Chalcone isoprenyl transferase gene GiPT16, GiPT16 protein, amplification primer set and application

Abstract

This invention provides a chalcone isopentenyltransferase gene. GiPT16 This invention relates to the GiPT16 protein, amplification primer set, and applications, belonging to the field of biogenetics technology. It is based on *Glycyrrhiza inflata* (GiPT16 protein, amplification primer set, and applications). Glycyrrhiza inflata Using whole-genome sequencing data and a reverse genetics strategy, the key aromatic isopentenyltransferase GiPT16, involved in the isopentenylation modification of chalcone active ingredients, was successfully identified and functionally characterized. This enzyme was confirmed to specifically catalyze the biosynthesis of psoralen and glycyrrhizin C using DMAPP as a donor. It is the first chalcone isopentenyltransferase characterized in *Glycyrrhiza inflata*, filling a gap in the study of key enzymes in the chalcone isopentenylation metabolic pathway of this species and providing important evidence for further elucidating the molecular mechanisms of quality formation in *Glycyrrhiza inflata* medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a chalcone isopentenyltransferase gene. GiPT16 GiPT16 protein, amplification primer set and its applications. Background Technology

[0002] Licochalcones are a class of characteristic secondary metabolites mainly found in *Glycyrrhiza inflata*. Structurally, they consist of two aromatic rings linked by α,β-unsaturated ketones to form a "trans" chalcone skeleton, often modified with isopentenyl groups. Licochalcones A, B, and C exhibit significant antitumor and antiviral (e.g., inhibition of respiratory syncytial virus) biological activities, and their structural derivatives also show good pharmaceutical potential. However, the current acquisition of licorice chalcones still heavily relies on plant extraction, which is not only inefficient and costly but also exacerbates the pressure on wild *Glycyrrhiza inflata* resources, making it difficult to meet the needs of high-value development and industrialization.

[0003] Studies have shown that isopentenylation modification of glycyrrhizin chalcone compounds in *Glycyrrhiza inflata* is a key step in enhancing its structural diversity and bioactivity. This type of reaction is mainly catalyzed by UbiA superfamily aromatic isopentenyltransferases (PHB-type UbiA prenyltransferases, PT) in plants. These membrane-bound enzymes can use dimethyl allyl pyrophosphate (DMAPP) as a donor in Mg... 2+ With the assistance of TA, isopentenyl groups are transferred to electron-rich aromatic acceptors. Although UbiA-PT has been studied in metabolic pathways of flavonoids and coumarins, its role in the isopentenylation of chalcones, especially glycyrrhizin, has not been reported.

[0004] It is noteworthy that UbiA-PTs typically exhibit high substrate or donor specificity, and their functions vary significantly across different species. For example, HpPT4px in *Hypericum perforatum* recognizes only xanthones and specifically utilizes DMAPP; while two closely related PTs in *Murraya paniculata* show drastically different substrate preferences. These characteristics make functional predictions based on homology comparisons unreliable, requiring individual experimental verification. Compared to glycosyltransferases or methyltransferases, UbiA-PTs present greater challenges in gene screening and functional identification due to their difficult membrane localization, expression, and complex functions, resulting in very limited related research.

[0005] Therefore, to achieve sustainable and efficient production of glycyrrhizin chalcones, it is urgent to systematically identify and characterize the key UbiA-PT gene involved in their isopentenylation modification. This will not only help elucidate the biosynthetic mechanism of glycyrrhizin chalcones, but also lay the foundation for the green manufacturing of these high-value active ingredients through synthetic biology methods (such as microbial heterologous synthesis or plant metabolic engineering), which is of great significance for protecting wild resources and promoting the modernization of traditional Chinese medicine. Summary of the Invention

[0006] The purpose of this invention is to provide a chalcone isopentenyltransferase gene. GiPT16 The study of GiPT16 protein, amplification primer set, and its application fills the terminal gap in the biosynthetic pathway of glycyrrhizin chalcone in Glycyrrhiza inflata.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a chalcone isopentenyltransferase gene. GiPT16 The chalcone isopentenyltransferase gene GiPT16 Includes one or more of the following sequences:

[0009] (1) A DNA molecule having the gene sequence shown in SEQ ID NO.1;

[0010] (2) DNA molecules that have more than 80% homology with the gene sequence shown in SEQ ID NO.1;

[0011] (3) A DNA molecule that can hybridize with the gene sequence shown in SEQ ID NO.1.

[0012] This invention also provides a chalcone isopentenyltransferase gene. GiPT16 The expressed GiPT16 protein includes one or more of the following sequences:

[0013] (1) A protein having the amino acid sequence shown in SEQ ID NO.2;

[0014] (2) A protein derived from (1) that has more than 80% sequence similarity to the amino acid sequence shown in SEQ ID NO.2 and that catalyzes the synthesis of chalcone compounds.

[0015] This invention also provides an application of the chalcone isopentenyltransferase GiPT16 protein as an isopentenyltransferase.

[0016] This invention also provides a chalcone isopentenyltransferase gene. GiPT16 Or the application of GiPT16 protein in catalyzing the conversion of the substrate isoglycyrrhizin to psoralen.

[0017] This invention also provides a chalcone isopentenyltransferase gene. GiPT16 Or the application of GiPT16 protein in catalyzing the conversion of the substrate glycyrrhizin chalcone to glycyrrhizin C.

[0018] This invention also provides a method for amplifying the chalcone isopentenyltransferase gene. GiPT16 The primer pair is characterized in that the sequences of the primer pair are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0019] This invention also provides a gene containing chalcone isopentenyltransferase. GiPT16 Expression cassettes, recombinant expression vectors, transgenic cell lines, or recombinant microorganisms.

[0020] The beneficial effects of this invention compared to the prior art are as follows:

[0021] (1) This invention is based on Glycyrrhiza uralensis (Glycyrrhiza inflata) Glycyrrhiza inflata Using whole-genome sequencing data and a reverse genetics strategy, the key aromatic isopentenyltransferase GiPT16, involved in the isopentenylation modification of chalcone active ingredients, was successfully identified and functionally characterized. This enzyme was confirmed to specifically catalyze the biosynthesis of isobavachalcone and licorice chalcone C using DMAPP as a donor. It is the first chalcone isopentenyltransferase characterized in *Glycyrrhiza inflata*, filling a gap in the study of key enzymes in the chalcone isopentenylation metabolic pathway of this species and providing important evidence for further elucidating the molecular mechanisms of quality formation in *Glycyrrhiza inflata* medicinal materials.

[0022] (2) As a key element of synthetic biology, GiPT16 can be applied to microbial cell factories or plant metabolic engineering systems to achieve sustainable and efficient heterologous synthesis of high-value chalcone compounds such as psoralen and glycyrrhizin C, thereby reducing dependence on wild licorice resources and promoting the green development and industrial application of traditional Chinese medicine resources. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The molecular structural formulas of isoliquiritin, psoralen, licorice chalcone, and glycyrrhizin C of this invention are shown below.

[0025] Figure 2 This is the phylogenetic tree of the PT gene in Example 1 of the present invention, with the red pentagram marking the position of GiPT16 in the phylogenetic analysis;

[0026] Figure 3 The images shown are agarose gel electrophoresis images of colony PCR after GiPT16 gene cloning and vector construction in Example 2 of the present invention. A is an agarose gel electrophoresis image of GiPT16 gene cloning, and B is an agarose gel electrophoresis image of colony PCR after pESC-His-GiPT16 vector was constructed and transformed into Trans1-T1 host.

[0027] Figure 4 This is an agarose gel electrophoresis image of the successful transformation of the pESC-His-GiPT16 recombinant plasmid into YPH499 Saccharomyces cerevisiae colonies for PCR in Example 3 of this invention.

[0028] Figure 5 This is an example of LC-Q-TOF-MS identification of the catalytic product of isoliquiritigenin by GiPT16 of Glycyrrhiza uralensis in Example 3 of the present invention;

[0029] Figure 6 This is an example of LC-Q-TOF-MS identification of the catalytic product of GiPT16 of Glycyrrhiza uralensis var. inflata, which is catalyzed by chalcone of Glycyrrhiza uralensis. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] GiPT16 Gene (SEQ ID NO.1):

[0036]

[0037] GiPT16 protein (SEQ ID NO.2):

[0038] MDSLVVGSFPKASSINSGGNLLRGENRTKSYYATSSYTPKASLRKRKTQKEYNFLRSQQTSLKHLYKGVEGGFTYQEYNRKYVVKTAPKTSFVSDPRSIEWK NILESVKTFLDAFYMFITPYSVIATVLSIISACLLAVEKLSDISSLFFTGVLQAIIPHLFMSIYVNGINQLGDIEIDKINKPYLPLASGKISFTTGAIIVAS SLILSLWLAWIVGSWPSIWALISIAMIWGAYSVNVPLLRWKRYPVLAAMVIVGSFSIVFPIGYFLHMQTFVFKRPAFFSRPLIFTTTFTSFFSLVIALFKDI PDIEGDQAFGVQSFAARLGQKRVFWICISLLEMAYGVALLMGLTSSCLWSKTVTVLGHTVLASVVLYRAKSVDLRSKASIVSFYMLIWKLLSVEYFLMPLVR. Example 1

[0039] (1) Licorice root with inflated fruit ( Glycyrrhiza inflata The genomes were collected from Yuli County, Bayingolin Mongol Autonomous Prefecture, Xinjiang Uygur Autonomous Region, and were sequenced using the Illumina and PacBio platforms.

[0040] The PT protein sequence of the plant was downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and Blast homology analysis was performed on it against the full-length transcriptomics database of *Glycyrrhiza inflata*. The Hidden Markov Model (HMM) file of the PT protein was downloaded from Pfam (http: / / pfam-legacy.xfam.org / ), and the HMMER (http: / / hmmer.janelia.org / static / binaries / hmmer3.0) software was used to search for protein sequences containing the PT.HMM (IPR000537) domain. The results of the Blast homology analysis and the HMMER domain search were combined, and sequences with a similarity of less than 30% and an e-value of less than 10 were excluded. -5The transcripts were sequences less than 300 amino acids in length. These transcripts were submitted to NCBI-CDD (https: / / www.ncbi.nlm.nih.gov / Structure / bwrpsb / bwrpsb.cgi) to verify the conserved domains of PT. The protein sequences obtained through the above screening steps were identified as members of the PT gene family of *Glycyrrhiza inflata*. GiPTs ).

[0041] Based on the genome data of *Glycyrrhiza inflata*, a total of 18 PTs sequences with the conserved UbiA membrane-bound protein domain were screened. Phylogenetic analysis of these 18 GiPTs with other PTs reported to have functions in other species revealed that GiPT16 clustered with isopentenyltransferases, which have been reported to catalyze flavonoid components (e.g.,...). Figure 2 (As shown) ， It is speculated that it may have isopentenyltransferase activity, and further functional characterization will be carried out. Example 2

[0042] Example 2 of this invention tested candidates in Glycyrrhiza uralensis var. inflata. GiPT16 The specific steps for gene cloning and expression vector construction are as follows:

[0043] (1) Primer design and reaction system:

[0044] Obtained by homologous recombination pESC-His-GiPT16 The expression vector was designed with primer sequences (see Table 1). Using cDNA from the root of *Glycyrrhiza inflata* as a template, KOD One was employed. TM PCR Master Mix (purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., No. KMM-201) cloning GiPT16 Gene fragment (KOD high-fidelity enzyme PCR system total volume: 50 μL : 25 μL KODOne) TM PCR Master Mix, 1.5 μL primers (10 mM), 1 μL template and 21 μL water, program as shown in Table 2).

[0045]

[0046] The target gene was constructed into the pESC-His vector using the ClonExpress II One Step Cloning Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., product catalog number C112), with the restriction enzyme sites being: Bam HI and SalI. According to the restriction endonuclease instructions (purchased from New England Biolabs, catalog number E8200S), the optimal molar ratio of cloning vector to insert fragment in this reaction system is 1:2. The optimal amount is calculated using the following formula:

[0047] Optimal cloning vector usage = [0.02 × number of cloning vector base pairs] ng (0.03 pmol);

[0048] Optimal amount of insert fragment used = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol);

[0049] The composition of this reaction system is shown in Table 3 after calculation:

[0050]

[0051] After all components have been added, gently aspirate to mix the reaction solution, then briefly centrifuge and incubate at 37°C for 30 minutes to obtain the final product. pESC-GiPT16 Recombination product.

[0052] (2) Transformation of recombinant products:

[0053] 1) Take Trans1 -T1 competent cells (purchased from Beijing TransGen Biotech Co., Ltd., product catalog number CD501-02) were thawed on ice. 50 μL of the thawed cells were then used. Trans1 -T1 competent cells were aliquoted into pre-chilled 1.5 mL centrifuge tubes.

[0054] 2) Add 5 μL of the recombinant product to the centrifuge tube, mix gently, and incubate on ice for 30 minutes.

[0055] 3) After heat shock in a 42℃ water bath for 30 seconds, quickly transfer the centrifuge tube to an ice bath for 2 minutes without shaking it.

[0056] 4) Take 50 μL of transformed competent cells, spread them evenly on ampicillin-resistant LB agar medium, and invert the plate to incubate at 37°C for 12-16 hours.

[0057] (3) Screening for positive clones:

[0058] 1) Pick 4 single clones grown on the recombinant plate and incubate them in 200 μL LB liquid medium (ampicillin resistant) at 37°C for 2 hours (200 rpm).

[0059] 2) Colony PCR identification was performed using Green Taq Mix DNA polymerase (purchased from Nanjing Novizan Biotechnology Co., Ltd., product catalog number P131-01). The amplification primer sequences designed using Primer 6.0 were: GAL1-F (SEQ ID NO.5): ATTTTCGGTTTGTATTACTTC; GAL1-R (SEQ ID NO.6): GTTCTTAATACTAACATAACT.

[0060] The standard PCR reaction system is shown in Table 4. The standard PCR reaction procedure is shown in Table 5. (3) Take 5 μL of PCR product for agarose gel electrophoresis detection. Refer to the position of the DNA Marker (purchased from Beijing TransGen Biotech Co., Ltd., product catalog number BM111-01), pick bacterial solutions with reasonable band positions and send them to Beijing Nuosai Genome Research Center Co., Ltd. for detection. Compare with RNAseq sequences to obtain the final sequence information.

[0061]

[0062] Cloning was performed using the primers in Table 1. GiPT16 The results were detected using agarose gel electrophoresis. Figure 3 As shown in the figure. Sequencing yielded its sequence, and BLAST alignment revealed a 67% similarity between the sequenced nucleotide sequence and the genome annotation sequence. The actual sequencing result was used as the definitive answer. The actual sequencing result, as shown in SEQ ID NO.1, contains 1227 nucleotides, encoding the protein shown in SEQ ID NO.2, which consists of 408 amino acids. This gene was named... GiPT16 The protein it encodes is named GiPT16.

[0063] Example 3

[0064] Embodiment 3 of the present invention for candidates GiPT16 The gene underwent functional verification, and the specific steps are as follows:

[0065] (1) Transformation of recombinant plasmids:

[0066] The engineered Saccharomyces cerevisiae YPH499 was selected as the heterologous expression host for PT. The specific steps for transforming the expression strain are as follows:

[0067] 1) Carrier DNA pretreatment: Insert the carrier DNA into a 95℃ metal bath for 5 min, then quickly insert it into ice after heating;

[0068] 2) Take 100 μL of YPH499 competent cells thawed on ice, add 2-5 μg of pre-cooled target plasmid, 10 μL of pretreated carrier DNA, and 500 μL of PEG / LiAc, mix with a pipette, and incubate in a 30℃ water bath for 30 min (invert 6-8 times at 15 min to mix).

[0069] 3) Place the test tube in a 42℃ water bath for 15 min (invert 6-8 times every 7.5 min to mix thoroughly);

[0070] 4) Centrifuge at 5000 rpm for 40 s, then discard the supernatant;

[0071] 5) Resuspend in 400 μL ddH2O, centrifuge at 5000 rpm for 30 s and discard the supernatant;

[0072] 6) Resuspend in 50 μL ddH2O, spread on SD-His solid medium, and incubate at 30℃ for 48-96 h;

[0073] 7) Positive clone verification: Select a single clone and incubate it in SC-His liquid medium at 200 rpm and 30℃ for about 24 h. Verify the positive clone by bacterial PCR.

[0074] (2) Induced expression:

[0075] 1) Transfer the yeast culture to 500 mL of YPD medium (containing 1% glucose and 2% galactose), incubate for 16 h, and then centrifuge at 4000 rpm for 6 min.

[0076] 2) The yeast cells collected by centrifugation were washed sequentially with 40 mL TES buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA and 0.6 mM D-sorbitol) and 30 mL TES-M buffer (TES buffer solution with 30 mM β-mercaptoethanol added), and resuspended with 25 mL extraction buffer (TES buffer solution with 1% bovine serum albumin and 1 mM PMSF added).

[0077] 3) Lyse yeast cells using an ultra-high pressure cell disruptor, and then perform the lysis at 5000 mg / L pressure at 4°C. × g and 10,000 × After centrifugation for 10 min, the supernatant was collected and then subjected to 120,000 rpm at 4°C. × (4) The particles containing microsomes were resuspended in 1 mL of TEG-M buffer (containing 20% ​​(v / v) glycerol) and stored at -80°C.

[0078] (4) In vitro enzyme activity detection:

[0079] The obtained microsomes were functionally validated according to the following reaction system: 200 μL microsomes, 0.5 mM MgCl2, 0.5 mM DMAPP and 2 μL substrate (1 mg / mL), incubated at 30℃ for 2 h. After terminating the reaction with an equal volume of methanol, the sample was filtered through a 0.22 μm microporous membrane and transferred to a liquid chromatography vial for detection and analysis. The sample was detected using an Agilent 1290 Infinity II-6430 Q-TOF. The specific liquid chromatography conditions were: (1) Column: Ultimate® XB-C18 3 μm 2.1×100 mm; (2) Mobile phase: Phase A was 0.1% formic acid water, and Phase B was chromatographic acetonitrile; (3) Flow rate: 0.3 mL / min; (4) Column temperature: 40℃; (5) Injection volume: 1.0 μL; (6) Elution conditions are shown in Table 6. The mass spectrometry conditions were: ion mode was negative ion, and the scanning range was m / z 100-3000; Mode: Automated MS / MS; Ion fragment collision energy: 20V; Source temperature: 350°C; Atomizer pressure: 30 psi. Results are as follows: Figures 4-6 As shown.

[0080]

[0081] Figure 4 show, pESC-His-GiPT16 The recombinant plasmids were successfully transformed into *Saccharomyces cerevisiae*, and further enzyme activity analysis was conducted to identify their functions. The donor for the enzyme activity reaction was DMAPP, and the acceptors were isoliquiritin (C...). 15 H 12 O4, purchased from Shanghai Yuanye Biotechnology Co., Ltd., product catalog number: S31477) or licorice chalcone (C 16 H 14 O4, purchased from Shanghai Yuanye Biotechnology Co., Ltd., product catalog number: B20213). Figure 5 , 6 Mass spectrometry was used to identify the enzyme activity products, and it was found that GiPT16 produced product peaks (corresponding to P1 and P2, respectively) for enzyme activity reactions with isoglycyrrhizin and glycyrrhizin chalcone as receptors.

[0082] Compared with the detection results of the microparticle catalytic system containing pESC-His empty support, when isoliquiritigenin was used as the substrate and DMAPP was used as the isopentenyl donor, product P1 exhibited the same retention time and mass spectrometric fragmentation pattern as psoralen ([MH)). - : m / z=323.1289); When glycyrrhizin chalcone is used as the substrate and DMAPP is the isopentenyl donor, product P2 has the same retention time and mass spectrometric fragmentation pattern as glycyrrhizin chalcone C ([MH)). - : m / z =337.1445).

[0083] In summary, based on mass spectrometry data, it was determined that the isopentenyltransferase GiPT16 has the functions of catalyzing the conversion of isoliquiritigenin to psoralen and catalyzing the conversion of glycyrrhizin chalcone to glycyrrhizin C.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A GiPT16 protein expressed by the chalcone isopentenyltransferase gene GiPT16, characterized in that, The amino acid sequence of the GiPT16 protein is shown in SEQ ID NO.

2.

2. A chalcone isopentenyltransferase gene, GiPT16, the nucleotide sequence of which is shown in SEQ ID NO.

1.

3. The use of the GiPT16 chalcone isopentenyltransferase protein of claim 1 as a chalcone isopentenyltransferase.

4. The use of the GiPT16 protein of claim 1 in catalyzing the conversion of the substrate isoglycyrrhizin to psoralen.

5. The use of the GiPT16 protein of claim 1 in catalyzing the production of glycyrrhizin C from the substrate glycyrrhizin chalcone.

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