Strain for producing taurine by one-step method and construction method thereof

Abstract

The invention discloses a strain for producing taurine by a one-step method and a construction method thereof, and belongs to the field of gene engineering. Corynebacterium glutamicum ATCC13032 is used as a chassis bacterium, by weakening gpmA gene expression in a TCA path, knocking out serB and ldh genes in L-serer and acetic acid-lactic acid synthesis paths and enhancing serA and serC gene overexpression in a phosphoserine synthesis path, a genetically engineered bacterium for efficiently synthesizing taurine by taking glucose as a substrate is successfully constructed, and the yield of the strain is up to 1.5 g / L after the strain is fermented for 96 hours in a shake flask. The taurine yield of the genetically engineered bacterium is further improved by optimizing a fermentation culture medium, and the yield reaches 1.8 g / L.

Description

Technical Field

[0001] This invention relates to a strain for one-step taurine production and its construction method, belonging to the field of genetic engineering. Background Technology

[0002] Taurine is a sulfur-containing β-aminosulfonic acid that is widely found in animal tissues, especially in the heart, skeletal muscle, brain, and retina. As an essential nutrient for the human body, taurine has many uses, including improving nerve conduction and visual function, improving endocrine function, enhancing immunity, and acting as an antioxidant.

[0003] Taurine can be synthesized by chemical or biological methods. Chemical synthesis typically involves reacting chloroacetic acid with ammonia to produce 2-aminoacetic acid, which then reacts with sodium bisulfite to generate taurine. Alternatively, ethylene oxide reacts with ammonia to produce 2-aminoethanol, which then reacts with sodium bisulfite to produce taurine. However, chemical taurine production suffers from high raw material costs, demanding reaction conditions, low yields, difficult separation and purification, and significant pollution. For example, the ethylene oxide method requires high temperature and pressure, with a yield of only about 40%. Biological methods also have drawbacks, including low production efficiency, high substrate and enzyme costs, difficulty in product separation, and stringent fermentation conditions.

[0004] In recent years, several domestic and international publications have reported on the production of taurine using microorganisms. Currently, the highest yield is achieved by Joo YC through modification of Corynebacterium glutamicum, reaching 0.5 g / L. However, the synthases CS, CDO1, and CSAD involved in this strain increase substrate consumption during metabolism, leading to high costs and hindering commercial application. Summary of the Invention

[0005] This invention provides an engineered strain of Corynebacterium glutamicum that weakens the TCA and acetic acid-lactic acid synthesis pathways and enhances the phosphoserine synthesis pathway.

[0006] In one embodiment, the weakening of TCA is to reduce the expression of the phosphoglycerate mutase gene gpmA.

[0007] In one embodiment, reducing the expression of the phosphoglycerate mutase gene gpmA involves replacing the start codon of the phosphoglycerate mutase gene gpmA with GTG.

[0008] In one embodiment, the weakening of the acetate-lactic acid synthesis pathway includes knocking out the phosphoserine phosphatase gene serB and the lactate dehydrogenase gene ldh.

[0009] In one embodiment, the genes of the phosphoserine synthesis pathway include, but are not limited to, the phosphoglycerate dehydrogenase gene serA and the phosphoserine transaminase gene serC.

[0010] In one embodiment, the genetically engineered bacteria have the serB gene, ldh gene, and glyR gene knocked out, and the PGK gene, serA gene, and serC gene overexpressed.

[0011] In one embodiment, the PGK gene is expressed under the regulation of the promoter Ptuf shown in SEQ ID NO.1.

[0012] In one embodiment, the engineered Corynebacterium glutamicum is a substrate bacterium in (a) or (b), wherein:

[0013] (a) Corynebacterium glutamicum ATCC13032;

[0014] (b) Recombinant bacteria constructed by integrating the PFKA gene at the PTA site on the genome of (a).

[0015] In one embodiment, the engineered Corynebacterium glutamicum integrates and expresses the phosphoglycerate kinase gene PGK at the SerB site on the genome of the Corynebacterium glutamicum.

[0016] In one embodiment, the engineered Corynebacterium glutamicum integrates and expresses the phosphoglycerate dehydrogenase gene SerA at the lactate dehydrogenase gene ldh (Gene ID: 1020853) site on the genome of the Corynebacterium glutamicum.

[0017] In one embodiment, the engineered Corynebacterium glutamicum also knocked out the transcriptional regulator glyR (Gene ID: 1021220) and overexpressed the phosphoserine transaminase SerC (Gene ID: 1018823).

[0018] The present invention also provides a method for increasing taurine production by culturing taurine-producing microorganisms in a culture medium containing corn steep liquor powder and nicotinamide.

[0019] In one embodiment, the culture medium is CGXII medium containing 16-32 g / L corn steep liquor powder and 16-32 mg / L nicotinamide.

[0020] The present invention also provides a method for preparing taurine by fermentation, which involves culturing the engineered strain of Corynebacterium glutamicum in a culture medium with glucose as a carbon source for a period of time and collecting the taurine from the fermentation broth.

[0021] In one embodiment, the culture medium is CGXII medium containing corn steep liquor powder and nicotinamide.

[0022] In one embodiment, the concentration of nicotinamide is 16-32 mg / L, and the concentration of corn steep liquor powder is 16-24 g / L.

[0023] In one embodiment, the culture medium contains 16 g / L of corn steep liquor powder and 16 mg / L of nicotinamide.

[0024] In one embodiment, the CGXII culture medium contains 50 g / L glucose, 20 g / L (NH4)2SO4, 5 g / L urea, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4·7H2O, 13.3 mg / L CaCl2·2H2O, 42 g / L MOPS, 0.2 mg / L biotin, and 1 ml / L trace element solution. The pH is adjusted to 7.0 using KOH. The trace element solution contains: 10 g / L FeSO4·7H2O, 10 g / L MnSO4·1H2O, 1 g / L ZnSO4·7H2O, 313 mg / L CuSO4·5H2O, and 20 mg / L NiCl·6H2O.

[0025] The present invention also provides the application of the engineered strain of Corynebacterium glutamicum or the method thereon in the preparation of products containing taurine.

[0026] Beneficial Effects: This invention uses Corynebacterium glutamicum ATCC13032 as the substrate bacteria. By weakening the expression of the gpmA gene in the TCA pathway, knocking out the serB and ldh genes in the L-ser and acetate-lactic acid synthesis pathways, and enhancing the overexpression of the serA and serC genes in the phosphoserine synthesis pathway, a genetically engineered bacterium capable of efficiently synthesizing taurine using glucose as a substrate was successfully constructed. This strain achieved a yield of 1.5 g / L after 96 hours of shake-flask fermentation. Furthermore, this invention further improved the taurine yield of the genetically engineered bacterium by optimizing the fermentation medium, reaching a yield of 1.8 g / L. Attached Figure Description

[0027] Figure 1 This study outlines the taurine synthesis pathway and the genes involved.

[0028] Figure 2 This is the result of liquid chromatography detection of taurine.

[0029] Figure 3 The results are from the shake-flask fermentation of taurine. Detailed Implementation

[0030] Culture medium:

[0031] CGXII medium: glucose 50 g / L, (NH4)2SO4 20 g / L, urea 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.25 g / L, CaCl2·2H2O 13.3 mg / L, MOPS 42 g / L, biotin 0.2 mg / L, trace element solution 1 ml / L, pH adjusted to 7.0 with KOH; wherein, the trace element solution: FeSO4·7H2O 10 g / L, MnSO4·1H2O 10 g / L, ZnSO4·7H2O 1 g / L, CuSO4·5H2O 313 mg / L, NiCl·6H2O 20 mg / L.

[0032] LYS medium: glucose 5g / L, urea 3g / L, yeast extract 10g / L, soybean meal 15g / L, NaCl 2.5g / L, KH2PO4 1g / L, succinic acid 0.5g / L.

[0033] Detection method:

[0034] Taurine detection method: Taurine was detected using a high-performance liquid chromatograph (HPLC) 1290 equipped with a photodiode array detector. The chromatographic column was a ZORBAX RR Eclipse Plus C18 HPLC column (3.5 μm, 2.1 × 100 mm). The mobile phase was sodium acetate solution (containing 0.1% formic acid). Isocratic elution with pure methanol was performed for 5 min. The flow rate was 0.3 mL / min. The detection wavelength was 330-360 nm. The column temperature was 40 °C. The injection volume was 2 μL.

[0035] Example 1: Construction of chassis cells of a high-taurine-producing Corynebacterium glutamicum engineered strain

[0036] Figure 1 To synthesize taurine, the lactate and L-serine synthesis pathways were knocked out or weakened, while the expression of some key genes in these pathways was enhanced, resulting in the construction of a Corynebacterium glutamicum capable of synthesizing taurine. Details are as follows:

[0037] (1) Constructing the recombinant plasmid pK18-ΔPTA::PFKA, knocking out the phosphoacetyltransferase gene PTA, and enhancing the synthesis of 6-phosphofructokinase PFKA: Using the genome of Corynebacterium glutamicum ATCC13032 as a template, clone the homologous arms of the phosphoacetyltransferase gene PTA (Gene ID: 1020698) 1000bp upstream and downstream, clone the 6-phosphofructokinase gene PFKA (Gene ID: 3344017), construct the PFKA gene fragment between the upstream and downstream homologous arms of the PTA gene, and link it to the Pk18mobsacB backbone using the Gibson method to construct the recombinant plasmid pK18-ΔPTA::PFKA. Transform the obtained recombinant plasmid pK18-ΔPTA::PFKA into competent cells of Corynebacterium glutamicum Erg2 (disclosed in the patent application document with publication number CN117247881A) to obtain the strain Erg2 / ΔPTA::PFKA, named T8.

[0038] (2) Construct the recombinant plasmid pK18-ΔSerB::PGK, knock out the phosphoserine phosphatase gene SerB (Gene ID: 1020471), and enhance the expression of phosphoglycerate kinase PGK (Gene ID: 1019555). Construction method: Using the genome of Corynebacterium glutamicum ATCC13032 as a template, clone the homologous arms of the SerB gene upstream and downstream of each other, clone the phosphoglycerate kinase gene PGK fragment containing the promoter Ptuf (nucleotide sequence as shown in SEQ ID NO.1), construct it between the upstream and downstream homologous arms of the SerB gene, and link it with the Pk18mobsacB backbone using the Gibson method to construct the recombinant plasmid pK18-ΔSerB::PGK. Transform the obtained recombinant plasmid pK18-ΔSerB::PGK into competent cells of the recombinant strain T8 constructed in step (1) to obtain the recombinant strain T8 / ΔSerB::PGK, named T9.

[0039] (3) Following the same strategy described above, the recombinant plasmid pK18-Δldh::SerA was constructed to knock out the lactate dehydrogenase gene ldh (Gene ID: 1020853) and enhance the expression of the phosphoglycerate dehydrogenase gene SerA (Gene ID: 1019265). The constructed recombinant plasmid pK18-Δldh::SerA was transformed into competent cells of the recombinant strain T9 constructed in step (2) to obtain the recombinant strain T9 / Δldh::SerA, named T10.

[0040] (4) Following the same strategy described above, the recombinant plasmid pK18-ΔglyR::SerC was constructed to knock out the transcriptional regulator glyR (Gene ID: 1021220) and overexpress the phosphoserine transaminase gene SerC (Gene ID: 1018823) to enhance phosphoserine synthesis. The constructed recombinant plasmid pK18-ΔglyR::SerC was transformed into competent cells of the recombinant strain T10 constructed in step (3) to obtain the recombinant strain T10 / ΔglyR::SerC, named T11.

[0041] (5) Following the same strategy described above, a recombinant plasmid pK18-ΔgpmA::gpmA(ATG→GTG) was constructed to replace the start codon of the phosphoglycerate mutase gpmA (Gene ID: 23502190), thereby weakening the expression of the TCA-cycle phosphoglycerate mutase. The constructed recombinant plasmid pK18-ΔgpmA::gpmA(ATG→GTG) was transformed into competent cells of the recombinant strain T11 constructed in step (4), resulting in the recombinant strain T11 / ΔgpmA::gpmA(ATG→GTG), named T12.

[0042] Example 2: Production of taurine by shake-flask fermentation using engineered strains of Corynebacterium glutamicum.

[0043] The recombinant taurine-producing bacteria constructed in Example 1 were used for a one-step fermentation process to produce taurine from glucose. Strains T9, T10, T11, and T12 constructed in Example 1 were cultured in LYS medium in test tubes at 30°C for 12 hours to obtain seed culture. The seed culture was inoculated into 250 mL shake flasks containing 30 mL of CGXII medium, allowing the initial OD after inoculation to reach a certain level. 600 =6, fermentation temperature is 30℃, shaker speed is 200rpm, fermentation time is 96 hours.

[0044] Taurine yield was determined using liquid chromatography, such as... Figure 2 As shown, compared with the standard of taurine, a peak of taurine was detected in the sample, indicating that the constructed strain can produce taurine.

[0045] Figure 3 The results show the taurine yield after shake-flask fermentation. After 72 hours of fermentation, the 50 g / L glucose was nearly exhausted, and the OD reached its maximum value of 45. Finally, at 96 hours, taurine levels of 1.1 g / L, 1.15 g / L, 1.4 g / L, and 1.58 g / L were detectable in the fermentation broths of recombinant strains T9, T10, T11, and T12, respectively. The yield of recombinant strain T12 was increased by 43.64%, 37.39%, and 12.86% compared to recombinant strains T9, T10, and T11, respectively.

[0046] Example 3: Optimization of Culture Medium for Engineered Corynebacterium glutamicum

[0047] Using a single-factor experimental method, the composition of the CGXII medium was optimized, with three parallel experiments performed in each group to increase the yield of the target product, taurine.

[0048] (1) Nitrogen source optimization

[0049] Based on the components of CGXII medium, corn steep liquor powder, sugarcane molasses, or wheat peptone were added at a concentration of 8 g / L. The recombinant taurine-producing strain T12 seed culture constructed in Example 1 was inoculated into 250 mL shake flasks containing 30 mL of CGXII-1 medium with different nitrogen sources, and transferred at a rate of 0.2 OD / mL to achieve an initial OD of [missing value]. 600 The fermentation temperature was 30℃, the shaking speed was 200 rpm, and the fermentation time was 96 hours. Samples were taken to measure OD600 and taurine yield. The results are shown in Table 1.

[0050] Table 1. Fermentation effects of recombinant strain T12 on different culture media

[0051]

[0052] (2) Optimization of corn steep liquor dry powder concentration

[0053] Based on the CGXII medium composition, corn steep liquor powder at concentrations of 8 g / L, 16 g / L, and 24 g / L was added respectively. The recombinant taurine-producing strain T12 seed culture constructed in Example 1 was inoculated into 250 mL shake flasks containing 30 mL of CGXII-YMJ medium with different concentrations of corn steep liquor powder. The inoculation was performed at a rate of 0.2 OD / mL, resulting in an initial total OD of 6 in the shake flasks. The fermentation temperature was 30℃, the shaker speed was 200 rpm, and the fermentation time was 96 hours. Samples were taken to measure OD600 and taurine yield. The results are shown in Table 2.

[0054] Table 2. Fermentation results of recombinant strain T12 in CGXII medium with different concentrations of corn steep liquor powder.

[0055]

[0056] (3) Optimization of nicotinamide concentration

[0057] Based on the composition of CGXII-YMJ medium, nicotinamide concentrations of 16 mg / L, 32 mg / L, and 48 mg / L were added. The seed culture of the recombinant taurine-producing strain T12 constructed in Example 1 was inoculated into 250 mL shake flasks containing 30 mL of CGXII-YY medium with different nicotinamide concentrations. The inoculation was carried out at a rate of 0.2 OD / mL, resulting in an initial total OD of [missing value]. 600The fermentation temperature was 30℃, the shaking speed was 200 rpm, and the fermentation time was 96 hours. Samples were taken to measure OD600 and taurine yield. The OD and taurine yield of different nicotinamide concentrations at 96 hours are shown in Table 3.

[0058] Table 3. Fermentation results of recombinant strain T12 in CGXII medium containing different concentrations of nicotinamide.

[0059]

[0060]

[0061] The results of the single-factor optimization above show that adding 16 g / L of corn steep liquor powder and 16 mg / L of nicotinamide to the CGII medium significantly improved the taurine yield of the recombinant strain T12. Compared with the original CGII medium, the taurine yield increased by 18.71%. The optimized medium was named CGXII-YY.

[0062] 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. Engineered Corynebacterium glutamicum, characterized in that, The expression of phosphoserine phosphatase gene serB, lactate dehydrogenase gene ldh, and phosphoglycerate mutase gene gpmA was reduced, while the expression of genes in the phosphoserine synthesis pathway was enhanced; the phosphoserine synthesis pathway includes phosphoglycerate dehydrogenase gene SerA and phosphoserine transaminase gene SerC.

2. The engineered Corynebacterium glutamicum according to claim 1, characterized in that, The reduction of phosphoglycerate mutase gene gpmA expression is achieved by replacing the start codon of gene gpmA with GTG.

3. The engineered Corynebacterium glutamicum according to claim 1 or 2, characterized in that, The transcriptional regulator glyR was also knocked out.

4. The engineered Corynebacterium glutamicum according to any one of claims 1 to 3, characterized in that, It also overexpresses the phosphoglycerate kinase gene PGK.

5. The engineered Corynebacterium glutamicum according to any one of claims 1 to 4, characterized in that, Using (a) or (b) as the basal bacteria, where: (a) Corynebacterium glutamicum ATCC13032; (b) Recombinant Corynebacterium glutamicum constructed by integrating the PFKA gene at the PTA site on the genome of (a).

6. A method for increasing taurine yield, characterized in that, The taurine-producing microorganisms were cultured in a medium containing corn steep liquor powder and nicotinamide.

7. A method for preparing taurine by fermentation, characterized in that, The engineered strain of Corynebacterium glutamicum according to any one of claims 1 to 5 is cultured in a culture medium for a period of time using glucose as a carbon source, and the taurine in the fermentation broth is collected.

8. The method according to claim 7, characterized in that, The culture medium is CGXII medium containing corn steep liquor powder and nicotinamide.

9. The method according to claim 7 or 8, characterized in that, The concentration of nicotinamide is 16–32 mg / L, and the concentration of corn steep liquor powder is 16–24 g / L.

10. The use of the engineered Corynebacterium glutamicum according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9 in the preparation of products containing taurine.

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