Prenyltransferase and use thereof for catalyzing synthesis of lavandulyl flavones

Abstract

The present application provides a kind of prenyltransferase and the application of catalyzing synthesis lavandulyl flavone.The specific relates to a new prenyltransferase, including the nucleic acid sequence of the enzyme, expression vector, the recombinant cell of the enzyme production and the preparation method of recombination enzyme, also relates to the application of the prenyltransferase preparation lavandulyl flavone.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and pharmaceutical and chemical manufacturing, specifically to a recombinant isopentenyltransferase derived from Sophora flavescens and its application in the synthesis of lavender-based flavonoids. Background Technology

[0002] Lavandulyl flavonoids are a class of flavonoids characterized by isopentenyl (lavandyl) substitution. Common structural types include lavandyl dihydroflavonoids, dihydroflavonols, chalcones, and their derivatives. Their primary natural source is the rhizome of *Sophora flavescens* Ait., a plant in the Fabaceae family. Modern research has revealed that lavandyl flavonoids derived from *Sophora flavescens* possess a variety of significant biological activities. For example, sophoraflavanone G (SFG) exhibits antibacterial, anti-inflammatory, antitumor, antidiabetic, and antimalarial activities, making it a highly promising candidate molecule in the field of natural drug development.

[0003] Currently, the development and utilization of lavender-based flavonoids such as SFG face two challenges: firstly, the low content of target components in natural plants and the large number of structural analogs lead to low separation and purification efficiency; secondly, the complex isopentenyl substitution structure results in lengthy chemical synthesis routes, low yields, and significant environmental pollution. Against this backdrop, biosynthetic technology, due to its advantages of being green, environmentally friendly, and highly controllable, has become an important strategy for overcoming the bottleneck of large-scale production of lavender-based flavonoids. By developing novel enzyme catalytic systems or reconstructing biosynthetic pathways, the targeted biomanufacturing of target molecules can be achieved. Early biochemical studies have shown that the direct biosynthetic precursor of SFG is leachianone G (LG, 5,7,2′,4′-tetrahydroxy-8-isopentenyl dihydroflavonoid), which may be further introduced into the LG isopentenyl side chain by isopentenyl transferase catalysis, thereby completing the construction of the lavender-based structure. However, the key isopentenyl transferase that catalyzes the formation of lavenderyl in the SFG biosynthetic pathway has not been identified for a long time. The lack of this key isopentenyl transferase element has severely restricted the development and application of lavenderyl flavonoids prepared by biosynthesis. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide a novel isopentenyltransferase comprising its nucleic acid molecule, an expression vector of the nucleic acid molecule, and a recombinant cell comprising the vector, and the use of the isopentenyltransferase nucleic acid molecule, recombinant enzyme, or recombinant cell in the preparation of lavender-based flavonoids, their analogues, or derivatives.

[0005] In a first aspect, the present invention provides an isopentenyltransferase having the following amino acid sequence:

[0006] (1) The amino acid sequence shown in SEQ ID NO.1.

[0007] (2) The amino acid sequence obtained by similar variation of the amino acid sequence shown in SEQ ID NO.1, that is, the amino acid sequence with more than 80% consistency and equivalent function formed by replacing, deleting or adding one or more amino acids.

[0008] As a preferred embodiment, the present invention also provides a truncated form of the isopentenyltransferase. The truncated form is the isopentenyltransferase with the signal peptide sequence removed, retaining only amino acids 15 to 407 of the isopentenyltransferase.

[0009] The recombinant isopentenyltransferase described above can be enzymatically modified or fused with peptides for detection, purification, or alteration of enzymatic properties.

[0010] The enzymatic modifications performed on isopentenyltransferases as described above include, but are not limited to, acetylation, amidation, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, and immobilization modification; the fusion peptides include, but are not limited to, eGFP, n*His, GST, Strep, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the isopentenyltransferase described in the first aspect of the present invention, preferably the nucleic acid sequence shown in SEQ ID NO.2.

[0012] Thirdly, the present invention provides a recombinant expression vector containing the nucleic acid molecule described in the second aspect of the present invention.

[0013] As a preferred embodiment, the vector may be an expression vector capable of expressing the target protein in bacterial, fungal, plant, or animal cells. More preferably, the vector is an expression vector capable of expressing the target protein in Escherichia coli, actinomycetes, Pichia pastoris, Saccharomyces cerevisiae, Arabidopsis thaliana, tobacco, tomato, or insect cells.

[0014] Fourthly, the present invention provides a recombinant cell carrying the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect. The recombinant cell may be a bacterium, fungus, plant cell, or animal cell.

[0015] Fifthly, the present invention provides the application of synthesizing lavender-based flavonoids using the nucleic acid molecule, the carrier, or the recombinant cell.

[0016] As a specific approach, the isopentenyl transferase can be obtained by using the nucleic acid molecule, the vector, or the recombinant cell through heterologous expression and other common techniques in the field of this invention.

[0017] Sixthly, the present invention provides the application of the isopentenyltransferase described in the first aspect. Specifically, it refers to obtaining the isopentenyltransferase through recombinant cell expression, and using the isopentenyltransferase, through in vitro or in vivo catalysis, including co-expression of the isopentenyltransferase with other enzymes having catalytic or regulatory functions or by constructing a cell factory, to catalyze the introduction of a new isopentenyl group onto the isopentenyl side chain of a compound having the core structure of Formula I, thereby forming lavender-based flavonoids with the core structure of Formula II; or for the preparation of formulations in which isopentenyl groups are introduced onto the isopentenyl side chain of a compound having the core structure of Formula I, thereby forming lavender-based flavonoids with the core structure of Formula II. In the above core structure, the dashed lines indicate the presence or absence of chemical bonds, which is a method of representation commonly used by those skilled in the art.

[0018]

[0019] The application of the isopentenyltransferase includes expressing the isopentenyltransferase alone or in combination with other enzymes having catalytic or regulatory functions, or constructing a cell factory. The expression host can be bacteria, fungi, plant cells, or animal cells.

[0020] The other enzymes with catalytic or regulatory functions may be selected from, but are not limited to, oxidases, reductases, kinases, glycosyltransferases, methyltransferases, acyltransferases, isopentenyltransferases, etc. These other enzymes with catalytic or regulatory functions may be single enzymes or combinations of multiple enzymes.

[0021] The isopentenyl donors described above include exogenously added or endogenously derived isopentenyl-containing compounds and their chemical variations. Preferably, the isopentenyl donor is selected from one or more of the following: dimethylallyl pyrophosphate (DMAPP), isopentenyl pyrophosphate (IPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP).

[0022] Supplementary Explanation

[0023] Unless otherwise stated, the technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. This invention may also be practiced using any methods and materials similar to or equivalent to those described herein. While specific embodiments and preferred methods and materials are described herein, they do not impose any limitation on the invention.

[0024] Beneficial technical effects:

[0025] This invention provides an isopentenyltransferase that can catalyze the synthesis of lavender-based flavonoids, providing a necessary functional enzyme element for the biosynthesis of structurally complex and pharmacologically diverse lavender-based flavonoids. Attached Figure Description

[0026] Figure 1 Schematic diagram of the transport peptide cleavage site and conserved motif of the SfLFPT amino acid sequence.

[0027] Figure 2 Transmembrane structure analysis of SfLFPT amino acid sequence

[0028] Figure 3 Image of yeast recombinant plasmid expressing the SfLFPT gene

[0029] Figure 4 HPLC-HRMS analysis of the recombinant isopentenyltransferase reaction. (A) Comparison of HPLC chromatograms of the substrate standard, product standard, and enzyme reaction; 1 represents substrate LG, and 1a represents product SFG; (B) High-resolution mass spectrum of product SFG. Detailed Implementation

[0030] The following embodiments are provided to further illustrate various aspects of the present invention. These embodiments are non-limiting and should not be construed as limiting any aspect of the invention. The scope of protection of the present invention is limited only by the claims. Various modifications and improvements can be made to various aspects of the present invention without departing from the scope of the claims, and these modifications and improvements also fall within the scope of protection of the present invention. For example, the expression vector and host bacteria used in the embodiments can be replaced with other commonly used expression vectors and host bacteria in the art; furthermore, partial substitution, deletion, or addition of the amino acid sequence of the isopentenyltransferase described in the present invention without changing the enzyme function is something that those skilled in the art can understand and implement.

[0031] In addition, it should be noted that, unless otherwise specified, the various materials and reagents used in the following embodiments are commonly used in the art and can be obtained through conventional commercial channels; the methods used are all conventional methods adopted by those skilled in the art.

[0032] Example 1: Cloning of the SfLFPT gene

[0033] 1. Extraction of total RNA and synthesis of cDNA first strand

[0034] Sophora flavescens root was selected as the biological material, and the plant RNA extraction kit EZNA was used. TMTotal RNA was extracted using the Plant RNA Kit (OMEGA). The sample was ground in liquid nitrogen and transferred to a centrifuge tube. 500 μL of Buffer RCL was added per 100 mg of tissue, and the mixture was vortexed. The mixture was incubated at 55°C for 3 min and centrifuged at 15,000 g for 5 min. The supernatant was transferred to a filter column and centrifuged at 14,000 g for 2 min. The filtrate was transferred to a new centrifuge tube, and an equal volume of Buffer RCB was added. The tube was inverted 5–10 times. The liquid was transferred to an RNA adsorption column and centrifuged at 12,000 g for 1 min. The filtrate was discarded, and 400 μL of RNA Wash Buffer was added to the adsorption column. The column was centrifuged at 10,000 g for 1 min. The filtrate was discarded, and 500 μL of RNA Wash was added to the adsorption column. The column was centrifuged at 10,000 g for 1 min, and this step was repeated once. The adsorption column was air-dried at room temperature, and the total RNA was obtained by elution with 50 μL of DEPC water. The integrity of total RNA was detected by agarose gel electrophoresis, and the OD was measured by ultraviolet spectrophotometer. 260 / OD 280 Ratio and RNA concentration. Using SMARTScribe TM Reverse Transcriptase (Clontech) synthesizes the first strand of cDNA.

[0035] 2. PCR amplification of the target gene fragment

[0036] By comparing and analyzing the transcriptome data of Sophora flavescens roots and leaves, a novel isopentenyltransferase gene specifically expressed in the roots of Sophora flavescens was discovered and named SfLFPT. Specific primers (SEQ ID NO.3 and SEQ ID NO.4) were designed based on the gene sequence of SfLFPT, and KOD One was used. TM PCR amplification was performed using PCR Master Mix to obtain the CDS sequence of the SfLFPT gene. The PCR product (target gene fragment) was recovered from the gel, ligated into the TOPO vector (LabpO), transformed into DH5α competent cells, and screened by colony PCR. Positive clones were selected for sequencing verification.

[0037] 2.1 PCR reaction system

[0038]

[0039] 2.2 PCR Cycle

[0040] 1) 94℃ 2min; 2) 98℃ 10sec; 3) 56℃ 5sec; 4) 68℃ 10sec; 4) go to 2) for39cycles; 5) 68℃ 1min.

[0041] Example 2: Truncated expression of SfLFPT, recombinase isolation, purification and activity analysis

[0042] 1. Construction of recombinant expression strains

[0043] The open reading frame of the SfLFPT gene contains 1224 nucleotides, encoding 407 amino acids. The encoded protein contains two characteristic conserved motifs of plant isopentenyltransferases (NQxxDxxxD and KDxxDxxGD), and has a 14-amino acid signal peptide sequence at the N-terminus. Figure 1 The SfLFPT-encoded protein not only possesses a signal peptide sequence that can potentially guide to the plasmid, but is also a strongly membrane-bound protein. Figure 2 To enhance the expression of the SfLFPT gene in yeast, the sequence encoding the N-terminal signal peptide in the SfLFPT gene was truncated using PCR before being ligated into the pESC-His expression vector. First, truncated expression primers containing homologous arms (SEQ ID NO. 5 and SEQ ID NO. 6) were designed based on the pESC-His vector sequence. Homologous arm sequences were introduced at both ends of the gene using PCR. The PCR products were recovered using a gel, and the target gene fragment was ligated to the pESC-His vector, which had been linearized by double digestion with restriction endonucleases EcoRI and SpeI, using homologous recombinase to construct the truncated expression plasmid pESC-His-SfLFPT. 15AA ( Figure 3 The expression plasmid was truncated and transformed into the DH5α cloning host. Positive transformants were screened by PCR, and the correct reading frame was verified by sequencing. The correctly sequenced transformants were amplified and cultured, plasmids were extracted, and transformed into Saccharomyces cerevisiae YPH499 competent cells. Transformants were picked and cultured in liquid culture, and yeast plasmids were extracted for PCR verification and sequencing identification.

[0044] 2. Recombinase-induced expression and microsomal preparation

[0045] Like most plant isopentenyltransferases, SfLFPT is a strongly membrane-bound protein with multiple transmembrane structures. The recombinant enzyme is attached to the yeast inner membrane system, and it is necessary to obtain the recombinant enzyme by preparing yeast microsomes for in vitro activity analysis. The steps for recombinase induction expression, microsomal enzyme solution preparation and detection are as follows: (1) Transformants verified by sequence sequencing are inoculated into 15 mL SD-H medium and cultured at 30℃ and 200 rpm for 20-24 h with shaking; (2) Seed culture is inoculated into 100 mL SD-H medium and cultured at 30℃ and 200 rpm for 20-24 h with shaking; (3) The cells are collected by centrifugation at 3,000 g for 2 min and transferred to 1000 mL SG-H medium (OD600 reaches 0.8-1.0) to induce protein expression and cultured at 30℃ and 240 rpm for 24-36 h with shaking; (4) The cells are collected by centrifugation at 3,000 g for 5 min, the supernatant is discarded, and the cells are washed twice with protein extraction buffer (100 mM Tris-HCl, pH 9.0) and centrifuged to discard the supernatant; (5) 3-5 times the volume of 1 mM protein extract is added to each gram of cells. Add the protein extraction buffer of PMSF to the pre-cooled glass beads of equal volume to the bacterial cells, vortex vigorously for 30 seconds, and let stand on ice for 30 seconds. Repeat 12 times. (6) Centrifuge at 10,000g for 40 minutes to remove cell debris. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 160,000g for 90 minutes. Discard the supernatant. (7) Resuspend the precipitate in 500 μL of reaction buffer (100 mM Bis-Tris, pH 7.0) to obtain the recombinant yeast microsomal enzyme solution.

[0046] 3. The reaction catalyzed by recombinase to produce lavender flavonoids

[0047] Using DMAPP as the isopentenyl donor, through recombination of SfLFPT 15AA Yeast microsomes catalyze the isopentenylation of LG. The reaction system is as follows: 0.4 mM LG, 0.8 mM DMAPP, 10 mM MgCl2, and microsomal enzyme solution to a final volume of 50 μL. The reaction was carried out overnight in a 40°C water bath, and then an equal volume of methanol was added to terminate the reaction. The mixture was thoroughly vortexed, incubated at -20°C for 10 min, and centrifuged. The supernatant was then analyzed by HPLC-HRMS. Comparison with standards using HPLC and HRMS confirmed that the catalytic product was lavenderyl flavonoid SFG. Figure 4 ).

Claims

1. An isopentenyltransferase, characterized in that, The amino acid sequence of the isopentenyltransferase is as follows: (1) The amino acid sequence shown in SEQ ID NO.1; (2) The amino acid sequence obtained by similar variation of the amino acid sequence shown in SEQ ID NO.1, that is, the amino acid sequence with more than 80% consistency and equivalent function formed by replacing, deleting or adding one or more amino acids.

2. The isopentenyltransferase according to claim 1, characterized in that, The isopentenyltransferase can be enzymatically modified; or a fusion peptide for detection, purification or alteration of enzymatic properties can be fused to the isopentenyltransferase.

3. The isopentenyltransferase according to claim 2, characterized in that, The enzymatic modifications include, but are not limited to, acetylation, amidation, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, and immobilization modification; the fusion peptides include, but are not limited to, eGFP, n*His, GST, Strep, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

4. A nucleic acid molecule, characterized in that, It encodes the isopentenyltransferase according to any one of claims 1 to 3, and preferably has the nucleotide sequence shown in SEQ ID NO.

2.

5. An expression carrier, characterized in that, The vector sequence comprises the nucleotide sequence of claim 4, or is capable of expressing the isopentenyltransferase of any one of claims 1 to 3.

6. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A method for preparing an isopentenyltransferase, characterized in that, The isopentenyl transferase is prepared using the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant cell of claim 6.

8. The application of the isopentenyltransferase according to claim 1, characterized in that, A new isopentenyl group is introduced onto the isopentenyl side chain of a compound having the core structure of Formula I using the isopentenyl transferase of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant cell catalysis of claim 6, thereby forming lavender-based flavonoids with the core structure of Formula II; or a formulation for preparing a compound having the core structure of Formula I by introducing isopentenyl groups onto the isopentenyl side chain of the compound, thereby forming lavender-based flavonoids with the core structure of Formula II.

9. The application according to claim 8, characterized in that, The isopentenyltransferase is obtained by recombinant cell expression. Using the isopentenyltransferase, in vitro or in vivo catalysis is employed, including co-expression of the isopentenyltransferase with other enzymes with catalytic or regulatory functions or construction of cell factories.

10. The application according to any one of claims 8 and 9, characterized in that, The target compound can be prepared by co-expressing the isopentenyltransferase with other enzymes having catalytic or regulatory functions; preferably, the other enzymes having catalytic or regulatory functions are selected from one or more of the following: oxidases, reductases, kinases, glycosyltransferases, methyltransferases, acyltransferases, and isopentenyltransferases.

11. The application according to any one of claims 8 and 9, characterized in that, The isopentenyl group is used as the isopentenyl group donor, including exogenously added or endogenous isopentenyl group-containing compounds and their chemical variations, preferably: dimethylallyl pyrophosphate DMAPP and isopentenyl pyrophosphate IPP.

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