CN119876057B - CYP71P2 enzyme, biomaterials and applications - Google Patents

Abstract

The invention discloses CYP71P2 enzyme, biological material and application, wherein the CYP71P2 enzyme is derived from rhizoma atractylodis which is subjected to stem development mutation in advance, the key effect of the CYP71P2 enzyme in the biosynthesis pathway of sesquiterpenoids is cloned and verified for the first time, the enzyme and the biological material can be used for synthesizing the sesquiterpenoids, a novel pathway is provided for synthesizing the sesquiterpenoids, genes encoding the CYP71P2 enzyme are introduced into squalene synthase to knock out saccharomyces cerevisiae, the efficient production of the sesquiterpenoids nerolidol can be realized, and the yield can reach 500mg/L.

Description

CYP71P2 enzyme, biological material and application thereof

Technical Field

The invention relates to cytochrome P450 enzyme, in particular to CYP71P2 enzyme, biological material and application thereof.

Background

Rhizoma Atractylodis (Atractylodes lancea) is perennial herb of Atractylodes of Compositae, and its dried rhizome is traditional Chinese medicine rhizoma Atractylodis. According to the description of Chinese pharmacopoeia, rhizoma atractylodis has the effects of eliminating dampness and strengthening spleen, dispelling wind and cold, improving eyesight and the like, and is widely used for treating symptoms such as spleen deficiency and excessive dampness, rheumatalgia and the like. Modern researches have shown that the main medicinal active ingredient of rhizoma atractylodis lanceae is sesquiterpenoids, and the sesquiterpenoids have various biological activities and are widely applied to the fields of food, daily chemicals, medical treatment and the like.

Nerolidol (Nerolidol) is one of the active ingredients of rhizoma atractylodis sesquiterpenes, and has various biological activity functions of sterilization, disinsection, antioxidation, anti-inflammatory, anticancer and the like. Has wide application prospect in the fields of medicine, food additive, cosmetics, agriculture and the like. However, there are a number of limitations to the current main production of nerolidol. The traditional production method of nerolidol mainly depends on plant extraction, and the method generally adopts means such as distillation and organic solvent extraction, but has the defects that firstly, the content of the nerolidol in rhizoma atractylodis lanceae is low, so that the large-scale extraction of the nerolidol is large in required raw material amount, the extraction cost is high, secondly, the method is influenced by climate and soil conditions, and germplasm resources of the method are gradually degraded, so that the yield and quality are unstable, and the plant growth period is long, so that the market demand cannot be responded quickly.

Although the yield of nerolidol can be improved to a certain extent in chemical synthesis, the method has a plurality of problems that on one hand, raw materials such as linalool and the like required by chemical synthesis are expensive, the production cost is greatly increased, on the other hand, catalysts are required to be used in the synthesis process, toxic byproducts and wastes are possibly generated, the risk of environmental pollution exists, and moreover, the chemical synthesis conditions are harsh, the reaction conditions are required to be accurately controlled, the process is complex, and the industrialized application is limited.

In contrast, microbial fermentation is an ideal way for producing nerolidol due to its high efficiency, green and sustainable characteristics. The key synthetase is led into engineering strain by genetic engineering means, so that the biosynthesis of nerolidol can be realized. However, the prior microbial fermentation production of nerolidol still has a bottleneck, mainly the synthesis way of the nerolidol involves a plurality of key enzymes, the metabolic pathway is complex and is not completely resolved and optimized, and the catalytic efficiency and stability of the sesquiterpene synthesis related enzyme are low, thus further limiting the application of the sesquiterpene synthesis related enzyme. In the existing genetically engineered microbial fermentation system, the yield of nerolidol is only 200-400mg/L, and the increasing market demands can not be met.

Disclosure of Invention

The invention aims to provide a cytochrome P450 enzyme-CYP 71P2 enzyme and related biological materials, and a second aim to provide application of the enzyme and the biological materials in high-efficiency synthesis of sesquiterpenoids.

The CYP71P2 enzyme is cytochrome P450 enzyme derived from rhizoma atractylodis with advanced stem development mutation, and the amino acid sequence of the enzyme is shown as SEQ ID NO. 1.

The nucleotide sequence of the invention codes for the CYP71P2 enzyme.

Preferably, the nucleotide sequence is the sequence shown in SEQ ID NO. 2.

The recombinant vector comprises the nucleotide sequence.

The recombinant microorganism comprises the nucleotide sequence or the recombinant vector.

The product of the 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.

The CYP71P2 enzyme, or a nucleotide sequence, or a recombinant vector, or a recombinant microorganism, or an application of a product in preparation of sesquiterpenoids.

Preferably, the step of preparing the sesquiterpenoids comprises the following steps:

(1) Connecting a CYP71P2 enzyme nucleotide sequence to a vector plasmid to obtain a recombinant vector;

(2) Transforming the recombinant expression vector into host microorganism, and screening to obtain recombinant microorganism;

(3) Culturing recombinant microorganism, and extracting and purifying to obtain sesquiterpenoids.

Preferably, the host microorganism is squalene synthase knockout Saccharomyces cerevisiae, and the sesquiterpene compound is nerolidol.

Compared with the prior art, the method has the advantages that 1, CYP71P2 enzyme from Atractylodes lancea obtained through cloning and separation can be used for synthesizing sesquiterpene compounds, a new way is provided for synthesizing the compounds, a new thought is provided for breeding excellent varieties of Atractylodes lancea, 2, CYP71P2 is introduced into squalene synthase to knock out saccharomyces cerevisiae, thereby realizing high-efficiency production of nerolidol, and the yield can reach 500mg/L.

Drawings

FIG. 1 is a graph showing comparison of sesquiterpene content of wild rhizoma Atractylodis and rhizoma Atractylodis with advanced stem development mutation;

FIG. 2 is a heat map of P450 genes related to sesquiterpene synthesis differentially expressed by wild type rhizoma Atractylodis and stem advanced development mutant rhizoma Atractylodis;

FIG. 3 is a graph showing the analysis of the expression level of CYP71P2 enzyme in wild rhizoma Atractylodis and in rhizoma Atractylodis which is a stem advanced development mutation;

FIG. 4 is a three-dimensional structure diagram of CYP71P2 enzyme protein;

FIG. 5 is an agarose gel electrophoresis diagram of CYP71P2 enzyme, wherein M is Marker, and lane 1 and lane 2 are CYP71P2 enzyme coding genes;

FIG. 6 is a phylogenetic tree diagram of the CYP71P2 enzyme;

FIG. 7 is a graph showing Western-Blot results of the expression of CYP71P2 enzyme recombinant squalene synthase knockdown Saccharomyces cerevisiae induced by CuSO ₄, wherein M represents Marker, lane 1 represents protein extracted from an uninduced recombinant strain, lane 2 represents protein extracted from a recombinant strain induced by 50mM CuSO ₄, and lane 3 represents protein extracted from a recombinant strain induced by 100mM CuSO ₄;

FIG. 8 is a total ion flow diagram of yeast Saccharomyces cerevisiae and control strain fermentation extracts, with or without CYP71P2 enzyme recombinant squalene synthase knockdown, wherein A is total ion flow induced by adding 50mM CuSO ₄ to the recombinant strain, B is total ion flow of the uninduced recombinant strain, C is total ion flow induced by the control strain, and red arrows indicate differential compounds at retention time 15.965;

FIG. 9 is a mass spectrum of the difference compound and nerolidol standard at retention time 15.965, where A is the difference compound and B is the nerolidol standard.

Detailed Description

The technical scheme of the invention is further described below.

FastPure Universal Plant Total RNA Isolation Kit、HiScript IV 1st Strand cDNA Synthesis Kit(+gDNA wiper)、2×Phanta Flash Master Mix、2×Rapid Taq Master Mix、FastPure Plasmid Mini Kit、 E.coli DH 5. Alpha. Competent cells used in the following examples,Anti-His Mouse Monoclonal Antibody,Goat Anti-Mouse IgG (H+L), HRP Conjugate is a commercially available product. Wild type and mutant atractylis lancea specimens were stored in the rare animal and plant museum at the university of south Beijing (wild type accession number S01240, mutant accession number S01240-1).

EXAMPLE 1 cloning of the CYP71P2 enzyme-encoding Gene

1. Screening CYP450 genes for efficiently synthesizing sesquiterpenes by mediating mutant rhizoma atractylodis lanceae:

1.1, respectively taking 6 strains of wild rhizoma atractylodis lanceae and rhizoma atractylodis which are cultivated for 3 months and are subjected to development mutation in advance, washing with flowing water, absorbing water, placing a sample in a baking oven at 37 ℃ for drying for 36 hours, taking out and grinding after the sample is completely dried, transferring the ground sample into an EP tube, adding 10 times of n-hexane with volume (v: w) for leaching for 12 hours in a dark place, centrifuging at 8000rpm for 10 minutes after ultrasonic vibration for 30 minutes, taking supernatant, filtering by a filter membrane with the aperture of 0.22 mu m, and transferring into a brown sample injection vial.

The above samples were tested for sesquiterpene content using an Agilent 7890A gas chromatograph, the column was an HP-5 capillary column (30 m 0.32mm 0.1 μm), the detector was a Flame Ion Detector (FID), the carrier gas was high purity nitrogen, the temperature program was 70℃for 1min,8℃for 200℃and 20℃for 300℃for 5min.

The content of 12 sesquiterpenes was calculated according to a standard curve, and the results are shown in FIG. 1, wherein CK represents wild rhizoma Atractylodis, var represents mutant rhizoma Atractylodis. The results show that the total sesquiterpene content of the mutant rhizoma atractylodis is improved by 1.39 times, wherein the content of the amaranth alcohol is improved by 1.99 times, the content of the caryophyllene oxide is improved by 1.65 times, the content of the cedrol is improved by 1.67 times, the content of the beta-eucalyptol is improved by 1.58, and the content of the atractylone is improved by 1.42 times, wherein the content of the amaranth alcohol is improved by 1.99 times, the content of the nerolidol is improved by 1.74 times.

1.2, Extracting total RNA of overground parts of wild rhizoma atractylodis and stem advanced development mutant rhizoma atractylodis respectively by FastPure Universal Plant Total RNAIsolation Kit, carrying out transcriptome sequencing, screening out 28P 450s possibly involved in synthesizing sesquiterpenes by adopting a Blast method according to a sequencing result, wherein the result is shown in a figure 2, three P450 expression level changes show an up-regulation trend in mutant strains, the trend is the same as the content change of sesquiterpenes, in particular to CL31.Contig2_all, the expression level is up-regulated to the maximum, which suggests that the total RNA possibly plays a key role in the synthesis of the sesquiterpenes of rhizoma atractylodis;

1.3, designing a primer, wherein an upstream primer is 5'-ACCTATCGTTTGGCTGCTC-3', and a downstream primer is 5' -GCGGGTTACTTCCTCATCTC-3;

Extracting total RNA of overground parts of wild rhizoma atractylodis and stem advanced development mutant rhizoma atractylodis by FastPure Universal Plant Total RNA Isolation Kit respectively, taking 1 mug total RNA respectively, and performing reverse transcription into cDNA by using HISCRIPT IV 1st Strand cDNA Synthesis Kit (+ GDNAWIPER);

then preparing a reaction system of ChamQ Universal SYBR QPCR MASTER Mix 10 mu L, 1 mu L of template cDNA, 0.4 mu L of each of the upstream primer and the downstream primer, and 8.2 mu L of sterile water;

The result of gene expression level measurement by using Applied Biosystems ^TMQuantStudio^TM real-time fluorescence quantitative PCR system is shown in figure 3, the expression level of mutant atractylis lancea CL31.Contig2_all is improved by 2.12 times, the expression level change is obvious, the expression level of CL31.Contig2_all is closely related to the sesquiterpene content, and the protein structure is shown in figure 4, and the protein structure is named CYP71P 2.

2. Cloning of CYP71P2 enzyme coding Gene

Obtaining a cytochrome P450 gene CYP71P2 sequence from the full-length transcriptome data of the stem advanced development mutation rhizoma atractylodis obtained in the step 1.2, and designing an upstream primer 5'-ATGGACGATTTTACCCTTACAGTTGT-3' and a downstream primer 5'-TTATGTAGGTGAAACCCAAGCACCAT-3';

Extracting total RNA of atractylis lancea leaves with FastPure Universal Plant Total RNAIsolation Kit, taking 1 mug total RNA, and performing reverse transcription into cDNA by using HISCRIPT IV 1st Strand cDNASynthesis Kit (+ GDNAWIPER);

PCR amplification was performed using the Atractylodes lancea cDNA as a template in a specific amplification system of 2X PHANTA FLASH MASTER Mix 10. Mu.L, 1. Mu.L of template cDNA, 1. Mu.L of each of the upstream and downstream primers, and 7. Mu.L of sterile water under the following conditions of 98℃pre-denaturation 30s,98℃denaturation 10s,56℃annealing 5s,72℃extension 10s,35 cycles followed by 72℃extension 1min, and 4℃storage. The PCR product is loaded into 1% agarose gel for electrophoresis, the result is shown in figure 5, the size of the product fragment is 1566bp, after electrophoresis, fastPure Gel DNA Extraction Mini Kit is used for cutting gel, recovering and purifying the electrophoresis strip, and the CYP71P2 enzyme coding gene is obtained, the sequence of which is shown as SEQ ID NO. 2, and the amino acid sequence of the CYP71P2 enzyme is shown as SEQ ID NO. 1.

3. Bioinformatics analysis of CYP71P2 enzyme coding gene

The amino acid sequence of CYP71P2 enzyme is analyzed and compared, and by adopting a MEGA software (v 10.0), a sequence with higher similarity obtained by comparison and the amino acid sequence of CYP71P2 are used for constructing a phylogenetic tree, and the result shows that the genetic relationship between the CYP71P2 enzyme gene and the CYP71P1 gene of japonica rice is closer as shown in figure 6.

The result shows that CYP71P2 is a key factor for efficiently synthesizing sesquiterpenes by mutant rhizoma atractylodis lanceae, so that the gene can be also applied to an indicator factor for breeding of excellent rhizoma atractylodis varieties, and can contribute to the breeding of the excellent varieties of rhizoma atractylodis lanceae when the quality of wild rhizoma atractylodis is reduced.

EXAMPLE 2 construction of Yeast expression System of CYP71P2 enzyme

1. Constructing a recombinant expression vector by using a homologous recombination method, replacing GAL1,10promoter of a pESC-His vector with Cup1 promoter, adding His-tag and Flag-tag after the promoter to obtain a pESC-Cup-HF vector as an original vector, and designing a primer with a homology arm according to BamHI, sacI restriction sites and CYP71P2 gene sequences of the vector:

the upstream primer is 5'-gataaggtacccggatccATGGACGATTTTACCCTTACAGTTG-3';

The downstream primer is 5'-caacttctgttccatgtcgacTTATGTAGGTGAAACCCAAGCACC-3'.

Amplifying and purifying CYP71P2 gene with a homology arm by taking the gel recovery and purification DNA obtained in the step 2 of the example 1 as a template, connecting the fragment with a vector subjected to enzyme tangential linearization by using homologous recombinase to obtain a recombinant vector pESC-Cup-CYP71P2, and converting the recombinant vector into escherichia coli DH5 alpha competence by adopting a heat shock method, wherein the main operation conditions are that the temperature is 4 ℃ for 3min in ice bath, the temperature is 42 ℃ for 45s in heat shock, and the temperature is 4 ℃ for 3min in ice bath;

2. The transformed E.coli is evenly coated on an LB plate containing 30mg/L kanamycin, cultured overnight until single colony grows out, and the single colony is picked up to 10 mu L of sterile water, dispersed and evenly mixed to prepare a PCR system, wherein the PCR system comprises 2X RAPID TAQ MASTER Mix 10 mu L, 1 mu L of bacterial solution, 1 mu L of upper and lower primers and 7 mu L of sterile water, the PCR is carried out according to the following conditions of 5 ℃ pre-denaturation for 5min,95 ℃ denaturation for 30s,56 ℃ annealing for 15s,72 ℃ extension for 40s,30 cycles and 72 ℃ extension for 5min and 4 ℃ heat preservation, the upstream primer is 5'-AGCGATGCGTCTTTTCCGCT-3', and the downstream primer is 5'-TGCGTACACGCGTCTGTACAG-3';

after the product is checked by agarose gel electrophoresis, screening to obtain positive bacterial colony bacterial liquid, adding the rest 9 mu L bacterial liquid into 50mL LB liquid medium containing 30mg/L kanamycin for overnight culture, and extracting pESC-Cup-CYP71P2 plasmid by FastPure PLASMID MINI KIT;

1 mug of empty plasmid and recombinant plasmid pESC-Cup-CYP71P2 are respectively transformed into squalene synthase knocked out saccharomyces cerevisiae by adopting a lithium acetate transformation method, the transformed bacterial liquid is evenly coated on an SD-H solid culture medium containing 30mg/L ergosterol, positive clones are screened again by adopting a colony PCR method after single colonies grow out, the obtained positive clones are inoculated into the SD-H culture medium for overnight culture by adopting the same operation method and the same reagent, when OD ₆₀₀ = 0.9 of the bacterial liquid is added into CuSO ₄ with the final concentration of 50mM or 100mM for induction,

Wherein squalene synthase knockout Saccharomyces cerevisiae reference Zhuang,X.(2013).Engineering novel terpene production platforms in the yeast Saccharomyces cerevisiae. is constructed;

3. Protein expression in the recombinant squalene synthase knock-out saccharomyces cerevisiae strain is verified by Western-Blot, after CuSO ₄ induction is carried out for 24 hours, 500 mu L of bacterial liquid is taken, the supernatant is discarded after 8000rpm centrifugation for 5 minutes, 100 mu L of 1X Loadingbuffer is added into a 98 ℃ water bath kettle for heating for 10 minutes, the supernatant is taken after 8000rpm centrifugation for 5 minutes again, SDS-PAGE gel electrophoresis is carried out, membrane transfer and sealing are carried out after the electrophoresis is finished, wherein the primary antibody is Anti-His Mouse Monoclonal Antibody, the dilution ratio is 1:3000, the secondary antibody isGoat Anti-Mouse IgG (H+L), HRP Conjugate, dilution ratio was 1:5000. After incubation is complete, exposure verification is performed under a chemiluminescent instrument using Tanon ^TM Femto-sig ECL chemiluminescent substrate. As shown in FIG. 7, lanes 2 and 3 were induced by CuSO ₄ for recombinant strains, and the positions of the bands were consistent with the expected sizes of CYP71P2 enzyme, indicating that the enzyme can be expressed normally.

Example 3 functional verification of CYP71P2 enzyme

The strain of the squalene synthase knocked-out saccharomyces cerevisiae with no-load transformation is selected as a control strain, the control strain and the recombinant strain are inoculated into an SD-H culture medium for culture, and when the bacterial liquid OD ₆₀₀ = 0.9, cuSO ₄ with the final concentration of 50mM is added for induction for 7d. After induction, respectively taking 3mL of bacterial liquid, adding 3mL of acetone, vigorously shaking for 5min, standing at room temperature for 5min, adding 3mL of n-hexane, vigorously shaking for 5min, standing at room temperature for 10min, centrifuging at 8000rpm for 10min, taking an upper organic phase into a new centrifuge tube, placing a sample into a vacuum concentrator, concentrating to 50 mu L, storing in a brown liquid phase sample bottle containing an inner liner tube, and storing in a refrigerator at-20 ℃;

Samples were analyzed using an Agilent 5973 gas chromatograph-mass spectrometer, the column being HP-5, its specific specification being 30m x 0.25mm x 0.25 μm, the gas chromatograph conditions being 100 ℃ for 4min,5 ℃/min up to 280 ℃ for 6min. The mass spectrum conditions are that the ion source temperature is 230 ℃, the interface temperature is 280 ℃, and the scanning mass range is 35-500aum. The total ion flow chart is shown in figure 8, a differential compound with the retention time 15.965 exists, the mass spectrum of the differential compound is shown in figure 9, wherein A is a differential compound mass spectrum with the retention time 15.965, B is a standard mass spectrum of nerolidol, the result shows that the differential compound is the nerolidol, and the further calculation shows that the synthesis amount of the nerolidol reaches 500mg/L.

Claims (9)

1. The CYP71P2 enzyme is characterized in that the CYP71P2 enzyme is a cytochrome P450 enzyme derived from rhizoma atractylodis with advanced stem development mutation, and the amino acid sequence of the enzyme is shown in SEQ ID NO. 1.

2. A nucleic acid molecule encoding the CYP71P2 enzyme of claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID No. 2.

4. A recombinant vector comprising the nucleic acid molecule of claim 2 or 3.

5. A recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3 or the recombinant vector of claim 4.

6. A product comprising one or more of the CYP71P2 enzyme of claim 1, or the nucleic acid molecule of claim 2 or 3, or the recombinant vector of claim 4, or the recombinant microorganism of claim 5.

7. Use of a CYP71P2 enzyme according to claim 1, or a nucleic acid molecule according to claim 2 or 3, or a recombinant vector according to claim 4, or a recombinant microorganism according to claim 5, or a product according to claim 6, for the preparation of nerolidol.

8. The use according to claim 7, wherein the step of preparing nerolidol comprises:

(1) Connecting CYP71P2 enzyme nucleic acid molecules to a vector plasmid to obtain a recombinant vector;

(2) Transforming the recombinant vector into host microorganism, and screening to obtain recombinant microorganism;

(3) Culturing recombinant microorganism, extracting and purifying to obtain nerolidol.

9. The use according to claim 8, wherein the host microorganism is squalene synthase knockout saccharomyces cerevisiae.