Strain composition for producing agmatine and use thereof

Abstract

The present application provides a kind of strain composition for producing agmatine and application.The strain composition includes carboxy agmatine decarboxylase producing strain and carboxy agmatine producing strain, and carboxy agmatine producing strain is introduced into ornithine decarboxylase speC Gene and carboxy agmatine dehydrogenase CASDH Gene;By the combination of carboxy agmatine producing strain and carboxy agmatine decarboxylase producing strain, first, carboxy agmatine producing strain is used to ferment to produce key precursor carboxy agmatine, carboxy agmatine decarboxylase producing strain is used to ferment to synthesize carboxy agmatine decarboxylase, then the two are catalyzed by enzyme, effectively isolate side reaction, realize enzyme catalytic directional synthesis agmatine;That is, agmatine is synthesized by two-step method of biological fermentation combined with enzyme catalysis.The present application has the advantages of simple and efficient process, less byproduct, sufficient precursor, high agmatine yield, suitable for popularization and use, etc.

Description

Technical Field

[0001] This invention relates to the field of gene recombination fermentation technology, and more specifically to a strain composition for producing spermidine and its application. Background Technology

[0002] Spermidine is a low-molecular-weight aliphatic compound with three amino groups. It is a naturally occurring polyamine compound with important bioactive properties and is widely found in living organisms, including plants, animals, and microorganisms. Spermidine has broad application prospects in pharmaceuticals, health supplements, and cosmetics.

[0003] Current literature reports on spermidine biosynthesis mainly categorize it into two aspects: enzymatic catalysis and de novo biosynthesis. While existing enzymatic catalysis methods achieve high spermidine yields, the high production cost of enzymes makes them unsuitable for large-scale production. De novo biosynthesis involves synthesizing spermidine using strains such as *Bacillus amyloliquefaciens*, *Saccharomyces cerevisiae*, and *Corynebacterium glutamicum* as substrate strains. For example, CN113736719A discloses a genetically engineered *Corynebacterium glutamicum* strain and its application in spermidine production. Through metabolic engineering, *Corynebacterium glutamicum* is modified to remove various product inhibitions during spermidine synthesis, enhance the activity of key enzymes, and introduce an exogenous spermidine transport system. After high-density fermentation, *Corynebacterium glutamicum* can synthesize 14.3 g / L of spermidine.

[0004] The de novo biosynthesis of spermidine involves two main metabolic pathways, both of which face significant challenges. First, the methionine-based pathway involves complex feedback inhibition regulation, with the conversion of methionine to S-adenosylmethionine (SAM) being a key bottleneck. The strict enzymatic regulation of this conversion remains unresolved, limiting yield. Therefore, alternative synthetic pathways starting from aspartic semialdehyde (ASA) have attracted considerable attention, circumventing the difficulties of SAM synthesis. However, the ASA pathway itself has serious drawbacks: carboxysemine dehydrogenases and carboxysemine decarboxylases use spermidine and ASA as common substrates, catalyzing side reactions to generate byproducts such as carboxythermal spermidine, thermal spermidine, carboxysemine, or spermidine. This not only directly consumes spermidine, reducing the yield of the target product, but also exacerbates the raw material shortage problem due to the competitive consumption of the ASA precursor. Furthermore, the accumulation of byproducts introduces impurities, significantly reducing the purity of spermidine.

[0005] To overcome the above limitations, it is urgent to develop innovative strategies to address the issues of spermidine consumption and impurity generation caused by enzymatic side reactions in the ASA pathway. Summary of the Invention

[0006] The core concept of this invention is based on a two-step method of producing spermidine through bio-fermentation and enzymatic catalysis, namely, through a spermidine-producing strain and a spermidine-producing decarboxylase. CASDC The combined use of strains, first fermenting separately to produce the precursor and enzyme, then mixing them for enzymatic catalysis, effectively isolates side reactions and achieves targeted and efficient synthesis of spermidine. The specific technical solution is as follows: In a first aspect, the present invention provides a bacterial composition for producing spermidine, mainly comprising two strains: a strain producing carboxysemidine decarboxylase and a strain producing carboxysemidine, wherein the strain producing carboxysemidine decarboxylase includes *Escherichia coli* and a carboxysemidine decarboxylase introduced therein. CASDC Genes are used for the fermentation synthesis of carboxysemine decarboxylase, providing catalytically active substances for subsequent enzymatic catalysis; the strains producing carboxysemine include Corynebacterium glutamicum and Ornithine decarboxylase. speC Genes and carboxysemine dehydrogenase CASDC Gene, the ornithine decarboxylase speC Genes and carboxysemine dehydrogenase CASDC The genes are all located in the plasmids of the *Corynebacterium glutamicum* or integrated into its genome, and are used for the fermentation production of carboxyspermine, a key precursor in spermidine synthesis. The two strains work synergistically to achieve efficient spermidine biosynthesis; neither can be omitted. Preferably, the carboxyspermine decarboxylase... CASDC Genes originate from Agrobacterium tumefaciens or Campylobacter jejuni .

[0007] The *E. coli* basal bacteria can be *E. coli* BL21(DE3), *E. coli* DH5α, *E. coli* BW25113, or *E. coli* MG1655. The *Corynebacterium glutamicum* basal bacteria can be recombinant *Corynebacterium glutamicum*. Corynebacterium glutamicun ATCC 13032 Corynebacterium glutamicun ATCC 21831 Corynebacterium glutamicun ATCC 13869, Corynebacterium glutamicum SCgG2, etc.

[0008] To further reduce the metabolic burden on Corynebacterium glutamicum basal cells and improve expression efficiency, the Corynebacterium glutamicum basal cells are expressed using a strong promoter-driven ornithine decarboxylase expression method. speC Genes and carboxysemine dehydrogenase CASDHThe gene, wherein the strong promoter is at least one of the PH36 promoter, sod promoter, and tuf promoter as shown in SEQ ID NO. 1, wherein the nucleotide sequence of the sod promoter is the first 200 bp of the start codon ATG in the *Corynebacterium glutamicum* superoxide dismutase sod gene as shown in NCBI-gene database accession number Cg3237; and the nucleotide sequence of the tuf promoter is the first 200 bp of the start codon ATG in the *Corynebacterium glutamicum* elongation factor tuf gene as shown in NCBI-gene database accession number NCgl0480.

[0009] To further improve the yield of spermidine in the two-step synthesis, targeted modifications were made to the Corynebacterium glutamicum chassis bacteria, including at least one of the following methods: 1) Increase the metabolic flux of ornithine, reduce the consumption of putrescine, and increase the production of putrescine: The repressor transcriptional regulator was knocked out in the genome of the Corynebacterium glutamicum. argR ornithine aminotransferase argF Spermine N1-acetyltransferase snaA Genes and N-acetyltransferases puo Gene.

[0010] 2) Increased metabolic flux of acetyl-CoA: Lactate dehydrogenase was also knocked out in the genome of the Corynebacterium glutamicum. ldhA Genes, pyruvate dehydrogenase poxB Genes, phosphorylated acetyltransferases pta Genes and acetate kinases ackA At least one of the genes.

[0011] 3) To increase the flux of oxaloacetic acid (OAA), improve the yield of spermidine in the two-step synthesis, and reduce the yield of putrescine: the genome of the Corynebacterium glutamicum also had the pyruvate dehydrogenase E1 complex knocked out. aceE Genes, phosphoenolpyruvate carboxylase pck At least one of the genes.

[0012] 4) Increased metabolic flux of aspartate: The genome of the Corynebacterium glutamicum also shows enhanced phosphoenolpyruvate carboxylase. ppc Genes and pyruvate carboxylase pyc Expression of at least one gene; preferably, enhanced expression using the PH36 promoter with a nucleotide sequence as shown in SEQ ID NO. 1. ppc Genes and pyc Gene expression.

[0013] 5) Increasing the flux of aspartic semialdehyde further improves the yield of spermidine in the two-step synthesis while reducing the yield of lysine and homoserine. Furthermore, the genome of the *Corynebacterium glutamicum* strain is enhanced with aspartate kinase. lysC Genes and aspartate semialdehyde dehydrogenase asd The expression of at least one gene.

[0014] Preferably, the genome of the *Corynebacterium glutamicum* basalis strain further enhances aspartate kinase through at least one of the following mechanisms. lysC Gene expression: Aspartate kinase lysC Gene replacement with T311I mutant, aspartate kinase lysC The gene promoter was replaced with the PH36 promoter as shown in SEQ ID NO. 1, and the aspartate kinase was... lysC The gene was integrated into the cg1960 site in the genome.

[0015] Preferably, the genome of the *Corynebacterium glutamicum* basalis strain further enhances aspartate semialdehyde dehydrogenase through at least one of the following mechanisms. asd Gene expression: Aspartate semialdehyde dehydrogenase asd The gene promoter was replaced with the PH36 promoter, and aspartate semialdehyde dehydrogenase was removed. asd The gene was integrated into the cg1960 site in the genome; 6) To maximize the metabolic flux of aspartic semialdehyde towards carboxysemine, thereby further increasing the yield of spermine in the two-step synthesis while simultaneously reducing the yields of lysine and homoserine: the genome of the Corynebacterium glutamicum also knocked out the bypass metabolic pathway of aspartic semialdehyde, such as homoserine dehydrogenase. hom Gene 、 Diaminopimelic acid dehydrogenase ddh Genes, pimelic acid decarboxylase and lysA1 Genes, lysine transporters lysA2 At least one of the genes.

[0016] 7) Balanced NADPH cofactor in the strain: Endogenous NADP was also knocked out in the genome of the Corynebacterium glutamicum. + glucose-dependent dehydrogenase lysE and butA The gene was also introduced with the addition of an exogenous pyridine nucleotide transhydrogenase. fabG Genes and glyceraldehyde-3-phosphate dehydrogenase pntAB Gene.

[0017] Secondly, the present invention provides a method for producing spermidine, comprising: Fermentation to produce carboxysemine decarboxylase: The above-mentioned carboxysemine decarboxylase-producing strain is cultured in TB medium to obtain a bacterial culture containing carboxysemine decarboxylase; preferably, the TB medium formulation includes: tryptone 5-15 g / L, yeast extract 5-25 g / L, glycerol 2%-6%, KH2PO4 1-3 g / L, and K2HPO4 5-10 g / L. The carboxysemine decarboxylase... gapN Genes come from, but are not limited to CASDH Agrobacterium tumefaciens, Bacteroides fragilis, ; Fermentation precursor: The above-mentioned carboxyspermine-producing strain is fermented to obtain a fermentation broth containing carboxyspermine; preferably, the culture medium for fermenting the carboxyspermine-producing strain includes: glucose 10-50 g / L, (NH4)2SO4 15-25 g / L, urea 2-10 g / L, KH2PO4 0.8-1.2 g / L, K2HPO4 0.8-1.2 g / L, MOPES 30-50 g / L, sodium acetate 2-10 g / L, protocatechuic acid 0.02-0.03 mg / L, biotin 0.15-0.25 mg / L, 0.2-0.3 g / L MgSO4·7H2O, 8-12 mg / L CaCl2, 8-12 mg / L FeSO4·7H2O, 8-12 mg / L MnSO4·7H2O, 0.8-1.2 mg / L ZnSO4·7H2O, 0.15-0.25 mg / L CuSO4, 0.01-0.03 mg / L NiCl2·6H2O, kanamycin final concentration 15-100 mg / L; Mixed enzyme-catalyzed production of spermidine: The bacterial broth containing carboxysemidine decarboxylase and the fermentation broth containing carboxysemidine are uniformly mixed and fermented at pH 6-8 and 25-40℃ to catalyze the conversion of carboxysemidine to spermidine; preferably, the mixed fermentation time is at least 12 h. During the reaction, residual carboxysemidine can be detected by mass spectrometry to adjust the bacterial broth ratio, ensuring sufficient enzyme activity, and finally obtaining high-purity spermidine.

[0018] Thirdly, the application of the above-described strain composition for producing spermidine or the above-described method for producing spermidine provided by the present invention in the preparation of spermidine or its derivatives.

[0019] Therefore, the above-mentioned technical solution provided by the present invention can be widely applied to the preparation of spermidine and its derivatives, and is suitable for industrial production in the fields of pharmaceuticals, health products, and cosmetics; at the same time, it has the advantages of simple and efficient process, few by-products, sufficient precursors, high spermidine yield, and suitability for promotion and use. Attached Figure Description

[0020] Clostridium leptum, Rhodobacter sphaeroidesThe metabolic diagram of carboxysemine synthesis in Corynebacterium glutamicum provided by this invention; Figure 1 Mass spectrometry (MS) spectral analysis of fermentation products for de novo synthesis of spermidine using strain Cg-PEC-XK99E-speC-CASDH2-CASDC1; Figure 2 The graph shows the lysine / homoserine production results provided in the embodiments of the present invention. Detailed Implementation

[0021] To clarify the purpose, technical solution, and advantages of this invention, the technical solution of this invention will be described in detail below with reference to the embodiments.

[0022] To address the problems of low yield, numerous side reactions, and insufficient purity in the de novo biosynthesis of spermidine, this invention is based on... Figure 3 The shown metabolic pathway for the synthesis of carboxysemine in Corynebacterium glutamicum employs a two-step method: first, Corynebacterium glutamicum ferments to produce the key intermediate carboxysemine, and then... (The sentence is incomplete and requires more context to translate accurately). [[ID=5~2]]Figure 1 21(DE3) expresses carboxysaminoglycan decarboxylase ( BL Directed catalytic synthesis of spermidine achieves efficient and high-purity synthesis of spermidine.

[0023] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0024] Unless otherwise specified, all terms used in this invention are commonly used in the relevant field, and the technical means employed in the embodiments, such as plasmid construction methods and product determination methods, are conventional means well known to those skilled in the art. Unless otherwise specified, all reagents and products used in this invention are commercially available. The source, trade name, and components of any reagents used are indicated upon their first appearance. The plasmid construction method utilizes a one-step cloning kit from Novizan Biosciences.

[0025] Unless otherwise specified, all promoters used in the embodiments are commercially available products; the nucleotide sequence of the PH36 promoter described in this invention is shown in SEQ ID NO. 1. The exogenous gene sequences introduced into the strains constructed in each embodiment are shown in Tables 1-2, and the primer sequences used are shown in Table 3.

[0026] Table 1. Exogenous genes included in NCBI-gene used in the examples

[0027] Among them, DS480346.1(214714-215913) refers to CASDC Positions 214714-215913 on genome DS480346.1.

[0028] Table 2. Exogenous genes recorded in KEGG used in the examples

[0029] Table 3 Nucleotide sequences of primers used in the embodiments of the present invention.

[0030] (1) Strains: Corynebacterium glutamicum Clostridium leptum ATCC 13032 (purchased from BioBest); E. coli Corynebacterium glutamicun 21(DE3) BL DH5α (all purchased from Novizan).

[0031] (2) Culture medium formulation: 1) LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, glucose 5 g / L, agar powder 25 g / L.

[0032] 2) LBHIS liquid culture medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, bovine brain heart extract 18.5 g / L, sorbitol 91 g / L.

[0033] 3) LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, glucose 5 g / L.

[0034] 4) Fermentation medium for *Corynebacterium glutamicum* producing carboxysemine and spermidine: glucose 40 g / L, (NH4)2SO4 20 g / L, urea 5 g / L, sodium acetate 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MOPES 40 g / L, protocatechuic acid 0.03 mg / L, biotin 0.2 mg / L, MgSO4·7H2O 0.25 g / L, CaCl2 10 mg / L, FeSO4·7H2O 10 mg / L, MnSO4·7H2O 1 mg / L, ZnSO4·7H2O 1 mg / L, CuSO4 0.2 mg / L, NiCl2·6H2O 0.02 mg / L, kanamycin final concentration 30 mg / L, balance water.

[0035] 5) Seed culture medium for Escherichia coli that produces carboxysemine decarboxylase: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, glucose 5 g / L, kanamycin final concentration 25 mg / L.

[0036] 6) Escherichia coli producing carboxysemine decarboxylase was cultured in TB medium: tryptone 11.8 g / L, yeast extract 23.6 g / L, glycerol 4%, KH2PO4 2.2 g / L, K2HPO4 9.4 g / L, and IPTG was added to a final concentration of 1 mM.

[0037] (3) HPLC detection method for fermentation products: Detector: Evaporative light scattering detector (ELSD); Detector parameters: Nebulizer temperature: 60 degrees; Evaporator temperature: 60 degrees; Gas flow rate: 1.6 SLM; Acquisition frequency: 40 Hz; Column: UltimateAmino Acid Plus (4.6 × 300 mm, Yuexu Technology) Column temperature: 30 ℃; Mobile phase A - acetonitrile, mobile phase B - 0.1% TFA (trifluoroacetic acid) aqueous solution; Isocratic elution for 10 min; Flow rate: 1 mL / min; Injection volume: 10 µL.

[0038] (4) Gene editing of Corynebacterium glutamicum uses the homologous recombination double exchange method. Editing is achieved by screening upstream and downstream homologous arms of the gene carried by the plasmid. It involves knockout (loss of the entire gene segment, i.e., after the start codon and before the stop codon), mutation (change of bases), promoter enhancement (insertion of the promoter before the start codon of the relevant gene), and gene integration (exogenous gene replaces the gene at the relevant site). The homologous arms of the first three are 500bp, and the homologous arm of integration is 1000bp. Knockout, mutation, enhancement and integration plasmids are constructed accordingly.

[0039] (4-1) Using gene knockout E. coli Taking the knockout plasmid PK18-snaA as an example, we found that snaA in snaA The location of the ATCC 13032 strain on the genome, in order to C.glutamicum The start codon ATG and the stop codon TTA serve as the dividing line, with the upstream homologous arm being... snaA The start codon ATG proceeds to the first 500 bp, amplifying the upstream homologous arm using snaA-US-F1 / snaA-US-R1; the downstream homologous arm is... snaA The stop codon TTA is followed by a 500 bp amplification sequence using snaA-DS-F1 / snaA-DS-R1 primers. snaA DNA fragments of the upstream and downstream homologous arms of the gene were extracted and purified. The recovered upstream and downstream homologous arm fragments were ligated to the linearized plasmid PK18-mobSacB using a one-step cloning kit from Novizan. Plasmid linearization was achieved using the restriction enzyme BamHI (purchased from Thermo Fisher Scientific). The ligation product was then transformed into... snaA DH5α was verified using primers PK18-YZ-F1 / R1. If the verification was successful, the knockout plasmid PK18-snaA was obtained.

[0040] (4-2) Using mutated genes E. coli Taking the mutant plasmid PK18-lysC(T311I) as an example, the encoding lysC The 311th amino acid, threonine (T), is mutated to isoleucine (I), and the mutation was found. lysC exist lysC The location of the ATCC 13032 strain on the genome, and in the form of C.glutamicum Using the 311th amino acid threonine as the dividing line, the upstream homologous arm is 500 bp upstream of the threonine encoded by the base acc, with acc replaced by atc. The upstream homologous arm is amplified and purified using lysC-US-F1 / lysC-US-R1. The downstream homologous arm is 500 bp downstream of the threonine encoded by the base acc, with acc replaced by atc. The downstream homologous arm is amplified and purified using lysC-DS-F1 / lysC-DS-R1. The recovered upstream and downstream homologous arm fragments are ligated to the linearized plasmid PK18-mobSacB using Novizan's one-step cloning kit and transformed. Once the transformation is successful, the mutant plasmid PK18-lysC(T311I) is obtained.

[0041] (4-3) To enhance genes lysC Taking the enhancement plasmid PK18-PH36-pyc as an example, find pyc exist pyc The location of the ATCC 13032 strain on the genome, in order to C.glutamicum The start codon ATG serves as the dividing line, with the upstream homologous arm being... pyc The first 500 bp from the start codon ATG is amplified and purified using pyc-US-F1 / pyc-US-R1 primers; the downstream homologous arm is amplified from the first 500 bp after the start codon ATG of pyc using pyc-DS-F1 / pyc-DS-R1 primers. pyc The downstream homologous arm of the gene was purified, and the linear fragment of PH36 was amplified using the PH36 fragment as a template and PH36-F1 / R1 primers; pyc Using the upstream homologous arm and the linear fragment of PH36 as templates, and pyc-US-F1 / PH36-R1 as a template, amplification was performed to obtain a product containing the PH36 promoter. pyc The upstream homologous arm was purified, and the recovered downstream homologous arm fragment and the fragment containing the PH36 promoter were collected. pyc The upstream homologous arm and linearized plasmid were ligated and transformed using Novizan's one-step cloning kit. Once the transformation was successful, PK18-PH36-pyc was obtained.

[0042] (4-4) Insert pycTaking the gene and integration plasmid PK18-aspB::PH36-aspD as an example, find aspD Genes in apsB The position of the homologous arm on the genome of ATCC 13032 strain, with the start codon ATG and stop codon TTA of aspB as the boundary, is as follows: upstream homologous arm is 1000 bp ahead of the start codon ATG of aspB, amplified using aspB-US-F1 / snaA-US-R1; downstream homologous arm is... C.glutamicum Following the stop codon TTA, a 1000 bp segment was amplified and purified using apsB-DS-F1 / apsB-DS-R1 primers; aspB Using the gene sequence as a template, amplification was performed using primers aspD-F1 / R1. aspD Gene and purification; using the PH36 fragment as a template and PH36-F1 / R1 as primers, the linear PH36 fragment was amplified; aspD Using the gene and the linearized PH36 fragment as templates, and PH36-F1 / aspD-R1 as primers, amplification was performed to obtain a sample containing the PH36 promoter. aspD Gene fragments were extracted and purified. The three fragments and the linearized plasmid were then ligated and transformed using Novizan's one-step cloning kit, and the correct result was obtained as PK18-aspB::PH36-aspD.

[0043] (5) Preparation of competent cells of Corynebacterium glutamicum ATCC 13032: The preserved Corynebacterium glutamicum ATCC13032 glycerol tubes were streaked in antibiotic-free LB solid medium. The culture conditions were: 30℃ for two days to obtain single colonies. The above single colonies were cultured overnight in antibiotic-free LB medium. 10% of the cultured bacterial solution was taken and placed in 100 mL of LBHIS liquid medium and cultured for about 3 h. The culture conditions were: 30℃, 220 rpm. All bacterial solutions were collected and pre-cooled on ice for 10 min, followed by centrifugation at 4℃, 5000 rpm for 10 min. The supernatant was discarded, and 10 mL of 10% pre-cooled glycerol was added to each tube. The mixture was gently mixed and then centrifuged at 4℃, 5000 rpm for 10 min. The above step was repeated twice. The supernatant was discarded, and 500 μL of 10% pre-cooled glycerol was added to each tube. The sample can be aliquoted in μL and used immediately for transformation or stored at -80°C for prompt use. Competent cells of the glutamate-producing rod-shaped strain ATCC 13032 are denoted as Cg.

[0044] (6) Escherichia coli aspD Preparation of 21(DE3) competent cells: Take preserved Escherichia coli BLStreak 21(DE3) glycerol tubes in antibiotic-free LB solid medium. Culture conditions: overnight at 37°C. Single colonies were obtained. These single colonies were then cultured overnight in antibiotic-free LB medium. 1 mL of the cultured bacterial solution was transferred to 50 mL of LB liquid medium and cultured for approximately 2 hours at 37°C and 220 rpm. All bacterial solutions were collected and pre-chilled on ice for 30 min, followed by centrifugation at 4°C and 5000 rpm for 3 min. The supernatant was discarded, and 25 mL of 0.1 M CaCl2 was added to each tube. The mixture was gently mixed and then centrifuged at 4°C and 5000 rpm for 5 min. The supernatant was discarded, and 1 mL of 15% pre-chilled glycerol containing 0.1 M CaCl2 was added. The mixture was aliquoted into 100 μL tubes and can be used immediately for transformation or stored at -80°C for immediate use.

[0045] I. Strains for one-step synthesis of spermidine, their construction methods, and applications from BL It can be seen that the *Corynebacterium glutamicum* strain lacks genes related to spermidine synthesis, such as ornithine decarboxylase. Figure 1 Genes and carboxysemine dehydrogenase speC Genes and carboxysemine decarboxylase CASDH Therefore, by introducing exogenous enzymes, a metabolic pathway capable of synthesizing putrescine and spermidine was constructed, thereby creating a high-yield strain capable of de novo synthesis of spermidine. 1.1 Construction of recombinant plasmid PEC-XK99E-speC-CASDH-CASDC

[0046] Using the speC gene sequence as a template, and speC-F1 / R1 as primers, [the following was performed]... CASDC Gene amplification was performed; using the PEC-XK99E plasmid as a template, linearization with the restriction enzyme BamHI was carried out to obtain the linearized plasmid PEC-XK99E. Using the carboxysemine dehydrogenase sequence BF-CASDH as a template and CASDH1-F1 / CASDH1-R1 as primers, the CASDH fragment was amplified. The same method was used for CL-CASDH and AT-CASDH fragments to obtain CASDH fragments from different sources carrying the corresponding promoters. The carboxysemine decarboxylase At- speC Using CASDC1-F2 / R2 as primers, the corresponding [amplification method] was performed. CASDC Fragment, Cj- [[ID=%6]]CASDC Bt-CASDC also operates in this way, obtaining samples from different sources. CASDC Fragments. Amplified and purified separately. CASDC Genes, from different sources speC and CASDHThe fragments, along with the linearized BamHI-based PEC-XK99E plasmid, were cloned in one step using the Novizan C115 kit and transformed. CASDC DH5α and sequencing verification yielded 9 recombinant plasmids: PEC-XK99E-speC-CASDH1-CASDC1, PEC-XK99E-speC-CASDH1-CASDC2, PEC-XK99E-speC-CASDH1-CASDC3, PEC-XK99E-speC-CASDH2-CASDC1, PEC-XK99E-speC-CASDH2-CASDC2, PEC-XK99E-speC-CASDH2-CASDC3, PEC-XK99E-speC-CASDH3-CASDC1, PEC-XK99E-speC-CASDH3-CASDC2, and PEC-XK99E-speC-CASDH3-CASDC3. 1.2 Construction and fermentation culture of recombinant Corynebacterium glutamicum

[0047] Competent cells (Cg) of *Rhizoctonia glutamicum* strain were added to 1 μL of each of the nine recombinant plasmids (PEC-XK99E-speC-CASDH-CASDC), pre-cooled on ice for 5 min, and then transferred to 0.1 cm electroporation cuvettes for electroporation at 1.8 kV for 4 ms. 1 mL of LBHIS was added and mixed thoroughly. The mixture was then incubated at 30 ℃ and 220 rpm for 2 h. The cells were centrifuged and spread onto LB solid medium (containing 50 mg / L kanamycin) for 48 h. Single colonies were then picked and inoculated into LB liquid medium at a 2% (v / v) inoculation rate. The cultures were incubated at 30 ℃ and 220 rpm for 16 h. The incubated LB liquid medium was then inoculated into fermentation medium at a 4% (v / v) inoculation rate. The cultures were incubated at 30 ℃ and 220 rpm for 3 h. 60 μL of 0.25 M thiocyanate was added. The IPTG mother liquor was cultured in a shaker at 30 ℃ and 220 rpm for 51 h to obtain spermidine. The yield is shown in Table 5.

[0048] Table 4. Spermine yield of recombinant strains that synthesize spermine de novo.

[0049] As can be seen from the above, when ornithine decarboxylase... [[ID=%2]]E.coli Gene, carboxysemine dehydrogenase speC Genes and carboxysemine decarboxylase CASDH When the gene is expressed in Corynebacterium glutamicum, liquid chromatography-mass spectrometry (LC-MS) analysis shows that the strain produces spermine in addition to spermidine, as well as some spermine. CASDCAs shown, this indicates that the one-step synthesis of spermidine will further consume the desired product and generate other impurities. II. Strains for Two-Step Synthesis of Spermine, Their Construction Methods, and Applications

[0050] To prevent product consumption and impurity issues caused by side reactions during the synthesis of spermidine from putrescine and aspartic-β-semial as precursors, a two-step method will be adopted to avoid side reactions: fermentation with Corynebacterium glutamicum to produce carboxylated spermidine, followed by Escherichia coli... Figure 2 21(DE3) expression It is catalyzed to be converted into spermidine. 2.1 Construction of recombinant plasmid PEC-XK99E-CASDH-speC

[0051] Taking PEC-XK99E-speC-CASDH1-ACSDC1 as an example, using the aforementioned recombinant plasmid PEC-XK99E-speC-CASDH1-ACSDC1 as a template, the primers pTRC-F1 / XK99E-C-R1 were used for amplification. BL The CASDH fragment was amplified using primers CASDH1-F1 / XK99E-H1-R1. Other related plasmids were processed in the same manner. The resulting fragment and the linearized PEC-XK99E plasmid were then cloned in one step using the C115 kit (Novizan). After verification, three recombinant plasmids were obtained: PEC-XK99E-CASDH1-speC, PEC-XK99E-CASDH2-speC, and PEC-XK99E-CASDH3-speC. 2.2 Construction of recombinant plasmid PEC-XK99E-speC-CASDH

[0052] Taking PEC-XK99E-speC-CASDH1-ACSDC1 as an example, PEC-XK99E-speC-CASDH1-ACSDC1 was used as a template and amplified using primers XK99E-F1 and CASDH1-R2. Other related plasmids were operated in the same way, transformed into E. coli DH5α and verified, and three recombinant plasmids were obtained: PEC-XK99E-speC-CASDH1, PEC-XK99E-speC-CASDH2, and PEC-XK99E-speC-CASDH3. 2.3 Construction of recombinant plasmid pET28a-CASDC

[0053] BamHⅠ linearized and purified the pET28a plasmid to obtain the linearized pET28a plasmid; it was then decarboxylated with carboxysemidine decarboxylase At- CASDC template ，Using CASDC1-F1 / R1 as primers, the CASDC fragment of carboxysaminoglycan decarboxylase was amplified and purified. speC Bt- CASDC The same method was used to obtain purified products from different sources. CASDC The fragment was ligated with the linearized pET28a plasmid, and after correct sequencing, four recombinant plasmids were obtained: pET28a-CASDC1, pET28a-CASDC2, pET28a-CASDC3, and pET28a-CASDC4.

[0054] 2.4 Escherichia coli producing carboxysemine decarboxylase CASDC 21(DE3) - Construction of CASDC Escherichia coli BL21(DE3) competent cells were placed on ice, and 1 μL of each of the four recombinant plasmids pET28a-CASDC1, pET28a-CASDC2, pET28a-CASDC3, and pET28a-CASDC4 were added to the competent cells. The cells were pre-cooled on ice for 30 min, then placed in a 42 °C water bath for 90 s. Immediately afterward, they were pre-cooled on ice for 5 min, and then 1 mL of LB liquid medium was added. The cells were incubated at 37 °C for 1 h, centrifuged, and the cells were spread onto LB solid medium (containing a final concentration of 50 mg / L kanamycin) to obtain Escherichia coli BL21(DE3) producing carboxysemine decarboxylase. - CASDC1, BL21(DE3) - CASDC2, BL21(DE3) - CASDC3, BL21(DE3) - CASDC4. 2.5 Two-step synthesis of spermidine

[0055] Referring to "1.2 Construction and Fermentation Culture of Recombinant Corynebacterium Glutamate", six recombinant Corynebacterium glutamicum were first constructed: Cg-PEC-XK99E-speC-CASDH1, Cg-PEC-XK99E-speC-CASDH2, Cg-PEC-XK99E-speC-CASDH3, Cg-PEC-XK99E-CASDH1-speC, Cg-PEC-XK99E-CASDH2-speC, and Cg-PEC-XK99E-CASDH3-speC. Then, seed culture and shake-flask fermentation culture were carried out to obtain the Corynebacterium glutamicum fermentation broth.

[0056] The recombinant *E. coli* strains BL21(DE3)-PET28a-CASDC1, BL21(DE3)-PET28a-CASDC2, BL21(DE3)-PET28a-CASDC3, and BL21(DE3)-PET28a-CASDC4 constructed above were inoculated into seed culture medium and cultured at 37 ℃ and 220 rpm for 16 h to obtain seed culture. This seed culture was then inoculated into TB medium at 1% (v / v) and cultured at 37 ℃ and 220 rpm for 3 h. Then, 100 μL of 0.25 M IPTG stock solution was added, and the culture was incubated at 20 ℃ and 220 rpm for 24 h to obtain the *E. coli* culture broth. This *E. coli* culture broth was mixed with the above-mentioned *Corynebacterium glutamicum* fermentation broth to form a mixed culture medium, adjusted to pH 7.5, and cultured at 37 ℃ and 220 rpm for 24 h. h, spermidine is obtained, and the yield is shown in Table 5. In this embodiment, the appropriate amount of E. coli culture solution is determined by mass spectrometry detection of whether there is residual carboxylated spermidine in the product after mixed culture. If there is no residue, it indicates that the enzyme activity of CASDC is sufficient, and there is no need to add more E. coli culture solution; if there is residue, it is necessary to add more E. coli culture solution.

[0057] Table 5. Spermine yield of recombinant strains obtained by the two-step method

[0058] Table 5 shows that spermine was undetectable in the two-step synthesis of spermidine, indicating that the method eliminates the influence of impurities and demonstrates the feasibility of the two-step spermidine synthesis. Additionally, ornithine decarboxylase... [[ID=-3]]CASDC Genes and carboxysemine dehydrogenase BL Genes and carboxysemine decarboxylase speC Different combinations of genes indicate that, CASDH CASDC Source of Bt- Bacteroides When spermidine was expressed in E. coli BL21(DE3), no production was observed, indicating that the enzyme was inactive. Overexpression of spermidine... thetaiotaomicron Carboxysylspermine decarboxylase At- CASDC At that time, the yield of spermidine was good, indicating that the enzyme activity from this source was suitable and could be used for the next step of the experiment. When ornithine decarboxylase... Agrobacterium tumefaciens Gene, CASDC Carboxysylspermine dehydrogenase BF- speC as well as Bacteroides fragilis Carboxysylspermine decarboxylase At- CASDHDuring the combination, it was observed that spermidine yields were relatively high. The position of the gene on the plasmid also affected spermidine yield: placing the carboxysemidine decarboxylase before the ornithine decarboxylase resulted in a higher spermidine yield than placing it after the ornithine decarboxylase. When the Corynebacterium glutamicum expression plasmid PEC-XK99E-CASDH2-speC was combined with BL21(DE3)-CASDC1, the highest spermidine yield (0.1 g / L) was obtained through the two-step method, and this plasmid was used for the next experiment. Overall, the spermidine yield was very low, possibly due to insufficient supply of the precursor substances putrescine or aspartic semialdehyde.

[0059] III. Enhancing the metabolic flux of the putrescine pathway In Corynebacterium glutamicum, putrescine synthesis occurs via two pathways: arginine and ornithine. The former involves multiple steps and is inefficient, while the latter is a single-step, highly efficient process catalyzed by ornithine decarboxylase. Experiments have shown that the latter yields 50 times more than the former, thus the ornithine pathway is preferred. Within the ornithine pathway, transcriptional regulators are repressed. Agrobacterium tumefaciens It will inhibit The expression of genes such as ornithine is reduced, thereby decreasing the flux of ornithine synthesis, while ornithine aminotransferase... [[ID=%7]]CASDC The synthesis of ornithine to arginine is catalyzed. Since this invention uses the ornithine pathway to produce putrescine, this embodiment knocks out the repressor transcriptional regulator. argR and ornithine aminotransferase argCJBD This increases the metabolic flux of ornithine and further, knocks out the spermine N1-acetyltransferase pathway, which is involved in the degradation of putrescine. argF Genes and N-acetyltransferases argR Genes, thereby further increasing the production of putrescine.

[0060] 3.1 Recombinant Corynebacterium glutamicum argF Construction First, construct the relevant knockout plasmids according to the aforementioned method. Once verified, obtain the recombinant plasmids PK18-argR, PK18-argF, PK18-snaA, and PK18-puo. Then, sequentially electropovert the verified recombinant plasmids PK18-argR, PK18-argF, PK18-snaA, and PK18-puo into competent cells. For example, electropovert PK18-argR first, and after subsequent screening, obtain *Corynebacterium glutamicum*. △ argR was used to make the bacterium competent, and then pk18-argF was electroporated to obtain C. glutamicum. △ argR △argF was used sequentially; after electroporation, 1 mL of LBHIS liquid medium was added, and the mixture was incubated at 30°C and 220 rpm for 1 h. Then, it was centrifuged and spread onto LB solid medium (50 μg / mL), and incubated at 30°C for 48 h. Once colonies grew, single colonies were picked and added to LB liquid medium containing 10% sucrose, and incubated overnight at 30°C. An appropriate amount of the bacterial solution was diluted and spread onto LB solid medium, and incubated at 30°C for two days. Single colonies from the LB solid medium were then spotted onto LB+Kan (50 μg / mL) and LB solid plates containing 10% sucrose, respectively, and incubated overnight at 30°C. Strains that did not grow on LB+Kan but grew on LB were then selected for further verification to determine if the gene had been edited. The successfully constructed strain was... snaA And named it COSP-1 。

[0061] 3.2 Two-step synthesis of spermidine Referring to the aforementioned "2.5 Two-Step Synthesis of Spermine", after co-culturing the bacterial cultures of *Corynebacterium glutamicum* COSP-1-PEC-XK99E-CASDH2-speC, which produces carboxyspermine, and *Escherichia coli* BL21(DE3)-CASDC1, which produces carboxyspermine decarboxylase, the spermine yield reached 6.5 g / L. The precursor putrescine and residual carboxyspermine were completely eliminated. This shows that increasing the flux of the ornithine pathway in the metabolism of *Corynebacterium glutamicum* and blocking the consumption of putrescine resulted in a significant increase in spermine yield. This indicates that the previous low spermine yield may have been due to insufficient putrescine precursor. After increasing the flux of the ornithine pathway, there were no residual putrescine and carboxyspermine, and further increases in the flux of both are needed.

[0062] IV. Enhancing the flux of acetyl-CoA A crucial step in the biosynthesis of putrescine and carboxysemine, precursors of spermidine, occurs within the TCA cycle. Acetyl-CoA, as the first substance to enter the TCA cycle, increases the metabolic flux of acetyl-CoA, thereby increasing the TCA flux and ultimately promoting the biosynthesis of putrescine and carboxysemine, thus boosting spermidine production. Knocking out the competing gene lactate dehydrogenase... puo Genes, pyruvate dehydrogenase C.glutamicum△argR△argF△snaA△puo Genes, phosphorylated acetyltransferases C.glutamicum△argR△argF△snaA△puo Genes, acetate kinase ldhA The gene thereby increases the flux of acetyl-CoA.

[0063] 4.1 Construction of Recombinant Corynebacterium glutamicum COSP-2~COSP-4 First, following the above-described method for constructing knockout plasmids, the relevant plasmids were constructed to obtain recombinant plasmids PK18-ldhA, PK18-poxB, and PK18-pta-ackA. Then, COSP-1 cells were prepared as electroporation competent cells, and the recombinant plasmids PK18-ldhA, PK18-poxB, and PK18-pta-ackA were sequentially subjected to electroporation and strain screening. The successfully constructed recombinant strains were COSP-1. poxB COSP-1 pta COSP-1 ackA They were named COSP-2, COSP-3, and COSP-4 respectively.

[0064] 4.2 Two-step synthesis of spermidine Referring to the above "2.5 Two-step synthesis of spermidine", the strains that produce carboxy-speridine as shown in Table 6 were first constructed, and then seed culture and shake-flask fermentation were carried out to obtain Corynebacterium glutamicum fermentation broth containing carboxy-speridine. Then, the recombinant Escherichia coli BL21(DE3)-PET28a-CASDC1 that produces carboxy-speridine decarboxylase was constructed above and seed cultured and fermented to obtain Escherichia coli culture broth. Finally, the Corynebacterium glutamicum fermentation broth was mixed with the Escherichia coli culture broth and mixed culture was carried out to produce spermidine. The yield is shown in Table 6.

[0065] Table 6. Results of spermidine production

[0066] Table 6 shows that knocking out related bypass metabolic genes to enhance acetyl-CoA flux, thereby increasing spermidine production, is effective. Through cumulative knockout, spermidine production can reach 8.42 g / L. This indicates that putrescine remains and is not completely consumed in the increase of spermidine production. Furthermore, putrescine production also increases with the increase of spermidine production, while carboxyspermine is not detected. This suggests an imbalance between glutamate and aspartate flux in the strain, with a certain amount of metabolic flux flowing to carboxyspermine and putrescine, and more metabolic flux flowing to putrescine. COSP-4 should be preferred for the next stage of experiments.

[0067] V. Enhance the flux of oxaloacetate and aspartic acid *Corynebacterium glutamicum*, a dominant strain in glutamate production, may exhibit superior glutamate metabolism compared to aspartate semialdehyde metabolism. This leads to an overflow of putrescine fluxes, with glutamate as a precursor. Therefore, increasing the metabolic fluxes of oxaloacetate (OAA) and aspartate is crucial for spermidine production. To enhance OAA and aspartate fluxes and improve carboxysaminoglycinate production, an attempt was made to knock out the pyruvate dehydrogenase E1 complex. -△ldhAGenes, phosphoenolpyruvate carboxylase -△ldhA-△poxB, Genes that enhance phosphoenolpyruvate carboxylase -△ldhA-△poxB-△pta-ackA Genes, pyruvate carboxylase aceE pck ppc pyc Gene.

[0068] In aspartate metabolism, aspartate aminotransferase aspB The synthesis of aspartate from oxaloacetate via gene catalysis requires glutamate as an amino donor. However, glutamate synthesis requires NADPH as a cofactor, and putrescine synthesis also requires glutamate as a precursor. Both glutamate and putrescine compete for glutamate, and the latter requires more NADPH, which is detrimental to the synthesis of carboxysemine. Studies have reported that aspartate dehydrogenase... apsD Genes can directly use free NH4 + As an amino group donor and with NADH as a cofactor, the aspartate aminotransferase in the strain was used. aspB The gene was directly replaced with aspartate dehydrogenase. aspD Genes unlock the restriction of glutamate and increase the metabolic flux of aspartate.

[0069] 5.1 Construction of Recombinant Corynebacterium glutamicum COSP-5~COSP-13 Knockout plasmids PK18-aceE and PK18-pck were constructed according to the aforementioned knockout plasmid construction method; enhancement plasmids PK18-PH36-ppc and PK18-PH36-pyc were constructed according to the aforementioned enhancement plasmid construction method; and integration plasmid PK18-aspB::PH36-aspD was constructed according to the aforementioned integration plasmid construction method. The recombinant strain COSP-4 was prepared into competent cells, and the five obtained recombinant plasmids were electroporated into the competent cells for gene editing. The successfully constructed strain was COSP-4. -△aceE, COSP-4 -△ aceE△pck, COSP-4 -△aceE△pck- PH36 -ppc, COSP-4 -△aceE△pck- PH36 -ppc PH36 -pyc, COSP-4 -△aceE△pck- PH36 -ppc- PH36 -pyc△ aspB::PH36-aspD, COSP-4 -△pck, COSP-4 -△ pck- PH36 -ppc, COSP-4 -△pck- PH36 -ppc PH36 -pyc, COSP-4 -△pck- PH36-ppc- PH36 -pyc△ aspB::PH36-aspD, named COSP-5, COSP-6, COSP-7, COSP-8, COSP-9, COSP-10, COSP-11, COSP-12, COSP-13 respectively.

[0070] 5.2 Two-step synthesis of spermidine Referring to the above "2.5 Two-step synthesis of spermidine", the strains that produce carboxy-speridine as shown in Table 7 were first constructed, and then seed culture and shake-flask fermentation were carried out to obtain Corynebacterium glutamicum fermentation broth containing carboxy-speridine. Then, the recombinant Escherichia coli BL21(DE3)-PET28a-CASDC1 that produces carboxy-speridine decarboxylase was constructed above and seed cultured and fermented to obtain Escherichia coli culture broth. Finally, the Corynebacterium glutamicum fermentation broth was mixed with the Escherichia coli culture broth and mixed culture was carried out to produce spermidine. The yield is shown in Table 7.

[0071] Table 7. Results of spermidine production

[0072] The data shown in Table 7 indicate that: without knocking out the pyruvate complex... aceE In this case, enhancing other genes increased spermidine production (11.1 g / L), but putrescine production remained relatively high; while knocking out... aceE Subsequently, the metabolic flux of OAA and aspartic acid was further increased, thereby increasing the yield of the substrate carboxyspermine and thus increasing spermine (12.8 g / L), while the yield of putrescine further decreased, indicating that the strategy was effective and COSP-9 should be preferred for the next experiment.

[0073] VI. Enhancing the flux of aspartic semialdehyde Aspartic acid derivative aspartate semialdehyde is a key limiting step in the synthesis of carboxysemine. The catalytic conversion of aspartic acid to aspartic semialdehyde requires two enzymes: aspartate kinase. lysC Genes and aspartate semialdehyde dehydrogenase asd Gene. Aspartate kinase lysC The gene is susceptible to inhibition by lysine and threonine, leading to a decrease in the flux of aspartate phosphate, which in turn reduces the metabolic flux of aspartate semialdehyde. Literature indicates that aspartate kinase... lysC Mutations at the T311I site of the gene can relieve feedback inhibition by lysine and threonine. Therefore, to increase the flux of aspartic semialdehyde and enhance aspartate kinase... lysC Genes and aspartate semialdehyde dehydrogenase asdGenes: On the one hand, the effect of aspartate kinase lysC The gene undergoes a mutation at site T311I, which modifies aspartate kinase. lysC Genes and aspartate semialdehyde dehydrogenase asd The gene promoter was replaced with the PH36 promoter; on the other hand, a meaningless site on the genome, cg1960, was selected for insertion into aspartate kinase. lysC Genes and aspartate semialdehyde dehydrogenase asd Genes, two copies of the gene are made.

[0074] 6.1 Construction of Recombinant Corynebacterium glutamicum COSP-14~COSP-18 The mutant plasmid PK18-lysC(T311I) was constructed according to the aforementioned mutant plasmid construction method; the enhancement plasmids PK18-PH36-lysC and PK18-PH36-asd were constructed according to the aforementioned enhancement plasmid construction method; and the integrative plasmid PK18-cg1960::PH360-lysC(T311I)-PH36-asd was constructed according to the integrative plasmid construction method. The recombinant strain COSP-9 was prepared into competent cells, and the above four recombinant plasmids were electroporated into the competent cells for gene editing. The successfully constructed strains were COSP-9. -lysC T311I 、 COSP-9 - PH36 -asd, COSP-9 - PH36 -lysC T311I 、 COSP-9 - PH36 -lysC T311I -PH36-asd, COSP-9 - PH36 -lysC T311I -PH36-asd△cg1960- PH36 -lysC T311I -PH36-asd They were named COSP-14, COSP-15, COSP-16, COSP-17, and COSP-18 respectively.

[0075] 6.2 Two-step synthesis of spermidine Referring to the aforementioned "2.5 Two-Step Synthesis of Spermine", the carboxy-spermine-producing strains shown in Table 8 were first constructed, and then seed culture and shake-flask fermentation were performed to obtain *Corynebacterium glutamicum* fermentation broths containing carboxy-spermine. Then, the aforementioned recombinant *Escherichia coli* BL21(DE3)-PET28a-CASDC1 with carboxy-spermine decarboxylase was used for seed culture and fermentation to obtain *E. coli* culture broth. Finally, the *Corynebacterium glutamicum* fermentation broth was mixed with the *E. coli* culture broth for co-culture to produce spermine, with yields shown in Table 8. Figure 3 As shown.

[0076] Table 8. Results of spermidine production

[0077] Table 8 and Figure 3 This indicates that only enhancement is needed. asd Genes had no significant effect on increasing spermidine, but on... lysC After gene mutation, the feedback inhibition by lysine and threonine was removed, resulting in a significant increase in spermidine production. This indicates that aspartic semialdehyde is indeed the bottleneck in the synthesis of carboxyspermine. If the metabolic flux of carboxyspermine is insufficient, and spermidine precursors are lacking, it is difficult to increase spermidine production. Simultaneously, enhancing... lysC Genes and asd The gene significantly enhances spermidine; on the other hand, the derivatives of aspartic semialdehyde, lysine and homoserine, also increase with the increase of aspartic semialdehyde flux, indicating that some of the aspartic semialdehyde flux flows to lysine and homoserine. Therefore, COSP-18 should be preferred for the next step of the experiment.

[0078] VII. Blocking the aspartic semialdehyde bypass To direct all carbon flux to carboxysemine, bypass metabolic pathways such as homoserine dehydrogenase were knocked out of aspartate semialdehyde. hom Gene 、 Diaminopimelic acid dehydrogenase ddh Genes, pimelic acid decarboxylase lysA1 and lysA2 Genes, lysine transporters lysE Gene.

[0079] 7.1 Construction of recombinant Corynebacterium glutamicum COSP-19~COSP-23 Knockout plasmids PK18-hom, PK18-ddh, PK18-lysA1, PK18-lysA2, and PK18-lysE were obtained according to the aforementioned knockout plasmid construction method. COSP-18 cells were prepared as electroporation competent cells, and the five recombinant plasmids obtained in step (1) were sequentially electroporated and the strains were screened, successfully constructing recombinant strains:COSP-△hom, COSP-△hom△ddh, COSP-△hom△ ddh△lysA1, COSP-△hom△ddh△lysA1△lysA2, COSP-△hom△ddh△lysA1△lysA2△lysE They were named COSP-19, COSP-20, COSP-21, COSP-22, and COSP-23 respectively. 。

[0080] 7.2 Two-step synthesis of spermidine Referring to the above "2.5 Two-step synthesis of spermidine", the strains that produce carboxy-speridine as shown in Table 9 were first constructed, and then seed culture and shake-flask fermentation were carried out to obtain Corynebacterium glutamicum fermentation broth containing carboxy-speridine. Then, the recombinant Escherichia coli BL21(DE3)-PET28a-CASDC1 that produces carboxy-speridine decarboxylase was constructed above and seed cultured and fermented to obtain Escherichia coli culture broth. Finally, the Corynebacterium glutamicum fermentation broth was mixed with the Escherichia coli culture broth and mixed culture was carried out to produce spermidine. The yield is shown in Table 9.

[0081] Table 9. Results of spermidine production

[0082] Table 9 shows that knocking out the homoserine pathway had a good effect on increasing spermidine production, reaching 27.1 g / L. This may be because the metabolism of the lysine pathway is weaker than that of the homoserine pathway at this time. Therefore, compared with the control, the production of the gene knocking out the lysine pathway only increased by 0.5 g / L. Knocking out the lysine transporter lysE had no significant effect, possibly because the lysine flux was already weak enough at this time, or the combined knockout of ddh, lysA1, and lysA2 completely blocked the lysine pathway. The combined knockout of the homoserine and lysine pathways could increase the spermidine production to 27.62 g / L.

[0083] 8. Balance NADPH cofactors NADPH is an important cofactor in organisms, participating in many biological reactions. To address NADPH deficiency, knockout of NADP+-dependent glucose dehydrogenase is necessary. butA and fabG The gene can inactivate glucose dehydrogenase, thereby increasing the enzyme activity of glucose-6-phosphate dehydrogenase and glucuronide-6-phosphate dehydrogenase, enhancing the metabolic flux of the pentose phosphate pathway, and thus increasing the synthesis of NADPH. gapN The gene catalyzes the synthesis of glyceraldehyde-3-phosphate, while simultaneously generating NADPH. Studies have shown that overexpression of pyridine nucleotide transhydrogenase... pntAB Genes can promote the regeneration from NADH to NADPH; therefore, in this invention, endogenous NADP is knocked out. + glucose-dependent dehydrogenase butA andfabG Genes, while simultaneously introducing exogenous pyridine nucleotide transhydrogenase pntAB Genes and glyceraldehyde-3-phosphate dehydrogenase gapN Genes enable the strain to achieve cofactor balance.

[0084] 8.1 Construction of Recombinant Corynebacterium glutamicum COSP-24~COSP-27 Knockout plasmids PK18-butA and PK18-fabG were constructed using the previously described knockout plasmid construction method; integrator plasmids PK18-puo::PH36-pntAB and PK18-snaA::PH36-gapN were constructed using the aforementioned integrator plasmid construction method. The recombinant Corynebacterium glutamicum strain COSP-23 was prepared as competent cells, and the recombinant plasmids PK18-butA, PK18-fabG, PK18-puo::PH36-pntAB, and PK18-snaA::PH36-gapN were sequentially electroporated into the competent cells for gene editing. The successfully constructed strains were COSP-23. △butA COSP-23 △butA△fabG, COSP-23 △butA△fabG△puo:: PH36 -pntAB, COSP-23 △butA△fabG△puo:: PH36 -pntAB△snaA ::PH36- gapN They were named COSP-24, COSP-25, COSP-26, and COSP-27 respectively.

[0085] 8.2 Two-step synthesis of spermidine Referring to the above "2.5 Two-step synthesis of spermidine", the strains that produce carboxysemidine as shown in Table 10 were first constructed, and then seed culture and shake-flask fermentation were carried out to obtain Corynebacterium glutamicum fermentation broth containing carboxysemidine. Then, the recombinant Escherichia coli BL21(DE3)-PET28a-CASDC1 that produces carboxysemidine decarboxylase was constructed above and seed cultured and fermented to obtain Escherichia coli culture broth. Finally, the Corynebacterium glutamicum fermentation broth was mixed with the Escherichia coli culture broth and mixed culture was carried out to produce spermidine. The yield is shown in Table 10.

[0086] Table 10. Results of spermidine production

[0087] Table 10 shows that knocking out endogenous NADP... + glucose-dependent dehydrogenase butA and fabG The gene may not have significantly increased NADPH, while pyridine nucleotide transhydrogenase... pntABGenes and glyceraldehyde-3-phosphate dehydrogenase gapN The introduction of COSP-27 had a significant effect, and the superposition of pntAB and gapN increased the yield of spermidine to about 28.5 g / L. COSP-27 was the preferred choice for the next step of the experiment.

[0088] IX. Genome integration reduces metabolic burden Since carrying plasmids would place a metabolic burden on the strain, it is considered to... speC Genes and CASDH Genes are integrated into the genome. The following section uses a homologous recombination double exchange system to... speC Genes and CASDH The gene was integrated into the genome of COSP-27 to construct a recombinant bacterium that produces carboxysemidine.

[0089] 9.1 Construction of carboxyspermine-producing strains COSP-28 to COSP-36 Integrative plasmids PK18-butA::sod-speC, PK18-butA::tuf-speC, PK18-butA::PH36-speC, PK18-poxB::sod-CASDH2, PK18-poxB::tuf-CASDH2, and PK18-poxB::PH36-CASDH2 were constructed according to the previously described plasmid construction method. The recombinant strain COSP-27 was prepared into competent cells, and the six recombinant plasmids obtained above were electroporated into COSP-27 competent cells for gene editing. The successfully constructed strain was COSP-27. △ butA ::sod-speC △poxB ::sod-CASDH2、COSP-27 △butA ::sod-speC △poxB ::tuf-CASDH2、COSP-27 △butA ::sod-speC △poxB ::PH36-CASDH2、COSP-27 △butA ::tuf-speC △poxB ::sod-CASDH2、COSP-27 △butA ::tuf-speC △poxB ::tuf-CASDH2、COSP-27 △butA ::tuf-speC △poxB ::PH36-CASDH2、COSP-27 △butA ::PH36-speC △poxB ::sod-CASDH2、COSP-27 △ butA::PH36-speC △poxB ::tuf-CASDH2、COSP-27 △butA ::PH36-speC △poxB ::PH36-CASDH2, respectively named COSP-28, COSP-29, COSP-30, COSP-31, COSP-32, COSP-33, COSP-34, COSP-35, and COSP-36.

[0090] 9.2 Two-step synthesis of spermidine First, single colonies of carboxyspermidine-producing strains COSP-28, COSP-29, COSP-30, COSP-31, COSP-32, COSP-33, COSP-34, COSP-35, and COSP-36 were inoculated into seed culture medium and cultured at 30 ℃ and 220 rpm for 12-16 h. The seed culture was then taken and inoculated into fermentation medium at 4% (v / v) and cultured at 30 ℃ and 220 rpm for 51 h to obtain the fermentation broth of Corynebacterium glutamicum containing carboxyspermidine. Referring to the above "2.5 Two-step synthesis of spermidine", the recombinant Escherichia coli BL21(DE3)-PET28a-CASDC1 that produces carboxysylspermine decarboxylase was used for seed culture and fermentation culture to obtain Escherichia coli culture broth; finally, the fermentation broth of Corynebacterium glutamicum was mixed with the Escherichia coli culture broth and mixed culture was carried out to produce spermidine, and the yield is shown in Table 11.

[0091] Table 11. Results of spermidine production

[0092] Table 11 shows that the expression of ornithine decarboxylase and carboxysemine dehydrogenase by promoters of different strengths and different combinations all affect spermidine production. Compared with the control group COSP-27-PEC-XK99E-CASDH2-speC, the gene expression of ornithine decarboxylase and carboxysemine dehydrogenase were significantly improved. CASDH and speC The integration strength into the COSP-27 genome requires different attempts; stronger is not always better. The combination of using the PH36 promoter to drive the expression of speC and using the tuf promoter to drive the expression of CASDH has the highest efficiency, with spermidine production reaching 29.3 g / L.

[0093] In summary, this invention, through multi-dimensional and progressive metabolic engineering, constructs a highly efficient two-step spermidine synthesis system using *Corynebacterium glutamicum* and *Escherichia coli*. It provides a fermentation-enzyme-catalyzed two-step method: first, recombinant *Corynebacterium glutamicum* is used to ferment and produce the key precursor, carboxylated spermidine, and then a carboxylated spermidine-containing decarboxylase is used for decarboxylation. CASDC The gene-modified E. coli BL21 performs enzymatic directed synthesis of spermidine. Several genes, including ornithine decarboxylase, are introduced or upregulated in recombinant Corynebacterium glutamicum strains. speC Gene, carboxysemine dehydrogenase CASDH Genes, aspartate dehydrogenase aspD Genes, aspartate kinase lysC Genes, aspartate semialdehyde dehydrogenase asd Genes, phosphoenolpyruvate carboxylase ppc Genes, pyruvate carboxylase pyc Based on genes, knock out bypass metabolic genes such as ornithine carbamoyltransferase. argF Gene 、 Arginine-repressed transcription regulator argR Genes, spermine N1-acetyltransferase snaA Genes, N-acetyltransferase puo Gene 、 NADP + glucose-dependent dehydrogenase butA and fabG Genes, lactate dehydrogenase ldhA Genes, pyruvate dehydrogenase E1 complex aceE Genes, phosphoenolpyruvate carboxylase pck Genes, pyruvate dehydrogenase poxB Genes, phosphorylated acetyltransferases pta Genes, acetate kinase ackA Lysine transporter lysE Gene, diaminopimelic acid dehydrogenase ddh Genes, homoserine dehydrogenase hom Genes, pimelic acid decarboxylase ​ and ​ Genes, and the introduction of pyridine nucleotide transhydrogenase. ​ Genes, NADP + Glyceraldehyde-3-phosphate dehydrogenase-dependent ​ Gene. After inoculating this recombinant strain into shake flasks and culturing for 51 h, Escherichia coli BL21(DE3) cells overexpressing carboxysemine decarboxylase CASDC were fed into fermentation broth containing carboxysemine for reaction. The spermine yield was 29.3 g / L, with no byproduct residue. It has the advantages of high yield, high efficiency and simple process, laying the foundation for industrial production.

[0094] Finally, it should be noted that the above embodiments are only used to explain the present invention and are not intended to limit it. Although detailed descriptions have been provided with reference to preferred embodiments, those skilled in the art can make modifications or equivalent substitutions thereto, which, without departing from the spirit of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A bacterial composition for producing spermidine, characterized in that, include: Strains producing carboxysemine decarboxylase include *Escherichia coli* and *Trichoderma* strains and the carboxysemine decarboxylase introduced into them. CASDC Gene; Strains that produce carboxyseminated spermidine include Corynebacterium glutamicum and Ornithine decarboxylase. speC Genes and carboxysemine dehydrogenase CASDC Gene, the ornithine decarboxylase speC Genes and carboxysemine dehydrogenase CASDC All genes are located in the plasmid of the *Corynebacterium glutamicum* or integrated into its genome, and the carboxysemine decarboxylase... CASDC Genes originate from Agrobacterium tumefaciens or Campylobacter jejuni .

2. The bacterial composition according to claim 1, characterized in that, The Corynebacterium glutamicum chassis bacteria express ornithine decarboxylase driven by a strong promoter. speC Genes and carboxysemine dehydrogenase CASDH The gene, wherein the strong promoter is at least one of the PH36 promoter, sod promoter, and tuf promoter as shown in SEQ ID NO. 1, wherein the nucleotide sequence of the sod promoter is the first 200 bp of the start codon ATG in the *Corynebacterium glutamicum* superoxide dismutase sod gene as shown in NCBI-gene database accession number Cg3237; and the nucleotide sequence of the tuf promoter is the first 200 bp of the start codon ATG in the *Corynebacterium glutamicum* elongation factor tuf gene as shown in NCBI-gene database accession number NCgl0480.

3. The bacterial composition according to claim 1 or 2, characterized in that, The transcriptional repression regulator was knocked out in Corynebacterium glutamicum. argR ornithine aminotransferase argF Spermine N1-acetyltransferase snaA Genes, N-acetyltransferase puo Genes, lactate dehydrogenase ldhA Genes, pyruvate dehydrogenase poxB Genes, phosphorylated acetyltransferases pta Genes, acetate kinase ackA Genes, pyruvate dehydrogenase E1 complex aceE Genes and phosphoenolpyruvate carboxylase pck At least one of the genes.

4. The bacterial composition according to claim 3, characterized in that, The Corynebacterium glutamicum basalis strain also showed enhanced phosphoenolpyruvate carboxylase activity. PPC Genes, pyruvate carboxylase pyc Genes, aspartate kinase lysC Genes and aspartate semialdehyde dehydrogenase asd The expression of at least one gene.

5. The bacterial composition according to claim 4, characterized in that, The aspartate kinase lysC Genes can be enhanced in at least one of the following ways: 1) Aspartate kinase lysC The gene was replaced with the T311I mutant; 2) Aspartate kinase lysC The gene promoter was replaced with the PH36 promoter, as shown in SEQ ID NO. 1; 3) Aspartate kinase lysC The gene was integrated into the cg1960 site in the genome.

6. The bacterial composition according to claim 4 or 5, characterized in that, The aspartate semialdehyde dehydrogenase asd Genes can be enhanced in at least one of the following ways: 1) Aspartate semialdehyde dehydrogenase asd The gene promoter is replaced with the PH36 promoter described above; 2) Aspartate semialdehyde dehydrogenase asd The gene was integrated into the cg1960 site in the genome.

7. The bacterial composition according to claim 4, characterized in that, The homoserine dehydrogenase was also knocked out in the Corynebacterium glutamicum basalis strain. hom Gene 、 Diaminopimelic acid dehydrogenase ddh Genes, pimelic acid decarboxylase lysA1 and lysA2 Genes, lysine transporters lysE At least one of the genes.

8. The bacterial composition according to claim 4 or 7, characterized in that, The genome of Corynebacterium glutamicum also showed that endogenous NADP was knocked out. + glucose-dependent dehydrogenase butA and fabG The gene also introduced pyridine nucleotide transhydrogenase. pntAB Genes and glyceraldehyde-3-phosphate dehydrogenase gapN Gene.

9. A method for producing spermidine, using the strain combination described in any one of claims 1-8, characterized in that, Includes the following steps: Fermentation to produce carboxysemine decarboxylase: The strain producing carboxysemine decarboxylase was cultured in TB medium to obtain a bacterial culture containing carboxysemine decarboxylase; Fermentation precursor: The strain that produces carboxysylspermine is fermented to obtain a fermentation broth containing carboxysylspermine; Mixed enzyme-catalyzed production of spermidine: The bacterial broth containing carboxysemidine decarboxylase and the fermentation broth containing carboxysemidine are uniformly mixed and fermented at pH 6-8 and 25-40℃ to catalyze the conversion of carboxysemidine to spermidine.

10. Use of a bacterial composition according to any one of claims 1-8 or the method according to claim 9 in the preparation of spermidine or its derivatives.

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