CYP716A236a gene and CYP72A1371a gene and their use in the biosynthesis of betulinic acid and 23-hydroxybetulinic acid

Abstract

This invention discloses CYP716A236a Genes and CYP72A1371a The gene and its application in the biosynthesis of betulinic acid and 23-hydroxybetulinic acid belong to the field of genetic engineering technology. CYP716A236a Genes and the CYP72A1371a The nucleotide sequence of the gene is shown in SEQ ID NO. 1-2. Overexpression in plants or yeast. CYP716A236a The gene can efficiently catalyze the oxidation of lupinol to betulin in plants or yeast; and overexpression of this gene... CYP716A236a Genes and CYP72A1371a The gene can catalyze the continuous oxidation of lupeol to synthesize 23-hydroxybetulic acid in plants or yeast. This invention provides new gene resources and catalytic elements for the production of betulic acid and 23-hydroxybetulic acid using synthetic biology methods.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to... CYP716A236a Genes and CYP72A1371a Genes and their application in the biosynthesis of betulinic acid and 23-hydroxybetulinic acid. Background Technology

[0002] Lupeol, betulinic acid, and 23-hydroxybetulinic acid are all pentacyclic triterpenoid compounds of the lupeane class (see...). Figure 1 In recent years, betulinic acid and 23-hydroxybetulinic acid have attracted great interest in drug development due to their broad biological activities, particularly their potential in antitumor, antiviral, anti-inflammatory, antibacterial, antioxidant, and neuroprotective effects. However, their content in plants is low, and extraction and separation require large amounts of plant materials, resulting in resource waste. Furthermore, the isolated betulinic acid and 23-hydroxybetulinic acid contain high levels of impurities that are difficult to remove, and purification requires large amounts of organic solvents. Therefore, industrial research has developed a multi-step chemical reaction involving protection, oxidation, and deprotection to oxidize lupeol, which is relatively abundant in nature, to obtain betulinic acid and 23-hydroxybetulinic acid.

[0003] However, chemical synthesis methods suffer from problems such as hazardous reagents, harsh reaction conditions, and cumbersome procedures. Therefore, research has begun to shift towards synthetic biology, a method characterized by milder processes, environmental friendliness, and high product specificity. Biosynthesis requires enzyme elements with high catalytic efficiency and specific catalytic function. However, currently, there are few reports on oxidases catalyzing the synthesis of betulinic acid from lupeol and hydroxylases catalyzing the subsequent synthesis of 23-hydroxybetulinic acid. Furthermore, their catalytic efficiency is low in heterogeneous production applications, leading to excessive accumulation of intermediate products. Summary of the Invention

[0004] The purpose of this invention is to provide CYP716A236a Genes and CYP72A1371a This invention addresses the problems existing in the prior art by investigating the genes and their applications in the biosynthesis of betulinic acid and 23-hydroxybetulinic acid. Specifically, it identifies the oxidase CYP716A236a from *Pulsatilla chinensis* that catalyzes the synthesis of betulinic acid from lupeol, and the 23-hydroxylase CYP72A1371a that subsequently catalyzes the synthesis of 23-hydroxybetulinic acid. Experiments have demonstrated that the CYP716A236a protein can efficiently catalyze the oxidation of lupeol at the C-28 position to betulinic acid, and the CYP72A1371a protein can catalyze the hydroxylation of betulinic acid at the C-23 position to 23-hydroxybetulinic acid, laying the foundation for the subsequent production of betulinic acid and 23-hydroxybetulinic acid through synthetic biology methods.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a catalytic method for the production of betulinic acid from lupeol. CYP716A236a Genes, the ones mentioned CYP716A236a The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] The present invention also provides a CYP716A236a protein that catalyzes the production of betulinic acid from lupeol, wherein the amino acid sequence of the CYP716A236a protein is shown in SEQ ID NO.3.

[0007] This invention also provides a catalytic method for the production of 23-hydroxybetulic acid from betulinic acid. CYP72A1371a Genes, the ones mentioned CYP72A1371a The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0008] The present invention also provides a CYP72A1371a protein that catalyzes the production of 23-hydroxybetulic acid from betulinic acid, wherein the amino acid sequence of the CYP72A1371a protein is shown in SEQ ID NO.4.

[0009] The present invention also provides the application of the above-mentioned CYP716A236a protein in the preparation of a betulinic acid synthesis catalyst, wherein the betulinic acid synthesis catalyst has the function of catalyzing the conversion of lupeol to betulinic acid.

[0010] The present invention also provides the application of the above-mentioned CYP72A1371a protein in the preparation of a 23-hydroxybetulic acid synthesis catalyst, wherein the 23-hydroxybetulic acid synthesis catalyst has the function of catalyzing the production of 23-hydroxybetulic acid from betulic acid.

[0011] The present invention also provides the above. CYP716A236a The application of the gene or the aforementioned CYP716A236a protein in increasing the content of betulinic acid in plants or yeast, wherein the method for increasing the content of betulinic acid in plants or yeast includes using genetic transformation technology to... CYP716A236a The gene is transferred into plants or yeast to increase the expression level of the CYP716A236a protein, thereby catalyzing the process of converting lupeol to betulin in plants.

[0012] The present invention also provides the above. CYP716A236a Gene combination of the above CYP72A1371a The application of the gene or the aforementioned CYP716A236a protein combined with the aforementioned CYP72A1371a protein in increasing the content of 23-hydroxybetulic acid in plants or yeast, wherein the method for increasing the content of 23-hydroxybetulic acid in plants or yeast includes using genetic transformation technology to... CYP716A236a Genes and the CYP72A1371aThe genes are co-transferred into plants or yeast to increase the expression levels of the CYP716A236a and CYP72A1371a proteins, thereby catalyzing the conversion of lupeol to betulinic acid in plants or yeast, and subsequently to 23-hydroxybetulinic acid.

[0013] The present invention also provides the above. CYP716A236a Application of the gene or the aforementioned CYP716A236a protein in the cultivation of plant strains or yeast strains that produce high levels of betulinic acid.

[0014] The present invention also provides the above. CYP716A236a Gene combination of the above CYP72A1371 Application of gene a or the above-mentioned CYP716A236a protein combined with the above-mentioned CYP72A1371a protein in cultivating plant strains or yeast strains that produce high yields of 23-hydroxybetulic acid.

[0015] Optionally, the plant includes tobacco.

[0016] Optionally, the yeast includes Saccharomyces cerevisiae.

[0017] Optionally, when the plant is tobacco or the yeast is Saccharomyces cerevisiae, the lupeol content in the tobacco or Saccharomyces cerevisiae is first increased using genetic transformation technology, and then the above-mentioned lupeol content is overexpressed in the tobacco or Saccharomyces cerevisiae using genetic transformation technology. CYP716A236a Genes and / or the above CYP72A1371a Genes are used to express large amounts of the above-mentioned CYP716A236a protein and / or the above-mentioned CYP72A1371a protein, thereby synthesizing betulinic acid and / or 23-hydroxybetulinic acid in tobacco or brewer's yeast, and cultivating tobacco strains and brewer's yeast strains that produce high amounts of betulinic acid and / or tobacco strains and brewer's yeast strains that produce high amounts of 23-hydroxybetulinic acid.

[0018] The present invention discloses the following technical effects: This invention addresses the pharmacological value of betulinic acid and 23-hydroxybetulinic acid, as well as the numerous problems encountered in their chemical synthesis. Through transcriptomics, content determination, co-expression analysis, protein sequence clustering, enzyme activity analysis, and identification, the oxidase CYP716A236a catalyzing the synthesis of betulinic acid from lupeol in *Pulsatilla chinensis*, and the subsequent hydroxylase CYP72A1371a catalyzing the synthesis of 23-hydroxybetulinic acid, were identified. Experiments have confirmed that overexpression of these enzymes in plants or yeast... CYP716A236a The gene can catalyze the synthesis of betulin from lupeol in plants or yeast; and overexpression of this gene... CYP716A236a Genes and CYP72A1371aThe gene can catalyze the continuous oxidation of lupeol to synthesize 23-hydroxybetulic acid in plants or yeast. This invention provides new gene resources and catalytic elements for the production of betulic acid and 23-hydroxybetulic acid using synthetic biology methods, and provides technical support for cultivating plant or yeast strains that naturally produce high yields of betulic acid and 23-hydroxybetulic acid. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the chemical structures of lupeol, betulinic acid, and 23-hydroxybetulinic acid. Figure 2 The differential expression of 168 CYP450-encoding genes in Pulsatilla chinensis at different ages and in different tissues; Figure 3 The differential expression of 170 CYP450 encoding genes in Pulsatilla chinensis at different ages and in different tissues; Figure 4 For overexpression CYP716A236a Genes and / or CYP72A1371a EIC diagram of betulin and 23-hydroxybetulin production in tobacco plants; compound 1 is lupeol, compound 2 is betulin, compound 3 is betulin, compound 4 is betulin, and compound 5 is 23-hydroxybetulin. Figure 5 A flowchart showing the production of betulinic acid from lupeol and its subsequent oxidation to 23-hydroxybetulinic acid; Figure 6 For overexpression CYP716A236a Genes and / or CYP72A1371a EIC diagram of betulin and 23-hydroxybetulin production in yeast with the gene; compound 1 is lupeol, compound 2 is betulin, compound 3 is betulin, compound 4 is betulin, and compound 5 is 23-hydroxybetulin. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0027] Example 1 This invention screens cytochromes based on third-generation and second-generation full-length transcriptome data from Pulsatilla chinensis. P450 Protein (CYP450) and phylogenetic analysis were conducted to screen for genes related to the synthesis of betulinic acid and 23-hydroxybetulinic acid. The specific process is as follows: 1. Experimental Methods Using the conserved domain of the CYP450 protein (Pfam ID: PF00067) as a seed file, HMM searches were performed on the full-length transcriptome protein sequence files of three generations of Pulsatilla chinensis to identify genes of this family. The obtained sequences were submitted to the NCBI database to verify the conserved domain, thereby identifying the CYP450 family members expressed in Pulsatilla chinensis. The expression levels of CYP450 family members were extracted from second-generation transcriptome data of different tissue parts of Pulsatilla chinensis of different ages (one-year-old Pulsatilla chinensis: leaves (NHL), petioles (NHS), rhizomes (NHN), and roots (NHR); perennial Pulsatilla chinensis: leaves (LHL), petioles (LHS), and roots (LHR)). This identified CYP450 family members highly expressed in sites rich in betulinic acid and 23-hydroxybetulinic acid.

[0028] 2. Results and Analysis Based on the full-length transcriptome data of three generations of Pulsatilla chinensis, a total of 338 domain-complete transcriptomes were screened. CYP450 Gene family members. Previous studies on metabolomics data from different ages and tissues of *Pulsatilla chinensis* have found that betulinic acid and 23-hydroxybetulinic acid are mainly enriched in rhizomes and roots. Analysis of the expression trends of these 338 PcCYP450s members in these tissues revealed that QC_transcript_18856 and QC_transcript_14713 are specifically highly expressed in rhizomes and roots (see...). Figure 2 and Figure 3 Therefore, it is speculated that they may be involved in the synthesis of betulinic acid and 23-hydroxybetulinic acid. After the protein sequences were submitted to the CYP450 International Nomenclature Committee, the proteins QC_transcript_18856 and QC_transcript_14713 were named CYP716A236a and CYP72A1371a, respectively. Their nucleotide and protein sequences are as follows: CYP716A236a The nucleotide sequence of the gene: CYP72A1371a The nucleotide sequence of the gene: CYP716A236a Protein sequence encoded by the gene: MDLILFSMLFTIVALFLSLPLMFLFTRSKSQSTSIPSSLFPPGKTGWPIIGESLEFLNTGRRGVPEKFIKDRMQKFSSQIFRTSLLGEAAAVMCGPAGNKFLFSNENKLVTAWWPSSVYKIFPSSAQTSSNEESIKMRKMLPGFFKPEALQRYVGIMDAIAKKHLRENWDNKKEVTVFPLSKSYTFSLACKIFMSIEDPVQLAKFADPFAILACGIISLPIDLPGTPFNKAIKASNLIRKDLLEIIKKRKIDLAEAKASPTQDVLSHMLLTSDESGQFMKEHDIADKILGLLVGGHDTASAAITFVVKYLAELPHIYDAVRKEQLEILKAKEPGAPLSWEDIQKMRYSWNVACEVMRLAPPLQGAFREALTDFSYNGFSIPKGWKLYWSTNSTHRNPVYFPEPEKFDPSRYEGNGPAPYTYVPFGGGPRMCPGKEYARLEILIFMHNVVTKFKWETILKDEKIVVDPMPMPAKGLPVRLKPQVY, SEQ ID NO. 3; CYP72A1371a Protein sequence encoded by the gene: MEMIAKAILTVCIVFFLRLAWNFLNWVWLKPKKMEKYLKEQGVQGPSYKFLFGNLKEFRSSTNEAKSKPMEFSHKILPRVMPFFQSLIQKYGKITYIWLGASPRVIITDPDMIRDIFSEKFASFRKSTAN PLGRLLVSGIVRLEGDKWAKHRKIINPAFHLEKLKLMLPAFYISCSEMVNKWQTTVGEGSAELDVYPFMKKLTADVISRTAFGSSYEDGQKIFELQTEQAKLIMQASQSINIPGFLHFPTKNNRRMKEIQ REVRSILWDMIMKREKAKEAGEVFNSDLLGLLLESNLQEIKEGGNSKKFGITIDEVIEECKLFYFAGQDTTMNLLNWTMILLSMHKEWQEIARKEVLQVFGSDKPDFDGLKHLKQITMILNEALRLYPPI SEQ IDNO.4.

[0029] Example 2 This invention constructs CYP716A236a Genes and CYP72A1371a The specific process of using a plant binary overexpression vector for genes is as follows: 1. Experimental Methods Overexpression vectors pEAQ-CYP716A236a and pEAQ-CYP72A1371a were obtained using a seamless cloning method. The primer sequences used in the construction process are shown in Table 1.

[0030] Using cDNA from perennial roots of Pulsatilla chinensis as a template, the resulting DNA was cloned using KOD-Plus-Neo DNA Polymerase high-fidelity enzyme (purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., product number KOD-401). CYP716A236a and CYP72A1371aGene fragment. The total volume of the cloning system was 50 μL: 25 μL KOD-Plus-Neo DNA Polymerase, 1 μL template, 1.5 μL forward primer, 1.5 μL reverse primer, and 21 μL water; the cloning procedure is shown in Table 2. The ClonExpressII One Step Cloning Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., product number C112) was used. The obtained gene fragment was seamlessly cloned into the pEAQ-HT vector, and the recombinant product was directly transformed into TOP10 E. coli competent cells (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B528412). Positive clones were selected for colony PCR verification and sequencing. The total volume of the colony PCR reaction system was 20 μL: 10 μL 2×Taq PCR Mix, 1 μL template, 1 μL pEAQ-HT-F primer, 1 μL pEAQ-HT-R primer, and 7 μL water; the reaction procedure is shown in Table 3. After expanding the culture of the correctly sequenced positive clones, the bacterial cells were collected and the plasmids were extracted using the SanPrep column-based plasmid DNA mini-extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B518191) to obtain the expression vectors pEAQ-CYP716A236a and pEAQ-CYP72A1371a.

[0031] Table 1 Primer sequences for plant binary overexpression vectors Table 2 KOD high-fidelity enzyme PCR reaction procedure Table 3 Colony PCR reaction procedure 2. Results and Analysis Sequencing and cloning CYP716A236a The gene sequence is shown in SEQ ID NO.1. CYP72A1371a The gene sequence is as shown in SEQ ID NO.3, the sequence is correct, and the expression vector has been successfully constructed.

[0032] Example 3 This embodiment utilizes the overexpression vector constructed in Example 2 to verify the efficacy of tobacco (which does not produce betulinic acid and 23-hydroxybetulinic acid) in a specific culture medium. CYP716A236a Genes and CYP72A1371a The gene catalyzes the synthesis of betulinic acid and 23-hydroxybetulinic acid. The specific process is as follows: 1. Experimental Methods The pEAQ-CYP716A236a, pEAQ-CYP72A1371a, and pEAQ-PchAS obtained above are used as the bases for further processing. PchAS The gene has been shown to have the ability to catalyze the synthesis of lupeol. PchAS The nucleotide sequence of the gene is shown in SEQ ID NO.11; the construction process of pEAQ-PchAS is as described in Example 2, with insertion sites at AgeI and XhoI sites. The pEAQ-HT empty binary expression vector was transformed into GV3101 Agrobacterium tumefaciens competent cells (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., product code ZC141). Positive colonies were picked and cultured overnight, then activated, resuspended, and had their OD adjusted using infection buffer (10 mM MgCl2, 10 mM 2-(N-morpholine)ethanesulfonic acid, and 100 μM acetylsyleugenone). 600 After the value was adjusted to 0.5 and allowed to stand for 4 hours, the mixed resuspension was injected into the tobacco leaves according to the system described in Table 4.

[0033] SEQ ID NO.11:

[0034] After 6 days of normal tobacco cultivation, leaves were collected and freeze-dried under negative pressure. 100 mg of the dried leaf powder was resuspended in 10 mL of 20% KOH / 90% ethanol solution and incubated at 90°C for 1 hour, followed by extraction three times with 10 mL of ethyl acetate. All ethyl acetate solutions were combined, dried under nitrogen, and then dissolved in 1 mL of ethyl acetate. Approximately 200 μL of the *Nicotiana benthamiana* leaf extract was dried under nitrogen, and 100 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added for derivatization at 70°C for 30 minutes. The derivatized extract was analyzed by gas chromatography-mass spectrometry (GC / MS). In GC-MS analysis, 2 μL of sample was injected into a Shimadzu QP2010 gas chromatograph equipped with a WM-5MS capillary column (30 m × 250 μm, 0.25 μm film thickness) in splitless mode. The injection port temperature was 250°C, the carrier gas was helium, and the flow rate was 1.2 mL / min. The GC column oven temperature program was as follows: from 170℃ to 300℃ at a rate of 20℃ / min, and held for 11.5 minutes. The ion trap temperature was 250℃. The electron energy was 70 eV. The mass spectrometry recording range was 50–800 m / z.

[0035] Table 4 Mixing ratio of Agrobacterium resuspension 2. Results and Analysis Catalytic results showed that, in the case of overexpression alone PchAS Only lupeol was detected in tobacco leaves containing the gene, with no oxidation products of lupeol detected; in single overexpression... CYP716A236a A large amount of betulinic acid, the C-28 oxidation product of lupeol, was detected in tobacco leaves, with trace amounts of intermediate oxidation products betulin and betulin. However, in single overexpression... CYP72A1371a No oxidation products of lupeol were found in tobacco leaves containing the gene, even with simultaneous overexpression. CYP716A236a Genes and CYP72A1371a The gene was detected in tobacco leaves containing 23-hydroxybetulic acid, the oxidation product of lupeol at positions C-23 and C-28 (see [link to article]). Figure 4 ).

[0036] The above results indicate that CYP716A236a is a lupeol C-28 oxidase and CYP72A1371a is a betulinic acid C-23 oxidase, and overexpression of these enzymes is beneficial. CYP716A236a The gene can efficiently synthesize betulinic acid in plants, and at the same time, it is overexpressed CYP716A236a Genes and CYP72A1371a The gene can synthesize 23-hydroxybetulic acid in plants. Furthermore, only overexpression... CYP72A1371aThe gene's inability to synthesize 23-hydroxylated lupeol indicates that CYP72A1371a cannot hydroxylate lupeol at position 23 unless position 28 is oxidized, suggesting a sequential process in the synthesis of 23-hydroxybetulic acid from lupeol in plants (see...). Figure 5 ).

[0037] Example 4 This invention constructs CYP716A236a Genes and CYP72A1371a The yeast-induced gene expression vector, the specific process is as follows: 1. Experimental Methods The yeast induced expression vectors pESC-Ura-AtATR1-CYP716A236a and pESC-Leu-AtATR1-CYP72A1371a were obtained using the seamless cloning method. The primer sequences used in the construction process are shown in Table 5.

[0038] Cloning was performed using Arabidopsis cDNA as a template. AtATR1 The gene fragment was then seamlessly cloned into the XhoI site of the pESC-Ura and pESC-Leu vectors to ensure correct expression under the regulation of the GAL1 promoter. Cloning, seamless cloning, and transformation procedures were the same as in Example 2. Antibiotic-positive clones were validated by colony PCR and sequenced using Gal1-F / R primers. After amplification of correctly sequenced positive clones, bacterial cells were collected to extract plasmids, yielding the pESC-Ura-AtATR1 and pESC-Leu-AtATR1 yeast inducible expression vectors.

[0039] Cloning was performed using cDNA from perennial roots of Pulsatilla chinensis as a template. CYP716A236a and CYP72A1371a Gene fragments. CYP716A236a and CYP72A1371a Gene fragments were seamlessly cloned into the EcoRI site of the pESC-Ura-AtATR1 and pESC-Leu-AtATR1 vectors, respectively, to ensure correct expression under the regulation of the GAL10 promoter. Cloning, seamless cloning, transformation steps, colony PCR reaction system, and procedure were the same as in Example 2. Colony PCR verification and sequencing of antibiotic-positive clones were performed using Gal10-F / R primers. After expansion culture of correctly sequenced positive clones, bacterial cells were collected and plasmids were extracted to obtain the yeast-induced expression vectors pESC-Ura-AtATR1-CYP716A236a and pESC-Leu-AtATR1-CYP72A1371a.

[0040] Table 5 Primer sequences for yeast-induced expression vectors 2. Results and Analysis Sequencing and cloning CYP716A236aThe gene sequence is shown in SEQ ID NO.1. CYP72A1371a Gene sequence as shown in SEQ ID NO.3, AtATR1 The gene sequence is shown in SEQ ID NO.22. The sequence is correct, and the expression vector has been successfully constructed.

[0041] SEQ ID NO.22:

[0042] Example 5 This embodiment utilizes the yeast-induced expression vector constructed in Example 4 to verify the expression in *Saccharomyces cerevisiae* (which does not produce betulinic acid and 23-hydroxybetulinic acid). CYP716A236a Genes and CYP72A1371a The gene catalyzes the synthesis of betulinic acid and 23-hydroxybetulinic acid. The specific process is as follows: 1. Experimental Methods The obtained pESC-Ura-AtATR1, pESC-Leu-AtATR1, pESC-Ura-AtATR1-CYP716A236a, pESC-Leu-AtATR1-CYP72A1371a, and pESC-His-PchAS are used in conjunction with the above-mentioned pESC-Ura-AtATR1, pESC-Leu-AtATR1-CYP72A1371a, and pESC-His-PchAS. PchAS The gene has been confirmed to catalyze the synthesis of lupeol. The nucleotide sequence of the PchAS gene is shown in SEQ ID NO.11. The construction process of pESC-His-PchAS is as described in Example 4. The yeast inducible expression vector was transformed into competent cells of Saccharomyces cerevisiae according to the combination described in Table 6. Positive colonies were screened using SD / -His / -Leu / -Ura yeast triple-deficient solid medium (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., product code ZC1781). Positive colonies were picked and inoculated into SD / -His / -Leu / -Ura yeast triple-deficient liquid medium and cultured overnight. After centrifugation to collect the cells, they were resuspended in 200 mL of SC- / -His / -Leu / -Ura medium containing 2% galactose and cultured at 30°C and 200 rpm for 2 days to induce protein expression. The extraction and product detection steps after cell collection were the same as in Example 3.

[0043] Table 6 Yeast plasmid combinations 2. Results and Analysis Catalytic results showed that, in the case of overexpression alone PchAS Only lupeol was detected in yeast containing the gene, and no oxidation products of lupeol were detected; in single overexpression CYP716A236a In yeast samples containing the gene, betulin and tuftate (the oxidation product of lupeol at C-28) were detected in equal proportions, with a small amount of betulin present; however, in single overexpression... CYP72A1371a No oxidation products of lupeol were found in yeast containing the gene, even with simultaneous overexpression. CYP716A236a Genes and CYP72A1371a The gene was detected in yeast containing 23-hydroxybetulic acid, the oxidation product of lupeol at C-23 and C-28 (see [link to gene]). Figure 6 ).

[0044] The above results indicate that the functions of CYP716A236a and CYP72A1371a are consistent in yeast and plants. The oxidation efficiency of CYP716A236a in yeast is lower than that in plants, but it is still significantly improved compared to the catalytic efficiency of other published lupeol C-28 oxidases (betulic acid accounts for only 21%).

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A catalytic method for the production of betulinic acid from lupeol. CYP716A236a Genes, characterized by, The CYP716A236a The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A CYP716A236a protein that catalyzes the conversion of lupeol to betulinic acid, characterized in that, The amino acid sequence of the CYP716A236a protein is shown in SEQ ID NO.

3.

3. A catalytic method for the production of 23-hydroxybetulic acid from betulinic acid. CYP72A1371a Genes, characterized by, The CYP72A1371a The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A CYP72A1371a protein that catalyzes the production of 23-hydroxybetulic acid from betulinic acid, characterized in that, The amino acid sequence of the CYP72A1371a protein is shown in SEQ ID NO.

4.

5. The application of the CYP716A236a protein according to claim 2 in the preparation of a catalyst for the synthesis of betulinic acid, characterized in that, The betulinic acid synthesis catalyst catalyzes the conversion of lupeol to betulinic acid.

6. The application of the CYP72A1371a protein according to claim 4 in the preparation of a catalyst for the synthesis of 23-hydroxybetulic acid, characterized in that, The 23-hydroxybetulic acid synthesis catalyst has the function of catalyzing the production of 23-hydroxybetulic acid from betulic acid.

7. The claim 1 CYP716A236a The application of the gene or the CYP716A236a protein of claim 2 in increasing the content of betulinic acid in plants or yeast, characterized in that... The method for increasing the betulinic acid content in plants or yeast includes using genetic transformation technology to... CYP716A236a The gene is transferred into plants or yeast to increase the expression level of the CYP716A236a protein, thereby catalyzing the process of converting lupeol to betulin in plants.

8. The claim 1 CYP716A236a Gene combination as described in claim 3 CYP72A1371a The application of the gene or the CYP716A236a protein of claim 2 combined with the CYP72A1371a protein of claim 4 in increasing the content of 23-hydroxybetulic acid in plants or yeast, characterized in that, The method for increasing the content of 23-hydroxybetulic acid in plants or yeast includes using genetic transformation technology to... CYP716A236a Genes and the CYP72A1371a The genes are co-transferred into plants or yeast to increase the expression levels of the CYP716A236a and CYP72A1371a proteins, thereby catalyzing the conversion of lupeol to betulinic acid in plants or yeast, and subsequently to 23-hydroxybetulinic acid.

9. The application of the CYP716A236a gene of claim 1 or the CYP716A236a protein of claim 2 in the cultivation of plant strains or yeast strains that produce high levels of betulinic acid.

10. The claim 1 CYP716A236a Gene combination as described in claim 3 CYP72A1371a The application of the gene or the CYP716A236a protein of claim 2 in combination with the CYP72A1371a protein of claim 4 in the cultivation of plant strains or yeast strains that produce high levels of 23-hydroxybetulic acid.

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