Escherichia coli transmembrane transporter gene and its application in vitamin b6 biosynthesis

Abstract

The application discloses an Escherichia coli transmembrane transporter gene and application thereof in vitamin B6 biosynthesis. The application weakens a vitamin B6 transporter gene coding in the Escherichia coli, and the ability of a genetically engineered strain to produce vitamin B6 is greatly improved, and the genetically engineered strain has great application and popularization value. In the best embodiment, the extracellular yield of vitamin B6 of the genetically engineered strain is increased by 33.7% compared with that of a wild type, and the effect is extremely remarkable.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and microbial metabolic engineering, specifically to an Escherichia coli transmembrane transporter gene and its application in the biosynthesis of vitamin B6. Background Technology

[0002] Vitamin B6 is an essential water-soluble vitamin containing six interconvertible pyridine compounds: pyridoxine, pyridoxal, pyridoxamine, and their phosphorylated forms—pyridoxal phosphate, pyridoxine phosphate, and pyridoxamine phosphate. As a key cofactor, vitamin B6 plays a central role in various physiological metabolic processes, including amino acid synthesis and metabolism, neurotransmitter production, and carbohydrate metabolism. Since animals and humans cannot synthesize vitamin B6 themselves and must obtain it through diet, it has significant commercial value in the food and livestock industries. Furthermore, vitamin B6 is widely used in the medical field to treat peripheral neuritis, dermatitis, pregnancy vomiting, tumor-related symptoms, and infantile seizures.

[0003] Currently, the main form of vitamin B6 produced on an industrial scale is pyridoxine hydrochloride, and its mainstream production technology is chemical synthesis. However, this method has certain drawbacks. The synthesis process requires the use of highly corrosive phosphorus oxychloride and the toxic solvent benzene, posing certain safety hazards. Simultaneously, the preparation process generates large amounts of wastewater, waste gas, and waste residue, placing significant pressure on the environment. In contrast, biosynthesis exhibits significant advantages, such as environmental friendliness, mild reaction conditions, and enormous potential for production efficiency. Therefore, developing efficient vitamin B6 biosynthesis technology is not only of significant scientific importance but also meets the urgent needs of green industrial production, and is expected to bring considerable economic and social benefits.

[0004] Currently, the biosynthetic yield of vitamin B6 remains low, and a key bottleneck restricting its increase lies in the unknown intracellular transport system of vitamin B6. Although studies in other bacteria have confirmed that certain transport proteins are involved in this process, such as Tpn1p in Saccharomyces cerevisiae and PUP1 in Arabidopsis thaliana, the function of vitamin B6 transport proteins in Escherichia coli, an important model factory microorganism, has not yet been explored and identified. Therefore, screening and identifying proteins involved in vitamin B6 transport in Escherichia coli is of great significance for improving the yield of vitamin B6 produced by microbial fermentation. Summary of the Invention

[0005] To identify membrane proteins involved in vitamin B6 transport, *E. coli* strains with a certain baseline vitamin B6 production are more helpful in highlighting differences in production. Previously, the inventors constructed a high-yielding *E. coli* strain (202410443221.X), using the promoter Ptrc to enhance the expression of the epd, pdxB, serC, and dxs genes in the vitamin B6 synthesis pathway. Preferably, this enhanced expression was achieved by constructing a plasmid vector, specifically the pTrc99a plasmid framework. The recombinant plasmid was transformed into the host bacteria to obtain a high-yielding *E. coli* strain containing the recombinant plasmid. Based on this strain (named LL1397), transcriptome analysis and attenuation studies were used to screen and identify vitamin B6 transport proteins in *E. coli*. Finally, using the existing strain LL07-serC13 (see Chinese patent application 202310145384.5) as a test strain, the universality of this transport protein in different chassis cells was demonstrated.

[0006] In view of this, the purpose of the present invention is to provide an Escherichia coli transmembrane transporter protein and its application, wherein weakening of the Escherichia coli transporter protein gene putP can increase the production of vitamin B6 in Escherichia coli.

[0007] The present invention provides an Escherichia coli transporter gene putP, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0008] The present invention also provides a protein encoded by the Escherichia coli transporter gene putP, the amino acid sequence of which is shown in SEQ ID No. 2.

[0009] The present invention also provides the application of weakening the Escherichia coli transporter gene putP in increasing the vitamin B6 production of engineered strains, wherein the strain is Escherichia coli.

[0010] This invention provides the application of recombinant putP, recombinant vector, and recombinant microorganisms in the preparation of vitamin B6.

[0011] This invention, through transcriptomics analysis during vitamin B6 accumulation, revealed significant changes in the transcription of the putP gene. A putP-attenuated plasmid was constructed using the plv-dCas9-sgRNA vector, and its positive regulatory effect on vitamin B6 production was verified after transformation in the chassis strain LL1397. This led to the screening of PutP as a transmembrane transport protein in *E. coli*, specifically as an internal transport protein. Comparative studies confirmed that attenuation of the internal transport protein PutP in this invention not only promotes biomass increase but also effectively enhances the vitamin B6 production capacity of *E. coli*. Furthermore, the introduction of the attenuated plasmid reduces PutP expression in the genome, thus achieving the attenuation effect and effectively improving the vitamin B6 production capacity of *E. coli*. However, directly knocking out the gene does not effectively enhance the vitamin B6 production capacity of *E. coli*. The function of this transport protein has also been confirmed in other *E. coli* strains such as LL07-serC13. Attached Figure Description

[0012] Figure 1 The spectrum is for the carrier plv-putP.

[0013] Figure 2 The spectrum of the carrier pBAD-putP.

[0014] Figure 3 This is a standard curve for vitamin B6.

[0015] Figure 4 This is a graph showing the yield of engineered strain LL1397 after 48 hours of fermentation.

[0016] Figure 5 The OD600 of the engineered strain LL1397 after 48 hours of fermentation. Detailed Implementation

[0017] The following embodiments and accompanying drawings are merely illustrative of specific implementation schemes for carrying out the present invention. These schemes and drawings should not be construed as limiting the present invention. Any changes made without departing from the principles and essence of the present invention shall fall within the protection scope of the present invention.

[0018] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional. Unless otherwise specified, all materials and reagents used in this embodiment can be obtained through legitimate commercial channels.

[0019] Example 1: Discovery of the putp gene and construction of a vector to weaken the putP gene

[0020] 1. Vitamin B6 was exogenously added to engineered strain LL1397 at concentrations of 0, 1, 5, and 10 g / L. After culturing in LB liquid medium for 12 hours, samples were taken for transcriptomics analysis. It was found that the transcription level of the transporter protein putP gene was increased to varying degrees in the samples with added vitamin B6, suggesting that it has the function of transporting vitamin B6. Further experimental verification of its function in vitamin B6 biosynthesis was carried out.

[0021] 2. The original putP gene of wild-type MG1655 is shown in SEQ ID NO: 1. Primers putP-F / putP-R were designed (Table 1).

[0022] 3. Primer annealing, and concentration determination of the annealed product.

[0023] 4. Using the Plv-dCas9-sgRNA plasmid (the plasmid is derived from the article Li Lv, Yi-Lin Ren, Jin-ChunChen, Qiong Wu, Guo-Qiang Chen, Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: Controllable P(3HB-co-4HB) biosynthesis, Metabolic Engineering, 2015(29):160-168) as the vector backbone, the plasmid was digested with BspQ1 enzyme and incubated at 50℃ for 1 hour according to the instructions. The digested product was subjected to agarose gel electrophoresis to verify the size of the target band. The target band was cut and recovered by gel extraction and the concentration was determined.

[0024] 5. The annealing product and the plasmid digestion product were ligated using T4 DNA Ligase. 10 μL of the ligation product was used for transformation into DH5α *E. coli*. The transformed plasmid was plated on LB agar plates (containing 34 μg / mL chloramphenicol). Positive clones were screened and verified (using primers plv-TEST-F / plv-TEST-R). Sequencing was then performed to confirm the correct bands, yielding the verified recombinant plasmid Plv-putP (see diagram). Figure 1 ), extract plasmids for later use.

[0025] Table 1. Primers for constructing the attenuated putP gene vector

[0026]

[0027] Example 2: Construction of a vector overexpressing the putP gene

[0028] 1. The primers used to amplify the encoding amino acid sequence from putP of wild-type MG1655 were putP-F / putP-R. The backbone was amplified using the empty pBAD-HisA plasmid as a template (primers putP-VF / putP-VR). The putP gene was ligated to the plasmid backbone via Gibson assembly. The resulting plasmid was transformed into DH5α *E. coli*, plated on LB agar plates (containing 100 μg / mL ampicillin), and positive clones were screened and verified (primers pBAD test-F / pBAD test-R). Sequencing confirmed the correctness of the bands, yielding the verified recombinant plasmid pBAD-putP (see diagram). Figure 2 Extract plasmids for later use.

[0029] Table 2. Primers for constructing vectors overexpressing the putP gene

[0030]

[0031] Example 3: Construction of plasmid-containing engineered Escherichia coli strains

[0032] The plasmids Plv-putP and pBAD-putP obtained by the above method were transformed into the engineered Escherichia coli strain LL1397 by chemical transformation according to the following steps. Recombinant Escherichia coli strains were then screened on LB plates (containing 34 μg / mL chloramphenicol or 100 μg / mL ampicillin) to obtain the recombinant Escherichia coli strains.

[0033] (1) Activate Escherichia coli MG1655 derivative strain LL1397 from a -80℃ refrigerator, and culture it in a constant temperature incubator at 37℃ and 200 rpm / min for about 12 hours with shaking. The next day, under aseptic conditions, inoculate it into 5 mL of LB liquid medium with an initial OD of 0.1, and take 1 mL of bacteria with an OD600nm of about 0.5-0.6 into a 1.5 mL microcentrifuge tube.

[0034] (2) Centrifuge at 4,000 rpm for 4-5 minutes to completely remove the supernatant, and then add 0.1 ml of pre-cooled SSCS solution (Shanghai Generay Biotech Co., Ltd.) to gently suspend the bacterial cells.

[0035] (3) Add 10 ng of plasmid DNA for transformation.

[0036] (4) After mixing the DNA and cells, place on ice for 30 minutes, then place at 42°C for 90 seconds, and then place on ice for 15-20 minutes.

[0037] (5) Add 0.8 ml of LB medium to a centrifuge tube and then incubate at 37°C and 200 rpm for 1 hour on a shaker.

[0038] (6) Spread the cells on plates with the appropriate resistance.

[0039] (7) Select positive clones for verification and preservation. The engineered strains are 1397+Plv-putP and 1397+pBAD-putP.

[0040] Example 4: Fermentation and Pretreatment of Engineered Strains

[0041] Culture medium formulation:

[0042] LB medium (g / L): sodium chloride 10, tryptone 10, yeast extract 5, solid medium plus agar powder 15.

[0043] Seed culture medium (g / L): glycerol 10, tryptone 10, yeast extract 5, sodium chloride 5.

[0044] Fermentation medium (g / L): glycerol 15, acid-hydrolyzed casein 5, yeast extract 5, sodium chloride 5, glucose 1, MgSO4·7H2O 200 mg / L, FeSO4·7H2O 10 mg / L, MnSO4·5H2O 10 mg / L, pH controlled at 6.5 by KOH.

[0045] The operation steps are as follows:

[0046] (1) Take fresh and activated engineered strains into a test tube containing 5 mL of seed culture medium (containing 50 ug / mL chloramphenicol), and culture at 37℃ and 200 rpm for 15 h.

[0047] (2) The next day, the OD of the bacterial solution in the test tube was measured. 600 The culture medium was transferred to a 250 mL shake flask containing 30 mL of culture medium. The initial OD600 was 0.1. The culture was carried out at 37 °C on a shaker at 200 rpm for 48 h. Three replicates were performed for each fermentation strain.

[0048] (3) After fermentation, the OD600 of the bacterial solution was measured by a spectrophotometer.

[0049] (4) The yield of extracellular vitamin B6 was determined by fermentation broth supernatant. 1 mL of bacterial culture was taken into an Eppendorf tube, centrifuged at 12,000 rpm for 3 minutes, and the supernatant was diluted 20 times and filtered through a 0.22 μm filter membrane before being placed into a liquid chromatography vial for detection.

[0050] (5) The production of intracellular vitamin B6 was determined by weighing the wet weight of the cells, adding 5 times the volume of 0.7 M HClO4 (volume-to-mass ratio), mixing well, and letting stand on ice for 15 min; then adding 2.5 times the volume of pre-cooled 0.7 M K2CO3, incubating on ice for 15 min, centrifuging at 12000 rpm for 3 min, taking the supernatant, filtering it through a 0.22 μm filter membrane, and placing it in a liquid chromatography vial for detection.

[0051] Example 5: Detection of Vitamin B6

[0052] The detection was performed using a high-performance liquid chromatograph equipped with a fluorescence detector.

[0053] (1) Preparation of standard products

[0054] Prepare a gradient of vitamin B6 standards - pyridoxine (10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L).

[0055] (2) HPLC detection conditions

[0056] COSMOSIL 5C18-AR-II Packed Column (Cosmosil, 4.6 mm ID × 250 mm, 5 µm). Mobile phase A: 33 mM phosphoric acid, 8 mM sodium 1-octanesulfonate aqueous solution, pH adjusted to 2.5 with KOH.

[0057] Mobile phase B: 80% acetonitrile.

[0058] The liquid chromatography conditions were as follows: 0-5 min, gradient from 100% A to 99% A / 1% B; 5-10 min, gradient to 81% A / 19% B; 10-20 min, gradient to 72% A / 28% B; 20-25 min, gradient to 37% A / 63% B; 25-30 min, gradient back to 100% A. The total time for each sample was 30 min. The fluorescence detector was set to excitation wavelength of 293 nm, emission wavelength of 395 nm, column temperature of 35 °C, flow rate of 0.8 mL / min, and injection volume of 20 µL.

[0059] (4) Drawing the standard curve of vitamin B6

[0060] Standards of different concentrations were analyzed by HPLC under the conditions described above, and a standard curve was plotted based on peak area A versus vitamin B6 concentration. The measured peak area A was plotted on the ordinate, and the vitamin B6 concentration C (mg / L) on the abscissa to create a vitamin B6 standard curve. See... Figure 3 The regression equation is obtained as y = 128563x - 35432, R0. 2=0.9999, indicating a good linear relationship between absorbance and mass concentration. Sample yield was calculated based on the vitamin B6 standard curve after liquid chromatography.

[0061] (5) Sample testing and result analysis

[0062] Using LL1397 as the substrate strain, the putP gene was subjected to dual verification through attenuation and overexpression. The fermentation yield and biomass results are shown in Table 3. Figure 4 As shown in the figures, compared with the control strain, the engineered strain with attenuated putP exhibited a higher extracellular yield, reaching 440 mg / L, representing a 33.7% increase in yield. Intracellular yield decreased somewhat, while biomass increased. Conversely, overexpression of putP reduced extracellular yield while increasing intracellular yield, with biomass remaining almost unchanged. Therefore, attenuating putP significantly promoted the fermentation yield of vitamin B6, further enhancing cellular production of vitamin B6 by reducing its internal transport. This invention screened out a vitamin B6 transport protein, which has a significant promoting effect on increasing vitamin B6 production.

[0063] Table 3. Fermentation yield of vitamin B6 in engineered strain LL1397 after attenuation of putP and overexpression of putP.

[0064]

[0065] Example 6: Effect of putp attenuation on vitamin B6 production in other engineered strains

[0066] The plv-putP and pBAD-putP strains constructed in Examples 1 and 2, respectively, were transformed into another engineered strain LL07-serC13 (see Chinese Patent 202310145384.5), which can also produce vitamin B6, according to the method in Example 3. Experiments were then conducted using the fermentation, pretreatment, and detection procedures described in Examples 5 and 6. The experimental results were consistent with those obtained using LL1397 as the chassis (Table 4 and...). Figure 5 This demonstrates that the vitamin B6 transporter protein PutP screened in this invention has internal transport function, and certain metabolic modifications to it can affect the fermentation yield of the strain to a certain extent. Specifically, after weakening, it has a certain promoting effect on increasing vitamin B6 yield.

[0067] Table 4. Fermentation yield of vitamin B6 in engineered strain LL07-serC13 after attenuation of putP and overexpression of putP.

[0068]

[0069] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of an Escherichia coli transporter gene in the biosynthesis of vitamin B6, characterized in that, The amino acid sequence of the protein encoded by the Escherichia coli transporter gene is shown in SEQ ID No.

2. The purpose of increasing vitamin B6 production is achieved by weakening the Escherichia coli transporter gene in the starting strain, and the starting strain is an Escherichia coli that can produce vitamin B6.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the Escherichia coli transporter gene is shown in SEQ ID No. 1 or its degenerate sequence.

3. A recombinant Escherichia coli strain that produces vitamin B6, characterized in that, This is achieved by weakening the endogenous Escherichia coli transporter gene in the starting strain of Escherichia coli, the amino acid sequence of the protein encoded by the Escherichia coli transporter gene is shown in SEQ ID No. 2, wherein the starting strain is an Escherichia coli that produces vitamin B6.

4. The recombinant Escherichia coli as described in claim 3, characterized in that, The nucleotide sequence of the Escherichia coli transporter gene is shown in SEQ ID No. 1 or its degenerate sequence.

5. The recombinant Escherichia coli as described in claim 3, characterized in that, The originating strain also includes genetic modifications that enhance the expression of the epd, pdxB, serC, and / or dxs genes in the vitamin B6 synthesis pathway.

6. A method for biosynthesizing vitamin B6, characterized in that, The step of producing vitamin B6 by culturing recombinant Escherichia coli as described in any one of claims 3 to 5.

7. The method as described in claim 6, characterized in that, It also includes the step of separating or purifying the resulting vitamin B6.

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