CYP450 hydroxylase for catalyzing biosynthesis of DNJ and application of CYP450 hydroxylase

Abstract

The invention discloses CYP450 hydroxylase for catalyzing biosynthesis of DNJ and application of the CYP450 hydroxylase, and belongs to the field of medicinal plant genetic engineering. An enzyme catalysis experiment shows that the compound participates in a hydroxylation process of a DNJ alkaloid biosynthetic pathway, namely, C4-site stereoselective hydroxylation of (R)-2-methylpiperidine is catalyzed to generate (2R, 4R)-2-methylpiperidine-4-alcohol. The invention also relates to a gene for coding the enzyme, a cloning vector and an expression vector containing the gene, and a recombinant engineering bacterium containing the expression vector, which can be used for producing (2R, 4R)-2-methylpiperidine-4-alcohol. The method promotes the analysis of the biosynthetic pathway process of DNJ in mulberry leaves, and has important economic value and application prospect for realizing heterologous synthesis of DNJ and key intermediates.

Description

Technical Field

[0001] The present invention clones cytochrome P450 hydroxylase MaCYP71BG22 and its encoding product from mulberry leaf cDNA for the first time, which can catalyze the stereoselective hydroxylation of (R)-2-methylpiperidine at the C4 position to generate (2R, 4R)-2-methylpiperidin-4-ol. The hydroxylase involved can be used for the biosynthesis and application of 1-deoxynojirimycin (DNJ) in mulberry leaves, and belongs to the field of medicinal plant genetic engineering. Background Art

[0002] Mulberry leaf is the dried leaf of Morus alba L., a plant of the Moraceae family. It has the effects of dispersing wind-heat, clearing the lungs and moistening dryness, clearing the liver and improving eyesight. As a traditional Chinese medicine in my country, mulberry leaf has been used by Chinese medicine practitioners to treat diabetes since ancient times, which is diabetes in modern medicine. Modern pharmacological studies have shown that the main active ingredient of mulberry leaf for the prevention and treatment of diabetes is a polyhydroxy alkaloid component mainly composed of 1-deoxynojirimycin (DNJ). DNJ, as a potent α-glycosidase inhibitor, can significantly inhibit the increase of blood sugar, and also has the effects of lowering blood lipids, reducing atherosclerosis, anti-oxidation, anti-virus and anti-tumor. Therefore, DNJ has important and extensive application value in the design of new drugs for the prevention and treatment of diabetes and anti-viral infections, as well as health foods. However, the content of DNJ polyhydroxy alkaloids from natural sources is very low. The highest content in mulberry is only 0.1%-0.5%, which is far from meeting the current market demand. Chemical synthesis has problems such as difficulty in isomer separation and low yield, which seriously restricts the development and application of DNJ polyhydroxy alkaloids in the fields of medicine and food. Based on this, how to improve the sustainable utilization of mulberry medicinal plant resources or efficiently accumulate and produce DNJ is an urgent problem to be solved. In recent years, by analyzing the biosynthetic pathway of medicinal active ingredients, using the principles and strategies of synthetic biology, designing and constructing new biological systems, and thus establishing a new pathway for the production of natural products with high added value, it has been proven to be a very promising method, but the biosynthetic pathway of DNJ alkaloids has not yet been fully analyzed.

[0003] DNJ belongs to the piperidine polyhydroxy alkaloids with the structural formula of (2R,3R,4R,5S)-2-hydroxymethylpiperidine-3,4,5-triol. It has a hydroxymethyl group at the C2 position and a hydroxyl group at the C3, C4, and C5 positions respectively. The configuration of the hydroxyl group is the main site for the manifestation of its pharmacological activity. The research team initially explored the biosynthetic pathway of DNJ, excavated the key functional enzyme genes in the biosynthetic pathway, and analyzed the biosynthetic pathway from L-lysine to 2-methylpiperidine, that is, L-lysine is decarboxylated by lysine decarboxylase (LDC) to produce cadaverine, which is oxidized and deaminated by copper amine oxidases (CAO) to form 5-aminovaleraldehyde, which then automatically cyclizes to form 1-piperidinene, which is reduced by short-chain dehydrogenase / reductases (SDRs) to form piperidine, which is formed by methyltransferases (MTs) to form 2-methylpiperidine, which is then stereoselectively hydroxylated by cytochrome P450 enzymes (CYP450s) to form DNJ. However, the hydroxylation process in the downstream pathway of DNJ biosynthesis has not been reported. Exploring the CYP450s hydroxylases in the DNJ biosynthesis pathway and fully analyzing its synthesis pathway can provide a new way to alleviate the shortage of DNJ resources.

[0004] The present invention clones a MaCYP71BG22 hydroxylase from mulberry leaves for the first time. The in vitro functional verification results show that the MaCYP71BG22 hydroxylase can use (R)-2-methylpiperidine as a substrate and stereoselectively catalyze the hydroxylation reaction at the C4 position thereof to generate (2R, 4R)-2-methylpiperidin-4-ol. The hydroxylase involved can be used for the biosynthesis and application of DNJ in mulberry leaves. Before the present invention was published, there had been no disclosure or report on the mulberry leaf MaCYP71BG22 hydroxylase and its application mentioned in the present patent application. Summary of the invention

[0005] The present invention clones a novel cytochrome P450 hydroxylase (or mulberry leaf cytochrome P450 oxidase, hereinafter referred to as MaCYP71BG22) from mulberry leaves. The hydroxylase participates in the biosynthesis pathway of DNJ and can catalyze the stereoselective hydroxylation of C4 of (R)-2-methylpiperidine to generate (2R, 4R)-2-methylpiperidin-4-ol.

[0006] In a first aspect of the present invention, a MaCYP71BG22 hydroxylase gene is provided, and the nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0007] In a second aspect, the present invention provides the hydroxylase encoded by the above-mentioned MaCYP71BG22 gene, wherein the amino acid sequence of the hydroxylase encoded is shown in SEQ ID NO:2.

[0008] The variant forms of MaCYP71BG22 hydroxylase in the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and hydroxylases encoded by DNA that can hybridize with the MaCYP71BG22 hydroxylase encoding gene under high or low stringency conditions.

[0009] In the present invention, the terms "MaCYP71BG22 protein encoding gene" or "MaCYP71BG22 gene" can be used interchangeably; the terms "MaCYP71BG22", "cytochrome P450 hydroxylase MaCYP71BG22", and "MaCYP71BG22 protein" can be used interchangeably. The terms "nucleotide molecule" or "nucleotide sequence" can be used interchangeably.

[0010] The third aspect of the present invention provides an expression vector comprising the polynucleotide described in the second aspect of the present invention. The expression vector may be an expression vector selected from the pESC series.

[0011] In the present invention, various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, etc., can be selected. When producing the MaCYP71BG22 hydroxylase of the present invention, the nucleotide sequence of the MaCYP71BG22 hydroxylase encoding gene can be operably connected to the expression control sequence to form a MaCYP71BG22 hydroxylase expression vector. The "operably connected" when referring to DNA segments means that these segments are arranged in a certain way so that they can coordinately play their intended purpose, such as starting transcription in the promoter and moving forward through the coding segment to the terminator. It also refers to such a situation: that is, some parts of the linear DNA sequence can affect the activity of other parts of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a prerequisite and participates in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably connected to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably connected to the coding sequence; if the ribosome binding site is placed in a position that enables its translation, then it is operably connected to the coding sequence. Generally "operably linked" means contiguous, and, with respect to a secretory leader, means contiguous and in reading frame.

[0012] In a fourth aspect, the present invention provides a recombinant host bacteria comprising the polynucleotide described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention, wherein the host bacteria is a DH5α strain, a yeast such as a WAT ​​yeast or a plant cell.

[0013] According to ordinary methods, in a culture medium containing nutrients and other necessary components for the growth of selected host cells, the transformed or transfected host cells are cultivated. A variety of suitable culture mediums, including known component culture mediums and complex culture mediums, are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins and minerals. If necessary, the culture medium can also contain components such as growth factors or serum. Growth mediums are generally selected to contain cells containing exogenous added DNA by, for example, drug screening or lacking the necessary nutrients supplemented by the selection markers carried by the expression vector or cotransfected into the host cell. By conventional means, sufficient air is provided to liquid cultures as shaking triangular flasks.

[0014] In a fifth aspect of the present invention, a microsomal protein is obtained from the recombinant MaCYP71BG22 hydroxylase of Saccharomyces cerevisiae WAT11, which can catalyze the stereoselective hydroxylation of (R)-2-methylpiperidine at the C4 position to produce (2R, 4R)-2-methylpiperidin-4-ol. The subsequent step also includes a GC-MS analysis of the reaction product, wherein the product peak appearing at 11.98 minutes is (2R, 4R)-2-methylpiperidin-4-ol.

[0015] In a sixth aspect of the present invention, there is provided a method for preparing a (2R, 4R)-2-methylpiperidin-4-ol compound, which is carried out according to the following steps:

[0016] (1) inserting the MaCYP71BG22 hydroxylase gene into the eukaryotic expression vector pESC-TRP by restriction endonuclease and DNA ligase to prepare a recombinant yeast expressing the enzyme;

[0017] (2) (R)-2-methylpiperidine is used as a substrate and added to the culture medium of the recombinant yeast of the enzyme MaCYP71BG22 obtained in step (1) for shake flask fermentation. The fermentation medium is a SG-TRP medium containing 2% galactose (100 mL of SG-TRP liquid culture medium contains 2 g of galactose) (1 L of SG-TRP liquid culture medium contains 6.70 g of YNB (yeast nitrogen base), 0.10 g of L-arginine, 0.10 g of L-cysteine, and 0. 10g, L-threonine 0.10g, L-asparagine 0.05g, L-isoleucine 0.05g, L-phenylalanine 0.05g, L-proline 0.05g, L-serine 0.05g, L-tyrosine 0.05g, L-valine 0.05g, L-methionine 0.05g, L-histidine 0.10g, L-leucine 0.10g, uracil 0.10g, adenine 0.10g), thereby preparing (2R, 4R)-2-methylpiperidin-4-ol compounds;

[0018] After step (2) is completed, the method further comprises performing LC-MS analysis on the reaction product, wherein the product peak appearing at 17.12 minutes is (2R, 4R)-2-methylpiperidin-4-ol.

[0019] The MaCYP71BG22 hydroxylase and the gene encoded by the MaCYP71BG22 hydroxylase provided by the present invention are cloned and prepared from mulberry leaves for the first time, and catalyze the stereoselective hydroxylation of (R)-2-methylpiperidine at the C4 position to generate (2R, 4R)-2-methylpiperidin-4-ol. The MaCYP71BG22 hydroxylase of the present invention can be used to provide key gene elements for heterologous synthesis of DNJ in chassis cells using synthetic biology strategies, providing a new way to alleviate the resource shortage problem of DNJ. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the agarose gel electrophoresis diagram of mulberry leaf MaCYP71BG22 gene cloning; Lane M represents DNA Marker (nucleic acid molecular weight standard, the bands are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100 bp from top to bottom); Lanes 1-3 represent MaCYP71BG22 gene.

[0021] Figure 2 It is the agarose gel electrophoresis diagram of PCR verification of MaCYP71BG22 / pMD18-T / DH5α bacterial solution; Lane M represents DNA Marker (nucleic acid molecular weight standard, the bands are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp from top to bottom); Lanes 1-3 represent MaCYP71BG22 / pMD18-T / DH5α bacterial solution.

[0022] Figure 3 It is the agarose gel electrophoresis diagram of PCR verification of MaCYP71BG22 / pESC-TRP / DH5α bacterial solution; Lane M represents DNA Marker (nucleic acid molecular weight standard, the bands are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp from top to bottom); Lanes 1-4 represent MaCYP71BG22 / pESC-TRP / DH5α bacterial solution.

[0023] Figure 4It is the agarose gel electrophoresis diagram of PCR verification of MaCYP71BG22 / pESC-TRP / WAT11 bacterial solution; Lane M represents DNA Marker (nucleic acid molecular weight standard, the bands are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp from top to bottom); Lane 1 represents MaCYP71BG22 / pESC-TRP / WAT11 bacterial solution.

[0024] Figure 5 Verification of the in vitro enzyme activity of MaCYP71BG22. (A) GC-MS image of the catalysis of (R)-2-methylpiperidine to (2R,4R)-2-methylpiperidin-4-ol by MaCYP71BG22 microsomal protein. (B) Mass spectrum of the hydroxylation product of (R)-2-methylpiperidine.

[0025] Figure 6 LC-MS analysis of the products of the whole-cell catalytic system of MaCYP71BG22. (A) LC-RID chromatogram of the yeast fermentation broth extracted by feeding the recombinant yeast expressing MaCYP71BG22 with (R)-2-methylpiperidine as substrate. (B) Mass spectrum of the hydroxylation products of (R)-2-methylpiperidine. (C) High-resolution mass spectrum of the hydroxylation products of (R)-2-methylpiperidine.

[0026] Figure 7 (2R,4R)-2-methylpiperidin-4-ol 1 H NMR spectrum (400 MHz, solvent DMSO-d6).

[0027] Figure 8 (2R,4R)-2-methylpiperidin-4-ol 13 C NMR spectrum (101 MHz, solvent DMSO-d6).

[0028] Fig. 9 This is the HSQC spectrum of (2R,4R)-2-methylpiperidin-4-ol (solvent DMSO-d6).

[0029] Fig.10 This is the COSY spectrum of (2R,4R)-2-methylpiperidin-4-ol (solvent DMSO-d6).

[0030] Fig.11 This is the HMBC spectrum of (2R,4R)-2-methylpiperidin-4-ol (solvent DMSO-d6).

[0031] Fig.12 This is the NOESY spectrum of (2R,4R)-2-methylpiperidin-4-ol (solvent DMSO-d6). DETAILED DESCRIPTION

[0032] The specific implementation of the present invention is described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific implementation. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0033] The fresh mulberry leaves in the following examples were picked from the mulberry garden of Jiangsu University (32°12′13.50″N, 119°30′45.26″E, Zhenjiang, China); restriction endonucleases XhoⅠ, KpnⅠ and T4 DNA Ligase were purchased from Yangzhou Borui Biotechnology Co., Ltd.; TRIzol reagent, pMD18-T cloning vector, and DH5α Escherichia coli were all purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; gel recovery kit and plasmid extraction kit were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; DreamTaqDNA Polymerase and Revert Aid First Strand cDNA Synthesis Kit were purchased from Thermo Fisher Scientific; pESC-TRP vector was purchased from Hunan Fenghui Biotechnology Co., Ltd.; WAT11 was purchased from Beijing Coolbo Technology Co., Ltd.; Frozen-EZ Yeast Transformation II Kit TM Purchased from Zymo Research Biotechnology Company, USA.

[0034] Example 1 Cloning of the MaCYP71BG22 Hydroxylase Gene from Mulberry Leaves

[0035] The specific experimental operations are as follows:

[0036] (1) Primer Primer 5.0 software was used to design specific primers for the ORF region of the MaCYP71BG22 hydroxylase gene, as shown in Table 1.

[0037] Table 1 Specific primers for MaCYP71BG22 hydroxylase gene

[0038]

[0039] (2) The total RNA of mulberry leaves was extracted using the TRIzol reagent method, and the cDNA obtained by reverse transcription using the Revert Aid First Strand cDNA Synthesis Kit was used as a template. PCR amplification was performed using the specific primers designed above. The reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0040] Table 2 PCR reaction system

[0041]

[0042] Table 3 PCR reaction conditions

[0043]

[0044] (3) The above PCR products were detected by 1.5% agarose gel electrophoresis, and the bands of the correct size were cut out. The amplified products were recovered by gel electrophoresis according to the instructions of the gel recovery kit of Shanghai Sangon Biotechnology Co., Ltd., and then the recovered products were connected to the cloning vector according to the instructions of the pMD18-T vector ligation kit, and transformed into DH5α competent cells, plated, and the positive clones were sequenced for verification.

[0045] Example 1 Test results:

[0046] (1) Cloning of the MaCYP71BG22 hydroxylase gene from mulberry leaves

[0047] According to the CDS sequence of the MaCYP71BG22 hydroxylase gene in the transcriptome sequence, the cDNA obtained by reverse transcription of mulberry leaf total RNA was used as a PCR template, and MaCYP71BG22-F and MaCYP71BG22-R were used as specific primers for PCR amplification. The product had a single bright band at around 1500 bp, which was consistent with the expected size. The results are shown in Figure 1 The amplified gene sequence SEQ ID NO:1 was submitted to the CYP450 Nomenclature Committee and named MaCYP71BG22. The ORF of the gene is 1446 bp, encoding 481 amino acid residues. The amino acid sequence of the hydroxylase is SEQ ID NO:2.

[0048] (2) PCR verification of MaCYP71BG22 / pMD18-T bacterial culture

[0049] MaCYP71BG22-T was verified by PCR using universal primers M13-F and M13-R of pMD18-T vector. Figure 2 As shown. The bacterial samples all had a single bright band at around 1500bpp, which was consistent with the expected size, indicating that the target fragment was successfully connected to the pMD18-T vector. The cloning vector was named MaCYP71BG22 / pMD18-T, and the cloning strain was named MaCYP71BG22 / pMD18-T / DH5α, and was stored in a -80℃ refrigerator for future use.

[0050] (3) Sequencing and alignment of MaCYP71BG22-T

[0051] The sequencing results of MaCYP71BG22-T showed that the MaCYP71BG22 hydroxylase gene had a complete ORF reading frame, with an ORF length of 1446 bp and a CDS sequence length of 1443 bp. The coverage of the mulberry leaf transcriptome sequence c21932_g1 and MaCYP71BG22-T was 100%, and the similarity was 99.86%

[0052] Example 2 In vitro functional study of mulberry leaf MaCYP71BG22 hydroxylase

[0053] The specific experimental operations are as follows:

[0054] 1. Construction of pESC-TRP (MaCYP71BG22 / pESC-TRP) eukaryotic expression vector

[0055] (1) The strain preserved in step (4) of Example 1 and the purchased pESC-TRP / DH5α were streaked and activated on LB solid culture medium containing 100 ug / mLAmp (ampicillin) antibiotics, and single colonies were selected for culture. The single colonies were identified by PCR using pESC-TRP's own primers GalⅠ-F and GalⅠ-R. The positive single colonies successfully identified were expanded for culture, and plasmids were extracted from the MaCYP71BG22 / pMD18-T / DH5α and pESC-TRP / DH5α bacterial suspensions with reference to the plasmid extraction kit, and their concentrations were determined.

[0056] Table 4 GalⅠ specific primers

[0057]

[0058] (2) The plasmid extracted in step (1) of Example 2 was double-digested with restriction endonucleases XhoⅠ-KpnⅠ. The digestion system is shown in Table 3. The double-digested products were then gel-recovered using a SanPrep column DNA gel recovery kit. The target gene was recovered as a small fragment, and the vector was recovered as a large fragment.

[0059] Table 5 Double enzyme digestion reaction system

[0060]

[0061] (3) The plasmid recovered from the gel in step (2) of Example 2 was ligated overnight using T4 ligase. The ligation system is shown in Table 4 below, and transformed into DH5α competent cells. The cells were plated and the positive clones were sequenced for verification.

[0062] Table 6T4 DNA ligation reaction system

[0063]

[0064] (4) After sequencing and identification, the recombinant plasmid MaCYP71BG22 / pESC-TRP with no mutation in the sequenced nucleotide sequence was obtained.

[0065] 2. Transformation of MaCYP71BG22 / pESC-TRP eukaryotic expression vector into Saccharomyces cerevisiae WAT11

[0066] (1) Using Frozen-EZ Yeast TransformationⅡ TM The competent cells of Saccharomyces cerevisiae WAT11 were prepared by using the kit and identified by PCR.

[0067] (2) The plasmid was extracted from the successfully connected MaCYP71BG22 / pESC-TRP / DH5α positive single colony using the SanPrep column plasmid DNA small-scale extraction kit, and the concentration and quality of the plasmid solution were determined using a nucleic acid protein detector.

[0068] (3) Using Frozen-EZ Yeast TransformationⅡ TM The kit transforms the MaCYP71BG22 / pESC-TRP recombinant plasmid into the WAT11 strain, spreads it on the SC-TRP defective solid medium (containing 2% glucose), and cultures it at 30°C for 2-3 days. Pick a single colony, identify it by PCR with pESC-TRP self-primers GalⅠ-F and GalⅠ-R, and store it in a -80°C refrigerator for later use.

[0069] 3. Induced expression of MaCYP71BG22 / pESC-TRP / WAT11 strain, extraction of microsomal proteins and in vitro enzyme activity verification

[0070] Galactose was used to induce heterologous expression of the target gene in Saccharomyces cerevisiae WAT11. The specific steps are as follows:

[0071] (1) Take 2 mL of the fresh positive MaCYP71BG22 / pESC-TRP / WAT11 bacterial solution and add it to 100 mL of SC-TRP-deficient liquid culture medium. Cultivate the culture medium at 30°C and 200 rpm for 2 days.

[0072] (2) Collect the cells by centrifugation at 6000 rpm for 5 min and wash them three times with sterile water;

[0073] (3) Transferred into 100 mL of YPG liquid medium containing 2% galactose and cultured at 30°C, 200 rpm for 16 h to induce protein expression;

[0074] The glass bead method was used to extract the MaCYP71BG22 hydroxylase microsomal protein. The specific steps are as follows:

[0075] (1) Collect the cells by centrifugation at 9000 × g for 2 min at 4°C.

[0076] (2) Resuspend the cells in 10 mL of TEK buffer (1 / 10 the volume of YPG medium during induction) and place at room temperature for 5 min.

[0077] (3) Collect the cells by centrifugation at 9000 × g for 1 min, resuspend the cells in 1 mL of ice-cold TESB buffer, and transfer to a 50 mL centrifuge tube;

[0078] (4) Add glass beads with a diameter of 0.5 mm until they just cover the bacteria;

[0079] (5) Vortex and shake for 15 seconds to break up the bacteria, place on ice for 1 minute, and repeat 30 to 60 times;

[0080] (6) Add 3 mL of pre-cooled TESB buffer to the centrifuge tube to wash the glass beads, aspirate the supernatant, and repeat four times to combine the washing solutions;

[0081] (7) Centrifuge at 12,000 × g for 15 min at 4°C and transfer the supernatant to a new centrifuge tube;

[0082] (8) Add NaCl with a final concentration of 0.15 mol / L and 0.1 g / mL PEG4000 to precipitate the microsomes and place on ice for 20-30 min;

[0083] (9) Centrifuge at 12,000 × g for 15 min at 4°C and discard the supernatant;

[0084] (10) Add 500 μL TEG buffer to dissolve, divide into aliquots, and store at -80°C until use.

[0085] The in vitro enzymatic reaction of MaCYP71BG22 hydroxylase microsomal protein was verified, and the specific steps were as follows:

[0086] (1) Using (R)-2-methylpiperidine as substrate, 100 mM Tris-HCl (tris(hydroxymethyl)aminomethane-hydrochloric acid) as buffer system, and NADPH (tetrasodium salt of reduced coenzyme II) as electron donor of MaCYP71BG22 hydroxylase, the in vitro enzymatic activity of MaCYP71BG22 hydroxylase microsomal protein was verified. The enzymatic reaction system is as follows:

[0087]

[0088] (2) The reaction system was placed in a shaker at 30°C and 100 rpm for 70 min, then placed in -80°C to terminate the reaction, freeze-dried, reconstituted with 400 μL of methanol, filtered through a 0.22 μm filter membrane into a sample vial, and the product was analyzed by GC-MS.

[0089] (3) The above products were detected by gas chromatography-mass spectrometry (GC-MS). GC-MS chromatographic conditions: Rtx-5 chromatographic column (30m×0.25mm×0.25μm), helium as carrier gas, carrier gas flow rate of 1mL / min, injection port temperature of 250℃, transfer tube temperature of 280℃, programmed temperature rise mode: initial temperature of 60℃, maintained for 5min, increased to 100℃ at 10℃ / min, maintained for 3min, then increased to 280℃ at 20℃ / min, maintained for 7min. Split ratio of 1:30. Mass spectrometry conditions: ionization mode EI, electron energy 70eV, ion source temperature 230℃, interface temperature 250℃, scanning range 50-200m / z, solvent delay 3min.

[0090] 4. Product fermentation of MaCYP71BG22 / pESC-TRP / WAT11 strain

[0091] (1) The MaCYP71BG22 / pESC-TRP / WAT11 strain preserved above was streaked and activated, and a single colony was picked and inoculated into 10 mL of SC-TRP liquid medium (SD-TRP) containing 2% glucose and cultured at 30°C and 200 rpm for 16 h. The inoculation volume was transferred to 200 mL of SD-TRP liquid medium at 5% and cultured in a shaking incubator at 30°C and 200 rpm for 16 h.

[0092] (2) Centrifuge the cells at 5000 × g for 4 min at 4°C, carefully discard the supernatant, and collect the cells.

[0093] (3) Wash the cells with sterile water three times to remove as much glucose as possible.

[0094] (5) Resuspend the cells in an appropriate amount of SC-TRP liquid medium (SG-TRP) containing 2% galactose and adjust the OD 600 is 1.0.

[0095] (6) The yeast strains were induced and cultured in a shaking incubator at 30°C for 5 h. Then, 5.0 mM substrate (R)-2-methylpiperidine was added to each strain and the culture was continued at 30°C and 220 rpm for 72 h.

[0096] (7) The fermentation broth was centrifuged to collect the bacterial cells and the bacterial liquid. An equal volume of glass beads was added to the bacterial cells and vortexed for 30 min. 1 mL of methanol was added to wash the glass beads. The process was repeated three times. The washing liquid was collected and centrifuged at 9000 × g for 5 min. The upper organic phase was aspirated and dried together with the bacterial liquid. The bacterial liquid was vacuum dried at 57°C to the minimum volume, freeze-dried, and re-dissolved in methanol. The supernatant was aspirated and filtered through a 0.22 μm microporous filter membrane into a sample vial. The product was analyzed by LC / MS.

[0097] 5. Preparation and NMR Identification of MaCYP71BG22 Hydroxylase Products

[0098] (1) The MaCYP71BG22 / pESC-TRP / WAT11 strain successfully constructed in Example 2 was inoculated into 2 mL of SD-TRP liquid culture medium and cultured at 30°C and 200 rpm for 16 h;

[0099] (2) Transfer the above bacterial solution to 50 mL SD-TRP medium and culture with shaking at 30°C and 230 rpm for 16 h; further expand to 3 L SD-TRP medium and culture with shaking at 30°C and 230 rpm for 16 h;

[0100] (3) Then, the expression was induced according to the same method as above, and 3L SD-TRP was replaced with 3L SG-TRP medium, and the recombinant protein expression was induced at 30°C and 200rpm for 5h. Then, (R)-2-methylpiperidine was added to the SG-TRP medium to a final concentration of 5mM, and the fermentation was continued for another 96 hours, and the fermentation broth was collected by centrifugation;

[0101] (4) The fermentation broths are combined and concentrated under vacuum, and resuspended in an appropriate amount of methanol. The precipitate is removed by centrifugation, and the supernatant is collected again and concentrated under vacuum for later use;

[0102] (5) The concentrated fermentation broth was resuspended in methanol, first eluted by silica gel column chromatography (eluent: dichloromethane / methanol = 10:1), and then purified by preparative HPLC. Preparative HPLC separation used an LC-1500 instrument (JASCO) with a differential detector (JASCO, RID-1530), and the chromatographic column was a Hypersil NH2-S column (250 mm × 10 mm, 5 μm); the flow rate was 2 mL / min. Water-acetonitrile (v / v = 5:95) was used as the mobile phase, and the column oven was 30°C. The target fractions were manually collected, dried, and resuspended in DMSO-d6, and analyzed by nuclear magnetic resonance spectroscopy (NMR) and high resolution mass spectrometry (HRMS).

[0103] (6) The chemical structure of the purified samples was characterized using the data collected on a Bruker DRX 400 spectrometer.1 H-NMR (400MHz), 13 C-NMR (101 MHz) and 2D NMR spectra. Tetramethylsilane was used as an internal standard. Observed chemical shift values ​​are reported in ppm.

[0104] Example 2 Test results:

[0105] (1) MaCYP71BG22-T and pESC-TRP plasmids were double digested with XhoⅠ-KpnⅠ, and the digestion reaction products were recovered by gel. The recovered products were ligated with T4 DNA ligase and transformed into Escherichia coli. Single clones were selected for PCR verification. The bacterial solution was PCR verified by GalⅠ-F and GalⅠ-R for MaCYP71BG22 / pESC-TRP recombinant plasmid. The results are as follows: Figure 3 As shown, all samples had a single bright band at 1500 bp, indicating a positive recombinant.

[0106] (2) The sequencing results of the MaCYP71BG22 / pESC-TRP recombinant plasmid showed that the MaCYP71BG2 hydroxylase gene had a complete ORF reading frame, an ORF length of 1446, a CDS sequence length of 1443, and a 100% coverage rate and 99.86% similarity between the mulberry leaf transcriptome sequence c21932_g1 and MaCYP71BG22 / pESC-TRP, indicating that the MaCYP71BG22 / pESC-TRP eukaryotic expression vector was successfully constructed.

[0107] (3) The positive MaCYP71BG22 / pESC-TRP bacterial solution that was successfully identified was expanded and the plasmid was extracted to obtain the MaCYP71BG22 / pESC-TRP plasmid, which was then transformed into the competent cells of the yeast strain WAT11 and re-cultured. A single colony was picked for expansion and plasmid extraction. The PCR verification results showed that the recombinant plasmid had a single target band at around 1500 bp, such as Figure 4 As shown, it indicates that the positive MaCYP71BG22 / pESC-TRP / WAT11 strain was successfully constructed.

[0108] (4) The glass bead method was used to extract the MaCYP71BG22 microsomal protein, and the in vitro enzyme activity was determined using (R)-2-methylpiperidine as the substrate and 100 mM Tris-HCl as the buffer system. The GC-MS results showed that the MaCYP71BG22 microsomal protein could catalyze the hydroxylation reaction of (R)-2-methylpiperidine. The retention time of the product was 11.98 minutes, and the mass fragment was m / z = 115, such as Figure 5 In yeast transformed with the empty vector, no corresponding new peak was observed at the same retention time.

[0109] (5) The recombinant plasmid that was successfully sequenced and verified was transformed into the S. cerevisiae strain WAT11 to obtain the expression strain MaCYP71BG22 / pESC-TRP / WAT11, and the expression strain pESC-TRP / WAT11 was used as a control. The HPLC-RID results showed that the MaCYP71BG22 engineered yeast could catalyze the hydroxylation reaction of (R)-2-methylpiperidine, with a new peak retention time of 17.12 and mass spectrometry data of m / z = 116, which was not observed in the empty vector engineered yeast. The precise molecular formula was confirmed by HRMS to be m / z = 116.1071, as shown in Figure 6 In order to obtain sufficient amount of compound for NMR analysis, MaCYP71BG22 / pESC-TRP / WAT11 engineered yeast was cultured and fermented at a scale of 3 L, and the new peak was purified by preparative LC to enrich 2.8 mg of product.

[0110] (6) The purified fermentation product was dissolved in DMSO-d6 for NMR analysis. The results showed that the purified compound was identified as (2R, 4R)-2-methylpiperidin-4-ol, and all NMR signals were assigned as follows: Figure 7-12 shown. 13 The C chemical shift (d 65.1) confirmed that C4 was an oxygen-carrying site, and the splitting of the chemical shift of the H3 and H5 hydrogen atoms proved that the hydroxyl group connected to C4 had a stereo configuration. (2R,4R)-2-methylpiperidin-4-ol: 1 H NMR (400MHz, DMSO-d6): δ3.60-3.53(m,1H),3.17-3.12(m,1H),3.02-2.96(m,1H),2.78-2.71(m,1H), 1.91-1.82(m,2H),1.40(tdd,J=13.3,10.9,4.4Hz,1H),1.23(t,J=12.3Hz,1H),1.18(d,J=6.4Hz,3H). 13 CNMR (101MHz, DMSO-d6): δ65.1, 50.4, 41.9, 40.3, 31.7, 19.1. In addition, HSQC proved the correlation of the same CH atom, HMBC, 1 H- 1H COSY further confirmed the connectivity of C3-C4-C5 atoms and the position of hydroxyl group in the piperidine ring. NOESY spectrum showed that H-7 (δH=1.19) was correlated with H-2 (δH=2.99), H-6 (δH=3.14) was correlated with H-5 (δH=1.91) and H-3 (δH=1.88), and all lacked strong signals with H-4. Combined with the coupling constant of H-5 (1.40tdd, J=13.3,10.9,4.4Hz,1H), it was confirmed that 4-OH was in R configuration. In summary, the hydroxylation product of MaCYP71BG22 hydroxylase was determined to be (4R)-2-methylpiperidin-4-ol, and the absolute configuration of C-4 position was the same as that in DNJ structure.

[0111]

[0112]

[0113]

Claims

1. A MaCYP71BG22 hydroxylase gene that catalyzes the hydroxylation at the C4 position of DNJ biosynthesis, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A hydroxylase encoded by the MaCYP71BG22 gene that catalyzes the hydroxylation at the C4 position of DNJ biosynthesis, characterized in that The amino acid sequence encoding the hydroxylase is shown in SEQ ID NO:

2.

3. A method for preparing (2R, 4R)-2-methylpiperidin-4-ol compounds, characterized in that Follow the steps below: (1) inserting the MaCYP71BG22 hydroxylase gene into the eukaryotic expression vector pESC-TRP by restriction endonuclease and DNA ligase to prepare a recombinant yeast expressing the enzyme; (2) using (R)-2-methylpiperidine as a substrate, adding it to the culture medium of the recombinant yeast of the enzyme MaCYP71BG22 obtained in step (1) for shake flask fermentation, wherein the fermentation medium is an SG-TRP medium () containing 2% galactose by mass to volume ratio, thereby preparing a (2R, 4R)-2-methylpiperidin-4-ol compound; After step (2) is completed, the method further comprises performing LC-MS analysis on the reaction product, wherein the product peak appearing at 17.12 minutes is (2R, 4R)-2-methylpiperidin-4-ol.

4. A method for preparing (2R, 4R)-2-methylpiperidin-4-ol compounds according to claim 3, characterized in that The composition of the SG-TRP culture medium is as follows: 1L of SG-TRP liquid culture medium contains 6.70g of YNB (yeast nitrogen base), 0.10g of L-arginine, 0.10g of L-cysteine, 0.10g of L-lysine, 0.10g of L-threonine, 0.05g of L-asparagine, 0.05g of L-isoleucine, 0.05g of L-phenylalanine, 0.05g of L-proline, 0.05g of L-serine, 0.05g of L-tyrosine, 0.05g of L-valine, 0.05g of L-methionine, 0.10g of L-histidine, 0.10g of L-leucine, 0.10g of uracil, and 0.10g of adenine.

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