Oxymethyltransferase fsr2 mutant and application thereof
Through the transformation and recombinant expression of the oxygen methyltransferase FSR2, the problem of insufficient catalyzed hydroxyoxymethylation activity of 4' position of yamphetamine was solved, and efficient biomanufacturing of yamphetamine to icariin was achieved, providing A sustainable green and clean production method has been developed, which has enhanced the application value of biomanufacturing technology.
Patent Information
- Application Number
- CN202411440003.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-15
- Publication Date
- 2024-12-10
AI Technical Summary
The existing oxygen methyltransferase FSR2 is insufficient in catalyzing the hydroxyoxymethylation reaction of 4' position of yamphetol, which is difficult to meet the needs of industrial applications. Moreover, the method of plant extraction and production of icariin has caused damage to wild plant resources. A sustainable and efficient method of biomanufacturing is needed.
By transforming the oxygen methyltransferase FSR2 found in rice seedling bacteria, a mutant that efficiently catalyzes the hydroxymethylation of 4' position of yeast was obtained, and a recombinant plasmid that could be expressed in yeast was constructed and in yeast was constructed. Heterologous expression transformation was carried out to achieve efficient synthesis of 4'oxymethyl pyrophenol.
The obtained oxygen methyltransferase mutant can efficiently catalyze the hydroxyoxymethylation of 4' position of yamphetamine, realize the key steps from yamphetamine to icariin, promote the advancement of biomanufacturing technology, and provide a A sustainable green and clean production method.
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Figure CN119101674A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to an oxygen methyltransferase fsr2 mutant and application thereof in catalyzing the oxygen methylation of kaempferol 4' hydroxyl. Background Art
[0002] Fungal biotransformation is a metabolic reaction that uses specific enzymes in cells to modify and transform the structure of substrates to obtain more valuable compounds. Kaempferol is a common natural flavonoid plant secondary metabolite, which is widely found in the roots, leaves and fruits of many plants. Kaempferol has antioxidant, anti-inflammatory, tumor growth inhibition, liver cell protection, platelet aggregation and adhesion inhibition activities, so it has attracted widespread attention. In addition, it is also an important intermediate product for the synthesis of a variety of flavonoid active substances, such as icariin.
[0003] Icariin is the main active ingredient of Epimedium, which is an 8-isopentenyl flavonoid glycoside compound. Icariin can increase cardiovascular and cerebrovascular blood flow, promote hematopoiesis, immune function and bone metabolism, and also has the effects of invigorating the kidney and strengthening yang, anti-aging, etc. At present, icariin is mainly extracted from the dried stems and leaves of Epimedium sagittatum, Epimedium pubescens, Epimedium wushanense, and Epimedium koreana. However, the long plant growth cycle and low product content cause serious damage to wild plant resources. How to produce these high-value-added natural products in a sustainable, green and clean way is crucial to ensure the sustainable development of social economy. Biomass is a renewable clean resource. Through biomanufacturing technology, biomass can be converted into fuels, bulk chemicals and natural products, thereby replacing petrochemical refining and plant resource extraction. The core technology of biomanufacturing is to build an efficient microbial cell factory to convert biomass raw materials into various end products. How to expand the types of chemicals produced by microbial cells and how to improve the production efficiency of cells are two key technical issues that limit the industrialization of cell factories.
[0004] Oxymethylation of the 4' hydroxyl group is one of the key steps in synthesizing icariin from kaempferol; one of the key technologies for constructing efficient synthesis of icariin using biomass is to be able to efficiently and specifically carry out the methylation reaction of the 4' hydroxyl group of kaempferol, and one of the ways to achieve this is to explore the oxygen methyltransferase with this biological function. The patent CN202210132042 applied by the inventor previously disclosed an oxygen methyltransferase fsr2, and also disclosed its application in catalyzing the oxygen methylation of the 4' hydroxyl group of kaempferol. However, the activity of the oxygen methyltransferase fsr2 is not high enough, and there is still a certain gap from the actual industrial application. It needs to be modified to obtain a more active oxygen methyltransferase. Summary of the invention
[0005] The purpose of the present invention is to obtain a methyltransferase mutant gene capable of efficiently and specifically carrying out the 4'-hydroxymethylation reaction of kaempferol, so as to realize the efficient synthesis of 4'-oxymethyl kaempferol.
[0006] The present invention transforms the oxygen methyltransferase fsr2 (Uniprot S0DQQ0) excavated from the rice seedling pathogen Fusarium fujikuroi to obtain a mutant that efficiently catalyzes the 4'-hydroxymethylation of kaempferol. The fsr2 mutant is obtained by site-directed mutagenesis of the oxygen methyltransferase fsr2, and a recombinant plasmid that can be expressed in yeast is constructed, and heterologous expression transformation is performed in the yeast. By exogenously adding kaempferol, 4'-oxymethyl kaempferol (i.e. kaempferol) is extracted from the fermentation product without other conversion products.
[0007] An oxygen methyltransferase fsr2 mutant, the amino acid sequence of which is shown in the sequence list SEQ ID NO: 1-4.
[0008] The invention discloses a gene of an oxygen methyltransferase fsr2 mutant, and the nucleotide sequence thereof is shown in the sequence list as SEQ ID NO: 5-8.
[0009] A tag sequence is connected to the amino terminus or carboxyl terminus, preferably Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, more specifically RRRRR, HHHHHH, DYKDDDDK, WSHPQFEK, EQKLISEEDL. In order to facilitate protein purification, a tag as shown in Table 1 can be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1-4.
[0010] Table 1 Tag sequences
[0011] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHQ c-myc 10 EQKLISEEDL
[0012] Primers for amplifying any fragment of the oxygen methyltransferase fsr2 mutant gene.
[0013] A recombinant expression vector containing the oxygen methyltransferase fsr2 mutant gene.
[0014] A method for preparing 4'-oxymethyl kaempferol is achieved by using kaempferol as a substrate and catalyzing the methyltransferase fsr2 mutant according to claim 1.
[0015] The method for preparing 4'oxymethyl kaempferol is prepared by fermentation of a recombinant strain containing the methyltransferase fsr2 mutant gene.
[0016] The recombinant strain is Saccharomyces cerevisiae.
[0017] The oxygen methyltransferase fsr2 mutant is used in the preparation of icariin, using kaempferol as a substrate, and 4'-oxymethyl kaempferol is obtained by oxygen methylation of the 4'-hydroxyl group of kaempferol, and then further converted into icariin.
[0018] Beneficial effects of the present invention: the oxygen methyltransferase fsr2 mutant obtained by the present invention can efficiently catalyze the oxygen methylation reaction of the 4'-hydroxyl group of kaempferol, which is a key step in the synthesis of icariin from kaempferol. Therefore, the biocatalytic method provided by the present invention can realize the fermentation synthesis of icariin in a cell factory, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Predicted three-dimensional structure for FSR2.
[0020] Figure 2 The amplification results of fsr2-F158W and fsr2-K328M gene fragments; 1 is pRS425M digestion, 2 and 3 are fsr2-F158W amplification results, 4 and 5 are fsr2-K328M amplification results.
[0021] Figure 3 The physical maps of the recombinant vectors pRS425M-F158W, pRS425M-K328M, and pRS425M-F158W-K328M.
[0022] Figure 4 Construction of mutant enzyme plasmid map.
[0023] Figure 5 This is the liquid chromatogram before kaempferol modification.
[0024] Figure 6 This is the mass spectrum of kaempferol before modification.
[0025] Figure 7 This is the liquid chromatogram of the kaempferol standard sample.
[0026] Figure 8 This is the mass spectrum of the kaempferol standard sample.
[0027] Fig. 9 This is the HPLC liquid chromatogram of kaempferol modified by mutants F158W, K328M, and F158W-K328M (Note: the [M+H]+ of compound 1 is 287, which is consistent with the molecular weight of kaempferol; the [M+H]+ of compound 2 is 301, which is consistent with the molecular weight of kaempferol).
[0028] Fig.10 This is the mass spectrum of a methylated compound produced after kaempferol was modified by a mutant.
[0029] Fig.11 The bar graph shows the kaempferol conversion efficiency catalyzed by fsr2 (WT) and 20 mutants.
[0030] Fig.12 This is a bar graph showing the conversion efficiency of kaempferol catalyzed by fsr2 (WT), mutant F158W and mutant K328M under the optimal fermentation conditions. DETAILED DESCRIPTION
[0031] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0032] The experimental materials used in the following examples are described and their sources are as follows:
[0033] Strains and vectors:
[0034] The rice seedling pathogen (Fusarium fujikuroi), yeast S.cerevisiae BJ5464-NpgA, and yeast expression vector PRS425m were all from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.
[0035] Enzymes and kits: restriction endonucleases and T4 DNA ligase were purchased from NEB; RNA reverse transcription kit was purchased from TAKARA; Quick-Fusion Cloning Kit was purchased from Biotool; hot-start high-fidelity DNA amplification kit was purchased from Biotool; DNA polymerase and DNA marker were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; plasmid mini-extraction kit and universal DNA purification gel recovery kit were purchased from Tiangen Company; frozen yeast transformation kit was purchased from YMO RESEARCH Biological Company; Escherichia coli DH5α competent culture was purchased from Kangwei Century Company; other reagents were domestic analytical grade products.
[0036] Culture medium: E. coli culture medium is LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). SC--Leu-deficient medium (1% glucose, 6.7% Difco TMYeast Nitrogen Base w / o AminoAcids, -Leu / -Trp DO Supplement). YPD medium (1% yeast extract, 2% Peptone peptone, 2% glucose). YPD low sugar medium (1% yeast extract, 2% Peptone peptone, 1% glucose.) If making solid medium, add 2% agar powder.
[0037] Other experimental methods without specific conditions in the examples were carried out according to conventional methods, and molecular cloning was carried out according to the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the manufacturer.
[0038] Example 1 Screening of mutation sites of oxygen methyltransferase Fsr2
[0039] In previous studies, Fsr2 showed a high conversion rate from kaempferol to kaempferol and strict regiospecificity. Remodeling the active pocket may further improve its activity for 4'-O-methylation of kaempferol. Since Fsr2 has no resolved crystal or NMR structure, its three-dimensional structural model was obtained using AlphaFold2 ( Figure 1 ). According to the prediction of active pockets by Caver Web 1.2 and the molecular docking program Rosetta Ligand_docking, the complex structure of Fsr2-substrate (kaempferol and SAM) was obtained, and the amino acids within 5 angstroms near the substrate kaempferol were selected as potential sites affecting substrate binding and enzyme activity. Visual analysis was performed using Pymol, and 22 amino acid sites were found to be within 5 angstroms of the substrate. At the same time, PSI BLAST2.13.0+ was used to screen similar sequences of oxygen methyltransferase fsr2, with parameters set to 3 iterations, E-value less than 0.01, and uniref90 database selected; the sequence data used the PSSM matrix (position-specific scoring matrix) to analyze the amino acid conservation of potential sites of fsr2. The higher the PSSM value, the more conservative the amino acid at the site, proving that the amino acid may have a greater impact on the activity. Some of the amino acids have the highest PSSM value at this position, proving that the site is extremely conservative and may have a greater impact on the catalytic activity, so no mutation was performed. The amino acids with relatively low PSSM values were selected to mutate into more conservative amino acids to increase the catalytic activity of Fsr2. Further combined with rational design ideas, reasonable mutants were selected. The screening process is shown in Table 1.
[0040] Table 1
[0041]
[0042] Example 2 Construction of expression vector for fsr2 mutant enzyme of oxygen methyltransferase
[0043] The oxygen methyltransferase in the genome of rice seedling pathogen (Fusarium fujikuroi) was analyzed by relevant bioinformatics software, and an oxygen methyltransferase gene fsr2 (its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 5) was found through comparison. The nucleotide sequence of fsr2 was predicted and analyzed by various software, and the introns were removed and spliced to obtain the coding region sequence of fsr2. RNA of rice seedling pathogen was extracted and cDNA was obtained by reverse transcription, and the coding gene of fsr2 was amplified using designed primers, and the target fragment was recovered and submitted to a sequencing company for sequencing and comparison, and the oxygen methyltransferase fsr2 shown in SEQ ID NO: 1 was obtained.
[0044] Restriction sites were introduced during synthesis. NdeI restriction site was added before the promoter and PmeI restriction site was added after the terminator. The fragment length was 0.75Kb. The amplified fragments of fsr2 and its mutant genes are shown in Figure 2 .
[0045] The shuttle vector PXW06F was double-digested with NdeI and PmeⅠ to recover a fragment of 6114 bp, and the two fragments were connected using a seamless ligation cloning kit.
[0046] Heat shock method was used to transform into competent E. coli DH5α. r Resistance screening, plasmid extraction, ScaI and NdeⅠ enzyme digestion identification. Recombinant plasmid PRS425M-fsr2 and mutant enzyme map are shown in Figure 3 , Figure 4 .
[0047] Saccharomyces cerevisiae BJ5464-NpgA was inoculated into YPD medium and cultured at 30°C and 200 r / min until the OD value was 0.8-1.0. Saccharomyces cerevisiae competent cells were prepared and the recombinant plasmids were transformed according to the instructions provided by ZYMO. The recombinant plasmids of PRS425M-fsr2 and PRS425M-fsr2-mutase containing the fsr2 gene were transformed into Saccharomyces cerevisiae and spread on leucine-deficient SC solid defective medium and cultured at 30°C for about 3 days. The obtained yeast transformants were streaked and cultured to a new SC- - Incubate on Leu-deficient medium in a 30°C incubator for about 2 days.
[0048] Example 3: Conversion of kaempferol to kaempferol using the oxygen methyltransferase recombinant vector PRS425M-fsr2-mutant enzyme
[0049] The metabolites modified by oxygen methyltransferase fsr2 were obtained by HPLC high performance liquid phase separation and their molecular structures were analyzed. The experimental method is as follows:
[0050] 1) Fermentation culture
[0051] The two-step fermentation technique was adopted. First, an appropriate amount of Saccharomyces cerevisiae transformant cells were inoculated into the corresponding 25-mL - Leu liquid deficiency medium (1% glucose, 6.7% Difco TM Yeast Nitrogen Base w / o AminoAcids, -Trp / -ura DO Supplement), 30℃, 200r·min -1 After culturing for about 16 hours, 25 mL of YPD low-sugar medium (1% yeast extract, 2% Peptone peptone, 1% glucose) was added, and 5 mg of pure kaempferol was added and the culture was continued for 48 hours; the fermentation product was extracted with ethyl acetate, and the ratio of ethyl acetate to fermentation liquid was 1:1, that is, 50 mL of ethyl acetate was used to extract the fermentation product; the ethyl acetate dried extract was recovered by a rotary evaporator, and the extract was re-dissolved with 1 mL of methanol.
[0052] 2) High performance liquid chromatography (HPLC)
[0053] The fermentation product obtained above was detected by high performance liquid chromatography after high speed centrifugation.
[0054] HPLC detection conditions are as follows: chromatographic column RRHD Eclipse Plus C18, 4.6x100mm, acetonitrile-H2O as mobile phase for gradient elution, gradient elution conditions: 0-5min, acetonitrile 10%; 5-8.5min, acetonitrile from 50%→95%; 8.5-9.5min, acetonitrile 95%; 9.5-10min, acetonitrile from 95%→10%. Flow rate 3.mL·min -1 , the detection wavelength is 280nm.
[0055] HPLC analysis revealed that kaempferol was converted into another peak without other obvious product peaks, and its retention time was consistent with that of the kaempferol standard sample (see Figure 5 , Figure 7 and Fig. 9 ). After high-resolution mass spectrometry, the [M+H] + The mass-to-charge ratio is 301.0690, and the predicted molecular formula is C16H13O6 (error is 5.55ppm), which is consistent with kaempferol. After secondary mass spectrometry analysis, the spectrum of the product is highly similar to that of kaempferol (see Figure 6 , Figure 8 and Fig.10). Based on the above results, it was determined that the product was kaempferol.
[0056] The conversion rate of the mutant enzyme was calculated by UV peak area, and the mutant enzymes F158W (whose amino acid sequence is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 6) and K328M (whose amino acid sequence is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 7) with significantly improved catalytic efficiency were screened out from 20 mutant enzymes. Fig.11 F158W-K328M (its amino acid sequence is shown in SEQ ID NO: 4, and its nucleotide sequence is shown in SEQ ID NO: 8). After the fermentation time was extended and the substrate concentration was increased, the efficiency of methylation of kaempferol catalyzed by oxygen methyltransferase fsr2 and mutant enzymes F158W, K328M, and F158W-K328M was as follows: Fig.12 As shown, the conversion rate of the mutant enzyme F158W-K328M was significantly improved compared with the wild-type fsr2.
[0057] The above results prove that the oxygen methyltransferase involved in the present invention can specifically convert kaempferol into kaempferol, and can be used for the preparation of kaempferol derivative compounds such as icariin.
[0058] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. An oxygen methyltransferase fsr2 mutant, characterized in that The amino acid sequence thereof is shown in the sequence listing SEQ ID NO: 1-4.
2. A gene of an oxygen methyltransferase fsr2 mutant, characterized in that: The nucleotide sequences thereof are shown in the sequence listing as SEQ ID NO: 5-8.
3. Primers for amplifying any fragment of the oxygen methyltransferase fsr2 mutant gene according to claim 2.
4. A recombinant expression vector containing the oxygen methyltransferase fsr2 mutant gene according to claim 2.
5. A method for preparing 4'oxymethylkaempferol, characterized in that: The process is achieved by using kaempferol as a substrate and catalyzed by the methyltransferase fsr2 mutant described in claim 1.
6. The method for preparing 4'-oxymethylkaempferol according to claim 5, characterized in that: The method is prepared by fermenting a recombinant strain containing the methyltransferase fsr2 mutant gene of claim 2.
7. The method for preparing 4'-oxymethylkaempferol according to claim 6, characterized in that: The recombinant strain is Saccharomyces cerevisiae.
8. The use of the oxygen methyltransferase fsr2 mutant according to claim 1 in the preparation of icariin, characterized in that: Using kaempferol as a substrate, 4'-oxymethyl kaempferol is obtained by oxymethylation of the 4'-hydroxyl group of kaempferol, and then further converted into icariin.