Methyltransferase mutant and its encoding gene, vector, recombinant bacteria and application

Abstract

This invention relates to a methyltransferase mutant targeting L-tryptophan methylation, its encoding gene, vector, recombinant strain, and applications. This invention involves mutating a methyltransferase from *Mycolicibacterium smegmatis* ATCC700084, changing Thr at position 163 to Gly, Met at position 252 to Val, and Glu at position 282 to Ala. This enables the synthesis of L-ephedrine from L-tryptophan via a three-stage iterative methylation process, potentially enabling the synthesis of L-ephedrine using whole-cell *E. coli*. This provides a theoretical and technical basis for establishing an industrial-scale L-ephedrine production process with low production costs, high production intensity, and minimal environmental pollution.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a methyltransferase mutant and its encoding gene, vector, recombinant bacteria and application. Background Art

[0002] L-Hypaphorine (L-HYP), molecular formula is C 14 H 18 N2O2 is a natural small molecule indole alkaloid found in Erythrina plants, also known as trimethyl betaine and yunganding. It is produced by iterative three methylation under the action of methyltransferase with L-tryptophan as the substrate. L-erythrina alkaloid is slightly soluble in organic solvents such as methanol, ethanol, and DMSO, and its solubility is 5 mg / mL in PBS solution with a pH of 7.2. Studies have found that L-erythrina alkaloid has many medicinal values. It can act on the nervous system of rodents and lower blood sugar. It has potential significance for cancer treatment by affecting the organization of actin and inhibiting the action of indole-3-acetic acid. Secondly, it can significantly prolong the non-rapid eye movement sleep time and promote the sleep of mice. In addition, it also affects the central nervous system and cognitive function, can inhibit the activity of acetylcholinesterase (AchE) in the cerebellum, and no toxic effects on normal human cells have been observed. It can be used to treat Alzheimer's disease. [3] Given the medical effects of L-erythrina alkaloids in promoting sleep, reducing inflammation and affecting cognition, L-erythrina alkaloids have great research potential in the clinical application and formulation development of the treatment of diseases such as insomnia, cancer and Alzheimer's disease.

[0003] The process of L-erythrine directed from L-tryptophan requires the use of S-adenosyl-L-methionine (SAM)-dependent methyltransferases to iteratively catalyze three transmethylation reactions, in which SAM is the methyl donor and L-tryptophan and its derivatives are the methyl acceptors. Therefore, in order to achieve the synthesis of L-erythrine using L-tryptophan as a substrate, it is crucial to screen or construct a methyltransferase that can specifically catalyze the methylation of L-tryptophan. The research group of Alastair Murchie and Chen Dongrong used the artificial screening SELEX technology to screen a ribozyme with SAM as a methyl donor, which can catalyze the methylation of the 7-position N atom of guanine in a specific sequence. In addition, Blei et al. reported the function and phylogenetic characteristics of the SAM-dependent methyltransferase TrpM of Psilocybe ocybe, which catalyzes the formation of tertiary amines through iterative N-methylation of L-tryptophan, which is of great significance in the synthesis of L-erythrine. A study reported that the methyltransferase EgtD from MycolicibacteriumsmegmatisATCC700084 uses L-histidine as a substrate for iterative methylation to synthesize ergothioneine. It is worth noting that the site-directed mutation of the 252nd and 282nd amino acids in the EgtD amino acid sequence from M.smegmatisATCC700084 to Val and Ala can enable it to use L-tryptophan as a substrate for iterative methylation to synthesize L-erythrine. However, EgtD carrying the 252nd and 282nd amino acid mutations (i.e., EgtD M252V,E282A ) Although L-tryptophan can be used as a substrate to synthesize L-erythrine, its catalytic efficiency is too low to meet industrial applications.

[0004] At present, there are three production methods of L-erythrine: processing method, chemical synthesis method and plant extraction method, among which plant extraction method is the main one. However, plant extraction method has problems such as cumbersome extraction steps, low production efficiency and large environmental pollution. In view of the advantages of microbial pollution, low cost, fast reproduction and the ability to change the substrate preference of key enzymes by site-directed mutagenesis, this study aims to screen the best methyltransferase EgtD mutant and improve the efficiency of the mutant in catalyzing the synthesis of L-erythrine from L-tryptophan, so as to realize the in vitro synthesis of L-erythrine using whole microbial cells. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a methyltransferase mutant, characterized in that the methyltransferase mutant is EgtD M252V,E282A or EgtD T163a,M252V,E282A , the EgtD M252V,E282A The EgtD parent sequence shown in SEQ ID NO.1 is mutated from Met at position 252 to Val and from Glu at position 282 to Ala; T163a,M252V,E282A The method comprises mutating Thr at position 163 into a, Met at position 252 into Val, and Glu at position 282 into Ala of the EgtD parent sequence as shown in SEQ ID NO.1; wherein a is Ala or Val or Leu or Ile or Gly or Pro or Ser or Cys or Asp.

[0008] As a preferred embodiment of the methyltransferase mutant of the present invention, wherein: the EgtD M252V,E282A The sequence is shown in SEQ ID NO.2, the EgtD T163a,M252V,E282A The sequences are shown as SEQ ID NO.3 to SEQ ID NO.11 respectively.

[0009] Another object of the present invention is to overcome the deficiencies in the prior art and provide a gene encoding the methyltransferase mutant.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide an expression vector carrying the encoding gene.

[0011] As a preferred embodiment of the expression vector of the present invention, the expression vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide a recombinant bacterium expressing the methyltransferase mutant.

[0013] As a preferred embodiment of the recombinant bacteria of the present invention, the recombinant bacteria uses bacteria, fungi, plants, insects or animal cells as host cells.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of the methyltransferase mutant in catalyzing the synthesis of L-erythrine using L-tryptophan as a substrate.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and provide an enzyme preparation comprising the methyltransferase mutant.

[0016] As a preferred embodiment of the enzyme preparation of the present invention, the enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

[0017] Beneficial effects of the present invention:

[0018] The present invention mutates the methyltransferase EgtD from M. smegmatis ATCC700084, mutates Met at position 252 to Val and Glu at position 282 to Ala, so that the methyltransferase EgtD can use L-tryptophan as a substrate, significantly improves the catalytic efficiency, and efficiently synthesizes L-erythrinaine. This may be expected to realize the synthesis of L-erythrinaine using whole E. coli cells, and provides a theoretical and technical basis for establishing an industrialized L-erythrinaine production process with low production cost, high production intensity and low environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0020] Figure 1 The figure shows the expression of egtD in BL21(DE3).

[0021] Figure 2 The enzyme activity and L-erythrine yield of the 163 amino acid residue saturation mutant.

[0022] Figure 3 Structural analysis of EgtD methyltransferase and its mutants. DETAILED DESCRIPTION

[0023] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0024] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0025] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0026] In the present invention, the starting bacteria is Escherichia coli BL21 (DE3) which is a wild-type Escherichia coli.

[0027] Qualitative and quantitative analysis of substrates and products and monitoring of bacterial growth: Determination of bacterial solution concentration: Take the sample bacterial solution, dilute it with distilled water, use distilled water as a blank control, and use a spectrophotometer to measure OD at 600nm and 1cm pathlength 600 The contents of L-tryptophan, L-abrin and L-erythrine were determined by high performance liquid chromatography (HPLC) and referenced to the standard curve. The crude enzyme solution was purified by affinity chromatography according to the His tag on pET-28a.

[0028] Example 1: Construction of expression plasmid pET-egtD

[0029] The methyltransferase EgtD encoding gene egtD from M. smegmatis ATCC700084 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the sequence was codon optimized and connected to the pET-28a plasmid through the restriction enzyme cutting sites BamHI and SalI. The sequence of EgtD is shown in SEQ ID NO.1.

[0030] Example 2: Transformation of plasmid into E. coli BL21 (DE3) for expression

[0031] The recombinant expression plasmid pET-egtD was transformed into E. coli BL21 (DE3), and the recombinant expression strain was selected by culturing in LB+Kan solid medium at 37°C. The starting strain and the recombinant strain were inoculated into liquid TB medium, and the cells were collected after IPTG induction and ultrasonically disrupted. The supernatant and precipitate were subjected to SDS-PAGE electrophoresis, and a specific band with a molecular weight of about 35 kDa was detected ( Figure 1 ), which was consistent with the reported target protein size, indicating that egtD could be correctly expressed in Escherichia coli BL21(DE3).

[0032] Example 3: Site-directed mutagenesis of amino acid residues 252 and 282 of egtD encoded protein EgtD

[0033] Using the expression plasmid pET-egtD as a template, PCR amplification was performed using MT252-F / MT252-R and ET282-F / ET282-R (primer sequences are shown in Table 1) as primers, and the plasmid template was removed using DpnI digestion enzyme. Subsequently, the PCR product was purified and transformed into Escherichia coli BL21 (DE3) competent cells for plating culture. The target recombinant strain was screened and sequenced using the test primers (i.e., EgtD-F / EgtD-R; the primer sequences are shown in Table 1) to obtain the target recombinant plasmid pET-egtD M252V,E282A and target recombinant strain E.coli / pET-egtD M252V,E282A .

[0034] Table 1 Primer sequences required for PCR amplification

[0035]

[0036]

[0037]

[0038] Example 4: Site-directed saturation mutagenesis of amino acid residue 163 of egtD encoded protein EgtD

[0039] The expression plasmid pET-egtD M252V,E282A As a template, TT X -163-F / TT X -163-R (Note: X represents 19 amino acids other than Thr; the primer sequences are shown in Table 1) was used as primer for PCR amplification, and the plasmid template was removed with DpnI digestion enzyme. Subsequently, the PCR product was purified and transformed into Escherichia coli BL21 (DE3) competent cells for plating culture. The target recombinant strain was screened and sequenced using the test primers (i.e., EgtD-F / EgtD-R; the primer sequences are shown in Table 1) to obtain the target recombinant plasmid pET-egtD T163x,M252V,E282A and target recombinant strain E.coli / pET-egtD T163x,M252V,E282A .

[0040] Example 5: Enzyme activity assay of wild-type EgtD and EgtD mutants

[0041] The target recombinant strain E.coli / pET-egtD M252V,E282A and E. coli / pET-egtD T163x,M252V,E282A Culture in a 10 mL LB liquid vial for 9-10 h, then transfer 1 mL into TB medium and culture at 37°C until OD 600Between 0.5 and 0.6, add 0.1mmol / L IPTG to induce expression at 16℃ for 24h. After expression, collect the cells, wash twice with PBS buffer, suspend the cells and control them at the same OD 600 Then, the bacteria were broken by ultrasonic disruptor and the supernatant was obtained by centrifugation to obtain crude enzyme solution.

[0042] Enzyme activity assay reaction system: 200 μL reaction solution contains Tris·HCl 50 mM (pH 7), NaCl 50 mM, MnBr2 100 μM, SAM 150 μM, S-adenosylhomocysteine ​​hydrolase (SAHH) 10 μM, L-tryptophan 50 μM and 100 μL crude enzyme solution, react at 35 °C and monitor at 265 nm for 10 min. The enzyme activity unit (U) is defined as the amount of enzyme required to consume 1 μmol L-tryptophan per minute under the above reaction conditions. The enzyme activities of wild-type EgtD and different EgtD mutants are shown in Figure 2. Figure 2 As shown. Figure 2 It was found that the wild-type EgtD had no ability to catalyze the synthesis of L-tryptophan into L-erythrine, but when the 252nd and 282nd amino acid residues were changed to Val and Ala, the EgtD mutant (i.e., EgtD M252V,E282A ) showed an enzyme activity of 13.3±0.82U / mL. M252V,E282A The mutation of Thr at position 163 to Ala or Gly can significantly enhance the ability to catalyze the synthesis of L-erythrine from L-tryptophan, while mutation to other amino acid residues has a negative effect.

[0043] Example 6: Effects of different EgtD on the synthesis of L-erythrine

[0044] Enzyme conversion system: 200 μL reaction solution contains Tris·HCl 50 mM (pH 7), SAM 1.5 mM, S-adenosylhomocysteine ​​hydrolase (SAHH) 1.5 mM, L-tryptophan 500 mM and 100 μL crude enzyme solution, and reacts at 35°C for 2 h. After the reaction, the content of L-erythrine was determined by high performance liquid chromatography (HPLC). The results are as follows: Figure 2 shown.

[0045] from Figure 2 It can be seen that the L-erythrine production is consistent with the enzyme activity level, that is, the EgtD mutant with high enzyme activity also shows high L-erythrine production. T163G,M252V,E282A The mutant EgtD (i.e., the 163rd Thr mutated to Gly, the 252nd Met mutated to Val, and the 282nd Glu mutated to Ala) showed the highest enzyme activity (25.1±0.6 U / mL) and L-erythrine production (22.6±0.9 mg / L).M252V,E282A , EgtD T163A,M252V,E282A , EgtD T163V,M252V,E282A , EgtD T163L,M252V,E282A , EgtD T163I,M252V,E282A , EgtD T163P,M252V,E282A , EgtD T163S,M252V,E282A , EgtD T163C,M252V,E282A , EgtD T163D,M252V,E282A It also showed better enzyme activity and L-erythrine production;

[0046] Among them, mutant EgtD M252V,E282A The sequence is shown in SEQ ID NO.2; the mutant EgtD T163A,M252V,E282A The sequence is shown in SEQ ID NO.3; mutant EgtD T163V,M252V,E282A The sequence is shown in SEQ ID NO.4; the mutant EgtD T163L,M252V,E282A The sequence is shown in SEQ ID NO.5; the mutant EgtD T163I,M252V,E282A The sequence is shown in SEQ ID NO.6; the mutant EgtD T163G,M252V,E282A The sequence is shown in SEQ ID NO.7; mutant mutant EgtD T163P,M252V,E282A The sequence is shown in SEQ ID NO.8; mutant EgtD T163S,M252V,E282A The sequence is shown in SEQ ID NO.9; mutant EgtD T163C,M252V,E282A The sequence is shown in SEQ ID NO.10; mutant EgtD T163D,M252V,E282A The sequence is shown in SEQ ID NO.11.

[0047] Example 7: Purification of wild-type EgtD and EgtD mutants and analysis of their kinetic parameters

[0048] The crude enzyme solution obtained by expressing the mutant enzyme was purified by affinity chromatography. L-tryptophan solutions with mass 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 and 100 mmol / L SAM were added to 1 mg of the purified enzyme, and the Michaelis constant (K) of EgtD and its mutants when L-tryptophan was used as a substrate was calculated according to the Lineweaver-Burk double reciprocal method. m ), maximum reaction rate (V max ) and the catalytic constant (k cat). The results showed that mutation of Thr at position 163 to Gly, Met at position 252 to Val, and Glu at position 282 to Ala can effectively improve the ability of methyltransferase to catalyze the synthesis of L-tryptophan into L-erythrine, as shown in Table 2. According to the above results, positions 163, 252, and 282 are important substrate recognition sites of EgtD, which are closely related to the iterative methylation of the substrate. Molecular docking of the mutants with the substrates showed the following results. Figure 3 As shown. The substrate binding pocket after mutation has changed significantly. After Thr at position 163 mutates to Gly, the steric hindrance to the cavity is reduced, and the substrate has a more spacious binding pocket, which is more conducive to the direction of the intermediate to accept the methyl group, thereby improving the enzyme activity and yield. Therefore, mutating its site to other simple amino acids can change the enzyme cavity structure and have a great impact on improving the catalytic efficiency of the enzyme with L-tryptophan as the substrate, which has a great impact on enzyme research and industrial production.

[0049] Table 2 Kinetic parameters of transmethylase and its mutants in SAM transmethylation reaction

[0050]

[0051] Note: ND was not detected

[0052] In summary, the present invention is the first to mutate Thr 163 to Gly, Met 252 to Val, and Glu 282 to Ala in the amino acid sequence of the methyltransferase EgtD from M. smegmatis ATCC700084, and overexpress it in Escherichia coli BL21 (DE3). The results show that the EgtD mutant significantly improves the ability to catalyze the synthesis of L-erythrine using L-tryptophan as a substrate.

[0053] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A methyltransferase mutant, characterized in that The methyltransferase mutant is EgtD T163a,M252V,E282A , the EgtD T163a,M252V,E282A The method comprises mutating Thr at position 163 into a, Met at position 252 into Val, and Glu at position 282 into Ala in the EgtD parent sequence encoded by SEQ ID NO.1; wherein a is Ala or Gly.

2. The methyltransferase mutant according to claim 1, characterized in that: Encoding the EgtD T163a,M252V,E282A The gene sequences are shown as SEQ ID NO.3 or SEQ ID NO.7 respectively.

3. A gene encoding the methyltransferase mutant according to claim 1 or 2.

4. An expression vector carrying the coding gene according to claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector is a bacterial plasmid. A recombinant bacterium expressing the methyltransferase mutant according to claim 1.

7. The recombinant bacterium according to claim 6, characterized in that The recombinant bacteria use bacteria as host cells.

8. An enzyme preparation comprising the methyltransferase mutant according to claim 1.

9. The enzyme preparation according to claim 8, characterized in that The enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

10. Use of the methyltransferase mutant according to claim 1 in catalyzing the synthesis of L-erythrine using L-tryptophan as a substrate.

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