Application of camptothecin O-methyltransferase CaOMT3 in O-methylation modification of hydroxyl camptothecin and quercetin

Abstract

This invention provides an application of the camptothecin oxygen methyltransferase CaOMT3 in catalyzing the oxygen methylation modification of hydroxycamptothecin and quercetin. Through transcriptomic and genomic analysis, four oxygen methyltransferases (CaOMT3) were identified. BECAUSE OF Genes were found to be clustered together; further analysis of metabolite and transcriptome co-expression revealed a gene significantly positively correlated with rhamnine accumulation. BECAUSE OF Genes, that is CaOMT3 Biochemical functional verification showed that CaOMT1, CaOMT2, and CaOMT3 can all catalyze the reaction of 9- and 10-hydroxycamptothecin. O Methylation yields the corresponding methoxycamptothecin. When quercetin is used as a substrate, CaOMT1 and CaOMT3 exhibit bifunctional catalytic activity, capable of simultaneously catalyzing the methylation of the 4'- and 7'-hydroxyl sites. O α-methylation yields rhamnosin, tamarindin, and phytoflavin; while CaOMT2 only catalyzes the 4'-hydroxyl site. Enzymatic kinetic analysis showed that CaOMT3's catalytic efficiency for quercetin ( k cat / K (m) is the highest. This invention provides a new enzyme tool and technological foundation for the synthetic biology research and industrial fermentation production of rhamnine and methoxycamptothecin.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, specifically relating to the application of a camptothecin oxygen methyltransferase CaOMT3 in the oxygen methylation modification of hydroxycamptothecin and quercetin. Background Technology

[0002] Camptotheca acuminata (Camptotheca acuminata) Camptotheca acuminata Camptotheca acuminata is a deciduous tree endemic to China. Due to its upright shape, lush foliage, unique fruit, and Chinese name implying "joy and auspiciousness," it is often considered an excellent landscape tree. Modern research shows that Camptotheca acuminata is rich in camptothecin, a monoterpenoid indole alkaloid with significant antitumor activity, thus attracting widespread attention. Currently, camptothecin derivatives irinotecan and topotecan are widely used in the global market for the treatment of malignant tumors. Camptothecin compounds can exert their anticancer effects by specifically binding to topoisomerase I and forming a stable ternary complex with the DNA double helix, causing irreversible DNA breakage, thereby inhibiting DNA replication and RNA synthesis, and ultimately inducing apoptosis. However, camptothecin has shown strong toxic side effects in clinical trials, such as hemorrhagic cystitis and spinal cord toxicity, limiting its direct application. In contrast, its derivative 9-methoxycamptothecin maintains significant anticancer activity while having lower toxic side effects, showing greater potential for clinical application.

[0003] Besides containing the antitumor active ingredient camptothecin, Camptotheca acuminata also contains abundant flavonoids. Currently, more than 60 flavonoid compounds have been identified from Camptotheca acuminata. Among them, quercetin is considered to have multiple effects, including potent antioxidant, anti-inflammatory, and antitumor activity, thus becoming a focus of modern medical research and health care. Rhamnetin is a methylated flavonol. Its methoxy substitution at the 7-position of ring A is a key structural feature distinguishing it from its parent compound, quercetin. This modification enhances rhamnetin's lipophilicity and metabolic stability, making it less susceptible to rapid degradation by phase II metabolic enzymes such as glucuronidation and sulfation, thus exhibiting superior pharmacokinetic properties. Rhamnetin's unique mechanism of action, including dual regulation of oxidative stress, inhibition of key signaling pathways (such as Chk2 and Notch-1), and reversal of EMT, makes it not only a promising monotherapy but also a potential adjuvant drug to enhance the effects of conventional chemotherapy and radiotherapy.

[0004] This invention lays the foundation for the synthetic biology research and industrial fermentation production of rhamnine and methoxycamptothecin. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an oxymethyltransferase derived from Camptotheca acuminata, and provides its role in catalyzing the reaction of hydroxycamptothecin and quercetin. O Applications in methylation modification.

[0006] In a first aspect, the present invention provides a camptothecin O-methyltransferase CaOMT3, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] Secondly, the present invention provides the aforementioned camptothecin O-methyltransferase. CaOMT3 The encoding gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0008] Thirdly, the present invention provides a recombinant vector containing the coding gene.

[0009] Fourthly, the present invention provides a genetically engineered bacterium containing the recombinant vector.

[0010] Fifthly, the present invention provides an application of the aforementioned camptothecin oxygen methyltransferase CaOMT3 in catalyzing the oxygen methylation of hydroxycamptothecin and / or quercetin.

[0011] Furthermore, hydroxycamptothecin and / or quercetin react with the methyl donor under the catalysis of the camptothecin oxygen methyltransferase CaOMT3.

[0012] Furthermore, the methyl donor includes S -Adenosylmethionine.

[0013] Furthermore, the catalysis is carried out in a cell-free system.

[0014] Furthermore, the hydroxycamptothecin oxymethylation product is 9-methoxycamptothecin or 10-methoxycamptothecin.

[0015] Furthermore, the quercetin oxymethylation product includes rhamnine, tamarindin, or phytolaccaflavin.

[0016] The beneficial effects of this invention are: (1) This invention, through tissue expression transcriptome and genomic data analysis, and further screening through correlation analysis between metabolism and tissue expression transcriptome, obtained... CaOMT3 , CaOMT3 Capable of catalyzing 9-hydroxycamptothecin, 10-hydroxycamptothecin, and quercetin O -Methylation yields 9-methoxycamptothecin, 10-methoxycamptothecin, rhamnoside, tamarindin, and phytolaccaflavin.

[0017] (2) The CaOMT3 obtained by screening in this invention can catalyze the 4' and 7' hydroxyl sites of quercetin. O -Methylation produces rhamnine, tamarindine, and phytolaccaflavin. Compared with other oxygen methyltransferases, CaOMT3 has a wider range of methylation site selectivity and is a highly efficient bifunctional oxygen methyltransferase.

[0018] (3) The catalytic efficiency of CaOMT3 obtained in this invention for the substrate quercetin ( k cat / K m) reached 7.54±0.63mmol- 1 ·L·s- 1 This invention provides a novel biocatalyst for the highly efficient conversion of quercetin to rhamnine, significantly outperforming other oxygen methyltransferases. It also opens up new avenues for the industrial production of rhamnine.

[0019] (4) This invention constructs a recombinant vector of the CaOMT3 gene and an engineered strain, laying the foundation for large-scale fermentation and enzyme production. At the same time, it establishes an in vitro catalytic reaction system, providing a new method for the synthesis of rhamnine and methoxycamptothecin. Attached Figure Description

[0020] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 for CaOMTs Analysis diagram of the gene cluster related to Camptotheca acuminata.

[0021] Figure 2 Methylated flavonoids, methoxycamptothecin and CaOMTs Correlation analysis heatmap.

[0022] Figure 3 To utilize a tobacco expression system to express CaOMTs and catalyze the reaction of 9-hydroxycamptothecin and 10-hydroxycamptothecin. O Mass spectrometry image after methylation reaction.

[0023] Figure 4 for pCold - TF - CaOMTs SDS-PAGE electrophoresis image of enzyme proteins obtained from shake-flask fermentation.

[0024] Figure 5 To utilize CaOMTs to catalyze the reaction of 9-hydroxycamptothecin and 10-hydroxycamptothecin O Liquid phase detection image of methylation.

[0025] Figure 6 To utilize CaOMTs to catalyze the reaction of 9-hydroxycamptothecin and 10-hydroxycamptothecin O Mass spectrometry detection of methylation.

[0026] Figure 7 This is a liquid phase detection image of the reaction of quercetin to rhamnoside and minor products tamarind and phytolaccalin catalyzed by CaOMTs.

[0027] Figure 8 UPLC-Q-TOF / MS was used to detect the extraction ion chromatogram of the reaction of quercetin to rhamnoside and minor products tamarind and phytolaccalin catalyzed by CaOMTs.

[0028] Figure 9 MS fragment information diagram of the reaction of quercetin to rhamnoside and minor products tamarind and phytolaccalin catalyzed by CaOMTs.

[0029] Figure 10 To utilize CaOMTs to catalyze rhamnoside and tamarind O Liquid phase detection graph of methylation reaction.

[0030] Figure 11 The effects of reaction temperature and buffer pH on the activity of recombinant CaOMTs enzymes were investigated.

[0031] Figure 12 The graph shows the Michaelis-Menten equation curves of CaOMTs against the substrate quercetin. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope defined by the claims of this application. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0033] Example 1: Camptotheca acuminata CaOMTs Gene screening 1. Transcriptome and genome analysis of Camptotheca acuminata tissue This study used transcriptome sequencing of seven tissues (young leaves, mature leaves, stem xylem, stem phloem, taproot, lateral roots, and fibrous roots) from an annual Camptotheca acuminata. The transcriptome data is available in the NCBI BioProject database under the number PRJNA1377334. Combined with annotation and analysis of the publicly available Camptotheca acuminata genome on NCBI, a 106.3 kb gene cluster was identified, containing one 1-deoxy-D-xylitol-5-phosphate synthase involved in the MEP pathway, and four oxygen methyltransferase genes, named as follows: CaOMT1 , CaOMT2 , CaOMT3 and CaOMT4This suggests that four-oxymethyltransferases may play a role in the biosynthesis of camptothecin. Figure 1 ).

[0034] 2. Metabolites and CaOMTs Correlation analysis Further metabolite analysis was performed on seven tissues of Camptotheca acuminata. Spearman correlation coefficient analysis was used to analyze the relationship between methylated metabolites and... CaOMTs The relationship between transcriptional levels showed that... CaOMT3 The expression level was positively correlated with rhamnosin accumulation, suggesting that it may be involved in quercetin metabolism. O -Methylation process ( Figure 2 ).

[0035] Example 2: CaOMTs Obtaining nucleotide sequences and their amino acid sequences The entire seedling of Camptotheca acuminata was used as a tissue sample, and total RNA was extracted from it. After isolating and enriching the total RNA, it was reverse transcribed into cDNA to construct a cDNA library. This library was then used as a template for PCR amplification using forward and reverse primers (see Table 1). CaOMTs The sequences before and after ORF are identical, but the sequences in the middle are different (therefore, the same pair of primers is used for amplification). CaOMT1 , CaOMT2 , CaOMT3 The gene sequence. CaOMT4 The gene sequence was synthesized by Zhejiang Shangya Biotechnology Co., Ltd. CaOMT3 The gene sequence is shown in SEQ ID NO.1. CaOMT1 The gene sequence is shown in SEQ ID NO.3. CaOMT2 The gene sequence is shown in SEQ ID NO.4. CaOMT4 The gene sequence is shown in SEQ ID NO.5.

[0036] The specific PCR amplification system and conditions are as follows: A 50 μL PCR amplification system includes 1 μL cDNA, 25 μL 2×PrimeSTAR Max DNA Polymerase, 1 μL each of forward and reverse primers (10 μmol / L), and 22 μL sterile ultrapure water. The PCR reaction conditions are: 98 °C. o C pre-denaturation for 5 minutes; 98 o C mutation 30 seconds, 56 o Annealing C for 30 seconds, 72 o C extends for 2 minutes, 35 cycles; final 72 o C extends for 10 minutes.

[0037] Table 1. Based on the transcriptome of Camptotheca acuminata. CaOMTs Sequence synthesis of the following primers

[0038] The obtained gene sequence was translated into the amino acid sequence of CaOMT3 through DNA sequence translation, as shown in SEQ ID NO.2.

[0039] The PCR products obtained above were respectively combined with the intermediate vector. pLB Ligation. The ligation product was transformed into *E. coli* Top10 competent cells for plasmid amplification. After colony PCR verification, positive clones were selected and sent to a biotechnology company for sequencing verification. Plasmid DNA sequencing confirmed the correct plasmid construction, and subsequent experiments were then conducted using this plasmid as a template.

[0040] use pCold - TF The vector was used to construct an in vitro heterologous expression vector. Based on the above experiments, CaOMT1 - CaOMT4 The gene coding region sequence was used to design specific primers containing linearized vector terminal homologous arms (primers used are listed in Table 2). CaOMT1 , CaOMT2 and CaOMT3 Amplification was performed using forward primer 1 and reverse primer 2, while CaOMT4 Amplification is then performed using forward primer 2 and the same reverse primer. The target gene fragment is inserted using a one-step cloning method. pCold - TF Recombinant plasmids were constructed in the vectors respectively. pCold - TF - CaOMT1 , pCold - TF - CaOMT2 , pCold - TF - CaOMT3 and pCold - TF - CaOMT4 The restriction endonuclease selected for vector construction is... Bam HI and Eco RI, after linearization, was used to perform homologous recombination with the target gene fragment containing a homologous arm. The recombinant product was transformed into competent E. coli cells, and after verification by colony PCR and double enzyme digestion, positive clones were selected and sent to a biotechnology company for sequencing verification. After confirming the correct plasmid construction, it was then transformed into E. coli Rosetta (DE3) for prokaryotic expression.

[0041] Table 2 Primers for constructing prokaryotic expression vectors

[0042] Example 3: CaOMTs Tobacco transient expression catalytic reaction use pHB - YFP The vector was used to construct a plant expression vector. Based on the above experiments, CaOMT1 - CaOMT4 The gene coding region sequence was determined, and specific primers containing linearized vector terminal homologous arms were designed using SnapGene software. The target gene fragment was then inserted using a one-step cloning method. pHB - YFP Recombinant plasmids were constructed in the vectors respectively. pHB - CaOMT1 - YFP , pHB - CaOMT2 - YFP , pHB - CaOMT3 - YFP and pHB - CaOMT4 - YFP The restriction endonuclease selected for vector construction is... Bam HI and Spe I. After linearizing the vector, homologous recombination was performed with the target gene fragment containing a homologous arm. The recombinant product was transformed into competent *E. coli* cells. After colony PCR and double enzyme digestion verification, positive clones were selected and sent to a biotechnology company for sequencing verification. The correctly sequenced recombinant plasmids were used for subsequent *Agrobacterium* transformation and transient expression experiments in tobacco. The primers used are shown in Table 3 below. CaOMT1 , CaOMT2 and CaOMT3 Amplification was performed using forward primer 1 and reverse primer 2, while CaOMT4 Then, amplification is performed using forward primer 2 and the same reverse primer.

[0043] Table 3 Primers for constructing plant overexpression vectors

[0044] Subsequently, the correctly sequenced recombinant plasmid ( pHB-CaOMT1-YFP , pHB-CaOMT2-YFP , pHB-CaOMT3- YFP , pHB-CaOMT4-YFP The cells were transformed into GV3101 Agrobacterium competent cells (Shanghai Weidi Biotechnology Co., Ltd.) and cultured on YEB plates containing rifampicin and kanamycin. The plates were then placed at 28°C. o Incubate upside down in a constant temperature incubator for 2-3 days until single colonies appear.

[0045] Take the GV3101 Agrobacterium bacterial suspension that has been successfully identified positive, and inoculate it at a ratio of 1:100 into 50 mL of YEB liquid medium containing the corresponding antibiotics (rifampin 50 mg / L, kanamycin 75 mg / L), and incubate at 28°C. o In a constant temperature shaker at 200 rpm, the bacterial culture was incubated until the OD of the bacterial culture was reached. 600 The OD value was adjusted to 0.6-0.8. The cultured bacterial culture was transferred to a 50 mL sterile centrifuge tube and centrifuged at 4,000 rpm for 10 min at room temperature. The supernatant was discarded. The bacterial cells were resuspended in an appropriate amount of tobacco transient expression injection buffer, and the OD value was adjusted accordingly. 600 To approximately 0.6. Place the resuspended bacterial solution at 28°C. o Activate by static incubation in C for 2 hours before use.

[0046] Select tobacco plants that are in good growth condition, have dark green leaves, and are approximately 5 weeks old. Nicotiana benthamiana Use 1 mL sterile syringe needle to gently poke a small hole on the back of the leaf (avoid penetrating the leaf). After removing the needle, press the syringe tip tightly against the hole and slowly inject the bacterial solution until it saturates most of the leaf mesophyll area. Inject 3-4 leaves per tobacco plant. After injection, place the tobacco plants in a dark, protected cardboard box for 2 days of incubation. After the dark incubation, use a microsyringe to inject the corresponding substrates (9-hydroxycamptothecin, 10-hydroxycamptothecin, final concentration 100 μM) into the injection areas. Continue incubation for 3 days before collecting leaf samples.

[0047] The experimental groups were as follows: (1) CaOMT1 (containing 100 μM hydroxycamptothecin); (2) CaOMT2 (containing 100 μM hydroxycamptothecin); (3) CaOMT3 (containing 100 μM hydroxycamptothecin); (4) CaOMT4 (containing 100 μM hydroxycamptothecin). Simultaneously, an empty vector was injected ( pHB-YFP () as a negative control.

[0048] The collected tobacco leaf samples were immediately placed in a freeze dryer and dried for 24 h until constant weight. 20 mg of the dried sample was accurately weighed and placed in a 2 mL centrifuge tube. 1 mL of methanol was added, and the sample was thoroughly pulverized using a grinder and then ultrasonically extracted for 1 h. The extract was centrifuged at 12,000 rpm for 10 min at room temperature. The supernatant was filtered through a 0.22 μm microporous membrane and transferred to a sample vial. The sample was then incubated at -20°C. o The samples were stored at C for later use. UPLC-Q-TOF / MS system was used for analysis.

[0049] The UPLC-Q-TOF / MS detection method is as follows: Instrument: Waters SYNAPT G2-Si; Column: C18 column (2.1 × 50 mm, 1.7 µm); Injection volume: 2 µL; Column temperature: 35°C. o C; The mobile phase consisted of an aqueous solution (A) containing 0.1% formic acid and acetonitrile (B), with the following gradient elution program: 0-2 min (5% B), 2-30 min (5%-100% B), 30-31 min (100% B), 31-33.5 min (100%-5% B), 33.5-35 min (5% B); the flow rate was 0.3 mL / min. Mass spectrometry detection was performed in positive ion mode, with the following parameters: capillary voltage: 2.5-3.0 kV, cone voltage: 40 V, nitrogen as the drying gas, and desolvation gas flow rate: 800 L / h (temperature: 400°C). o C), Conical orifice gas flow rate: 50 L / h, ion source temperature: 100 o C; Quality scan range: 50–1,000 Da.

[0050] The test results showed that monomethylation products (m / z 379.1295) were detected in leaves expressing CaOMT1, CaOMT2, and CaOMT3, and their retention times were consistent with those of 9-methoxycamptothecin and 10-methoxycamptothecin standards, respectively. Figure 3 No methylation activity was detected in CaOMT4 within plants. This indicates that CaOMT4 cannot catalyze hydroxycamptothecin. O -Methylation, while CaOMT1, CaOMT2, and CaOMT3 catalyze 9- and 10-hydroxycamptothecin in plants. O - The function of methylation.

[0051] Example 4: Expression and purification of CaOMTs protein The recombinant plasmid was transformed into *E. coli* Rosetta (DE3) competent cells. Transformant clones were picked and transferred into an appropriate amount of LB medium (with ampicillin added to a final concentration of 50 μg / mL). 37 o Culturing at 600 nm until the absorbance reached 0.3, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.02%. oC was induced for 18 h. After centrifugation at 5,000 rpm for 10 min to collect the bacterial cells, they were resuspended in buffer solution: 20 mM PBS, pH 6.0, at a ratio of 1 (g): 10 (mL). The resuspended cells were then lysed using sonication at 60% power for 3 s, followed by a 5 s interval, for a total of 15 min. The resuspended cells were then placed on an ice pack on a tray. After lysis, the cells were centrifuged at 12,000 rpm for 40 min to remove precipitate and other particulate impurities. The supernatant was conjugated with Ni-NTA affinity medium and then washed with buffer solution containing 500 mM sodium chloride, 100 mM phosphate, and 20 mM imidazole to remove contaminating proteins. Finally, the target protein was eluted from the affinity medium using an elution buffer containing 500 mM sodium chloride, 100 mM phosphate buffer, and 250 mM imidazole. The eluted proteins from each group were collected and analyzed by SDS-PAGE electrophoresis. The SDS-PAGE results are shown below. Figure 4 As shown in the figure. M in the figure represents the protein molecular weight standard. The protein is recombinant CaOMTs purified by nickel column chromatography.

[0052] Example 5: CaOMTs catalyze hydroxycamptothecin O -In vitro functional validation of methylation Take 100 μL of CaOMTs-purified protein into a 1.5 mL EP tube, and add the following sequentially: S -Sysylmethionine (SAM, final concentration 1 mM) and 9-hydroxycamptothecin or 10-hydroxycamptothecin (final concentration 1 mM) were used to set up the following experimental groups: (1) CaOMT1 (containing 1 mM SAM and 1 mM hydroxycamptothecin); (2) CaOMT2 (containing 1 mM SAM and 1 mM hydroxycamptothecin); (3) CaOMT3 (containing 1 mM SAM and 1 mM hydroxycamptothecin); (4) CaOMT4 (containing 1 mM SAM and 1 mM hydroxycamptothecin).

[0053] Three negative control groups were also set up: pCold-TF Empty protein (containing 1 mM SAM and 1 mM hydroxycamptothecin), CaOMTs mixed boil-inactivated protein (containing 1 mM SAM and 1 mM hydroxycamptothecin), and CaOMTs mixed protein (containing only 1 mM hydroxycamptothecin, without SAM).

[0054] The above reaction system was placed at 37°C. oThe mixture was incubated in a shaker at 200 rpm in the dark for 5 hours. After the reaction was complete, an equal volume of chromatographically pure methanol was added to terminate the reaction. After shaking and mixing, the mixture was centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and dried under vacuum. After vacuum drying, 100 μL of chromatographically pure methanol was added to reconstitute the mixture. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was collected and analyzed by HPLC and UPLC-Q-TOF / MS. The HPLC results are shown below. Figure 5 , Figure 5 A represents the HPLC results of the reaction between 10-hydroxycamptothecin and CaOMTs. Figure 5 B represents the HPLC results of the reaction between 9-hydroxycamptothecin and CaOMTs. The UPLC-Q-TOF / MS detection results are as follows: Figure 6 As shown, Figure 6 A is the extraction ion diagram of CaOMTs catalyzing the conversion of 10-hydroxycamptothecin to 10-methoxycamptothecin. Figure 6 B is the extraction ion diagram for the CaOMTs-catalyzed formation of 9-hydroxycamptothecin from 9-methoxycamptothecin. Figure 6 C is the ion fragment diagram of the reaction product 10-methoxycamptothecin. Figure 6 Ion fragment diagram of 9-methoxycamptothecin, the product of reaction D.

[0055] The HPLC detection method is as follows: Instrument: Agilent 1260 HPLC; Column: COSMOSIL Packed Column (C 18 -AR-II, 3.5 μm, 4.6 × 150 mm); column temperature: 30 o C; Mobile phase: A (acetonitrile), B (water + 0.1% formic acid); Gradient elution conditions: 0-25 min, 10-100% A; 25-30 min, 100% A; 30-35 min, 100-10% A; Flow rate: 0.8 mL / min; Detection wavelength: 254 nm; Injection volume: 10 μL. UPLC-Q-TOF / MS detection was performed as in Example 3.

[0056] HPLC-DAD analysis showed that new product peaks corresponding to methoxycamptothecin appeared in both the CaOMT1 and CaOMT3 reaction systems. Figure 5 The UPLC-Q-TOF / MS detection results are as follows: Figure 6 As shown. After extracting the ion chromatogram with a mass-to-charge ratio of 379.1295, chromatographic peaks of CaOMT1, CaOMT2, and CaOMT3 were observed at approximately 11.1 min, consistent with the retention time of the methoxycamptothecin standard. No chromatographic peak was observed for CaOMT4. Figure 6A and 6B). Further observation of the ion fragmentation diagrams of each product reveals the parent ion fragment (m / z 379.1294) and the fragment ion peak generated after the loss of a methyl molecule (m / z 335.1383). Figure 6 (C and 6D). In summary, CaOMT4 cannot catalyze the reaction of 9- and 10-hydroxycamptothecin. O -Methylation, and CaOMT1, CaOMT2, and CaOMT3 can all catalyze the methylation of 9- and 10-hydroxycamptothecin. O -Methylation, in which CaOMT1 and CaOMT3 have comparable activity, while CaOMT2 has weaker activity.

[0057] Example 6: CaOMTs catalyze quercetin O -In vitro functional validation of methylation Take 100 μL of CaOMTs-purified protein into a 1.5 mL EP tube, and add the following sequentially: S - Adenosylmethionine (final concentration 1 mM) and quercetin (final concentration 1 mM) were used to set up the following experimental groups: (1) CaOMT1 (containing 1 mM SAM and 1 mM quercetin); (2) CaOMT2 (containing 1 mM SAM and 1 mM quercetin); (3) CaOMT3 (containing 1 mM SAM and 1 mM quercetin); (4) CaOMT4 (containing 1 mM SAM and 1 mM quercetin).

[0058] Three negative controls were set up: pCold-TF empty vector protein (containing 1 mM SAM and 1 mM quercetin), CaOMTs mixed boil-inactivated protein (containing 1 mM SAM and 1 mM quercetin), and CaOMTs mixed protein (containing only 1 mM quercetin, without SAM).

[0059] The above reaction system was placed at 37°C. o The mixture was incubated in a shaker at 200 rpm in the dark for 5 hours. After the reaction was complete, an equal volume of chromatographically pure methanol was added to terminate the reaction. After shaking and mixing, the mixture was centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and dried under vacuum. After vacuum drying, 100 μL of chromatographically pure methanol was added to reconstitute the mixture. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was collected and analyzed by HPLC and UPLC-Q-TOF / MS. The reaction results are as follows: Figure 7 , 8 As shown in Figure 9, Figure 7 HPLC detection and analysis were performed on each group after the reaction. Figure 8 A shows the extracted ion diagrams for the reactions of quercetin to rhamnoside and tamarind in each group. Figure 8 B is the extraction ion diagram of the reaction catalyzing the conversion of quercetin to phytolaccaflavin in each group; Figure 9A is the ion fragment diagram of the reaction product rhamnoside. Figure 9 B is an ion fragment diagram of the reaction product tamarinin. Figure 9 C is the ion fragment diagram of the reaction product, phytoflavin.

[0060] HPLC-DAD analysis showed that three product peaks appeared at 13.5 min, 14.8 min, and 17.2 min in the CaOMT1 and CaOMT3 reaction systems, while only one product peak was detected at 13.5 min in CaOMT2. Figure 7 After comparison with standard samples, the three products were identified as: tamarinin (4'- O 7-methylquercetin, rhamnine (7- O 4',7-di-O-methylquercetin and phytoflavin (4',7-di-O-methylquercetin). UPLC-Q-TOF / MS further confirmed that CaOMT1 and CaOMT3 generated two monomethylated derivatives (m / z 317.0650) and one dimethylated product (m / z 331.0819). The characteristic fragment ions of the product at 13.7 min, m / z 285.0408 and 153.0180, were consistent with the tamarind standard; the retention time and characteristic fragment ion m / z 302.1451 of the product at 14.6 min matched rhamnoside; the key fragments m / z 301.1400 and 285.0753 of the dimethylated product were consistent with the phytoflavin standard. Only tamarind was detected in the CaOMT2 reaction system, and no product was detected in the CaOMT4 reaction system. Figure 8 , 9 This demonstrates that different oxygen methyltransferases have different catalytic sites for their substrates, resulting in different catalytic products. Compared to CaOMT2, CaOMT1 and CaOMT3 have a wider range of catalytic sites.

[0061] Example 7: CaOMTs catalyze tamarinin and rhamnoside O -In vitro functional validation of methylation Take 100 μL of CaOMTs-purified protein into a 1.5 mL EP tube, and add the following sequentially: S - Adenosylmethionine (SAM, final concentration 1 mM) and tamarind or rhamnine (final concentration 1 mM) were used to set up the following experimental groups: (1) CaOMT1 (containing 1 mM SAM and 1 mM tamarind or rhamnine); (2) CaOMT2 (containing 1 mM SAM and 1 mM tamarind or rhamnine); (3) CaOMT3 (containing 1 mM SAM and 1 mM tamarind or rhamnine); (4) CaOMT4 (containing 1 mM SAM and 1 mM tamarind or rhamnine).

[0062] Three negative control groups were also set up: pCold-TF Empty protein (containing 1 mM SAM and 1 mM tamarinin or rhamnosine), CaOMTs mixed boiled protein (containing 1 mM SAM and 1 mM tamarinin or rhamnosine), and CaOMTs mixed protein (containing only 1 mM tamarinin or rhamnosine, without SAM).

[0063] The above reaction system was placed at 37°C. o In a C-type shaker, the mixture was incubated at 200 rpm in the dark for 5 hours. After the reaction was complete, an equal volume of chromatographically pure methanol was added to terminate the reaction. After shaking and mixing, the mixture was centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and dried under vacuum. After vacuum drying, 100 μL of chromatographically pure methanol was added to reconstitute the mixture. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was collected and analyzed by HPLC. The reaction results are shown below. Figure 10 As shown, Figure 10 A represents the catalytic tamarind of each group. O Liquid phase detection image of methylation reaction Figure 10 B represents the catalytic rhamnosides of each group. O Liquid phase detection graph of methylation reaction.

[0064] The test results showed that both CaOMT1 and CaOMT3 could catalyze the conversion of rhamnin and tamarindin into phytolaccaflavin (Phytolacca flavin). Figure 10 ( ), while CaOMT2 has no activity against either.

[0065] Example 8: Effect of reaction pH on the oxygen methyl transfer activity of CaOMTs Determination of CaOMT1, CaOMT2, and CaOMT3 using paraquercetin as a substrate O -Methyl transfer activity. The pH of the reaction buffer was set to 3, 4, 5, 6, 7, 8, 9, and 10, respectively, and the reaction system was consistent with the above. The group with the highest activity in the reaction was set as 100% relative enzyme activity. Sodium citrate buffer was used for pH 3, 4, and 5, phosphate buffer was used for pH 6, 7, and 8, and carbonate buffer was used for pH 9 and 10.

[0066] The results are as follows Figure 11 As shown in A, C, and E, among which Figure 11 Figures A, C, and E show the test results for CaOMT1, CaOMT2, and CaOMT3, respectively. Test results: The optimal reaction pH for CaOMT1 and CaOMT3 is 8.0, while the optimal reaction pH for CaOMT2 is 7.0. Figure 11 A, C, E).

[0067] Example 9: Effect of reaction temperature on the oxygen methyl transfer activity of CaOMTs Determination of CaOMT1, CaOMT2, and CaOMT3 using paraquercetin as a substrate O -Methyl transfer activity. The reaction temperature was set at 30°C. o C, 35 o C, 40 o C, 45 o C, 50 o C, 55 o C, 60 o C. The reaction system is the same as described above. The group with the highest activity in the reaction is set as having a relative enzyme activity of 100%.

[0068] The results are as follows Figure 11 As shown in B, D, and F, among them Figure 11 Figures B, D, and F show the test results for CaOMT1, CaOMT2, and CaOMT3, respectively. Test results: The optimal reaction temperature for CaOMT1 is 45°C. o C, below 45 o At temperature C, the activity increases with increasing temperature, exceeding 45°C. o The activity gradually decreases after C. The optimal reaction temperature for CaOMT2 is 35°C. o The optimal reaction temperature for CaOMT3 is 40°C. o C ( Figure 11 B, D, F).

[0069] Example 10: Determination of kinetic parameters of CaOMTs The reaction system consisted of 95 µL of purified CaOMTs protein dissolved in PBS buffer, followed by the addition of quercetin to a final concentration of 0.2–2 mM, bringing the total volume to 100 µL. The reaction was carried out at 37°C. o After reacting for 15 minutes, an equal volume of methanol was added to terminate the reaction, and the product was analyzed by HPLC.

[0070] Test results: The results show that the Michaelis constant of CaOMT1 is... K m is 0.108 ± 0.001 mmol·L -1 , conversion number k cat was 0.343 ± 0.004 s -1 catalytic efficiency k cat / K m was 3.18 ± 0.04 mmol -1 ·L·s -1 CaOMT3 K m is 0.120 ± 0.010 mmol·L -1 , k cat was 0.905 ± 0.010 s -1 , k cat / K m was 7.54 ± 0.63 mmol -1 ·L·s -1 CaOMT2 K m was 1.243 ± 0.197 mmol·L. -1 , k cat is (1.41±0.14)×10 -3 s -1 , k cat / K m is (4.75±0.46)×10 -3 mmol -1 ·L·s -1 ( Figure 12 CaOMT1 and CaOMT3 have similar and smaller... K The m-value indicates that both have a high affinity for quercetin. CaOMT3 exhibits the highest m-value. k cat and k cat / K The m-value corresponds to the highest catalytic efficiency. CaOMT2's... Km maximum, k cat and k cat / K The smallest m indicates the lowest affinity for quercetin and the weakest catalytic efficiency.

[0071] 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.

[0072] sequence list Zhejiang University of Traditional Chinese Medicine SEQ ID NO. 1 Application of camptothecin O-methyltransferase CaOMT3 in the O-methylation Modification of Hydroxycamptothecin and Quercetin Camptotheca acuminata CaOMT3 Zhejiang University of Traditional Chinese Medicine SEQ ID NO. 2 Application of camptothecin O-methyltransferase CaOMT3 in the O-methylation Modification of Hydroxycamptothecin and Quercetin Camptotheca acuminata CaOMT3 MANDERYTELLEAQAQLWNRTGNFIYSASLKCAVELGIPDVIHSHGKPMTLPELVSALPINPSKAHCIYRLMRTLVHAGFFVQQKEGTNEQENGGYLLTRASRLLLKEEPLNARAFLLAVINPIMTKPWHFLSDWFKNDDPTAFNTAHGRSFWDYLAQEPRLGNFFDEAMASDSCLI TNVLINECKEVFEGLTSLVDVGGGTGTVGRAIAKTFPHLKCTVFDLPHVIADLQGTENLDFVGGDMFEAIPPANAILLKWILHNWSEEECVKILKRCKEAIPSKDGGKVIIIDMIMEKVKADDESVEAQLSSDMTMMVLFRGQERSEKEWEKLFLAAGFSHYKITSALGLRSLIEVYP Zhejiang University of Traditional Chinese Medicine SEQ ID NO.3 Application of camptothecin O-methyltransferase CaOMT3 in the O-methylation Modification of Hydroxycamptothecin and Quercetin Camptotheca acuminata CaOMT1 Zhejiang University of Traditional Chinese Medicine SEQ ID NO.4 Application of camptothecin O-methyltransferase CaOMT3 in the O-methylation Modification of Hydroxycamptothecin and Quercetin Camptotheca acuminata CaOMT2 Zhejiang University of Traditional Chinese Medicine SEQ ID NO.5 Application of camptothecin O-methyltransferase CaOMT3 in the O-methylation Modification of Hydroxycamptothecin and Quercetin Camptotheca acuminata CaOMT4 ATGGCTAATGAGGAGAGATATACTGAGCTACTTGAAGCTCAAGCTCACGTCTGGAACCGAACATTCAACTTCATAAACTCTGCATCTCTAAAATGCGCAGTTGAATTAGGCATACCAGATGTCATCCACAGCCATGGTAAACCCATGACTCTTCCAGAGCTTGACAGCGCCCTTAACATTAACCCTTCAAAAGCCCATTGCATATATCGCCTCATGCGCACTTTAGTTCACTCTGGCTTCTTTGTTCAGCAAAAAGAAGGCACAAATGAGCAAGAAAATGTTATCGATCCAATCCTAACAAAACCATGGCACTTCTTGAGTGATTGGTTCAAGAATGATGATCCCACTGCATTTAATACAACACAAGGAAGAAGTTTTTGGGATTATCAAGCTCAAGAGCCAAGACTAGGAAATTTTTTTGACGAAGCCATGGCAAGTGACTCCCGCTTGATCACAAATGTGATGATCAATGAATGCAAGGAGGTGTTTGAGGGCTTGACATCAATAGTGGATGTTGGGGGTGGCACTGGGACTGTGGGGAGGGCCATCGCCAAAACTTTCCCACACCTCAAGTGCACTGTGTTTGATCTTCCACATGTGATTGCTGACTTGCAAGGGACTGAAAACTTGGACTTTGTTGGAGGGGATATGTTCGAAGCAATTCCTCCTGCAAATGCAATTTTACTCAAGTGGATTCTGCACGATTGGACTGAGGAAGAATGTGTGAAGATATTGAAGCGGTGCAAAGAAGCACTTCCAAGCAAGGACGGTGGGAAGGTGATTATCATAGACATGATTATGGAGAAGGTCAAAGCAGATGATGAGTCAGTGGAGGCACAGCTCTCTTTTGACATGATGATGATGGTTCTGTTAAGAGGTCAAGAGAGAAGTGAGAAAGAATGGGAAAAACTCTTCTTGGCTGCGGGCTTCAGTCACTACAAAATAACTTCAGCTTTGGGTTTAAGGTCACTCATTGAGGTTTATCCTTAA。

Claims

1. A camptothecin-oxymethyltransferase CaOMT3, characterized in that, The amino acid sequence of the camptothecin oxygen methyltransferase CaOMT3 is shown in SEQ ID NO:

2.

2. A gene encoding the Camptotheca oxygen methyltransferase CaOMT3 as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

1.

3. A recombinant vector, characterized in that, It includes the encoding gene as described in claim 2.

4. A genetically engineered bacterium, characterized in that, It includes the recombinant vector as described in claim 3.

5. The use of the camptothecin oxygen methyltransferase CaOMT3 of claim 1 in catalyzing the oxygen methylation of hydroxycamptothecin and / or quercetin.

6. The application according to claim 5, characterized in that, Hydroxycamptothecin and / or quercetin react with methyl donors under the catalysis of the camptothecin oxygen methyltransferase CaOMT3.

7. The application according to claim 6, characterized in that, The methyl donor includes S -Adenosylmethionine.

8. The application according to claim 6, characterized in that, The catalysis was carried out in a cell-free system.

9. The application according to claim 6, characterized in that, The hydroxycamptothecin oxymethylation products include 9-methoxycamptothecin or 10-methoxycamptothecin.

10. The application according to claim 6, characterized in that, The quercetin oxymethylation products include rhamnine, tamarindin, or phytolaccaflavin.

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