CN115927218B - CYP450 enzyme protein for catalyzing beta-amyrin 21-position hydroxylation, coding gene and application thereof - Google Patents

Abstract

The invention discloses CYP450 enzyme protein catalyzing beta-amyrin 21-bit hydroxylation, a coding gene and application thereof. The protein is the protein of the following a) or b): a) A protein consisting of an amino acid sequence shown as a sequence 2 in a sequence table; b) And a protein which is derived from a) and has the enzymatic activity of catalyzing the hydroxylation of the C-21 position of beta-amyrin alcohol through the substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table. The invention utilizes WGCNA analysis to dig out key enzyme catalyzing beta-amyrin C-21 position hydroxylation based on high-throughput sequencing of different tissue parts of horse chestnut leaves, branches, flowers, epicarp and kernels and horse chestnut fruit space metabonomics results, thereby providing key gene modules for the synthesis biology research of aescin and key gene loci for molecular design breeding of horse chestnut.

Description

CYP450 enzyme protein for catalyzing beta-amyrin 21-position hydroxylation, coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to CYP450 enzyme protein catalyzing beta-amyrin alcohol 21-position hydroxylation, and a coding gene and application thereof.

Background

Cytochrome P450 is the first group of enzymes classified as "superfamily" and is found in almost all prokaryotic and eukaryotic organisms, even in thermophilic archaea. The CYP450 gene family is also an ancient super gene family, comprising more than 1000 families and 2500 subfamilies. CYP450 is involved in the biosynthesis of a variety of terpenoids. Terpenes are widely found in nature and comprise oxygen-containing derivatives of alcohols, aldehydes, ketones, carboxylic acids, esters and the like, which are used in a number of fields including industry, medicine, hygiene and the like. The diversity of terpenes is largely dependent on the modification of many specific chemical groups, rearrangement of the backbone structure and post-modification reactions, most of which are catalyzed by cytochrome P450 monooxygenases. Up to now, more than 55P 450 enzymes have been identified, which modify the pentacyclic triterpene skeleton of plants. Most of them belong to the family of CYP716 members, CYP51, CYP71, CYP72, CYP87, CYP88, CYP93 are other families of P450 involved in pentacyclic triterpene modification. Recent studies of the phylogenetic analysis of CYP716 from plants greater than 200 have shown that in dicots the CYP716 family has evolved towards triterpene biosynthesis.

The aesculus seed is an important traditional Chinese medicine recorded in Chinese pharmacopoeia, and aescine is a main active ingredient of the aesculus seed, has various pharmacological activities of diminishing inflammation, detumescence, resisting tumor, resisting virus and the like, and is clinically used for treating chronic venous insufficiency, venous embolism, hemorrhoids, postoperative edema and the like. Although aescin has important medical value, analysis and research on the biosynthesis pathway are relatively weak, and new technology and new method are urgently needed to analyze the biosynthesis pathway of aescin.

Triterpenes belong to the class of isoprenoids, the synthesis precursor farnesyl pyrophosphate of which is mainly derived from the mevalonate pathway (Mevalonate Pathway, MVA), which is in turn catalyzed by squalene synthase and squalene monooxygenase to form 2, 3-oxidized squalene, which is a common precursor for the biosynthesis of triterpenes and sterols, which under the catalysis of 2, 3-oxidized squalene cyclase form different triterpene frameworks, of which β -amyrin is the most common triterpene framework. CYP450 enzymes can catalyze the oxidation of beta-amyrin, and a great deal of researches show that the CYP716 family enzymes can catalyze the oxidation of other forms such as C-28, C-16, C-22 or C-12 hydroxylations of pentacyclic triterpene frameworks. The above results indicate that the CYP716 family plays an important role in the hydroxylation modification of pentacyclic triterpenes, and that different CYPs 716 have different substrate specificities and different catalytic activities.

Because the aescin has good pharmacological activities such as anti-inflammatory, detumescence and antivirus, and the like, the supply of aescin resources is tension caused by the shortage of clinical supply, so that the problem of the tension of the aescin resources is urgently needed to be solved by using a new technology and a new method.

Disclosure of Invention

The discovery and identification of plant CYP450 genes plays a vital role in the exploration of triterpene biosynthesis. CYP450 catalyzed beta-amyrin alcohol hydroxylation can provide sites for further glycosylation and acylation to form triterpene compounds with various structures, and provides abundant lead compounds for triterpene drug development. The CYP450 gene for catalyzing the hydroxylation of the C-21 position of the beta-amyrin is analyzed, so that a key gene module is provided for the synthesis biology research of aescin, a key site is provided for the cultivation of new varieties of aesculus, and an important reference is provided for researching the hydroxylation modification of pentacyclic triterpene compounds.

In view of the above, the main object of the present invention is to provide CYP450 enzyme protein catalyzing beta-amyrin C-21 hydroxylation, and coding gene and application thereof.

The technical scheme of the invention is as follows:

the invention provides a protein which is the protein of the following a) or b):

a) A protein consisting of an amino acid sequence shown as a sequence 2 in a sequence table;

b) And a protein which is derived from a) and has the enzymatic activity of catalyzing the hydroxylation of the C-21 position of beta-amyrin alcohol through the substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table.

The catalytic beta-amyrin C-21 position hydroxylation is the catalytic beta-amyrin to generate 21 beta-hydroxy-beta-amyrin.

The coding genes of the proteins also belong to the protection scope of the invention.

The coding gene is as follows 1) or 2) or 3):

1) The nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in 1);

3) A DNA molecule having a homology of 90% or more with the DNA molecule defined in 1) or 2).

The gene was namedAcCYP716A278，The protein encoded by it was designated AcCYP716a278. The specific information is as follows:AcCYP716A278the gene sequence is shown as a sequence 1 in a sequence table, contains 1422 nucleotides, encodes a protein shown as a sequence 2 in the sequence table, and the sequence 2 consists of 473 amino acids.

Expression cassettes, recombinant expression vectors, transgenic cell lines or recombinant microorganisms containing the coding genes also belong to the scope of protection of the invention.

The application of the protein in the key enzyme for catalyzing the hydroxylation of the C-21 position of beta-amyrin alcohol also belongs to the protection scope of the invention.

The catalytic beta-amyrin C-21 position hydroxylation is the catalytic beta-amyrin to generate 21 beta-hydroxy-beta-amyrin.

The application of the protein and the coding gene in catalyzing beta-amyrin to generate 21 beta-hydroxy-beta-amyrin also belongs to the protection scope of the invention.

The invention utilizes WGCNA analysis to dig out key enzyme catalyzing beta-amyrin C-21 position hydroxylation based on high-throughput sequencing of different tissue parts of horse chestnut leaves, branches, flowers, epicarp and kernels and horse chestnut fruit space metabonomics results, thereby providing key gene modules for the synthesis biology research of aescin and key gene loci for molecular design breeding of horse chestnut.

The invention comprehensively utilizes the combination analysis of space metabonomics and transcriptomics, digs CYP450 genes catalyzing the hydroxylation of the C-21 position of beta-amyrin, verifies that AcCYP716A28 can catalyze the formation of 21 beta-hydroxy-beta-amyrin by utilizing a tobacco transient expression system, not only provides important gene elements for the biosynthesis of aescin A, but also provides key gene sites for the molecular design breeding of aesculus by the research result, and the structural formula of 21 beta-hydroxy-beta-amyrin and aescin A is shown in figure 1.

Drawings

For purposes of illustration and not limitation, the invention will now be described in accordance with its preferred embodiments, particularly with reference to the accompanying drawings, in which:

FIG. 1 shows the molecular structural formula of 21 beta-hydroxy-beta-amyrin alcohol and aescin A.

Fig. 2 is a mass spectrometry imaging diagram of a cross section of a horse chestnut fruit.

FIG. 3 shows the results of cloning and constructing the vector of AcCYP716A278 gene.

FIG. 4 is a PCR gel diagram of the objective gene Agrobacterium tumefaciens bacterial liquid.

FIG. 5 is a GC-MS diagram for identification of tobacco products injected.

Detailed Description

EXAMPLE 1 cloning of a Gene encoding a CYP450 enzyme protein catalyzing hydroxylation of beta-amyrin alcohol at position 21

1. Method and procedure for cloning of genes

The horse chestnut is drawn on the slice by using the MALDI-MSI imaging technologyAesculus chinensis) Spatial distribution map of aescin and coumarin in fruit (Sparvero LJ, AA Amoscano, CE Dixon, JB Long, PM Kochanek, BR Pitt, H Bay ı r)&VE Kagan (2012) Mapping of phospholipids by MALDI imaging (MALDI-MSI) realizations and predictions CHEM PHYS LIPIDS 165:165:545-562. The result shows that the aescin component is obviously enriched in the seedsInternally, coumarin is mainly distributed in the pericarp (fig. 2).

And (3) carrying out transcriptome sequencing on different tissue parts of the horse chestnut leaves, branches, flowers, epicarp and kernel by utilizing an Illumina platform. Conventional bioinformatics analysis and Weighted Gene Co-expression network analysis (Weighted Gene Co-Expression Network Analysis, WGCNA) were performed on transcriptome data from different tissue sites. A module was found in the seed that correlated very significantly with acoc 6 (P < 0.01) and included AcCYP716a278 in addition to acoc 6.

Collecting mature seeds of horse chestnut, extracting RNA in the horse chestnut seeds by using an RNA extraction kit (Aidelai, RN38 EASYspin plus), and carrying out reverse transcription after the quality detection of the RNA is qualified to obtain cDNA with qualified quality. Designing primer sequence (shown in Table 1), cloning with KOD-Plus-Neo high-fidelity enzyme using cDNA as templateAcCYP716A278、AcOSC6Gene fragment (KOD high-fidelity enzyme PCR system total volume 50. Mu.L: 5. Mu.L 10 XBuffer, 3. Mu.L MgSO4, 5. Mu.L dNTP (2 mM), 1.5. Mu.L forward primer (10. Mu.M), 1.5. Mu.L reverse primer (10. Mu.M), 1. Mu.L template, 1. Mu.L KOD enzyme and 32. Mu.L water) was prepared as described in Table 2. With the help of pEASY-Blunt vector, successful implementation will be achievedAcCYP716A278The fragments were ligated into the vector (total ligation system volume: 3. Mu.L, 0.5. Mu.L pEASY-Blunt vector and 2.5. Mu.L cDNA template, ligation reaction at 25℃for 1 h). The ligation system was directly transformed into TransT1 competent, and positive clone sequencing (colony PCR system total volume was 12.5. Mu.L: 6.25. Mu.L 2 XTaq PCR Mix, 1. Mu.L template, 0.25. Mu.L forward primer, 0.25. Mu.L reverse primer and 4.75. Mu.L water, procedure as in Table 3, PCR as shown in FIG. 3A) was selected, and the nucleotide sequence was found to be 100% similar to the original data by alignment with RNAseq sequence.

The front 417: 417 bp base sequence of the transmembrane region of AstHMGR (GeneBank ID: KY 284573) was deleted, and the entire length of the gene coding sequence of the remaining 1272: 1272 bp base sequence and GmCYP72A69 (GeneBank ID: LC 143440) gene was subjected to gene synthesis by the Shanghai Biotechnology service Co., ltd, and then ligated into pUC118p cloning vector.

Construction of a tobacco transient expression vector by referring to Invitrogen Gateway cloning technology, which comprises the following specific steps:

(1) Designing primer sequence, adding attB1 sequence at 5 'end and attB2 sequence at 3' end of target fragment (Table 1), and performing KOD-PCR (KOD high-fidelity enzyme PCR system total volume is 50. Mu.L: 5. Mu.L 10X Buffer, 3. Mu.L MgSO) with the target fragment plasmid obtained above as template ₄ mu.L dNTP (2 mM), 1.5. Mu.L forward primer (10. Mu.M), 1.5. Mu.L reverse primer (10. Mu.M), 1. Mu.L template, 1. Mu.L KOD enzyme and 32. Mu.L water, were used as in Table 2.

(2) BP reaction: 25ng of attB PCR recovery and 75ng of pDONR207 entry vector (Biovector NTCC collection) were mixed to 4. Mu.L with water, then L. Mu.L of BP Clonase II enzyme (Thermo Fisher, gateway ™ BP close ™ enzyme mixture) were added, mixed, incubated at 25℃for L h, 0.5. Mu.L of protease K was added, incubated at 37℃for 10min, transferred to TransT1 competence, positive clones were screened on 15 mg/L Kan resistant LB solid medium and PCR tested (gel diagram results are shown in FIG. 3B), and positive clones sequenced successfully extracted recombinant plasmids.

(3) LR reaction: pDONR207 recombinant plasmid of 75ng and 75ng pEAQ-HT-DEST vector plasmid (Biovector NTCC collection) were mixed to 4. Mu.L with deionized water, then 0.4. Mu.L of LR Clonase II enzyme (Thermo Fisher, gateway ™ LR close ™ enzyme mixture) was added, mixed, incubated at 25℃for L h, 0.5. Mu.L of protease K was added, incubated at 37℃for 10min, transferred to TransT1 competence, positive clones were selected on 50 mg/mL Kan-resistant LB solid medium and PCR tested (Table 3) (gel diagram results are shown in FIG. 3C), positive clones sequenced successfully were preserved and plasmids were extracted, and finally plasmids were obtained in which four genes AcCYP716A28, acOSC6, astHMGR and Gm72A 69 were ligated to pEAQ-HT-DEST vector.

2. Obtaining a gene sequence and a protein sequence encoded by the gene sequence

Sequencing results show that the gene amplified by using the primer in Table 1 contains 1422 nucleotides (shown as sequence 1 in the sequence table), encodes 473 amino acid protein (shown as sequence 2 in the sequence table), and the gene is named by official nameAcCYP716A278The protein encoded by it was designated AcCYP716a278.

Gene amplified by using the primers in Table 1AcOSC6Contains 2298 nucleotides (shown as sequence 3 in a sequence table) and codes for 765 amino acid protein AcOSC6 (shown as sequence 4 in the sequence table).

3. Verification of Gene function

The test of the full gold treatment extraction kit EasyPure HiPure Plasmid MiniPrep Kit is referred to for extracting plasmids in positive escherichia coli:

(1) 2mL of the overnight cultured bacterial liquid was centrifuged at 10,000 g for 1 minute, and the supernatant was removed (as completely as possible).

(2) 250. Mu.L of colorless solution RB (containing RNase A) was added, and the bacterial pellet was suspended by shaking, leaving no small clumps.

(3) 250 mu L of blue solution LB is added, and the mixture is gently turned up and down and mixed for 4 to 6 times, so that the thalli are fully cracked, and a blue transparent solution is formed.

(4) 350. Mu.L of yellow solution NB was added and gently mixed 5-6 times until a compact yellow agglomerate formed and allowed to stand at room temperature for 2 minutes.

(5) Centrifuge 12,000 g for 5 minutes, carefully aspirate the supernatant and load into the column. Centrifuge at 12,000 g for 1 min, discard effluent.

(6) 650. Mu.L of WB solution was added, and the mixture was centrifuged at 12,000 g for 1 minute, and the effluent was discarded.

(7) Centrifugation at 12,000 g for 2 min, the residual WB was removed thoroughly.

(8) The centrifuge column was placed in a clean centrifuge tube, 40 μl deionized water was added to the center of the column, and the column was allowed to stand at room temperature for 1 minute.

(9) 10,000 g was centrifuged for 1 min and the DNA eluted for the next Agrobacterium transformation experiment.

The agrobacterium transformation was performed with reference to the experimental description of the local organism EH105 agrobacterium electrotransformation:

(1) Taking out the 0.1 cm electric shock cup and the cup cover from the storage liquid, pouring the electric shock cup and the cup cover on clean water-absorbing paper for 5 minutes, draining ethanol until the electric shock cup and the cup cover are drained, standing for 5 minutes, enabling the ethanol to volatilize fully, inserting the electric shock cup and the cup cover into ice immediately after the ethanol volatilizes cleanly, compacting the ice surface, enabling the top of the electrode cup to be away from the ice surface by 0.5 cm so as to cover the cup cover conveniently, standing in the ice for 5 minutes, and cooling fully.

(2) EH105 Agrobacterium competent cells were removed from the-80℃refrigerator, inserted into ice for 5 min, after thawing, 0.01-1. Mu.g pEAQ-HT-DEST positive plasmid DNA (volume not more than 6. Mu.l) linked to the gene of interest was added, the tube bottom was stirred by hand, mixed well, immediately inserted into ice, the competent-plasmid mixture was rapidly transferred into a cuvette with 200. Mu.l gun head, covered with a cup lid, and the hollow tube was kept ready for use.

(3) Starting an electrotransport device, rapidly placing an electric shock cup into an electrotransport tank, rapidly inserting electric shock into ice, adding 700 mu l of LB without antibiotics, transferring the LB without antibiotics into a competent empty tube, and culturing at 28 ℃ for 2-3 hours in an oscillating way.

(4) After centrifugation at 6000 rpm for one minute, about 100. Mu.l of supernatant was left to gently blow the resuspended pellet and spread on LB plates containing 50 mg/L Kan and 50 mg/L Rif antibiotics, and the pellet was placed upside down in a28℃incubator for 2-3 days.

(5) Single colonies on LB plates were picked with sterilized toothpicks, and were subjected to PCR identification (results shown in FIG. 4) in 500. Mu.L of liquid LB medium containing 50 mg/L Kan and 50 mg/L Rif antibiotics, shaking culture at 28℃for 8-12 hours, selection of the forward primer of pEAQ-HT-DEST (Table 4) and the reverse primer of the target fragment (AcOSC 6-R and AcCYP716A278-R, see Table 1, and AstHMGR-R and GmCYP72A69-R, see Table 4), respectively, and the positive strains were subjected to the next transient expression test of tobacco.

Tobacco transient expression and product identification were as follows:

(1) Agrobacterium EHA105 injection Benshi tobacco leaf

EHA105 containing recombinant plasmid was inoculated in 5 ml LB liquid medium containing 50 mg/L Rif and 50 mg/L Kan antibiotic, shake cultured at 28℃to OD600 of about 1.0; 4000 g centrifuging for 5 min, discarding supernatant, collecting bacterial precipitate, and concentrating with MMA solution (10 mM MES,10 Mm MgCl concentration) ₂ 200 mu M mixed solution of acetosyringone) is resuspended to an OD600 of 0.8-1.2 and left at room temperature of 3 h; selecting good-growth Nicotiana benthamiana (Goodin MM, zaitlin D, naidu RA, lommel SA (2008): nicotiana benthamiana: its History and Future as a Model for Plant-Pathen interactions. Molecular Plant-Microbe Interactions:21:1015-1026.), lightly puncturing the small holes on the surface of the leaf with a syringe needle, and facilitating injection of the Agrobacterium MMA suspension; dark culturing 1 d, taking out, culturing with light again 4 d, collecting leaves injected with Agrobacterium, and preserving at-80deg.C.

(2) Analysis and identification of catalytic products

Grinding the tobacco leaves into powder in liquid nitrogen, taking about 0.1 and g of the powder into a 500uL leaching agent (extraction reagent: methanol: water: potassium hydroxide=9:1:1, v: w), leaching for 2 hours at 65 ℃, vibrating for 1 time every half hour, then adding 250ul of water, vibrating and mixing uniformly, adding 500ul of normal hexane, vibrating and mixing uniformly, centrifuging for 1 min at 12000 g, taking 100ul of upper normal hexane solution, and concentrating to dryness by a vacuum centrifugal concentrator (Concentrator plus, eppendor); then 50uL of ammonium N-methyl-N-trimethylsilyl trifluoroacetate (aladine, china) was added for derivatization for 30 min at 80 ℃, diluted with 50uL ethyl acetate, and transferred to agilent sample bottles for GC-MS detection.

GC-MS detection was performed using Agilent 7890, gas chromatography column hb-5 (30 mX0.25 mm X0.25 um, aglient), temperature program: maintaining at 170deg.C for 2 min, heating to 300deg.C at 20deg.C/min, and maintaining at 300deg.C for 10min (total 20 min); carrier gas (He) flow rate 1mL/min; the sample injection amount is 1 mu L; the temperature of the gasification chamber is 250 ℃; the mass spectrum detector is an Agilent 7000C triple quaternary rod, and the mass spectrum scanning range is 60-800 u.

The GC-MS analysis results are shown in FIG. 5, in FIG. 5AstHMGRResearch from oat, geneBank ID KY284573, reed et al foundAstHMGRCan increase the content of 2, 3-oxidation squalene in tobacco leaves, and provide more substrate for beta-amyrin alcohol synthesis (Thimmappa, R., et al, triterpene biosynthesis in plants Annual Review of Plant Biology, 2014.65: p.225-257); in horse chestnutAcOSC6Can catalyze the formation of beta-amyrin alcohol, and provide a substrate for further hydroxylation modification;GmCYP72A69from soybean, can catalyze the hydroxylation of the C-21 position of beta-amyrin, and can be used for assisting in identifying 21 beta-hydroxy-beta-amyrin products. Will beAstHMGR+AcOSC6、AstHMGR+ AcOSC6+AcCYP716A28、AstHMGR+AcOSC6/GmCYP72A69And respectively co-injecting in tobacco, wherein beta-amyrin can be detected by all three injection modes, and a substrate is provided for the next hydroxylation reaction. It was found that GmCYP72A69 in soybean catalyzes the hydroxylation of the C21-position of beta-amyrin to 21 beta-hydroxy-beta-amyrin, whose fragment ions are 496, 306, 291, 190 when examined by GC-MS (Yano, R., et al, metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in cytochrome P450A 69. The Plant Journal, 2017.89 (3): p.527-539.; leveau, A., et al, towards take-all control: a C-21 beta oxidase required for acylation of triterpene defence compounds in oat. New bacteriology, 2019.221 (3): p.1544-1555.). In the present invention, co-injection is performed in tobaccoAstHMGR+AcOSC6+GmCYP72A69A peak with characteristic ion 306 was detected and the retention time was 13.572 min. Co-injection in tobaccoAstHMGR+AcOSC6+ AcCYP716A28Special can also be detectedThe peak with the characteristic ion of 306 and the peak outlet time of 13.572 are compared, and mass spectrum fragment information of the two is completely consistent (figure 5), which shows that AcCYP716A28 in horse chestnut can catalyze beta-amyrin to generate 21 beta-hydroxy-beta-amyrin.

The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives can occur depending upon design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims (7)

1. A protein has an amino acid sequence shown in SEQ ID NO. 2.

2. The protein-encoding gene of claim 1, wherein: the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

3. An expression cassette, recombinant expression vector, transgenic cell line or recombinant microorganism comprising the coding gene of claim 2.

4. Use of the protein of claim 1 as a key enzyme for catalyzing hydroxylation of beta-amyrin at C-21 position.

5. The use according to claim 4, characterized in that: the catalytic beta-amyrin C-21 position hydroxylation is the catalytic beta-amyrin to generate 21 beta-hydroxy-beta-amyrin.

6. Use of the protein of claim 1 for catalyzing the production of 21 beta-hydroxy-beta-amyrin.

7. The use of the coding gene of claim 2 for catalyzing the production of 21 beta-hydroxy-beta-amyrin from beta-amyrin.