Robinine methyltransferase mutant as well as coding gene, vector, recombinant bacterium and application thereof

Abstract

The invention discloses a ormosine methyltransferase mutant as well as a coding gene, a vector, recombinant bacteria and application thereof, and belongs to the technical field of enzyme engineering. According to the invention, mutagenesis is carried out on variants EgtDM252V and E282A of methyltransferase EgtD from M. smegmais ATCC700084, Pro at the 34 site is mutated into Lys, Thr at the 213 site is mutated into Ser, Ser at the 284 site is mutated into Ala, the methylation catalytic efficiency with L-ormosin as a substrate is obviously improved, and the L-erythrinine is efficiently synthesized. On the basis that the L-erythrinine is synthesized by using E. coli whole cells, a further theoretical and technical basis is provided for establishing an industrial production process of the L-erythrinine, which is low in production cost, high in production intensity and small in environmental pollution.

Description

Technical Field

[0001] This invention relates to a red bean methyltransferase mutant, its encoding gene, vector, recombinant bacteria, and applications, belonging to the field of enzyme engineering technology. Background Technology

[0002] L-Hypaphorine (L-HYP) is a natural alkaloid found in certain plants such as *Caragana korshinskii* and *Pomacea canaliculata*. It is produced by iterative three-stage methylation of L-tryptophan as a substrate under the action of methyltransferases. It not only regulates plant growth processes—such as regulating fungal-plant symbiosis and increasing cytoplasmic calcium concentration to modulate cell signaling pathways—but also has applications in animal and human disease treatment, including alleviating dexamethasone-induced insulin resistance and acting as an acetylcholinesterase inhibitor for the treatment of Alzheimer's disease. These rich physiological functions and therapeutic potential highlight the important value of L-Hypaphorine in various fields. It is widely obtained through extraction from exogenous organisms or chemical synthesis. However, plant extraction yields are low, and chemical synthesis is highly polluting. Microbial synthesis, on the other hand, offers advantages such as low pollution, low cost, rapid reproduction, and the ability to alter substrate preference through site-directed mutagenesis of key enzymes. Therefore, developing a catalytic synthesis system and whole-cell transformation platform based on this enzyme holds significant promise for the efficient and green preparation of L-Hypaphorine.

[0003] Methyltransferases (MTases) are a class of enzymes that specifically catalyze the transfer of methyl groups from a methyl donor (mainly S-adenosylmethionine, SAM) to a specific atom (oxygen, nitrogen, carbon, etc.) in a substrate molecule. MSE is a SAM-dependent methyltransferase that transfers methyl groups to nitrogen atoms; it does so by targeting Mycobacterium smegma (…). Mycobacterium smegmatisThe engineered variant of the methyltransferase EgtD derived from [a specific enzyme name] was obtained through site-directed mutagenesis at sites M252V and E282A. The substitution of these two key residues significantly altered the enzyme's substrate specificity, transforming it from a native enzyme catalyzing histidine methylation into an engineered enzyme capable of efficiently catalyzing L-tryptophan aminotrimethylation, thereby generating the product L-ketoalkaloid. The reaction mechanism involves L-tryptophan binding to the enzyme's active site, which attacks the positively charged methylthionium ion in the SAM molecule via the nucleophilic center of the substrate molecule. MsE then sequentially transfers the methyl group from SAM to the amino site of L-tryptophan, completing three consecutive N-methylation reactions, ultimately generating the trimethylated product L-ketoalkaloid. However, this "single binding-continuous methylation" catalytic mode easily leads to the dissociation of the intermediate methylation product from the enzyme molecule, causing the methylation reaction to be interrupted. This results in a large accumulation of the monomethylation intermediate L-redoxaline in the reaction system (L-redoxaline, i.e., N,α-methyl-L-tryptophan, is a monomethyl product of L-Trp methylation by MSE). Wang et al. focused on the active pocket in EgtD... M252V,E282A Based on this, the Thr mutation at position 163 is changed to Gly, resulting in the mutant EgtD. T163G,M252V,E282A The conversion of erythrine to erythrine catalyzed by E. coli cells was achieved. However, the increased catalytic efficiency of this mutant was accompanied by a simultaneous increase in the accumulation of L-erythrine, indicating that the large accumulation of intermediate products may be a key bottleneck limiting the complete methylation reaction.

[0004] To further improve the dimethylation efficiency of the enzyme, this study aims to improve the dimethylation efficiency of EgtD. M252V,E282A By modifying the enzyme and screening for the optimal multiple mutant of the methyltransferase EgtD, the efficiency of this mutant in catalyzing the synthesis of L-erythrine from L-red alkaloids was improved. This mutant was then used in combination with existing methyltransferases that catalyze the conversion of L-tryptophan to L-erythrine, thereby achieving efficient in vitro synthesis of L-erythrine using whole microbial cells. Summary of the Invention

[0005] To address the problem in existing technologies where the intermediate methylation product dissociates from the enzyme molecule during L-tryptophan methylation, interrupting the methylation reaction and leading to the accumulation of the monomethylation intermediate L-caryophylline in the reaction system, this invention, for the first time, introduces the mutant EgtD... M252V,E282A The amino acid sequence of the mutant was modified by mutating Pro at position 34 to Lys, Thr at position 213 to Ser, and Ser at position 284 to Ala, and it was overexpressed in Escherichia coli BL21(DE3). The results showed that the EgtD mutant significantly improved the ability of L-ethynoline to be synthesized using L-ethynoline as a substrate.

[0006] This invention is achieved through the following technical solution: The first objective of this invention is to provide a daidzein methyltransferase mutant, wherein the daidzein methyltransferase mutant is formed by mutating the following amino acid sequences as shown in SEQ ID NO.1: proline (Pro) at position 34 to lysine (Lys), threonine (Thr) at position 213 to serine (Ser), methionine (Met) at position 252 to valine (Val), glutamic acid (Glu) at position 282 to alanine (Ala), and serine (Ser) at position 284 to alanine (Ala).

[0007] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the parental sequence is shown in SEQ ID NO. 2.

[0008] A second objective of this invention is to provide a gene encoding the red bean methyltransferase mutant.

[0009] A third objective of this invention is to provide an expression vector carrying the encoded gene.

[0010] In one embodiment of the present invention, the expression vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, or mammalian cell virus.

[0011] A fourth objective of this invention is to provide a recombinant bacterium expressing the said red bean methyltransferase mutant.

[0012] In one embodiment of the present invention, the recombinant bacteria uses bacteria, fungi, plants, insects or animal cells as host cells.

[0013] The fifth objective of this invention is to provide the application of the said erythrine methyltransferase mutant in the biocatalytic synthesis of L-erythrine.

[0014] In one embodiment of the present invention, the application uses L-erythrine as a substrate and the erythrine methyltransferase mutant as a catalyst to catalyze the synthesis of L-erythrine.

[0015] In one embodiment of the present invention, the application is to synthesize L-ketoalkaloid using L-tryptophan as a substrate by co-catalysis of the red bean methyltransferase mutant and the methyltransferase that catalyzes L-tryptophan-L-ketoalkaloid.

[0016] In one embodiment of the present invention, the red bean methyltransferase mutant and the methyltransferase catalyzing L-tryptophan L-erythrine are catalyzed by a fusion protein.

[0017] A sixth object of the present invention is to provide an enzyme preparation comprising the said red bean methyltransferase mutant.

[0018] In one embodiment of the present invention, the enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

[0019] The beneficial effects of this invention are: This invention, through the analysis of materials from... M. smegmatis The EgtD variant of the methyltransferase EgtD of ATCC700084 M252V,E282A Mutations were performed, mutating Pro at position 34 to Lys, Thr at position 213 to Ser, and Ser at position 284 to Ala, significantly improving the methylation catalytic efficiency using L-erythrine as a substrate, thus enabling the efficient synthesis of L-erythrine. This has already been achieved using... E. coli Based on the whole-cell synthesis of L-erythrin, this study provides a further theoretical and technical foundation for establishing an industrialized L-erythrin production process with low production costs, high production intensity, and minimal environmental pollution. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 For mutants in BL21 (DE3) The expression in the text; lane description: Lane M is the protein molecular weight standard marker; lane 1 is... BL21 (DE3) pET- ectD M252V,E282A Lane 2 is BL21 (DE3) pET- ectD T163G,M252V,E282A Lane 3 is BL21 (DE3) pET- ectD P34K,T213S,S284A,M252V,E282A ; Figure 2 For EgtD M252V,E282A The relative enzyme activity of the saturated mutant at amino acid residue 34.

[0022] Figure 3 For EgtD P34K,M252V,E282A The relative enzyme activity of the saturated mutant at amino acid residue 213.

[0023] Figure 4 For EgtD P34K,M252V,E282A The relative enzyme activity of the saturated mutant at amino acid residue 284.

[0024] Figure 5 For EgtDM252V,E282A The relative enzyme activity was determined by combining mutations of Pro at position 34 to Lys, Thr at position 213 to Ser, and Ser at position 284 to Ala.

[0025] Figure 6 For EgtD T163G,M252V,E282A With EgtD P34K,T213S,S284A,M252V,E282A Enzyme activity and L-etholin yield using erythrine as a substrate.

[0026] Figure 7 For EgtD T163G,M252V,E282A With EgtD P34K,T213S,S284A,M252V,E282A Structural analysis. Detailed Implementation

[0027] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] In this invention, the initiating bacterium is Escherichia coli. BL21(DE3) It is wild-type Escherichia coli; Qualitative and quantitative analysis of substrates and products, and monitoring of bacterial growth: Determination of bacterial concentration: Sample bacterial suspension was taken and diluted with distilled water at a certain ratio, using distilled water as a blank control. OD was measured using a spectrophotometer at 600 nm and 1 cm path length. 600 The contents of L-caryophylline and L-ethionine were determined by high-performance liquid chromatography (HPLC) with reference to a standard curve. The crude enzyme solution was purified by affinity chromatography based on the His tag on pET-28a. Table 1 Primer sequences required for PCR amplification

[0029] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0030] Example 1: ectD Site-directed saturation mutation at amino acid residue 38 of the protein EgtD To express plasmid pET- ectD M252V,E282AUsing Pro34X-F / Pro34X-R (Note: X represents the other 19 amino acids besides Pro; primer sequences are shown in Table 1) as primers for PCR amplification, and using... Dpn The plasmid template was removed using digestive enzymes. The PCR product was then purified and transformed into *E. coli* BL21(DE3) competent cells for plating and culture. Target recombinant strains were screened using test primers (i.e., EgtD-F / EgtD-R; primer sequences are shown in Table 1) and sequenced to obtain the target recombinant plasmid pET- ectD P34X,M252V,E282A and target recombinant strain E. coli / pET- ectD P34X,M252V,E282A .

[0031] Example 2: ectD Iterative saturation mutations and combinatorial mutations at amino acid residues 213 and 284 encoding protein EgtD To express plasmid pET- ectD P34K,M252V,E282A Using Thr213X-F / Thr213X-R (Note: X represents the other 19 amino acids besides Thr; primer sequences are shown in Table 1) as primers for PCR amplification, and using... Dpn The plasmid template was removed using digestive enzymes. The PCR product was then purified and transformed into *E. coli* BL21(DE3) competent cells for plating and culture. Target recombinant strains were screened using test primers and sequenced to obtain the target recombinant plasmid pET- ectD P34K,T213X,M252V,E282A and target recombinant strain E. coli / pET- ectD P34K,T213X,M252V,E282A Similarly, using the expression plasmid pET- ectD P34K,T213S,M252V,E282A Using Ser284X-F / Ser284X-R (Note: X represents the other 19 amino acids besides Ser; primer sequences are shown in Table 1) as primers, PCR amplification was performed. The target recombinant plasmid pET- was obtained. ectD P34K,M252V,E28-2A,S284X and target recombinant strain E. coli / pET- ectD P34K,M252V,E282A,S284X Similarly, using the expression plasmid pET- ectD P34K,T213S,M252V,E282A Using Ser284Ala-F / Ser284Ala-R as primers, PCR amplification was performed to obtain the target recombinant plasmid pET- ectD P34K,T213S,M252V,E282A,S284A and target recombinant strain E. coli / pET- ectD P34K,T213S,M252V,E282A,S284A .

[0032] Example 3: Plasmid was transformed into E. coli BL21(DE3) for expression. The recombinant expression plasmid mutant was transformed into *E. coli* BL21(DE3), and the recombinant expression strain was screened by culturing on LB+Kan solid medium at 37℃. The starting strain and the recombinant strain were inoculated into liquid TB medium, and after IPTG induction, the bacterial cells were collected, sonicated, and the supernatant and precipitate were subjected to SDS-PAGE electrophoresis. The results showed that the protein levels of the mutant and... ectD T163G,M252V,E282A Comparability Figure 1 The protein size is 36 kDa, consistent with the reported target protein size, indicating that the change in catalytic ability is not caused by changes in protein expression.

[0033] Example 4: Enzyme activity assay of wild-type EgtD and EgtD mutant target recombinant strain E. coli / pET- ectD T163G,M252V,E282A and E. coli / pET- ectD P34K,T213S,M252V,E282A,S284A Incubate in 10 mL LB broth vials for 9–10 h, then transfer 1 mL to TB medium and incubate at 37°C until OD (open-circuit retardation). 600 IPTG was added to a final concentration of 0.1 mmol / L between 0.5 and 0.6, and expression was induced at 16°C for 24 h. After expression, the bacterial cells were collected, washed twice with PBS buffer, and the cells were resuspended and kept at the same OD value. 600 Then, the bacterial cells were broken up using an ultrasonic disruptor, and the supernatant was collected by centrifugation to obtain crude enzyme solution.

[0034] Enzyme activity assay reaction system: 200 µL of reaction solution contained 50 mM Tris·HCl (pH 8), 50 mM NaCl, 100 μM MnBr2, 150 μM SAM, 10 μM S-adenosylhomocysteine ​​hydrolase (SAHH), 50 μM L-tryptophan, and 100 μL crude enzyme solution. The reaction was carried out at 35℃ and monitored at 265 nm for 10 min. Enzyme activity unit (U) is defined as the amount of enzyme required to consume 1 µmol of L-tryptophan per minute under the above reaction conditions. EgtD M252V,E282A The relative activity of different mutants, such as Figure 2 As shown, EgtD M252V,E282A The catalytic activity for L-HYP formation was set to 100%. Figure 2 EgtD was found in M252V,E282AA mutation at position 34 (Pro) to Lys can enhance the catalytic ability to synthesize L-erythrine from L-redoybene. Using the same method, such as... Figure 3-5 As shown, in the iterative saturation mutation, when Thr at position 213 mutates into Ser and Ser at position 284 mutates into Ala, the final mutant EgtD is obtained. P34K,T213S,M252V,E282A,S284A It can significantly enhance the ability of catalyzing the synthesis of L-erythrine from L-red alkaloids.

[0035] Example 5: Effects of different EgtD mutants on L-etholin synthesis Enzyme conversion system: 200 µL of reaction solution contained 50 mM Tris·HCl (pH=8), 1.5 mM SAM, 1.5 mM S-adenosylhomocysteine ​​hydrolase (SAHH) 1.5 mM, 500 mM L-erythrine, and 100 μL of crude enzyme solution. The reaction was carried out at 35℃ for 2 h. After the reaction, the L-erythrine content was determined by high performance liquid chromatography (HPLC), and the results are as follows. Figure 6 As shown. From Figure 6 It can be seen that the yield of L-erythrine is consistent with the enzyme activity level, meaning that the EgtD mutant with high enzyme activity also exhibits high L-erythrine yield. In summary, the EgtD mutant... P34K,T213S,M252V,E282A,S284A It exhibited the highest enzyme activity (35.02±0.4 U / mL) and L-erythrine production (64.04±0.12 mg / L).

[0036] Example 6: Purification and Kinetic Parameter Analysis of Wild-Type EgtD and EgtD Mutants The crude enzyme solution obtained from the above mutant enzyme expression was purified using affinity chromatography. L-caryophylline solutions with concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, and 3.0 mg / mL were prepared, and 1 mg of the purified enzyme was added to 100 mmol / L SAM. Using the Lineweaver-Burk double reciprocal method, the Michaelis constants of EgtD and its mutants with L-caryophylline as the substrate could be calculated. K m ), maximum reaction rate ( V max ) and catalytic constant ( k cat The results showed that the mutant EgtD M252V,E282A In the amino acid sequence, mutating Pro at position 34 to Lys, Thr at position 213 to Ser, and Ser at position 284 to Ala effectively enhances the ability of methyltransferase to catalyze the synthesis of L-ethynine from L-caryophylline, as shown in Table 2. Based on these results, positions 34, 213, and 284 are EgtD... M252V,E282AThe docking with L-caryophylline at important sites within a 4 Å range can significantly affect the conformation of the substrate active pocket. Therefore, it is possible to mutate these sites into amino acids with other structures, which may alter the enzyme cavity structure and have a significant impact on improving the catalytic efficiency of enzymes using L-caryophylline as a substrate.

[0037] Table 2 Kinetic parameters of transmethylases and their mutants in the SAM transmethylation reaction.

[0038] In summary, this invention, through the analysis of materials from... M. smegmatis The EgtD variant of the methyltransferase EgtD of ATCC700084 M252V,E282A Based on this, the 34-position Pro was mutated to Lys, the 213-position Thr was mutated to Ser, and the 284-position Ser was mutated to Ala. Overexpression was performed in *E. coli* BL21(DE3), and the results showed that the mutant EgtD... P34K,T213S,M252V,E282A,S284A It significantly improved the catalytic efficiency of methylation using L-red alkaloid as a substrate, enabling the efficient synthesis of L-erythrine.

[0039] The sequences used in this invention are as follows: Parental sequence of methyltransferase SEQ ID NO.1: MTLSLANYLAADSAAAEALRRDVRAGLTAAPKSLPPKWFYDAVGSDLFDQITRLPEYYPTRTEAQILRTRSAEIIAAAGADTLVELGSGTSEKTRMLLDAMRDAELLRRFIPFDVDAGVLRSAGAAIGAEYPGIEIDAVCGDFEEHLGKIPHVGRRLVVFL GSTIGNLTPAPRAEFLSTLADTLQPGDSLLLGTDLVKDTGRLVRAYDDAAGVTAAFNRNVLAVVNRELSADFDLDAFEHVAKWNSDEERIEMWLRARTAQHVRVAALDLEVDFAAGEEMLTEVSCCKFRPENVVAELAEAGLRQTHWWTDPAGDFGLSLAVR The parental sequence of the methyltransferase encodes the gene sequence SEQ ID NO.2: ATGACCCTGAGCCTGGCGAATTACCTGGCGGCGGATAGCGCGGCGGAAGCGTTACGTCGCGATGTTCGCGCGGGCCTGACCGCGGCGCCGAAAAGCCTGCCGCCGAAATGGTTTTATGATGCGGTGGGCAGCGATCTGTTTGATCAGATTACCCGCCTGCCGGAATATTATCCGACCCGCACCGAAGCGCAGATTCTGCGCACCCGCAGCGCGGAAATTATTGCGGCCGCGGGCGCGGACACCCTGGTGGAACTGGGCAGCGGCACGAGCGAAAAAACCCGCATGCTGCTGGATGCGATGCGCGATGCGGAACTGCTGCGCCGCTTTATTCCGTTTGATGTGGATGCGGGCGTGCTGCGCAGCGCGGGCGCGGCGATTGGCGCGGAATATCCGGGCATTGAAATTGATGCGGTGTGCGGCGATTTTGAAGAACATCTGGGCAAAATTCCGCATGTGGGCCGCCGCCTGGTGGTGTTTCTGGGCAGCACCATTGGCAACCTGACCCCGGCGCCGCGCGCGGAATTTCTGAGCACCCTGGCGGATACCCTGCAGCCGGGCGATAGCCTGTTACTGGGCACCGATCTGGTGAAAGATACCGGCCGCCTGGTGCGCGCGTATGATGATGCGGCGGGCGTGACCGCGGCGTTTAACCGCAACGTGCTGGCGGTGGTGAACCGCGAACTGAGCGCGGATTTTGATCTGGATGCGTTTGAACATGTGGCGAAATGGAACAGCGATGAAGAACGCATTGAAATGTGGCTGCGCGCGCGCACCGCGCAGCATGTGCGCGTGGCGGCGCTGGATCTGGAAGTGGATTTTGCGGCGGGCGAAGAAATGCTGACCGAAGTGAGCTGCAAATTTCGCCCGGAAAACGTGGTGGCGGAACTGGCGGAAGCGGGCCTGCGTCAGACCCATTGGTGGACCGATCCGGCGGGCGATTTTGGCCTGAGCTTAGCGGTGCGCTAA The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A red bean methyltransferase mutant, characterized in that, The aforementioned red bean methyltransferase mutant is obtained by mutating proline at position 34 to lysine, threonine at position 213 to serine, methionine at position 252 to valine, glutamic acid at position 282 to alanine, and serine at position 284 to alanine in the parental amino acid sequence shown in SEQ ID NO.

1.

2. The encoding gene of the red bean methyltransferase mutant according to claim 1.

3. An expression vector carrying the encoding gene of claim 2.

4. The expression vector according to claim 3, characterized in that, The expression vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, or mammalian cell virus.

5. A recombinant bacterium expressing the red bean methyltransferase mutant of claim 1.

6. The recombinant bacteria according to claim 5, characterized in that, The recombinant bacteria use bacteria, fungi, plants, insects, or animal cells as host cells.

7. The application of the red bean methyltransferase mutant of claim 1 in the biocatalytic synthesis of L-erythrine.

8. The application according to claim 7, characterized in that, The application uses L-erythrine as a substrate and the erythrine methyltransferase mutant as a catalyst to catalyze the synthesis of L-erythrine; or, The application involves the synthesis of L-ketoalkaloid using L-tryptophan as a substrate, through the co-catalysis of the aforementioned red bean methyltransferase mutant and a methyltransferase that catalyzes L-tryptophan-L-ketoalkaloid.

9. An enzyme preparation comprising the red bean methyltransferase mutant of claim 1.

10. The enzyme preparation according to claim 9, characterized in that, The enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

Images