Engineering strain for producing urolithin A and application thereof

Abstract

The invention discloses an engineering strain for producing urolithin A and application of the engineering strain. The engineering strain contains a tannin acyl hydrolase gene tah, an esterase gene lip, a gallic acid decarboxylase gene gdo and a dopamine dehydroxylase gene ascd. The four artificially synthesized genes are jointly constructed on an escherichia coli expression vector, so that a brand-new vector containing four co-expression genes is constructed, then the brand-new vector is transferred into a chassis strain cell to realize co-expression of the four genes, and four enzymes expressed in the cell all have activity; and the obtained heavy engineering strain can stably convert the extracellular added ellagic tannin into the urolithin A, so that the whole-cell catalysis process of producing the urolithin A by taking the ellagic tannin as a substrate is realized.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to an engineered strain for producing urolithin A and its application. Background Technology

[0002] Urolithin-A (Uro-A) is a natural compound with various biological activities and is considered to have potential health benefits, such as anti-aging, antioxidant, and anti-inflammatory effects. Currently, there are two main methods for preparing urolithin-A: chemical synthesis and biosynthesis. The chemical synthesis method is relatively mature, using 3-methoxybenzoic acid as a starting material and achieving mass production of Uro-A through chemical reactions such as bromination, esterification, and demethylation. Additionally, patent CN202311496048.1 discloses a method for preparing urolithin-A using urolithin B as a raw material and undergoing Elbs oxidation with potassium persulfate under alkaline conditions to obtain urolithin-A. While the chemical synthesis method involves complex purification processes and has been applied in industrial production, it is not the optimal production method because Uro-A prepared by chemical synthesis cannot be used as a food additive in China. Biosynthesis, on the other hand, offers higher safety, lower cost, simpler operation, and the potential to achieve the goal of using Uro-A and other uro compounds as functional food ingredients, making it a potential production method for Uro-A.

[0003] Ellagic acid (EA) is a dietary polyphenol that exists primarily in nature in its condensed form—ellagitannin (ETs), mainly derived from berries and nuts. It is known that ellagic acid can be converted into Uro under the influence of gut microbiota. Little is known about the bacterial species involved in this process of EA generating Uro. To date, two strains of *Gordonella* capable of producing Uro-C have been identified. Gordonibacter genus Strain: *Gordonella urolithiasis* ( G. urolithinfaciens DSM 27213T=CCUG 64261T), Pamela Gordonella ( G. pamelaeae DSM 19378T=CCUG 55131T); A strain of *Ellagibacter genus* with iso-Uro-A production capacity: *Ellagibacter genus* ( E.isourolithinifaciens DSM 104140T=CCUG 70284T). Additionally, patents CN202210972288.3, ​​CN202211134634.7, and CN202211409720.4 respectively provide strains of *Lactococcus gasseri* that produce Uro-A (…). Lactococcus garvieae FUA009), Enterococcus faecalis (Enterococcus faecium FUA027) and Lactobacillus plantarum ( Lactiplantibacillus plantarum (CCFM1290). The body produces Uro-A through intestinal flora metabolism, so its yield is influenced by the flora and is accompanied by the generation of other types of Uro. In contrast, in vitro fermentation using artificially cultured microorganisms to prepare Uro-A has the potential for large-scale production and reduced byproducts. Therefore, in vitro fermentation of Uro-A has significant potential applications and can provide more comprehensive technical support for research and commercial development in related fields. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, the present invention provides an engineered strain for the production of urolithin A and its application.

[0005] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an engineered strain for producing urolithin A, wherein the engineered strain contains a tannic acid hydrolase gene. tah、 esterase gene lip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd .

[0006] As a specific implementation scheme, the tannin acyl hydrolase gene tah The nucleotide sequence is shown in SEQ ID NO.1; The esterase gene lip The nucleotide sequence is shown in SEQ ID NO.2; The gallic acid decarboxylase gene gdo The nucleotide sequence is shown in SEQ ID NO.3; The dopamine dehydroxylase gene ascd The nucleotide sequence is shown in SEQ ID NO.4.

[0007] As a specific implementation plan, the chassis strain of the engineered strain is Escherichia coli. E.coli BL21 (DE3).

[0008] Secondly, the present invention provides a vector containing a tannic acid hydrolase gene. tah、 esterase gene lip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd .

[0009] As a specific implementation scheme, the vector is a tannic acid hydrolase gene. tah、esterase gene lip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd The vector pET28a-UroA, which contains four co-expressed genes, was co-constructed on the E. coli expression vector pET-28a(+).

[0010] Thirdly, the present invention provides a method for constructing the engineered strain, comprising the following steps: Genes containing tannic acid hydrolases tah、 esterase gene lip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd The vector was introduced into the chassis strain to obtain the engineered strain.

[0011] Fourthly, the present invention provides the application of the engineered strain in the biosynthesis of urolithin A.

[0012] Fifthly, the present invention provides a method for biosynthesizing urolithin A, which utilizes the engineered strain to perform whole-cell catalysis to convert extracellularly added ellagitannins into urolithin A.

[0013] As a specific implementation plan, the method for biosynthesizing urolithin A includes the following steps: (1) The engineered strain was inoculated into liquid LB medium, kanamycin was added and cultured in a shaker, and then a portion of the bacterial culture was inoculated into liquid LB medium and cultured in a shaker for a longer period of time. (2) When OD 600 When OD = 0.6, add isopropyl-β-D-thiogalactoside and culture in a shaker. 600 When the concentration reaches 4, collect the bacterial cells, resuspend them in tris(hydroxymethyl)aminomethane buffer, add ellagitannin, and continue to culture in a shaker. (3) After the conversion is completed, filter the whole cell catalytic solution, recover the whole cell cells, concentrate the filtrate, and then obtain urolithin A.

[0014] As a further option: In step (1), the conditions for shaking culture are 35-39℃ and 200-300 rpm; preferably 37℃ and 220 rpm.

[0015] In step (2), the conditions for the shaker culture are 28-35℃ and 200-300 rpm, preferably 30℃ and 220 rpm; the pH of the tris(hydroxymethyl)aminomethane buffer is 7.5 and the concentration is 40-60 nmol / L, preferably 50 nmol / L.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following advantages: 1. Based on the known synthetic pathway of urolithin A, this invention utilizes microbial metabolic engineering and synthetic biology methods to construct a novel vector pET28a-UroA containing four co-expressed genes. The pET28a-UroA vector is transformed into... E.coli BL21(DE3) enables co-expression of genes after intracellular synthesis, and all four enzymes expressed intracellularly exhibit high activity, resulting in recombinant... E.coli The BL21(DE3) strain was able to stably convert extracellularly added ellagitannins into urolithin A, realizing a whole-cell catalytic process for the production of urolithin A using ellagitannins as a substrate.

[0017] 2. The carrier pET28a-UroA constructed in this invention can achieve the effect of large-scale synthesis of urolithin A, becoming a microbial cell factory for the synthesis of urolithin A. Furthermore, the construction method of this invention is based on the modification of microorganisms and uses recombinant Escherichia coli strains to synthesize urolithin A, avoiding the large-scale extraction, separation, and purification processes in industry. It does not require high temperature and high pressure synthesis conditions, nor does it require the use of catalysts. It is not only environmentally friendly, but also easy to control production costs, and has good prospects for industrial application. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the construction of the pET28a-UroA vector based on gene co-expression according to the present invention.

[0019] Figure 2 This is the technical route for the biosynthesis of urolithin A in this invention. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0022] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0023] Unless otherwise specified, the experimental methods described in the following examples are conventional methods. Example

[0024] 1. Construction of pET28a-UroA plasmid for gene co-expression The four target genes in this invention were synthesized by GenScript Biotech Ltd.: tannin acyl hydrolase gene. tah、 esterase genelip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd The enzymes were then sequentially digested and introduced into the E. coli expression vector pET-28a(+) using a double enzyme digestion method.

[0025] 2. Plasmid transformation The chemically competent Escherichia coli BL21(DE3) cells used for cloning were thawed on ice. Add 2 μL (100 ng / μL) of plasmid to 100 μL of competent cells, gently tap the tube wall to mix (do not shake to mix), and let stand on ice for 30 min. After heat shock in a 42℃ water bath for 45 seconds, immediately place on ice to cool for 2-3 minutes. Add 900 μL of LB liquid medium (without antibiotics) and incubate at 37°C for 1 h (200-250 rpm). Preheat the Kan-resistant LB solid medium plates in an incubator at 37°C. After 1 h of culture, the bacterial culture was centrifuged at 5,000 rpm (2,500 × g) for 5 min, and 900 μL of supernatant was discarded. The bacterial culture was resuspended in the remaining culture medium and gently spread evenly on a plate containing Kan resistance using a sterile spreader. Incubate upside down in a 37℃ incubator for 12-16 h. 3. Carrier connectivity test Single colonies were picked with sterile toothpicks and placed in 1 mL of LB liquid medium containing kanamycin (final concentration of 50 μg / mL). The culture was carried out at 37°C and 200 rpm with shaking for 4-6 hours until the bacterial culture became turbid. Then, bacterial PCR was performed to verify whether the vector was successfully ligated based on the electrophoresis bands and sequencing results. 4. Fermentation production of urolithiasis A (1) Inoculate the Escherichia coli strain containing the pET28a-UroA vector into 5 mL of liquid LB medium, add 5 μL of kanamycin antibiotic, and culture in a shaker at 37℃ and 220 rpm for 12 h. Then take 1% of the bacterial solution and inoculate it into 50 mL of liquid LB in a 250 mL Erlenmeyer flask, and culture in a shaker at 37℃ and 220 rpm. (2) When OD 600 When OD = 0.6, add 25 μL of 1 mol / L isopropyl-β-D-thiogalactoside (IPTG) and continue culturing at 30℃ and 220 rpm. 600When the concentration reaches 4, collect the bacterial cells, resuspend them in 50 mL of buffer (50 mM Tris, pH 7.5), pour them into a 250 mL Erlenmeyer flask, add 50 mg / L ellagitannin, and continue to culture in a shaker at 30℃ and 220 rpm for 48 h. (3) After the culture and transformation are completed, filter the whole cell catalytic solution in the triangular flask, recover the whole cell cells, and concentrate the filtrate through a vacuum concentrator to obtain urolithin A.

[0026] 5. Determination of urolithiasis A (1) Take 1.5 mL of the whole cell catalytic solution from step 4, centrifuge at 8500 rpm for 3 min, and transfer the supernatant to a liquid phase bottle after filtering through a 0.22 μm filter membrane; (2) The yield of urolithin A was detected by high performance liquid chromatography (HPLC) using a reversed-phase C18 column and a UV detector at 306 nm. The mobile phase consisted of a gradient elution of 0.1% formic acid and water with methanol at a flow rate of 1 mL / min and an injection volume of 10 μL. The yield of urolithin A in the filtrate obtained in step 4 was 14.48 mg / L, and the conversion rate of ellagitannin was 28.96%. The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. An engineered bacterial strain for producing urolithin A, characterized in that, The engineered strain contains a tannin acyl hydrolase gene. tah、 esterase gene lip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd .

2. The engineered strain for producing urolithin A according to claim 1, characterized in that, The tannic acid hydrolase gene tah The nucleotide sequence is shown in SEQ ID NO.1; The esterase gene lip The nucleotide sequence is shown in SEQ ID NO.2; The gallic acid decarboxylase gene gdo The nucleotide sequence is shown in SEQ ID NO.3; The dopamine dehydroxylase gene ascd The nucleotide sequence is shown in SEQ ID NO.

4.

3. The engineered strain for producing urolithin A according to claim 1, characterized in that, The substrate strain of the engineered strain is Escherichia coli. E. coli BL21 (DE3).

4. A carrier, characterized in that, The vector contains a tannic acid hydrolase gene. tah、 esterase gene lip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd .

5. The carrier according to claim 4, characterized in that, The vector is a tannic acid hydrolase gene. tah、 esterase gene lip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd The vector pET28a-UroA, which contains four co-expressed genes, was co-constructed on the E. coli expression vector pET-28a(+).

6. The method for constructing the engineered strain according to any one of claims 1-3, characterized in that, Includes the following steps: Genes containing tannic acid hydrolases tah、 esterase gene lip Gallic acid decarboxylase gene gdo and dopamine dehydroxylase gene ascd The vector was introduced into the chassis strain to obtain the engineered strain.

7. The use of the engineered strain according to any one of claims 1-3 in the biosynthesis of urolithin A.

8. A method for biosynthesizing urolithin A, characterized in that, Using the engineered strain described in any one of claims 1-3, whole-cell catalysis is performed to convert extracellularly added ellagitannins into urolithin A.

9. The method for biosynthesizing urolithin A according to claim 8, characterized in that, Includes the following steps: (1) The engineered strain was inoculated into liquid LB medium, kanamycin was added and cultured in a shaker, and then a portion of the bacterial culture was inoculated into liquid LB medium and cultured in a shaker for a longer period of time. (2) When OD 600 When OD = 0.6, add isopropyl-β-D-thiogalactoside and culture in a shaker. 600 When the concentration reaches 4, collect the bacterial cells, resuspend them in tris(hydroxymethyl)aminomethane buffer, add ellagitannin, and continue to culture in a shaker. (3) After the conversion is completed, filter the whole cell catalytic solution, recover the whole cell cells, concentrate the filtrate, and then obtain urolithin A.

10. The method for biosynthesizing urolithin A according to claim 9, characterized in that, In step (1), the conditions for shaking culture are 35-39℃ and 200-300 rpm; In step (2), the conditions for the shaker culture are 28-35℃ and 200-300 rpm; the pH of the tris(hydroxymethyl)aminomethane buffer is 7.5 and the concentration is 40-60 nmol / L.

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