A scopolamine 6β-hydroxylase mutant, gene and application

Abstract

The present invention discloses a hyoscine 6β-hydroxylase mutant, gene and application, relating to the field of biotechnology. Compared with the wild-type hyoscine 6β-hydroxylase, the mutant has the following mutations: the 43rd amino acid is mutated from histidine to aspartic acid, the 247th amino acid is mutated from glutamine to glutamate, and the 324th amino acid is mutated from aspartic acid to glutamate. The amino acid sequence of the mutant is shown in SEQ ID NO: 4. The nucleotide sequence encoding the hyoscine 6β-hydroxylase mutant is shown in SEQ ID NO: 8. The present invention obtains a HnH6H mutant with significantly improved catalytic activity, and proves that it has stronger production performance than the wild-type HnH6H in Escherichia coli fermentation, in vitro enzyme kinetic analysis and plant metabolic engineering, and has important application value in scopolamine metabolic engineering and synthetic biology.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a scopolamine 6β-hydroxylase mutant, a gene and an application thereof. Background Art

[0002] Tropane alkaloids (TAs) are a class of natural anticholinergic drugs commonly used in clinical practice, including hyoscyamine, anisodamine, and scopolamine. They exert a wide range of anticholinergic effects in the peripheral and central nervous systems by non-selectively and competitively antagonizing muscarinic acetylcholine receptors (mAChR). Compared with other cholinergic receptor antagonists, scopolamine has better blood-brain barrier permeability, so it can better act on the central nervous system. It is widely used to relieve symptoms of organophosphorus pesticide poisoning, postoperative nausea and vomiting, motion sickness, and other symptoms of the peripheral and central systems. At present, the medicinal use of tropane alkaloids still depends entirely on the production of drugs from belladonna ( Atropa belladonna )、Mandala( Datura stramonium )、Three-thirds-three( Anisodus acutangulus ) and Henbane ( Hyoscyamus niger ) and a few other Solanaceae plants. However, the content of tropane alkaloids in wild plants is extremely low, making them difficult to obtain, which seriously hinders the production of related drugs.

[0003] Scopolamine 6 β -Hydroxylase (hyoscyamine 6 β -hydroxylase (H6H) is a bifunctional enzyme that catalyzes the 6-carbon hydroxylation of hyoscyamine to synthesize anisodamine, and further catalyzes its 6- and 7-carbon epoxidation to synthesize scopolamine ( Figure 1 ). Overexpression of the H6H gene in TAs resource plants can effectively increase the production of hyoscine and scopolamine. When the H6H gene is expressed in Escherichia coli and hyoscine is fed in the culture medium, the formation of hyoscine and scopolamine can be detected. Related research has laid an important foundation for the application of H6H in scopolamine production, but the researchers currently use wild-type H6H from plants, and its catalytic activity still has room for improvement. CN118726283A discloses a hyoscine 6β-hydroxylase mutant, a preparation method and an application, constructs a three-thirds source AaH6H random mutant library, and screens out mutants AaH6H with improved catalytic activity. S14P+K97A The mutant showed superior scopolamine production performance compared to wild-type AaH6H in E. coli fermentation. Therefore, obtaining highly active H6H mutants through protein engineering could provide more efficient components for scopolamine metabolic engineering and synthetic biology.

[0004] Studies have found that Hyoscyamus chinensis is a plant with a high proportion of scopolamine, while Belladonna, Datura, and Tripterygium wilfordii are all plants with a high proportion of scopolamine. Based on this speculation, compared with H6H from other species with a high proportion of scopolamine, Hyoscyamus chinensis HnH6H with a high proportion of scopolamine has a stronger ability to synthesize scopolamine. Therefore, this application compares HnH6H from Hyoscyamus chinensis with the reported mutant AaH6H. S14P+K97A Conduct activity comparisons and carry out protein design. Summary of the Invention

[0005] To address the deficiencies in the prior art, the present invention provides a scopolamine 6β-hydroxylase mutant, gene, and application, which significantly improve the catalytic activity of the enzyme.

[0006] In order to achieve the purpose of the present invention, the following scheme is proposed:

[0007] A scopolamine 6β-hydroxylase mutant has the following mutation compared to wild-type scopolamine 6β-hydroxylase: amino acid 43 is mutated from histidine to aspartic acid, the amino acid sequence of the mutant is shown in SEQ ID NO: 1. The nucleotide sequence of the corresponding gene is shown in SEQ ID NO: 5.

[0008] A scopolamine 6β-hydroxylase mutant has the following mutations compared to wild-type scopolamine 6β-hydroxylase: amino acid 43 is mutated from histidine to aspartic acid, and amino acid 247 is mutated from glutamine to glutamate. The amino acid sequence of the mutant is shown in SEQ ID NO: 2. The nucleotide sequence of the corresponding gene is shown in SEQ ID NO: 6.

[0009] A scopolamine 6β-hydroxylase mutant has the following mutations compared to wild-type scopolamine 6β-hydroxylase: amino acid 43 is mutated from histidine to aspartic acid, and amino acid 324 is mutated from aspartic acid to glutamic acid. The amino acid sequence of the mutant is shown in SEQ ID NO: 3. The nucleotide sequence of the corresponding gene is shown in SEQ ID NO: 7.

[0010] A scopolamine 6β-hydroxylase mutant having the following mutations compared to wild-type scopolamine 6β-hydroxylase: amino acid 43 is mutated from histidine to aspartic acid, amino acid 247 is mutated from glutamine to glutamic acid, and amino acid 324 is mutated from aspartic acid to glutamic acid. The amino acid sequence of the mutant is shown in SEQ ID NO: 4. The nucleotide sequence of the corresponding gene is shown in SEQ ID NO: 8.

[0011] The scopolamine 6β-hydroxylase mutant of the present invention is used in the following two aspects:

[0012] (1) Used to synthesize hyoscine / scopolamine in Escherichia coli expression system.

[0013] (2) Used for metabolic engineering of belladonna to synthesize hyoscine / scopolamine.

[0014] The beneficial effects of the present invention are: obtaining an HnH6H mutant with significantly improved catalytic activity, and proving that it has stronger production performance than wild-type HnH6H in Escherichia coli fermentation, in vitro enzyme kinetic analysis and plant metabolic engineering, and having important application value in scopolamine metabolic engineering and synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is scopolamine 6 β -Hyoscyamine catalyzed by hyoscyamine reaction to scopolamine;

[0016] Figure 2 The figure shows the sequence consistency analysis results of the amino acid mutation at position 11;

[0017] Figure 3 The sequence consistency analysis results of the amino acid mutation at position 12 are shown;

[0018] Figure 4 The sequence consistency analysis results of the amino acid mutation at position 22 are shown;

[0019] Figure 5 The figure shows the sequence consistency analysis results of the amino acid mutation at position 43;

[0020] Figure 6 The sequence consistency analysis results of the amino acid mutation at position 45 are shown;

[0021] Figure 7 The sequence consistency analysis results of the amino acid mutation at position 71 are shown;

[0022] Figure 8 The sequence consistency analysis results of the amino acid mutation at position 74 are shown;

[0023] Figure 9 The sequence consistency analysis results of the amino acid mutation at position 94 are shown;

[0024] Figure 10 The sequence consistency analysis results of the amino acid mutation at position 95 are shown;

[0025] Figure 11 The sequence consistency analysis results of the amino acid mutation at position 100 are shown;

[0026] Figure 12The figure shows the sequence consistency analysis results of the amino acid mutation at position 141;

[0027] Figure 13 The figure shows the sequence consistency analysis results of the amino acid mutation at position 153;

[0028] Figure 14 The sequence consistency analysis results of the amino acid mutation at position 242 are shown;

[0029] Figure 15 The figure shows the sequence consistency analysis results of the amino acid mutation at position 247;

[0030] Figure 16 The sequence consistency analysis results of the amino acid mutation at position 279 are shown;

[0031] Figure 17 The figure shows the sequence consistency analysis results of the amino acid mutation at position 307;

[0032] Figure 18 The sequence consistency analysis results of the amino acid mutation at position 320 are shown;

[0033] Figure 19 The figure shows the sequence consistency analysis results of the amino acid mutation at position 324;

[0034] Figure 20 The sequence consistency analysis results of the amino acid mutation at position 335 are shown;

[0035] Figure 21 A graph showing anisodamine production in an E. coli fermentation screening experiment is shown;

[0036] Figure 22 A graph showing scopolamine production in an E. coli fermentation screening experiment is shown;

[0037] Figure 23 Shown is a diagram of the purification results of SDS-PAGE analysis;

[0038] Figure 24 The figures show the Michaelis-Menten plot (A) of the synthesis of anisodamine using hyoscyamine as a substrate and the Michaelis-Menten plot (B) of the synthesis of scopolamine using anisodamine as a substrate in the enzyme kinetic constant determination experiment;

[0039] Figure 25 Shown are graphs of hyoscyamine content (A), anisodamine content (B), and scopolamine content (C) in a plant metabolic engineering experiment. DETAILED DESCRIPTION

[0040] Example 1

[0041] Design of HnH6H mutants by sequence consistency analysis:

[0042] First, this study used the method of amino acid sequence consistency analysis to improve enzyme activity by identifying evolutionary information in homologous or isoenzyme sequences. This theory holds that at a given site, consistent amino acids in homologous or isoenzymes contribute more to protein function and stability than non-conserved amino acids. Therefore, this study first compared and analyzed the amino acid sequences of H6H from 13 different species of Solanaceae, analyzed the conservation of all amino acid sites based on the Position-Specific Scoring Matrix (PSSM), and mutated HnH6H to highly conserved sites. This analysis targeted 19 sites and designed single-point mutations, including: HnH6H K11N 、HnH6H S12N 、HnH6H Q22E 、HnH6H H43D 、HnH6H H45L 、HnH6H E71K 、HnH6H L74A 、HnH6H F94L 、HnH6H K95Q 、HnH6H A100P 、HnH6H Q141E 、HnH6H K153T 、HnH6H T242N 、HnH6H Q247E 、HnH6H D279N 、HnH6H N307S 、HnH6H S320A 、HnH6H D324E and HnH6H S335A ( Figure 2-Figure 20 ).

[0043] Example 2

[0044] Screening of HnH6H mutants with improved catalytic activity by E. coli fermentation:

[0045] Entrust a biological company to synthesize the above-mentioned HnH6H mutants and AaH6H S14P+K97A , and constructed into pET28a plasmid, transformed into BL21, and obtained a series of mutant prokaryotic expression strains. The above-mentioned engineered strains and wild-type HnH6H engineered bacteria were inoculated into LB liquid medium, cultured at 37°C until OD600 was 0.5, 0.25 mM IPTG and 1 mM substrate (hyoscyamine) were added, and cultured at 18°C for 12 h. Take 1 ml of culture medium, centrifuge and filter the bacteria, and use HPLC to analyze the product (anisodamine / scopolamine) content. The results showed that AaH6H S14P+K97AThe production capacity is much lower than that of wild-type HnH6H ( Figure 21 and Figure 22 ). Compared with wild-type HnH6H, HnH6H H43D 、HnH6H A100P 、HnH6H T242N 、HnH6H Q247E 、HnH6H N307S 、HnH6H D324E and HnH6H S335A The content of anisodamine in the fermentation product of the mutant was significantly increased ( Figure 21 );HnH6H H43D 、HnH6H A100P 、HnH6H Q247E 、HnH6H S335A The yield of scopolamine in the fermentation product increased significantly ( Figure 22 Next, a series of double mutants and triple mutants were constructed using the same method described above, and their fermentation product contents were analyzed. The results showed that HnH6H H43D+Q247E+D324E The best, anisodamine production is wild-type HnH6H and AaH6H S14P+K97A The scopolamine production of wild-type HnH6H and AaH6H was 6.11 times and 12.3363 times higher than that of wild-type HnH6H and AaH6H. S14P +K97A 14.42 times and 26.48 times ( Figure 21 and Figure 22 ).

[0046] Next, for AaH6H S14P+K97A , wild-type HnH6H and mutant HnH6H H43D+Q247E+D324E Enzyme kinetic analysis was performed. The prokaryotic expression plasmids of the aforementioned genes were transformed into competent Escherichia coli BL21 (DE3) to obtain prokaryotic expression strains. Protein expression was induced overnight at 16°C with 0.25 mM IPTG in LB liquid medium. The 6×His-tagged H6H protein was then purified using HisPur-Ni-NTA resin (Thermo Fisher Scientific). The target band was approximately 40 kDa, consistent with the theoretical value ( Figure 23After desalting, the fresh protein was immediately used for enzyme activity determination. Based on existing literature reports, enzyme kinetic constant determination was performed at 30°C in this project. The reaction system consisted of: pH 7.6 Tris / HCl buffer, 0.4 mM FeSO₄, 4 mM sodium ascorbate, 1 mM α-ketoglutarate, 2 mg / mL catalase, a gradient of hyoscyamine / anisodamine (0.005 mM, 0.01 mM, 0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.1 mM, 0.15 mM, 0.20 mM), and 0.6 μM H6H protein. The target product content in the reaction samples was determined by LC-MS.

[0047] The results showed that AaH6H S14P+K97A of K The m value is the highest, V Max is the lowest, and the overall catalytic efficiency is the lowest. H43D +Q247E+D324E The catalytic activity is the highest. H43D+Q247+D324E Catalyzes the first and second steps of the reaction K cat / K The m values are 2.90 and 1.97 times that of HnH6H, respectively. Figure 24 and Table 1).

[0048] Table 1 Enzyme kinetic constants determination table

[0049]

[0050] Example 3

[0051] The value of HnH6H mutants in plant metabolic engineering:

[0052] A pair of primers with BamHI and SacI restriction sites (BamHI-F-H6H: cgcGGATCCATGGCTACTTTTGTGTCGAACTG; SacI-R-H6H: cgcGAGCTCTTAGACATTGATTTTATATGGCT) were designed to clone HnH6H and HnH6H. H43D+Q247E+D324E The coding regions were ligated to the plant binary expression plasmid pBI121 to obtain engineered plasmids. The pBI121 original plasmid (control group) and the engineered plasmids were transformed into Agrobacterium rhizogenes C58C1 to obtain engineered bacteria. The engineered bacteria were then used to infect belladonna leaves to obtain transgenic hairy roots. The specific method for constructing hairy roots is as follows:

[0053] (1) Inoculate the above positive monoclonal engineered strain into 1 mL of antibiotic-containing (Kan 100 mg.L -1 , Rif 50mg.L-1 ) in YEP liquid medium (centrifuge tube or test tube), culture at 28 °C overnight for activation; then inoculate the bacteria in good growth state into 10 mL of new YEP liquid medium containing antibiotics and culture until the OD600 is about 0.6-0.8.

[0054] (2) Place the Agrobacterium culture solution in a 50 mL EP tube under sterile conditions, centrifuge at 3000 rpm for 10 min at room temperature, and remove the supernatant (as clean as possible); then use the transformation solution (containing 20 mg.L acetosyringone) to -1 After resuspending in MS liquid medium, centrifuge at 3000 rpm at room temperature for 10 min, remove the supernatant, and repeat at least twice.

[0055] (3) Resuspend the bacteria in the transformation solution to a final concentration of OD600 of about 0.3 and let it stand at room temperature for 30 minutes. Cut the leaves of sterile belladonna seedlings with good growth as explants and poke appropriate holes on the leaf surface with scissors or a scalpel. Add the explants to the Agrobacterium resuspension solution and soak for 5 minutes. Then remove the explants and absorb the residual bacterial liquid on the surface with sterilized absorbent paper, and place them in the co-cultivation medium (containing 20 mg.L AS). -1 The cells were grown on MS solid medium for 2 days and then transferred to sterile medium (containing Cef200 mg.L -1 and Kan 100 mg.L -1 Continue growing on MS solid medium. After approximately 15 days, new white hairy roots will emerge from the leaf wound. After 30 days, the hairy roots can be cut and inoculated onto new sterilized medium for growth. Subculture once a month, and sterilization is generally complete after two rounds.

[0056] Fifteen independently transformed root lines of each transgenic type were randomly selected for liquid culture. Hairy roots from each single clone were inoculated into flasks containing 100 mL of MS liquid medium and incubated in the dark at 120 rpm and 25°C for 28 days before harvest. Hairy roots were freeze-dried and analyzed for hyoscyamine, anisodamine, and scopolamine using UPLC-MS.

[0057] The results are as follows Figure 25 As shown in the figure, CK represents the control strain transformed with the original plasmid, OE-HnH6H represents the transgenic strain overexpressing wild-type HnH6H, and OE-HnH6H 3M Represents HnH6H H43D+Q247E+D324E Overexpression transgenic lines; each group consists of 15 independent transformed lines, ** represents independent sample T test p The value was less than or equal to 0.01. The results showed that overexpression of HnH6H and HnH6H H43D+Q247E+D324EThe scopolamine content in the strains was significantly reduced, only 42.31% and 25.17% of the control group ( Figure 25 A). Overexpression of HnH6H significantly increased the production of anisodamine and scopolamine in A. belladonna, which was 1.69 times and 4.8 times that of the control group ( Figure 25 B). HnH6H H43D+Q247E+D324E The levels of anisodamine and scopolamine in the overexpression group were significantly higher than those in the HnH6H overexpression group, which were 2.87 times and 10.81 times that of the control group ( Figure 25 C). In summary, HnH6H H43D+Q247E+D324E The metabolic engineering effect of HnH6H is better than that of HnH6H.

[0058] The above embodiments are only used to illustrate the technical ideas and features of the present invention and are not intended to be exclusive or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention are within the scope of protection of the present invention.

Claims

1. A scopolamine 6β-hydroxylase mutant, characterized in that: Compared with the wild-type scopolamine 6β-hydroxylase, the following mutation exists: the 43rd amino acid is mutated from histidine to aspartic acid. The amino acid sequence of the mutant is shown in SEQ ID NO:

1.

2. A scopolamine 6β-hydroxylase mutant, characterized in that: Compared with the wild-type scopolamine 6β-hydroxylase, the following mutations exist: the 43rd amino acid is mutated from histidine to aspartic acid, and the 247th amino acid is mutated from glutamine to glutamate. The amino acid sequence of the mutant is shown in SEQ ID NO:

2.

3. A scopolamine 6β-hydroxylase mutant, characterized in that: Compared with the wild-type scopolamine 6β-hydroxylase, the following mutations exist: the 43rd amino acid is mutated from histidine to aspartic acid, and the 324th amino acid is mutated from aspartic acid to glutamic acid. The amino acid sequence of the mutant is shown in SEQ ID NO:

3.

4. A scopolamine 6β-hydroxylase mutant, characterized in that: Compared with the wild-type scopolamine 6β-hydroxylase, the following mutations exist: the 43rd amino acid mutates from histidine to aspartic acid, the 247th amino acid mutates from glutamine to glutamate, and the 324th amino acid mutates from aspartic acid to glutamate. The amino acid sequence of the mutant is shown in SEQ ID NO:

4.

5. A gene, characterized in that Used to encode the scopolamine 6β-hydroxylase mutant according to claim 1, the nucleotide sequence of the gene is shown in SEQ ID NO:

5.

6. A gene, characterized in that Used to encode the scopolamine 6β-hydroxylase mutant according to claim 2, the nucleotide sequence of the gene is shown in SEQ ID NO:

6.

7. A gene, characterized in that Used to encode the scopolamine 6β-hydroxylase mutant according to claim 3, the nucleotide sequence of the gene is shown in SEQ ID NO:

7.

8. A gene, characterized in that Used to encode the scopolamine 6β-hydroxylase mutant according to claim 4, the nucleotide sequence of the gene is shown in SEQ ID NO:

8.

9. Use of the scopolamine 6β-hydroxylase mutant according to any one of claims 1 to 4, characterized in that: Used to synthesize anisodamine and / or scopolamine in Escherichia coli expression system.

10. Use of the scopolamine 6β-hydroxylase mutant according to any one of claims 1 to 4, characterized in that: Used for metabolic engineering improvement of belladonna to synthesize anisodamine and / or scopolamine.

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