CYP71P2 enzyme, biomaterials and applications

Abstract

The present invention discloses a CYP71P2 enzyme, a biomaterial and applications thereof. The CYP71P2 enzyme is derived from a stem premature development mutant of Atractylodes lancea. The present invention clones and verifies for the first time the key role of the CYP71P2 enzyme in the biosynthesis pathway of sesquiterpenoid compounds. The enzyme and the biomaterial thereof can be used for the synthesis of sesquiterpenoid compounds, providing a new pathway for the synthesis of such compounds. The gene encoding the CYP71P2 enzyme is introduced into squalene synthase-knockout cerevisiae yeast to achieve efficient production of the sesquiterpenoid compound nerolidol, with a yield of up to 500 mg / L.

Description

Technical Field

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

[0002] Atractylodes lancea is a perennial herbaceous plant in the Asteraceae family. Its dried rhizome is the traditional Chinese medicine "Atractylodes lancea." According to the Chinese Pharmacopoeia, Atractylodes lancea has the effects of dispelling 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, rheumatic pain, and rheumatic arthralgia. Modern research shows that the main active pharmaceutical ingredient in Atractylodes lancea is sesquiterpenoids, which possess various biological activities and are widely used in food, daily chemicals, and medical applications.

[0003] Nerolidol, one of the active sesquiterpenoids in Atractylodes macrocephala, possesses a variety of bioactive properties, including antibacterial, insecticidal, antioxidant, anti-inflammatory, and anti-cancer properties. It holds broad application prospects in medicine, food additives, cosmetics, and agriculture. However, the current main production methods for nerolidol have numerous limitations. Traditional nerolidol production relies primarily on plant extraction, typically employing distillation and organic solvent extraction. These methods suffer from the following drawbacks: First, the low content of nerolidol in Atractylodes macrocephala results in large quantities of raw materials required for large-scale extraction, leading to high extraction costs. Second, the plant's long growth cycle prevents it from responding quickly to market demand due to the influence of climate and soil conditions, as well as the gradual degradation of its germplasm resources.

[0004] Although chemical synthesis can increase the yield of nerolidol to a certain extent, many problems still exist: 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, posing an environmental pollution risk; and chemical synthesis conditions are harsh, requiring precise control of reaction conditions, the process is complex, and industrial application is limited.

[0005] In contrast, microbial fermentation, due to its high efficiency, environmental friendliness, and sustainability, has become an ideal pathway for nerolidol production. By genetically engineering key enzymes into engineered strains, nerolidol biosynthesis can be achieved. However, microbial fermentation for nerolidol production currently faces bottlenecks. This is primarily due to the complex metabolic pathways involved in the nerolidol synthesis pathway, which involve multiple key enzymes and have yet to be fully elucidated and optimized. Furthermore, the low catalytic efficiency and stability of enzymes involved in sesquiterpene synthesis further limit its application. Existing genetically engineered microbial fermentation systems only produce nerolidol at a rate of 200-400 mg / L, 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, nucleotide sequence, recombinant vector, 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 include:

[0015] (1) ligating 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) Cultivate the recombinant microorganisms and extract and purify 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 existing technology, 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 superior Atractylodes lancea varieties; 2. By introducing CYP71P2 into squalene synthase-knockout 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 sesquiterpenoid content between the wild type Atractylodes lancea and the Atractylodes lancea with premature stem development mutation;

[0021] Figure 2 This is a heat map of differentially expressed P450 genes related to sesquiterpenoid biosynthesis in 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 of the CYP71P2 enzyme protein;

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

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

[0026] Figure 7 This is a Western-blot result of the expression of recombinant squalene synthase knockout yeast CYP71P2 after CuSO4 induction. 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 50 mM CuSO4, and lane 3 represents the protein extracted from the recombinant strain induced with 100 mM CuSO4.

[0027] Figure 8 The total ion currents of the fermentation extracts of recombinant squalene synthase knockout Saccharomyces cerevisiae and the control strain 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 after induction. The red arrow indicates the differential compound at retention time 15.965;

[0028] Figure 9 The figure shows the mass spectra of the differential compound and the nerolidol standard at the retention time of 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 commercially available. Wild-type and mutant Atractylodes lancea specimens are stored at the Museum of Rare Animals and Plants of Nanjing Normal University (wild-type accession number S01240, mutant accession number S01240-1).

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

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

[0033] 1.1. Take 6 plants of wild-type Atractylodes lancea and 6 plants of Atractylodes lancea with premature stem development mutation that have been 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 to EP tubes, add 10 volumes (v:w) of n-hexane and extract them in the dark for 12 hours, sonicate in an ultrasonic oscillator for 30 minutes, centrifuge at 8000 rpm for 10 minutes, take the supernatant, filter it through a 0.22 μm pore size filter membrane, and transfer it to 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 as follows: 70℃ for 1min, then heated to 200℃ at 8℃ / min, and then heated to 300℃ at 20℃ / min and held for 5min.

[0035] The contents of 12 sesquiterpenes were calculated based on the standard curve. The results are as follows: Figure 1As shown in the figure, CK represents the wild type Atractylodes lancea and Var represents the mutant Atractylodes lancea. The results showed that the total amount of sesquiterpenes in the mutant Atractylodes lancea increased by 1.39 times, and all except zingiberene showed significant increases. In particular, the increase in oxygenated sesquiterpenes in the mutant strain was more significant: the content of turmericol 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. Total RNA from the aerial parts of wild-type Atractylodes lancea and the Atractylodes lancea mutant with premature stem development was extracted using the 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 in the figure, the expression levels of three P450s showed an up-regulation trend in the mutant strains, which was the same as the change in the content of sesquiterpenes. In particular, CL31.Contig2_All had the largest up-regulation, 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] The total RNA from the aerial parts of wild-type Atractylodes lancea and Atractylodes lancea with premature stem development mutation was extracted using the FastPure Universal Plant Total RNA Isolation Kit. 1 μg of total RNA was taken from each strain and reverse transcribed into cDNA using the 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 sesquiterpenoid content. It was named CYP71P2, and its protein structure is shown in Figure 2. Figure 4 shown.

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

[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] Total RNA from Atractylodes lancea leaves was extracted using the FastPure Universal Plant Total RNA Isolation Kit, and 1 μg of total RNA was reverse transcribed into cDNA using the 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: 10 μL of 2× Phanta Flash MasterMix, 1 μL of template cDNA, 1 μL of upstream and downstream primers, and 7 μL of sterile water. The reaction conditions were as follows: 98°C pre-denaturation for 30 seconds, 98°C denaturation for 10 seconds, 56°C annealing for 5 seconds, 72°C extension for 10 seconds, 35 cycles of 72°C extension for 1 minute, and storage at 4°C. The PCR product was loaded onto a 1% agarose gel for electrophoresis. The results are shown in Figure 2. Figure 5 As shown, the product fragment size was 1566 bp. After the electrophoresis, the electrophoresis band was cut and recovered and purified using the FastPure Gel DNA Extraction Mini Kit to obtain the CYP71P2 enzyme encoding gene, whose sequence is shown in SEQ ID NO: 2, and the amino acid 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 sequences of CYP71P2 enzymes were 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 2. 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 a key factor in 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. Add a His-tag and a Flag-tag after the promoter to obtain the pESC-Cup-HF vector as the original vector. Based on 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 and purified DNA obtained in step 2 of Example 1 as a template, the CYP71P2 gene with homology arms was amplified and purified. This fragment was ligated with the linearized vector using a homologous recombinase to obtain the recombinant vector pESC-Cup-CYP71P2. The recombinant vector was then transformed into competent Escherichia coli DH5α using a heat shock method. The main operating conditions were: ice incubation at 4°C for 3 minutes, followed by heat shock at 42°C for 45 seconds, and then ice incubation at 4°C for 3 minutes.

[0053] 2. The transformed E. coli was evenly spread on LB plates containing 30 mg / L kanamycin and cultured overnight until a single colony grew. The single colony was picked and dispersed in 10 μL of sterile water and mixed thoroughly. 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, 30 cycles of extension at 72°C for 5 min, and incubation 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, positive colonies were screened and the remaining 9 μL of the bacterial solution was added to 50 mL of LB liquid medium containing 30 mg / L kanamycin and cultured overnight. The pESC-Cup-CYP71P2 plasmid was extracted using the FastPure Plasmid MiniKit.

[0055] The lithium acetate transformation method was used to transform 1 μg of the empty plasmid and the recombinant plasmid pESC-Cup-CYP71P2 into squalene synthase knockout Saccharomyces cerevisiae. The transformed bacterial solution was evenly spread on SD-H solid medium containing 30 mg / L ergosterol. After a single colony was grown, the positive clone was screened again by colony PCR. The operation method and reagents were the same as above. The obtained positive clone was inoculated into SD-H medium for overnight culture. When the OD of the bacterial solution reached 0. 600 =0.9, add CuSO4 with a final concentration of 50mM or 100mM for induction.

[0056] The squalene synthase knockout yeast Saccharomyces cerevisiae 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 yeast strain was verified by Western-Blot. After 24 hours of CuSO4 induction, 500 μL of bacterial solution was taken, centrifuged at 8000 rpm for 5 minutes, 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 minutes. The supernatant was taken again and subjected to SDS-PAGE gel electrophoresis. After the electrophoresis, the membrane was transferred and blocked. The primary antibody was Anti-His Mouse Monoclonal Antibody, 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 represent the recombinant strains induced by CuSO4. The positions of the bands shown are 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 to a final concentration of 50 mM for 7 days of induction. After induction, take 3 mL of bacterial solution, add 3 mL of acetone, shake vigorously for 5 minutes, and let it stand at room temperature for 5 minutes. Then add 3 mL of n-hexane, shake vigorously for 5 minutes, let it stand at room temperature for 10 minutes, and centrifuge at 8000 rpm for 10 minutes. The upper organic phase is collected and transferred to a new centrifuge tube. The sample is placed in a vacuum concentrator, concentrated to 50 μL, and stored in a brown liquid phase injection bottle with an inner tube and stored in a -20°C refrigerator.

[0060] The samples were analyzed using an Agilent 5973 gas chromatograph-mass spectrometer with an HP-5 column (30 m × 0.25 mm × 0.25 μm). The gas chromatography conditions were as follows: 100°C for 4 min, then ramped to 280°C at 5°C / min and held for 6 min. The mass spectrometry conditions were as follows: ion source temperature 230°C, interface temperature 280°C, and scan mass range 35-500 μm. The resulting total ion current is shown in the figure below. Figure 8 As shown in FIG, there is a differential compound with a retention time of 15.965, and the mass spectrum of the differential compound is as shown in FIG. Figure 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. Further calculation shows 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 nucleic acid molecule, characterized in that The nucleic acid molecule encodes the CYP71P2 enzyme according to claim 1.

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

2.

4. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid molecule according to claim 2 or 3.

5. A recombinant microorganism, characterized in that The recombinant microorganism comprises the nucleic acid molecule according to claim 2 or 3 or the recombinant vector according to claim 4.

6. A product, characterized in that The product comprises one or more of the CYP71P2 enzyme according to claim 1, or the nucleic acid molecule 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 nucleic acid molecule 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 nerolidol.

8. The application according to claim 7, characterized in that: The step of preparing nerolidol comprises: (1) Connecting the CYP71P2 enzyme nucleic acid molecule to the vector plasmid to obtain a recombinant vector; (2) Transforming the recombinant vector into a host microorganism and screening to obtain the recombinant microorganism; (3) Cultivate the recombinant microorganisms and extract and purify nerolidol.

9. The application according to claim 8, characterized in that: The host microorganism is squalene synthase knockout Saccharomyces cerevisiae.

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