Algae moss flavone synthetic route related protein and coding gene and application thereof

Abstract

The invention discloses algal moss flavone synthetic route related protein as well as a coding gene and application of the algal moss flavone synthetic route related protein. The invention discloses algal moss flavone synthesis route related proteins, which are named as Tl4CL, TlCHS and TlCHI, and the amino acid sequences of the algal moss flavone synthesis route related proteins are respectively shown as SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6. A test proves that the Tl4CL can efficiently catalyze p-coumaric acid to generate p-coumaryl CoA. TlCHS and TlCHI are chalcone synthetase and chalcone isomerase in algae moss respectively, p-coumaroyl CoA can be converted into naringenin under the catalysis of the two enzymes, and the yield reaches 574ng / ml through fermentation of recombinant bacteria.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to proteins and their encoding genes related to the flavonoid synthesis pathway in algae and mosses, and their applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Algae moss ( Takakia lepidozioides Belonging to the order Phylobryophytes and family Phylobryophytes, *Takakia* is an ancient bryophyte species found only in high-altitude or high-latitude regions. In these extreme habitats, it exhibits unique adaptive evolutionary characteristics. From an evolutionary perspective, *Takakia* is the sister group to all extant bryophytes, possibly sharing characteristics with the last common ancestor (LCA) of terrestrial plants, and providing important insights into the evolution of early terrestrial plants.

[0004] Flavonoids are important secondary metabolites in nature, possessing functions such as anti-free radical and antioxidant properties, and have extremely high application value. Their basic structure consists of a C6-C3-C6 carbon skeleton composed of two 6-carbon benzene rings (rings A and B) connected by a 3-carbon heterocycle (ring C). Although flavonoids vary in structure, their biosynthesis follows the same basic skeleton construction process: naupliine chalcone is generated via the shikimic acid-polyketide pathway, followed by cyclization of the naupliine chalcone molecule under the catalysis of chalcone isomerase (CHI) to generate naringenin, the flavonoid core. This C15 skeleton can be further modified through oxidation, methylation, and other processes to form a wide range of different flavonoid structures.

[0005] Naringenin, a scaffold component in the synthesis of flavonoids, has seen rapid development in its synthesis in recent years. Significant progress has been made in the biosynthesis of flavonoids from *E. coli*, but the problem of insufficient plant genetic resources and low yields remains. The genetic resources for heterologous synthesis of naringenin in *E. coli* are mostly obtained from fungi and Lamiaceae plants, with very little utilization of algae and bryophytes. Summary of the Invention

[0006] The purpose of this invention is to develop gene resources of algae, provide a key rate-limiting enzyme in the pathway of naringenin synthesis from algae flavonoids, and apply it to Escherichia coli, thereby achieving the synthesis of naringenin in Escherichia coli B21 using genes from algae. This strain is named T4CC.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the invention provides proteins named Tl4CL, TlCHS, and TlCHI, derived from the algae *Takakia lepidozioides* S. Hatt. Experimental verification has shown that Tl4CL can efficiently catalyze the conversion of p-coumaric acid to p-coumaryl-CoA. TlCHS and TlCHI are a chalcone synthase and a chalcone isomerase, respectively, found in the algae. Under the catalysis of these two enzymes, p-coumaryl-CoA can be converted into naringenin.

[0008] The amino acid sequence of Tl4CL is shown in SEQ ID No. 2, consisting of 562 amino acid residues; the amino acid sequence of TlCHS is shown in SEQ ID No. 4, consisting of 402 amino acid residues; and the amino acid sequence of TlCHI is shown in SEQ ID No. 6, consisting of 126 amino acid residues.

[0009] In a second aspect, the present invention provides genes encoding Tl4CL1, TlCHS8, and TlCHI1, wherein the nucleotide sequence of the gene encoding Tl4CL is shown in SEQ ID No. 1; the nucleotide sequence of the gene encoding TlCHS is shown in SEQ ID No. 3; and the nucleotide sequence of the gene encoding TlCHI is shown in SEQ ID No. 5.

[0010] In a third aspect, recombinant expression vectors, recombinant engineered bacteria, recombinant cell lines or transformants containing the above-mentioned coding genes are also within the scope of protection of this invention.

[0011] In one specific embodiment of the present invention, the recombinant expression vector may be: 1) pCDFduet-Tl4CL: The nucleotide sequence is shown in SEQ ID No. 1. Tl4CL Gene inserted into pCDFduet-1 plasmid Nde I and Xho Between the I enzyme recognition sites, the expression of the enzyme is obtained. Tl4CL The recombinant expression vector for the gene was named pCDFduet-Tl4CL; 2) pET-TlCHS: The nucleotide sequence is as shown in SEQ ID No. 3. TlCHS Gene inserted into the pETduet-1 vector EcoR I and Hind Between the III enzyme recognition sites, the expression of the TlCHS The recombinant expression vector for the gene was named pET-TlCHS; 3) pET-TlCHS-TlCHI: The nucleotide sequence is as shown in SEQ ID No. 5. TlCHI Gene insertion into the pET-TlCHS vector Nde I and Xho Between the I enzyme recognition sites, the expression of the enzyme is obtained. TlCHI The recombinant expression vector for the gene was named pET-TlCHS-TlCHI.

[0012] The recombinant engineered bacteria can be co-transformed with pCDFduet-Tl4CL and pET-TlCHS-TlCHI into bacterial cells to obtain recombinant engineered bacteria expressing the Tl4CL protein with the amino acid sequence shown in SEQ ID No. 2, the TlCHS protein with the amino acid sequence shown in SEQ ID No. 4, and the TlCHI protein with the amino acid sequence shown in SEQ ID No. 6.

[0013] Preferably, the bacterial cells are Escherichia coli or Agrobacterium; more preferably, they are Escherichia coli BL21(DE3).

[0014] The bacterial cells include Escherichia coli (such as BL21(DE3)), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, etc.

[0015] The application of the recombinant engineered bacteria in the preparation of naringenin is also within the scope of protection of this invention.

[0016] In a fourth aspect of the present invention, the use of the proteins Tl4CL, TlCHS, and TlCHI as 4-coumaric acid coenzyme A ligase, chalcone synthase, and chalcone ligase, respectively, should also be within the scope of protection of the present invention.

[0017] A fifth aspect of the invention provides the use of the coding gene, recombinant expression vector, and transformed cells in the preparation of strains that synthesize naringenin.

[0018] In a sixth aspect, the present invention provides a protein composition for synthesizing naringenin, comprising a protein with an amino acid sequence as shown in SEQ ID No. 2, a protein with an amino acid sequence as shown in SEQ ID No. 4, and a protein with an amino acid sequence as shown in SEQ ID No. 6.

[0019] The application of the Tl4CL protein or its encoding gene with the amino acid sequence shown in SEQ ID No. 2, the TlCHS protein or its encoding gene with the amino acid sequence shown in SEQ ID No. 4, and the TlCHI protein or its encoding gene with the amino acid sequence shown in SEQ ID No. 6 in the preparation of naringenin is also within the scope of protection of this invention.

[0020] A seventh aspect of the present invention provides a method for preparing naringenin, comprising fermenting recombinant engineered bacteria expressing the amino acid sequences of TlCHS protein as shown in SEQ ID No. 2, TlCHS protein as shown in SEQ ID No. 4, and TlCHI protein as shown in SEQ ID No. 6 in a fermentation medium containing tyrosine substrate and IPTG to obtain a culture medium containing naringenin.

[0021] Specifically, pCDFduet-Tl4CL and pET-TlCHS-TlCHI were co-transformed into bacterial cells to obtain recombinant engineered bacteria expressing the Tl4CL protein (as shown in SEQ ID No. 2), the TlCHS protein (as shown in SEQ ID No. 4), and the TlCHI protein (as shown in SEQ ID No. 6).

[0022] In the recombinant engineered bacteria, pCDFduet-Tl4CL catalyzes the linkage of coumaric acid to coenzyme a; pET-TlCHS-TlCHI catalyzes the decarboxylation condensation of 3 molecules of malonyl-CoA and 1 molecule of coumaroyl-CoA to synthesize 2′,4,4′,6′-tetrahydroxychalcone, which is then converted into dihydroflavonoids by chalcone isomerase.

[0023] The eighth aspect of the present invention is the application of the above-mentioned recombinant engineered bacteria in the actual production of naringenin.

[0024] The beneficial effects of one or more of the above technical solutions: The Tl4CL from algae provided by the above technical solution is a key rate-limiting enzyme, 4-coumaric acid-CoA ligase, discovered for the first time in this species, in the synthesis of naringenin. It catalyzes the conversion of p-coumaric acid to p-coumaryl-CoA, a high-energy intermediate that is a common precursor for the synthesis of all downstream phenylpropane derivatives. TlCHS and TlCHI are provided as chalcone synthase and chalcone isomerase from algae, respectively. Under the catalysis of these two enzymes, p-coumaryl-CoA can be converted into naringenin. The full-length sequences of three genes were obtained from cDNA using PCR technology. Protein expression strains were obtained by constructing pCDFduet-1 and pETduet-1 protein expression vectors and transforming them into *E. coli* BL21(DE3). Exogenous application of tyrosine enabled the strains to directly produce naringenin, thus demonstrating high economic value and broad application prospects. This invention utilizes genes from the flavonoid synthesis pathway of *Takakia* in *E. coli* to directly generate naringenin, with a yield of 574 ng / ml. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 Electrophoresis diagram of the ORF amplification products of the target genes Tl4CL1c07268, TlCHS2c05338 and TlCHI1c00625.

[0027] Figure 2 Western blot results for proteins Tl4CL1c07268, TlCHS2c05338, and TlCHI1c00625. Where: M: protein molecular weight standard; left side: Anti-His tag; right side: Anti-S-tag tag. Figure 3 HPLC chromatogram of the main catalytic reaction product of strain T4CC using tyrosine as a donor. Figure 3 (A) LC-MS spectrum ( Figure 3 (B and C in the middle). Detailed Implementation

[0028] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of this invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of this invention is for describing specific embodiments and not for limiting the scope of protection of this invention. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0031] Example 1. Obtaining the key rate-limiting enzyme gene in the naringenin synthesis pathway in algae. This invention constructs a phylogenetic tree on Figtree using the TAIR and Phytozome databases, employing a neighbor-joining model. Then, TBtools is used to analyze the gene structure of all gene sequences, and the expression levels of screened genes before and after UV treatment are analyzed. Genes with high expression levels after UV treatment and predicted 4-coumaric acid-coenzyme A ligase function based on their domains are identified. Through extensive functional screening, key rate-limiting enzyme genes and their encoded proteins in the naringenin synthesis pathway are obtained, named Tl4CL, TlCHS, and TlCHI. Tl4CL is the first discovered 4-coumaric acid-coenzyme A ligase in this species, catalyzing the conversion of p-coumaric acid to p-coumaryl-CoA, a high-energy intermediate that is a common precursor for the synthesis of all downstream phenylpropane derivatives. TlCHS and TlCHI are provided as chalcone synthase and chalcone isomerase, respectively, found in algae. Under the catalysis of these two enzymes, p-coumaryl-CoA can be converted into naringenin.

[0032] Tl4CL, TlCHS and TlCHI The methods for obtaining genes are as follows: RNA was extracted from Tibetan algae and reverse transcribed to synthesize Tibetan algae cDNA. The diluted reverse-transcribed algae cDNA was used as a template and amplified using the primers listed below.

[0033] Tl4CL Gene amplification primer sequences: pCDF-Tl4CL-F: 5'-GTATAAGAAGGAGATATACATATGGCCCCTTGTGAAACAG-3'; pCDF-Tl4CL-R: 5'-CTTTACCAGACTCGAGTAAATCCATCAGATTCAGTTTAT-3'.

[0034] TlCHS Gene amplification primer sequences: pET-TlCHS-F: 5'-CACAGCCAGGATCCGAATTCAATGGCACCAAGCGGGGACC-3'; pET-TlCHS-R: 5'-CATTATGCGGCCGCAAGCTTCTAGCTAGTTGGCATGCTCCGCAG-3'.

[0035] TlCHI Gene amplification primer sequences: pET-TlCHI-F: 5'-GTATAAGAAGGAGATATACATATGGTTTCCGATAGCCTGG-3'; pET-TlCHI-R: 5'-GTTTCTTTACCAGACTCGAGTTCGGGCTCCTCATATTTGTC-3'.

[0036] The amplification system and amplification program are as follows: 25 μL KOD One PCR Master Mix, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, 2 μL DNA template, and ddH2O to 50 μL; add the above components to a 200 μL PCR tube, mix well, centrifuge at low speed, and then place in a PCR instrument for amplification according to the following program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 50℃ annealing for 15 s, 65℃ extension for 45 s, 33 cycles.

[0037] The PCR reaction products were detected by agarose gel electrophoresis. Figure 1 The target size bands were cut into gels, recovered, and sequenced. The results showed that... Tl4CL Gene fragments, TlCHS gene fragments 、TlCHI Gene fragments. Tl4CL The gene has the nucleotide sequence of SEQ ID No. 1 in the sequence listing, consists of 1686 nucleotides, and encodes the protein shown in SEQ ID No. 2 in the sequence listing, named Tl4CL; TlCHS The gene has the nucleotide sequence of SEQ ID No. 3 in the sequence listing, consists of 1209 nucleotides, and encodes the protein shown in SEQ ID No. 4 in the sequence listing, named TlCHS; TlCHI The gene fragment has the nucleotide sequence of SEQ ID No. 5 in the sequence listing, consists of 378 nucleotides, and encodes the protein shown in SEQ ID No. 6 in the sequence listing, named TlCHI.

[0038] Example 2: Construction of vectors for pCDFduet-Tl4CL and pET-TlCHS-TlCHI The pCDFduet-1 plasmid obtained in Example 1 was digested with NdeI and XhoI double enzymes. Tl4CL The fragment was ligated to the enzyme-digested vector via homologous recombination, and the ligated product was transformed into *E. coli* DH5α competent cells. Plasmid was extracted to obtain expression. Tl4CLThe recombinant vector was sequenced, and the results showed that Tl4CL is as shown in SEQ ID No. 1 of the sequence listing, and the sequence of the protein it encodes is Tl4CL as shown in SEQ ID No. 2. The recombinant vector expressing this Tl4CL gene was named pCDFduet-Tl4CL.

[0039] The pETduet-1 vector (purchased from Beyotime) was digested with EcoRI and Hind III. The TlCHS fragment from Example 1 was ligated to the digested vector via homologous recombination. The ligated product was transformed into E. coli DH5α competent cells. Plasmid was extracted to obtain a recombinant vector expressing TlCHS. Sequencing showed that TlCHS is as shown in SEQ ID No. 3 in the sequence listing, and the sequence of the protein it encodes is TlCHS2c05338 as shown in SEQ ID No. 4. The recombinant vector expressing this TlCHS was named pET-TlCHS, and the strain containing this recombinant vector was named the pET-TlCHS strain. pET-TlCHS was digested with NdeI and XhoI. The TlCHI fragment obtained in Example 1 was ligated to the digested vector via homologous recombination. The ligated product was transformed into E. coli DH5α competent cells. Plasmids were extracted to obtain recombinant vectors expressing TlCHS and TlCHI. Sequencing showed that TlCHI is as shown in SEQ ID No. 5 of the sequence listing, and the sequence of the protein it encodes is TlCHI as shown in SEQ ID No. 6. The recombinant vector expressing the TlCHS and TlCHI genes obtained above was named pET-TlCHS-TlCHI, and the recombinant engineered bacteria containing this recombinant vector was named strain CC.

[0040] The constructed pCDFduet-Tl4CL and pET-TlCHS-TlCHI vectors were simultaneously transformed into DH5α competent cells. Plasmids were extracted to obtain recombinant vectors expressing Tl4CL, TlCHS, and TlCHI. Sequencing showed that Tl4CL, TlCHS, and TlCHI are as shown in SEQ ID Nos. 1, 3, and 5 of the sequence listing, and the sequences of their encoded proteins are shown in SEQ ID Nos. 2, 4, and 6. The recombinant engineered strain containing the pCDFduet-Tl4CL and pET-TlCHS-TlCHI vectors was named strain T4CC.

[0041] Example 2. Expression and detection of Tl4CL, TlCHS, and TlCHI proteins 2.1 Induction of target protein expression (1) Select T4CC strain and inoculate it into 5 mL of LB medium containing Sm, and shake it overnight at 37℃ and 220 rpm.

[0042] (2) Transfer the shaken bacterial culture 1:100 to the culture flask until the OD value is 0.05-0.1, at 37℃ and 220 rpm until the OD600 is between 0.6-0.8. Add IPTG to a final concentration of 1 mM in a clean bench and incubate at 30℃ and 200 rpm for 2, 3, and 4 h respectively. (3) Collect bacterial cells at room temperature for 5 minutes at 4000 rpm.

[0043] (4) Centrifuge at 4℃ and 4000 rpm for 10 min. Take 20 μL of the supernatant after centrifugation, add 5×SDS Loading Buffer and 80 μL ddH2O, mix well, and boil at 100℃ for 10 min.

[0044] The expression, isolation, and purification of the target protein were represented by immunoblotting.

[0045] 2.2 Immunoblotting of the target protein (1) Vertical plate electrophoresis is used to fix the glass plate on the rubber frame.

[0046] (2) Prepare a 10% separating gel, add it to the electrophoresis apparatus, add ethanol liquid seal, and let stand until the separating gel solidifies.

[0047] (3) Prepare a 3% concentrated gel and discard the top layer of ethanol. Mix the prepared 5% concentrated gel thoroughly and pour it in immediately. Insert the comb between the glass plates (to avoid the formation of air bubbles). After the gel solidifies, pull out the comb.

[0048] (4) Centrifuge at 13,000 rpm for 10 min, take 10 μL of supernatant for spotting, and at the same time take 5 μL of protein marker for spotting.

[0049] (5) Add an appropriate amount of electrophoresis buffer to the electrophoresis tank, and perform electrophoresis at a constant voltage of 60V. When the sample is electrophoresed to the separating gel... Switch to 120V constant voltage electrophoresis until bromophenol blue reaches the bottom edge of the gel, then stop electrophoresis.

[0050] (6) Gently pry open the prepared gel glass plate and cut off the stacking gel and any unwanted areas around it. Place the gel in the transfer buffer, ensuring the gel remains intact.

[0051] (7) Soak the PVDF membrane in methanol for 30 seconds (from opaque to transparent), and then put the membrane and filter paper into the transfer buffer.

[0052] (8) Place the sandwich jacket with the black side down and the transparent side (or red side) up on a clean table. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in that order from bottom to top. Gently roll the top foam pad with a roller to ensure there are no air bubbles between each layer.

[0053] (9) Insert the jacket into the transfer electrophoresis core, place it in the transfer tank, add enough transfer buffer, set it to constant voltage, 100 V, and transfer for 1 hour.

[0054] (10) After the transfer is completed, take out the PVDF membrane and seal it with 5% BSA. Seal it in a shaker at room temperature for 45 min.

[0055] (11) Place the sealed PVDF membrane in a prepared liquid containing primary antibody and incubate overnight at 4°C on a four-dimensional rotator.

[0056] (12) After the primary antibody incubation is completed, the primary antibody is recovered, a small box is taken and PBST washing solution is poured in, the membrane is taken out and placed in the box for washing, 3-4 times, 10 min each time.

[0057] (13) After washing the membrane, add secondary antibody to 5% skim milk powder and incubate at room temperature for 1 h.

[0058] (14) After the secondary antibody incubation is completed, take out the membrane and put it into the washing box. Wash it 3-4 times, 10 min each time.

[0059] (15) Prepare the luminescent solution at a 1:1 ratio and mix well. Place the film horizontally on the luminescent plate, coat it evenly with the luminescent solution, and begin development.

[0060] Test results as follows Figure 2 As shown, the results indicate that all three target proteins could be successfully induced to express in the induction expression system. TlCHS and Tl4CL carry His tags and can be specifically detected by Anti-His antibodies; while Tl4CL and TlCHI carry S-tag tags and can be specifically detected by Anti-S-tag antibodies. All proteins were successfully expressed after induction. The band positions were largely consistent with the labeled predicted molecular weights (TlCHS ~45.1kDa, Tl4CL ~63.2kDa, TlCHI ~16.2kDa), indicating that the proteins did not undergo large-scale degradation. Furthermore, all proteins were not expressed or expressed at extremely low levels before induction, began to be expressed 2 hours after induction, and reached even higher levels after 4 hours.

[0061] Example 3: Fermentation of T4CC strain and detection of naringenin in fermentation product 1. Fermentation culture of T4CC strain (1) Select positive clones of T4CC strain and inoculate them into 5 mL of LB medium containing streptomycin (Sm) at a final concentration of 100 μg / mL and ampicillin (Amp) at 50 μg / mL. Shake gently at 220 rpm overnight at 37°C.

[0062] (2) Transfer the shaken bacterial solution 1:100 to the culture flask until the OD value is 0.05-0.1, and add Sm at a final concentration of 100 μg / mL and ampicillin (Amp) at a final concentration of 50 μg / mL. (3) Cultivate the bacterial culture at 37°C and 220 rpm until the OD600 is between 0.6 and 0.8, induce with a final concentration of 1 mM IPTG, and incubate at 30°C and 220 rpm for 3 h.

[0063] (4) Collect bacterial cells by centrifugation at 5000 g for 15 min at 4℃; (5) When the pre-cooled MOPS medium (pH 7.4) was resuspended to an OD600 of 1.8-2.0, IPTG was added again to a final concentration of 1 mM for secondary induction, and tyrosine substrate was added to a final concentration of 3 mM. The genetically engineered bacteria were cultured for 36 h and the final product was identified.

[0064] The preparation method for 10x MOPS stock solution (250ml) is as follows: (1) Weigh 20.93g MOPS and 1.79g Tricine into 150ml ddH2O. (2) Adjust the pH value to 7.4 with 4M KOH, the amount added is approximately 25-50ul. (3) Add freshly prepared FeSO4·7H2O to a final concentration of 0.1 mM. (4) Add inorganic salts in sequence. The specific components and final concentrations are: NH4Cl 95 mM, K2SO4 2.76 mM, CaCl2 5 μM, MgCl2 5.28 mM, NaCl 500 mM.

[0065] (5) Add 250 μL of trace element stock solution, prepared as follows: (NH4)6Mo7O 24 3 μM, H3BO3 0.4 mM, CoCl2·6H2O 30 μM, CuSO4·5H2O 10 μM, MnCl2·4H2O 80 μM, ZnSO4·7H2O 10 μM. (6) Adjust the volume of the solution to 250 ml with ddH2O and sterilize by filtering through a 0.22 μm filter membrane.

[0066] Naringenin was detected using LC-MS (high performance liquid chromatography-tandem mass spectrometry), as follows: Centrifuge the culture medium at 4℃ and 4000 g for 15 min to collect the E. coli cell pellet. Take 10 mL of the supernatant and add an equal volume of ethyl acetate (chromatographic grade). Vortex for 1 min to mix thoroughly. Sonicate for 30 min to enhance extraction efficiency. Centrifuge again (4000 g, 15 min, 4℃) to separate the organic and aqueous phases. Rotary evaporate the organic phase to dryness. Redissolve the residue in 300 μL of methanol (chromatographic grade) and filter through a 0.22 μm nylon membrane for later use.

[0067] The obtained solution was analyzed by high-performance liquid chromatography (HPLC), with the column temperature controlled at 30°C. The flavonoid skeleton was separated by gradient elution with 0.1% formic acid in acetonitrile / water. The elution conditions were: 0-3 min, 10-40% acetonitrile (v / v); 3-5 min, 40-60% acetonitrile (v / v); 5-7 min, 60-90% acetonitrile.

[0068] UPLC-ESI-Q-TOF MS and MS2 data analysis were performed on a Waters MassLysn V4.1 system. Full-scan mass spectrometry was performed using a two-stage tandem mass spectrometer in negative ion mode, employing a Wasters Q-TOF mass spectrometer with quadrupole and time-of-flight technology, equipped with an ion trap mass spectrometer passing through an ESI source. ESI mass spectra of positive ions (PI) and negative ions (NI) were obtained. ESI was performed with a capillary voltage of 3 kV, a gas (N2) temperature of 300 °C, and a flow rate of 8.0 L min⁻¹. MS and MS2 mass spectra were recorded in the m / z range of 50 to 1000.

[0069] The results are as follows Figure 3 As shown, the fermentation product naringenin of the strain eluted at 3.127 min ( Figure 3 In the case of A), the mass-to-charge ratio of the parent ion (m / z 271.2) and the two daughter ions (m / z 151 and m / z 119) of the substance eluting at this time is consistent with that of naringenin. Figure 3 (B and C) The substance was further identified as naringenin based on the ratio of daughter ions to mother ions. Liquid chromatography analysis was performed using naringenin standards of different concentrations to construct a standard curve. Finally, the concentration of naringenin in the fermentation broth was determined based on the peak area, and the yield reached 574 ng / ml.

[0070] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims

1. A protein related to the synthesis pathway of alginate flavonoids, as shown in A1), A2) or A3) below: A1) the amino acid sequence shown in SEQ ID No. 2; A2) the amino acid sequence shown in SEQ ID No. 4; A3) the amino acid sequence shown in SEQ ID No.

6.

2. A gene encoding the protein related to the synthesis pathway of alginate flavonoids shown in claim 1, as shown in B1), B2) or B3) below: B1) the nucleotide sequence shown in SEQ ID No. 1; B2) the nucleotide sequence shown in SEQ ID No. 3; B3) the nucleotide sequence shown in SEQ ID No.

5.

3. A recombinant vector containing the gene encoding shown in claim 2, a recombinant engineering bacterium containing the gene encoding shown in claim 2 or the recombinant vector shown in claim 3, a recombinant cell line containing the gene encoding shown in claim 2 or the recombinant vector shown in claim 3.

4. The use of the protein with the amino acid sequence shown in SEQ ID No. 2 as a 4-coumaric acid coenzyme A ligase, the use of the protein with the amino acid sequence shown in SEQ ID No. 4 as a chalcone synthase, and the use of the protein with the amino acid sequence shown in SEQ ID No. 6 as a chalcone ligase.

5. A protein composition for synthesizing naringenin, consisting of the protein with the amino acid sequence shown in SEQ ID No. 2, the protein with the amino acid sequence shown in SEQ ID No. 4, and the protein with the amino acid sequence shown in SEQ ID No.

6.

6. A method for preparing an engineering bacterium for synthesizing naringenin, comprising the following steps: 1) constructing pCDFduet-Tl4CL: The nucleotide sequence as shown in SEQ ID No. 1 Tl4CL The gene was inserted into the pCDFduet-1 plasmid Nde I and Xho I enzyme recognition site, to obtain a recombinant expression vector expressing the Tl4CL gene, named pCDFduet-Tl4CL; 2) construction of pET-TlCHS-TlCHI: the nucleotide sequence as shown in SEQ ID No. 3 TlCHS gene into the pETduet-1 vector EcoR I and Hind III enzyme recognition site, to obtain a recombinant expression vector expressing the TlCHS gene, named pET-TlCHS; the nucleotide sequence as shown in SEQ ID No. 5 TlCHI gene into the pET-TlCHS vector Nde I and Xho I enzyme recognition site, to obtain a recombinant expression vector expressing the TlCHI gene, named pET-TlCHS-TlCHI; 3) co-transferring pCDFduet-Tl4CL and pET-TlCHS-TlCHI into bacterial cells to obtain a recombinant engineering bacterium expressing Tl4CL protein with the amino acid sequence shown in SEQ ID No. 2, TlCHS protein with the amino acid sequence shown in SEQ ID No. 4, and TlCHI protein with the amino acid sequence shown in SEQ ID No. 6; Preferably, the bacterial cells are Escherichia coli or Agrobacterium; more preferably, Escherichia coli BL21 (DE3).

7. The recombinant engineering bacterium obtained by the method shown in claim 6.

8. The use of the recombinant engineering bacterium shown in claim 3 or 7 in the preparation of naringenin.

9. The use of Tl4CL protein with the amino acid sequence shown in SEQ ID No. 2 or its encoding gene, TlCHS protein with the amino acid sequence shown in SEQ ID No. 4 or its encoding gene, and TlCHI protein with the amino acid sequence shown in SEQ ID No. 6 or its encoding gene in the preparation of naringenin.

10. A method for preparing naringenin, wherein a recombinant engineering bacterium expressing a TlCHS protein having an amino acid sequence as shown in SEQ ID No. 2, a TlCHS protein having an amino acid sequence as shown in SEQ ID No. 4, and a TlCHI protein having an amino acid sequence as shown in SEQ ID No. 6 is fermented in a fermentation medium containing a tyrosine substrate and IPTG to obtain a culture solution containing naringenin.

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