Recombinant pichia pastoris with phenolic acid decarboxylase displayed on surface as well as preparation method and application of recombinant pichia pastoris

Abstract

The invention belongs to the technical field of enzyme engineering, genetic engineering and food engineering, and discloses recombinant pichia pastoris with phenolic acid decarboxylase displayed on the surface as well as a construction method and application of the recombinant pichia pastoris. The recombinant pichia pastoris capable of displaying phenolic acid decarboxylase on the surface is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.33253. The recombinant pichia pastoris capable of displaying phenolic acid decarboxylase on the surface can efficiently remove carboxyl from hydroxycinnamic acid to generate vinyl derivatives, reaction conditions are mild and green, and the recombinant pichia pastoris can be used for preparing a whole-cell catalyst for catalyzing conversion of hydroxycinnamic acid into vinyl derivatives. The recombinant pichia pastoris strain disclosed by the invention can be used for efficiently immobilizing and repeatedly using the phenolic acid decarboxylase, solves the problems of instability and low catalytic activity of the phenolic acid decarboxylase in the process of preparing a vinyl derivative by using a biological enzyme method catalyst, and has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical fields of enzyme engineering, genetic engineering and food engineering, and relates to a recombinant Pichia pastoris, in particular to a recombinant Pichia pastoris displaying phenolic acid decarboxylase on its surface, and a construction method and use thereof. Background Art

[0002] 4-Vinyl derivatives are volatile and have a unique aroma. They are important flavor substances in seasonings, white wine, red wine and other beverages. They are often used in high-end spices and are also important precursors for the synthesis of other active substances (such as vanillin and phenolic pyranoanthocyanins). At present, the synthesis of 4-vinyl derivatives mostly adopts chemical synthesis. The biosynthesis method has a single substance, is green and mild, and has broad prospects. Phenolic acid decarboxylase is a kind of enzyme that can catalyze the conversion of common phenolic acid substances in plants such as p-coumaric acid, ferulic acid, caffeic acid, and sinapic acid into corresponding 4-vinyl derivatives. However, the low secretion level of phenolic acid decarboxylase under natural conditions and the poor acid stability of phenolic acid decarboxylase have seriously hindered the development of 4-vinyl derivatives. Summary of the invention

[0003] In order to overcome the above obstacles, the primary purpose of the present invention is to provide a recombinant Pichia pastoris displaying phenolic acid decarboxylase on its surface, which can achieve efficient immobilization and repeated use of phenolic acid decarboxylase and enhance the catalytic activity of phenolic acid decarboxylase in an acidic environment, while providing a whole-cell catalyst for the enzymatic preparation of vinyl derivatives, and has good prospects for industrial application.

[0004] The recombinant Pichia pastoris displaying phenolic acid decarboxylase on its surface described in the present invention is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC) with a deposit number of CGMCC No.33253, a classification name of Pichia pastoris, a strain of dLPPAD-GS115, and a deposit date of December 30, 2024.

[0005] According to a further feature of the recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface of the present invention, the phenolic acid decarboxylase is the phenolic acid decarboxylase LPPAD of Lactobacillus plantarum.

[0006] According to a further feature of the recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface of the present invention, the nucleotide sequence of the phenolic acid decarboxylase is a codon-optimized nucleotide sequence, as shown in SEQ ID NO.1.

[0007] According to a further feature of the recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface of the present invention, the recombinant Pichia pastoris uses an anchor protein to display phenolic acid decarboxylase on the cell surface, and the anchor protein is GCW61.

[0008] According to a further feature of the recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface of the present invention, the anchor protein nucleotide sequence is a codon-optimized nucleotide sequence, as shown in SEQ ID NO.2.

[0009] The second object of the present invention is to provide a method for preparing the recombinant Pichia pastoris displaying phenolic acid decarboxylase on its surface.

[0010] The method for preparing the recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface of the present invention comprises the following steps: A. The phenolic acid decarboxylase (LPPAD) gene from Lactobacillus plantarum was codon optimized, and the optimized nucleotide sequence was shown in SEQ ID NO.1; the anchor protein GCW61 gene was codon optimized, and the optimized nucleotide sequence was shown in SEQ ID NO.2; the plasmid pPIC9K was used as an expression vector, and the LPPAD-GCW61 fusion protein gene was inserted to construct a surface display phenolic acid decarboxylase vector pPIC9K-LPPAD-GCW61; B. Transform the recombinant plasmid pPIC9K-LPPAD-GCW61 obtained in step A into Pichia pastoris GS115 competent cells to obtain the recombinant Pichia pastoris strain dLPPAD displaying phenolic acid decarboxylase on its surface; C. Inoculate the recombinant Pichia pastoris obtained in step B into YPG medium and culture until OD 600 Between 2 and 6 hours, the cells were collected by centrifugation and resuspended in BMMY medium. Pure methanol with a final concentration of 1% (v / v) was added every 24 hours to induce expression for 96 hours, and the cells were collected by centrifugation to obtain the recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface.

[0011] The third object of the present invention is to provide the use of the recombinant Pichia pastoris displaying phenolic acid decarboxylase on its surface, specifically the use of the recombinant Pichia pastoris displaying phenolic acid decarboxylase on its surface for preparing a whole-cell catalyst for catalyzing the conversion of hydroxycinnamic acid into vinyl derivatives.

[0012] The present invention is based on Pichia pastoris ( Pichia pastoris ) surface display technology, the principle of which is to use the anchoring protein on the cell surface to display the exogenous protein on the surface of Pichia pastoris cells, eliminating the process of separation and purification of phenolic acid decarboxylase, saving costs, and being able to efficiently decarboxylate hydroxycinnamic acid to generate vinyl derivatives. The reaction conditions are mild and green.

[0013] The phenolic acid decarboxylase displayed by the recombinant Pichia pastoris of the present invention retains an independent quaternary structure and biological activity, can be recycled and used multiple times, and can also enhance the robustness of the phenolic acid decarboxylase. Experiments show that the highest decarboxylation activity of the recombinant Pichia pastoris displaying the phenolic acid decarboxylase of the present invention reaches 3.38 U / mg dry cell, and can still retain 70% of the activity after being reused 5 times. It can still retain 70% of the activity under acidic conditions, enhancing the robustness of the phenolic acid decarboxylase.

[0014] The recombinant Pichia pastoris strain of the present invention can efficiently fix and reuse phenolic acid decarboxylase, solves the problems of instability and low catalytic activity of phenolic acid decarboxylase in the process of preparing vinyl derivatives by bioenzymatic catalyst, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 for pPIC9K-LPPAD-GCW61 Gene map.

[0016] Figure 2 Flow cytometric analysis results of recombinant Pichia pastoris cells are shown.

[0017] Figure 3 Shown are the results of flow cytometric analysis of wild-type Pichia pastoris cells.

[0018] Figure 4 The optimal temperature of recombinant Pichia pastoris cells is shown.

[0019] Figure 5 The optimal pH for recombinant Pichia pastoris cells is shown.

[0020] Figure 6 Demonstrating the thermostability of recombinant Pichia pastoris cells.

[0021] Figure 7 The pH stability of recombinant Pichia pastoris cells was shown.

[0022] Figure 8 The number of times the recombinant Pichia cells were reused is shown. DETAILED DESCRIPTION

[0023] The terms used herein, unless otherwise specified, have the meanings commonly understood by those skilled in the art. The following provides definitions of some special terms used in the present invention.

[0024] "LPPAD" stands for Lactobacillus plantarum phenolic acid decarboxylase, and its gene is in italics LPPAD express.

[0025] "GCW61" indicates the anchoring protein GCW61, whose gene is GCW61 express.

[0026] "dLPPAD" indicates a recombinant Pichia pastoris strain displaying phenolic acid decarboxylase on its surface.

[0027] YPG medium: yeast extract 10 g / L, soy peptone 20 g / L, glycerol 10 g / L.

[0028] MD medium: yeast basal nitrogen source 13.4 g / L, glucose 20 g / L, biotin 10 -5 g / L.

[0029] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, YNB 13.4 g / L, potassium phosphate buffer (pH 6.0) 100 mmol / L, biotin 4×10 -4 g / L, glycerol 10 g / L.

[0030] BMMY medium: yeast extract 10 g / L, peptone 20 g·L -1 , YNB 13.4 g / L, potassium phosphate buffer (pH 6.0) 100 mmol / L, biotin 4×10 -4 g / L, methanol 1% (v / v).

[0031] In this experiment, Pichia pastoris ( Pichia pastoris ) GS115 is a commercial product and can be purchased from Invitrogen. The vector pPIC9K is a commercial product.

[0032] High performance liquid chromatography (HPLC) detection of hydroxycinnamic acid and 4-vinyl derivatives: Shimadzu HPLC LC-2050 with C18 analytical column and PDA detector was used for analysis. The mobile phase was 2% formic acid water-acetonitrile, used at a flow rate of 1.0 mL / min at 30°C column temperature. The injection volume of all samples was 10 μL. The detection wavelength was set to 280 nm. The peak areas of hydroxycinnamic acid and 4-vinyl derivatives in the HPLC spectrum are proportional to their concentrations, and standard curves of hydroxycinnamic acid and 4-vinyl derivatives were drawn based on this, respectively. The concentrations of hydroxycinnamic acid and 4-vinyl derivatives in the samples were calculated using the standard curves.

[0033] Example 1: Surface display of phenolic acid decarboxylase carrier ( pPIC9K-LPPAD-GCW61 ) Construction of plasmid The phenolic acid decarboxylase (LPPAD) gene from Lactobacillus plantarum was codon optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.1. The anchor protein GCW61 gene was codon optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.2. LPPADA Flag tag is added to the C-terminus of the protein. The Flag tag C-terminus is immediately connected to the anchor protein gene. GCW61, Constructed into a fusion protein gene, the 5′ and 3′ ends of the fusion protein gene were added EcoR I and Not I restriction endonuclease cleavage site, select plasmid pPIC9K as an expression vector, and insert the fusion protein gene sequence into pPIC9K. The present invention uses α-factor signal peptide and AOX1 promoter to achieve its inducible secretory expression in Pichia pastoris GS115. Obtain a recombinant surface display phenolic acid decarboxylase vector pPIC9K-LPPAD-GCW61 ,like Figure 1 As shown, the gene was synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0034] Example 2: Construction of a recombinant Pichia pastoris strain displaying phenolic acid decarboxylase on its surface The recombinant plasmid obtained in Example 1 pPIC9K-LPPAD-GCW61 Restriction enzyme Sal After linearization by enzyme digestion with I, the linearized plasmid was electroporated into Pichia pastoris GS115 competent cells using the electric pulse method to obtain the recombinant Pichia pastoris strain dLPPAD displaying phenolic acid decarboxylase on the surface.

[0035] Specific steps of electroporation: (1) Use Sal I linearize the expression vector of Example 1, enzyme digestion system: 44 μL plasmid, 5 μL Q.cut buffer, 1 μL Q.cut Sal I, incubate at 37°C for 30 min. Analyze the enzyme digestion system using 1% agarose gel electrophoresis, and recover the linearized plasmid using the gel recovery kit.

[0036] (2) Add 10 μL of linearized vector (about 300 ng) to 80 μL of competent yeast cells, transfer the mixture to the bottom of a pre-cooled 0.2 cm electroporation cuvette, and place on ice for 5 min.

[0037] (3) Immediately after one electric shock at 1500 V for 5 ms, add 500 μL of pre-cooled 1 M sorbitol solution into the electroporation cup, mix quickly, and place on ice for 5 min.

[0038] (4) Take 200 μL of the transformation solution and spread it on the MD plate and culture it at 30℃ for 3 days. Pick a single clone for colony PCR, and the corresponding band amplified is preliminarily judged as a positive transformant.

[0039] Example 3: Inducible expression of recombinant Pichia pastoris strain displaying phenolic acid decarboxylase The recombinant Pichia pastoris obtained in Example 2 was inoculated into a YPG test tube culture medium, cultured at 28°C and 200 rpm for 24 h, and then transferred to 50 mL of BMGY culture medium at a 1% inoculum volume and cultured until OD 600 Between 2 and 6, the bacterial solution was centrifuged at 4000 g for 5 min to collect the cells and resuspended in 50 mL BMMY medium. Pure methanol was added every 24 h to a final concentration of 1% (v / v), and the expression was induced for 96 h, and the cells were collected by centrifugation.

[0040] Example 4: Flow cytometric analysis of recombinant Pichia pastoris strains displaying phenolic acid decarboxylase on their surface The recombinant Pichia pastoris obtained in Example 3 was washed three times with PBS buffer and then resuspended in PBS buffer containing 1% BSA to an OD of 600 The cell suspension was incubated with Flag primary antibody at room temperature for 2 h, washed three times with PBS buffer after incubation, and then 200 μL PBS buffer containing Flag secondary antibody was added to resuspend the cells, incubated at room temperature in the dark for 1 h, during which the cells were kept mixed, washed three times with PBS buffer after incubation, and analyzed by flow cytometry after washing.

[0041] like Figure 2 As shown in FIG. 1 , the average fluorescence signal intensity of the recombinant Pichia pastoris strain displaying phenolic acid decarboxylase on its surface at 488 nm was 2373. Figure 3 As shown, the average fluorescence signal intensity of wild-type Pichia pastoris GS115 is 91, which can confirm that phenolic acid decarboxylase is successfully displayed on the surface of yeast cells. The successfully verified surface display strain is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC No.33253, the classification name is Pichia pastoris, the strain is dLPPAD-GS115, and the deposit date is December 30, 2024.

[0042] Example 5: Preparation of lyophilized powder of recombinant Pichia pastoris strain displaying phenolic acid decarboxylase on its surface The recombinant Pichia yeast cells obtained in Example 3 were washed three times with 40 mM Britton-Robinson buffer at pH 6.0, washed with 0.2 M trehalose solution (as a freeze-drying protective agent), and centrifuged at 5000 g for 5 min to collect the cells. The cells were frozen at -80°C for 30 min, and then freeze-dried for 20 h, and the freeze-dried enzyme powder was collected as the whole cell catalyst.

[0043] Example 6: Determination of the decarboxylase activity of the recombinant Pichia pastoris strain displaying phenolic acid decarboxylase on its surface The standard assay mixture contained 1.0 mg / mL hydroxycinnamic acid in 0.2 mL of 200 mM Na2HPO4-citrate buffer (pH 5.0), 1 mg of the whole cell catalyst obtained in Example 4 was added, incubated at 35°C for 30 minutes, and then 50 μL of methanol was added to terminate the reaction mixture. The formed product was quantified by high performance liquid chromatography (HPLC). One unit of enzyme activity was defined as the whole cell catalyst required to consume 1 μmoL hydroxycinnamic acid per minute.

[0044] PAD activity (IU / mg) = V2X / M1T Where M1 represents the mass of whole-cell catalyst added, X represents the content of hydroxycinnamic acid calculated according to the standard curve, V2 represents the final volume of the reaction, and T represents the reaction time of the enzyme.

[0045] The results showed that the decarboxylase activities of the recombinant Pichia pastoris strain displaying phenolic acid decarboxylase on different hydroxycinnamic acids were: p-coumaric acid, 3.381 U / mg stem cell; caffeic acid, 3.082 U / mg stem cell; ferulic acid, 0.572 U / mg stem cell; sinapinic acid, 0.005 U / mg stem cell.

[0046] Example 7: Determination of the enzymatic properties of the recombinant Pichia pastoris strain displaying phenolic acid decarboxylase on its surface 1. Optimum temperature of whole-cell catalyst The whole cell catalyst obtained in Example 4 was tested for enzyme activity at 4° C., 15° C., 25° C., 35° C., 45° C., 55° C., and 65° C. The group with the highest activity was defined as 100% activity.

[0047] like Figure 4 As shown, the optimum temperature of the whole-cell catalyst is 45°C, and the enzyme activity of the whole-cell catalyst can be maintained at more than 50% in the temperature range of 25°C-55°C.

[0048] 2. Optimal pH of whole-cell catalyst The whole cell catalyst obtained in Example 4 was tested for enzyme activity at pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively. The group with the highest activity was defined as 100% activity.

[0049] like Figure 5 As shown, the optimum pH of the whole-cell catalyst is 7.0, and the enzyme activity of the whole-cell catalyst can be maintained at more than 80% under the condition of pH range of 6.0-8.0, and the enzyme activity can be maintained at more than 70% under the condition of pH 3.0.

[0050] 3. Thermal stability of whole-cell catalysts The whole cell catalyst obtained in Example 4 was incubated at 4° C., 15° C., 25° C., 35° C., 45° C., 55° C., and 65° C. for 60 minutes and then the enzyme activity was measured. The group with the highest activity was defined as 100% activity.

[0051] like Figure 6 As shown, the whole-cell catalyst can maintain more than 70% enzyme activity when incubated at a temperature range of 4°C-45°C.

[0052] 4. pH stability of whole-cell catalysts The whole cell catalyst obtained in Example 4 was incubated at pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 for 60 minutes and then the enzyme activity was measured. The group with the highest activity was defined as 100% activity.

[0053] like Figure 7 As shown, the whole-cell catalyst can maintain more than 70% enzyme activity when incubated in the pH range of 5.0-8.0.

[0054] 5. Reuse of whole-cell catalysts The whole cell catalyst in Example 5 was centrifuged at 5000 g for 5 min to collect the bacterial cells and reused 4 times. The enzyme activity of the first test was defined as 100% enzyme activity.

[0055] like Figure 8 As shown, the whole-cell catalyst can maintain more than 60% of the enzyme activity after 5 repeated uses, indicating that the strain has good reproducibility.

[0056] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change or other variation of the above embodiment based on the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A recombinant Pichia pastoris displaying phenolic acid decarboxylase on its surface, characterized in that: It is deposited in China General Microbiological Culture Collection Center with the deposit number CGMCC No.33253.

2. The recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface according to claim 1, characterized in that: The phenolic acid decarboxylase is the phenolic acid decarboxylase LPPAD of Lactobacillus plantarum.

3. The recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface according to claim 2, characterized in that: The phenolic acid decarboxylase nucleotide sequence is a codon-optimized nucleotide sequence, as shown in SEQ ID NO.

1.

4. The recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface according to claim 1, characterized in that: The recombinant Pichia pastoris uses an anchor protein to display phenolic acid decarboxylase on the cell surface, and the anchor protein is GCW61.

5. The recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface according to claim 4, characterized in that: The anchor protein nucleotide sequence is a codon-optimized nucleotide sequence, as shown in SEQ ID NO.

2.

6. The method for preparing a recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface according to claim 1, characterized in that: The following steps are involved: A. The phenolic acid decarboxylase (LPPAD) gene from Lactobacillus plantarum was codon optimized, and the optimized nucleotide sequence was shown in SEQ ID NO.1; the anchor protein GCW61 gene was codon optimized, and the optimized nucleotide sequence was shown in SEQ ID NO.2; the plasmid pPIC9K was used as an expression vector, and the LPPAD-GCW61 fusion protein gene was inserted to construct a surface display phenolic acid decarboxylase vector pPIC9K-LPPAD-GCW61; B. Transform the recombinant plasmid pPIC9K-LPPAD-GCW61 obtained in step A into Pichia pastoris GS115 competent cells to obtain the recombinant Pichia pastoris strain dLPPAD displaying phenolic acid decarboxylase on its surface; C. Inoculate the recombinant Pichia pastoris obtained in step B into YPG medium and culture until OD 600 Between 2 and 6 hours, the cells were collected by centrifugation and resuspended in BMMY medium. Pure methanol with a final concentration of 1% (v / v) was added every 24 hours to induce expression for 96 hours, and the cells were collected by centrifugation to obtain the recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface.

7. Use of the recombinant Pichia pastoris displaying phenolic acid decarboxylase on the surface of claim 1 for preparing a whole-cell catalyst for catalyzing the conversion of hydroxycinnamic acid into vinyl derivatives.

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