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

Abstract

The invention discloses a 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, variants EgtDT163G, M252V and E282A of methyltransferase EgtD from M. smegmais ATCC700084 are mutated, Pro at the 35 site is mutated into Cys, Lys at the 36 site is mutated into Arg, Phe at the 38 site is mutated into Leu, Thr at the 168 site is mutated into Phe, the methylation catalytic efficiency with L-tryptophan 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 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), with the molecular formula C0... 14 H 18 N2O2, also known as trimethyl betaine or yungan alkaloid, is a natural small-molecule indole alkaloid found in plants of the genus *Erythrina*. It is produced by iterative three-stage methylation of L-tryptophan as a substrate under the action of methyltransferases. L-Erythrina alkaloid is slightly soluble in organic solvents such as methanol, ethanol, and DMSO, and has a solubility of 5 mg / mL in PBS solution at pH 7.2. Due to its unique indole ring and trimethyl substituent in its molecular structure, L-Erythrina alkaloid exhibits diverse physiological activities. It can regulate plant growth processes—such as regulating fungal-plant symbiosis and increasing cytoplasmic calcium concentration to regulate cell signaling pathways—and can also be used to treat diseases in animals and humans, including alleviating dexamethasone-induced insulin resistance and acting as an acetylcholinesterase inhibitor for the treatment of Alzheimer's disease. Most importantly, recent studies have identified L-Erythrina alkaloid as a novel antiviral compound against dengue virus. These rich physiological functions and therapeutic potential highlight the important value of L-Erythrina alkaloid in various fields. It is widely obtained through extraction from exogenous organisms or chemical synthesis. However, plant extraction yields are low, and chemical synthesis results in significant pollution. 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 erythrine.

[0003] The directed synthesis of L-erythrine from L-tryptophan requires the iterative catalysis of three transmethylation reactions by an S-adenosyl-L-methionine (SAM)-dependent methyltransferase, where SAM is the methyl donor and L-tryptophan and its derivatives are methyl acceptors. Therefore, to achieve the synthesis of L-erythrine from L-tryptophan, screening or constructing methyltransferases that specifically catalyze the methylation of L-tryptophan is crucial. Studies have reported that... Mycobacterium smegmatis The methyltransferase EgtD of ATCC700084 synthesizes ergothionein via iterative methylation using L-histidine as a substrate. It is worth noting that... M. smegmatisSite-directed mutations at amino acid positions 252 and 282 of the EgtD amino acid sequence in ATCC700084 to Val and Ala, respectively, allow it to synthesize L-erythrine via iterative methylation using L-tryptophan as a substrate. However, EgtD carrying mutations at positions 252 and 282 (i.e., EgtD: EgtD) M252V,E282A Although L-erythrine can be synthesized using L-tryptophan as a substrate, its catalytic efficiency is too low to meet industrial application requirements. 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 It can still achieve the basic conversion of erythrine to be catalyzed by Escherichia coli cells, but the conversion rate is still at a low level. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention improves upon EgtD. T163G,M252V,E282A Further modifications were made to improve the efficiency of this mutant in catalyzing the synthesis of L-erythrin from L-tryptophan, thereby achieving efficient in vitro synthesis of L-erythrin using whole microbial cells. This invention utilizes the mutant EgtD... T163G,M252V,E282A The amino acid sequence of the mutant was modified by mutating proline (Pro) at position 35 to cysteine ​​(Cys), lysine (Lys) at position 36 to arginine (Arg), phenylalanine (Phe) at position 38 to leucine (Leu), and threonine (Thr) at position 168 to phenylalanine (Phe), and it was overexpressed in *Escherichia coli* BL21(DE3). The results showed that the EgtD mutant significantly enhanced the ability to catalyze the synthesis of L-erythrine using L-tryptophan as a substrate.

[0005] This invention is achieved through the following technical solution: The first objective of this invention is to provide a methyltransferase mutant, wherein the methyltransferase mutant is formed by mutating the following amino acid sequences as shown in SEQ ID NO.1: proline (Pro) at position 35 to cysteine ​​(Cys), lysine (Lys) at position 36 to arginine (Arg), phenylalanine (Phe) at position 38 to leucine (Leu), threonine (Thr) at position 168 to phenylalanine (Phe), threonine (Thr) at position 163 to glycine (Gly), methionine (Met) at position 252 to valine (Val), and glutamic acid (Glu) at position 282 to alanine (Ala).

[0006] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the methyltransferase parental sequence is shown in SEQ ID NO.2.

[0007] A second objective of this invention is to provide a gene encoding the methyltransferase mutant.

[0008] A third objective of this invention is to provide an expression vector carrying the encoded gene.

[0009] In one embodiment of the present invention, the expression vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, or mammalian cell virus.

[0010] A fourth objective of this invention is to provide a recombinant bacterium expressing the methyltransferase mutant.

[0011] In one embodiment of the present invention, the recombinant bacteria uses bacteria, fungi, plants, insects or animal cells as host cells.

[0012] A fifth objective of this invention is to provide the use of the methyltransferase mutant or the recombinant bacteria in the catalytic synthesis of L-erythrine from L-tryptophan or its derivatives.

[0013] In one embodiment of the present invention, the application uses S-adenosyl-L-methionine as a methyl donor, L-tryptophan or its derivative as a methyl acceptor, and the methyltransferase mutant or the recombinant bacteria as a catalyst to catalyze the synthesis of L-erythrine.

[0014] A sixth object of the present invention is to provide an enzyme preparation comprising the methyltransferase mutant described above.

[0015] In one embodiment of the present invention, the enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

[0016] 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 T163G,M252V,E282A Mutations were performed to mutate Pro at position 35 to Cys, Lys at position 36 to Arg, Phe at position 38 to Leu, and Thr at position 168 to Phe, significantly improving the methylation catalytic efficiency using L-tryptophan as a substrate and enabling the efficient synthesis of L-erythrine. This has already been achieved in the utilization of... 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

[0017] 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.

[0018] 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,E284A Lane 2 is BL21 (DE3) pET- ectD T163G,M252V,E284A Lane 3 is BL21 (DE3) pET- ectD P35C,K36R,F38L,T163G,T168F,M252V,E284A ; Figure 2 For EgtD T163G,M252V,E282A Based on the relative enzyme activity of the saturated mutant at amino acid residue 38, EgtD T163G,M252V,E282A The activity for catalyzing the production of L-erythrine from L-tryptophan was set at 100%.

[0019] Figure 3 For EgtD F38L,T163G,M252V,E282A Based on the relative enzyme activity of the saturated mutant at amino acid residue 35, EgtD F38L,T163G,M-252V,E282A The activity for catalyzing the production of L-erythrine from L-tryptophan was set at 100%.

[0020] Figure 4 For EgtD P35C,F38L,T163G,M252V,E282A Based on the relative enzyme activity of the saturated mutant at amino acid residue 36, EgtD P35C,F38L,T163G,M252V,E282A The activity for catalyzing the production of L-erythrine from L-tryptophan was set at 100%.

[0021] Figure 5 For EgtD P35C,K36R,F38L,T163G,M252V,E282A Based on the relative enzyme activity of the saturated mutant at amino acid residue 168, EgtD P35C,K36R,F38L,T163G,M252V,E282A The catalytic conversion of L-tryptophan to L-erythrine was set at 100%.

[0022] Figure 6 For EgtD T163G,M252V,E282A With EgtD P35C,K36R,F38L,T163G,T168F，M252V,E282A Enzyme activity and L-erythrine yield using L-tryptophan as a substrate.

[0023] Figure 7 For EgtD T163G,M252V,E282A With EgtDP35C,K36R,F38L,T163G,T168F，M252V,E282A Three-dimensional structure diagram. Detailed Implementation

[0024] 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.

[0025] In this invention, the starting bacterium is Escherichia coli BL21(DE3), which is a 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-tryptophan 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 according to the His tag on pET-28a.

[0026] Table 1 Primer sequences required for PCR amplification

[0027] 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.

[0028] Example 1: ectD Site-directed saturation mutation at amino acid residue 38 of the protein EgtD To express plasmid pET- ectD T163G,M252V,E282A Using Phe38X-F / Phe38X-R (Note: X represents the other 19 amino acids besides Phe; 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 F38X,T163G,M252V,E282Aand target recombinant strain E. coli / pET- ectD F38X,T163G,M252V,E282A .

[0029] Example 2: ectD Iterative saturation mutations at amino acid residues 35, 36, and 168 of the protein EgtD. To express plasmid pET- ectD F38L,T163G,M252V,E282A Using Phe38Leu,Pro35X-F / Phe38Leu,Pro35X-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 and sequenced to obtain the target recombinant plasmid pET- ectD P35X,F38L,T163G,M252V,E282A and target recombinant strain E. coli / pET- ectD P35X, F38L,T163G,M252V,E282A Similarly, using the expression plasmid pET- ectD P35C,F38L,T163G,M252V,E282A Using Phe38Leu,Pro35Cys,Lys36X-F / Phe38Leu,Pro35Cys,Lys36X-R (Note: X represents the other 19 amino acids besides Lys; primer sequences are shown in Table 1) as primers, PCR amplification was performed. The target recombinant plasmid pET- was obtained. ectD P35C,K36X,F38L,T163G,M252V,E282A and target recombinant strain E. coli / pET- ectD P35C,K36X,F38L,T163G,M252V,E282A To express the pET- plasmid ectD P35C,K36R,F38L,T163G,M252V,E282A Using Thr168X-F / Thr168-R as a template, PCR amplification was performed using primers (Note: X represents the other 19 amino acids besides Thr; primer sequences are shown in Table 1). The target recombinant plasmid pET- was obtained. ectD P35C,K36R,F38L,T163G,T168X,M252V,E282A and target recombinant strain E. coli / pET- ectD P35C,K36R,F38L,T163G,T168X,M252V,E282A .

[0030] 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 crude enzyme solution was subjected to SDS-PAGE electrophoresis. The results showed that the protein levels of the mutant in the expression system 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.

[0031] 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 P35C,K36R,F38L,T163G,T168F,M252V,E282A 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.

[0032] 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 T163G,M252V,E282A The relative activity of different mutants, such as Figure 2 As shown, EgtD T163G,M252V,E282A The catalytic activity for L-HYP formation was set to 100%. Figure 2 EgtD was found in T163G,M252V,E282A The Phe mutation at position 38, replacing it with Leu, significantly enhances the catalytic ability to synthesize L-erythrine from L-tryptophan. Using the same method, such as... Figure 3-5As shown, in the iterative saturation mutation, when Pro at position 35 mutates to Cys, Lys at position 36 mutates to Arg, and Thr at position 168 mutates to Phe, the final mutant EgtD is obtained. P35C,K36R,F38L,T163G,T168F,M252V,E282A It can significantly enhance the ability to catalyze the synthesis of L-erythrine from L-tryptophan.

[0033] 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-tryptophan, 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... P35C,K36R,F38L,T163G,T168F,M252V,E282A It exhibited the highest enzyme activity (45.23±0.6 U / mL) and L-erythrine production (71.02±0.22 mg / L).

[0034] 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-tryptophan 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. The Michaelis constants of EgtD and its mutants were calculated using the Lineweaver-Burk double reciprocal method with L-tryptophan as the substrate. K m ), maximum reaction rate ( V max ) and catalytic constant ( k cat The results showed that the mutant EgtD T163G,M252V,E282A Mutations at positions 35 (Pro) to Cys, 36 (Lys) to Arg, 38 (Phe) to Leu, and 168 (Thr) to Phe in the amino acid sequence can effectively enhance the ability of methyltransferase to catalyze the synthesis of L-erythrine from L-tryptophan, as shown in Table 2. Based on these results, positions 35, 36, 38, and 168 are EgtD... T163G / M252V / E282ASites on important loops near the active pocket 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 change the enzyme cavity structure and have a great impact on improving the catalytic efficiency of enzymes using L-tryptophan as a substrate.

[0035] Table 2 Kinetic parameters of transmethylases and their mutants in the SAM transmethylation reaction.

[0036] In summary, this invention, through the analysis of materials from... M. smegmatis The EgtD variant of the methyltransferase EgtD of ATCC700084 T163G,M252V,E282A Mutations were performed, with Pro at position 35 mutated to Cys, Lys at position 36 mutated to Arg, Phe at position 38 mutated to Leu, and Thr at position 168 mutated to Phe. These mutations were then overexpressed in *E. coli* BL21(DE3), and the results showed that the mutant EgtD… P35C,K36R,F38L,T163G,T168F,M252V,E282A It significantly improved the catalytic efficiency of methylation using L-tryptophan as a substrate, enabling the efficient synthesis of L-erythrine.

[0037] The sequence used in the examples is 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 methyltransferase mutant, characterized in that, The methyltransferase mutant is formed by mutating the following amino acid sequences as shown in SEQ ID NO.1: proline at position 35 to cysteine, lysine at position 36 to arginine, phenylalanine at position 38 to leucine, threonine at position 163 to glycine, threonine at position 168 to phenylalanine, methionine at position 252 to valine, and glutamic acid at position 282 to alanine.

2. The encoding gene of the methyltransferase mutant of 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 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 use of the methyltransferase mutant of claim 1 or the recombinant bacteria of claim 5 or 6 in the catalytic synthesis of L-erythrine from L-tryptophan or its derivatives.

8. The application according to claim 7, characterized in that, The application uses S-adenosyl-L-methionine as a methyl donor, L-tryptophan or its derivative as a methyl acceptor, and the methyltransferase mutant or the recombinant bacteria as a catalyst to catalyze the synthesis of L-erythrine.

9. An enzyme preparation comprising the 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.

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