Engineering strain for producing p-hydroxybenzoic acid as well as construction method and application of engineering strain

Abstract

The invention relates to an engineering strain for producing p-hydroxybenzoic acid as well as a construction method and application thereof, and belongs to the technical field of synthesis of p-hydroxybenzoic acid. The engineering strain can be used for producing p-hydroxybenzoic acid through shikimic acid way fermentation by taking glucose as a substrate, and the used starting strain is lactococcus lactis NZ3900; the expression mode is plasmid-induced heterologous expression of the p-hydroxybenzoic acid catalytic gene; by adding a natural food preservative Nisin, an exogenous gene is subjected to induced expression, so that the safety of the strain is improved; the engineering strain heterologously synthesizes p-hydroxybenzoic acid through a shikimic acid way, the aromatic compound is successfully synthesized in the lactococcus lactis for the first time, and the application of the lactococcus lactis in the aspect of biosynthesis of the aromatic compound is expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of p-hydroxybenzoic acid synthesis, and specifically relates to an engineering strain producing p-hydroxybenzoic acid, a construction method and application thereof. Background Art

[0002] Para-hydroxybenzoic acid is an aromatic compound, which is usually a white or slightly yellow crystalline powder with a special aromatic smell. It is widely used in the synthesis of plant growth regulators, dyes, fragrances and other chemicals. The existing production process of para-hydroxybenzoic acid mostly starts from petroleum products and is carried out under high temperature and high pressure conditions. It not only consumes a lot of energy and has high costs, but also has low production efficiency.

[0003] Synthetic biology is a technology that uses genetic engineering to redesign, construct and synthesize organisms. That is, it uses known metabolic regulation mechanisms to transform various microorganisms to construct a "cell factory" for the production of various raw materials, drugs, etc. Among them, the current research on the biosynthesis of aromatic compounds such as p-hydroxybenzoic acid is mainly centered on the shikimic acid pathway. The shikimic acid pathway synthesizes chorismate through 7 steps and is an important aromatic amino acid synthesis metabolic pathway commonly found in plants, fungi and bacteria. The disadvantage of using plants to synthesize p-hydroxybenzoic acid is that p-hydroxybenzoic acid is a glucosylated or esterified product in plants. , which brings difficulties to the downstream purification project, especially some steps in the phenylpropanoid synthesis pathway are still unclear. The use of bacteria as a "cell factory" for the synthesis of p-hydroxybenzoic acid has the following advantages: 1) Bacteria grow and reproduce quickly and have a short metabolic cycle; 2) The synthesis of p-hydroxybenzoic acid can partially utilize the bacterial aromatic amino acid synthesis metabolic pathway; 3) p-hydroxybenzoic acid is less toxic to bacteria and can be accumulated and produced in high concentrations; 4) No intermediates harmful to the environment and humans are generated; From both an environmental and economic perspective, the use of microorganisms to synthesize p-hydroxybenzoic acid through glucose, a renewable resource, is theoretically superior to other methods.

[0004] As a probiotic, lactic acid bacteria plays an important regulatory role in the healthy metabolism of the human body. At the same time, since the plasmid used is a special plasmid of food-grade lactic acid bacteria, the development of engineered lactic acid bacteria strains will be of great convenience in the food and cosmetics industries. However, there are currently no reports on the use of food-grade lactic acid bacteria to synthesize parahydroxybenzoic acid, and technical improvements are urgently needed. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide an engineered strain for producing p-hydroxybenzoic acid and a construction method and application thereof.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A p-hydroxybenzoic acid-producing engineered strain, the engineered strain is Lactococcus lactis MZLc-UA; the engineered strain co-expresses a chorismate lyase gene and a 3-deoxy-arabinoheptulose-7-phosphate synthase gene.

[0008] As a preferred technical solution of the present invention, the chorismate lyase gene is the chorismate lyase gene ubiC of Escherichia coli BL21 (DE3).

[0009] As a preferred technical solution of the present invention, the 3-deoxy-arabinoheptulose-7-phosphate synthase gene is the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF of Lactococcus lactis NZ3900.

[0010]

[0011] The engineered strain has a strong flux in the shikimic acid metabolic pathway and can be used for the development and utilization of aromatic compounds. The engineered strain is a generally recognized as safe (GRAS) microorganism that can be used to solve the health and safety risks in the biosynthetic production of p-hydroxybenzoic acid.

[0012] A method for constructing an engineered strain that produces p-hydroxybenzoic acid, the method comprising the following steps:

[0013] (1) The chorismate lyase gene ubiC and the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF were cloned;

[0014] (2) connecting the chorismate lyase gene ubiC and the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF to a plasmid vector to obtain a recombinant plasmid;

[0015] (3) Introducing the recombinant plasmid into the starting strain.

[0016] As a preferred technical solution of the present invention, the construction method comprises the following steps:

[0017] Step A: Using a single colony of Escherichia coli BL21 (DE3) as the first template, PCR amplify the chorismate lyase gene ubiC using primers ubiCF (SEQ ID NO.1) / ubiC-R (SEQ ID NO.2);

[0018] Step B: using the genome of Lactococcus lactis NZ3900 as the second template, PCR amplifying the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF using primers aroF-F (SEQ ID NO.3) / aroF-R (SEQ ID NO.4);

[0019] Step C: using the chorismate lyase gene ubiC and the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF as the third template, and using primers ubiC-F (SEQ ID NO.1) / aroF-R (SEQ ID NO.4) to PCR amplify the ubiC-rbs-aroF gene;

[0020] Step D: using plasmid pNZ8148 as the fourth template, using primers 8149-F (SEQ ID NO.5) / 8149-R (SEQ ID NO.6) for reverse PCR amplification to obtain a linearized pNZ8148 vector;

[0021] Step E: using seamless cloning ligase to connect the linearized pNZ8148 vector and the ubi C-rbs-aroF gene, and transforming to obtain the recombinant plasmid pNZ8148-ubiC-rbs-aroF;

[0022] Step F: Introduce the recombinant plasmid pNZ8148-ubiC-rbs-aroF into Lactococcus lactis NZ3900, and the construction is completed.

[0023] The invention discloses an application of an engineered strain for producing p-hydroxybenzoic acid, wherein the engineered strain is applied in the production of p-hydroxybenzoic acid.

[0024] As a preferred technical solution of the present invention, the production comprises the following steps:

[0025] The engineered strain was inoculated into GM17 medium at an inoculum rate of 2 wt % and then cultured at 30 °C until OD 600nm The concentration of Nisin was 0.4, and then 10 ng / mL of Nisin was added, and static culture was continued at 30°C for 12 h.

[0026] Furthermore, the GM17 medium contains the following components in weight percentage: 0.5% glucose, 5% soytone, 2.5% peptone, 2.5% casein peptone, 2.5% yeast extract powder, 5% beef extract powder, 5% lactose, 0.5% sodium ascorbate, 19% sodium β-glycerophosphate, 0.25% magnesium sulfate and the balance deionized water.

[0027] Beneficial effects of the present invention:

[0028] (1) The engineered strain of the present invention has higher chorismate lyase and 3-deoxy-arabinoheptulose-7-phosphate synthase activities than the starting strain.

[0029] (2) The engineered strain of the present invention can ferment and produce p-hydroxybenzoic acid using glucose as a substrate, and the yield is increased by 53.6% compared with the original strain.

[0030] (3) The engineered strain of the present invention is a GRAS microorganism, which overcomes the disadvantage that most of the fermentation strains for biosynthesizing p-hydroxybenzoic acid are conditionally pathogenic bacteria.

[0031] (4) The present invention has guiding significance for the development of raw materials for aromatic compounds.

[0032] (5) Lactococcus lactis is a probiotic that has no special requirements for fermentation equipment and can be widely used in the field of green fermentation production.

[0033] (6) The engineered strain of the present invention can directly use the fermentation product, which is simple and efficient.

[0034] (7) The present invention has positive significance for developing new strains of aromatic compounds. The engineered strain uses the lactic acid bacteria food-grade expression vector pNZ8148 and induces the expression of exogenous genes by adding the natural food preservative Nisin, thereby improving the safety of lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0036] Figure 1 This is a colony morphology diagram of the engineering strain in Example 1 of the present invention;

[0037] Figure 2 This is a growth curve of the engineering strain in Example 1 of the present invention;

[0038] Figure 3 This is a graph verifying the protein expression of the chorismate lyase gene ubiC and the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF in Example 1 of the present invention.

[0039] Figure 4 This is the standard curve of p-hydroxybenzoic acid in Example 1 of the present invention. DETAILED DESCRIPTION

[0040] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0041] Example 1

[0042] A p-hydroxybenzoic acid-producing engineered strain, the engineered strain is Lactococcus lactis MZLc-UA; the engineered strain co-expresses a chorismate lyase gene and a 3-deoxy-arabinoheptulose-7-phosphate synthase gene.

[0043] The chorismate lyase gene is the chorismate lyase gene ubiC of Escherichia coli BL21 (DE3).

[0044] The 3-deoxy-arabinoheptulose-7-phosphate synthase gene is the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF of Lactococcus lactis NZ3900.

[0045]

[0046] A method for constructing an engineered strain that produces p-hydroxybenzoic acid, the method comprising the following steps:

[0047] Step A: Using a single colony of Escherichia coli BL21 (DE3) as the first template, PCR amplified the chorismate lyase gene ubiC using primers ubi CF (SEQ ID NO.1) / ubiC-R (SEQ ID NO.2); the PCR reaction system is shown in Table 1; the PCR amplification conditions are 98°C pre-denaturation for 3min; 98°C denaturation for 10s, 55°C annealing for 20s, 72°C extension for 30s, and amplification for 30 cycles; 72°C reaction for 5min; after the completion of PCR amplification, agarose gel electrophoresis was performed, and the target gene fragment was recovered using a Zhuangmeng biogel recovery kit.

[0048] Table 1

[0049] Reagents volume 2×FidCycleEvo High-Fidelity PCRMix 10μL ubiC-F (SEQ ID NO.1) 1μL ubiC-R (SEQ ID NO.2) 1μL E.coliBL21(DE3) single colony 1μL <![CDATA[ddH2O]]> To20μL

[0050] Step B: Using the Lactococcus lactis NZ3900 genome as the second template, PCR amplified the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF using primers aroF-F (SEQ ID NO.3) / aroF-R (SEQ ID NO.4); the PCR reaction system is shown in Table 2; the PCR amplification conditions are 98°C pre-denaturation for 3min; 98°C denaturation for 10s, 55°C annealing for 20s, 72°C extension for 40s, and amplification for 30 cycles; 72°C reaction for 5min; after the PCR amplification is completed, agarose gel electrophoresis is performed, and the target gene fragment is recovered using the Zhuangmeng biogel recovery kit.

[0051] Table 2

[0052]

[0053]

[0054] Step C: using the chorismate lyase gene ubiC and the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF as the third template, using primers ubiC-F (SEQ ID NO.1) / aroF-R (SEQ ID NO.4) to PCR amplify the ubiC-rbs-aroF gene; the PCR reaction system is shown in Table 3; the PCR amplification conditions are 98°C pre-denaturation for 3min; 98°C denaturation for 10s, 55°C annealing for 20s, 72°C extension for 60s, and amplification for 30 cycles; 72°C reaction for 5min; after the PCR amplification is completed, agarose gel electrophoresis is performed, and the target gene fragment is recovered using a Zhuangmeng biogel recovery kit.

[0055] Table 3

[0056]

[0057] Step D: Using plasmid pNZ8148 as the fourth template, reverse PCR amplification was performed using primers 8149-F (SEQ ID NO.5) / 8149-R (SEQ ID NO.6) to obtain a linearized pNZ8148 vector; the PCR reaction system is shown in Table 4; the PCR amplification conditions were 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 55°C annealing for 20 s, 72°C extension for 100 s, and 30 cycles of amplification; 72°C reaction for 5 min; after the PCR amplification was completed, agarose gel electrophoresis was performed, and the linearized vector fragment was recovered using the Zhuangmeng biogel recovery kit.

[0058] Table 4

[0059] Reagents volume 2×FidCycleEvo High-Fidelity PCRMix 10μL 8149-F (SEQ ID NO.5) 1μL 8149-R (SEQ ID NO.6) 1μL Plasmid pNZ8148 1μL <![CDATA[ddH2O]]> To20μL

[0060] Step E: Use seamless cloning ligase and ClonExpress Ultra One Step Cloning Kit to connect the linearized pNZ8148 vector and the ubiC-rbs-aroF gene. The connection reaction system is shown in Table 5. After the connection reaction system is prepared, mix it evenly, centrifuge it briefly, and incubate it at 37°C for 30 minutes. Then, take 5 μL of the connection reaction system to Lactococcus lactis NZ3900 competent cells for electroporation transformation, incubate it at 30°C for 2 hours, then spread it on a GM17 plate containing chlorine resistance, and culture it at 30°C overnight.

[0061] Table 5

[0062] Reagents volume Linearized pNZ8148 vector fragment 0.4μL ubiC-rbs-aroF gene fragment 0.5μL 5×CEⅡBuffer 4μL ExnaseⅡ 2μL <![CDATA[ddH2O]]> To20μL

[0063] The primer sequences used to construct the engineered strains are shown in Table 6.

[0064] Table 6

[0065]

[0066]

[0067] Positive clone verification

[0068] A single colony was randomly picked from the plate and inoculated into 5 mL of GM17 liquid culture medium containing chloride resistance, and cultured at 30°C overnight. The plasmid was extracted using a plasmid extraction kit, and then the plasmid fragment was double-digested with two restriction endonucleases, BamHⅠ / SalⅠ. After the digestion, agarose gel electrophoresis was performed for verification, and the strain was sent to Aiki Biotechnology Co., Ltd. for sequencing. The strain with the correct sequencing was the engineered strain, named MZLc-UA.

[0069] Bacterial species identification

[0070] The engineered strain MZLc-UA was morphologically identified, such as Figure 1 As shown, the colonies of this strain are medium-sized, convex, slightly white, moist, with neat edges, round in shape, and 3.0 mm ± 1 mm in diameter.

[0071] Biological characteristics research

[0072] The growth curve of the engineered strain MZLc-UA was determined. During the static culture in a 30°C incubator, samples were taken every 2 hours to measure the OD 600nm Value, by Figure 2 It can be seen that the growth is slow in the first 2 hours of culture, enters the logarithmic growth phase after 3 hours, is in the stable growth phase from 8 to 16 hours, and enters the decline phase after 16 hours.

[0073] Fermentation Validation

[0074] A single colony of the engineered strain MZLc-UA was selected and inoculated into 5 mL of GM17 liquid medium containing chlorine resistance and cultured at 30°C overnight. Then, 2 wt% of the inoculum was inoculated into 50 mL of GM17 medium containing chlorine resistance and cultured at 30°C. When the OD of the engineered strain reached 0. 600nm When the concentration of Nisin reached 0.4, 10 ng / mL Nisin was added for induction, and the culture was continued at 30°C. The fermentation was terminated after 12 hours. Lactococcus lactis NZ3900 / pNZ8148 was set as the blank control group. After the fermentation, the content of p-hydroxybenzoic acid in the fermentation broth was determined, and the results showed that the yield increased by 53.6% compared with the original strain.

[0075] Verification of ubiC and aroF protein expression

[0076] SDS-PAGE verification of induced expression of engineered strain MZLc-UA protein:

[0077] a. Prepare 12% SDS-PAGE protein gel;

[0078] b. Pipette 1mL of fermentation broth into a centrifuge tube, centrifuge at 4℃, 12000r / min for 1min, take the precipitate and resuspend it in 40μL protein loading buffer, heat it for 10min, take 10μL and load it for SDS-PAGE protein gel electrophoresis. The results are as follows: Figure 3 As shown, the protein of about 39.3 kDa is aroF, and the protein of about 18.8 kDa is ubiC.

[0079] Separation and Detection of p-Hydroxybenzoic Acid

[0080] Preparation of standard curve of p-hydroxybenzoic acid:

[0081] a. Prepare standard solutions of p-hydroxybenzoic acid of different concentrations (25, 50, 100, 200, 800 mg / L).

[0082] b. Preparation of mobile phase: Phase A, 0.1% phosphoric acid aqueous solution: add 100 μL of phosphoric acid solution to 1L of pure water and shake thoroughly to mix; Phase B, 100% methanol solution.

[0083] c. HPLC detection method: Gradient elution was used with the following procedure: 0-2 min, 2% B phase; 2-6 min, 15% B phase; 6-13 min, 40% B phase; 13-18 min, 2% B phase.

[0084] d. Use high performance liquid chromatography to detect the absorption value at 283nm of ultraviolet light, draw a standard curve with the concentration of standard solution (mg / L) as the horizontal axis and the absorption peak area as the vertical axis, and make a linear recovery equation. Figure 4 As shown, the regression equation is y=5631.34x, and the correlation coefficient is R2=0.999, which indicates that the two have a good linear relationship.

[0085] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An engineered strain producing p-hydroxybenzoic acid, characterized in that: The engineered strain is Lactococcus lactis MZLc-UA; the engineered strain co-expresses a chorismate lyase gene and a 3-deoxy-arabinoheptulose-7-phosphate synthase gene.

2. The engineered strain for producing p-hydroxybenzoic acid according to claim 1, characterized in that: The nucleotide sequence of 16S rDNA of the engineered strain is shown in SEQ ID NO.

7.

3. The engineered strain for producing p-hydroxybenzoic acid according to claim 1, characterized in that: The engineered strain produces p-hydroxybenzoic acid by fermentation using glucose as a substrate.

4. A method for constructing an engineered strain producing p-hydroxybenzoic acid according to any one of claims 1 to 3, characterized in that: The construction method comprises the following steps: (1) The chorismate lyase gene ubiC and the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF were cloned; (2) connecting the chorismate lyase gene ubiC and the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF to a plasmid vector to obtain a recombinant plasmid; (3) Introducing the recombinant plasmid into the starting strain.

5. The method for constructing an engineered strain producing p-hydroxybenzoic acid according to claim 4, characterized in that: The construction method comprises the following steps: Step A: Using a single colony of Escherichia coli BL21 (DE3) as the first template, PCR amplification of the chorismate lyase gene ubiC using primers ubiC-F / ubiC-R; Step B: using the genome of Lactococcus lactis NZ3900 as the second template, PCR amplification of the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF using primers aroF-F / aroF-R; Step C: using the chorismate lyase gene ubiC and the 3-deoxy-arabinoheptulose-7-phosphate synthase gene aroF as the third template, and using primers ubiC-F / aroF-R to perform PCR amplification of the ubiC-rbs-aroF gene; Step D: using plasmid pNZ8148 as the fourth template, using primers 8149-F / 8149-R for reverse PCR amplification to obtain a linearized pNZ8148 vector; Step E: using seamless cloning ligase to connect the linearized pNZ8148 vector and the ubiC-rbs-aroF gene, and transforming to obtain the recombinant plasmid pNZ8148-ubiC-rbs-aroF; Step F: Introduce the recombinant plasmid pNZ8148-ubiC-rbs-aroF into Lactococcus lactis NZ3900, and the construction is completed.

6. The method for constructing an engineered strain producing p-hydroxybenzoic acid according to claim 4, characterized in that: The starting strain in step (3) is Lactococcus lactis NZ3900.

7. Use of an engineered strain for producing p-hydroxybenzoic acid as claimed in any one of claims 1 to 3, characterized in that: The engineered strain is applied in the production of p-hydroxybenzoic acid.

8. The use of an engineered strain producing p-hydroxybenzoic acid according to claim 7, characterized in that: The production comprises the following steps: The engineered strain was inoculated into GM17 medium at an inoculum rate of 2 wt % and then cultured at 30 °C until OD 600nm The concentration of Nisin was 0.4, and then 10 ng / mL of Nisin was added, and static culture was continued at 30°C for 12 h.

9. The use of an engineered strain producing p-hydroxybenzoic acid according to claim 8, characterized in that: The GM17 culture medium comprises the following components in weight percentage: 0.5% glucose, 5% soytone, 2.5% peptone, 2.5% casein peptone, 2.5% yeast extract powder, 5% beef extract powder, 5% lactose, 0.5% sodium ascorbate, 19% sodium β-glycerophosphate, 0.25% magnesium sulfate and the balance deionized water.

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