Screening identification and application of corydaline 9-O-methyltransferase in amur corktree
By screening and validating the PaOMT1 gene in Phellodendron amurense, the problem of unclear biosynthetic pathway of berberine in Phellodendron amurense was solved, and the efficient synthesis of berberine in Phellodendron amurense was realized, providing a molecular basis for its large-scale production.
Patent Information
- Application Number
- CN202410875824.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-01
- Publication Date
- 2025-12-05
AI Technical Summary
The biosynthetic pathway of berberine in Phellodendron bark is unclear, and the S9OMT enzyme is the rate-limiting enzyme, making it difficult to achieve efficient and large-scale production of berberine with existing technologies.
By analyzing genomic, transcriptomic, and metabolomic data, the PaOMT1 gene in Phellodendron amurense involved in the (S)-corydaline 9-O-methyltransferase was screened out, and its function was verified in a prokaryotic expression system, confirming its ability to catalyze the formation of (S)-tetrahydrotetrahydroafricanine from (S)-corydaline.
This study provides the molecular basis for the biosynthetic pathway of berberine in Phellodendron amurense, verifies the function of PaOMT1, promotes the efficient synthesis of BIAs such as berberine in Phellodendron amurense, and lays the foundation for large-scale production.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to screening, identification and application of a key enzyme PaOMT1 catalyzing (S)-tetrahydrocoptisine to be produced from (S)-coptisine C9 hydroxymethylation in a biosynthesis pathway of main active components berberine and other BIAs in Phellodendron amurense Rupr. BACKGROUND
[0002] Phellodendron amurense Rupr. is a deciduous tree of Rutales Rutaceae, and its dried bark is a traditional Chinese medicine with the name of Guanhuangbai. It has anti-inflammatory, antibacterial, anticancer, hypoglycemic, uric acid-lowering, and liver and kidney protection, and other pharmacological effects. It mainly contains protoberberines (such as berberine, palm leaf jatamansi alkaloids) and aporphine alkaloids (such as magnoflorine). Berberine is a protoberberine type of benzylisoquinoline alkaloids (BIAs), which has a wide therapeutic potential for various diseases such as diabetes, hypertension, depression, obesity, inflammation and cancer, and is considered as a promising candidate for the treatment of cancer, diabetes, Alzheimer's disease and other diseases.
[0003] The biosynthesis pathway of berberine has been studied in Ranunculales plants, and the pathway has been completely elucidated in Coptis chinensis. The upstream pathway is conserved, and like all BIAs, the dopamine and 4-hydroxyphenylacetaldehyde are condensed by norlaudanosine synthase to form the first BIAs, norlaudanosine. Then, under the action of two O-methyltransferases (6OMT and 4'OMT), one N-methyltransferase (CNMT), one CYP450 (CYP80B) and one FAD-dependent enzyme (BBE), the key intermediate coptisine is produced. Coptisine produces tetrahydrocoptisine under the action of S9OMT (coptisine 9-O-methyltransferase), and then produces tetrahydroberberine under the catalysis of CYP719A, and finally produces berberine under the action of BBE-like enzyme. At present, berberine has been produced from scratch in Saccharomyces cerevisiae, but the yield is low, and further research is needed to realize large-scale production.
[0004] S9OMT is a key enzyme in the biosynthesis of berberine and is the main rate-limiting enzyme in the biosynthesis of berberine compounds. The biosynthesis pathway of berberine in Phellodendron amurense Rupr. is not clear, and the purpose of the present study is to characterize the enzyme S9OMT involved in catalyzing coptisine to produce tetrahydrocoptisine in Phellodendron amurense Rupr., and to lay a foundation for the biosynthesis pathway analysis and large-scale application of main active components berberine and other BIAs in Phellodendron amurense Rupr. SUMMARY
[0005] The present application aims to provide a gene of coptisine 9-O-methyltransferase involved in the synthesis of (S)-tetrahydrocoptisine in Phellodendron amurense Rupr. and a protein encoded by the gene.
[0006] Another object of the present application is to provide a screening method of a key enzyme gene for BIAs biosynthesis in P. bungeana.
[0007] A third object of the present application is to provide a functional verification method of the key enzyme gene S9OMT screened above.
[0008] The PaOMT1 gene provided by the present application has a nucleotide sequence as shown in SEQ ID No. 1, or a mutant sequence thereof.
[0009] The protein encoded by the PaOMT1 gene provided by the present application has an amino acid sequence as shown in SEQ ID No. 2, or a mutant sequence thereof.
[0010] The object of the present application can be achieved by the following technical solutions,
[0011] Technical solution one: screening of a key enzyme gene for BIAs biosynthesis in P. bungeana based on genomic, transcriptomic and metabolomic data. Co-expression analysis is performed on the metabolomic data of the main component berberine in each tissue of P. bungeana and the transcriptomic data of the gene, and genes with a Pearson correlation coefficient greater than 0.8 and an expression greater than 50 in roots and bark are screened. Annotation is performed by CDD breach, and (S)-chondodendrine 9-O-methyltransferase is further screened, which is a key enzyme gene involved in the catalysis of (S)-chondodendrine to form (S)-tetrahydrocoptisine in the BIAs biosynthesis pathway of berberine and other BIAs.
[0012] Technical solution two: functional verification of the key enzyme gene PaOMT1. The function of chondodendrine 9-O-methyltransferase PaOMT1 is identified in vitro by using a prokaryotic expression system and (S)-chondodendrine as a substrate.
[0013] The present application discloses a screening and functional identification method of a key enzyme gene OMTs for catalyzing (S)-chondodendrine to form (S)-tetrahydrocoptisine in P. bungeana, which verifies that PaOMT1 has the function of catalyzing (S)-chondodendrine to form (S)-tetrahydrocoptisine, provides a molecular basis for elucidating the BIAs biosynthesis pathway of berberine and other BIAs in P. bungeana, and has great application value. BRIEF DESCRIPTION OF DRAWINGS
[0014] Figure 1 : Figure of Pearson correlation coefficient (r>0.80) of the transcriptomic data of each tissue of P. bungeana and the content of berberine.
[0015] Figure 2 : In vitro functional verification of PaOMT1 of P. bungeana. PaOMT1 catalyzes (S)-chondodendrine to form (S)-tetrahydrocoptisine, and CcS9OMT is a positive control. Specific embodiments
[0016] The present application is described in detail below with reference to Examples. The embodiments are presented to provide a better understanding of the present application, and are not intended to limit the present application. The experimental methods in the following embodiments are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.
[0017] Example 1 Screening of BIA biosynthesis key enzyme genes in P. amarus based on genomic, transcriptomic and metabolomic data
[0018] Based on the genomic annotation information of P. amarus, the transcriptome and metabolome data of 18 tissues of P. amarus prepared by the research group in the early stage, the Pearson correlation coefficient (r) was calculated and sorted. Genes with r greater than 0.8 were screened, and the gene family was annotated using the online website CD-Search (https: / / www.ncbi.nlm.nih.gov / Structure / bwrpsb / bwrpsb.cgi).
[0019] 1.2 Results and analysis
[0020] Based on the Pearson correlation analysis of transcriptome and metabolome, it was shown that 237 genes had a correlation coefficient greater than 0.8. Annotation of these 237 genes showed that only 2 OMT genes were found, and they were significantly highly expressed in the tissues rich in berberine (roots) and bark (trunk). These two genes were listed as candidate genes, PaOMT1 (Pamu08G000115) and PaOMT2 (Pamu06G000283). Figure 1 ).
[0021] Example 2 Functional verification of candidate OMTs encoding genes
[0022] 2.1 Experimental method
[0023] The OMT encoding gene was cloned into the pET28a vector to form a pET28a-OMTs recombinant expression vector, and the vector was transformed into a BL21 (DE3) Escherichia coli expression strain. 2 mL of overnight induced bacterial solution was added to 100 mL of LB liquid medium (containing 50 μg / mL kanamycin-resistant medium), activated at 37°C, 200 rpm for 3 h, and then OD 600To 0.6-0.8, add IPTG to a final concentration of 0.3 mM for induction, 16 °C, 180 rpm. After induction for 12-16 h, centrifuge at 5000 rpm, and resuspend the bacterial cells with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole). After ultrasonic disruption, centrifuge at 12000 rpm at 4 °C for 15 min. The supernatant is purified by Ni-NTA: gradient imidazole concentration (20, 60 and 80 mM) buffer to wash away impurities, and the target protein is eluted with high imidazole concentration (250 mM). The target protein eluate is desalted and concentrated by ultrafiltration, and the protein concentration is determined by BCA protein concentration determination kit.
[0024] The obtained pure enzyme is functionally verified in vitro according to the following reaction system (100 μL): Gly-NaOH buffer (pH 9.0), 20 μg of purified protein, 1 mM of substrate (S)-Corydine and 2 mM of methyl donor SAM. After termination of the reaction with 1 / 2 volume of methanol, mix well, centrifuge at 12000 rpm for 20 min, and detect the reaction product by UPLC-MS / MS. Instrument model, SCIEX TripleTOF 6600+. Injection volume 1 μL, chromatographic column: Kinetex C18 100A analytical column (4.6 mm x 150 mm, 2.6 μm), column temperature: 35 °C. Mobile phase: (A): 0.1% formic acid water, (B): acetonitrile, flow rate: 0.5 mL / min, elution program: 0-1 min, 10% B; 1-10 min, 10%-95% B; 12.3-13 min, 95% B; 13-15 min, 95%-10% B.
[0025] 2.2 OMT functional verification results and analysis
[0026] The liquid phase detection results show that PaOMT1 can catalyze (S)-Corydine to form (S)-Tetrahydrocolumbamine, and further catalyze (S)-Tetrahydrocolumbamine to form (S)-Tetrahydropalmatine. Figure 2
[0027] Other candidate PaOMT2s show no catalytic activity, and the above description is based on liquid chromatography-mass spectrometry detection, that is, compared with the standard product, it has the same mass spectrometry behavior (such as: retention time, secondary fragment ion, etc.).
Claims
1. An OMT1 gene in P. fulvotum, characterized in that It is the following gene (a) or gene (b): Gene (a1): a gene having the nucleotide sequence shown in SEQ ID No.
1. Gene (b1): a gene having the nucleotide sequence shown in SEQ ID No. 1 obtained by replacing, deleting or adding one or more bases, which has the action of methylating (S)-chondodendrine C9 hydroxyl to generate (S)-tetrahydrocolumbamine, and further methylating C2 to generate (S)-tetrahydropalmatine.
2. A protein encoded by OMTl gene in P. fulvotum, characterized in that It is the following protein (a) or protein (b): Protein (a1): a protein having the amino acid sequence shown in SEQ ID No.
2. Protein (b1): a protein having the amino acid sequence shown in SEQ ID No. 2 obtained by replacing, deleting or adding one or more amino acids, which has the action of methylating (S)-chondodendrine C9 hydroxyl to generate (S)-tetrahydrocolumbamine, and further methylating C2 to generate (S)-tetrahydropalmatine.
3. A method for screening OMT genes in the biosynthesis pathway of BIAs in B. fortunei, which methylate (S)-chondodendrine C9 hydroxyl to generate (S)-tetrahydrocolumbamine, using genomic, transcriptomic and metabolomic data, comprising the following steps: 1) Based on the transcriptomic data and metabolomic data of different tissue parts of B. fortunei, Pearson correlation analysis is used to screen genes with a Pearson correlation coefficient greater than 0.
8. 2) The genes screened in the previous step are further classified and annotated by gene family using CDD breach, and O-methyltransferase genes with expression greater than 50 in roots and bark are further screened as candidate genes.
4. In vitro enzyme activity identification of OMT in B. fortunei, comprising the following steps: 1) The candidate OMT genes are respectively cloned into pET28a expression vector and transformed into E. coli BL21(DE3) expression strain, and kanamycin is used for screening. Protein expression is induced by 0.3mM IPTG at 16℃, 200rpm in the dark for 16h. The protein is purified by Ni-NDA, the protein expression is detected by SDS-PAGE, and the protein concentration is determined by BCA protein concentration determination kit. 2) The purified protein is used for functional verification in vitro according to the following reaction system (100μL): Gly-NaOH buffer (pH9.0), 20μg purified protein, 2μL substrate (S)-chondodendrine (1mg / mL) and 2mM methyl donor SAM. Shake overnight at 37℃, 200rpm, terminate the reaction with 1 / 2 volume of methanol, mix well, centrifuge at 12000rpm for 20min, and detect the reaction product by UPLC-MS / MS. 3) UPLC-MS / MS instrument model, SCIEX TripleTOF 6600+. Injection volume 1 μL, column: Kinetex C18 100A analytical column (4.6 mm x 150 mm, 2.6 μm), column temperature: 35 °C. Mobile phase: (A): 0.1% formic acid water, (B): acetonitrile, flow rate: 0.5 mL / min, elution program: 0-1 min, 10% B; 1-10 min, 10%-95% B; 12.3-13 min, 95% B; 13-15 min, 95%-10% B.
5. The use of the gene encoding the key enzyme involved in the biosynthesis of methylated Coptisine in the BIAs biosynthetic pathway in Coptis teeta or its mutant gene in plant genetic engineering according to claim 1, characterized in that Application of the PaOMT1 in P. henryi to synthesize BIAs in bacteria, fungi and higher plants by genetic engineering.
6. The use of claim 6, wherein the introduction of the foreign gene into the host cell requires a plasmid carrying the PaOMTl gene of P. armandii, characterized in that: The plasmid can be selected from pET series, pGEX series, pCold series and other prokaryotic expression vectors, pPIC9, pHIL-D2, pPIC3.5 and other yeast expression vectors, and PBI series, pCAMBIA series and other plant expression vectors, and contains the nucleotide sequence or mutant sequence of claim 1.