Plectranthus amboinicus-derived flavone synthase and its application in synthesis of apigenin in recombinant pichia pastoris

Abstract

The application discloses a flavone synthase from pachouli and application of the flavone synthase in synthesis of apigenin in recombinant Pichia pastoris, the amino acid sequence of the flavone synthase is shown as SEQ ID NO. 1, and the application also provides application of the flavone synthase or the recombinant Pichia pastoris in catalytic synthesis of apigenin, wherein the catalysis is that the recombinant Pichia pastoris is induced and cultured by using a methanol solution containing naringenin as the only carbon source, and the naringenin is converted into apigenin by the flavone synthase after entering the cell. The application has the advantages that the flavone synthase from pachouli and the application of the flavone synthase in synthesis of apigenin in recombinant Pichia pastoris do not need 6-8 genes in a de novo synthesis pathway of apigenin, are convenient for subsequent optimization, regulation and modification, and can increase yield to an entry-level industrial application.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a patchouli-derived flavonoid synthase and its application in the synthesis of apigenin by recombinant Pichia pastoris. Background Technology

[0002] Apogenin is a natural flavonoid compound (4',5,7-trihydroxyflavone). Its biological activity mainly comes from the phenolic hydroxyl structure in the molecule, and it has antioxidant (scavenging free radicals), anti-inflammatory (inhibiting the release of inflammatory factors), anti-tumor (inducing tumor cell apoptosis), antibacterial and antiviral effects.

[0003] The invention patent with application number 202510060473.9, entitled "Yeast engineered strain for producing apigenin and its application", discloses a yeast engineered strain for producing apigenin and its application. The method uses Saccharomyces cerevisiae or Yeast lipolyticis as chassis cells, introduces an NADPH supply module and a de novo synthesis pathway for apigenin, and constructs a recombinant strain for whole-cell catalytic synthesis of apigenin, using glucose as a carbon source and yeast extract as a nitrogen source. 1. Although de novo synthesis of apigenin is low-cost, it involves a long pathway, many genes, and is difficult to regulate and optimize, making it challenging to improve yield and conversion rate. 2. The culture medium of Saccharomyces cerevisiae or Yersinia lipolytica is mainly composed of hydrophilic and polar substances, while apigenin and its intermediate substrate naringenin are both poorly soluble in water, making it difficult to add large amounts of exogenous substances. 3. Most plant flavonoids such as apigenin and naringin are alcohol-soluble, while common methanol and ethanol are highly toxic to Saccharomyces cerevisiae and Yeast lipolytica cells. Therefore, the production of apigenin synthesized by these two yeasts as chassis cells is easily limited.

[0004] The research paper "Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside" established a technique for the collaborative synthesis of apigenin by Y. lipolytica and E. coli expressing apigenin synthase. However: 1. Yeast extract and Escherichia coli worked together to synthesize apigenin de novo, resulting in a significant increase in yield (168 mg / L), but there is still a large gap between the yield and the requirements for industrial production and application. 2. The optimization of mixed-culture fermentation process is quite complex and involves many parameters, making it difficult to apply in actual production. 3. The construction of the recombinant strain involves 6 genes (up to apigenin), which is a large number, making further regulation and optimization difficult in the later stages; 4. Naringin and apigenin have significant adverse effects on Yersinia lipolytica and Escherichia coli due to their alcohol solubility and cytotoxicity, and their disadvantages are the same as those of the prior art mentioned above.

[0005] In summary, by identifying flavonoid synthases that can efficiently catalyze the synthesis of apigenin from naringenin in existing plants rich in apigenin, introducing them into Pichia pastoris cells, and using exogenously added naringenin methanol solution as a carbon source, a more efficient and environmentally friendly method for the biosynthesis of apigenin is expected to be established, with promising prospects for production and application. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the above-mentioned technical defects and provide a patchouli-derived flavonoid synthase with 6-8 genes that do not require a de novo apigenin synthesis pathway, which is convenient for subsequent optimization, regulation and modification, and increases the yield to entry-level industrial applications, and its application in the synthesis of apigenin by recombinant Pichia pastoris.

[0007] To solve the above-mentioned technical problems, the present invention provides a flavonoid synthase derived from patchouli, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Another aspect of the present invention discloses a gene encoding the aforementioned flavonoid synthase, the DNA sequence of which is shown in SEQ ID NO: 2.

[0009] Another aspect of the present invention discloses an expression vector carrying the above-mentioned coding gene, wherein the coding gene is operatively linked between the promoter and terminator of Pichia pastoris alcohol oxidase, wherein the expression vector is a pPICZ series vector, including pPICZA, pPICZB, and pPICZC.

[0010] Another aspect of the present invention discloses a recombinant Pichia pastoris strain using Pichia pastoris X-33 as the host cell and integrating the above-mentioned expression vector for expressing the above-mentioned flavonoid synthase.

[0011] Another aspect of the present invention discloses a biocatalyst containing the aforementioned flavonoid synthase.

[0012] Another aspect of the present invention discloses the application of the flavonoid synthase or the recombinant Pichia pastoris in the catalytic synthesis of apigenin.

[0013] Preferably, the application includes the production of apigenin by flavonoid synthase catalysis using naringenin as a substrate under methanol-induced conditions; The applications include the production of apigenin in the fields of feed, food, or medicine.

[0014] Another aspect of the present invention discloses a method for preparing recombinant Pichia pastoris strains, comprising the following steps: S1: The mRNA of a flavonoid synthase gene and its translated protein sequence, which are actively expressed in leaf tissues, were extracted from the spliced ​​transcripts of patchouli transcriptome sequencing data; S2: Based on the codon preference of Pichia pastoris, the flavonoid synthase gene sequence obtained in S1 was codon optimized and the optimized gene was synthesized; S3: The flavonoid synthase gene synthesized based on S2 was ligated with the pPICZ series expression vectors to obtain the recombinant plasmid pPICZ-FSII; S4: Transform the recombinant plasmid pPICZ-FSII constructed in S3 into Pichia pastoris.

[0015] The advantages of this invention compared to the prior art are: 1. By combining transcriptomic and metabolomic analyses of different tissues of patchouli rich in apigenin and its glycosides, a previously unstudied, actively expressed flavonoid synthase gene (FSII) closely related to changes in apigenin content was discovered. The enzyme encoded by this gene alone can catalyze the direct conversion of naringenin to apigenin without the need for 6-8 genes in the de novo apigenin synthesis pathway, which facilitates subsequent optimization, regulation, and modification. Using Pichia pastoris as the chassis cell, taking advantage of its metabolic characteristics and ability to use methanol as the sole carbon source, as well as the solubility of naringenin and apigenin in methanol, the upper limit of the host cell's ability to synthesize and accumulate large amounts of apigenin was significantly increased. The Pichia pastoris alcohol oxidase promoter (PAOX1) is strictly induced and regulated by external methanol concentration and has a strong ability to drive the expression of exogenous genes. When the FSII gene is expressed by PAOX1, the FSII enzyme protein can be coupled with the substrate level of naringenin when an exogenous methanol solution of naringenin is added. That is, the more exogenous naringenin is added, the more FSII enzyme is expressed in the cell, thus simultaneously increasing the yield and conversion rate of apigenin. Under the conditions of shake-flask fermentation and multiple additions of naringenin (total 800 mg / L) methanol solution, the recombinant Pichia pastoris constructed in this invention achieved a apigenin yield of 410 mg / L, with a conversion rate exceeding 50%. If combined with high-density fermentation, fermenter control, and extended culture time, the yield is expected to be increased several times further, reaching the entry level required for industrial production. Attached Figure Description

[0016] Figure 1 A schematic diagram illustrating the discovery of patchouli flavonoid synthase and its intracellular catalytic synthesis of apigenin from naringenin in Pichia pastoris.

[0017] Figure 2 This is a schematic HPLC chromatogram of the synthesis of apigenin, a natural product, by recombinant Pichia pastoris expressing flavonoid synthase.

[0018] Figure 3This is a schematic diagram showing the cumulative yield changes of apigenin synthesized by recombinant Pichia pastoris under methanol-induced culture at different time points.

[0019] Figure 4 This is a schematic diagram of existing technology.

[0020] Figure 5 This is a schematic diagram of existing technology.

[0021] Figure 6 This is a schematic diagram of existing technology.

[0022] As shown in the figure: Figure 5 The metabolic pathway for de novo synthesis of apigenin, and gene design for recombinant Yersinia lipolytica and recombinant Escherichia coli. Green represents the overexpressed gene in Yersinia lipolytica, purple represents the foreign gene introduced into Yersinia lipolytica, blue represents the foreign gene introduced into Escherichia coli, and UF6CGT1 is the glycosyltransferase that catalyzes the conversion of apigenin to vitexin; Figure 6 This study describes the yield and conversion rate of apigenin synthesized by co-fermentation of *Yersinia lipolyticis* and *Escherichia coli* on a small-scale fermenter scale. a) shows the yield of naringenin and apigenin during shake-flask fermentation (as a control). Recombinant *Yersinia lipolyticis* (Yl_Nar, the recombinant strain for naringenin synthesis) was cultured for 10 days, followed by the addition of recombinant *E. coli* (WT, the original recombinant strain; E12, the selected optimal mutant strain) and cultured for another day. The yields of naringenin and apigenin were measured at different time points. b) shows the conversion rate of naringenin to apigenin in different strains of recombinant *E. coli* (the conversion rate of the E12 preferred mutant strain was significantly improved). c) shows the final fermenter-scale culture process and synthesis yield, similar to a. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings.

[0024] Combined with appendix Figures 1-3 As shown, the first objective of this invention is to provide a plant-derived flavonoid synthase having the amino acid sequence shown in SEQ ID NO.1.

[0025] In one embodiment, the flavonoid synthase is derived from patchouli, whose amino acid sequence is shown in SEQ ID NO.1.

[0026] A second object of the present invention is to provide a gene encoding the flavonoid synthase, the DNA sequence of which is shown in SEQ ID NO.2.

[0027] A third objective of this invention is to provide an expression vector carrying the gene.

[0028] In one embodiment, the expression vector includes, but is not limited to, pPICZSeries of carriers, such as pPICZA, pPICZB, pPICZC .

[0029] A fourth object of the present invention is to provide a microorganism expressing the flavonoid synthase.

[0030] In one embodiment, the microorganism is recombinant Pichia pastoris.

[0031] In one embodiment, the recombinant Pichia pastoris uses Pichia pastoris X-33 as the host.

[0032] A fifth objective of this invention is to provide a method for constructing the above-mentioned recombinant Pichia pastoris, the method comprising the following steps: (1) The mRNA and its translated protein sequence of the flavone synthase II (FSII) gene, which is actively expressed in leaf tissue, were extracted from the transcriptome of patchouli sequencing data. (2) Based on the codon preference of Pichia pastoris, the flavonoid synthase gene sequence obtained in step (1) was designed with codon optimization and the optimized gene was synthesized; (3) Combine the flavonoid synthase gene synthesized in step (2) with... pPICZ A series of expression vectors were ligated to obtain recombinant plasmids. pPICZ-FSII ; (4) The recombinant plasmid constructed in step (3) pPICZ-FSII Transformed into Pichia pastoris.

[0033] A sixth object of the present invention is to provide the use of the flavonoid synthase or the recombinant Pichia pastoris in the catalytic synthesis of apigenin.

[0034] In one embodiment, the catalysis involves inducing the recombinant Pichia pastoris to culture using a methanol solution containing naringenin as the sole carbon source. After entering the cells, naringenin is catalyzed by flavonoid synthase to be converted into apigenin.

[0035] This invention claims protection for the use of the flavonoid synthase and its enzyme preparation, or the recombinant Pichia pastoris and its microbial preparation in the synthesis and production of apigenin in feed, food, and pharmaceutical fields.

[0036] In specific implementation of the present invention, (a) Culture medium YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 20 g / L agar (for solid plates only).

[0037] Z+YPDS medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, sorbitol 182.2 g / L, zeocin 100 mg / L, agar 20 g / L (for solid plates only).

[0038] BMGY medium: peptone 20 g / L, yeast extract 10 g / L, glycerol 10 mL / L, pH 6.0 0.1M potassium phosphate buffer (autoclaved separately), YNB 13.4 g / L (filter sterilized), biotin 0.4 mg / L (filter sterilized).

[0039] BMY medium: peptone 20 g / L, yeast extract 10 g / L, pH 6.0 0.1M potassium phosphate buffer (autoclaved separately), YNB 13.4 g / L (filter sterilized), biotin 0.4 mg / L (filter sterilized).

[0040] (II) Example 1: Gene mining and synthesis of flavonoid synthase from patchouli Based on the reference genome of patchouli and transcriptome sequencing data from different organs such as roots, stems, leaves, and flowers, transcript assembly, expression quantification, and functional annotation were performed. Correlation analysis between gene expression profiles and flavonoid content in different organs was also conducted to identify enzyme genes related to the apigenin synthesis pathway.

[0041] Flavonoid synthase II, which is highly expressed in flavonoid-rich leaves and directly catalyzes the synthesis of apigenin from naringenin, was selected. FSII Its amino acid sequence is shown in SEQ ID NO.1.

[0042] Codon optimization was performed for the Pichia pastoris expression host, and the coding gene for FSII was artificially synthesized. Its DNA sequence is shown in SEQ ID NO.2.

[0043] pass EcoR I and Not I Double enzyme digestion, FSII Gene inserted into Pichia pastoris expression vector pPICZA promoter PAOX1 With Termination AOX1 TT Between these steps, recombinant expression plasmids were obtained. pPICZA-FSII .

[0044] (III) Construction of a recombinant Pichia pastoris strain expressing flavonoid synthase FSII in Example 2 Recombinant plasmid pPICZA-FSII First use PmeI Linearization was achieved by single enzyme digestion, followed by precipitation with ethanol-sodium acetate at low temperature overnight, washing, resuspending in sterile water, and detection of DNA concentration to ensure that the total amount in 10 μL was 30-50 μg.

[0045] Pichia pastoris strain X-33 was inoculated into YPD medium and cultured at 30℃ for 16 h to ensure OD600 was 1.3-1.5. Competent cells were prepared by centrifugation and resuspending in sorbitol solution at 0℃, with a total volume of about 1000 μL.

[0046] Take 9 portions of X-33 competent cells and 1 portion pPICZA-FSII Mix well in an electroporation cup, incubate on ice, then electroporate at 2 kV. Immediately add sorbitol and transfer to a sterile tube.

[0047] Transformed X-33 cells were plated on Z+YPDS solid plates and cultured at 30°C for 2-4 days.

[0048] Ten single colonies were selected and propagated on YPD medium, and genomic DNA was extracted. 5' AOX1 and 3' AOX1 Primer amplification, agarose gel electrophoresis, and Sanger sequencing confirmed that the FSII gene was successfully integrated into the chromosome of Pichia pastoris strain X-33, obtaining a recombinant strain. X-33-FSII .

[0049] (iv) Example 3 Synthesis of apigenin from recombinant Pichia pastoris Recombinant Pichia pastoris strain with integrated flavonoid synthase gene X-33-FSII 300 μL of seed culture was inoculated into 40 mL of BMGY liquid medium and cultured with shaking at 30 °C and 220 rpm for 48 h.

[0050] Collect bacterial cells by centrifugation at room temperature, inoculate into 30 mL of BMY liquid medium, and incubate with shaking at 30℃ and 220 rpm for 30 min.

[0051] Add 300 μL of methanol solution containing 2% naringenin, and culture at 30℃ and 220 rpm for 12 h with shaking. Then, add the same concentration and volume of methanol solution containing naringenin every 12 h, for a total of 3 additions, and then stop the culture after 48 h.

[0052] During methanol-induced culture FSII Genes in PAOX1 Intracellular expression is driven by the promoter. Naringenin enters Pichia pastoris cells with methanol and is converted into apigenin under the catalysis of flavonoid synthase FSII.

[0053] The Pichia pastoris culture induced by methanol was aliquoted into sterile centrifuge tubes, centrifuged at 11,000 rpm for 5 min, and the supernatant and cells were separated. The samples were weighed and stored at -20℃.

[0054] (v) Extraction and detection of naringin and apigenin Weigh 1.2 g of the above Pichia pastoris cells, add 20 mL of methanol + 2% acetic acid 1:1 mixed extraction solution, vortex mix and then sonicate for 30 min.

[0055] After cooling to room temperature, bring the volume to 25 mL with the same mixed extract, mix well, centrifuge at 8000 rpm for 5 min, take 5 mL of the supernatant and dilute it 5 times with ultrapure water to obtain the crude extract.

[0056] Activate the HLB solid-phase extraction column sequentially with 5 mL of methanol and 5 mL of ultrapure water. Add the crude extract and pass it through the column at a flow rate of 1 mL / min. After complete elution, wash the column sequentially with 5 mL of ultrapure water and 6 mL of methanol, and collect the eluent.

[0057] The eluent was dried under nitrogen in a 45°C water bath and dissolved in 1 mL of methanol. After sonication for 10 min, the mixture was vortexed and filtered through a 0.22 μm organic filter membrane. The filtrate was the sample solution to be tested.

[0058] Qualitative and quantitative determinations were performed using Shimadu high-performance liquid chromatography. The chromatographic column was an xBridge C18 column (150 mm × 4.6 mm, 3.5 μm), the column temperature was 35℃, the injection volume was 10 μL, and the detector wavelength was 290 nm.

[0059] Mobile phase A was 0.1% acetic acid, and mobile phase B was methanol. The flow rate was 0.8 mL / min. The gradient elution program was as follows: 0–2 min, A 92% + B 8%; 2–11 min, A decreased to 50%, B increased to 50%; 11–16 min, A decreased to 35%, B increased to 65%; 16.1–17 min, A 10% + B 90%; 17.1–20 min, A 92% + B 8%.

[0060] This invention involves ligating the gene sequence encoding patchouli flavonoid synthase into the Pichia pastoris pPICZ series expression vector, driven by the alcohol oxidase promoter PAOX1, and expressing it in Pichia pastoris strain X-33. Induction culture using naringenin methanol solution as the sole carbon source enables efficient intracellular expression of the aforementioned flavonoid synthase, facilitating the efficient synthesis of apigenin from naringenin that enters the cell with methanol. Under shake-flask fermentation and periodically fed-methanol induction culture conditions, the recombinant Pichia pastoris can convert 800 mg / L of exogenously added naringenin into 410 mg / L apigenin, with a molar conversion rate exceeding 50%. Further combining this method with high-density fermentation technology holds great promise for achieving the requirements of industrial-scale synthesis and production of apigenin.

[0061] in: SEQ 1 Flavonoid synthase II derived from patchouli MDLVEITLYAALFLLSAAFLLTLLAGKRRQYLSSAPGPFPLPLIGHLHLLGPRLHYTFHKMAQRYGPIMQIRLGSIPCVVVSSPELAKEFLKTHELVFSSRKHSTAIDIVTYESSFAFSPYGPYWKYIKKLCTYELLGARNLSHFQPIRTSEVTSFLEGLMQKGISGEGFNATEELVKLTSNVISHMMLSIRCSETEGEAEAARTVIREVTQIFGEFDVADIIWICKNFDLQGIKRRSVDIQKRYDALLEKIITDREEQRRSHGGDARDFLDMFLDIMEGGNSEVKFTREHLKALILDFFTAGTDTTAITIEWAIAELINNPDVLKKAQNEIDKVIGSTRLLQESDAPNLPYLQAIIKETFRLHPPIPMLSRKSISDCVIDGYKIPAHCLLFVNIWSMGRNPKYWESPMSFRPDRFLETKNASIDIKGQHFELLPFGSGRRGCPGMLLGIQEVTHIIGTMIQCFEWELVDGSGRVDMTERHGLTAPRANDLVCRVVPRVDRAIISGQ SEQ 2 Flavonoid synthase II coding gene optimized for codons of Pichia pastoris

[0062] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0063] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0064] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A patchouli-derived flavonoid synthase, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding a gene, characterized in that: It encodes the flavonoid synthase described in claim 1.

3. The encoding gene as described in claim 2, characterized in that: Its DNA sequence is shown in SEQ ID NO:

2.

4. An expression vector, characterized in that: It carries the coding gene as described in claim 2, and the coding gene is operatively linked between the Pichia pastoris alcohol oxidase promoter and terminator.

5. The expression vector as described in claim 4, characterized in that: The expression vector is a pPICZ series vector, including pPICZA, pPICZB, and pPICZC.

6. A recombinant Pichia pastoris strain, characterized in that: Using Pichia pastoris X-33 as the host cell and integrating the expression vector as described in claim 4, the flavonoid synthase as described in claim 1 is expressed.

7. A biocatalyst, characterized in that: The biocatalyst contains the flavonoid synthase as described in claim 1.

8. The use of the flavonoid synthase as described in claim 1, the recombinant Pichia pastoris strain as described in claim 6, or the biocatalyst as described in claim 7 in the synthesis of apigenin.

9. The application as described in claim 8, characterized in that: The application includes the production of apigenin by flavonoid synthase catalysis using naringenin as a substrate under methanol-induced conditions; The applications include the production of apigenin in the fields of feed, food, or medicine.

10. A method for preparing the recombinant Pichia pastoris strain as described in claim 6, characterized in that: Includes the following steps: S1: The mRNA of a flavonoid synthase gene and its translated protein sequence, which are actively expressed in leaf tissues, were extracted from the spliced ​​transcripts of patchouli transcriptome sequencing data; S2: Based on the codon preference of Pichia pastoris, the flavonoid synthase gene sequence obtained in S1 was codon optimized and the optimized gene was synthesized; S3: The flavonoid synthase gene synthesized based on S2 was ligated with the pPICZ series expression vectors to obtain the recombinant plasmid pPICZ-FSII; S4: Transform the recombinant plasmid pPICZ-FSII constructed in S3 into Pichia pastoris.

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