A recombinant Escherichia coli for producing heparin precursors, and its construction and application

Abstract

The present invention discloses a recombinant Escherichia coli for producing heparin precursors and its construction and application. The present invention uses the probiotic Escherichia coli Nissle 1917 as the host, designs to increase the yield of heparin precursors by studying the role of KfiB in the synthesis of heparin precursors and studying the quantitative change between KfiB and the heparin precursor synthase KfiAC, clarifies the optimal structure of kfiACB expression, optimizes the heparin precursor yield to 0.349 g / L through different combinations, and further optimizes the expression level of kfiACB by screening RBS in the mutant library. The heparin precursor yield can reach 0.97 g / L in shake flasks, which is more than 6 times higher than that of the wild-type strain.

Description

Technical Field

[0001] The present invention relates to a recombinant Escherichia coli for producing heparin precursors, its construction and application, belonging to the technical field of bioengineering. Background Art

[0002] EcN (Escherichia coli Nissle 1917, O6:K5:H1) is a natural probiotic. Currently, EcN has been used as an over-the-counter probiotic drug for more than 100 years. And EcN has natural capsular polysaccharide (CPS, heparin precursor) and a complete ABC transport system. At the same time, EcN also has advantages such as biosafety, clear genetic background, and short culture cycle. Heparin precursor is the most important substrate for the chemical enzymatic method of heparin, but currently, it is impossible to extract and prepare heparin precursor from tissues. Heparin precursor exists in the form of a capsule on the surface of a few bacteria, and the current production mainly relies on bacterial fermentation. The wild-type Escherichia coli that naturally produces heparin precursor generally has a low yield, unstable fermentation level, and some are also pathogenic.

[0003] The synthesis of heparin precursor chains in EcN is mainly completed by KfiA (α - N - acetylglucosaminyltransferase) and KfiC (β - glucuronyltransferase). These two genes are both located in the gene cluster II region of EcN that produces heparin precursor. It is known that KfiB is also located on the synthetic region gene cluster of the heparin precursor synthesis gene cluster, but its specific function is still unknown. Clarifying what role it plays in EcN and its connection with KfiA and KfiC on the same synthetic region is crucial for further increasing the yield of heparin precursor.

[0004] In addition, the effect of different expression intensities of KfiABC on the synthesis of heparin precursor in EcN is unknown. How to coordinate the expression levels of KfiA and KfiC through KfiB is crucial for the synthesis of heparin precursor in EcN. The ribosome binding site (RBS) is necessary for prokaryotic initiation of transcription. Different intensities of RBS-expressed proteins will optimize the transcription level of this protein to a certain extent. Optimizing RBS can not only increase the expression level of genes, but also coordinate the transcription between genes to a certain extent, thereby increasing the yield of the target product.

[0005] Therefore, it is necessary to explore the role of KfiB, coordinate the expression of KfiABC, construct a more efficient microbial cell factory, and improve the yield and quality of heparin precursor. Summary of the Invention

[0006] To solve the above problems, the present invention explores the function of KfiB, optimizes the different combination orders of KfiABC, and introduces RBSs with different strengths to coordinate the expression levels of KfiABC, thereby increasing the production of heparin precursors in EcN from a different perspective compared with the prior art.

[0007] The first object of the present invention is to provide a recombinant Escherichia coli for efficiently producing heparin precursors, wherein the recombinant Escherichia coli includes the following modifications: overexpressing the α - N - acetylglucosaminyltransferase KfiA - encoding gene kfiA, the KfiB - encoding gene kfiB, and the β - glucuronosyltransferase KfiC - encoding gene kfiC, with the gene kfiC located between the gene kfiA and the gene kfiB and the gene kfiA located upstream of the gene kfiB.

[0008] Furthermore, the NCBI accession number of the α - N - acetylglucosaminyltransferase KfiA is AXY46007.1, the NCBI accession number of KfiB is AXY46006.1, and the NCBI accession number of the β - glucuronosyltransferase KfiC is AXY46005.1.

[0009] Furthermore, the expression of the α - N - acetylglucosaminyltransferase KfiA - encoding gene kfiA, the KfiB - encoding gene kfiB, and the β - glucuronosyltransferase KfiC - encoding gene kfiC is respectively regulated by RBS sequences.

[0010] The present invention designs a random mutation library of degenerate base - pair RBS0 (AAGGAGC), characterizes the strengths of 96 randomly mutated RBSs through the intensity of the fluorescent protein sfGFP, and finally selects 9 RBSs for the construction of kfiACB to verify that RBS can significantly increase the production of heparin precursors in the fermentation products. Most preferably, the α - N - acetylglucosaminyltransferase KfiA - encoding gene kfiA is regulated by the GAGCAGCG sequence, and the expression of the KfiB - encoding gene kfiB and the β - glucuronosyltransferase KfiC - encoding gene kfiC is regulated by the TGGGCTCG sequence.

[0011] Furthermore, the above - mentioned genes are expressed under the initiation of the P tac promoter.

[0012] Furthermore, the starting strain of the above - mentioned recombinant Escherichia coli includes but is not limited to Escherichia coli Nissle 1917.

[0013] Furthermore, the plasmid pETDuet - 1 is used as the expression vector.

[0014] The second object of the present invention is to provide a method for constructing the above - mentioned recombinant Escherichia coli, comprising the following steps:

[0015] S1. Connect the RBS coding gene, the α-N-acetylglucosaminyltransferase KfiA coding gene kfiA, the RBS coding gene, the β-glucuronosyltransferase KfiC coding gene kfiC, the RBS coding gene, and the KfiB coding gene kfiB to the plasmid backbone in sequence to construct a recombinant plasmid.

[0016] S2. Transform the above recombinant plasmid into an Escherichia coli host to obtain the above recombinant Escherichia coli.

[0017] The third object of the present invention is to provide a method for producing a heparin precursor, including the step of fermenting and producing by using the above recombinant Escherichia coli.

[0018] Further, the fermentation is to culture the above recombinant Escherichia coli to obtain a seed solution, and transfer the seed solution to a fermentation medium containing glucose for fermentation production.

[0019] Further, the fermentation medium used includes the following components: KH 2 PO 4 10 - 15 g / L, (NH 4 ) 2 HPO 4 3 - 5 g / L, citric acid 1 - 3 g / L, MgSO 4 1 - 3 g / L, thiamine 0.1 - 0.3 g / L, CaCl 2 1 - 3 g / L, ZnSO 4 ·7H 2 O 1 - 4 g / L, MnSO 4 ·4H 2 O 0.2 - 0.8 g / L, FeSO 4 ·7H 2 O 8 - 12 g / L, NaB 4 O 7 ·10H 2 O 0.01 - 0.03 g / L, CuSO 4 ·5H 2 O 0.5 - 1.5 g / L, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.05 - 0.2 g / L, D-glucose 15 - 25 g / L, pH 7.0 - 7.2.

[0020] Further, when the OD 600 of the bacterial solution is 0.8 - 1, induce with 0.2 mmol / L IPTG, and end the shake flask culture when the glucose is exhausted during the shake flask culture process.

[0021] Advantages of the present invention:

[0022] The present invention clarifies the function of KfiB in Region II of the EcN gene cluster and explores its role in the production of heparin precursor. By modifying the key genes kfiA, kfiB, and kfiC for synthesizing heparin precursor, the accumulation and expression of heparin precursor in recombinant bacteria are promoted. On this basis, the RBS regulatory sequence is further optimized to obtain an engineered strain with high yield of heparin precursor, and the yield can reach 0.97 g / L in shake flasks. Brief Description of the Drawings

[0023] Figure 1 It is a graph showing the yield of heparin precursor produced by Escherichia coli Nissle 1917 after knocking out kfiB and plasmid complementation.

[0024] Figure 2 It is a graph showing the effect of knocking out kfiB on the expression levels of kfiACD genes on the Region II gene cluster in Escherichia coli Nissle 1917, and the effect of different combinations of kfiABCD expression on the yield of heparin precursor.

[0025] Figure 3 It is a graph showing the intracellular protein localization of KfiB with KfiA, KpsC, and KpsT respectively in Escherichia coli Nissle 1917.

[0026] Figure 4 It is a graph showing the effect of different combinations of KfiABC on the yield of heparin precursor in Escherichia coli Nissle 1917.

[0027] Figure 5 It is a graph showing the effect of overexpressing KfiACB with different strength RBS combinations on the yield of heparin precursor in Escherichia coli Nissle 1917. Detailed Embodiments

[0028] The present invention will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0029] The solutions involved in the present invention are as follows:

[0030] (1) Using the CRISPER-Cas9 method to knock out KfiABC in the Region II gene cluster of the EcN genome. The Escherichia coli after knocking out KfiABC is named EcNΔKfiABC, and EcNΔKfiABC, as a negative control, no longer produces heparin precursor. Using the CRISPER-Cas9 method to knock out KfiB in the Region II gene cluster of the EcN genome. The Escherichia coli after knocking out KfiB is named EcNΔKfiB, and it is detected whether KfiB affects the synthesis of heparin precursor.

[0031] (2) The KfiABCD on the gene cluster in Region II of the Escherichia coli Nissle 1917 genome was constructed on the plasmid pETDuet separately or in combination using the tac promoter and RBS0 (AAGGAGC) in a homologous recombination ligation manner to form plasmids pET-kfiA, pET-kfiB, pET- N kfiB, pET- C kfiB, pET-kfiC, pET-kfiAC, pET-kfiCD, pET-kfiBC, and pET-kfiBCD.

[0032] (3) The plasmids pET-kfiB, pET- N kfiB, and pET- C kfiB were transformed into EcNΔKfiB by chemical transformation and named EcN pET-kfiB,ΔKfiB, EcN pET- N kfiB,ΔKfiB, and EcN pET- C kfiB,ΔKfiB. The plasmids pET-kfiA, pET-kfiC, pET-kfiAC, pET-kfiCD, pET-kfiBC, and pET-kfiBCD were transformed into EcNΔKfiB by chemical transformation and named EcN pET-kfiA,ΔKfiB, EcN pET-kfiC,ΔKfiB, EcNpET-kfiAC,ΔKfiB, EcN pET-kfiCD,ΔKfiB, EcN pET-kfiBC,ΔKfiB, and EcN pET-kfiBCD,ΔKfiB.

[0033] (4) Different combinations of kfiABC were constructed on the plasmid pETDuet-p tacO strengthened, and the best combination was determined to be pET-kfiACB by detecting the extracellular production of heparin precursors. RBS0 (AAGGAGC) was randomly mutated, and 96 randomly mutated RBSs were constructed on the pETDuet-p tacO -sfGFP plasmid by homologous recombination and then transformed into EcN by chemical transformation. Different intensities of RBS were distinguished by different fluorescence intensities and constructed on the plasmid pET-kfiACB, and then transformed into Escherichia coli Nissle 1917 by chemical transformation.

[0034] The materials and methods involved in the following examples:

[0035] 1. RNA reverse transcription kit, PrimeSTAR DNA polymerase, phosphorylase, DNA Marker, and enzyme reagents such as fluorescence quantitative PCR were purchased from Baorui Biotechnology (Beijing) Co., Ltd.

[0036] The Seamless Cloning Kit was purchased from Beyotime (Shanghai).

[0037] The gel extraction kit was purchased from Thermo fisher Scientific.

[0038] The plasmid extraction kit was purchased from Biotech (Shanghai) Co., Ltd.

[0039] The total bacterial RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0040] Various analytical pure reagents were purchased from the Sinopharm Group.

[0041] 2. Culture media

[0042] LB solid medium (g / L): Tryptone 10, Yeast extract 5, Sodium chloride 10, Agar powder 20.

[0043] LB liquid medium (g / L): Tryptone 10, Yeast extract 5, Sodium chloride 10.

[0044] Flask fermentation medium (g / L): Also known as glucose - limited medium, KH 2 PO 4 13.5, (NH 4 ) 2 HPO 4 4.0, Citric acid 1.7, MgSO 4 1.4, Thiamine 0.1, CaCl 2 2.0, ZnSO 4 ·7H 2 O 2.2, MnSO 4 ·4H 2 O 0.5, FeSO 4 ·7H 2 O10.0, NaB 4 O 7 ·10H 2 O 0.02, CuSO 4 ·5H 2 O 1.0, (NH 4 ) 6 Mo 7 O 24 ·4H 2O 0.1, D-glucose 20.0, adjust the pH to 7.0 - 7.2.

[0045] 3. Heparin precursor yield measurement: Add 1 mL of boric acid sulfate (dissolve 4.77 g of borax in 500 mL of concentrated H 2 SO 4 ) to a 50 mL glass test tube and place it on ice. Add 200 μL of the appropriately diluted sample to the glass test tube, mix well, and place it in a boiling water bath for 15 min. After cooling to room temperature on ice, add 50 μL of the carbazole reagent (dissolve 0.625 g of carbazole in 500 mL of absolute ethanol), mix well, and boil for 15 min. Measure the absorbance at a wavelength of 530 nm. Perform the same reaction with D-glucuronic acid standards at different concentrations (10, 20, 30, 40, 50 μg mL -1 ) and plot the standard curve. Standard curve equation: y = 133.41x - 6.9236, R2 = 0.9971 (x, absorbance A530; y, glucuronic acid content in the sample (μg mL -1 ).

[0046] Heparin precursor yield calculation formula: Heparin precursor yield (g / L) = (concentration measured from the standard curve * dilution factor * 2.067) / 1000.

[0047] 4. Preparation of competent cells of E. coli Nissle 1917, E. coli Nissle 1917ΔKfiABC, and E. coli Nissle 1917ΔKfiB:

[0048] (1) Prepare CaCl 2 solution:

[0049] (2) Separate single colonies of E. coli Nissle 1917, E. coli Nissle 1917ΔKfiB, or E. coli Nissle 1917ΔKfiABC from the LB plate and inoculate them into 5 mL of LB liquid medium. Incubate overnight at 37 °C and 220 rpm;

[0050] (3) Transfer the bacterial solution at 2% to a 250 mL flask containing 50 mL of LB liquid medium and culture at 37 °C and 220 rpm until OD 600 = 0.4 - 0.6;

[0051] (4) Place on ice for 15 min, then centrifuge to collect the bacterial cells (4000 rpm, 4 °C, 10 min);

[0052] (5) Resuspend the bacterial cells in the ice-bathed CaCl 2 solution, let stand for 30 min, and then centrifuge (4000 rpm, 4 °C, 10 min);

[0053] (6) Discard the supernatant, and re - add 1 - 2 mL of CaCl 2 solution, mix well, aliquot 50 - 100 μL per tube into 1.5 ml sterile centrifuge tubes, and store at - 80 °C or perform transformation.

[0054] Table 1 Primer sequence list used in the examples

[0055]

[0056]

[0057]

[0058] Example 1: Construction of E. coli Nissle 1917ΔKfiB, pETDuet - p tacO -kfiB

[0059] (I) Knockout of kfiB on the genome of Escherichia coli Nissle 1917

[0060] (1) Extract the genome of Escherichia coli Nissle 1917, design primers for amplification. Use primers pT - ΔB - F / pT - ΔB - R to circularize and amplify the linearized pTarget - ΔkfiB vector containing the homologous recombination fragment by loop - mediated isothermal amplification (LAMP) in Escherichia coli JM109, and construct the pTarget - ΔkfiB plasmid.

[0061] (2) Use the CRISPER - Cas9 method to knockout KfiB in the gene cluster of region II on the EcN genome. Construct the PUM site recognizing NGG of N20 on the pTargetF plasmid, and ligate the homologous recombination fragments at both ends by fusion PCR. Transform the constructed homologous recombination fragment and pTargetF plasmid into the competent cells of EcN - Cas9 by electroporation at a ratio of 1400 ng:700 ng, and use primers ΔkfiB - F / R to verify whether kfiB is successfully knocked out.

[0062] (II) Construction of the E. coli Nissle 1917ΔKfiB, pETDuet - p tacO -kfiB plasmid

[0063] (1) Use primers kfiB - F / R, N kfiB - F / R and C kfiB - F / R with the EcN genome as a template, perform PCR amplification to obtain kfiB, N kfiB andC The kfiB fragment (wherein, N kfiB consists of the 1st to 280th amino acids of the KfiB protein, C kfiB consists of the 450th to 562nd amino acids of the KfiB protein).

[0064] (2) Using plasmid pETDuet-1 as a template and primers pET(B)-F / R, PCR amplification was carried out to obtain the vector pETDuet-1 linearized at the tac promoter expression frame, which can enable the sequences amplified in step (1) to be ligated to the corresponding vectors respectively;

[0065] (3) The fragments obtained in steps (1) and (2) were ligated using seamless ligation cloning enzymes to construct the recombinant plasmids pET-kfiB, pET- N kfiB and pET- C kfiB;

[0066] (4) Competent cells of E. coli Nissle 1917ΔKfiB constructed in step (1) were prepared, and the plasmids obtained in step (3) above were transformed into the competent cells. Positive clones were screened through kanamycin-resistant plates to obtain the E. coli Nissle 1917ΔKfiB recombinant bacteria containing the kfiB gene, namely EcN pET-kfiB,ΔKfiB, EcN pET- N kfiB,ΔKfiB and EcN pET- C kfiB,ΔKfiB;

[0067] (5) The recombinant bacteria EcN pET-kfiB,ΔKfiB, EcN pET- N kfiB,ΔKfiB and EcNpET- C kfiB,ΔKfiB and EcNΔKfiB obtained in step (4) were cultured at the shake flask level: The recombinant bacteria were cultured in LB medium for 10 h, and the bacterial solution was added to the fermentation medium to make the OD of the system 600= 0.1, incubate for 24 h, take 10 mL of the bacterial liquid and centrifuge it at 12000 rpm for 10 min. Precipitate the heparin precursor in the supernatant of the fermentation broth with ethanol at 4 times the volume of the fermentation broth supernatant; then wash the precipitate of the centrifuged bacterial liquid twice with 10 mL of deionized water. Finally, resuspend the precipitated cells after centrifugation with 10 mL of ultrapure water and disrupt them with a high-pressure homogenizer. The disrupted cell suspension is left standing overnight in an oven at 60 °C to remove the impurity proteins. Centrifuge the cell disruption suspension after removing the impurity proteins at 12000 rpm for 10 min, and precipitate the heparin precursor with ethanol at 4 times the volume of the supernatant after centrifugation; centrifuge the supernatant and the heparin precursor precipitated intracellularly at 8000 rpm for 5 min, discard the supernatant after centrifugation, and leave the precipitate in the fume hood until the ethanol has completely evaporated. Add an appropriate amount of ultrapure water to dissolve the heparin precursor, and finally detect the yield of the heparin precursor by the carbazole sulfate method.

[0068] The measurement results show that the recombinant EcN pET-kfiB,ΔKfiB can detect an extracellular heparin precursor yield of only 0.08 g / L. However, when expressing only the N-terminal or C-terminal of KfiB alone, such as strains EcNΔKfiB, EcN pET- N kfiB,ΔKfiB and EcN pET- C kfiB,ΔKfiB do not produce heparin precursors. However, when overexpressing kfiB based on wild-type EcN, the heparin precursor yield is increased from 0.15 g / L to 0.20 g / L ( Figure 1 ).

[0069] Example 2: Effect of knocking out kfiB on the expression levels of kfiACD in the gene cluster in Region II of the E. coli Nissle 1917 genome

[0070] (1) Effect of knocking out kfiB on the expression levels of kfiACD in the gene cluster in Region II

[0071] (1) Refer to the method of the Tiangen RNA extraction kit to extract the total RNA of the recombinant strain E. coli Nissle 1917ΔKfiB obtained in step (4), and then quickly reverse transcribe the extracted total RNA into cDNA with reference to the method of the Takara RNA reverse transcription kit.

[0072] (2) Using the cDNA obtained in step (1) as a template and recA as an internal reference gene, design primers RT-recA-F / R, RT-kfiA-F / R, RT-kfiC-F / R, and RT-kfiD-F / R for qPCR respectively, and then calculate the change in the expression levels of the kfiACD genes according to 2- ΔΔt ( Figure 2 A). It shows that knocking out kfiB in E. coli Nissle 1917 will affect the gene expression of kfiACD on the gene cluster.

[0073] (2) Effects of complementing different combinations of kfiABCD on the production of heparin precursor by E. coli Nissle 1917

[0074] (1) Design primers kfiA-F / kfiA-R, kfiC-F / kfiC-R, kfiAC-F / kfiAC-R, kfiCD-F / kfiCD-R, kfiBC-F / kfiBC-R, kfiBCD-F / kfiBCD-R. Amplify fragments using the E. coli Nissle 1917 genome as a template;

[0075] (2) Linearize plasmid pETDuet-1 and ligate it with the fragments in (1) using seamless cloning ligase to construct recombinant plasmids pET-kfiA, pET-kfiC, pET-kfiAC, pET-kfiCD, pET-kfiBC, and pET-kfiBCD;

[0076] (3) Transform the recombinant plasmids constructed in step (2) into E. coli Nissle 1917ΔKfiB competent cells respectively to construct recombinant bacteria EcN pET-kfiA,ΔKfiB, EcN pET-kfiC,ΔKfiB, EcNpET-kfiAC,ΔKfiB, EcNpET-kfiCD,ΔKfiB, EcN pET-kfiBC,ΔKfiB, and EcNpET-kfiBCD,ΔKfiB.

[0077] (4) According to the shake flask level culture in Example 1, culture the recombinant bacteria obtained in step (3) in LB medium for 10 h, add the bacterial liquid to the fermentation medium to make the OD of the system 600 = 0.1, culture for 24 h, and after the end, detect that the extracellular heparin precursor yields are 0.007 ± 0.002 g / L, 0.010 ± 0.001 g / L, 0.026 ± 0.01 g / L, 0.018 ± 0.008 g / L, 0.172 ± 0.025 g / L, and 0.162 ± 0.02 g / L( Figure 2 B). It shows that knocking out kfiB in E. coli Nissle 1917 and complementing kfiBC or kfiBCD can restore the ability of E. coli Nissle 1917 to produce heparin precursor.

[0078] Example 3: Intracellular positional relationship between KfiB and heparin precursor synthesis pathway proteins, Kdo synthesis pathway proteins, and ABC transport pathway proteins in E. coli Nissle 1917

[0079] (1) According to the method of knocking out kfiB in Example 1, the red fluorescent protein mCHERRY was fused to the C-terminus of KfiB and integrated into E. coli Nissle 1917, and the strain was named E. coli Nissle 1917kfiB-mCherry;

[0080] (2) Referring to the method in (1), the green fluorescent protein sfGFP was fused to the C-terminus of KfiA, KpsC, and KpsT respectively, and integrated into the genome of the strain E. coli Nissle 1917kfiB-mCherry. The strains were named E. coli Nissle 1917kfiB-mCherry kfiA-sfgfp, E. coli Nissle 1917kfiB-mCherry kpsC-sfgfp, and E. coli Nissle 1917kfiB-mCherry kpsT-sfgfp respectively;

[0081] (3) The recombinant strains constructed in step (2) were cultured at the shake flask level as in Example 1, and the intracellular positions of the green and red fluorescent proteins in different strains were observed under a fluorescence microscope. By merging the green fluorescence image and the red fluorescence image in the same field of view ( Figure 3 ), the protein relationships between KfiB and KfiA, KpsC, and KpsT were judged respectively.

[0082] (4) By merging the fluorescence micrographs, it was found that the protein KfiB and the protein KfiA were completely non-overlapping, and the protein KfiB and the protein KpsC were also completely non-overlapping. However, the protein KfiB and the protein KpsT showed an overlapping and similar brightness relationship. This result also indicates that the role of KfiB may be more involved in polysaccharide transport.

[0083] Example 4: Effects of different expressions of medium-level optimized KfiACB in recombinant E. coli Nissle 1917 on the yield of heparin precursor

[0084] (1) Primers kfiABC-F / R, kfiACB-F / R, and kfiBAC-F / R were designed, and fragments were amplified using the E. coli Nissle 1917 genome as a template;

[0085] (2) Design primers pET(kfiABC)-F / R, pET(kfiACB)-F / R and pET(kfiBAC)-F / R to linearize plasmid pETDuet-1, and ligate it with the fragment in (1) using seamless cloning ligase to construct recombinant plasmids pET-kfiABC, pET-kfiACB and pET-kfiBAC respectively. Transfer the plasmids into competent E. coli Nissle 1917 to obtain strains EcN pET-kfiABC, EcN pET-kfiACB and EcN pET-kfiBAC;

[0086] (3) Cultivate the recombinant strains constructed in step (2) according to the shake flask culture in Example 1. After the cultivation, detect the production of heparin precursors inside and outside the cells. The yields of EcN pET-kfiABC, EcN pET-kfiACB and EcN pET-kfiBAC are 0.244 ± 0.055 g / L, 0.349 ± 0.06 g / L and 0.328 ± 0.04 g / L respectively ( Figure 4 );

[0087] (4) The highest yield is screened in step (3) for EcN pET-kfiACB. Through an orthogonal experiment (L 9 3 3 ) with three different strengths of RBS design, as shown in Table 2, optimize the expression level of KfiACB with three different strength levels of RBS. Nine recombinant strains with different strengths of RBS are obtained, namely EcN pET-kfiACB1, EcN pET-kfiACB2, EcN pET-kfiACB3, EcNpET-kfiACB4, EcN pET-kfiACB5, EcN pET-kfiACB6, EcN pET-kfiACB7, EcN pET-kfiACB8 and EcN pET-kfiACB9;

[0088] Specifically, the sequences of three different strengths of RBS (RBS1-3) are:

[0089] RBS1: GAGCAGCG

[0090] RBS2: GATTGACG

[0091] RBS3: TGGGCTCG

[0092] (5) Cultivate the 9 strains in (4) at the shake flask level as in Example 1. After the cultivation is completed, the yields of heparin precursors inside and outside the cells are detected to be 0.66±0.02 g / L, 0.68±0.04 g / L, 0.97±0.01 g / L, 0.77±0.01 g / L, 0.76±0.01 g / L, 0.66±0.04 g / L, 0.71±0.01 g / L, 0.68±0.01 g / L, and 0.57±0.05 g / L respectively ( Figure 5 ). It shows that EcNpET-kfiACB3 has the most obvious improvement in the yield of heparin precursors (the nucleotide sequence of kfiACB3 is shown in SEQ ID NO.1).

[0093] Table 2 Orthogonal design table of different strengths of RBS

[0094]

[0095] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A recombinant Escherichia coli for producing heparin precursor, characterized in that, the recombinant Escherichia coli includes the following modifications: S1. Connect RBS1, the encoding gene kfiA of α - N - acetylglucosaminyltransferase KfiA, RBS3, the encoding gene kfiC of β - glucuronyltransferase KfiC, RBS3 and the encoding gene kfiB of KfiB to the plasmid backbone in sequence to construct a recombinant plasmid; S2. Transform the above recombinant plasmid into an Escherichia coli host to obtain the recombinant Escherichia coli; the Escherichia coli host is Escherichia coli Nissle 1917, the sequence of RBS1 is GAGCAGCG, and the sequence of RBS3 is TGGGCTCG, the NCBI accession number of α - N - acetylglucosaminyltransferase KfiA is AXY46007.1, the NCBI accession number of KfiB is AXY46006.1, and the NCBI accession number of β - glucuronyltransferase KfiC is AXY46005.

1.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The expression vector is plasmid pETDuet - 1.

3. A method for producing heparin precursor, characterized in that: It includes the step of fermenting and producing using the recombinant Escherichia coli according to claim 1 or 2.

4. The method according to claim 3, characterized in that: The fermentation is to culture the recombinant Escherichia coli to obtain a seed solution, and transfer the seed solution to a fermentation medium containing glucose for culture.

5. The method according to claim 4, characterized in that, The fermentation medium comprises the following components: KH 2 PO 4 10 - 15 g / L, (NH 4 ) 2 HPO 4 3 - 5 g / L, citric acid 1 - 3 g / L, MgSO 4 1 - 3 g / L, thiamine 0.1 - 0.3 g / L, CaCl 2 1 - 3 g / L, ZnSO 4 ·7H 2 O 1 - 4 g / L, MnSO 4 ·4H 2 O 0.2 - 0.8 g / L, FeSO 4 ·7H 2 O 8 - 12 g / L, NaB 4 O 7 ·10H 2 O 0.01 - 0.03 g / L, CuSO 4 ·5H 2 O 0.5 - 1.5 g / L, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.05 - 0.2 g / L, D - glucose 15 - 25 g / L, pH 7.0 - 7.2.

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