 30%-45%.

[0053] Preferably, the method further comprises extracting 1,4-butanediamine from the fermentation broth obtained after fermentation, comprising the following steps: (1) Membrane filtration: centrifuging the fermentation broth to collect the supernatant, and filtering the supernatant with an ultrafiltration membrane and then a nanofiltration membrane to obtain a 1,4-butanediamine salt solution; (2) Alkalization: adding sodium hydroxide or potassium hydroxide to the 1,4-butanediamine salt solution obtained in step (1) for alkalization, stirring to carry out desalination reaction, and obtaining a 1,4-butanediamine aqueous solution after the desalination reaction is complete; (3) Extraction and distillation: n-butanol is added to the aqueous solution of 1,4-butanediamine obtained in step (2), and the mixture is stirred for extraction. After the extraction is completed, the mixture is allowed to stand and the layers are separated. The n-butanol phase extract is subjected to reduced pressure distillation to collect 1,4-butanediamine.

[0054] The fermentation liquid membrane filtration-alkalization-extraction distillation process provided by the present invention realizes the efficient recovery of 1,4-butanediamine, and the total recovery rate of 1,4-butanediamine reaches 67.1%-78.5%.

[0055] More preferably, in step (1), the fermentation broth is centrifuged at 4°C and 10,000 rpm for 10 min to collect the supernatant; and the supernatant is filtered through an ultrafiltration membrane with a molecular weight cutoff of 10 KDa and a nanofiltration membrane with a molecular weight cutoff of 300 Da.

[0056] More preferably, in step (2), sodium hydroxide or potassium hydroxide is added to the 1,4-butanediamine salt solution to make the pH of the 1,4-butanediamine salt solution reach 7.0-10.0, and the solution is alkalized at 70° C. and 100 rpm, and stirred for 5 h to carry out the desalting reaction.

[0057] More preferably, in step (3), n-butanol is added to the 1,4-butanediamine aqueous solution, the volume of n-butanol being 3 times the volume of the 1,4-butanediamine aqueous solution, and the mixture is stirred and extracted at 50-70° C. After the extraction is completed, the mixture is allowed to stand and separate into layers; the n-butanol phase extract is subjected to reduced pressure distillation at a pressure of -0.098 MPa and a temperature of 50° C. to collect 1,4-butanediamine.

[0058] More preferably, in step (2), the pH of the 1,4-butanediamine salt solution is adjusted to 9.0; and in step (3), the extraction is carried out under stirring at 70°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0060] Figure 1 The schematic diagram of the biosynthetic pathway of 1,4-butanediamine in Escherichia coli W3110 and the genetic engineering modification involved in the present invention; Figure 2 This is the process curve of the recombinant strain DIA-18 in Example 10 producing 1,4-butanediamine in a 5 L fermenter. DETAILED DESCRIPTION

[0061] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] The Escherichia coli W3110, Bacillus subtilis 168, Corynebacterium glutamicum ATCC13032, Bifidobacterium adolescentis ATCC 15703, Enterobacter cloacae ATCC 130473, Mythopodiella porcine host-associated Lactobacillus ATCC33222, Pasteurella multocida ATCC 12948 and Pseudomonas aeruginosa ATCC 700383 used in the examples of the present invention are all from the Metabolic Engineering Laboratory of Tianjin University of Science and Technology.

[0063] The recombination kit ClonExpress II One Step Cloning Kit was purchased from Nanjing Novogene Medical Technology Co., Ltd. The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0064] The detection method of 1,4-butanediamine and other organic acids in the present invention is: The fermentation broth was centrifuged at 12000×g for 3 min and the supernatant was collected for detection. Dansyl chloride (5 g·L -1, dissolved in acetone) to derivatize the fermentation broth, and the content of 1,4-butanediamine and other organic acids was determined by high performance liquid chromatography. The detection conditions were: Agilent C18 (150 mm×4.6 mm, 5 μm), injection volume 20 μL, mobile phase A was ultrapure water, mobile phase B was chromatographic grade acetonitrile, binary gradient elution was used (gradient elution program was set to 0-4 min, 55%-70%B; 4-6 min, 70%B; 6-11 min, 70%-95%B; 11-12 min, 95%B; 12-13 min, 95%-55%B; 13-16 min, 55%B), flow rate 0.7mL·min -1 , column temperature 33°C, detection wavelength 254 nm.

[0065] The gene sequences involved in the embodiments of the present invention can be encoded and synthesized according to the sequence information corresponding to the Protein ID number in NCBI provided in the above-mentioned Summary of the Invention section, or amplified using the microbial genome of the corresponding source as a template. The primers used in the embodiments of the present invention are shown in Table 1: Table 1 Primer sequences used in the examples of the present invention

[0066] The culture medium used in Examples 7-10 of the present invention is: Seed culture medium composition: 30 g / L glucose, 5 ml / L corn steep liquor, 2 g / L peptone, 2 g / L arginine, 2 g / L KH2PO4, 1.5 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, 1.3 mg / L B1 , 0.3 mg / LV H ; pH 7.0-7.2.

[0067] Fermentation medium composition: 10 g / L glucose, 3 ml / L corn steep liquor, 1 g / L glutamic acid, 2 g / L arginine, 3 g / L KH2PO4, 1.8 g / L MgSO4·7H2O, 2 g / L sodium citrate dihydrate, 10 mg / L FeSO4·7H2O, 10 mg / LMnSO4·7H2O, 2 mg / LV B1 , 0.2 mg / LV H ; pH 7.0-7.2.

[0068] Example 1 This embodiment provides a genetically engineered bacterium for producing 1,4-butanediamine, and the construction method thereof comprises the following steps: (1) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers lac-1 / lac-2 and lac-3 / lac-4 were used to amplify lacI The upstream homology arm and downstream homology arm of the gene were recovered and overlapped PCR was performed to obtain lacI Fusion fragment U of the upstream homology arm and the downstream homology arm lacI -D lacI .

[0069] (2) Based on lacI Sequence design and synthesis of gRNA 20 bp forward sequence PG-lac1 and reverse sequence PG-lac2, the two were annealed and connected to plasmid pGRB using the recombination kit ClonExpress II One Step Cloning Kit and transformed into E. coli The recombinant plasmid pGRB- lacI .

[0070] (3) Recombinant plasmid pGRB- lacI and fusion fragment U lacI -D lacI Electroporation into the pREDcas9 plasmid E. coli After resuscitation, W3110 competent cells were plated on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and cultured at 32°C overnight. The next day, colony PCR was performed using primers lac-1 / lac-4 to screen positive transformants. The transformants were activated, and arabinose was added at a final concentration of 0.2 mmol / L. The cells were shaken and cultured at 32°C overnight to make pGRB- lacI Loss; then shake culture at 42°C overnight to lose the pREDcas9 plasmid and obtain knockout lacI Recombinant strains E. coli W3110 ∆ lacI .

[0071] (4) Using the E. coli W3110 genome as a template, primers yjiP-1 / yjiP-2 and yjiP-3 / yjiP-4 were used to amplify yjiP The upstream homology arm and downstream homology arm of the gene were amplified using the genome of Enterobacter cloacae ATCC 13047 as a template and primers speF-1 / speF-2. trcPromoter driven, T trc Terminator-terminated speF Gene fragment P trc - speF -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yjiP Upstream homology arm, downstream homology arm and P trc - speF -T trc Fusion fragment U yjiP -P trc - speF -T trc -D yjiP .

[0072] (5) Based on yjiP Sequence design and synthesis of gRNA 20 bp forward sequence PG-1 and reverse sequence PG-2, and obtain recombinant plasmid pGRB-1 using the same method as step (2).

[0073] (6) Recombinant plasmid pGRB-1 and fusion fragment U yjiP -P trc - speF -T trc -D yjiP Electroporation into the pREDcas9 plasmid E. coli W3110 ∆lacI After recovery, the competent cells were plated on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin and cultured at 32°C overnight. The next day, colony PCR was performed using primers yjiP-1 / yjiP-4 to screen positive transformants. The pGRB-1 plasmid and pREDcas9 plasmid were lost in the positive transformants using the same method as step (3) to obtain overexpression speF The recombinant strain DIA-1.

[0074] (7) Using the E. coli W3110 genome as a template, primers argI-1 / argI-2 and argI-3 / argI-4 were used to amplify argI The upstream homology arm and downstream homology arm of the gene were recovered and overlapped PCR was performed to obtain argI The upstream homology arm and the downstream homology arm fusion fragment U argI -D argI .

[0075] (8) According to argI Sequence design and synthesis of gRNA 20 bp forward sequence PG-3 and reverse sequence PG-4, and obtain recombinant plasmid pGRB-2 using the same method as step (2).

[0076] (9) Recombinant plasmid pGRB-2 and fusion fragment U argI -D argI Electroporate into DIA-1 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers argI-1 / argI-4 to screen positive transformants. Use the same method as step (3) to make the pGRB-2 plasmid and pREDcas9 plasmid in the positive transformants lose and obtain knockout argI The recombinant strain DIA-2.

[0077] (10) Using the Escherichia coli W3110 genome as a template, primers argF-1 / argF-2 and argF-3 / argF-4 were used to amplify argF The upstream homology arm and downstream homology arm of the gene were recovered and overlapped PCR was performed to obtain argF Fusion fragment U of the upstream homology arm and the downstream homology arm argF -D argF .

[0078] (11) According to argF Sequence design and synthesis of gRNA 20 bp forward sequence PG-5 and reverse sequence PG-6, and obtain recombinant plasmid pGRB-3 using the same method as step (2).

[0079] (12) Recombinant plasmid pGRB-3 and fusion fragment U argF -D argF Electroporate into DIA-2 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers argF-1 / argF-4 to screen positive transformants. Use the same method as step (3) to make the pGRB-3 plasmid and pREDcas9 plasmid in the positive transformants lose and obtain knockout argF The recombinant strain DIA-3.

[0080] (13) Using the E. coli W3110 genome as a template, primers ycgH-1 / ycgH-2 and ycgH-3 / ycgH-4 were used to amplify ycgH The upstream homology arm and downstream homology arm of the gene were amplified using primers argJBD-1 / argJBD-2 with the genome of Corynebacterium glutamicum ATCC 13032 as a template. trc Promoter driven, T trc Terminator-terminated argJBD Gene fragment Ptrc - argJBD -T trc After the PCR products were recovered, overlapping PCR was performed to obtain ycgH Upstream homology arm, downstream homology arm and P trc - argJBD -T trc Fusion fragment U ycgH -P trc - argJBD -T trc -D ycgH .

[0081] (14) According to ycgH Sequence design and synthesis of gRNA 20 bp forward sequence PG-7 and reverse sequence PG-8, and obtain recombinant plasmid pGRB-4 using the same method as step (2).

[0082] (15) The recombinant plasmid pGRB-4 and the fusion fragment U ycgH -P trc - argJBD -T trc -D ycgH Transform into competent cells of DIA-3 containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers ycgH-1 / ycgH-4 for colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB-4 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression argJBD The recombinant strain DIA-4.

[0083] (16) Using the E. coli W3110 genome as a template, primers argR-1 / argR-2 and argR-3 / argR-4 were used to amplify argR The upstream homology arm and downstream homology arm of the gene were recovered and overlapped PCR was performed to obtain argR Fusion fragment U of the upstream homology arm and the downstream homology arm argR -D argR .

[0084] (17) According to argR Sequence design and synthesis of gRNA 20 bp forward sequence PG-9 and reverse sequence PG-10, and obtain recombinant plasmid pGRB-5 using the same method as step (2).

[0085] (18) The recombinant plasmid pGRB-5 and the fusion fragment U argR -D argRElectrotransform into DIA-4 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers argR-1 / argR-4 to screen positive transformants. Use the same method as step (3) to make the pGRB-5 plasmid and pREDcas9 plasmid in the positive transformants lose and obtain knockout argR The recombinant strain DIA-5.

[0086] (19) Using the E. coli W3110 genome as a template, primers yciQ-1 / yciQ-2 and yciQ-3 / yciQ-4 were used to amplify yciQ The upstream and downstream homology arms of the gene were amplified by primers argA-1 / argA-2. trc Promoter driven T trc Terminator-terminated argA Gene fragment P trc - argA -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yciQ Upstream homology arm, downstream homology arm and P trc - argA -T trc Fusion fragment U yciQ -P trc - argA -T trc -D yciQ .

[0087] (20) According to yciQ Gene sequence design and synthesis of gRNA 20 bp forward sequence PG-11 and reverse sequence PG-12, and the recombinant plasmid pGRB-6 was obtained using the same method as step (2).

[0088] (21) The recombinant plasmid pGRB-6 and the fusion fragment U yciQ -P trc - argA -T trc -D yciQ Transform into competent cells of DIA-5 containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yciQ-1 / yciQ-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB-6 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression yciQ The recombinant strain DIA-6.

[0089] (22) Using the E. coli W3110 genome as a template, primers yghX-1 / yghX-2 and yghX-3 / yghX-4 were used to amplify yghX The upstream and downstream homology arms of the gene were amplified using the genome of Bifidobacterium adolescentis ATCC 15703 as a template and primers fxpK-1 / fxpK-2. trc Promoter driven T trc Terminator-terminated fxpK The gene fragment P trc - fxpK -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yghX Gene upstream homology arm, downstream homology arm and P trc - fxpK -T trc Fusion fragment U yghX -P trc - fxpK -T trc -D yghX .

[0090] (23) According to yghX Gene sequence design and synthesis of gRNA 20 bp forward sequence PG-13 and reverse sequence PG-14, and the recombinant plasmid pGRB-7 was obtained using the same method as step (2).

[0091] (24) The recombinant plasmid pGRB-7 and the fusion fragment U yghX -P trc - fxpK -T trc -D yghX Transform into competent cells of DIA-6 containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yghX-1 / yghX-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB-7 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression fxpK The recombinant strain DIA-7.

[0092] (25) Using the E. coli W3110 genome as a template, primers yjiT-1 / yjiT-2 and yjiT-3 / yjiT-4 were used to amplify yjiT The upstream and downstream homology arms of the gene were amplified using primers gltA-1 / gltA-2. trc Promoter driven T trc Terminator-terminated gltAThe gene fragment P trc - gltA -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yjiT Gene upstream homology arm, downstream homology arm and P trc - gltA -T trc Fusion fragment U yjiT -P trc - gltA -T trc -D yjiT .

[0093] (26) According to yjiT Gene sequence design and synthesis of gRNA 20 bp forward sequence PG-15 and reverse sequence PG-16, and the recombinant plasmid pGRB-8 was obtained using the same method as step (2).

[0094] (27) The recombinant plasmid pGRB-8 and the fusion fragment U yjiT -P trc - gltA -T trc -D yjiT Transform into competent cells of DIA-7 containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification with primers yjiT-1 / yjiT-4 to screen positive transformants. Use the same method as step (3) to make the pGRB-8 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression gltA The recombinant strain DIA-8.

[0095] (28) Using the E. coli W3110 genome as a template, primers lfhA-1 / lfhA-2 and lfhA-3 / lfhA-4 were used to amplify lfhA The upstream and downstream homology arms of the gene were amplified by primers icd-1 / icd-2. trc Promoter driven T trc Terminator-terminated icd Gene fragment P trc - icd -T trc After the PCR products were recovered, overlapping PCR was performed to obtain lfhA Gene upstream homology arm, downstream homology arm and P trc - icd -T trc Fusion fragment U lfhA -P trc - icd -Ttrc -D lfhA .

[0096] (29) According to icd The promoter gene sequence was designed and the gRNA 20 bp forward sequence PG-17 and reverse sequence PG-18 were synthesized, and the recombinant plasmid pGRB-9 was obtained using the same method as step (2).

[0097] (30) The recombinant plasmid pGRB-9 and the fusion fragment U lfhA -P trc - icd -T trc -D lfhA Transform into competent cells of DIA-8 containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers lfhA-1 / lfhA-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB-9 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression icd The recombinant strain DIA-9.

[0098] (31) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers ygaY-1 / ygaY-2 and ygaY-3 / ygaY-4 were used to amplify ygaY The upstream and downstream homology arms of the gene were amplified by primers rocG-1 / rocG-2 using the genome of Bacillus subtilis 168 as a template. trc Promoter driven T trc Terminator-terminated rocG Gene fragment P trc - rocG -T trc After the PCR products were recovered, overlapping PCR was performed to obtain ygaY Upstream homology arm, downstream homology arm and P trc - rocG -T trc Fusion fragment U ygaY -P trc - rocG -T trc -D ygaY .

[0099] (32) According to ygaY Sequence design and synthesis of gRNA 20 bp forward sequence PG-19 and reverse sequence PG-20, and obtain recombinant plasmid pGRB-10 using the same method as step (2).

[0100] (33) The recombinant plasmid pGRB-10 and the fusion fragment U ygaY -P trc - rocG -T trc -D ygaY Electroporate into DIA-9 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers ygaY-1 / ygaY-4 to screen positive transformants. Use the same method as step (3) to make the pGRB-10 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression rocG The recombinant strain DIA-10.

[0101] (34) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers ackA-1 / ackA-2 and ackA-3 / ackA-4 were used to amplify ackA The upstream homology arm and downstream homology arm of the gene were obtained by overlapping PCR after the PCR product was recovered. ackA Fusion fragment U of the upstream homology arm and the downstream homology arm ackA -D ackA .

[0102] (35) According to ackA Sequence design and synthesis of gRNA 20 bp forward sequence PG-21 and reverse sequence PG-22, and obtain recombinant plasmid pGRB-11 using the same method as step (2).

[0103] (36) The recombinant plasmid pGRB-11 and the fusion fragment U ackA -D ackA Transform into DIA-10 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers ackA-1 / ackA-4 to screen positive transformants. Use the same method as step (3) to make the pGRB-11 plasmid and pREDcas9 plasmid in the positive transformants lose and obtain knockout ackA The recombinant strain DIA-11 was constructed.

[0104] (37) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers poxB-1 / poxB-2 and poxB-3 / poxB-4 were used to amplify poxBThe upstream homology arm and downstream homology arm of the gene were obtained by overlapping PCR after PCR product recovery. poxB Fusion fragment U of the upstream homology arm and the downstream homology arm poxB -D poxB .

[0105] (38) According to poxB Sequence design and synthesis of gRNA 20 bp forward sequence PG-23 and reverse sequence PG-24, and obtain recombinant plasmid pGRB-12 using the same method as step (2).

[0106] (39) The recombinant plasmid pGRB-12 and the fusion fragment U poxB -D poxB Electroporate into DIA-11 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers poxB-1 / poxB-4 to screen positive transformants. Use the same method as step (3) to make the pGRB-12 plasmid and pREDcas9 plasmid in the positive transformants lose and obtain knockout poxB The recombinant strain DIA-12 was constructed.

[0107] (40) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers ldhA-1 / ldhA-2 and ldhA-3 / ldhA-4 were used to amplify ldhA The upstream homology arm and downstream homology arm of the gene were recovered and overlapped PCR was performed to obtain l dhA Fusion fragment U of the upstream homology arm and the downstream homology arm ldhA -D ldhA .

[0108] (41) According to ldhA Sequence design and synthesis of gRNA 20 bp forward sequence PG-25 and reverse sequence PG-26, and obtain recombinant plasmid pGRB-13 using the same method as step (2).

[0109] (42) The recombinant plasmid pGRB-13 and the fusion fragment U ldhA -D ldhAElectrotransform into DIA-12 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers ldhA-1 / ldhA-4 to screen positive transformants. Use the same method as step (3) to make the pGRB-13 plasmid and pREDcas9 plasmid in the positive transformants lose and obtain knockout ldhA The recombinant strain DIA-13 was constructed.

[0110] (43) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers yjgX-1 / yjgX-2 and yjgX-3 / yjgX-4 were used to amplify yjgX The upstream and downstream homology arms of the gene were cleaved using primer potE Eco -1 / potE Eco -2 amplification by P trc Promoter driven T trc Terminator-terminated potE Gene fragment P trc - potE Eco -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yjgX Upstream homology arm, downstream homology arm and P trc - potE Eco -T trc Fusion fragment U yjgX -P trc - potE Eco -T trc -D yjgX .

[0111] (44) According to yjgX Sequence design and synthesis of gRNA 20 bp forward sequence PG-27 and reverse sequence PG-28, and obtain recombinant plasmid pGRB-14 using the same method as step (2).

[0112] (45) The recombinant plasmid pGRB-14 and the fusion fragment U yjgX -P trc - sweat Eco -T trc -D yjgXElectrotransform into DIA-13 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yjgX-1 / yjgX-4 for colony PCR identification to screen positive transformants. Use the same method as step (3) to make the pGRB-14 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression sweat The recombinant strain DIA-14 is a genetically engineered bacterium that produces 1,4-butanediamine.

[0113] Schematic diagram of the biosynthetic pathway of 1,4-butanediamine in Escherichia coli W3110 and the genetic modification of genetically engineered bacteria for producing 1,4-butanediamine Figure 1 shown.

[0114] Example 2 This embodiment provides a genetically engineered bacterium for producing 1,4-butanediamine, and the construction method thereof comprises the following steps: Steps (1) to (42) are the same as those in Example 1.

[0115] (43) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers yjgX-1 / yjgX-2 and yjgX-3 / yjgX-4 were used to amplify yjgX The upstream and downstream homology arms of the gene were cleaved using the genome of the mythical pig host-associated Lactobacillus ATCC 33222 as a template and primers potE Lsi -1 / potE Lsi -2, amplified by P trc Promoter driven T trc Terminator-terminated sweat Gene fragment P trc - sweat Lsi -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yjgX Upstream homology arm, downstream homology arm and P trc - sweat Lsi -T trc Fusion fragment U yjgX -P trc - sweat Lsi -T trc -D yjgX .

[0116] (44) According to yjgXSequence design and synthesis of gRNA 20 bp forward sequence PG-27 and reverse sequence PG-28, and obtain recombinant plasmid pGRB-14 using the same method as step (2).

[0117] (45) The recombinant plasmid pGRB-14 and the fusion fragment U yjgX -P trc - sweat Lsi -T trc -D yjgX Electrotransform into DIA-13 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yjgX-1 / yjgX-4 for colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB-14 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression sweat The recombinant strain DIA-15 is a genetically engineered bacterium that produces 1,4-butanediamine.

[0118] Example 3 This embodiment provides a genetically engineered bacterium for producing 1,4-butanediamine, and the construction method thereof comprises the following steps: Steps (1) to (42) are the same as those in Example 1.

[0119] (43) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers yjgX-1 / yjgX-2 and yjgX-3 / yjgX-4 were used to amplify yjgX The upstream and downstream homology arms of the gene were synthesized using the genome of Pasteurella multocida ATCC12948 as a template and potE Pm -1 / potE Pm -2, amplified by P trc Promoter driven T trc Terminator-terminated sweat Gene fragment P trc - sweat Pm -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yjgX Upstream homology arm, downstream homology arm and P trc - sweat Pm -T trc Fusion fragment U yjgX -P trc - sweat Pm -T trc -DyjgX .

[0120] (44) According to yjgX Sequence design and synthesis of gRNA 20 bp forward sequence PG-27 and reverse sequence PG-28, and obtain recombinant plasmid pGRB-14 using the same method as step (2).

[0121] (45) The recombinant plasmid pGRB-14 and the fusion fragment U yjgX -P trc - sweat Pm -T trc -D yjgX Electrotransform into DIA-13 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yjgX-1 / yjgX-4 for colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB-14 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression sweat The recombinant strain DIA-16 is a genetically engineered bacterium that produces 1,4-butanediamine.

[0122] Example 4 This embodiment provides a genetically engineered bacterium for producing 1,4-butanediamine, and the construction method thereof comprises the following steps: Steps (1) to (42) are the same as those in Example 1.

[0123] (43) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers yjgX-1 / yjgX-2 and yjgX-3 / yjgX-4 were used to amplify yjgX The upstream and downstream homology arms of the gene were cloned using the genome of Pseudomonas auriculatus ATCC 700383 as a template and using potE Ppa -1 / potE Ppa -2, amplified by P trc Promoter driven T trc Terminator-terminated sweat Gene fragment P trc - sweat Ppa -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yjgX Upstream homology arm, downstream homology arm and P trc - sweat Ppa -T trc Fusion fragment U yjgX -Ptrc - sweat Ppa -T trc -D yjgX .

[0124] (44) According to yjgX Sequence design and synthesis of gRNA 20 bp forward sequence PG-27 and reverse sequence PG-28, and obtain recombinant plasmid pGRB-14 using the same method as step (2).

[0125] (45) The recombinant plasmid pGRB-14 and the fusion fragment U yjgX -P trc - sweat Ppa -T trc -D yjgX Electrotransform into DIA-13 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yjgX-1 / yjgX-4 for colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB-14 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression sweat The recombinant strain DIA-17 is a genetically engineered bacterium that produces 1,4-butanediamine.

[0126] Example 5 This embodiment provides a genetically engineered bacterium for producing 1,4-butanediamine, and the construction method thereof comprises the following steps: Steps (1) to (42) are the same as those in Example 1.

[0127] (43) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers yjgX-1 / yjgX-2 and yjgX-3 / yjgX-4 were used to amplify yjgX The upstream and downstream homology arms of the gene were amplified using primers lpp-1 / lpp-2. lpp promoter, using primer potE lpp-Eco -1 / potE Eco -2 amplification by T trc Terminator-terminated sweat Gene fragment sweat Ppa -T trc After the PCR products were recovered, overlapping PCR was performed to obtain yjgX Upstream homology arm, downstream homology arm, P lpp and sweat Ppa -Ttrc Fusion fragment U yjgX -P lpp - sweat Ppa -T trc -D yjgX .

[0128] (44) According to yjgX Sequence design and synthesis of gRNA 20 bp forward sequence PG-27 and reverse sequence PG-28, and obtain recombinant plasmid pGRB-14 using the same method as step (2).

[0129] (45) The recombinant plasmid pGRB-14 and the fusion fragment U yjgX -P lpp - sweat Ppa -T trc -D yjgX Electrotransform into DIA-13 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid culture medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yjgX-1 / yjgX-4 for colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB-14 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression sweat The recombinant strain DIA-18 is a genetically engineered bacterium that produces 1,4-butanediamine.

[0130] Example 6 Shake flask fermentation (1) Seed cultivation The recombinant strains obtained in Examples 1-5 were respectively E. coli W3110∆ lacI , DIA-1, DIA-2, DIA-3, DIA-4, DIA-5, DIA-6, DIA-7, DIA-8, DIA-9, DIA-10, DIA-11, DIA-12, DIA-13, DIA-14, DIA-15, DIA-16, DIA-17 and DIA-18 were inoculated onto LB solid medium slants. E. coli W3110∆ lacIAs a control, culture at 37°C for 12 h to obtain seed culture. Then pick a single colony and inoculate it into 30 mL seed culture medium, and culture it at 37°C, 220 rpm on a shaker for 8 h. The composition of the seed culture medium used is 20 g / L glucose, 10 ml / L corn steep liquor, 6 g / L peptone, 2 g / L arginine, 1.2 g / L KH2PO4, 1 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4, 1.3 mg / LV B1 , 0.3 mg / LV H . Add 20 mL / L phenol red as a pH indicator to the seed culture medium, adjust the pH to 7.0-7.2, and sterilize by high-pressure steam at 115°C for 15 min before use.

[0131] (2) Fermentation culture The seed culture obtained in step (1) was inoculated into the fermentation medium at a 1% inoculation rate, and the culture was shaken at 35°C and 220 rpm for 24 h. During the fermentation process, 60% glucose was supplemented 2-3 times according to the situation to maintain the residual sugar concentration at 0.1%-0.5%, and 1 mL was supplemented each time. The pH was adjusted to 6.9-7.1 with ammonia water. After the fermentation, the content of 1,4-butanediamine in the fermentation broth was detected. The content of 1,4-butanediamine in the fermentation broth of each recombinant strain is shown in Table 2. The fermentation medium used was composed of 20 g / L glucose, 2 ml / L corn steep liquor, 4 g / L peptone, 2 g / L arginine, 2 g / L KH2PO4, 0.7 g / L MgSO4, 1 g / L sodium citrate dihydrate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4, and 0.8 mg / L B1 , 0.3 mg / LV H . Add 20 mL / L phenol red as a pH indicator to the fermentation medium, adjust the pH to 7.0-7.2, and sterilize with high-pressure steam at 115°C for 15 min before use.

[0132] Table 2 1,4-Butanediamine content in the fermentation broth of the recombinant strain

[0133] The results show that through gradual genetic engineering, the content of 1,4-butanediamine in the fermentation broth of the recombinant strain has shown an upward trend, and the ability of the recombinant strain to produce 1,4-butanediamine has gradually improved. The production of 1,4-butanediamine in shake flask fermentation can reach 19.9-23.9 mg / L. DIA-14, DIA-15, DIA-16, DIA-17 and DIA-18 are all based on DIA-13 through overexpression sweat Genetically modified, the difference is sweat The source of the gene and the promoter used, among which the one from Escherichia coli was used sweat The yields of the recombinant strains DIA-14 and DIA-18 were relatively high. DIA-18 used P lpp Promoter drive sweat The gene expression showed that the ability to produce 1,4-butanediamine was the strongest, about 5 times that of DIA-1. Therefore, the DIA-18 recombinant strain was selected for fermentation in a 5 L fermenter to produce 1,4-butanediamine.

[0134] Example 7 This embodiment provides a method for producing 1,4-butanediamine by fermentation using a 5 L fermenter.

[0135] (1) Seed culture: The activated recombinant strain DIA-18 (provided in Example 5) was inoculated into a 5 L fermenter containing 2.5 L seed culture medium at a 10% inoculation rate, and 25% ammonia water was added to adjust the pH of the fermentation liquid to 7.0-7.5. The dissolved oxygen was maintained at 25%, and the ventilation volume was 2-4 m 3 / h, stirring speed 200-800 rpm, culturing at 37℃ for 10 h to obtain seed culture. (2) Fermentation in fermenter: Inoculate the seed culture of step (1) with 10% inoculation amount into a 5 L fermenter filled with 3 L fermentation medium for fermentation culture, fermentation temperature 37℃, ventilation volume 2-4 m 3 / h, stirring speed 300-900 rpm, tank pressure 0.06-0.10 MPa, dissolved oxygen maintained at 30-40%, 80% glucose solution was added to maintain residual sugar concentration at 0.1%-0.5%, 25% ammonia water was added to adjust the pH of the fermentation liquid to 7.0-7.5, and the fermentation cycle was 48 h.

[0136] After the fermentation, the content of 1,4-butanediamine and other organic acids in the fermentation broth was detected. The content of 1,4-butanediamine in the fermentation broth of the recombinant strain DIA-18 reached 46.4 g / L, and no other organic acids were detected.

[0137] Example 8 This embodiment provides a method for producing 1,4-butanediamine by fermentation using a 5 L fermenter.

[0138] (1) Seed culture: The activated recombinant strain DIA-18 (provided in Example 5) was inoculated into a 5 L fermenter containing 2.5 L seed culture medium at a 10% inoculation rate, and 25% ammonia water was added to adjust the pH of the fermentation liquid to 7.0-7.5. The dissolved oxygen was maintained at 25%, and the ventilation volume was 2-4 m 3 / h, stirring speed 200-800 rpm, and culturing at 37°C for 10 h to obtain seed culture.

[0139] (2) Fermentation in a fermenter: The seed culture of step (1) was inoculated with 5% of the inoculum into a 5L fermenter containing 3L of fermentation medium for fermentation at a temperature of 32°C and a ventilation volume of 2-3 m 3 / h, stirring speed 400-800 rpm, dissolved oxygen maintained at 25-40%, 80% glucose solution was fed to maintain residual sugar concentration at 0.1%-0.5%, 25% ammonia water was fed to adjust the pH of the fermentation liquid to 7.0-7.5, and the fermentation period was 48 h.

[0140] After the fermentation, the content of 1,4-butanediamine and other organic acids in the fermentation broth was detected. The content of 1,4-butanediamine in the fermentation broth of the recombinant strain DIA-18 reached 39.2 g / L, and no other organic acids were detected.

[0141] Example 9 This embodiment provides a method for producing 1,4-butanediamine by fermentation using a 5 L fermenter.

[0142] (1) Seed culture: The activated recombinant strain DIA-18 (provided in Example 5) was inoculated into a 5 L fermenter containing 2.5 L seed culture medium at a 10% inoculation rate, and 25% ammonia water was added to adjust the pH of the fermentation liquid to 7.0-7.5. The dissolved oxygen was maintained at 25%, and the ventilation volume was 2-4 m 3 / h, stirring speed 200-800 rpm, and culturing at 37°C for 10 h to obtain seed culture.

[0143] (2) Fermentation in a fermenter: The seed culture of step (1) was inoculated with 8% of the inoculum into a 5 L fermenter containing 3 L of fermentation medium for fermentation. During the first 16 h of fermentation (the first stage), the fermentation temperature was set at 37 °C and the ventilation volume was 2-4 m 3 / h, stirring speed 300-900 rpm, tank pressure 0.05-0.1 MPa, dissolved oxygen level maintained at 20%-30%; after 16 hours of fermentation (second stage), set the fermentation temperature to 37°C and the ventilation volume to 3-5 m 3 / h, stirring speed to 400-900 rpm, tank pressure 0.1-0.15 MPa, dissolved oxygen level maintained at 30%-45%, the whole fermentation cycle 48 h, during the fermentation process by adding 25% ammonia water to maintain the pH of the fermentation liquid 7.0-7.5 by adding 80% glucose solution to maintain the residual sugar concentration of the fermentation liquid 0.1%-0.5% (W / V).

[0144] After the fermentation, the content of 1,4-butanediamine and other organic acids in the fermentation broth was detected. The content of 1,4-butanediamine in the fermentation broth of the recombinant strain DIA-18 reached 51.8 g / L, and no other organic acids were detected.

[0145] Example 10 This embodiment provides a method for producing 1,4-butanediamine by fermentation using a 5 L fermenter.

[0146] (1) Seed culture: The activated recombinant strain DIA-18 (provided in Example 5) was inoculated into a 5 L fermenter containing 2.5 L seed culture medium at a 10% inoculation rate, and 25% ammonia water was added to adjust the pH of the fermentation liquid to 7.0-7.5. The dissolved oxygen was maintained at 25%, and the ventilation volume was 2-4 m 3 / h, stirring speed 200-800 rpm, and culturing at 37°C for 10 h to obtain a seed culture.

[0147] (2) Fermentation in a fermenter: The seed culture of step (1) was inoculated with 8% of the inoculum into a 5 L fermenter containing 3 L of fermentation medium for fermentation. During the first 16 h of fermentation (the first stage), the fermentation temperature was set at 37 °C and the ventilation volume was 2-4 m 3 / h, stirring speed 300-900 rpm, tank pressure 0.05-0.1 MPa, so that the dissolved oxygen level is maintained at 20%-30%; after 16 hours of fermentation (second stage), the fermentation temperature is set to 32℃, the ventilation volume is 3-5 m 3 / h, stirring speed to 400-900 rpm, tank pressure 0.1-0.15 MPa, dissolved oxygen level maintained at 30%-45%, the whole fermentation cycle 48 h, during the fermentation process, the pH of the fermentation liquid was maintained at 7.0-7.5 by adding 25% ammonia water, and the residual sugar concentration of the fermentation liquid was maintained at 0.1%-0.5% (W / V) by adding 80% glucose solution.

[0148] Sampling was performed every 4 hours from the beginning of fermentation in the fermenter until the end of fermentation to detect the contents of 1,4-butanediamine and other organic acids in the fermentation broth at different fermentation time points. Figure 2 The curve of 1,4-butanediamine concentration changing with time during fermentation is shown. Figure 2 The data showed that in the first stage of fermentation, i.e. the first 16 h, the fermentation temperature was set at 37°C and the oxygen level was maintained at 20%-30%. The environmental conditions at this stage were conducive to the rapid growth of the strain and the activation of metabolic activity, and the concentration of 1,4-butanediamine gradually increased from 0. In the second stage of fermentation, i.e. 16 to 48 h, the fermentation temperature was reduced to 32°C and the dissolved oxygen level was increased to 30%-45%. At this stage, the concentration of 1,4-butanediamine increased rapidly. At this stage, the recombinant strain DIA-18 mainly metabolized and produced 1,4-butanediamine. This staged fermentation strategy effectively improves the yield and production efficiency of 1,4-butanediamine by optimizing the growth and metabolic processes of the strain.

[0149] Embodiment 11 This embodiment provides a method for extracting 1,4-butanediamine.

[0150] (1) Membrane filtration: The 1,4-butanediamine fermentation broth (provided in Example 10) was centrifuged at 4°C and 10,000 rpm for 10 min using a high-speed refrigerated centrifuge to collect the supernatant, and then the collected supernatant was subjected to membrane filtration treatment using an organic membrane separation test machine. Specifically, the supernatant was pre-treated with an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the recovery rate of the 1,4-butanediamine salt solution was 98.3%. On the basis of the ultrafiltration treatment, the supernatant was treated with a nanofiltration membrane with a molecular weight cutoff of 300 Da, and the recovery rate of the 1,4-butanediamine salt solution was 96.8%. After the membrane filtration treatment, the final recovery rate of the 1,4-butanediamine salt solution was 95.2%.

[0151] (2) Alkalization: Sodium hydroxide was added to the 1,4-butanediamine salt solution obtained after membrane filtration to adjust the pH to 8.0. The solution was alkalized at 70°C and 100 rpm and stirred for 5 h for desalination reaction. After the reaction, an aqueous solution of 1,4-butanediamine was obtained. The recovery rate of alkalized 1,4-butanediamine was 88.6%.

[0152] (3) Extraction and distillation: n-butanol was added to the obtained 1,4-butanediamine aqueous solution, with the volume of n-butanol being 3 times the volume of the 1,4-butanediamine aqueous solution, and the mixture was stirred and extracted at 50°C for 2 h. After the extraction was completed, the mixture was allowed to stand for stratification. The upper n-butanol phase was taken, and the extraction rate of 1,4-butanediamine was 91.4% after testing. Subsequently, the n-butanol phase extract was subjected to reduced pressure distillation at a pressure of -0.098 MPa and a temperature of 50°C to separate and collect n-butanol and 1,4-butanediamine. The recovery rate of 1,4-butanediamine was 84.3% after testing.

[0153] During the whole extraction process, the total recovery rate of 1,4-butanediamine was 71.1%.

[0154] Example 12 This embodiment provides a method for extracting 1,4-butanediamine. The difference from embodiment 11 is that in step (2), potassium hydroxide is added to the 1,4-butanediamine salt solution obtained after membrane filtration to adjust the pH to 8.0, the solution is alkalized at 70°C and 100 rpm, and stirred for 5 h for desalination reaction. After the reaction, a 1,4-butanediamine aqueous solution is obtained, and the recovery rate of alkalized 1,4-butanediamine is 83.6%.

[0155] During the whole extraction process, the total recovery rate of 1,4-butanediamine was 67.1%.

[0156] Embodiment 13 This embodiment provides a method for extracting 1,4-butanediamine.

[0157] (1) Membrane filtration: The method is the same as in Example 11.

[0158] (2) Alkalization: Sodium hydroxide was added to the 1,4-butanediamine salt solution obtained after membrane filtration to adjust the pH to 9.0. The solution was alkalized at 70°C and 100 rpm and stirred for 5 h for desalination reaction. After the reaction, an aqueous solution of 1,4-butanediamine was obtained. The recovery rate of alkalized 1,4-butanediamine was 93.4%.

[0159] (3) Extraction and distillation: n-butanol was added to the obtained 1,4-butanediamine aqueous solution, the volume of n-butanol being 3 times the volume of the 1,4-butanediamine aqueous solution, and the mixture was stirred and extracted at 70°C for 2 h. After the extraction was completed, the mixture was allowed to stand for stratification. The upper n-butanol phase was taken, and the extraction rate of 1,4-butanediamine was 95.2% after testing. Subsequently, the n-butanol phase extract was subjected to reduced pressure distillation at a pressure of -0.098 MPa and a temperature of 50°C to separate and collect n-butanol and 1,4-butanediamine. The recovery rate of 1,4-butanediamine was 88.3% after testing.

[0160] During the whole extraction process, the total recovery rate of 1,4-butanediamine was 78.5%.

[0161] Embodiment 14 This embodiment provides a method for extracting 1,4-butanediamine. The difference from embodiment 13 is that in step (2), sodium hydroxide is added to the 1,4-butanediamine salt solution obtained after membrane filtration to make the pH reach 10.0, and the solution is alkalized at 70°C and 100 rpm, and stirred for 5 h for desalination reaction. After the reaction, a 1,4-butanediamine aqueous solution is obtained, and the recovery rate of alkalized 1,4-butanediamine is 90.1%.

[0162] During the whole extraction process, the total recovery rate of 1,4-butanediamine was 75.7%.

[0163] In summary, the present invention significantly improves the production efficiency and extraction recovery rate of 1,4-butanediamine through comprehensive metabolic engineering means, provides an efficient and feasible technical solution for the industrial production of 1,4-butanediamine, and has important application prospects and economic value.

[0164] 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 principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium for producing 1,4-butanediamine, characterized in that: The genetically engineered bacteria is obtained by using Escherichia coli W3110 as a starting strain and performing the following gene editing on its genome: the genetically engineered bacteria does not express lactose operon repressor protein, ornithine carbamoyltransferase 1, ornithine carbamoyltransferase 2, arginine repressor protein, acetate kinase, pyruvate dehydrogenase and lactate dehydrogenase; and overexpresses ornithine decarboxylase, ornithine synthesis operon, N-acetylglutamate synthetase, phosphoketolase, citrate synthase 、 Isocitrate dehydrogenase, glutamate dehydrogenase, and putrescine transporter.

2. The genetically engineered bacterium according to claim 1, characterized in that: Ornithine decarboxylase, ornithine synthesis operon, N-acetylglutamate synthetase, phosphoketolase, citrate synthase 、 At least one of the genes encoding isocitrate dehydrogenase, glutamate dehydrogenase and putrescine transporter uses a promoter of P trc Promoter or P lpp promoter; the P trc The nucleotide sequence of the promoter is shown in the sequence table SEQ ID NO.

1. lpp The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.

2.

3. The genetically engineered bacterium according to claim 1, characterized in that The nucleotide sequence of the ornithine decarboxylase encoding gene is shown in SEQ ID NO.3; and / or The nucleotide sequence of the gene encoding the ornithine biosynthesis operon is shown in SEQ ID NO.4-6; and / or The nucleotide sequence of the N-acetylglutamate synthetase encoding gene is shown in SEQ ID NO.7; and / or The nucleotide sequence of the phosphoketolase encoding gene is shown in SEQ ID NO.8; and / or The nucleotide sequence of the citrate synthase encoding gene is shown in SEQ ID NO.9; and / or The nucleotide sequence of the isocitrate dehydrogenase encoding gene is shown in SEQ ID NO.10; and / or The nucleotide sequence of the gene encoding glutamate dehydrogenase is shown in SEQ ID NO.11; and / or The nucleotide sequence of the putrescine transporter encoding gene is one of the following nucleotide sequences: the nucleotide sequence is shown in SEQ ID NO.12; the nucleotide sequence is shown in SEQ ID NO.13; the nucleotide sequence is shown in SEQ ID NO.14; and the nucleotide sequence is shown in SEQ ID NO.

15.

4. The genetically engineered bacterium according to claim 3, characterized in that: The nucleotide sequence of the putrescine transporter encoding gene is shown in SEQ ID NO.

17. The promoter used for overexpression of the putrescine transporter encoding gene is P lpp Promoter.

5. A method for constructing a genetically engineered bacterium for producing 1,4-butanediamine, characterized in that: The following steps are involved: Using Escherichia coli W3110 as the starting strain, the genes encoding the lactose operon repressor protein, the genes encoding ornithine carbamoyltransferase 1, the genes encoding ornithine carbamoyltransferase 2, the genes encoding the arginine repressor protein, the genes encoding acetate kinase, the genes encoding pyruvate dehydrogenase and the genes encoding lactate dehydrogenase were knocked out from its genome; the genes encoding ornithine decarboxylase, the genes encoding the ornithine biosynthesis operon, the genes encoding N-acetylglutamate synthetase, the genes encoding phosphoketolase and the genes encoding citrate synthase were integrated 、 The genes encoding isocitrate dehydrogenase, glutamate dehydrogenase and putrescine transporter; there is no particular order in which the above genes are edited.

6. Use of the genetically engineered bacteria for producing 1,4-butanediamine according to any one of claims 1 to 4 in the fermentation production of 1,4-butanediamine.

7. A method for producing 1,4-butanediamine by fermentation, characterized in that: The genetically engineered bacteria according to any one of claims 1 to 4 or the genetically engineered bacteria obtained by the construction method according to claim 5 are used to carry out fermentation using glucose or a medium containing glucose as a substrate.

8. The method according to claim 7, characterized in that The following steps are involved: (1) inoculating the activated genetically engineered bacteria onto a seed culture medium to perform seed culture to obtain a seed culture; (2) inoculating the seed culture obtained in step (1) onto a fermentation medium at an inoculation rate of 5%-10%, performing aerobic fermentation, feeding a glucose solution, maintaining the residual sugar concentration in the fermentation broth at 0.1%-0.5%, and obtaining a fermentation broth containing 1,4-butanediamine; The seed culture medium comprises 30 g / L glucose, 5 ml / L corn steep liquor, 2 g / L peptone, 2 g / L arginine, 2 g / L KH2PO4, 1.5 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, 1.3 mg / L B1 , 0.3 mg / LV H ; pH 7.0-7.2; The fermentation medium comprises: 10 g / L glucose, 3 ml / L corn steep liquor, 1 g / L glutamic acid, 2 g / L arginine, 3 g / L KH2PO4, 1.8 g / L MgSO4·7H2O, 2 g / L sodium citrate dihydrate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, 2 mg / L B1 , 0.2 mg / LV H ; pH is 7.0-7.

2.

9. The method according to claim 8, characterized in that The fermentation cycle of the aerobic fermentation in step (2) is 42-48 hours. During the fermentation process, the pH of the fermentation liquid is maintained at 7.0-7.5 and the residual sugar concentration is maintained at 0.1%-0.5%. The aerobic fermentation is divided into two stages. In the first stage, the fermentation temperature is set at 32-37°C and the dissolved oxygen level in the fermentation liquid is maintained at 20%-30%. In the second stage, the fermentation temperature is set at 32-37°C and the dissolved oxygen level in the fermentation liquid is maintained at 30%-45%.

10. The method according to any one of claims 8 or 9, characterized in that: The method also includes extracting 1,4-butanediamine from the fermentation liquid obtained after fermentation, including the following steps: (1) Membrane filtration: centrifuging the fermentation broth to collect the supernatant, and filtering the supernatant with an ultrafiltration membrane and then a nanofiltration membrane to obtain a 1,4-butanediamine salt solution; (2) Alkalization: adding sodium hydroxide or potassium hydroxide to the 1,4-butanediamine salt solution obtained in step (1) for alkalization, stirring to carry out desalination reaction, and obtaining a 1,4-butanediamine aqueous solution after the desalination reaction is complete; (3) Extraction and distillation: n-butanol is added to the aqueous solution of 1,4-butanediamine obtained in step (2), and the mixture is stirred for extraction. After the extraction is completed, the mixture is allowed to stand and the layers are separated. The n-butanol phase extract is subjected to reduced pressure distillation to collect 1,4-butanediamine.

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