CYP71P2 enzyme, biological material and application

Abstract

The invention discloses a CYP71P2 enzyme, a biological material and application, the CYP71P2 enzyme is derived from Atractylodes lancea with advanced stem development mutation, the key effect of the CYP71P2 enzyme in the biosynthetic pathway of sesquiterpenoids is cloned and verified for the first time, the CYP71P2 enzyme and the biological material thereof can be used for synthesis of sesquiterpenoids, and the CYP71P2 enzyme can be applied to biosynthesis of sesquiterpenoids. A new way is provided for synthesis of the compounds; the gene for coding CYP71P2 enzyme is introduced into squalene synthase knockout saccharomyces cerevisiae, so that efficient production of sesquiterpenoids, nerolidol, can be realized, and the yield can reach 500mg / L.

Description

Technical Field

[0001] The invention relates to cytochrome P450 enzyme, in particular to a CYP71P2 enzyme, a biomaterial and an application thereof. Background Art

[0002] Atractylodes lancea is a perennial herbaceous plant of the genus Atractylodes in the Asteraceae family. Its dried rhizome is the traditional Chinese medicine "Atractylodes". According to the "Chinese Pharmacopoeia", Atractylodes lancea has the effects of removing dampness and strengthening the spleen, dispelling wind and cold, and improving eyesight. It is widely used to treat symptoms such as spleen deficiency and dampness, rheumatism and pain. Modern research shows that the main medicinal active ingredients of Atractylodes lancea are sesquiterpenoid compounds, which have multiple biological activities and are widely used in food, daily chemicals and medical fields.

[0003] Nerolidol is one of the active ingredients of sesquiterpenes in Atractylodes macrocephala, which has multiple biological activities such as bactericidal, insecticidal, antioxidant, anti-inflammatory and anti-cancer. It has broad application prospects in the fields of medicine, food additives, cosmetics and agriculture. However, the main production methods of nerolidol currently have many limitations. The traditional production method of nerolidol mainly relies on plant extraction, which usually uses distillation and organic solvent extraction, but has the following disadvantages: first, the content of nerolidol in Atractylodes macrocephala is low, resulting in a large amount of raw materials required for large-scale extraction and high extraction costs; second, it is affected by climate and soil conditions, and its germplasm resources gradually degenerate, resulting in unstable yield and quality; and the plant growth cycle is long, and it cannot respond quickly to market demand.

[0004] Although chemical synthesis can increase the yield of nerolidol to a certain extent, there are still many problems: on the one hand, the raw materials such as linalool required for chemical synthesis are expensive, which greatly increases the production cost; on the other hand, catalysts are required in the synthesis process, which may produce toxic by-products and waste, and there is a risk of environmental pollution; and chemical synthesis conditions are harsh, and the reaction conditions need to be precisely controlled. The process is complex and its industrial application is limited.

[0005] In contrast, microbial fermentation has become an ideal way to produce nerolidol because of its high efficiency, greenness and sustainability. The biosynthesis of nerolidol can be achieved by introducing key synthetic enzymes into engineered strains using genetic engineering methods. However, there are still bottlenecks in the production of nerolidol by microbial fermentation. The main reason is that the nerolidol synthesis pathway involves multiple key enzymes, the metabolic pathway is complex, and it has not been fully analyzed and optimized; and the catalytic efficiency and stability of enzymes related to sesquiterpene synthesis are low, which further limits its application. In the existing genetically engineered microbial fermentation system, the output of nerolidol is only 200-400 mg / L, which is far from meeting the growing market demand. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a cytochrome P450 enzyme - CYP71P2 enzyme and related biomaterials; the second purpose is to provide the application of the enzyme and biomaterials in the efficient synthesis of sesquiterpenoid compounds.

[0007] Technical solution: The CYP71P2 enzyme described in the present invention is a cytochrome P450 enzyme derived from a stem premature development mutation of Atractylodes lancea, and its amino acid sequence is the sequence shown in SEQ ID NO: 1.

[0008] The nucleotide sequence of the present invention encodes the aforementioned CYP71P2 enzyme.

[0009] Preferably, the nucleotide sequence is the sequence shown in SEQ ID NO:2.

[0010] The recombinant vector of the present invention comprises the aforementioned nucleotide sequence.

[0011] The recombinant microorganism of the present invention comprises the aforementioned nucleotide sequence or recombinant vector.

[0012] The product of the present invention comprises one or more of the aforementioned CYP71P2 enzyme, or the aforementioned nucleotide sequence, or the aforementioned recombinant vector, or the aforementioned recombinant microorganism.

[0013] The CYP71P2 enzyme, or nucleotide sequence, or recombinant vector, or recombinant microorganism, or product of the present invention is used in the preparation of sesquiterpenoid compounds.

[0014] Preferably, the steps of preparing the sesquiterpenoid compound for use include:

[0015] (1) connecting the CYP71P2 enzyme nucleotide sequence to a vector plasmid to obtain a recombinant vector;

[0016] (2) transforming the recombinant expression vector into a host microorganism and screening to obtain the recombinant microorganism;

[0017] (3) Cultivating the recombinant microorganisms and extracting and purifying the sesquiterpenoid compounds.

[0018] Preferably, the host microorganism is squalene synthase knockout Saccharomyces cerevisiae, and the sesquiterpenoid compound is nerolidol.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The cloned and isolated CYP71P2 enzyme derived from Atractylodes lancea can be used for the synthesis of sesquiterpenoid compounds, providing a new approach for the synthesis of such compounds and a new idea for the breeding of excellent varieties of Atractylodes lancea; 2. By introducing CYP71P2 into squalene synthase-knocked-out Saccharomyces cerevisiae, efficient production of nerolidol can be achieved, with a yield of up to 500 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison of the sesquiterpenoid content of wild-type Atractylodes lancea and Atractylodes lancea with premature stem development mutation;

[0021] Figure 2 The heat map of P450 genes related to sesquiterpene synthesis that are differentially expressed between wild-type Atractylodes lancea and Atractylodes lancea with premature stem development mutation;

[0022] Figure 3 This is an analysis diagram of the expression of CYP71P2 enzyme in wild-type Atractylodes lancea and Atractylodes lancea with premature stem development mutation;

[0023] Figure 4 This is the three-dimensional structure diagram of the CYP71P2 enzyme protein;

[0024] Figure 5 It is the agarose gel electrophoresis diagram of CYP71P2 enzyme, where M is a marker, and lane 1 and lane 2 are both CYP71P2 enzyme encoding genes;

[0025] Figure 6 This is the phylogenetic tree diagram of CYP71P2 enzyme;

[0026] Figure 7 The Western-Blot result of the recombinant squalene synthase knockout yeast of CYP71P2 enzyme after induction with CuSO4, where M represents Marker; Lane 1 represents the protein extracted from the uninduced recombinant strain, Lane 2 represents the protein extracted from the recombinant strain induced with 50mM CuSO4, and Lane 3 represents the protein extracted from the recombinant strain induced with 100mM CuSO4;

[0027] Figure 8 The total ion current diagram of the fermentation extracts of recombinant squalene synthase knockout Saccharomyces cerevisiae and control strains with or without induced CYP71P2 enzyme, where A is the total ion current of the recombinant strain after induction with 50 mM CuSO4, B is the total ion current of the uninduced recombinant strain, and C is the total ion current of the control strain induced. The red arrow indicates the differential compound at the retention time of 15.965;

[0028] Fig. 9 It is the mass spectrum of the differential compound and the nerolidol standard at the retention time 15.965, wherein A is the differential compound and B is the nerolidol standard. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below.

[0030] The following examples used FastPure Universal Plant Total RNA Isolation Kit, HiScript IV 1st Strand cDNA Synthesis Kit (+gDNA wiper), 2×Phanta Flash MasterMix, 2×Rapid Taq Master Mix, FastPure Plasmid Mini Kit, Escherichia coli DH5α competent cells, Anti-His Mouse Monoclonal Antibody, Goat Anti-Mouse IgG (H+L) and HRP Conjugate are both commercially available products. Wild-type and mutant specimens of Atractylodes lancea are preserved in the Rare Animal and Plant Museum of Nanjing Normal University (the wild-type is preserved as S01240, and the mutant is preserved as S01240-1).

[0031] Example 1: Cloning of the gene encoding the CYP71P2 enzyme

[0032] 1. Screening of CYP450 genes that mediate efficient synthesis of sesquiterpenes in mutant Atractylodes lancea:

[0033] 1.1. Take 6 plants of wild-type Atractylodes lancea and 6 plants of Atractylodes lancea with premature stem development mutation cultivated for 3 months, rinse them with running water and dry them, place the samples in an oven at 37°C for 36 hours, take them out and grind them after they are completely dry, transfer the ground samples into EP tubes, add 10 times the volume (v:w) of n-hexane and extract them in the dark for 12 hours, sonicate them in an ultrasonic oscillator for 30 minutes, centrifuge them at 8000 rpm for 10 minutes, take the supernatant, filter it through a 0.22 μm pore size filter membrane, and transfer it into a brown injection vial.

[0034] The sesquiterpene content of the above samples was detected using an Agilent 7890A gas chromatograph. The chromatographic column was an HP-5 capillary column (30m×0.32mm×0.1μm), the detector was a flame ionization detector (FID), the carrier gas was high-purity nitrogen, and the heating program was: 70°C for 1 min, 8°C / min to 200°C, 20°C / min to 300°C and held for 5 min.

[0035] The contents of 12 sesquiterpenes were calculated according to the standard curve. The results are as follows: Figure 1As shown, CK represents the wild type Atractylodes lancea and Var represents the mutant Atractylodes lancea. The results showed that the amount of total sesquiterpenes in the mutant Atractylodes lancea increased by 1.39 times, and all except gingerene showed significant increases, especially the increase of oxygenated sesquiterpenes in the mutant strain was more significant: the content of turmeric alcohol increased by 1.99 times, the content of nerolidol increased by 1.74 times; the content of caryophyllene oxide increased by 1.65 times, the content of cedrol increased by 1.67 times, the content of β-eudesmol increased by 1.58, and the content of atractylodesone increased by 1.42 times.

[0036] 1.2. The total RNA of the aerial parts of wild-type Atractylodes lancea and Atractylodes lancea with premature stem development mutation was extracted using FastPure Universal Plant Total RNA Isolation Kit, and transcriptome sequencing was performed. Based on the sequencing results, 28 P450s that may be involved in the synthesis of sesquiterpenes were screened using the Blast method. The results are as follows: Figure 2 As shown, the expression changes of three P450s showed an upward trend in the mutant strains, which was the same as the change in the content of sesquiterpenes, especially CL31.Contig2_All, which had the largest upward expression, suggesting that it may play a key role in the synthesis of sesquiterpenes in Atractylodes lancea.

[0037] 1.3. Design primers, wherein the upstream primer is 5'-ACCTATCGTTTGGCTGCTC-3', and the downstream primer is 5'-GCGGGTTACTTCCTCATCTC-3;

[0038] FastPure Universal Plant Total RNA Isolation Kit was used to extract the total RNA of the aerial parts of wild-type Atractylodes lancea and Atractylodes lancea with premature stem development mutation, and 1 μg of total RNA was taken from each, and reverse transcribed into cDNA using HiScript IV 1stStrand cDNA Synthesis Kit (+gDNAwiper);

[0039] Then prepare the following reaction system: ChamQ Universal SYBR qPCR Master Mix 10 μL, template cDNA 1 μL, upstream and downstream primers 0.4 μL each, sterile water 8.2 μL;

[0040] Using Applied Biosystems TM QuantStudio TM 5. Real-time fluorescence quantitative PCR system was used to measure gene expression. The results were as follows Figure 3As shown in Figure 2, the expression level of the mutant Atractylodes lancea CL31.Contig2_All increased by 2.12 times, and the expression level changed significantly, indicating that the expression level of CL31.Contig2_All was closely related to the sesquiterpene content. It was named CYP71P2, and its protein structure is shown in Figure 2. Figure 4 shown.

[0041] 2. Cloning of the gene encoding CYP71P2 enzyme

[0042] The cytochrome P450 gene CYP71P2 sequence was obtained from the full-length transcriptome data of the premature stem development mutant Atractylodes lancea obtained in step 1.2, and the upstream primer: 5'-ATGGACGATTTTACCCTTACAGTTGT-3', and the downstream primer: 5'-TTATGTAGGTGAAACCCAAGCACCAT-3' were designed;

[0043] The total RNA of Atractylodes lancea leaves was extracted using FastPure Universal Plant Total RNA Isolation Kit, and 1 μg of total RNA was reverse transcribed into cDNA using HiScript IV 1st Strand cDNA Synthesis Kit (+gDNAwiper);

[0044] PCR amplification was performed using Atractylodes macrocephala cDNA as a template. The specific amplification system was as follows: 2×Phanta Flash MasterMix 10μL, template cDNA 1μL, upstream and downstream primers 1μL each, sterile water 7μL; reaction conditions were as follows: 98℃ pre-denaturation for 30s, 98℃ denaturation for 10s, 56℃ annealing for 5s, 72℃ extension for 10s, 72℃ extension for 1min after 35 cycles, and 4℃ storage. The PCR product was loaded onto a 1% agarose gel for electrophoresis. The results were as follows: Figure 5 As shown, the product fragment size is 1566bp. After the electrophoresis, the electrophoresis band is cut and recovered and purified using FastPure Gel DNA Extraction Mini Kit to obtain the CYP71P2 enzyme encoding gene, whose sequence is shown in SEQ ID NO: 2, and the nucleotide sequence of the CYP71P2 enzyme is shown in SEQ ID NO: 1.

[0045] 3. Bioinformatics analysis of CYP71P2 enzyme encoding genes

[0046] The amino acid sequence of CYP71P2 enzyme was analyzed and compared. The neighbor-joining method was used to construct a phylogenetic tree using the sequences with the highest similarity obtained from the comparison and the amino acid sequence of CYP71P2. The results are shown in Figure 6As shown, the CYP71P2 enzyme gene is closely related to the CYP71P1 gene of japonica rice.

[0047] The above results indicate that CYP71P2 is the key factor for the efficient synthesis of sesquiterpenes in mutant Atractylodes lancea. Therefore, this gene can also be used as an indicative factor for the selection and breeding of excellent Atractylodes lancea varieties. At a time when the quality of wild Atractylodes lancea is declining, it can contribute to the selection and breeding of excellent varieties of Atractylodes lancea.

[0048] Example 2: Construction of yeast expression system for CYP71P2 enzyme

[0049] 1. Use homologous recombination to construct a recombinant expression vector, replace the GAL1,10 promoter of the pESC-his vector with the Cup1 promoter, and add His-tag and Flag-tag after the promoter to obtain the pESC-Cup-HF vector as the original vector. According to the BamHI and SacI restriction sites in its sequence and the gene sequence of CYP71P2, design primers with homology arms:

[0050] The upstream primer was: 5′-gataaggtacccggatccATGGACGATTTTACCCTTACAGTTG-3′;

[0051] The downstream primer was: 5′-caacttctgttccatgtcgacTTATGTAGGTGAAACCCAAGCACC-3′.

[0052] Using the gel-recovered purified DNA obtained in step 2 of Example 1 as a template, the CYP71P2 gene with homology arms was amplified and purified, and the fragment was connected to the linearized vector by enzyme digestion using a homologous recombinase; the recombinant vector pESC-Cup-CYP71P2 was obtained, and the recombinant vector was transformed into the competent Escherichia coli DH5α by heat shock method. The main operating conditions were: 4°C ice bath for 3 minutes, then heat shock at 42°C for 45 seconds, and then 4°C ice bath for 3 minutes;

[0053] 2. The transformed E. coli was evenly spread on an LB plate containing 30 mg / L kanamycin and cultured overnight until a single colony grew out. The single colony was picked up and dispersed in 10 μL of sterile water and mixed evenly. The following PCR system was prepared: 10 μL of 2× Rapid Taq Master Mix, 1 μL of bacterial solution, 1 μL of upper and lower primers, and 7 μL of sterile water; PCR was performed according to the following conditions: pre-denaturation at 5°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 15 s, extension at 72°C for 40 s, extension at 72°C for 5 min after 30 cycles, and insulation at 4°C. The upstream primer was 5'-AGCGATGCGTCTTTTCCGCT-3'; the downstream primer was 5'-TGCGTACACGCGTCTGTACAG-3';

[0054] After the product was tested by agarose gel electrophoresis, the positive colony liquid was screened and the remaining 9 μL of the bacterial liquid was added to 50 mL of LB liquid medium containing 30 mg / L kanamycin for overnight culture. The pESC-Cup-CYP71P2 plasmid was extracted using FastPure Plasmid MiniKit;

[0055] Using the lithium acetate transformation method, 1 μg of the empty plasmid and the recombinant plasmid pESC-Cup-CYP71P2 were transformed into squalene synthase knockout Saccharomyces cerevisiae, and the transformed bacterial solution was evenly spread on SD-H solid medium containing 30 mg / L ergosterol. After a single colony grew, the colony PCR method was used to screen the positive clones again. The operation method and reagents were the same as above. The obtained positive clones were inoculated into SD-H medium for overnight culture. When the OD of the bacterial solution reached 600 =0.9, add CuSO4 with a final concentration of 50mM or 100mM for induction.

[0056] The squalene synthase knockout yeast was constructed according to the literature Zhuang, X. (2013). Engineering novel terpene production platforms in the yeast Saccharomyces cerevisiae.

[0057] 3. The protein expression in the recombinant squalene synthase knockout Saccharomyces cerevisiae strain was verified by Western-Blot. After 24 h of CuSO4 induction, 500 μL of bacterial solution was taken, centrifuged at 8000 rpm for 5 min, and the supernatant was discarded. 100 μL of 1× Loading buffer was added, and the mixture was heated in a 98°C water bath for 10 min. The supernatant was taken and subjected to SDS-PAGE gel electrophoresis. After the electrophoresis, the membrane was transferred and blocked. The primary antibody was Anti-His Mouse MonoclonalAntibody, dilution ratio is 1:3000; secondary antibody is Goat Anti-Mouse IgG (H+L), HRP Conjugate, dilution ratio is 1:5000. After incubation, use Tanon TM Femto-sig ECL chemiluminescent substrate was exposed and verified under a chemiluminescence instrument. Figure 7 As shown, lanes 2 and 3 are the recombinant strains induced by CuSO4, and the position of the bands shown is consistent with the expected size of the CYP71P2 enzyme, indicating that the enzyme can be expressed normally.

[0058] Example 3: Functional verification of CYP71P2 enzyme

[0059] The untransformed squalene synthase knockout Saccharomyces cerevisiae strain was selected as the control strain. The control strain and the recombinant strain were inoculated into SD-H medium for culture. 600 =0.9, add CuSO4 with a final concentration of 50mM to induce for 7 days. After the induction, take 3mL of bacterial solution and add 3mL of acetone, shake vigorously for 5min, and then stand at room temperature for 5min, add 3mL of n-hexane, shake vigorously for 5min, and then stand at room temperature for 10min, centrifuge at 8000rpm for 10min, take the upper organic phase into a new centrifuge tube; place the sample in a vacuum concentrator, concentrate to 50μL, store in a brown liquid injection bottle with an inner liner and store in a refrigerator at -20℃;

[0060] The samples were analyzed using an Agilent 5973 gas chromatograph-mass spectrometer. The chromatographic column was HP-5, with specific specifications of 30m×0.25mm×0.25μm. The gas chromatography conditions were as follows: 100℃ for 4min, 5℃ / min to 280℃, and maintained for 6min. The mass spectrometry conditions were as follows: ion source temperature 230℃, interface temperature 280℃, and scanning mass range 35-500aum. The total ion current obtained is shown in the figure below. Figure 8 As shown in FIG. 1 , there is a differential compound with a retention time of 15.965. The mass spectrum of the differential compound is as follows: Fig. 9 As shown, A is the mass spectrum of the differential compound with a retention time of 15.965, and B is the standard mass spectrum of nerolidol; the results show that the differential compound is nerolidol, and further calculations show that the synthesis amount of nerolidol reaches 500 mg / L.

Claims

1. A CYP71P2 enzyme, characterized in that The CYP71P2 enzyme is a cytochrome P450 enzyme derived from a stem premature development mutation of Atractylodes lancea, and its amino acid sequence is the sequence shown in SEQ ID NO:

1.

2. A nucleotide sequence, characterized in that The nucleotide sequence encodes the CYP71P2 enzyme according to claim 1.

3. The nucleotide sequence according to claim 2, characterized in that: The nucleotide sequence is the sequence shown in SEQ ID NO:

2.

4. A recombinant vector, characterized in that: The recombinant vector comprises the nucleotide sequence of claim 2 or 3.

5. A recombinant microorganism, characterized in that The recombinant microorganism comprises the nucleotide sequence of claim 2 or 3 or the recombinant vector of claim 4.

6. A product, characterized in that The product comprises one or more of the CYP71P2 enzyme according to claim 1, or the nucleotide sequence according to claim 2 or 3, or the recombinant vector according to claim 4, or the recombinant microorganism according to claim 5.

7. Use of the CYP71P2 enzyme according to claim 1, or the nucleotide sequence according to claim 2 or 3, or the recombinant vector according to claim 4, or the recombinant microorganism according to claim 5, or the product according to claim 6 in the preparation of sesquiterpenoid compounds.

8. The use according to claim 7, characterized in that: The steps of preparing the sesquiterpenoid compound for use include: (1) connecting the CYP71P2 enzyme nucleotide sequence to a vector plasmid to obtain a recombinant vector; (2) transforming the recombinant expression vector into a host microorganism and screening to obtain the recombinant microorganism; (3) Cultivating the recombinant microorganisms and extracting and purifying the sesquiterpenoid compounds.

9. The method for preparing sesquiterpenoid compounds according to claim 8, characterized in that: The host microorganism is squalene synthase knockout Saccharomyces cerevisiae.

10. The method for preparing sesquiterpenoid compounds according to claim 8, characterized in that: The sesquiterpenoid compound is nerolidol.

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