Construction and application of DNA scaffold assisted escherichia coli producing tetrahydropyrimidine

Abstract

The application discloses a DNA scaffold assisted Escherichia coli for producing tetrahydropyrimidine and construction and application thereof, and belongs to the field of genetic engineering and fermentation engineering. The application is characterized in that: by fusing tetrahydropyrimidine synthesis pathway enzymes (EctA, EctB and EctC) and zinc finger domain (ZFa, ZFb and ZFc), replacing a suitable replicon for an expression plasmid, selecting a suitable enzyme binding site interval and enzyme binding direction for a DNA scaffold structure, optimizing a repeated unit of the DNA scaffold, adjusting the stoichiometric number of different enzyme binding sites on the DNA scaffold, increasing the expression amount of a rate-limiting enzyme EctB and changing a fermentation temperature conversion time, a high-yield tetrahydropyrimidine synthesis strain MWZ003 / pFV30 is obtained. Under the condition that the temperature is converted at 4h after the fermentation is started, the extracellular tetrahydropyrimidine yield of the strain reaches 22.79g·L ‑1 , which is close to 1.92 times higher than that of an initial strain MWZ003 / pFV4, and the glucose conversion rate is 0.65g·g ‑1 . The application provides a new strategy for improving the tetrahydropyrimidine yield in Escherichia coli.

Description

Technical Field

[0001] This invention relates to the construction and application of a DNA scaffold-assisted Escherichia coli production of tetrahydropyrimidine, belonging to the fields of genetic engineering and fermentation engineering. Background Technology

[0002] With the development of metabolic engineering and synthetic biology, numerous biotechnologies have been applied to introduce heterologous biosynthetic pathways to produce high-value-added products. Recombinant strains are mainly constructed through overexpression of heterologous enzymes, disruption of competing pathways, and weakening of unnecessary feedback regulation. However, heterologous biosynthesis still faces many challenges, including inherent defects in metabolic reactions, byproduct formation, accumulation of toxic intermediates, and competition between intermediates and bypass pathways. Recently, researchers have developed a novel approach called scaffold systems, which makes enzymatic reactions more efficient both spatially and temporally by increasing the local concentration of intermediates and reducing the diffusion of toxic intermediates. Using scaffold systems to precisely assemble metabolic pathways for product synthesis avoids interference from other adverse metabolic reactions, significantly improving the efficiency of product synthesis. For example, Dueber et al. developed a protein scaffold based on metazoan signal protein interaction domains to assemble metabolic enzymes, which significantly increased the production of mevalonic acid and gluconic acid. Delebecque et al. designed an RNA scaffold to link two metabolic enzymes involved in biohydrogen production, increasing hydrogen production by 48-fold compared to not using a scaffold. Conrado et al. constructed a DNA scaffold system by fusing target bioenzymes with zinc finger (ZF) domains to specifically target them onto the DNA double helix. This system was used to improve the production of resveratrol, 1,2-propanediol, and mevalonic acid.

[0003] Compared to protein and RNA scaffolds, DNA scaffolds offer several advantages. First, DNA-based scaffolds allow enzymes to be arranged in a predetermined sequence based on the highly predictable local structure of DNA. Since the DNA double helix rotates 360° every 10 bp, adding a 1 bp spacing between ZF binding sites on the scaffold adds 36° of rotation in an axial view. When the distance between enzymes on the scaffold is set to a multiple of 10 bp, the enzymes will lie in the same plane, which facilitates the efficient conversion of intermediate metabolites. Therefore, the spatial orientation of binding proteins can be adjusted by fine-tuning the spacing between enzymes on the scaffold. Second, the in vivo stability of DNA scaffolds is largely sequence-independent. This means that numerous structures of almost any sequence and length can be generated without reducing scaffold usability, a promising approach that avoids the fragility of protein and RNA-based scaffolds, including misfolding, degradation, and aggregation. Meanwhile, even subtle variations in protein and RNA sequences can have a significant adverse impact on their folding and stability. Third, many different DNA-binding proteins exist in nature, such as zinc finger proteins (ZFPs). ZFP consists of multiple individual zinc finger domains, each of which typically recognizes a 3-bp sequence in double-stranded DNA, and has the ability to specifically recognize and bind to a particular DNA sequence.

[0004] Tetrahydropyrimidine, also known as 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, is a heterocyclic amino acid derived from aspartic acid with a relative molecular weight of 142.16. It was first discovered in the halophilic phototrophic bacterium *Ectothiorhodospira halochloris*. Tetrahydropyrimidine is a valuable stabilizer and protectant widely used in biopharmaceuticals and cosmetic additives. A technique known as "bacterial milking" has been developed to produce tetrahydropyrimidine on a large scale using the halophilic bacterium *Halomonas elongate*, but the high-salt environment accelerates equipment corrosion and complicates downstream processing. Although subsequent fermentation with non-halophilic strains (*Escherichia coli* and *Corynebacterium glutamicum*) successfully produced tetrahydropyrimidine, glucose conversion and tetrahydropyrimidine yield remain major challenges. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention utilizes a DNA scaffold system to assist in the synthesis of tetrahydropyrimidine in *E. coli*. Through a series of modifications and optimizations to the DNA scaffold system, a final strain with an optimal DNA scaffold was obtained, enabling efficient synthesis of tetrahydropyrimidine.

[0006] The first objective of this invention is to provide a recombinant *Escherichia coli* that overexpresses ectA-ZFa expression cassettes, ectB-ZFb expression cassettes, ectC-ZFc expression cassettes, the gene ppc encoding phosphoenolpyruvate carboxylase, the gene aspDH encoding aspartate dehydrogenase, and the gene EclysC* encoding aspartate kinase III; the recombinant *Escherichia coli* also contains a DNA scaffold structure.

[0007] The DNA scaffold structure includes 1 to 12 repeating units, each repeating unit consisting of an ectA enzyme binding site, an ectB enzyme binding site, and an ectC enzyme binding site.

[0008] The DNA scaffold structure is located downstream of the ectC-ZFc expression cassette and upstream of the ppc gene.

[0009] In one embodiment, there is a 7-15 bp spacer sequence between the ectA enzyme binding site, the ectB enzyme binding site, and the ectC enzyme binding site.

[0010] In one embodiment, the stoichiometric ratios between the ectA enzyme binding site, the ectB enzyme binding site, and the ectC enzyme binding site are 1:1:1, 1:2:1, 1:1:2, 2:1:1, 1:2:2, 2:1:2, or 2:2:1.

[0011] In one embodiment, the ectA-ZFa expression cassette binds to the ectA enzyme binding site, the ectB-ZFb expression cassette binds to the ectB enzyme binding site, and the ectC-ZFc expression cassette binds to the ectC enzyme binding site.

[0012] In one embodiment, the ectA-ZFa expression cassette binds positively to the ectA enzyme binding site, the ectB-ZFb expression cassette binds positively to the ectB enzyme binding site, and the ectC-ZFc expression cassette binds positively to the ectC enzyme binding site.

[0013] In one embodiment, the ectA-ZFa expression cassette binds in reverse to the ectA enzyme binding site, the ectB-ZFb expression cassette binds in reverse to the ectB enzyme binding site, and the ectC-ZFc expression cassette binds in reverse to the ectC enzyme binding site.

[0014] In one embodiment, the DNA scaffold structure further includes a B1006 terminator.

[0015] In one embodiment, the recombinant Escherichia coli uses pFT24 as a vector.

[0016] In one embodiment, the 7-15 bp interval sequences are ATTACCC, CTGGCTGAA, AGCTGGCTGAA, AGCTGATGCTGAA, and GATGGATATAGCTTT, respectively.

[0017] In one embodiment, the vector pFT24 contains the gene encoding the temperature-sensitive protein CI857 and the replicon pMB1.

[0018] In one implementation, P is used R -P L Promoter expression ectA-ZFa expression frame, using P R The promoter expresses the ectB-ZFb expression frame, using P J23115 Promoter expression of gene ppc, using P R Promoter expression of the aspDH gene, using P R Promoter expression of gene EclysC*.

[0019] In one implementation, Escherichia coli MWZ003 is used as the host.

[0020] In one embodiment, the nucleotide sequence of the ectA-ZFa expression cassette is shown in SEQ ID NO.23, the nucleotide sequence of the ectB-ZFb expression cassette is shown in SEQ ID NO.24, and the nucleotide sequence of the ectC-ZFc expression cassette is shown in SEQ ID NO.25.

[0021] In one embodiment, the nucleotide sequence of the ectA enzyme binding site is GTGCGATGCC, the nucleotide sequence of the ectB enzyme binding site is GCGGCTGGG, and the nucleotide sequence of the ectC enzyme binding site is GAGGACGGC.

[0022] In one embodiment, the nucleotide sequence of the gene ppc is shown as positions 1916877 to 1919528 of NCBI accession number AP023230, the nucleotide sequence of the gene aspDH is shown as positions 3906908 to 3907711 of NCBI accession number CP034908, and the nucleotide sequence of the gene EclysC* is shown as SEQ ID NO.26.

[0023] In one implementation, the P R The nucleotide sequence of the promoter is shown in SEQ ID NO 27, the nucleotide sequence of the replicon pMB1 is shown in SEQ ID NO 28, and the nucleotide sequence of the gene encoding the temperature-sensitive protein CI857 is shown in SEQ ID NO 29.

[0024] The second objective of this invention is to provide a method for synthesizing tetrahydropyrimidine, using the aforementioned recombinant Escherichia coli as the fermentation strain to prepare tetrahydropyrimidine through fermentation.

[0025] In one embodiment, the method uses the recombinant E. coli with an initial OD 600 0.2–0.4 g of the solution was inoculated into the fermentation medium and grown at 35–38 °C for 3–5 h. Then the temperature was raised to 40–45 °C for fermentation to prepare tetrahydropyrimidine.

[0026] In one embodiment, the fermentation medium contains 30–40 g / L of glucose. -1 Yeast extract 1-5 g / L -1 Citric acid 1-5 g / L -1 (NH4)2SO4 20~30g·L -1 KH2PO4 6~8g·L -1 MgSO4·7H2O 1~5g·L -1 FeSO4·7H2O 1~10mg·L -1 MnSO4·4H2O 1~10mg·L -1 CaCO3 15~25g·L -1 Vitamin B1 0.5–1.0 mg / L -1 Vitamin H 0.1–0.5 mg / L -1 .

[0027] A third objective of this invention is to provide a DNA scaffold structure comprising 1 to 12 repeating units, wherein each repeating unit consists of an ectA enzyme binding site, an ectB enzyme binding site, and an ectC enzyme binding site.

[0028] In one embodiment, there is a 7-15 bp spacer sequence between the ectA enzyme binding site, the ectB enzyme binding site, and the ectC enzyme binding site.

[0029] In one embodiment, the stoichiometric ratios between the ectA enzyme binding site, the ectB enzyme binding site, and the ectC enzyme binding site are 1:1:1, 1:2:1, 1:1:2, 2:1:1, 1:2:2, 2:1:2, or 2:2:1.

[0030] In one embodiment, the nucleotide sequence of the ectA enzyme binding site is GTGCGATGCC, the nucleotide sequence of the ectB enzyme binding site is GCGGCTGGG, and the nucleotide sequence of the ectC enzyme binding site is GAGGACGGC.

[0031] In one embodiment, the ectA-ZFa enzyme binding site binds positively to the ectA enzyme, the ectB-ZFb enzyme binding site binds positively to the ectB enzyme, and the ectC-ZFc enzyme binding site binds positively to the ectC enzyme.

[0032] In one embodiment, the ectA-ZFa enzyme binding site binds in reverse to the ectA enzyme, the ectB-ZFb enzyme binding site binds in reverse to the ectB enzyme, and the ectC-ZFc enzyme binding site binds in reverse to the ectC enzyme.

[0033] In one embodiment, the DNA scaffold structure further includes a B1006 terminator.

[0034] In one implementation, the pFT24 plasmid is used as a vector.

[0035] A fourth object of the present invention is to provide a method for increasing the yield of tetrahydropyrimidine, wherein the method comprises inserting the above-mentioned DNA scaffold structure into cells that produce tetrahydropyrimidine.

[0036] The present invention also provides the use of the recombinant Escherichia coli or the DNA scaffold structure in the synthesis of tetrahydropyrimidine or products containing tetrahydropyrimidine.

[0037] The present invention also provides the application of the recombinant Escherichia coli or the DNA scaffold structure or the above method in the fields of fermentation, drug preparation, materials or environmental protection.

[0038] Beneficial effects

[0039] This invention designs a relatively complete DNA scaffold system. This DNA scaffold system carries ectA enzyme binding sites, ectB enzyme binding sites, ectC enzyme binding sites, and a terminator. The unit formed by the ectA enzyme binding sites, ectB enzyme binding sites, and ectC enzyme binding sites is repeated 1 to 12 times. There is a 7 to 15 bp spacer sequence between the ectA enzyme binding sites, ectB enzyme binding sites, and ectC enzyme binding sites, and the stoichiometry between the ectA enzyme binding sites, ectB enzyme binding sites, and ectC enzyme binding sites is 1:1:1, 1:2:1, 1:1:2, 2:1:1, 1:2:2, 2:1:2, or 2:2:1. The DNA scaffold system provided by this invention can be applied to cells producing tetrahydropyrimidine, promoting the synthesis of tetrahydropyrimidine in recombinant cells. Furthermore, by fusing tetrahydropyrimidine synthesis pathway enzymes (EctA, EctB, and EctC) with zinc finger domains (ZFa, ZFb, and ZFc), and replacing the replicon in the expression plasmid with a suitable one, the expression level of the rate-limiting enzyme EctB is increased, resulting in a series of high-yielding tetrahydropyrimidine recombinant strains. Among them, the recombinant strain MWZ003 / pFV30 produced 22.79 g·L⁻¹ of tetrahydropyrimidine in fermentation medium, which is 1.92 times that of the initial strain MWZ003 / pFV4, and the glucose conversion rate was 0.65 g·g⁻¹. -1 This invention provides a novel strategy for increasing the production of tetrahydropyrimidine in Escherichia coli. Attached Figure Description

[0040] Figure 1 Schematic diagram of different replicon plasmids;

[0041] Figure 2 Schematic diagram of DNA scaffold plasmids with different enzyme binding site spacing and enzyme binding direction;

[0042] Figure 3 Schematic diagram of a DNA scaffold plasmid that binds to different enzyme binding sites with reverse spacing;

[0043] Figure 4 Schematic diagram of different DNA scaffold repeat unit plasmids;

[0044] Figure 5 Schematic diagram of DNA scaffold plasmids with different enzyme binding site ratios;

[0045] Figure 6 : Schematic diagram of plasmids that increase the expression level of the rate-limiting enzyme EctB;

[0046] Figure 7 Schematic diagram of DNA scaffold plasmids with different enzyme binding site ratios after increasing the expression level of the rate-limiting enzyme EctB.

[0047] Figure 8The effect of different replicon strains on tetrahydropyrimidine production;

[0048] Figure 9 The effects of different enzyme binding site spacing and enzyme binding direction on the production of tetrahydropyrimidine by DNA scaffold strains;

[0049] Figure 10 The effect of reverse binding of enzymes, different enzyme binding site spacing, and DNA scaffold strains on tetrahydropyrimidine production;

[0050] Figure 11 The effect of DNA scaffold strains with different repeating units on tetrahydropyrimidine production;

[0051] Figure 12 The effect of DNA scaffold strains with different enzyme binding site stoichiometry on tetrahydropyrimidine production;

[0052] Figure 13 Effects of increasing the expression level of the rate-limiting enzyme EctB on the stoichiometry of different enzyme binding sites in DNA scaffold strains on tetrahydropyrimidine production;

[0053] Figure 14 : Optimize the fermentation temperature variation time of the final strain MWZ003 / pFV30. Detailed Implementation

[0054] (1) Culture medium composition:

[0055] LB liquid medium: 10 g / L tryptone -1 5g·L yeast extract -1 NaCl 10 g·L -1 Used for the activation and culture of Escherichia coli strains.

[0056] LB solid medium: 10 g / L tryptone -1 5g·L yeast extract -1 NaCl 10 g·L -1 15g / L agar powder -1 Used for the activation and culture of Escherichia coli strains.

[0057] STF medium: (NH4)2SO4 15 g·L -1 10g·L of sucrose -1 Tryptone 20g / L -1 5g·L yeast extract -1 MgSO4 1 g·L -1 The pH was adjusted to 7.3 with KOH. The sterilization temperature was 115℃, and the sterilization time was 15 minutes. This was used for the cultivation of the bacterial strain's fermentation seed culture.

[0058] Shake-flask fermentation medium: glucose 35 g·L -1 Yeast extract 2g·L -1 Citric acid 2g·L -1 , (NH4)2SO4 25 g·L -1 KH2PO4 7.46 g·L -1 MgSO4·7H2O 2g·L -1 FeSO4·7H2O 5mg·L -1 MnSO4·4H2O 5mg·L -1 20 g·L⁻¹ CaCO₃ -1 Vitamin B1 0.8 mg·L -1 and Vitamin H 0.2 mg·L -1 The pH was adjusted to 7.1 with KOH. The sterilization temperature was 115℃, and the sterilization time was 15 minutes. This was used for shake-flask fermentation culture of the strain.

[0059] (2) Determination of cell biomass: The fermentation sample was mixed on a vortex mixer, and 50 μL of the fermentation broth was added to 950 μL of 1 mol·L⁻¹ hydrochloric acid. -1 Dissolve in hydrochloric acid and mix by blowing and aspiration. Preheat the UV spectrophotometer with 1 mol·L⁻¹ water. -1 Hydrochloric acid at OD 600 The absorbance was calibrated, and then each sample was measured. Finally, the result was multiplied by the dilution factor to obtain the final bacterial concentration.

[0060] (3) Determination of residual glucose concentration in fermentation broth: The fermentation sample was heated at 12000 r·min -1 Centrifuge for 15 min, then transfer 10 μL of the fermentation supernatant to 990 μL of ultrapure water to complete sample preparation. The SBA40 reducing sugar analyzer was calibrated with a standard beforehand. Then, 25 μL of each sample was taken for analysis, and the remaining glucose content was recorded.

[0061] (4) Tetrahydropyrimidine content determination: The fermentation sample was heated at 12000 r·min -1 Centrifuge for 15 min, then dilute an appropriate amount of the fermentation supernatant in ultrapure water to a suitable concentration to complete sample preparation. Filter the sample through a 0.22 μm aqueous syringe filter. The filtered sample is then ready for determination of tetrahydropyrimidine content using high-performance liquid chromatography (HPLC). The HPLC instrument used in this invention is an Agilent 1260 / 1200 HPLC system, using a Thermo ODS-2HYPERSIL C18 column, with 70% acetonitrile as the mobile phase, and a flow rate of 0.8 mL / min. -1Under the following conditions, the column temperature was set to 35℃, the UV detection wavelength was set to 210nm, and the retention time was set to 20min.

[0062] Table 1 Primers used in the following examples

[0063]

[0064] Example 1: Construction of Zinc Finger Domain Plasmid

[0065] The specific construction process is as follows: The designed zinc finger domain ZFa fragment was inserted into the EcoRV restriction site of the commercial vector pUC57-Kan using primers ZFa-F and ZFa-R. After chemical transformation into strain JM109, the plasmid was plated onto LB agar plates containing kanamycin resistance. Following colony PCR verification, the correct strain was screened, and the plasmid was sequenced to verify its integrity, ensuring the absence of point mutations, thus obtaining plasmid pZFa. Using the same steps, the designed zinc finger domain ZFb and ZFc fragments were inserted into the EcoRV restriction site of the commercial vector pUC57-Kan using primers ZFb-F / ZFb-R and ZFc-F / ZFc-R, respectively, to construct plasmids pZFb and pZFc.

[0066] Example 2: Construction of plasmids containing different DNA scaffold fragments

[0067] The specific construction process is as follows: Using primers TZF-F1 and TZF-R1, a 4.1bp DNA scaffold fragment (SEQ ID NO. 4) containing a B1006 Terminator, a 1bp forward binding (+) spacing, four repeat units (n), and an EctA:EctB:EctC binding site ratio of 1:1:1 was inserted into the EcoRV restriction site of the commercial vector pUC57-Kan. After chemical transformation into strain JM109, the fragment was plated onto LB agar plates containing kanamycin resistance. Following colony PCR and sequencing verification, positive transformants were screened, yielding plasmid pUC57-1 containing the 4.1bp (1:1:1) DNA scaffold fragment.

[0068] The DNA scaffold fragments (1:1:1) 4,-1bp (SEQ ID NO.5), (1:1:1) 4,11bp (SEQ ID NO.6), (1:1:1) 4,-11bp (SEQ ID NO.7), (1:1:1) 4,21bp (SEQ ID NO.8), (1:1:1) 4,-21bp (SEQ ID NO.9), (1:1:1) 4,-7bp (SEQ ID NO.10), (1:1:1) 4,-9bp (SEQ ID NO.11), (1:1:1) 4,-13bp (SEQ ID NO.12), (1:1:1) 4,-15bp (SEQ ID NO.13), and (1:1:1) 1,-11bp (SEQ ID NO.14) of this invention were constructed using the same steps. NO.14), DNA scaffold fragment (1:1:1) 8,-11 bp (SEQ ID NO.15), DNA scaffold fragment (1:1:1) 12 Plasmids pUC57-2 to pUC57-13, containing 4,-11 bp (SEQ ID NO.16), were prepared. Using primers TZF-F2 and TZF-R2, a designed DNA scaffold fragment (1:2:1) 4,-11 bp (SEQ ID NO.17) containing a B1006 Terminator, an 11 bp reverse binding (-) spacing, four repeat units (n), and an EctA:EctB:EctC binding site ratio of 1:2:1 was inserted into the EcoRV restriction site of the commercial vector pUC57-Kan. After chemical transformation into strain JM109, the culture was plated onto LB agar plates containing kanamycin resistance. Colony PCR and sequencing were then performed to screen for positive transformants, yielding plasmid pUC57-14 containing the 4,-11 bp (1:2:1) DNA scaffold fragment.

[0069] Plasmids pUC57-15 to pUC57-19 containing the DNA scaffold fragment (2:1:1)4,-11bp (SEQ ID NO.18), DNA scaffold fragment (1:1:2)4,-11bp (SEQ ID NO.19), DNA scaffold fragment (2:2:1)4,-11bp (SEQ ID NO.20), DNA scaffold fragment (1:2:2)4,-11bp (SEQ ID NO.21), and DNA scaffold fragment (2:1:2)4,-11bp (SEQ ID NO.22) were constructed using the same steps.

[0070] Example 3 Construction of tetrahydropyrimidine expression plasmid

[0071] The specific construction process is as follows: Using FW18-X-1 as the upstream primer and FW18-X-3 as the downstream primer, and using the pFT28-ectABC-EclysC*-aspDH-ppc3 plasmid (published in Chinese invention application document with publication number CN112501102A) as a template, a 3097bp plasmid containing P was amplified. R -P L The tetrahydropyrimidine synthesis gene cluster ectABC of the promoter was inserted into the SacI site of pFT24 (disclosed in Chinese invention application CN111363757A) using a one-step cloning ligation method. The plasmid pFT24-ectABC was successfully constructed. Using plasmid pFT24-ectABC as a template, a 6928 bp linear vector fragment was amplified in reverse using primers ectA-ZFa-F and ectA-ZFa-R, followed by digestion of the original plasmid template with DpnI enzyme. Simultaneously, using plasmid pZFa constructed in Example 1 as a template, a 361 bp DNA fragment was amplified using primers ZFa-F and ZFa-R. The two fragments were ligated using a one-step cloning ligation method. After chemical transformation into strain JM109, the sample was plated onto LB agar plates containing triclosan resistance. Positive transformants were screened by colony PCR and sequencing, successfully constructing plasmid pFT24-ectABC-ZFa.

[0072] Using plasmid pFT24-ectABC-ZFa as a template, a 7270 bp linear vector fragment was amplified in reverse using primers ectB-ZFb-F and ectB-ZFb-R, followed by digestion of the original plasmid template with DpnI enzyme. Simultaneously, using plasmid pZFb constructed in Example 1 as a template, a 363 bp DNA fragment was amplified using primers ZFb-F and ZFb-R. The two fragments were then ligated using a one-step cloning ligation method. Plasmid pFT24-ectABC-ZFab was successfully constructed. Using plasmid pFT24-ectABC-ZFab as a template, a 7574 bp linear vector fragment was amplified in reverse using primers ectC-ZFb-F and ectC-ZFb-R, followed by digestion of the original plasmid template with DpnI enzyme. Simultaneously, using plasmid pZFc constructed in Example 1 as a template, a 388 bp DNA fragment was amplified using primers ZFc-F and ZFc-R. The two were ligated using a one-step cloning ligation method. The plasmid pFT24-ectABC-ZFabc was successfully constructed. The tetrahydropyrimidine biosynthesis pathway fusion enzymes (EctA-ZFa, EctB-ZFb, and EctC-ZFc) were formed by linking the tetrahydropyrimidine biosynthesis pathway enzymes with their corresponding ZF domains via G4S linkage. Primer P was used... J4The gene encoding EclysC* (C1055T) for E. coli aspartate kinase III, the gene aspDH for aspartate dehydrogenase, and the gene ppc encoding phosphoenolpyruvate carboxylase under the control of the BBa_J23115 promoter were amplified from plasmid pFT28-ectABC-EclysC*-aspDH-ppc3 by pppc-F and lysC-R. The recovered 5289 bp DNA fragment was ligated into the linearized plasmid pFT24-ectABC-ZFabc, obtained by double digestion with EcoRI and KpnI, using a one-step cloning ligation method. Plasmid pFV1 was successfully constructed.

[0073] Example 4: Construction of different replicon plasmids

[0074] The specific construction process is as follows: Using plasmid pFV1 constructed in Example 3 as a template, a linear vector fragment pFV1 with a length of 12579 bp was amplified using primers FW36-F and FW36-R. Then, using plasmid pCOLADuet-1 as a template, a ColA replicon fragment with a length of 728 bp was amplified using primers ColA-F and ColA-R. The two were then ligated using a one-step cloning ligation method, thus replacing the original p15A replicon of the plasmid with the ColA replicon, resulting in plasmid pFV2.

[0075] Using the same method, plasmids pRSFDuet-1 and pTargetF were used as templates. The 845 bp RSF1030 replicon fragment amplified using primers RSF1030-F and RSF1030-R, and the 931 bp pMB1 replicon fragment amplified using primers pMB1-F and pMB1-R, were respectively ligated to the linearized vector fragment pFV1 using a one-step cloning ligation method to obtain plasmids pFV3 and pFV4 containing RSF1030 and pMB1 replicons. Figure 1 ).

[0076] Example 5: Construction of DNA scaffold plasmids with different enzyme binding site spacing and enzyme binding direction

[0077] The specific construction process is as follows: Plasmid pFV4, constructed in Example 4, was digested with SpeI, and a linearized vector of 13510 bp was recovered. pUC57-1, constructed in Example 2, was amplified using primers TZF-F1 and TZF-R1 to obtain a 342 bp DNA scaffold fragment (1:1:1)4,1 bp (SEQ ID NO. 4) containing a B1006 Terminator, a 1 bp forward binding (+) spacing, 4 repeat units (n), and an EctA:EctB:EctC binding site ratio of 1:1:1. Then, the DNA scaffold fragment (1:1:1)4,1 bp was ligated to the linearized vector pFV4 obtained by SpeI digestion using a one-step cloning ligation method. After a series of screening and verification processes, plasmid pFV5 was successfully constructed.

[0078] Using the same method, plasmids pFV6-pFV10 containing DNA scaffold fragments with different enzyme binding site spacing and enzyme binding orientations were successfully constructed: (1:1:1) 4,-1bp (SEQ ID NO.5), (1:1:1) 4,11bp (SEQ ID NO.6), (1:1:1) 4,-11bp (SEQ ID NO.7), (1:1:1) 4,21bp (SEQ ID NO.8), and (1:1:1) 4,-21bp (SEQ ID NO.9). Figure 2 ).

[0079] Example 6: Construction of DNA scaffold plasmids with reverse binding sites of different enzyme binding sites

[0080] The specific construction process is as follows: Plasmid pFV4, constructed in Example 4, was digested with SpeI enzyme, and a linearized vector of 13510 bp was recovered. A 392 bp DNA scaffold fragment (1:1:1) 4,-7 bp (SEQ ID NO. 10), containing a B1006 Terminator, a 7 bp reverse binding (-) spacing, 4 repeat units (n), and an EctA:EctB:EctC binding site ratio of 1:1:1, was amplified using primers TZF-F1 and TZF-R1. Then, the DNA scaffold fragment was ligated to the linearized vector pFV4 obtained by SpeI digestion using a one-step cloning ligation method. After a series of screening and verification processes, plasmid pFV11 was successfully constructed.

[0081] Using the same method, plasmid pFV12-pFV14 was successfully constructed containing DNA scaffold fragments (1:1:1) 4,-9 bp (SEQ ID NO.11), (1:1:1) 4,-13 bp (SEQ ID NO.12), and (1:1:1) 4,-15 bp (SEQ ID NO.13) with reverse binding distances between different enzyme binding sites. Figure 3 ).

[0082] Example 7: Construction of plasmids with different DNA scaffold repeat units

[0083] The specific construction process is as follows: Plasmid pFV4, constructed in Example 4, was digested with SpeI, and a linearized vector of 13510 bp was recovered. A 205 bp DNA scaffold fragment (1:1:1)1,-11 bp (SEQ ID NO. 14), containing a B1006 Terminator, an 11 bp reverse binding (-) spacing, one repeat unit (n), and an EctA:EctB:EctC binding site ratio of 1:1:1, was amplified using primers TZF-F1 and TZF-R1. Then, the DNA scaffold fragment was ligated to the linearized vector pFV4 obtained by SpeI digestion using a one-step cloning ligation method. After a series of screening and verification processes, plasmid pFV15 was successfully constructed.

[0084] DNA scaffold fragments (1:1:1) 8,-11 bp (SEQ ID NO.15) and DNA scaffold fragments (1:1:1) containing different DNA scaffold repeat units were successfully constructed using the same method. 12 ,-11bp (SEQ ID NO.16) plasmids pFV16 and pFV17 ( Figure 4 ).

[0085] Example 8: Construction of DNA scaffold plasmids with different enzyme binding site stoichiometry

[0086] The specific construction process is as follows: Plasmid pFV4, constructed in Example 4, was digested with SpeI, and a linearized vector of 13510 bp was recovered. A 506 bp DNA scaffold fragment (1:2:1)4,-11 bp (SEQ ID NO. 17), containing a B1006 Terminator, an 11 bp reverse binding (-) spacing, four repeat units (n), and an EctA:EctB:EctC binding site ratio of 1:2:1, was amplified using primers TZF-F2 and TZF-R2. Then, the DNA scaffold fragment was ligated to the linearized vector pFV4 obtained by SpeI digestion using a one-step cloning ligation method. After a series of screening and verification processes, plasmid pFV18 was successfully constructed.

[0087] Using the same method, plasmid pFV19-pFV23 was successfully constructed containing DNA scaffold fragments with different ratios of enzyme binding sites: (2:1:1) 4,-11bp (SEQ ID NO.18), (1:1:2) 4,-11bp (SEQ ID NO.19), (2:2:1) 4,-11bp (SEQ ID NO.20), (1:2:2) 4,-11bp (SEQ ID NO.21), and (2:1:2) 4,-11bp (SEQ ID NO.22). Figure 5 ).

[0088] Example 9: Construction of a plasmid to increase the expression level of the rate-limiting enzyme EctB

[0089] The specific construction process is as follows: P is added before the EctB-ZFb fusion enzyme. R The promoter was used to further increase the expression level of the rate-limiting enzyme EctB. Using plasmid pFV4 constructed in Example 4 as a template, primer P was used... R -ectB-mF and mR1 amplified a 6420bp DNA fragment. Then, using plasmid pFV4 as a template, primer P... R -ectB-mR and mF1 amplified a vector fragment of 7180 bp. After recovery, the two were ligated using a one-step cloning ligation method. Positive transformants were screened by colony PCR and sequencing, and plasmid pFV24 was successfully constructed. Figure 6 ).

[0090] Example 10: Construction of DNA scaffold plasmids with different enzyme binding site stoichiometry after increasing the expression level of the rate-limiting enzyme EctB.

[0091] The specific construction process is as follows: Plasmid pFV24, constructed in Example 9, was digested with SpeI enzyme, and a linearized vector of 13560 bp in length was recovered. The designed 426 bp DNA scaffold fragment (1:1:1)4,-11 bp (SEQ ID NO. 7), containing a B1006 Terminator, an 11 bp reverse binding (-) spacing, 4 repeat units (n), and an EctA:EctB:EctC binding site ratio of 1:1:1, was amplified using primers TZF-F1 and TZF-R1. Then, the DNA scaffold fragment was ligated to the linearized vector pFV24 obtained by SpeI digestion using a one-step cloning ligation method. After a series of screening and verification processes, plasmid pFV25 was successfully constructed.

[0092] The plasmid pFV24 constructed in Example 9 was digested with SpeI, and a linearized vector of 13560 bp was recovered. A 506 bp DNA scaffold fragment (1:2:1)4,-11 bp (SEQ ID NO. 17) containing a B1006 Terminator, an 11 bp reverse binding (-) spacing, four repeat units (n), and EctA:EctB:EctC binding sites in a 1:2:1 ratio was amplified using primers TZF-F2 and TZF-R2. Then, the DNA scaffold fragment was ligated to the linearized vector pFV24 obtained by SpeI digestion using a one-step cloning ligation method. After a series of screening and verification processes, plasmid pFV26 was successfully constructed.

[0093] Using the same method, plasmid pFV27-pFV31 was successfully constructed containing DNA scaffold fragments with different stoichiometry at different enzyme binding sites after increasing the expression level of the rate-limiting enzyme EctB: (2:1:1) 4,-11bp (SEQ ID NO.18), (1:1:2) 4,-11bp (SEQ ID NO.19), (2:2:1) 4,-11bp (SEQ ID NO.20), (1:2:2) 4,-11bp (SEQ ID NO.21), and (2:1:2) 4,-11bp (SEQ ID NO.22). Figure 7 ).

[0094] Example 11 Application of recombinant Escherichia coli in the synthesis of tetrahydropyrimidine

[0095] Using E. coli MWZ003 (disclosed in Chinese invention application CN112501102A) as the expression host for tetrahydropyrimidine production, the plasmids pFV1 to pFV31 constructed in Examples 3-10 were introduced into electrocompetent cells of MWZ003 via electroporation to construct recombinant strains MWZ003 / pFV1 to MWZ003 / pFV31. After recovery culture at 37°C and 200 rpm for 1 h, the bacterial culture was plated on LB agar plates containing the corresponding antibiotics, and the LB agar plates were inverted and incubated at 37°C for at least 12 h. Single colonies were then verified by colony PCR. Correct strains were selected for glycerol preservation for later use, or for shake-flask fermentation to prepare tetrahydropyrimidine.

[0096] The recombinant bacterial strain was removed from a -80℃ freezer. An appropriate amount of bacterial culture was streaked onto LB agar plates containing triclosan-resistant culture medium. After overnight incubation, single colonies with regular morphology and appropriate size were picked and cultured overnight in 5 mL of LB liquid medium containing triclosan-resistant culture medium at 37℃ and 200 rpm. An appropriate amount of the activated bacterial culture was evenly spread onto LB agar plates containing triclosan-resistant culture medium and incubated overnight at 37℃ to obtain abundant bacterial flora as fermentation seed. Bacterial flora was scraped and cultured in 5 mL of LB liquid medium containing triclosan-resistant culture medium at 37℃ and 200 rpm for 2 hours as primary seed. All primary seed was transferred to 50 mL of STF medium containing triclosan-resistant culture medium and cultured for 2 hours at 37℃ and 200 rpm as secondary seed. The initial OD was used as the starting point for the fermentation. 600 Secondary seed culture was inoculated into 30 mL shake-flask fermentation medium at an inoculum size of 0.3. Cell biomass was accumulated by growth at 37°C for 3 h, followed by raising the fermentation temperature to 42°C and turning on the temperature control to produce tetrahydropyrimidine. The fermentation cycle was set to 36 h or longer. 1 mL of fermentation sample was collected each time for determining cell biomass, sugar consumption, and tetrahydropyrimidine yield.

[0097] Shake-flask fermentation experiments were conducted on strains containing plasmids with different copy numbers of replicons. The results are as follows: Figure 8 As shown. The strain MWZ003 / pFV4 (11.85 g·L⁻¹) possesses the highest copy number of pMB1 replicon. -1 The pMB1 replicon produced higher tetrahydropyrimidine yields than strains possessing other copy numbers of replicons, indicating that the pMB1 replicon is more suitable for tetrahydropyrimidine production than other replicons. This also demonstrates a close correlation between tetrahydropyrimidine production and enzyme expression levels; higher enzyme expression levels lead to higher tetrahydropyrimidine yields. Specifically, the strain MWZ003 / pFV2, possessing the ColA replicon, produced 1.04 g·L⁻¹ of tetrahydropyrimidine. -1Slightly lower than strain MWZ003 / pFV1, which possesses the p15A replicon (1.26 g·L⁻¹). -1 The reason for this might be that strain MWZ003 / pFV2 used a large amount of substrate for its growth during the early stages of fermentation. After comparing the fermentation results of four strains with different replicon plasmids, strain MWZ003 / pFV4, which had the highest tetrahydropyrimidine production and the highest copy number of the pMB1 replicon, was selected for further experimental research.

[0098] Shake-flask fermentation experiments were conducted on strains containing plasmids with different enzyme binding site spacing and enzyme binding direction. The results are as follows: Figure 9 As shown in the figure, using strain MWZ003 / pFV4 without a DNA scaffold structure as a control, it was observed that after 24 hours of fermentation, the biomass of strain MWZ003 / pFV4 without a DNA scaffold structure was higher than that of strain MWZ003 / pFV4 containing a DNA scaffold structure. Simultaneously, it was observed that cell biomass increased with increasing enzyme binding site spacing, in the order of control > 21 bp > 11 bp > 1 bp. These results indicate that the addition of a DNA scaffold structure has a certain impact on cell growth, and this impact decreases with increasing enzyme binding site spacing. Observations on the tetrahydropyrimidine production of different strains revealed that the tetrahydropyrimidine production of strains containing a DNA scaffold structure was higher than that of the control strain MWZ003 / pFV4 to varying degrees, directly demonstrating that the DNA scaffold plays a positive role in tetrahydropyrimidine production. Among them, the tetrahydropyrimidine production of three groups of bacteria with the same enzyme binding site spacing but different enzyme binding directions (Group 1: MWZ003 / pFV5 and MWZ003 / pFV6; Group 2: MWZ003 / pFV7 and MWZ003 / pFV8; Group 3: MWZ003 / pFV9 and MWZ003 / pFV10) was observed and compared. It was found that within the same enzyme binding site spacing, the reverse binding effect of the enzyme was better than the forward binding effect (Group 1: MWZ003 / pFV6 was better than MWZ003 / pFV5; Group 2: MWZ003 / pFV8 was better than MWZ003 / pFV7; Group 3: MWZ003 / pFV10 was better than MWZ003 / pFV9). Next, the tetrahydropyrimidine yields of three strains (MWZ003 / pFV6, MWZ003 / pFV8, and MWZ003 / pFV10) with reverse enzyme binding and different enzyme binding site spacings were further observed and compared. It was found that strain MWZ003 / pFV8, which possesses reverse enzyme binding and has an enzyme binding site spacing of 11 bp, has a higher tetrahydropyrimidine yield and is suitable for tetrahydropyrimidine production. Compared with the control strain MWZ003 / pFV4, the extracellular tetrahydropyrimidine yield of strain MWZ003 / pFV8 increased by 24.3%, reaching 14.73 g·L⁻¹. -1 .

[0099] Shake-flask fermentation experiments were conducted on strains containing DNA scaffold enzymes that bind to plasmids with different enzyme binding site spacings in reverse. The experimental results are as follows: Figure 10 As shown. Using strain MWZ003 / pFV8 as a control, strains with different enzyme binding sites spaced approximately 11 bp apart were constructed and compared with it. Observation of the experimental results revealed little difference between the experimental and control strains in biomass, glucose consumption, and tetrahydropyrimidine production. The tetrahydropyrimidine yields of strains MWZ003 / pFV11–MWZ003 / pFV14 were 14.49 g·L⁻¹, respectively. -1 13.75 g·L -1 14.7 g·L -1 14.75 g·L -1 .

[0100] Shake-flask fermentation experiments were conducted on strains containing plasmids with different repeating units of DNA scaffolds. The results are as follows: Figure 11 As shown. Using strain MWZ003 / pFV8 with 4 repeat units on the DNA scaffold as a control, the experimental results showed that after 24 hours of fermentation, the biomass of the strains increased with the increase of repeat units (n). Strain MWZ003 / pFV17 (n=12) had the highest biomass, while strain MWZ003 / pFV15 (n=1) had the lowest. The reason for this might be that fewer repeat units on the scaffold mean fewer enzymes that can bind to them, leaving most enzymes free in the cell and placing a burden on the cell. With the increase of repeat units on the scaffold, more and more enzymes are immobilized in the cell, forming substrate channels, rapidly completing the conversion of intermediate products, and reducing the burden on the cell. Analysis of the tetrahydropyrimidine yield showed that strain MWZ003 / pFV8 with 4 repeat units on the DNA scaffold (14.58 g·L⁻¹) produced the highest yield. -1 The highest yield was achieved by strain MWZ003 / pFV16 (n=8), followed by strain MWZ003 / pFV16 (13.72 g·L⁻¹). -1 The strain MWZ003 / pFV15 (12.8 g·L⁻¹) with n=1 -1 ) and strain MWZ003 / pFV17 (10.28 g·L⁻¹) with n=12 -1 The tetrahydropyrimidine yield results indicate that when the DNA scaffold has 4 repeat units, the enzyme binding sites on the DNA scaffold are sufficient to accommodate the fusion enzyme expressed in the cell. However, the fermentation results also show that when the scaffold has too many repeat units, the use of the DNA scaffold not only does not increase the yield of tetrahydropyrimidine, but actually reduces its synthesis. This may be because too many repeat units on the scaffold lead to redundancy of enzyme binding sites, resulting in dispersed and distant binding sites for the fusion enzyme on the scaffold, thus reducing the efficiency of the enzymatic reaction instead of increasing it.

[0101] Shake-flask fermentation experiments were conducted on strains containing different stoichiometric numbers of enzyme binding sites on DNA scaffolds. The results are as follows: Figure 12 As shown. Using the DNA-free strain MWZ003 / pFV4 as a control, observations were made on seven strains with different enzyme binding site stoichiometry. The results showed that all strains exhibited similar growth throughout the fermentation process, with little difference in final biomass. Furthermore, all strains consumed all glucose within 24 hours of fermentation. Subsequently, analysis of tetrahydropyrimidine production revealed that, compared to the control strain MWZ003 / pFV4, all strains containing the DNA scaffold showed varying degrees of increased tetrahydropyrimidine production. Among them, strain MWZ003 / pFV20, with an enzyme binding site ratio of 1:1:2, showed the best performance, achieving an extracellular tetrahydropyrimidine production of 17.97 g·L⁻¹. -1 Compared with the control strain MWZ003 / pFV4, the tetrahydropyrimidine yield of strain MWZ003 / pFV20 increased by 51.65%. Compared with strain MWZ003 / pFV8 (enzyme binding site ratio 1:1:1), the tetrahydropyrimidine yield of strain MWZ003 / pFV20 increased by 22%. During the analysis of fermentation results, strains MWZ003 / pFV20 (enzyme binding site ratio 1:1:2) and MWZ003 / pFV22 (enzyme binding site ratio 1:2:2, 16.99 g·L⁻¹) showed the following yields: -1 The strain MWZ003 / pFV23 (16.93 g·L⁻¹) has an enzyme binding site ratio of 2:1:2. -1 Both have high tetrahydropyrimidine yields.

[0102] Shake-flask fermentation experiments were conducted on strains containing different enzyme binding sites on the DNA scaffold after increasing the expression level of the rate-limiting enzyme EctB. The experimental results are as follows: Figure 13 As shown in the figure. Analysis of the biomass changes of the entire fermentation strain revealed that the biomass of strain MWZ003 / pFV27 increased rapidly in the early stages of fermentation, far exceeding that of other strains, reaching its peak at 18 hours. Simultaneously, the glucose of strain MWZ003 / pFV27 was also depleted at 18 hours. Strains with increased EctB enzyme binding sites (MWZ003 / pFV26, MWZ003 / pFV29, and MWZ003 / pFV30) consumed all their glucose at 30 hours of fermentation, while the remaining strains (MWZ003 / pFV24, MWZ003 / pFV25, MWZ003 / pFV28, and MWZ003 / pFV31) consumed all their glucose at 36 hours of fermentation. Overall observation of the glucose consumption process of the strains revealed that the addition of P before the EctB-ZFb fusion enzyme... RAfter the promoter was activated, the glucose consumption rate decreased in all strains except for strain MWZ003 / pFV27. Therefore, the fermentation time was extended from 36 h to 48 h.

[0103] Next, P was added before the EctB-ZFb fusion enzyme. R Tetrahydropyrimidine production was analyzed in DNA scaffold strains with promoters and different enzyme binding site stoichiometry. (The text then abruptly shifts to a seemingly unrelated topic: "using P...") R The promoter but without a DNA scaffold structure, strain MWZ003 / pFV24 (15.23 g·L⁻¹) -1 As a control, the highest-producing strain was MWZ003 / pFV30 (19.95 g·L⁻¹), with an enzyme binding site ratio of 1:2:2. -1 Compared to the control strain MWZ003 / pFV24, the extracellular tetrahydropyrimidine production of strain MWZ003 / pFV30 was increased by 30.99%. Additionally, for EctB-ZFb fusion enzyme without the addition of P... R The strain MWZ003 / pFV22, which also possesses an increased number of EctB and EctC enzyme binding sites (enzyme binding site ratio of 1:2:2), also exhibited relatively high yields. Strain MWZ003 / pFV27 had the lowest tetrahydropyrimidine yield, at only 11.72 g·L⁻¹. -1 This may be because the strain uses a large amount of substrate for growth, and it also indicates that simply adding EctA enzyme binding sites to the DNA scaffold is not conducive to the production of tetrahydropyrimidine.

[0104] Example 12 Optimization of fermentation time

[0105] In this invention, a temperature control switch is used to switch the growth and production modes of the strain; the strain accumulates biomass by growing at 37°C, and produces tetrahydropyrimidine when the temperature is raised to 42°C. This method avoids the need for the addition of other inducers. Therefore, selecting an appropriate temperature switching time may have a significant impact on improving the yield of tetrahydropyrimidine. Shake-flask fermentation experiments with different temperature switching times were conducted using strain MWZ003 / pFV30 as the fermentation strain.

[0106] The method for preparing tetrahydropyrimidine by shake-flask fermentation was the same as in Example 11, except that the temperature transition times were set at 3h, 4h, and 5h after the start of fermentation, and the initial temperature of 37℃ was increased to 42℃. All other fermentation conditions remained the same. Each group was divided into three replicates. During the culture process, 1mL of fermentation sample was taken every 6 hours to determine cell biomass, the concentration of glucose in the fermentation broth, and the concentration of tetrahydropyrimidine. Fermentation lasted for a total of 48h. The fermentation performance of strain MWZ003 / pFV30 at different temperature transition times was observed.

[0107] The results of shake-flask fermentation experiments of strain MWZ003 / pFV30 at different temperature transition times are as follows: Figure 14 As shown. Observation of the strain's biomass revealed that the strain's growth status was similar under the temperature transition conditions of 3 and 4 hours after fermentation began. However, under the temperature transition condition of 5 hours after fermentation began, the strain's biomass accumulated significantly in the early stages of fermentation, while the biomass in the later stages was lower than under the conditions of 3 and 4 hours after fermentation. Simultaneously, it was observed that the strain accumulated more biomass in the early stages as the temperature transition time was delayed. All strains under the three different conditions consumed all glucose at 30 hours. Next, by comparing the tetrahydropyrimidine production at different temperature transition times, it was found that strain MWZ003 / pFV30 had a relatively high tetrahydropyrimidine production under the temperature transition condition of 4 hours after fermentation began. This was compared to the tetrahydropyrimidine production under the temperature transition condition of 3 hours after fermentation began (20.66 g·L⁻¹). -1 Compared to the previous method, the tetrahydropyrimidine yield increased by 10.31% at the transition temperature 4 hours after fermentation began, reaching 22.79 g·L⁻¹. -1 Five hours after the start of fermentation, the yield of tetrahydropyrimidine increased by 7.26% at the transition temperature, reaching 22.16 g·L⁻¹. -1 .

[0108] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant Escherichia coli, characterized in that, overexpression ectA -ZFa expression box ectB -ZFb expression box ectC -ZFc expression cassette, gene encoding phosphoenolpyruvate carboxylase PPC Gene encoding aspartate dehydrogenase aspDH and the gene encoding aspartate kinase III EclysC The recombinant Escherichia coli also contains a DNA scaffold structure. The DNA scaffold structure includes 1, 4, or 8 repeat units, wherein the repeat units are composed of… ectA Enzyme binding site, ectB Enzyme binding sites and ectC Enzyme binding site composition; The ectA Enzyme binding site, ectB Enzyme binding site, ectC There are 7-15 bp spacer sequences between the enzyme binding sites; ectA Enzyme binding site, ectB Enzyme binding site, ectC The stoichiometry between enzyme binding sites is 1:1:1, 1:2:1, 1:1:2, 1:2:2, or 2:1:

2. The DNA scaffold structure is located downstream of the ectC-ZFc expression cassette and upstream of the ppc. The gene EclysC The nucleotide sequence is shown in SEQ ID NO.

26.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The recombinant Escherichia coli used pFT24 as a vector, which was created by replacing the pMB1 replicon in plasmid pFW001 with the p15A replicon.

3. The recombinant Escherichia coli according to any one of claims 1 to 2, characterized in that, With Escherichia coli Escherichia coli MWZ003 is the host, and the Escherichia coli is described. Escherichia coli MWZ003 uses Escherichia coli MG1655 as the starting strain to knock out the crr, iclR, and thrA genes.

4. A method for synthesizing tetrahydropyrimidine, characterized in that, Using the recombinant Escherichia coli described in any one of claims 1 to 3 as the fermentation strain, tetrahydropyrimidine is prepared by fermentation.

5. The method according to claim 4, characterized in that, The method uses the recombinant Escherichia coli according to any one of claims 1 to 3 with an initial OD 600 0.2-0.4 g of the solution was inoculated into the fermentation medium and grown at 35-38°C for 3-5 h. Then the temperature was raised to 40-45°C for fermentation to prepare tetrahydropyrimidine.

6. A DNA scaffold structure, characterized in that, The DNA scaffold structure includes 1, 4, or 8 repeat units, wherein the repeat units are composed of… ectA Enzyme binding site, ectB Enzyme binding sites and ectC Enzyme binding site composition; The ectA Enzyme binding site, ectB Enzyme binding site, ectC There are 7-15 bp spacer sequences between the enzyme binding sites; ectA Enzyme binding site, ectB Enzyme binding site, ectC The stoichiometric ratios between enzyme binding sites are 1:1:1, 1:2:1, 1:1:2, 1:2:2, or 2:1:

2.

7. A method for increasing the yield of tetrahydropyrimidine, characterized in that, The method involves inserting the DNA scaffold structure of claim 6 into cells that produce tetrahydropyrimidine, wherein the cells express EctA-ZFa fusion protein, EctB-ZFb fusion protein, and EctC-ZFc fusion protein, which specifically bind to the ectA, ectB, and ectC enzyme binding sites on the DNA scaffold, respectively.

8. The use of the recombinant Escherichia coli according to any one of claims 1 to 3 or the DNA scaffold structure according to claim 6 in the synthesis of tetrahydropyrimidine or products containing tetrahydropyrimidine.

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