Method for efficiently screening functional enzyme and application

Abstract

The invention discloses a method for efficiently screening a functional enzyme and application of the functional enzyme, a demethylase is rapidly screened through matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) combined with non-targeted metabonomics and transcriptomics analysis, and the demethylase can be converted into dihydroberberine by taking palmatine as a substrate, so that the functional enzyme can be efficiently screened. And the enzyme mining efficiency and accuracy are obviously improved. The functional enzyme mining strategy provided by the invention solves the problem that more comprehensive information of natural metabolites and functional enzymes cannot be obtained by a traditional method, and is more beneficial to environmental protection and sustainable development.

Description

(I) Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method and application for efficiently screening functional enzymes. (II) Background Technology

[0002] Corydalis yanhusuo, also known as Xuanhu or Yuanhu, is the dried tuber of the poppy plant Corydalis yanhusuo W.T.Wang. It is one of the famous "Eight Zhejiang Herbs" and is mainly produced in Dongyang and Pan'an areas of Zhejiang Province. Its properties are pungent, bitter, and warm. It enters the liver and spleen meridians. It has the effects of promoting blood circulation, regulating qi, and relieving pain. It is mainly used to treat chest and rib pain, abdominal pain, angina pectoris, amenorrhea, dysmenorrhea, postpartum blood stasis, and swelling and pain from falls. The *Compendium of Materia Medica* states that it "can move qi stagnation in the blood and blood stagnation in the qi, therefore it specifically treats various pains throughout the body; its effects are indescribable." The *Lei Gong Pao Zhi Lun* states, "For severe heart pain, quickly seek Corydalis." This shows that the analgesic effect of Corydalis yanhusuo is recognized by physicians throughout history. Corydalis has a long history of medicinal use and is widely applied clinically. For example, products containing Corydalis, such as "Yuanhu Zhitong Pian" (Corydalis Analgesic Tablets), "Anwei Pian" (Stomach-Soothing Tablets), and "Tongjing Wan" (Menstrual Pain Relief Pills), have been on the market for many years, and their safety and efficacy have been clinically verified. In traditional Chinese medicine, Corydalis is often used in combination with other herbs. Modern experiments have shown that Corydalis has excellent analgesic, sedative, antihypertensive, and antiarrhythmic effects.

[0003] Characterization of the chemical constituents of Corydalis has been ongoing for nearly a century, resulting in the identification of a range of compounds, including sugars, amino acid derivatives, triterpenoids, anthraquinones, phenolic acids, steroids, and organic acids. Furthermore, over 60 alkaloids have been discovered in the Corydalis tuber, most of which produce major pharmacological effects. These compounds are synthesized from tyrosine and differentiate into various benzylisoquinoline alkaloids (BIAs) via a common precursor pathway for the synthesis of (S)-reticuline. Based on their chemical structures, most BIAs can be classified into proberberine and apophene-type BIAs, but proberberine and benzo[a]phenanthridine BIAs are also present in Corydalis bulb extracts. Currently, thanks to the tireless efforts of biochemists and phytochemists, the labyrinthine biosynthetic pathways of BIAs in the model species Papaver somniferum have been well elucidated, but the elucidation of the biosynthetic pathways of apophene-type BIAs still requires considerable further work. Furthermore, the amino acid sequences of some key enzymes in the protoberberine BIA pathway remain missing, which greatly hinders the molecular cloning and metabolic engineering of the protoberberine pathway. In addition, the biosynthesis of BIAs in Corydalis plants has received little attention, resulting in a lack of understanding of the biosynthesis of several pharmacologically active alkaloids in Corydalis, including papaverine, corydaline, and dehydrocorydaline.

[0004] The biosynthesis of protoberberine-type alkaloids begins with L-tyrosine, which is converted to tyramine and 4-hydroxyphenypyruvate via tyrosine decarboxylase (TYDC) and tyrosine transaminase (TyrAT), respectively. Tyramine is then catalyzed by polyphenol oxidase (3OHase) to produce dopamine, a precursor of the isoquinoline moiety. 4-hydroxyphenyruvate is converted to 4-hydroxyphenylacetaldehyde (4-HPAA) by 4-hydroxyphenylpyruvate decarboxylase (4HPPDC), forming the benzyl group. Dopamine and 4-hydroxyphenylacetaldehyde are then catalyzed by norcoclaurine synthase (NCS) to produce (S)-norcoclaurine, a Pictet-Spengler condensation reaction. Next, 6-O-methyltransferase (6OMT) catalyzes the synthesis of linderine ((S)-coclaurine), which is then catalyzed by linderine N-methyltransferase (CNMT) to produce (S)-N-methylcoclaurine. (S)-N-methylcoclaurine is then converted to (S)-3-hydroxy-N-methylcoclaurine by N-methylcoclaurine 3-hydroxylase (NMCH), which is subsequently catalyzed by 4-O-methyltransferase (4OMT) to produce S-type berberine, a common intermediate in the synthesis of most BIAs. The synthetic pathway of protoberberine-type alkaloids involves the conversion of S-type berberine to S-type coulerine via berberine briningase (BBE), thus forming the basic framework of protoberberine-type alkaloids. S-type corydaline is catalyzed by corydaline 9-O-methyltransferase (SOMT1) to produce S-type tetrahydrocolumbamine, which is then catalyzed by tetrahydroberberine synthase (CAS) to produce S-type tetrahydroberberine ((S)-canadine), and finally catalyzed by tetrahydroprotoperine oxidase (STOX) to produce berberine. In Coptis chinensis, S-type tetrahydrocolumbamine can also be catalyzed by THBO to produce columbamine, which is then catalyzed by columbamine O-methyltransferase (COOMT) to produce palmatine, another very important protoperine-type alkaloid in Coptis chinensis. Furthermore, (S)-tetrahydrocolumbamine can be converted into tetrahydropalmatine via the catalysis of tetrahydrocolumbamine O-methyltransferase (COOMT), while the biosynthetic pathway of converting palmatine into dihydroberberine has not been reported.

[0005] Traditional enzyme screening strategies are function-oriented experimental methods based on natural microbial resources. Their core lies in systematically collecting samples, isolating microorganisms, culturing them, and detecting their functions to identify and obtain enzymes with specific catalytic activities from nature. Researchers typically collect samples from diverse ecological environments, such as soil, water bodies, and extreme environments, and use selective culture media to enrich and purify the microorganisms within them, thereby obtaining single strains. During the culturing process, specific inducers are often added to stimulate the expression of the target enzyme; for example, casein is added when screening for proteases, and microcrystalline cellulose is added when screening for cellulases. Subsequently, preliminary screening is performed using visual phenomena such as clear zones and color changes on solid culture plates, followed by further determination of enzyme activity through liquid culture. Commonly used detection methods include colorimetry, spectrophotometry, and titration. While traditional screening methods are intuitive and reliable, suitable for the discovery of unknown enzymes, and still feasible under conditions of limited resources, their limitations are also quite obvious. For example, they rely on the culturability of microorganisms, resulting in the neglect of enzymes encoded by a large number of unculturable microorganisms; the screening efficiency is low and the cycle is long, making it difficult to meet the needs of modern biotechnology for the rapid development of enzyme resources; in addition, the limitations of detection methods and substrates also make it difficult to discover some novel or atypical enzymes.

[0006] Over the past two decades, the rapid development of next-generation sequencing (NGS) technology has led to an exponential increase in genomic data from medicinal plants, providing unprecedented resources for identifying key enzyme genes related to the synthesis of bioactive natural products. However, this explosive growth in sequence data has not significantly improved our efficiency in identifying key synthetic enzymes. One possible explanation is that current screening strategies often overlook the functional impact of the spatial specificity of gene expression at the tissue level. Different plant tissues have unique anatomical structures, and this difference is reflected not only in gene expression levels but also in the distribution patterns of metabolites. Increasing research shows a high degree of consistency between the spatial distribution of plant secondary metabolites and the expression locations of their synthesis-related genes, suggesting that tissue-specific information is crucial for accurately identifying functional genes. However, current mainstream metabolite analysis methods, such as liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS), typically require tissue homogenization before analysis, resulting in the loss of the original spatial location information of metabolites. This makes it difficult for sequence-based functional gene screening strategies to effectively integrate the spatial correspondence between metabolite distribution and gene expression, thus limiting the accurate identification of key enzyme genes. This neglect of spatial dimension information is a significant drawback of current sequence data-based filtering strategies.

[0007] Matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI) is an advanced, label-free molecular imaging technique characterized by high sensitivity, wide molecular coverage, and high spatial resolution. In recent years, it has demonstrated significant application potential in life sciences and pharmaceutical research. This technique can directly perform spatial localization and qualitative analysis of various molecules (such as metabolites, lipids, peptides, and small molecule drugs) in biological tissue sections without damaging the tissue structure, and is particularly suitable for studying the distribution patterns of secondary metabolites in plant tissues. In medicinal plant research, secondary metabolites are often natural products with important biological activities, and their synthesis and accumulation are usually regulated by enzymes expressed at specific times and spaces. Therefore, understanding the precise distribution of these metabolites in tissues helps to reveal their potential synthetic pathways and regulatory mechanisms. Although MALDI-MSI technology performs excellently in revealing the spatial distribution of secondary metabolites, its application in enzyme gene discovery and functional analysis remains relatively limited. Previous studies have shown that the distribution of secondary metabolites in plants often highly coincides with the expression locations of their synthesis-related genes. This spatial co-localization provides important clues for identifying key enzymes involved in metabolic pathways. Research indicates that MALDI-MSI technology not only provides spatial dimensionality information unavailable through traditional methods but also significantly improves the efficiency and accuracy of enzyme gene mining. However, most studies still rely primarily on traditional transcriptomics and metabolomics analyses, rarely integrating the spatial information obtained from MALDI-MSI into metabolism-gene expression association analyses.

[0008] To fill this gap, this invention, for the first time, introduces MALDI-MSI technology into the screening of functional enzymes in the traditional Chinese medicinal herb Corydalis yanhusuo. By mapping the spatial distribution of target metabolites in the medicinal plant tissue at high resolution and comparing it with region-specific transcriptional data, key candidate genes potentially involved in metabolic pathways are identified. Based on this, the functions of these candidate genes are further validated, ultimately leading to the successful identification of novel functional enzymes with catalytic activity. These enzymes are not only difficult to discover using traditional screening strategies, but also play a crucial role in the biosynthesis of natural products, providing new ideas and technical support for the elucidation of natural product metabolic pathways and their application in synthetic biology. (III) Summary of the Invention

[0009] The purpose of this invention is to provide a highly efficient method and application for screening functional enzymes. By combining matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) with non-targeted metabolomics and transcriptomics analysis, a demethylase was rapidly screened. This enzyme can convert palmatine into dihydroberberine (DHB) using palmatine as a substrate, significantly improving the efficiency and accuracy of enzyme discovery. The functional enzyme discovery strategy provided by this invention solves the problem that traditional methods cannot obtain more comprehensive information on natural metabolites and functional enzymes, and is also more environmentally friendly and conducive to sustainable development.

[0010] The technical solution adopted in this invention is:

[0011] This invention provides a method for efficiently screening functional enzymes, the method comprising the following steps: (1) Sample preparation: freezing and slicing Corydalis tubers to prepare samples; (2) Covering matrix: using α-cyano-4-hydroxycinnamic acid (CHCA) as the main matrix and 9-aminoacridine (9-AA) as the auxiliary matrix, and spraying the sample from step (1) using a matrix sprayer; (3) Data acquisition: using matrix-assisted laser desorption / ionization imaging (MALDI-MSI) technology to acquire mass spectrometry signals with different m / z values; after imaging, H&E staining or Nissl staining of the slices, and obtaining tissue morphology images by taking pictures under a microscope; (4) Data processing: the raw data of the mass spectrometry signals acquired in step (3) are processed by SCiLS. Lab software was used for preprocessing, including background subtraction and signal normalization. MsiReader software was used for image reconstruction and spatial registration analysis with tissue morphology images to make the distribution of metabolite signals correspond precisely with the tissue morphology structure, realize the visualization mapping of metabolite spatial distribution, and accurately delineate the target region (ROI) with high metabolic activity; (5) Non-targeted metabolomics analysis: Corydalis part corresponding to the single target region delineated in step (4) was extracted with 75% methanol aqueous solution. The relative ion abundance of the extract was detected by LC-MS / MS and compared with metabolite standards. The chemical structure of metabolites was confirmed by database retrieval, and metabolites with large distribution variations in different parts were selected as candidate substrates; (6) Transcriptomics analysis: RNA was extracted from the tissue where the candidate substrates in step (5) were located and transcriptome sequencing was performed. The transcriptome sequencing results of other parts were used as a control. Hidden Markov Model (HMM) was used to analyze the transcriptome results to screen potential functional genes. A phylogenetic tree of potential functional genes was constructed to screen candidate genes. The FPKM value of the candidate genes (Fragments Per Kilobase of exon model per Millionmapped) was used to screen the candidate genes. (fragments), select the functional enzyme gene with the highest FPKM value.

[0012] Further, the sample in step (1) was prepared as follows: Fresh or quick-frozen Corydalis tubers were selected as the research object, and they were quickly frozen in liquid nitrogen and stored in an environment of -80℃. Then, a cryostat was used to prepare continuous slices with a thickness of 8-12 μm to obtain Corydalis tuber slices as samples.

[0013] Further, in step (2), α-cyano-4-hydroxycinnamic acid was prepared into a 10 mg / mL solution using acetonitrile-0.1% trifluoroacetic acid at a volume ratio of 1:1, and 9-aminoacridine was prepared into a 5 mg / mL solution using methanol-water at a volume ratio of 7:3. After being filtered through a 0.22 μm filter membrane, the samples from step (1) were sprayed using a Bruker Autoflex Speed ​​matrix sprayer. The CHCA spraying parameters were: pressure 0.3 MPa, nozzle distance 3 cm from the sample, speed 5 mm / s, and 6 sprays (with an interval of 30 s). The 9-AA spraying parameters were adjusted to: pressure 0.25 MPa, speed 4 mm / s, and 8 sprays (with an interval of 40 s). The spraying environment was 25℃ and 40% humidity.

[0014] Further, in step (3), a time-of-flight mass spectrometer (Bruker, timsTOF flex) with a MALDI source is used for data acquisition; the sample covered by the matrix in step (2) is placed in the mass spectrometry imaging system, and the 10kHz laser scanning technology of SmartBeam 3D is used to perform the analysis in qTOF mode with a frequency-triple Nd:YAG laser (355nm); the pixel size is set to 50 μm, each pixel is used for 200 laser shots, and 35% of the laser power is at 10 kHz; in positive ion mode, a high-resolution and high-sensitivity spatial scan is performed from m / z 200-2000 to acquire mass spectrometry signals of different m / z values ​​and obtain the raw data of the mass spectrometry signal.

[0015] Furthermore, the preprocessing method in step (4) is as follows: the raw mass spectrometry signal data acquired is basically corrected using SCiLS Lab version 2019 software. The core steps include background subtraction (removing non-target signals such as instrument noise and matrix interference) and signal normalization (unifying the signal intensity benchmark of different pixels to ensure data comparability); the mass spectrometry image is reconstructed using MsiReader v1.00 software, and the processed mass spectrometry signal is converted into a visualized spatial distribution image; then the reconstructed mass spectrometry image is spatially registered with the previously acquired stained tissue image to make the distribution of metabolite signals accurately correspond to the tissue morphology and structure, and finally the tissue localization of metabolites is completed.

[0016] Furthermore, the metabolites of step (4) include benzyl isoquinoline alkaloids (BIAs).

[0017] Further, the LC-MS / MS detection method in step (5) is as follows: ACQUITY UPLC system; mobile phase A is acetonitrile; mobile phase B is 0.1% formic acid; liquid phase gradient elution program: 0-12 min, 7-18% A; 12-18 min, 18-22% A; 18-33 min, 22-25% A; 33-37 min, 25-40% A; 37-42 min, 40-85% A; 42-48 min, 85-100% A; flow rate is 1.0 mL / min, column temperature is 30℃, injection volume is 10 μL, and detection wavelength is 280 nm. Mass spectrometer: TripleTOF 6600; ion source temperature: 550.0℃; polarity mode: positive ion mode; ion source parameters: CUR 35.000; GS1 55.000; GS2 55.000; ISVF 5500.000; First-stage mass spectrometry (TOF MS): mass range m / z 100.0 ~ 2000.0; collision energy CE 10.000; declustering voltage DP 80.000; cycle time 840 ms; pulse frequency 15.165 kHz; accumulation time 150.0 ms; Second-stage mass spectrometry (TOF MS²): mass range m / z 50.0 ~ 1500.0; collision energy CE 40.000; collision energy broadening CES 20.000; declustering voltage DP 80.000; cycle time 840 ms; pulse frequency 15.165 kHz; kHz; cumulative time 80.0 ms.

[0018] Furthermore, the method for constructing the phylogenetic tree in step (6) is as follows: based on Evolview software, use MEGAX software to perform 1000 bootstrap tests to generate a phylogenetic tree of neighbor connections.

[0019] The present invention also provides a benzyl isoquinoline alkaloid synthase screened by the method described above, wherein the amino acid sequence of the benzyl isoquinoline alkaloid synthase is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0020] This invention also provides the application of the benzyl isoquinoline alkaloid synthase screened by the method described above in the catalytic synthesis of dihydroberberine from palmatine.

[0021] Furthermore, the application is as follows: using the enzyme solution extracted from the engineered bacteria expressing the benzylisoquinoline alkaloid synthase after induction as a catalyst, using berberine as a substrate, adding adenosylmethionine and sodium vitamin C, and using a buffer solution with pH 4-10 as a reaction medium to form a reaction system, and reacting at 4-60℃ (preferably 37℃) and 200rpm in a shaker for 5-10h to obtain a reaction solution containing dihydroberberine.

[0022] Furthermore, in the reaction system, the substrate, S-adenosylmethionine, and sodium vitamin C are dissolved in the reaction medium and then added. The concentration of the substrate is 50-125 mM (preferably 100 mM); the concentration of S-adenosylmethionine is 20-40 mM (preferably 28 mM); the concentration of sodium vitamin C is 50-125 mM (preferably 100 mM); and the amount of catalyst added is 0.5-1.5 g / L (preferably 1.1 g / L) based on the protein content.

[0023] Furthermore, the buffer solution is a pH 7.0, 100mM K2HPO4-KH2PO4 buffer solution.

[0024] This invention develops a novel functional enzyme gene mining strategy. First, spatial mass spectrometry (MALDI-MSI) analysis of Corydalis rhizomes was performed to preliminarily obtain the spatial distribution of alkaloids. Then, after segmentation of the Corydalis rhizomes, non-targeted metabolomics analysis was conducted to further quantify the spatial content distribution of alkaloids and obtain their fold ratios. Next, transcriptome sequencing was performed on different parts of the Corydalis rhizomes to obtain a large amount of differentially expressed gene information. Bioinformatics tools were used to screen candidate functional genes, followed by gene synthesis, plasmid construction, and other steps. Finally, the functional enzyme was introduced into the reaction, successfully converting palmatine to dihydroberberine, and its biochemical properties were evaluated. Compared with traditional methods, the method involved in this invention can provide a more accurate and comprehensive map of biomolecular activity. This combined strategy not only allows for high-resolution spatial localization of the distribution and activity of enzymes in organisms but also enables simultaneous analysis of gene expression of these enzymes at specific locations, greatly improving efficiency.

[0025] Compared with existing technologies, this invention has the following significant advantages: Firstly, the method of this invention can achieve in-situ, intuitive spatial localization of secondary metabolites in plant tissues without relying on traditional tissue homogenization, thus preserving the regional characteristics of metabolite distribution. This introduction of spatial information allows researchers to more accurately correlate regions enriched by specific metabolites with corresponding gene expression profiles, significantly improving the accuracy of screening target enzyme genes. Secondly, MALDI-MSI has high sensitivity and broad-spectrum detection capabilities, enabling simultaneous detection of multiple types of metabolites with wide coverage, which helps in the discovery of novel functional enzymes related to the synthesis of rare or novel natural products. Furthermore, combining this technology with modern transcriptomics, metabolomics, and other multi-omics approaches can construct a more systematic "metabolism-gene-enzyme" functional network, accelerating the identification and verification process of key enzymes. Compared with traditional screening methods, the strategy of incorporating MALDI-MSI not only improves screening efficiency but also expands the understanding of unknown metabolic pathways in non-model medicinal plants, providing strong technical support for the biosynthesis research of natural products and the mining of enzyme resources. The 16233c2g5 functional enzyme derived from Corydalis yanhusuo obtained by cloning and isolating in this method can be used to synthesize benzylisoquinoline alkaloids, providing a new route for the synthesis of this type of compound. This is the first time that dihydroberberine has been synthesized through biosynthesis, filling the gap in the biosynthesis of this benzylisoquinoline alkaloid and providing new ideas for the breeding of superior Corydalis yanhusuo varieties. (iv) Description of the attached drawings

[0026] Figure 1 Tissue localization diagram of important alkaloid components in Corydalis rhizome.

[0027] Figure 2 A bar chart comparing the relative ion abundance of important alkaloid components in different parts of Corydalis rhizome.

[0028] Figure 3 Transcriptome gene density map of Corydalis tuber, outer skin, and interior.

[0029] Figure 4 Volcano diagram showing differential genes in the tuber, outer skin, and interior of Corydalis rhizome.

[0030] Figure 5 Phylogenetic analysis diagram of 43 potential genes.

[0031] Figure 6 FPKM heatmap for 8 candidate genes.

[0032] Figure 7 The image shows the three-dimensional structure of the benzyl isoquinoline alkaloid synthase 16233c2g5 protein.

[0033] Figure 8A schematic diagram of the construction of the recombinant plasmid pET28a(+)-16233c2g5.

[0034] Figure 9 This is an SDS-PAGE image of the enzyme solution.

[0035] Figure 10 Mass spectra of the reaction product, dihydroberberine standard, and blank control.

[0036] Figure 11 The relative enzyme activities of benzyl isoquinoline alkaloid synthase 16233c2g5 at different pH values.

[0037] Figure 12 The relative enzyme activity of benzylisoquinoline alkaloid synthase 16233c2g5 at different temperatures.

[0038] Figure 13 The relative enzyme activities of benzyl isoquinoline alkaloid synthase 16233c2g5 under different metal ions are given.

[0039] Figure 14 This is the mass spectrum of the reaction solution in Example 5. (V) Detailed Implementation Methods

[0040] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: the Corydalis rhizome used in the embodiments of the present invention was collected and prepared in accordance with the 2020 edition of the Chinese Pharmacopoeia.

[0041] Example 1: MALDI-MSI-based discovery of the gene encoding the benzyl isoquinoline alkaloid synthase for the synthesis of dihydroberberine.

[0042] The timsTOF flex combines a MALDI source with TIMS (ion mobility spectrometry) separation technology, enabling high-throughput, high-precision detection of metabolites in complex samples while improving spatial resolution and structural analysis capabilities. Strict control of sample processing conditions is crucial throughout the experiment to avoid repeated freeze-thaw cycles and tissue damage, ensuring data reproducibility and accurate spatial localization.

[0043] 1. Sample preparation

[0044] Fresh or quick-frozen Corydalis tubers were selected as the research object. They were rapidly frozen in liquid nitrogen and stored at -80°C. Subsequently, continuous sections with a thickness of 8-12 μm were prepared using a cryostat to obtain Corydalis tuber sections (including the outer skin and the inside of the tuber) as samples.

[0045] 2. Covering substrate

[0046] Matrix coating: The sample prepared in step 1 was attached to a conductive glass slide, fixed with acetone at -20℃ and dried at room temperature, and then coated with a matrix using a matrix coating instrument to ensure metabolite ionization efficiency and spatial resolution. α-cyano-4-hydroxycinnamic acid (CHCA, 10 mg / mL, dissolved in acetonitrile-0.1% trifluoroacetic acid 1:1) was selected as the main matrix, and 9-aminoacridine (9-AA, 5 mg / mL, dissolved in methanol-water 7:3) was selected as the auxiliary matrix. After filtration through a 0.22 μm filter membrane, the sample from step 1 was coated using a Bruker Autoflex Speed ​​matrix coating instrument. The CHCA coating parameters were: pressure 0.3 MPa, nozzle distance 3 cm, speed 5 mm / s, 6 sprays (30 s interval); the 9-AA parameters were adjusted to: pressure 0.25 MPa, speed 4 mm / s, 8 sprays (40 s interval); and the coating environment was 25℃ and 40% humidity.

[0047] 3. Mass spectrometry data acquisition

[0048] Data acquisition was performed using a time-of-flight mass spectrometer (Bruker, time-of-flight flex) with a MALDI source. The sample covered with the matrix from step 2 was placed in the mass spectrometry imaging system, and 10 kHz laser scanning technology was used with a third-harmonic Nd:YAG laser (355 nm) in qTOF analysis mode. The pixel size was set to 50 μm, with 200 laser shots per pixel and 35% laser power at 10 kHz. High-resolution, high-sensitivity spatial scanning was performed in positive ion mode from m / z 200-2000 to acquire mass spectrometry signals at different m / z values, obtaining the raw mass spectrometry data.

[0049] 4. Data Processing

[0050] To aid in tissue localization of metabolites, the sections were H&E staining or Nissl staining and microscopic photography was performed after imaging. Spatial registration analysis was then performed with the mass spectrometry images. The specific steps are as follows:

[0051] (1) Image registration preprocessing: After mass spectrometry imaging is completed, H&E staining or Nissl staining is performed on the same Corydalis slices, and tissue morphology images are obtained by taking pictures under a microscope to provide morphological reference for subsequent spatial registration.

[0052] (2) Basic processing of raw data: The raw mass spectrometry data were corrected using SCiLS Labversion 2019 (SCiLS, Bremen, Germany) software. The core steps included background subtraction (removing non-target signals such as instrument noise and matrix interference) and signal normalization (unifying the signal intensity benchmark of different pixels to ensure data comparability).

[0053] (3) Image reconstruction and spatial registration: Mass spectrometry image reconstruction was performed using MsiReader v1.00 software, converting the raw mass spectrometry signal data processed in step (2) into a visualized spatial distribution image; subsequently, the reconstructed mass spectrometry image was spatially registered with the previously acquired stained tissue image to achieve a precise correspondence between metabolite signal distribution and tissue morphology, realize the visualized mapping of the spatial distribution of benzyl isoquinoline alkaloids (BIAs), and accurately delineate the target region (ROI) of high metabolic activity of BIAs, ultimately completing the tissue localization of metabolites. The results are shown in Figure 1 .

[0054] Figure 1 The results showed that the spatial distribution of various alkaloids (such as berberine, dihydroberberine, tetrahydroberberine, corydaline, corydaline A, palmatine, and other important corydaline alkaloid components) in the corydaline tuber was significantly different. There were important differences in the spatial distribution of alkaloid components in the outer skin and the inside of the tuber. The content of various alkaloids in the outer skin of the tuber was significantly higher than that in the inside of the tuber. Therefore, the enzymes that catalyze the transformation of these alkaloids may be differentially expressed in these two regions.

[0055] 5. Non-targeted metabolomics detection

[0056] Based on the results of step 4, the outer skin and interior of the Corydalis rhizome were separated using a scalpel, resulting in three sample groups: outer skin, interior, and intact rhizome. Each group was chopped and crushed using a mortar and pestle, ensuring each group started with a sample weight greater than 10g. 1.0g of each sample was randomly and accurately weighed into a 50mL centrifuge tube, and 10mL of 75% methanol was precisely added. The tube was extracted using ultrasonic oscillation at 250W for 30 minutes, followed by centrifugation at 8000rpm for 10 minutes. The filtrate was collected. The residue was extracted once more. The filtrates were combined and transferred to a 20mL volumetric flask, methanol was added to the mark, and the mixture was shaken well. The filtrate was then filtered through a 0.22μm organic filter membrane to obtain the extract. The extract was analyzed using liquid chromatography-time-of-flight mass spectrometry (Sciex, Triple TOF6600+).

[0057] Liquid chromatography conditions: ACQUITY UPLC System; Mobile phase A: acetonitrile; Mobile phase B: 0.1% formic acid; Gradient elution program: 0-12 min, 7-18% A; 12-18 min, 18-22% A; 18-33 min, 22-25% A; 33-37 min, 25-40% A; 37-42 min, 40-85% A; 42-48 min, 85-100% A. Flow rate: 1.0 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Detection wavelength: 280 nm.

[0058] The mass spectrometry detection conditions were as follows: mass spectrometer TripleTOF 6600; ion source temperature: 550.0℃; polarity mode: positive ion mode; ion source parameters: CUR 35.000; GS1 55.000; GS2 55.000; ISVF 5500.000.

[0059] First-order mass spectrometry (TOF MS): mass range m / z 100.0 ~ 2000.0; collision energy CE 10.000; declustering voltage DP 80.000; cycle time 840 ms; pulse frequency 15.165 kHz; accumulation time 150.0 ms.

[0060] Secondary mass spectrometry (TOF MS²): mass range m / z 50.0 ~ 1500.0; collision energy CE 40.000; collision energy broadening CES 20.000; declustering voltage DP 80.000; cycle time 840 ms; pulse frequency 15.165 kHz; accumulation time 80.0 ms.

[0061] The relative ion abundances detected by LC-MS / MS were compared with those of standards (dehydrocorydaline, corydaline A, corydaline B, palmatine, tetrahydroberberine, dihydroberberine, tetrahydroberberine, and tetrahydrotetrahydroafricanine). Non-targeted metabolomics analysis was performed on metabolites in specific regions, and their chemical identities were confirmed by searching databases (such as HMDB, KEGG, and METLIN). Results are shown below. Figure 2 .

[0062] Based on untargeted metabolomics analysis, 159 metabolites were identified inside the Corydalis rhizome, 170 metabolites were identified in the outer skin, and 168 metabolites were identified in the intact rhizome. The levels of most alkaloids, including tetrahydrotetracycline, dehydrotetracycline, dihydroberberine, and berberine, were significantly higher in the outer skin than inside the rhizome, and also higher than in the intact rhizome samples, differing only in the multiples of increase. For example, the content of dehydrotetracycline in the outer skin was 5-6 times that inside the rhizome, and the content of corydaline in the outer skin was about twice that inside the rhizome. Therefore, we believe that some components that are only slightly increased or even decreased inside the rhizome may serve as candidate substrates to participate in the biosynthetic pathway and be transformed into substances that are more abundant in the outer skin.

[0063] 6. RNA-seq sequencing

[0064] Based on the results of step 5, several dozen fresh or quick-frozen Corydalis tubers were taken, rinsed thoroughly with running water and dried. The samples were placed on dry ice, and the outer skin and interior of the tuber were separated using a scalpel. The outer skin, interior, and intact tuber were chopped separately and placed in 2ml spiral cryovials. The tubers were then quick-frozen in liquid nitrogen for 15 minutes and then transferred to a -80℃ freezer for storage. These were the frozen tissues used in subsequent experiments.

[0065] Total RNA was extracted from tissue using the TRIzol kit (Invitrogen, CA, USA): 100 mg of the frozen tissue was added to a centrifuge tube, along with 1 mL of Trizol, and incubated at room temperature for 5 minutes; 0.2 mL of chloroform was added, and the centrifuge tube was vigorously shaken to mix thoroughly, and incubated at room temperature for 5-10 minutes; after centrifugation at 12,000 rpm for 15 minutes, the upper aqueous phase (70%) was transferred to a new centrifuge tube, taking care not to aspirate the protein material between the two aqueous phases. Transfer the solution to a new tube, add an equal volume of -20°C pre-chilled isopropanol, mix thoroughly by inverting, and place on ice for 10 minutes. Centrifuge at 12,000 rpm for 15 minutes, carefully discard the supernatant, and wash the precipitate with 75% DEPC (diethyl pyrocarbonate) ethanol solution at a ratio of 1 ml / 1 ml Trizol (store at 4°C). Vortex to mix, and centrifuge at 12,000 rpm for 5 minutes at 4°C. Discard the ethanol solution, allow the precipitate to air dry at room temperature for 5 minutes, and dissolve the precipitate in DEPC-water solution. RNA quality was then assessed. RNA purity was measured using a NanoDrop spectrophotometer (Thermo Scientific, DE, USA); RNA integrity was measured using an Agilent 2100 (Agilent Technologies, CA, USA); and RNA degradation was detected by gel electrophoresis. Transcriptome sequencing was then performed.

[0066] Based on the results of transcriptome sequencing, with the outer skin as the experimental group and the tuber interior and intact tubers as control groups, density maps and volcano maps were analyzed. The results are as follows: Figure 3 , Figure 4 Most genes expressed in the outer skin of Corydalis rhizome were significantly elevated compared to the inner tuber and intact tuber. Transcriptome analysis using a Hidden Markov Model (HMM) identified 43 potential genes with methyl transfer-related functions. Phylogenetic analysis of these 43 potential genes was performed using Evolview software and MEGAX software for 1000 bootstrap tests to generate a neighbor-linked phylogenetic tree, as shown below. Figure 5Eight candidate genes were selected (denoted as DN18486-c0-g2, DN22180-c0-g2, DN22180-c0-g1, DN21754-c2-g1, DN16233-c2-g5, DN16233-c2-g4, DN16233-c2-g2, and DN21814-c3-g3, respectively) for subsequent experiments. Based on the FPKM value (Fragments Per Kilobase of exon model per Million mapped fragments) of these eight candidate genes, i.e., the number of fragments per thousand bases of transcript per million mapped fragments), the candidate gene DN16233-c2-g5 with the high FPKM value was selected as the functional gene. This gene is highly expressed in the tuber outer skin but lowly expressed in the tuber interior. See [link to details]. Figure 6 .

[0067] 7. Prediction of the protein structure of functional gene DN16233-c2-g5

[0068] Predict the protein structure of the functional gene DN16233-c2-g5 using Alphafold3, such as Figure 7 The encoded enzyme is benzyl isoquinoline alkaloid synthase, denoted as benzyl isoquinoline alkaloid synthase 16233c2g5.

[0069] The benzyl isoquinoline alkaloid synthase 16233c2g5 gene (nucleotide sequence as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.1) was synthesized by Beijing Qingke Biotechnology Co., Ltd., and ligated with pET28a(+) after digestion with BamHI and HindIII to construct a recombinant plasmid. Figure 8 Transformed Escherichia coli BL21(DE3) competent cells, inoculated into TB medium, cultured at 37℃ for 20 h, and screened for positive clones.

[0070] TB culture medium composition: 1L purified water, 11.8g tryptone, 23.6g yeast extract, 4ml / L glycerol, 2.2g KH2PO4, 9.4g K2HPO4.

[0071] SEQ ID NO.1

[0072] SVDLQKTIDTDRLYRLMRYLVHLKFFTTEEGSDHGEIKYGLLPLAKFLIRGWPKSMAGLLTAMDKDFIAPWHHLEDGLDGRTDAFEVAFGKKVFDYLSENPKSSQLFNDYMAIHSSLLALELVKCKNVFEDRIKTLVDAGGGTTTAKAIANAFPHIKCMVYELPHVNADAPVDPNIQRIDGDIFKSVPKADAILMQGVLHDWNDGECIQILKNCRESIPQDGGKVILIEVVVNANSNHPYAKLMLLADLEMVIYGGKERTDEEWKKLLEAAGFTRYKLTEISAMQSIIEAYPY

[0073] SEQ ID NO.2

[0074] TCTGTTGACCTCCAAAAAACCATTGATACCGACCGTCTATATCGATTGATGCGGTATCTGGTTCACTTGAAGTTCTTTACAACAGAAGAAGGATCTGATCATTTAGGGGAAATCAAGTATGGGCTGTTGCCACTGGCAAAATTCTTAATCAGAGGATGGCCGAAGTCCATGGCTGGTTTGTTAACAGCTATGGATAAGGATTTCATTGCACCTTGGCATCATCTCGAGGATGGTTTGGATGGTCGCACCGACGCTTTCGAAGTAGCATTTGGGAAGAAAGTTTTTGATTATTTGTCCGAAAACCCCAAAAGCAGTCAGCTTTTCAATGATTATATGGCTATTCATAGTAGTCTCCTTGCTTTGGAGTTGGTTAAGTGTAAGAATGTTTTTGAAGACCGGATTAAAACACTTGTTGATGCTGGTGGTGGCACCGGAACAACTGCTAAAGCAATTGCCAACGCTTTCCCACACATAAAATGCATGGTTTATGAACTTCCTCACGTGAATGCGGATGCTCCCGTTGATCCTAATATCCAACGAATCGATGGGGATATTTTCAAGTCTGTTCCCAAGGCAGATGCAATTTTAATGCAGGGGGTCCTCCACGATTGGAATGATGGCGAATGTATTCAAATATTAAAGAATTGCAGAGAATCAATACCCCAAGATGGAGGAAAAGTTATCCTCATTGAAGTAGTGGTGAATGCGAATTCAAATCATCCTTATGCAAAACTGATGTTATTGGCGGATTTAGAAATGGTGATCTACGGAGGGAAGGAGAGAACCGACGAGGAGTGGAAGAAACTACTCGAAGCTGCTGGTTTCACTAGATACAAACTCACTGAAATATCAGCCATGCAATCTATAATCGAAGCTTATCCTTATTAA

[0075] Example 2: Preparation of benzylisoquinoline alkaloid synthase enzyme solution

[0076] 1. Preparation of recombinant Escherichia coli: Take 15-25 μl of the positive clone culture from Example 1 in a glycerol tube and add it to 20 ml of LB medium containing 100 mg / L kanamycin (Kan) resistance. Incubate overnight at 37°C and 200 rpm on a shaker. Add 1.5 ml of the culture to centrifuge tubes, centrifuge at 12000 g for 1 min, and discard the supernatant.

[0077] 2. Plasmid Extraction: Plasmids were extracted using the Axyprep Plasmid Mini-Preparation Kit (Ag. AP-MN-50G). The specific steps are as follows: Add 250 μl of Buffer S1 to the precipitate from step 1 to suspend the bacterial precipitate. Ensure the suspension is uniform. Then add 250 μl of Buffer S2 and gently and thoroughly invert the container 4-6 times to mix thoroughly until the bacteria are fully lysed, forming a clear solution. This step should not exceed 5 minutes. Add 350 μl of Buffer S3 and gently and thoroughly invert the container 6-8 times. Centrifuge at 12000g for 10 minutes. Transfer the supernatant to a preparation tube and centrifuge at 12000g for 1 minute, discarding the supernatant. Place the preparation tube back into the centrifuge tube, add 500 μl of Buffer W1, centrifuge at 12000g for 1 minute, and discard the supernatant. Place the preparation tube back into the centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12000g for 1 min, and discard the supernatant. Wash again with 700 μl of Buffer W2 in the same manner, and discard the filtrate. Place the preparation tube back into a 2 ml centrifuge tube and centrifuge at 12000g for 1 min. Transfer the preparation tube to a new 1.5 ml centrifuge tube, add 60-80 μl of Eluent or deionized water to the center of the preparation tube, heat to 65°C, and let stand at room temperature for 1 min. Centrifuge at 12000g for 1 min to obtain the plasmid solution.

[0078] 3. Recombinant Escherichia coli: Incubate the plasmid solution and competent Escherichia coli (BL21)(DE3) cells separately on ice for 30 min. Then, add 10-15 μl of plasmid to the competent cells, incubate on ice for another 30 min, followed by a water bath at 42℃ for 90 seconds, then on ice for 3 min. Add 900 μl of LB medium to the tube, and incubate at 37℃ and 200 rpm for 1-2 hours. Centrifuge at 8000g for 5 min, discard most of the supernatant, and repeatedly aspirate and resuspend the solution until homogeneous. Spread the solution evenly onto LB agar plates containing 100 mg / L kanamycin. Seal the plates with a sealing film and incubate at 37℃ for 30 min (positive incubation), then reverse incubate until the next day. Recombinant Escherichia coli colonies are obtained.

[0079] 4. Shake flask fermentation: Remove the culture dish from overnight culture, scrape a single colony from the plate using a pipette tip, transfer it to 20 ml of LB medium, and mix well. Shake at 37°C and 200 rpm for 10-12 hours. After shaking, observe the culture status; the color should be uniform and turbid. Add 3 ml of the shaker-cultured bacterial solution to 100 ml of TB medium containing 100 mg / L kanamycin. Incubate at 37°C and 200 rpm for 1.5-2.5 h until the OD600 value reaches 0.5-0.8. Then add 100 mM IPTG to the final concentration and induce culture at 20°C and 200 rpm for 20 h. Centrifuge at 8000 rpm and 4°C for 10 min, discard the supernatant, add 10-20 ml of physiological saline to the precipitate, mix well, transfer to a 50 ml centrifuge tube, centrifuge at 8000 rpm and 4°C for 10 min, discard the supernatant, weigh the precipitate, and add 2-5 ml of non-denaturing lysis buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% HCl) per 1 g of precipitate. The precipitate was dissolved in glycerol, and then the bacterial cells were disrupted using an ultrasonic disruptor (70W, 20min, 3s on, 7s off). The centrifuge tubes were kept at a low temperature using an ice-water mixture. After complete disruption, the cells were centrifuged at 8000rpm and 4℃ for 10min, and the supernatant was collected to obtain the enzyme solution.

[0080] 5. Enzyme Solution Detection: The enzyme solution was detected using an SDS-PAGE gel preparation kit (Beyotime, product number P0012A). The specific steps are as follows: Take 64 μL of enzyme solution and add 16 μL of 5× Protein SDS-PAGE Loading Buffer. Heat at 100℃ or in a boiling water bath for 10 minutes to fully denature the protein, then allow it to cool. Prepare 10 mL of 10% SDS-PAGE lower gel solution (4.1 mL distilled water, 3.3 mL 30% Acr-Bis, 2.5 mL lower gel buffer, 100 μL 10% gel polymerization catalyst, 4 μL TEMED) according to the instructions. Pour the solution into the glass plate interlayer to 2 cm from the top, add 1 mL of isopropanol to isolate it from air, and let it stand at room temperature for 30 min until complete polymerization. Discard the isopropanol and prepare 4 mL of upper gel solution (2.33 mL distilled water, 670 μL 30% Acr-Bis, 1 mL upper gel buffer, 40 μL 10% gel polymerization catalyst, 2 μL TEMED). Fill the glass plate interlayer to the brim, insert the 10-well comb, and let it stand at room temperature for 30 min until completely solidified. Place the gel in the electrophoresis tank, pour in electrophoresis buffer to the corresponding mark, pull out the 10-well comb parallel to the grain, and add the sample to the loading wells, with a loading volume of 10 μL; leave one well for adding protein marker. Perform SDS-PAGE gel electrophoresis, stain with Coomassie Brilliant Blue, and then destain. The results are as follows. Figure 9 Lane 1 represents the marker, lane 2 represents a loading volume of 15 μL of enzyme solution, and lane 3 represents a loading volume of 10 μL of enzyme solution.

[0081] Example 3: In vitro enzyme activity analysis method for benzyl isoquinoline alkaloid synthase 162332c2g5

[0082] The 11 mL reaction system consisted of: 4.8 mL of enzyme solution prepared according to the method in Example 2 (enzyme concentration was 1.1 mg / mL based on protein content), 3.2 mL of palmatine (dissolved in pH 7, 100 mM K₂HPO₄-KH₂PO₄ buffer, initial concentration of 100 mM), 300 μL of S-adenosylmethionine (dissolved in pH 7, 100 mM K₂HPO₄-KH₂PO₄ buffer, concentration of 28 mM), and 2.7 mL of sodium vitamin C (dissolved in pH 7, 100 mM K₂HPO₄-KH₂PO₄ buffer, concentration of 100 mM). The reaction was incubated at 37°C and 200 rpm for 5 h. After incubation, an equal volume of methanol was added to quench the reaction. The mixture was centrifuged at 12000 g at 4°C for 10 min. After filtering through a 0.22 μm filter, the product peaks and ion information were detected by LC-MS and compared with standards to calculate enzyme activity. Mass spectra of the reaction solution, blank control (without enzyme solution), and dihydroberberine (DHB) standard are shown in [reference needed]. Figure 10 .

[0083] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmmol of product from the substrate per minute under reaction conditions of pH 7 and 37°C. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmmol of product from the substrate per minute.

[0084] The instrument for LC-MS is an Agilent 1290 Infinity II liquid chromatography system + 6545 Q-TOF mass spectrometer.

[0085] The HPLC detection conditions for in vitro enzyme activity analysis were as follows: mobile phase A was acetonitrile; mobile phase B was 0.1% formic acid; elution gradient: 0–12 min, 7–18% A; 12–18 min, 18–22% A; 18–33 min, 22–25% A; 33–37 min, 25–40% A; 37–42 min, 40–85% A; 42–48 min, 85–100% A. Flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 10 μL; detection wavelength: 280 nm. Sample solvent: methanol.

[0086] The mass spectrometry conditions were as follows: positive ion mode and AutoMS2 acquisition mode were used; the mass-to-charge ratio (m / z) scan range for both MS and MS / MS was set to 100-1000; the scan rate was 1.00 spectra / second; the MS / MS isolation width was set to medium (~4 amu); the collision energy was fixed at 20.00; the maximum number of precursors per cycle was 2; a peptide isotope model was used; dynamic exclusion was disabled; and precursors were sorted by abundance. Ion source parameters were set as follows: gas temperature 300℃, gas flow rate 8 L / min, nebulizer pressure 35 psig, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, capillary voltage 3100 V, nozzle voltage 1000 V, and fragmenter voltage 175 V.

[0087] Example 4: Optimization of reaction conditions for benzyl isoquinoline alkaloid synthase 162332c2g5

[0088] 1. Optimal reaction pH value

[0089] The reaction system and detection method of Example 3 were used, except that the pH was changed to 4, 5, 6, 7, 8, 9, and 10 respectively. All other operations remained the same. The pH values ​​were 4.0-6.0 (citric acid-sodium citrate buffer), 6.0-8.0 (K₂HPO₄-KH₂PO₄ buffer), 6.5-8.5 (Tirs-HCl buffer), and 9.0-11.0 (Na₂CO₃-NaHCO₃ buffer). The relative enzyme activity was calculated with optimal enzyme activity as 100%. The Origin graph is shown below. Figure 11 The optimal pH is 7.0.

[0090] 2. Optimal reaction temperature

[0091] The reaction system and detection method of Example 3 were used, except that the temperature was set to 4, 12, 20, 37, 50, and 60°C. All other operations were the same. The relative enzyme activity was observed... Figure 12 The optimal temperature is 37℃.

[0092] 3. The effect of divalent metal ions on enzyme activity

[0093] The reaction system and detection method of Example 3 were used, except that divalent metal ions: FeSO4 and MnCl4 were added to a final concentration of 5 mM. - NiCl - SrCl - CaCl - BaCl ￣ MgCl ￣ CoCl - CuSO4 and ZnSO4. Other procedures are the same; see relative enzyme activity... Figure 13The results showed that divalent metal ions had no significant effect on the enzyme activity.

[0094] The results showed that the 16233c2g5 enzyme exhibited different reactivity in different reaction buffers and at different reaction temperatures. It showed the highest reactivity at 37℃ and pH=7 (K2HPO4-KH2PO4 buffer).

[0095] Example 5: Biosynthesis of dihydroberberine

[0096] The 11ml reaction system consisted of: 4.8ml enzyme solution (enzyme concentration of 1.1mg / mL based on protein content), 3.2ml palmatine (dissolved in pH 7, 100mM K2HPO4-KH2PO4 buffer, initial concentration of 100mM), 300μL adenosylmethionine (dissolved in pH 7, 100mM K2HPO4-KH2PO4 buffer, concentration of 28mM), and 2.7ml sodium vitamin C (dissolved in pH 7, 100mM K2HPO4-KH2PO4 buffer, concentration of 100mM). The reaction was incubated at 37℃ and 200rpm for 5 hours. After incubation, an equal volume of methanol was added to quench the reaction. The mixture was centrifuged at 12000g at 4℃ for 10 minutes. After filtering through a 0.22μm filter, the mass spectrum was analyzed using the LC-MS method described in Example 3. (See attached image). Figure 14 The product yield was 7.13%.

Claims

1. A method for efficiently screening functional enzymes, characterized in that, The method includes the following steps: (1) Sample preparation: freezing and slicing Corydalis tubers to prepare samples; (2) Covering matrix: using α-cyano-4-hydroxycinnamic acid as the main matrix and 9-aminoacridine as the auxiliary matrix, the sample in step (1) is sprayed using a matrix sprayer; (3) Data acquisition: using matrix-assisted laser desorption / ionization imaging technology to acquire mass spectrometry signals with different m / z values; after imaging, the slices are stained with H&E or Nissl stain, and tissue morphology images are obtained by taking pictures under a microscope; (4) Data processing: the raw data of the mass spectrometry signals acquired in step (3) are processed by SCiLS. Lab software was used for preprocessing, including background subtraction and signal normalization. MsiReader software was used for image reconstruction and spatial registration analysis with tissue morphology images to make the distribution of metabolite signals correspond precisely with the tissue morphology structure, realize the visualization mapping of the spatial distribution of metabolites, and accurately delineate the target area with high metabolic activity; (5) Non-targeted metabolomics analysis: Corydalis part corresponding to the single target area delineated in step (4) was extracted with 75% methanol aqueous solution. The relative ion abundance of the extract was detected by LC-MS / MS and compared with metabolite standards. The chemical structure of metabolites was confirmed by database retrieval. Metabolites with large distribution variations in different parts were selected as candidate substrates; (6) Transcriptomics analysis: RNA was extracted from the tissue where the candidate substrates in step (5) were located and transcriptome sequencing was performed. The transcriptome sequencing results of other parts were used as a control. Hidden Markov model was used to analyze the transcriptome results to screen potential functional genes. A phylogenetic tree of potential functional genes was constructed to screen candidate genes. According to the FPKM value of the candidate genes, the functional enzyme gene with the high FPKM value was selected.

2. The method as described in claim 1, characterized in that, Step (1) Samples are prepared as follows: Fresh or quick-frozen Corydalis tubers are selected as the research object. They are quickly frozen in liquid nitrogen and stored in an environment of -80℃. Then, continuous slices with a thickness of 8-12 μm are prepared using a cryostat to obtain Corydalis tuber slices as samples.

3. The method as described in claim 1, characterized in that, Step (2) α-Cyano-4-hydroxycinnamic acid was prepared into a 10 mg / mL solution using acetonitrile-0.1% trifluoroacetic acid at a volume ratio of 1:1, and 9-aminoacridine was prepared into a 5 mg / mL solution using methanol-water at a volume ratio of 7:

3. After filtration through a 0.22 μm filter membrane, the samples from step (1) were sprayed using a Bruker Autoflex Speed ​​matrix sprayer. The spraying parameters for α-cyano-4-hydroxycinnamic acid were: pressure 0.3 MPa, nozzle distance 3 cm from the sample, speed 5 mm / s, 6 sprays with an interval of 30 s. The spraying parameters for 9-aminoacridine were adjusted to: pressure 0.25 MPa, speed 4 mm / s, 8 sprays with an interval of 40 s. The spraying environment was 25℃ and 40% humidity.

4. The method as described in claim 1, characterized in that, Step (3) Data acquisition is performed using a time-of-flight mass spectrometer with a MALDI source; the sample covered by the matrix in step (2) is placed in the mass spectrometry imaging system, and the 10kHz laser scanning technology of SmartBeam 3D is used to perform the analysis in qTOF mode with a third-harmonic Nd:YAG laser; the pixel size is set to 50 μm, each pixel is used for 200 laser shots, and 35% of the laser power is at 10 kHz; in positive ion mode, high-resolution and high-sensitivity spatial scanning is performed from m / z 200-2000 to acquire mass spectrometry signals of different m / z values ​​and obtain the raw mass spectrometry signal data.

5. The method as described in claim 1, characterized in that, The metabolites in step (4) include benzyl isoquinoline alkaloids.

6. The method as described in claim 1, characterized in that, Step (5) LC-MS / MS detection method: ACQUITY UPLC system; mobile phase A is acetonitrile; mobile phase B is 0.1% formic acid; liquid phase gradient elution program: 0-12 min, 7-18% A; 12-18 min, 18-22% A; 18-33 min, 22-25% A; 33-37 min, 25-40% A; 37-42 min, 40-85% A; 42-48 min, 85-100% A; flow rate is 1.0 mL / min, column temperature is 30℃, injection volume is 10 μL, detection wavelength is 280 nm; mass spectrometer: TripleTOF 6600, ion source temperature: 550.0℃, polarity mode: positive ion mode; ion source parameters: CUR 35.000; GS1 55.000; GS2 55.000; ISVF 5500.000; Primary mass spectrometry: mass range m / z 100.0 ~ 2000.0; collision energy CE 10.000; declustering voltage DP 80.000; cycle time 840 ms; pulse frequency 15.165 kHz; accumulation time 150.0 ms; Secondary mass spectrometry: mass range m / z 50.0 ~ 1500.0; collision energy CE 40.000; collision energy broadening CES 20.000; declustering voltage DP 80.000; cycle time 840 ms; pulse frequency 15.165 kHz; accumulation time 80.0 ms.

7. A benzylisoquinoline alkaloid synthase screened by the method of claim 1, characterized in that, The amino acid sequence of the benzyl isoquinoline alkaloid synthase is shown in SEQ ID NO.

1.

8. The application of a benzylisoquinoline alkaloid synthase screened by the method of claim 1 in the catalytic synthesis of dihydroberberine from palmatine.

9. The application as described in claim 8, characterized in that, The application is as follows: using the enzyme solution extracted from the engineered bacteria expressing the benzylisoquinoline alkaloid synthase after induction as a catalyst, using berberine as a substrate, adding adenosylmethionine and sodium vitamin C, and using a buffer solution with pH 4-10 as the reaction medium to form a reaction system, and reacting at 4-60℃ and 200rpm in a shaker for 5-10h to obtain a reaction solution containing dihydroberberine.

10. The application as described in claim 9, characterized in that, In the reaction system, the concentration of the substrate is 50-125 mM; the concentration of S-adenosylmethionine is 20-40 mM; the concentration of sodium vitamin C is 50-125 mM; and the amount of catalyst added is 0.5-1.5 g / L based on protein content.

Images