Hypolamine 6 beta hydroxylase mutant, gene and application

Abstract

The invention discloses a hyoscyamine 6 beta hydroxylase mutant, a gene and application, and relates to the technical field of biology, and compared with wild hyoscyamine 6 beta 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 glutamic acid, and the 43rd amino acid is mutated from glutamine to glutamic acid. The 324th amino acid is mutated into glutamic acid from aspartic acid, and the amino acid sequence of the mutant is as shown in SEQ ID NO: 4. A nucleotide sequence for coding the hyoscyamine 6 beta hydroxylase mutant is shown as SEQ ID NO: 8. According to the invention, the HnH6H mutant with remarkably improved catalytic activity is obtained, and the HnH6H mutant is proved to have stronger production performance than wild type HnH6H in escherichia coli fermentation, in-vitro enzyme kinetic analysis and plant metabolism engineering, and has important application value in scopolamine metabolism engineering and synthetic biology.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a hyoscyamine 6β-hydroxylase mutant, gene and application. Background Art

[0002] Tropane alkaloids (TAs) are a class of natural anticholinergic drugs commonly used clinically, including hyoscyamine, anisodamine and scopolamine. By non-selectively and competitively antagonizing muscarinic acetylcholine receptors (mAChRs), they exert extensive anticholinergic effects in the peripheral and central nervous systems. Compared with other cholinergic receptor antagonists, scopolamine has better blood-brain barrier permeability, so it can better act on the central nervous system and is widely used to relieve symptoms such as organophosphorus pesticide poisoning, postoperative nausea and vomiting, and motion sickness, which are caused by the combined effects of the peripheral and central systems. At present, medicinal tropane alkaloids still completely rely on extraction from a few Solanaceae plants such as Atropa belladonna Atropa belladonna Datura stramonium Datura stramonium Anisodus acutangulus Anisodus acutangulus Hyoscyamus niger Hyoscyamus niger

[0003] However, the content of tropane alkaloids in wild plants is extremely low and difficult to obtain, which seriously hinders the production of related drugs. β Hyoscyamine 6 β -hydroxylase (H6H) is a bifunctional enzyme that can catalyze the hydroxylation of the 6-position carbon of hyoscyamine to synthesize anisodamine, and then further catalyze the epoxidation of its 6-position and 7-position to synthesize scopolamine ( Figure 1 ). Overexpressing the H6H gene in TAs resource plants can effectively increase the production of anisodamine and scopolamine. Expressing the H6H gene in Escherichia coli and feeding hyoscyamine in the culture medium can detect the formation of anisodamine and scopolamine. Related research has laid an important foundation for the application of H6H in scopolamine production, but currently, researchers have used wild-type H6H from plants, and its catalytic activity still has room for improvement. CN118726283A discloses a hyoscyamine 6β-hydroxylase mutant, preparation method and application, constructs a random mutant library of AaH6H from Anisodus acutangulus, and screens a mutant AaH6H with improved catalytic activity S14P+K97A which shows better scopolamine production performance than wild-type AaH6H in Escherichia coli fermentation. Therefore, obtaining highly active H6H mutants through protein engineering can provide more efficient components for scopolamine metabolic engineering and synthetic biology.

[0004] Previous studies have found that Hyoscyamus niger is a plant with a high proportion of scopolamine, while Atropa belladonna, Datura stramonium, and Anisodus acutangulus are all plants with a high proportion of hyoscyamine. Based on this speculation, compared with the H6H of other species with a high proportion of hyoscyamine, the HnH6H of Hyoscyamus niger with a high proportion of scopolamine has a stronger scopolamine synthesis ability. Therefore, in this application, the HnH6H from Hyoscyamus niger and the reported mutant AaH6H S14P+K97A were compared in terms of activity, and protein design was carried out. SUMMARY OF THE INVENTION

[0005] To solve the deficiencies of the prior art, the present invention provides a hyoscyamine 6β-hydroxylase mutant, gene, and application, and the catalytic activity of the enzyme is significantly improved.

[0006] To achieve the object of the present invention, the following scheme is proposed: A hyoscyamine 6β-hydroxylase mutant has the following mutations compared with the wild-type hyoscyamine 6β-hydroxylase: the amino acid at position 43 is mutated from histidine to aspartic acid, and the amino acid sequence of the mutant is as shown in SEQ ID NO:1. The nucleotide sequence of the corresponding gene is as shown in SEQ ID NO:5.

[0007] A hyoscyamine 6β-hydroxylase mutant has the following mutations compared with the wild-type hyoscyamine 6β-hydroxylase: the amino acid at position 43 is mutated from histidine to aspartic acid, and the amino acid at position 247 is mutated from glutamine to glutamic acid. The amino acid sequence of the mutant is as shown in SEQ ID NO:2. The nucleotide sequence of the corresponding gene is as shown in SEQ ID NO:6.

[0008] A hyoscyamine 6β-hydroxylase mutant has the following mutations compared with the wild-type hyoscyamine 6β-hydroxylase: the amino acid at position 43 is mutated from histidine to aspartic acid, and the amino acid at position 324 is mutated from aspartic acid to glutamic acid. The amino acid sequence of the mutant is as shown in SEQ ID NO:3. The nucleotide sequence of the corresponding gene is as shown in SEQ ID NO:7.

[0009] A hyoscyamine 6β-hydroxylase mutant has the following mutations compared with the wild-type hyoscyamine 6β-hydroxylase: the amino acid at position 43 is mutated from histidine to aspartic acid, the amino acid at position 247 is mutated from glutamine to glutamic acid, and the amino acid at position 324 is mutated from aspartic acid to glutamic acid. The amino acid sequence of the mutant is as shown in SEQ ID NO:4. The nucleotide sequence of the corresponding gene is as shown in SEQ ID NO:8.

[0010] The application of the hyoscyamine 6β-hydroxylase mutant of the present invention is in the following two aspects: (1) It is used for the synthesis of anisodamine / scopolamine in an Escherichia coli expression system.

[0011] (2) For metabolic engineering improvement of belladonna to synthesize anisodamine / scopolamine.

[0012] The beneficial effects of the present invention are as follows: An HnH6H mutant with significantly improved catalytic activity is obtained, and it is proved in Escherichia coli fermentation, in vitro enzyme kinetics analysis and plant metabolic engineering that it has stronger production performance than the wild-type HnH6H, and has important application value in scopolamine metabolic engineering and synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shows the reaction formula diagram of scopolamine 6 β - hydroxylase catalyzing the synthesis of scopolamine from hyoscyamine; Figure 2 Shows the sequence identity analysis result diagram of amino acid mutation at position 11; Figure 3 Shows the sequence identity analysis result diagram of amino acid mutation at position 12; Figure 4 Shows the sequence identity analysis result diagram of amino acid mutation at position 22; Figure 5 Shows the sequence identity analysis result diagram of amino acid mutation at position 43; Figure 6 Shows the sequence identity analysis result diagram of amino acid mutation at position 45; Figure 7 Shows the sequence identity analysis result diagram of amino acid mutation at position 71; Figure 8 Shows the sequence identity analysis result diagram of amino acid mutation at position 74; Figure 9 Shows the sequence identity analysis result diagram of amino acid mutation at position 94; Figure 10 Shows the sequence identity analysis result diagram of amino acid mutation at position 95; Figure 11 Shows the sequence identity analysis result diagram of amino acid mutation at position 100; Figure 12 Shows the sequence identity analysis result diagram of amino acid mutation at position 141; Figure 13 Shows the sequence identity analysis result diagram of amino acid mutation at position 153; Figure 14 Shows the sequence identity analysis result diagram of amino acid mutation at position 242; Figure 15 Shows the sequence identity analysis result diagram of amino acid mutation at position 247; Figure 16 The figure shows the sequence identity analysis results of the amino acid mutation at position 279; Figure 17 The figure shows the sequence identity analysis results of the amino acid mutation at position 307; Figure 18 The figure shows the sequence identity analysis results of the amino acid mutation at position 320; Figure 19 The figure shows the sequence identity analysis results of the amino acid mutation at position 324; Figure 20 The figure shows the sequence identity analysis results of the amino acid mutation at position 335; Figure 21 The figure shows the yield graph of anisodamine in the Escherichia coli fermentation screening experiment; Figure 22 The figure shows the yield graph of scopolamine in the Escherichia coli fermentation screening experiment; Figure 23 The figure shows the SDS-PAGE analysis purification results; Figure 24 The figure shows the Michaelis-Menten curves (A) for synthesizing anisodamine with tropine as the substrate and (B) for synthesizing scopolamine with anisodamine as the substrate in the enzyme kinetic constant determination experiment; Figure 25 The figure shows the tropine content graph (A), anisodamine content graph (B), and scopolamine content graph (C) in the plant metabolic engineering experiment. Detailed implementation mode

[0014] Example 1 Sequence identity analysis design of HnH6H mutants: First, this study used the method of amino acid sequence identity analysis to improve the enzyme activity by identifying the evolutionary information in homologous or isozyme sequences. This theory holds that at a given site, the identical amino acids in homologous or isozymes contribute more to protein function and stability than non-conserved amino acids. Therefore, this study first aligned and analyzed the amino acid sequences of H6H from 13 different Solanaceae species, analyzed the conservation of all amino acid sites according to the Position-Specific Scoring Matrix (PSSM), and mutated HnH6H to highly conserved sites. This analysis targeted 19 sites and designed single-point mutations, specifically 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 )。

[0015] Example 2 Screening for HnH6H mutants with improved catalytic activity by Escherichia coli fermentation: Entrust a biological company to synthesize the above HnH6H mutants and AaH6H S14P+K97A , and construct them onto the pET28a plasmid, transform BL21 to obtain a series of mutant prokaryotic expression strains. Inoculate the above engineering strains and the wild-type HnH6H engineering bacteria into LB liquid medium respectively, culture at 37°C until OD600 reaches 0.5, add 0.25 mM IPTG and 1 mM substrate (scopolamine), and culture at 18°C for 12 h. Take 1 ml of the culture medium, centrifuge and filter the bacteria, and use HPLC to analyze the content of the product (anisodamine / scopolamine). The results show that the production capacity of AaH6H S14P+K97A is much lower than that of the wild-type HnH6H ( Figure 21 and Figure 22 ). Compared with the wild-type HnH6H, the content of anisodamine in the fermentation products of HnH6H H43D , HnH6H A100P , HnH6H T242N , HnH6H Q247E , HnH6H N307S , HnH6H D324E and HnH6H S335A mutants increased significantly ( Figure 21 ); the production of scopolamine in the fermentation products of HnH6H H43D , HnH6H A100P , HnH6H Q247E , HnH6H S335A increased significantly ( Figure 22 ). Next, construct a series of double mutants and triple mutants by the same method as above, and analyze the content of their fermentation products. The results show that HnH6H H43D+Q247E+D324E is the best, and its anisodamine production is that of the wild-type HnH6H and AaH6H S14P+K97A6.11 times and 12.3363 times that of, and the scopolamine yield is 14.42 times and 26.48 times that of the wild-type HnH6H and AaH6H S14P +K97A respectively ( Figure 21 and Figure 22 ).

[0016] Next, enzyme kinetic analysis was performed on AaH6H S14P+K97A , wild-type HnH6H and mutant HnH6H H43D+Q247E+D324E . The prokaryotic expression plasmids of the above genes were respectively transformed into Escherichia coli BL21(DE3) competent cells to obtain prokaryotic expression strains. Protein expression was induced overnight at 16 °C with 0.25 mM IPTG in LB liquid medium. Then, HisPur-Ni-NTA resin (Thermo Fisher Scientific) was used to purify the 6×His-tagged H6H protein, and the target band was approximately 40 kDa, which was consistent with the theoretical calculated value ( Figure 23 ). After desalting, the fresh protein was immediately used for enzyme activity determination. Based on the reported literature, this project selected to measure the enzyme kinetic constants at 30 °C. The specific reaction system was as follows: Tris / HCl buffer at pH 7.6, 0.4 mM FeSO4, 4 mM sodium ascorbate, 1 mM α-ketoglutaric acid, 2 mg / mL catalase, scopolamine / anisodamine at gradient concentrations (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. LC-MS was used to determine the content of the target product in the reaction sample.

[0017] The results showed that the S14P+K97A m value of AaH6H K was the highest, and the V max was the lowest, with the overall catalytic efficiency being the lowest. The catalytic activity of HnH6H H43D +Q247E+D324E was the highest. The H43D+Q247+D324E cat K values of HnH6H catalyzing the first-step reaction and the second-step reaction were 2.90 times and 1.97 times that of HnH6H respectively ( / K and Table 1). Figure 24 and Table 1).

[0018] Table 1 Enzyme kinetic constant determination table Example 3 Value of HnH6H mutants in plant metabolic engineering: A pair of primers with BamHI and SacI restriction sites were designed (BamHI-F-H6H: cgcGGATCCATGGCTACTTTTGTGTCGAACTG; SacI-R-H6H: cgcGAGCTCTTAGACATTGATTTTATATGGCT) to clone HnH6H and HnH6H H43D+Q247E+D324E The coding regions were connected to the plant binary expression plasmid pBI121 to obtain the engineered plasmid. The pBI121 original plasmid (control group) and the above-mentioned engineered plasmid were transformed into Agrobacterium rhizogenes C58C1 to obtain the engineered bacteria, and then the engineered bacteria were used to infect the leaves of Belladonna to obtain transgenic hairy roots. The specific method of hairy root construction is as follows: (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 culture medium (centrifuge tube or test tube), culture at 28 °C overnight for activation; then inoculate the bacterial liquid with good growth into 10 mL of new YEP liquid culture medium containing antibiotics and culture until OD600 is about 0.6-0.8.

[0019] (2) Place the Agrobacterium culture solution in a 50 mL EP tube under sterile conditions and centrifuge at 3000 rpm for 10 min at room temperature. 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 for 10 min at room temperature, remove the supernatant, and repeat at least twice.

[0020] (3) Resuspend the cells in the transformation solution to a final concentration of about 0.3 at OD600 and leave at room temperature for 30 min. Cut the leaves of sterile belladonna seedlings in good growth as explants and poke appropriate holes on the leaf surface with scissors or a scalpel. Add the explants to the Agrobacterium resuspension and soak for 5 min. 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 MS solid medium) for 2 days and then transferred to sterile medium (containing Cef200 mg.L -1 and Kan 100 mg.L -1 After about 15 days, new white hairy roots can be seen growing from the wounds of the leaves. After 30 days, the grown hairy roots can be cut off and inoculated on a new sterilized medium for growth. Subculture is performed once a month, and sterilization is generally completed after 2 rounds.

[0021] For each transgenic type, 15 independently transformed roots were randomly selected for liquid culture. The hairy roots of each monoclonal line were inoculated into a triangular flask containing 100 mL of MS liquid medium and cultured at 25 °C with 120 rpm in the dark for 28 days before harvesting. After freeze-drying the hairy roots, the contents of hyoscyamine, anisodamine, and scopolamine were analyzed by UPLC-MS.

[0022] The results are as Figure 25 shown. In the figure, CK represents the control line transformed with the original plasmid, OE-HnH6H represents the transgenic line overexpressing wild-type HnH6H, and OE-HnH6H 3M represents the transgenic line overexpressing HnH6H H43D+Q247E+D324E . Each group consists of 15 independent transformed lines, and ** represents an independent sample T-test p value less than or equal to 0.01. The results show that the contents of hyoscyamine in the HnH6H- and HnH6H H43D+Q247E+D324E overexpressing lines were significantly reduced, only 42.31% and 25.17% of the control group ( Figure 25 A). Overexpression of HnH6H significantly increased the yields of anisodamine and scopolamine in Atropa belladonna, which were 1.69 times and 4.8 times that of the control group ( Figure 25 B). The contents of anisodamine and scopolamine in the HnH6H H43D+Q247E+D324E overexpressing group were significantly higher than those in the HnH6H overexpressing group, which were 2.87 times and 10.81 times that of the control group ( Figure 25 C). In summary, the metabolic engineering effect of HnH6H H43D+Q247E+D324E is better than that of HnH6H.

[0023] The above embodiments are only used to illustrate the technical ideas and characteristics of the present invention, and do not represent the only or limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within the scope of protection of the present invention.

Claims

1. A hyoscyamine 6β-hydroxylase mutant, characterized in that, Compared with the wild-type hyoscyamine 6β-hydroxylase, the following mutations exist: the amino acid at position 43 is mutated from histidine to aspartic acid, and the amino acid sequence of the mutant is as shown in SEQ ID NO:

1.

2. A hyoscyamine 6β-hydroxylase mutant, characterized in that, Compared with the wild-type hyoscyamine 6β-hydroxylase, the following mutations exist: the amino acid at position 43 is mutated from histidine to aspartic acid, and the amino acid at position 247 is mutated from glutamine to glutamic acid, and the amino acid sequence of the mutant is as shown in SEQ ID NO:

2.

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

3.

4. A hyoscyamine 6β-hydroxylase mutant, characterized in that, Compared with the wild-type hyoscyamine 6β-hydroxylase, the following mutations exist: the amino acid at position 43 is mutated from histidine to aspartic acid, the amino acid at position 247 is mutated from glutamine to glutamic acid, and the amino acid at position 324 is mutated from aspartic acid to glutamic acid, and the amino acid sequence of the mutant is as shown in SEQ ID NO:

4.

5. A gene, characterized in that, It is used to encode the hyoscyamine 6β-hydroxylase mutant described in claim 1, and the nucleotide sequence of the gene is as shown in SEQ ID NO:

5.

6. A gene, characterized in that, It is used to encode the hyoscyamine 6β-hydroxylase mutant described in claim 2, and the nucleotide sequence of the gene is as shown in SEQ ID NO:

6.

7. A gene, characterized in that, It is used to encode the hyoscyamine 6β-hydroxylase mutant described in claim 3, and the nucleotide sequence of the gene is as shown in SEQ ID NO:

7.

8. A gene, characterized in that, It is used to encode the hyoscyamine 6β-hydroxylase mutant described in claim 4, and the nucleotide sequence of the gene is as shown in SEQ ID NO:

8.

9. Use of the tropane alkaloid 6β-hydroxylase mutant according to any one of claims 1 to 4, characterized in that, It is used to synthesize anisodamine / scopolamine in an Escherichia coli expression system.

10. Use of the hyoscyamine 6β-hydroxylase mutant according to any one of claims 1 to 4, characterized in that, It is used for metabolic engineering improvement of Atropa belladonna to synthesize anisodamine / scopolamine.

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