Escherichia coli transmembrane transport protein gene and application thereof in biosynthesis of vitamin B6

Abstract

The invention discloses an escherichia coli transmembrane transport protein gene and application thereof in biosynthesis of vitamin B6. According to the invention, the vitamin B6 transporter gene is weakened and coded in escherichia coli, so that the capability of producing the vitamin B6 of the obtained genetic engineering strain is greatly improved, and the application and popularization value is relatively high. In the optimal embodiment, the extracellular yield of the vitamin B6 of the obtained genetic engineering strain is increased by 33.7% compared with that of a wild type, and the effect is extremely remarkable.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of synthetic biology and microbial metabolic engineering, and in particular to an Escherichia coli transmembrane transporter gene and its application in vitamin B6 biosynthesis. BACKGROUND

[0002] Vitamin B6 is an essential water-soluble vitamin, which contains six interconvertible pyridine compounds: pyridoxine, pyridoxal, pyridoxamine and their phosphorylated forms, pyridoxal 5'-phosphate, pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate. As a key cofactor, vitamin B6 plays a core role in various physiological metabolic processes, including the synthesis and metabolism of amino acids, the generation of neurotransmitters, and carbohydrate metabolism, etc. Since animals and humans cannot synthesize vitamin B6 themselves, they must rely on dietary intake, which makes it have important commercial value in the food industry and animal husbandry. In addition, vitamin B6 is also widely used in the medical field to treat diseases such as peripheral neuritis, dermatitis, pregnancy vomiting, tumor-related symptoms, and infantile convulsions, etc.

[0003] Currently, the main product form of vitamin B6 produced on an industrial scale is pyridoxine hydrochloride, and the mainstream production technology is chemical synthesis. However, this method has certain defects. In the synthesis process, strong corrosive phosphorus oxychloride and toxic solvent benzene are used, which has certain safety hazards. At the same time, a large amount of wastewater, waste gas and waste residue is generated during the preparation process, which causes great pressure on the environment. In contrast, biological synthesis method shows significant advantages, such as environmental friendliness, mild reaction conditions and great potential for production efficiency. Therefore, developing an efficient vitamin B6 biosynthesis technology not only has important scientific significance, but also can meet the urgent needs of green industrial production, and is expected to bring considerable economic and social benefits.

[0004] Currently, the yield of vitamin B6 biosynthesis is still low, and a key bottleneck restricting the yield improvement is that the transport system of vitamin B6 in cells is unknown. Although some studies have confirmed that certain specific transport proteins are involved in this process in other bacteria, 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 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 microbial fermentation production of vitamin B6. SUMMARY

[0005] In order to identify the membrane protein involved in the transport of vitamin B6, the E. coli with a certain amount of vitamin B6 basis production is more helpful to highlight the difference in production. The present inventors previously constructed an E. coli high-yield strain (202410443221.X), which utilized the promoter Ptrc to enhance the expression of the epd, pdxB, serC and dxs genes in the vitamin B6 synthesis pathway. Preferably, the enhanced expression is achieved by constructing a plasmid vector, and the specific plasmid framework is pTrc99a. The expression recombinant plasmid is transformed into the host bacteria to obtain an E. coli high-yield strain containing the recombinant plasmid. Here, the strain (named LL1397) is used as the basis for screening and identifying the vitamin B6 transport protein of E. coli through transcriptome analysis and weakening. Finally, the LL07-serC13 in the existing strain (see Chinese patent application 202310145384.5) is used as a test strain to prove the universality of the transport protein in different chassis cells.

[0006] Therefore, the purpose of the present application is to provide an E. coli transmembrane transport protein and its application, which can improve the yield of vitamin B6 in E. coli after weakening the putP gene of the E. coli transport protein.

[0007] The present application provides an E. coli transport protein gene putP, and the nucleotide sequence of the E. coli transport protein gene putP is shown in SEQ ID No. 1.

[0008] The present application also provides a protein encoded by the E. coli transport protein gene putP, and the amino acid sequence of the protein is shown in SEQ ID No. 2.

[0009] The present application also provides the application of weakening the E. coli transport protein gene putP in improving the yield of vitamin B6 in an engineering strain, and the strain is E. coli.

[0010] The present application provides the application of recombinant putP, recombinant vector and recombinant microorganism in preparing vitamin B6.

[0011] The present application finds that the transcription of putP gene has obvious changes in the process of vitamin B6 accumulation through transcriptomics. The plv-dCas9-sgRNA vector is used to construct the putP weakened plasmid, and the positive regulation effect of the plasmid on the yield of vitamin B6 is verified after the plasmid is transformed into the chassis strain LL1397. Further, PutP is screened as a kind of transmembrane transport protein in E. coli, especially as an endoporter. Through comparative study, it is confirmed that the endoporter PutP is weakened in the present application, which can not only promote the improvement of biomass, but also effectively improve the ability of E. coli to produce vitamin B6. Further, the expression of PutP on the genome is reduced by the introduction of the weakened plasmid, thereby achieving the effect of weakening, which can effectively improve the ability of E. coli to produce vitamin B6. However, if the gene is directly knocked out, the ability of E. coli to produce vitamin B6 cannot be effectively improved. The function of the transport protein is also confirmed in other E. coli such as LL07-serC13. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The figure is a map of the vector plv-putP.

[0013] Figure 2 The figure is a map of the vector pBAD-putP.

[0014] Figure 3 The figure is a standard curve of vitamin B6.

[0015] Figure 4 The figure is the yield of the engineering strain LL1397 after 48 hours of fermentation.

[0016] Figure 5 The figure is the OD600 of the engineering strain LL1397 after 48 hours of fermentation. DETAILED DESCRIPTION

[0017] The following examples and drawings of the present application only illustrate the specific implementation of the present application, and these examples and drawings cannot be understood as limiting the present application, and any changes made without departing from the principles and essence of the present application fall within the protection scope of the present application.

[0018] The experimental techniques and experimental methods used in the present example are all conventional technical methods unless otherwise specified. The materials, reagents and the like used in the present example can be obtained through the regular commercial channel unless otherwise specified.

[0019] Example 1: Mining of putp gene and construction of putP gene weakened vector

[0020] 1. 0, 1, 5, and 10 g / L of vitamin B6 were exogenously added to the engineered strain LL1397. After 12 hours of culture in LB liquid medium, samples were taken for transcriptomic analysis. It was found that the transcription level of the transport protein putP gene increased to varying degrees in the samples supplemented with vitamin B6. It was speculated that it has the function of transporting vitamin B6. Further experiments were conducted to verify its function in vitamin B6 biosynthesis.

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

[0022] 3. Anneal the primers and measure the concentration of the annealing products.

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

[0024] 5. Use T4 DNA Ligase to connect the annealed product and the enzyme digestion product of the plasmid. Take 10 μL of the enzyme ligation product for transformation, transform DH5α Escherichia coli, spread on LB plates (containing 34 μg / mL chloramphenicol), screen positive clones and verify (primers plv-TEST-F / plv-TEST-R) that the bands are correct. Then sequence and confirm to obtain the correct recombinant plasmid Plv-putP (see the map). Figure 1 ), extract the plasmid for later use.

[0025] Table 1. Primers for constructing weakened putP gene vectors

[0026] Example 2: Construction of a putP gene overexpression vector

[0027] 1、The primers used for amplifying the coding amino acid sequence from the putP of wild type MG1655 as a template are putP-F / putP-R. The backbone is amplified from the pBAD-HisA empty plasmid as a template (primers putP-V-F / putP-V-R). The putP gene is connected with the plasmid backbone through Gibson assembly. The DH5a Escherichia coli is transformed, spread on LB plates (containing 100 μg / mL ampicillin), and the positive clones are screened and verified (primers pBAD test-F / pBAD test-R) to confirm the correct band before sequencing. The recombinant plasmid pBAD-putP (schematic diagram see Figure 2 ) is obtained, and the plasmid is prepared for standby use.

[0028] Table 2, primers for constructing a vector overexpressing the putP gene

[0029] Example 3: Construction of Escherichia coli engineering strain containing plasmid

[0030] The plasmids Plv-putP and pBAD-putP obtained by the above method are transformed into the Escherichia coli engineering strain LL1397 by chemical transformation method according to the following steps, and the recombinant Escherichia coli strains are screened on LB plates (containing 34 μg / mL chloramphenicol or 100 μg / mL ampicillin), respectively.

[0031] (1) Activate the Escherichia coli MG1655 derivative strain LL1397 from the -80°C refrigerator, and shake culture in a constant temperature incubator at 37°C, 200 rpm / min for about 12 hours. The next day, under sterile conditions, inoculate 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 to a 1.5 ml microcentrifuge tube.

[0032] (2) Centrifuge at 4,000 rpm for 4-5 minutes, completely remove the supernatant, and add 0.1 ml of pre-cooled SSCS solution (Shanghai Generay Biotech Co., Ltd.) to suspend the bacteria.

[0033] (3) Add 10 ng of plasmid DNA for transformation.

[0034] (4) After mixing the DNA and cells, place them on ice for 30 minutes, then at 42°C for 90 seconds, and then on ice for 15-20 minutes.

[0035] (5) Add 0.8 ml of LB medium to the centrifuge tube, and then culture at 37°C, 200 rpm for 1 hour.

[0036] (6) Spread the cells on the corresponding resistant plates.

[0037] (7) Pick positive clones to verify the preservation, and the engineering strains are 1397+Plv-putP and 1397+pBAD-putP, respectively.

[0038] Example 4: Fermentation and pretreatment of the engineering strain

[0039] Culture medium formula:

[0040] LB medium (g / L): sodium chloride 10, tryptone 10, yeast extract 5, and agar powder 15 for solid medium.

[0041] Seed medium (g / L): glycerol 10, tryptone 10, yeast extract 5, and sodium chloride 5.

[0042] Fermentation medium (g / L): glycerol 15, acid hydrolysis 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, and pH controlled at 6.5 by KOH.

[0043] The operation steps are as follows:

[0044] (1) Take the freshly activated engineering strain in a test tube containing 5 mL of seed medium (containing 50 ug / mL chloramphenicol), and cultivate at 37°C on a shaker at 200 rpm for 15 h.

[0045] (2) The next day, measure the OD 600 of the bacterial solution in the test tube, transfer to a 250 mL flask containing 30 mL of medium, and cultivate at 37°C on a shaker at 200 rpm for 48 h, with 3 parallel samples for each fermentation strain.

[0046] (3) After the fermentation is completed, measure the OD600 of the bacterial solution by spectrophotometry.

[0047] (4) The yield of extracellular vitamin B6 is determined by measuring the supernatant of the fermentation solution. Take 1 mL of bacterial solution into an Ep tube, centrifuge at 12000 rpm for 3 minutes, dilute the supernatant 20 times, filter through a 0.22 μm filter membrane, and then place it into a liquid phase vial for detection.

[0048] (5) Intracellular vitamin B6 production was determined by weighing the wet weight of the bacterial cells, followed by the addition of 5 times the volume of 0.7 M HCIO4 (volume / mass ratio), mixing well, and then placing on ice for 15 min. Then 2.5 times the volume of pre-cooled 0.7 M K2CO3 was added, and the mixture was incubated on ice for 15 min. After centrifugation at 12,000 rpm for 3 min, the supernatant was filtered through a 0.22 μm filter and then placed in a liquid phase vial for detection.

[0049] Example 5: Detection of vitamin B6

[0050] A high-performance liquid chromatograph equipped with a fluorescence detector was used for detection.

[0051] (1) Preparation of standard

[0052] A gradient vitamin B6 standard-pyridoxine (10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L) was prepared.

[0053] (2) HPLC detection conditions

[0054] COSMOSIL 5C18-AR-II Packed Column chromatographic column (Cosmosil, 4.6 mm I.D. x 250 mm, 5 μm). Mobile phase A: 33 mM phosphoric acid, 8 mM 1-octane sulfonic acid sodium in water, pH = 2.5 adjusted with KOH.

[0055] Mobile phase B: 80% acetonitrile.

[0056] The liquid phase conditions were as follows: 0-5 min, 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 to 100% A, total time for each sample 30 min. The fluorescence detector was set to an excitation wavelength of 293 nm and an emission wavelength of 395 nm, the column temperature was set to 35°C, the flow rate was 0.8 mL / min, and the injection volume was 20 μL.

[0057] (4) Preparation of the vitamin B6 standard curve

[0058] Different concentrations of the standard were subjected to HPLC detection under the above conditions, and a peak area A-vitamin B6 concentration standard curve was prepared. The measured peak area A was taken as the ordinate, and the vitamin B6 mass concentration C (mg / L) was taken as the abscissa, and the vitamin B6 standard curve was prepared. See Figure 3 , the regression equation y = 128563x - 35432, R 2= 0.9999, the absorbance and mass concentration showed good linear relationship. After liquid phase ended, the sample yield was calculated according to the vitamin B6 standard curve.

[0059] (5) Sample detection and result analysis

[0060] With LL1397 as the chassis strain, the putP gene was subjected to dual verification of weakening and overexpression, and the fermentation yield and biomass are shown in Table 3 and Figure 4 From the data in the chart, it can be seen that, compared with the control strain, after weakening putP, the extracellular yield of the engineering strain is higher, reaching 440 mg / L, the yield is increased by 33.7%, the intracellular yield is decreased to a certain extent, and the biomass is increased to a certain extent; while after overexpression of putP, the extracellular yield is decreased, the intracellular yield is increased, and the biomass is almost unchanged. Therefore, weakening putP obviously promotes the increase of the fermentation yield of vitamin B6, and further promotes the production of vitamin B6 by the cell by reducing the intracellular transport of vitamin B6. In the present application, the vitamin B6 intracellular transport protein is screened, which has a great promoting effect on the increase of the yield of vitamin B6.

[0061] Table 3, fermentation yield of vitamin B6 of engineering strain LL1397 after weakening putP and overexpression of putP

[0062] Example 6: Effect of putP weakening on vitamin B6 yield in other engineering strains

[0063] The plv-putP and pBAD-putP respectively constructed in Examples 1 and 2 are transformed into another engineering strain LL07-serC13 (see Chinese Patent 202310145384.5) which can also produce vitamin B6 according to the method in Example 3, and experiments are carried out through the fermentation, pretreatment and detection processes in Examples 5 and 6, and the experimental results are consistent with the results of taking LL1397 as the chassis (Table 4 and Figure 5 ), which proves that the vitamin B6 transport protein PutP screened in the present application has the function of intracellular transport, and a certain metabolic modification thereof can affect the fermentation yield of the strain to a certain extent, specifically, it has a certain promoting effect on the increase of the yield of vitamin B6 after weakening.

[0064] Table 4, fermentation yield of vitamin B6 of engineering strain LL07-serC13 after weakening putP and overexpression of putP

[0065] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, and the like made within the spirit and principle of the present application should be included in the scope of protection of the present application.

Claims

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

2.

2. The use 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. The use according to claim 1, characterized in that The purpose of increasing vitamin B6 production is achieved by weakening the Escherichia coli transporter gene in the starting strain.

4. A recombinant Escherichia coli producing vitamin B6, characterized in that This is achieved by weakening the endogenous E. coli transporter gene in the starting E. coli strain. The amino acid sequence of the protein encoded by the E. coli transporter gene is shown in SEQ ID No.

2.

5. The recombinant Escherichia coli according to claim 4, characterized in that The starting bacteria is Escherichia coli which can produce vitamin B6.

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

7. The recombinant Escherichia coli according to claim 4, characterized in that The starting bacteria also include genetic modifications to enhance the expression of epd, pdxB, serC and / or dxs genes in the vitamin B6 synthesis pathway.

8. A method for biosynthesizing vitamin B6, characterized in that: The step of producing vitamin B6 by culturing the recombinant Escherichia coli according to any one of claims 4 to 7.

9. The method according to claim 8, wherein The process also includes the step of isolating or purifying the produced vitamin B6.

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