Method for generating isopentenyl polyphenol compound through biological catalysis
By mutation of the specific amino acid site of the isopentenyltransferase AtaPT, the selection and efficiency of existing enzymes in isopentenylated polyphenol compounds were solved, and a high chemical selectivity and high yield isopentenylation reaction was achieved.
Patent Information
- Application Number
- CN202510477235.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-03-21
- Filing Date
- 2025-04-16
- Publication Date
- 2025-07-08
AI Technical Summary
The existing soluble aromatic isoprenyl transferases have defects such as narrow range of acceptor or donor substrates, low efficiency and poor selectivity in the application of isoprenylated polyphenol compounds, which limits their industrial applications.
By protein engineering of the isoprenyltransferase AtaPT derived from Aspergillus turpermia, mutants G326W, S177G, M8 and M7 were designed to mutate specific amino acid sites to improve the chemoselectivity and regioselectivity of the enzyme.
The modified isoprenyltransferase mutants significantly improved the chemical selectivity and yield of isoprenylated products of polyphenol compounds such as quercetin, apigenin, luteolin, etc., and their activity was significantly improved.
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Figure CN120272455A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for biocatalytic production of isopentenyl polyphenolic compounds, belonging to the technical field of bioengineering. Background Art
[0002] Polyphenolic compounds are widely present in nature and have functions such as anti-inflammatory, antioxidant, enhancing physical resistance, lowering blood pressure, inhibiting the growth of bacteria and cancer cells, and have attracted increasing attention from the scientific community due to their importance to human health. These compounds themselves have characteristics such as low water solubility, poor stability, and rapid release, and produce low biological effects after in vivo metabolism. Therefore, improving the low in vivo bioavailability has become a difficult problem and challenge. With more and more in-depth research, a variety of modification methods have been developed to improve the bioavailability of these compounds, such as glycosylation modification, methylation modification, and isopentenylation modification, etc. Research shows that isopentenylated compounds show better bioactivity than precursors. Thanks to the enhanced lipophilicity of isopentenyl groups, the affinity of compound molecules with biological membranes is strengthened, thereby improving drug metabolism and pharmacokinetic properties.
[0003] It is precisely the recognition of the importance of such compounds that chemists have developed a variety of efficient isopentenylation methods, including Friedel-Crafts alkylation reactions (acid-catalyzed and base-catalyzed), cross-coupling reactions, and rearrangement reactions, etc., which have made many contributions to the field of isopentenylation. However, due to the complex and diverse structures of polyphenolic compounds themselves, containing multiple phenolic hydroxyl groups, resulting in multiple reaction sites, isopentenylation by chemical methods requires long protection-deprotection steps and it is difficult to control their regioselectivity. Enzymatic methods, as a supplement to chemical synthesis, provide new strategies for this. The isopentenylation reaction in natural product biosynthesis is catalyzed by isopentenyltransferases (PTs). These enzymes catalyze the transfer of isopentenyl molecules from activated isopentenyl donors - five-carbon units, such as dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), etc., to substrate receptors, and directly construct isopentenylated products in a simple one-step manner. Therefore, this type of enzyme has been widely recognized by scientists as a powerful isopentenylation tool.
[0004] Most prenylated polyphenolic compounds are derived from plants. However, the genetic backgrounds of many plants are unclear, most of the secondary metabolite genes are not clustered, and many plant prenyltransferases are multi-transmembrane membrane-bound proteins, making it difficult to find related genes. Moreover, when the related prenyltransferases from plants are expressed in vitro using microorganisms, there are problems such as low enzyme amount and low enzyme activity, or defects such as poor reaction efficiency and inhibition by high substrate concentration. Soluble aromatic prenyltransferases (aPTs) from microorganisms are a more suitable type of engineering enzyme with industrial potential, with high expression levels, mild catalytic conditions, a wide range of aromatic substrates, and good application prospects. However, most of the reported soluble aromatic prenyltransferases for prenylated polyphenolic compounds currently remain at the research stage of wild-type enzymes, and there is less mechanistic analysis. There are defects such as a narrow range of acceptor or donor substrates, low efficiency, and poor selectivity, which limit the application of PTs.
[0005] Therefore, there is an urgent need to develop an enzymatic prenylation platform to synthesize a variety of bioactive prenyl polyphenolic compounds with high chemo-selectivity and regioselectivity, laying a foundation for the development of new bioactive molecules or the customization of molecules with specific pharmacological properties. Summary of the Invention
[0006] Aiming at the deficiencies of the above-mentioned prior art, the present invention provides a method for biocatalytic synthesis of prenyl polyphenolic compounds, aiming to solve the technical problems that most of the currently reported soluble aromatic prenyltransferases for prenylated polyphenolic compounds remain at the research stage of wild-type enzymes, with less mechanistic analysis, and there are defects such as a narrow range of acceptor or donor substrates, low efficiency, and poor selectivity, which limit the application of PTs.
[0007] The first technical solution provided by the present invention is a mutant of prenyltransferase AtaPT, and the mutant is obtained by performing any one of the following mutations on the prenyltransferase AtaPT parent with the amino acid sequence as SEQ ID NO.1:
[0008] (1) Mutating glycine G at position 326 to tryptophan W;
[0009] (2) Mutating serine S at position 177 to glycine G;
[0010] (3) Mutating isoleucine I at position 167 to alanine A, mutating glutamate E at position 169 to alanine A, mutating serine S at position 170 to glutamine Q, mutating cysteine C at position 175 to methionine M, mutating serine S at position 177 to alanine A, mutating leucine L at position 323 to glutamine Q, mutating proline P at position 324 to histidine H, and mutating asparagine N at position 328 to arginine R;
[0011] (4) The isoleucine I at the 167th position is mutated to threonine T, the glutamic acid E at the 169th position is mutated to alanine A, the serine S at the 170th position is mutated to glutamine Q, the serine S at the 177th position is mutated to alanine A, the leucine L at the 323rd position is mutated to glutamine Q, the proline P at the 324th position is mutated to histidine H, and the asparagine N at the 328th position is mutated to methionine M.
[0012] In some embodiments, the gene sequence encoding the parent is as shown in SEQ ID NO.2.
[0013] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.
[0014] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.
[0015] In some embodiments, the recombinant vector uses plasmid pET28a as the expression vector.
[0016] The fourth technical solution provided by the present invention is a recombinant cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.
[0017] In some embodiments, the host cells of the recombinant cells include, but are not limited to, Escherichia coli.
[0018] The fifth technical solution provided by the present invention is a genetically engineered bacterium, which uses Escherichia coli as the host and a plasmid of the pET series as the vector to express the mutant described in the first technical solution.
[0019] In some embodiments, the construction of the genetically engineered bacterium specifically includes the following steps: ligating the gene with the pET28a expression vector and recombinantly expressing the isopentenyl transferase mutant in Escherichia coli E.coli BL21(DE3).
[0020] In some embodiments, the components of the fermentation medium are: TB medium, the inducer is 0.2 mM IPTG, the preferred temperature is 18 - 25 °C, the rotation speed is 200 - 220 rpm, and the expression duration is 18 - 20 h.
[0021] In some embodiments, about 0.05% glucose is added during inoculation.
[0022] In some embodiments, the protein purification of the genetically engineered bacterium specifically includes the following steps: collecting the expressed bacterial cells with a pre-cooled centrifuge, washing twice with a buffer solution, adding a small amount of lysozyme, quickly freezing in liquid nitrogen, thawing in an ice-water bath, ultrasonically disrupting, centrifuging at high speed, and performing His-nickel column affinity purification and desalting column desalting.
[0023] The sixth technical solution provided by the present invention is a method for improving the chemoselectivity of isopentenyltransferase AtaPT, and the method is to perform any one of the following mutations on the parent with the amino acid sequence as shown in SEQ ID NO.1:
[0024] (1) Mutate glycine G at position 326 to tryptophan W to obtain mutant G326W;
[0025] (2) Mutate serine S at position 177 to glycine G to obtain mutant S177G;
[0026] (3) Mutate isoleucine I at position 167 to alanine A, glutamate E at position 169 to alanine A, serine S at position 170 to glutamine Q, cysteine C at position 175 to methionine M, serine S at position 177 to alanine A, leucine L at position 323 to glutamine Q, proline P at position 324 to histidine H, and asparagine N at position 328 to arginine R to obtain mutant M8;
[0027] (4) Mutate isoleucine I at position 167 to threonine T, glutamate E at position 169 to alanine A, serine S at position 170 to glutamine Q, serine S at position 177 to alanine A, leucine L at position 323 to glutamine Q, proline P at position 324 to histidine H, and asparagine N at position 328 to methionine M to obtain mutant M7.
[0028] The seventh technical solution provided by the present invention is a method for synthesizing isopentenylated polyphenolic compounds. The method is to use the mutant described in the first technical solution or the genetically engineered bacterium described in the fifth technical solution as a catalyst, and use polyphenolic compounds as receptors to generate isopentenylated polyphenolic compounds.
[0029] In some embodiments, the polyphenolic compounds include quercetin, apigenin, luteolin, naringenin, etc.
[0030] In some embodiments, the method is carried out in a 40 - 60 mM Tris HCl buffer system, the catalyst addition amount is OD 80 (whole cells), pH = 7.0 - 8.0, 5 mM CaCl2, at 30 - 40 °C, the receptor substrate concentration is 2 mM, the donor substrate DMAPP or GPP concentration is 4 mM, the reaction rotation speed is 800 - 1200 rpm, and the reaction time is 6 - 8 h.
[0031] The eighth technical solution provided by the present invention is the application of the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the genetically engineered bacterium described in the fifth technical solution, or the method described in the sixth technical solution, or the method described in the seventh technical solution in the production of isoprenylated polyphenols.
[0032] The technical effects of the present invention are as follows:
[0033] (1) The present invention is based on the protein engineering transformation of the reaction of kaempferol and DMAPP catalyzed by the isopentenyltransferase AtaPT derived from Aspergillus terreus, and the obtained mutants G326W, S177G, M8 (I167A / E169A / S170Q / C175M / S177A / L323Q / P324H / N328R) and M7 (I167T / E169A / S170Q / S177A / L323Q / P324H / N328M) are used as biocatalysts. Compared with the wild enzyme AtaPT, using kaempferol as the substrate, highly chemoselective isoprenylated products can be obtained respectively, and both the activity and selectivity are improved.
[0034] (2) After the enzyme is transformed, its mutants G326W, M8 and M7 can synthesize different isoprenylated products with high chemoselectivity and yield for substrates such as quercetin, myricetin, apigenin, luteolin, naringenin, genistein, etc., as Figures 1 to 30 shown, which has important application value. Description of the Drawings
[0035] Figure 1 It is the HPLC chromatogram of the reaction mixture of kaempferol 1a with the mutants G326W, S177G, M8 of AtaPT and the wild type.
[0036] Figure 2 It is the HPLC chromatogram of the reaction mixture of kaempferol 1a with the mutant M7 of AtaPT and the wild type.
[0037] Figure 3 It is the 1H NMR spectrum of the product 2a - 8-(1,1 - Dimethyl - 2 - propenyl)kaempferol produced by the reaction of kaempferol 1a with the mutant M8 of AtaPT.
[0038] Figure 4 It is the 13C NMR spectrum of the product 2a - 8-(1,1 - Dimethyl - 2 - propenyl)kaempferol produced by the reaction of kaempferol 1a with the mutant M8 of AtaPT.
[0039] Figure 5 1H NMR spectrum of 3a-Isolicoflavonol, the product of the reaction of kaempferol 1a with the mutant G326W of AtaPT.
[0040] Figure 6 13C NMR spectrum of 3a-Isolicoflavonol, the product of the reaction of kaempferol 1a with the mutant G326W of AtaPT.
[0041] Figure 7 1H NMR spectrum of 5a-4'-(dimethylallyl)kaempferol, the product of the reaction of kaempferol 1a with the mutant S177G of AtaPT.
[0042] Figure 8 13C NMR spectrum of 5a-4'-(dimethylallyl)kaempferol, the product of the reaction of kaempferol 1a with the mutant S177G of AtaPT.
[0043] Figure 9 1H NMR spectrum of 6a-Robipseudin A, the product of the reaction of kaempferol 1a with the mutant M7 of AtaPT.
[0044] Figure 10 13C NMR spectrum of 6a-Robipseudin A, the product of the reaction of kaempferol 1a with the mutant M7 of AtaPT.
[0045] Figure 11 HPLC chromatogram of the reaction mixture of quercetin 1b with the mutants G326W, M8 and the wild type of AtaPT.
[0046] Figure 12 HPLC chromatogram of the reaction mixture of quercetin 1b with the mutants M7 and the wild type of AtaPT.
[0047] Figure 13 1H NMR spectrum of 2b-6′-prenylquercetin, the product of the reaction of quercetin 1b with the mutant M8 of AtaPT.
[0048] Figure 14 13C NMR spectrum of 2b-6′-prenylquercetin, the product of the reaction of quercetin 1b with the mutant M8 of AtaPT.
[0049] Figure 15 1H NMR spectrum of 3b-Denticulatain E, the product of the reaction of quercetin 1b with the mutant G326W of AtaPT.
[0050] Figure 16 It is the C spectrum of product 3b-Denticulatain E produced by the reaction of quercetin 1b with mutant G326W of AtaPT.
[0051] Figure 17 It is the H spectrum of product 4b-Denticulatain E produced by the reaction of quercetin 1b with mutant M7 of AtaPT.
[0052] Figure 18 It is the C spectrum of product 4b-Denticulatain E produced by the reaction of quercetin 1b with mutant M7 of AtaPT.
[0053] Figure 19 It is the HPLC chromatogram of the reaction mixture of apigenin 1e with mutants G326W, M8 and wild type of AtaPT.
[0054] Figure 20 It is the HPLC chromatogram of the reaction mixture of apigenin 1e with mutants M7 and wild type of AtaPT.
[0055] Figure 21 It is the H spectrum of product 2e-Yinyanghuo D produced by the reaction of apigenin 1e with mutant G326W of AtaPT.
[0056] Figure 22 It is the C spectrum of product 2e-Yinyanghuo D produced by the reaction of apigenin 1e with mutant G326W of AtaPT.
[0057] Figure 23 It is the H spectrum of product 3e-Kuwanon S produced by the reaction of apigenin 1e with mutant M7 of AtaPT.
[0058] Figure 24 It is the C spectrum of product 3e-Kuwanon S produced by the reaction of apigenin 1e with mutant M7 of AtaPT.
[0059] Figure 25 It is the HPLC chromatogram of the reaction mixture of naringenin 1g with mutants G326W, M8 and wild type of AtaPT.
[0060] Figure 26 It is the HPLC chromatogram of the reaction mixture of naringenin 1g with mutants M7 and wild type of AtaPT.
[0061] Figure 27 It is the H spectrum of product 2g-Licoflavanone produced by the reaction of naringenin 1g with mutant G326W of AtaPT.
[0062] Figure 28 It is the C spectrum of product 2g-Licoflavanone produced by the reaction of 1 g of naringenin with mutant G326W of AtaPT.
[0063] Figure 29 It is the H spectrum of product 3g-Propolin H produced by the reaction of 1 g of naringenin with mutant M7 of AtaPT.
[0064] Figure 30 It is the C spectrum of product 3g-Propolin H produced by the reaction of 1 g of naringenin with mutant M7 of AtaPT. Specific Embodiments
[0065] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.
[0066] Testing Method:
[0067] 1. Activity determination of isopentenyltransferase: The activity of isopentenyltransferase was determined in 300 μL of reaction buffer, which included 4 mM DMAPP / GPP, 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2 and mutant whole cells (OD 600nm = 80). Incubate at 37 °C for 8 h, then terminate the reaction by adding 3 volumes of methanol and analyze by HPLC.
[0068] 2. Product analysis method: High performance liquid chromatography and mass spectrometry analysis. The corresponding liquid phase detection method was a 5-μm C18 column, a PDA detector, and the mobile phase was: phase A (H2O containing 0.1% formic acid) and phase B (acetonitrile containing 0.1% formic acid). The gradient elution program was: 55% B for 4 min, 50%-60% B for 2 min, 60-90% B for 1 min, 90% B for 11 min, 90%-55% B for 6 min. The flow rate was 1 mL / min, and the detection wavelengths for 1a were 350 nm, 1b was 370 nm, 1e was 335 nm, and 1g was 288 nm. Raw materials used in the examples:
[0069] Solid medium formulation (1 L): 5 g of yeast extract, 10 g of peptone, 10 g of sodium chloride, 15 g of agar, made up to volume with deionized water and autoclaved.
[0070] LB medium formulation (1 L): 5 g of yeast extract powder, 10 g of peptone, 10 g of sodium chloride, made up to volume with deionized water and autoclaved.
[0071] TB medium formulation (1 L): 24 g of yeast extract, 12 g of peptone, 4 mL of glycerol. Add 900 mL of deionized water, dissolve and sterilize by autoclaving, then add 100 mL of 0.17 M KH2PO4 / 0.72 M K2HPO4 solution that has been sterilized by the same operation. (Note: Sodium chloride was purchased from Aladdin, and the rest were purchased from Sangon Biotech Co., Ltd.)
[0072] Example 1: Protein engineering modification of isopentenyl transferase AtaPT
[0073] (1) Design and preparation of mutants:
[0074] Using the parental gene with the sequence shown in SEQ ID NO.2 as a template, design primers to mutate sites I167, E169, S170, C175, S177, L323, P324, G326, N328, and obtain the coding genes of mutants G326W, S177G, M8 (I167A / E169A / S170Q / C175M / S177A / L323Q / P324H / N328R) and M7 (I167T / E169A / S170Q
[0075] / S177A / L323Q / P324H / N328M).
[0076] Table 1 Single-point mutation primers
[0077]
[0078] Table 2 Combinatorial mutation primers
[0079]
[0080] (2) Construction of recombinant plasmids:
[0081] Use the commercial One Step Cloning Kit to ligate the coding genes of the mutants prepared in step (1), as well as the parental gene, with the pET28a vector to obtain recombinant plasmids pET28a-AtaPT, pET28a-G326W, pET28a-S177G, pET28a-M8, pET28a-M7.
[0082] Example 2: Expression of isopentenyl transferase AtaPT mutants G326W, S177G, M8 and M7
[0083] The recombinant plasmids pET28a-AtaPT, pET28a-G326W, pET28a-S177G, pET28a-M8, and pET28a-M7 in Example 1 were chemically transformed into the competent cell BL21 DE3. An appropriate amount of the bacterial solution was spread on a solid medium containing kanamycin and cultured at 37°C for 12 - 15 h. One single colony containing the transformed recombinant plasmid was picked into an LB medium (containing 50 μg / mL -1 kanamycin), and cultured overnight at 37°C to obtain the genetically engineered bacteria BL21 / pET28a-AtaPT, BL21 / pET28a-G326W, BL21 / pET28a-S177G, BL21 / pET28a-M8, and BL21 / pET28a-M7.
[0084] 1% of the LB culture was inoculated into a TB medium (containing 50 μg / mL -1 kanamycin and 0.05% glucose that had been sterilized by membrane filtration), and then cultured at 37°C until the logarithmic growth phase, that is, the OD value was 0.6 - 0.8. 0.2 mM IPTG was added as an inducer, and the expression was carried out at 18°C and 220 rpm for 18 - 20 h.
[0085] The expressed bacterial cells were collected by a centrifuge pre-cooled at 18°C, washed twice with 50 mM (pH = 8.0) Tris-HCl buffer solution, then a small amount of buffer was added, and they were quickly frozen and stored in liquid nitrogen. After thawing in an ice-water bath, they could be used for the reaction.
[0086] Example 3: Isopentenyltransferase AtaPT mutants G326W, S177G, M8, and M7 catalyze the isopentenylation of kaempferol
[0087] In a 300 μL reaction system, the concentration of DMAPP / GPP was 4 mM, the concentration of the substrate kaempferol was 2 mM, DMSO (v / v, 5%), and the amount of the biocatalyst (the genetically engineered bacteria obtained in Example 2) was about OD 80. The corresponding isopentenylated or geranylated products were obtained by reacting at 37°C for 8 h. The reaction was terminated by adding 3 volumes of ice-cold methanol, and analyzed by high-performance liquid chromatography and mass spectrometry. It was scaled up to 50 mL for preparation, and after separation and purification by preparative liquid chromatography, nuclear magnetic resonance analysis was carried out to determine the product structure.
[0088] By Figures 1 to 10It can be seen that the mutants G326W, S177G, and M8 catalyze kaempferol to obtain prenylated products. Among them, M8 catalyzes the formation of 2a-8-(1,1-Dimethyl-2-propenyl)kaempferol, with the selectivity increased to 77.4% and the activity increased by about 45 times; G326W catalyzes the formation of 3a-Isolicoflavonol, with the selectivity increased to 95.8% and the activity increased by about 17 times, while the wild-type enzyme gives relatively mixed products with a yield of 1%; and S177G generates 5a-4'-(dimethylallyl)kaempferol, with the activity increased by about 17 times compared to the wild type. The mutant M7 catalyzes kaempferol to obtain the geranylated product 6a-Robipseudin A with a yield of 90.7%, while the yield of the wild-type enzyme is 14.6%. The mutants have higher chemo-selectivity and significantly increased yields compared to the wild-type enzyme.
[0089] Example 4: Prenylation of quercetin by AtaPT mutants G326W, M8, and M7 of prenyltransferase
[0090] In a 300 μL reaction system, the concentration of DMAPP / GPP is 4 mM, the concentration of the substrate quercetin is 2 mM, DMSO (v / v, 5%), and the amount of the biocatalyst (genetic engineering obtained in Example 2) is about OD 600nm 80. React at 37 °C for 8 h to obtain the corresponding prenylated or geranylated products. Add 3 volumes of ice methanol to terminate the reaction, and analyze by high-performance liquid chromatography and mass spectrometry. Scale up to 50 mL in equal proportion for preparation, and perform nuclear magnetic resonance analysis after separation and purification by preparative liquid chromatography to determine the product structure.
[0091] It can be seen that Figures 11 to 18 the mutants G326W and M8 can catalyze quercetin to obtain prenylated products. Among them, M8 catalyzes the formation of 2b-6′-prenylquercetin, with a slight decrease in selectivity but an increase in activity by about 20 times. G326W catalyzes the formation of 3b-Uralenol, with a slight decrease in selectivity but an increase in activity by about 43 times, while the wild-type enzyme gives relatively mixed products. The mutant M7 can catalyze quercetin to obtain the geranylated product 4b-Denticulatain E with a yield of about 80%, while the yield of the wild-type enzyme is less than 20%. The mutants have higher chemo-selectivity and significantly increased yields compared to the wild-type enzyme.
[0092] Example 5: Prenylation of apigenin by AtaPT mutants G326W and M7
[0093] In a 300 μL reaction system, the concentration of DMAPP / GPP was 4 mM, the concentration of the substrate apigenin was 2 mM, DMSO (v / v, 5%), and the amount of the biocatalyst (genetically engineered bacterium obtained in Example 2) was approximately OD 600nm = 80. The corresponding prenylated or geranylated product was obtained by reacting at 37 °C for 8 h. The reaction was terminated by adding 3 volumes of ice methanol, and high performance liquid chromatography and mass spectrometry analysis were performed. It was scaled up to 50 mL for preparation, and nuclear magnetic resonance analysis was performed after preparative liquid separation and purification to determine the product structure.
[0094] It can be seen from Figures 19 to 24 that the mutant G326W can catalyze apigenin to obtain the prenylated product 2e-Yinyanghuo D, with a 18-fold increase in activity while the selectivity is improved, while the wild enzyme has poor activity. The mutant M7 can catalyze apigenin to obtain the geranylated product 3e-Kuwanon S, with a chemical selectivity maintained and a yield increased by about 25-fold.
[0095] Example 6: Prenylation of naringenin by the mutants G326W and M7 of isopentenyltransferase AtaPT
[0096] In a 300 μL reaction system, the concentration of DMAPP / GPP was 4 mM, the concentration of the substrate naringenin was 2 mM, DMSO (v / v, 5%), and the amount of the biocatalyst (genetically engineered bacterium obtained in Example 2) was approximately OD 600nm = 80. The corresponding prenylated or geranylated product was obtained by reacting at 37 °C for 8 h. The reaction was terminated by adding 3 volumes of ice methanol, and high performance liquid chromatography and mass spectrometry analysis were performed. It was scaled up to 50 mL for preparation, and nuclear magnetic resonance analysis was performed after preparative liquid separation and purification to determine the product structure.
[0097] It can be seen from Figures 25 to 30 that the mutant G326W can catalyze naringenin to obtain the prenylated product 2g-Licoflavanone, with both selectivity and yield improved to a certain extent, while the wild enzyme has poor activity. The mutant M7 can catalyze naringenin to obtain the geranylated product 3g-Propolin H, with a yield greater than 95% and a selectivity greater than 97%, while the yield of the wild enzyme is 63.1% and the selectivity is 83.2%. The mutants have higher chemical selectivity and a significantly increased yield compared to the wild enzyme.
[0098] Comparative Example 1
[0099] The specific implementation method was the same as that of Example 1, except that only the site P324 was mutated to histidine. The enzyme mutant was prepared according to Example 2 and used for the catalytic reaction with 1a. The results showed that the product yield was 0.47%. Although the selectivity was twice that of the wild type, the activity was much lower than that of the wild type and about 95 times lower than that of the mutant M8.
[0100] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims
1. An isopentenyl transferase AtaPT mutant, characterized in that, The mutant is obtained by mutating the prenyltransferase AtaPT parent with the amino acid sequence as shown in SEQ ID NO.1 in any of the following ways: (1) Mutating glycine G at position 326 to tryptophan W; (2) Mutating serine S at position 177 to glycine G; (3) Mutating isoleucine I at position 167 to alanine A, mutating glutamate E at position 169 to alanine A, mutating serine S at position 170 to glutamine Q, mutating cysteine C at position 175 to methionine M, mutating serine S at position 177 to alanine A, mutating leucine L at position 323 to glutamine Q, mutating proline P at position 324 to histidine H, and mutating asparagine N at position 328 to arginine R; (4) Mutating isoleucine I at position 167 to threonine T, mutating glutamate E at position 169 to alanine A, mutating serine S at position 170 to glutamine Q, mutating serine S at position 177 to alanine A, mutating leucine L at position 323 to glutamine Q, mutating proline P at position 324 to histidine H, and mutating asparagine N at position 328 to methionine M.
2. A gene encoding the mutant according to claim 1.
3. A recombinant vector carrying the gene according to claim 2.
4. A recombinant cell expressing the mutant according to claim 1, or containing the gene according to claim 2, or transformed with the recombinant vector according to claim 3.
5. A genetically engineered bacterium, characterized in that, The genetically engineered bacterium expresses the mutant according to claim 1 using Escherichia coli as the host and a plasmid of the pET series as the vector.
6. A method for improving the chemo - selectivity of isopentenyl transferase AtaPT, characterized in that, The method is to mutate the prenyltransferase AtaPT parent with the amino acid sequence as shown in SEQ ID NO.1 in any of the following ways: (1) Mutating glycine G at position 326 to tryptophan W to obtain mutant G326W; (2) Mutating serine S at position 177 to glycine G to obtain mutant S177G; (3) Mutating isoleucine I at position 167 to alanine A, mutating glutamate E at position 169 to alanine A, mutating serine S at position 170 to glutamine Q, mutating cysteine C at position 175 to methionine M, mutating serine S at position 177 to alanine A, mutating leucine L at position 323 to glutamine Q, mutating proline P at position 324 to histidine H, and mutating asparagine N at position 328 to arginine R to obtain mutant M8; (4) Mutating isoleucine I at position 167 to threonine T, mutating glutamate E at position 169 to alanine A, mutating serine S at position 170 to glutamine Q, mutating serine S at position 177 to alanine A, mutating leucine L at position 323 to glutamine Q, mutating proline P at position 324 to histidine H, and mutating asparagine N at position 328 to methionine M to obtain mutant M7.
7. A method for synthesizing isoprenylated polyphenolic compounds, characterized in that, The method is to use the mutant according to claim 1 or the genetically engineered bacterium according to claim 5 as a catalyst, and polyphenolic compounds as acceptors to generate isoprenylated polyphenolic compounds.
8. The method according to claim 7, characterized in that The polyphenolic compounds include quercetin, apigenin, luteolin, and naringenin.
9. The method according to claim 7, wherein The method is carried out in a 40 - 60 mM Tris HCl buffer system, with the catalyst addition being whole cells with an OD 600nm = 80, pH = 7.0 - 8.0, 5 mM CaCl2, at 30 - 40 °C, with the receptor substrate concentration being 2 mM, the donor substrate DMAPP or GPP concentration being 4 mM, the reaction rotation speed being 800 - 1200 rpm, and the reaction time being 6 - 8 h.
10. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4, or the genetically engineered bacterium according to claim 5, or the method according to claim 6, or the method according to any one of claims 7 to 9 in the production of prenylated polyphenolic compounds.