Construction and application of a new tryptophan decarboxylase mutant and recombinant genetically engineered bacteria

Abstract

The present invention discloses a novel tryptophan decarboxylase mutant, the construction of a recombinant genetically engineered bacterium, and its application. By mutating the original tryptophan decarboxylase sequence, the activity of the mutated tryptophan decarboxylase is improved, which is manifested as a significant increase in the level of catalysis of tryptophan to produce tryptamine and catalysis of 5-hydroxytryptophan to produce 5-hydroxytryptamine. The present invention also provides an expression vector expressing the tryptophan decarboxylase mutant and a method for constructing an engineered Escherichia coli bacterium. The use of engineered bacteria expressing the tryptophan decarboxylase mutant can significantly improve catalytic efficiency, laying the foundation for the industrial production of tryptamine and 5-hydroxytryptamine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of a new tryptophan decarboxylase mutant and a recombinant genetic engineering bacterium. Background Art

[0002] Tryptamine and 5-hydroxytryptamine are both tryptophan derivatives with a similar structure to tryptophan. Tryptamine is present in the mammalian brain and has potential neuromodulatory functions. As an important intermediate, tryptamine can be further synthesized into various terpenoid indole alkaloids, such as vincamine, vinpocetine, and isostearyl glycosides, which are of great value in the pharmaceutical and chemical industries.

[0003] 5-Hydroxytryptamine (5-HT), also known as serotonin, was first discovered in serum. It is widely present in mammalian tissues, primarily in the pineal gland and hypothalamus, and regulates a variety of physiological functions, including sleep, appetite, mood, immune regulation, and pain perception. 5-HT also plays an important role in plants, including regulating flowering cycles, morphogenesis, aging, and resistance to biotic stress.

[0004] 5-Hydroxytryptamine can be obtained through chemical synthesis or extraction from plants and animals. Tryptamine primarily relies on chemical and biological synthesis. Chemical synthesis involves numerous toxic and hazardous reagents, and the process is lengthy, requires a wide variety of raw materials, and produces harsh conditions and a high level of waste. Nanjing Fine Chemical Co., Ltd., in its patent CN112300049B, utilizes 5-hydroxyindole and nitroethylene through a multi-step chemical reaction to produce 5-hydroxytryptamine, achieving a yield of up to 91%. However, this requires multiple organic solvents and harsh reaction conditions. Wu Jing et al. synthesized tryptamine using a chemical decarboxylation method, achieving a yield of 99.4%. However, the reaction required a high temperature of 120°C and the use of diphenyl ether, a toxic and hazardous reagent. Extraction technology from plants and animals is still immature, resulting in low yields and purity. Biosynthesis, on the other hand, is a green process with minimal waste, easy disposal, scalability, and a high cost-effectiveness, offering broad application prospects. Xintai Jiahe Biotechnology Co., Ltd. uses an enzymatic method in its patent CN113403351A to catalyze the conversion of L-tryptophan into tryptamine and D-tryptophan, with a conversion rate of 98.9%. However, the production process is relatively complex. For example, the crude enzyme solution needs to be subjected to high-pressure homogenization treatment and the substrate needs to be racemized before the reaction, which adds additional production costs.

[0005] The present invention explores the industrial production conditions of tryptamine and 5-hydroxytryptamine from the two aspects of improving decarboxylase activity and simplifying the production process, thereby reducing production costs and improving synthesis efficiency. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a new tryptophan decarboxylase (L-Tryptophandecarboxylase, TDC) mutant, the construction of recombinant genetic engineering bacteria and the application thereof.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a tryptophan decarboxylase mutant,

[0009] (a) The amino acid sequence is derived from the sequence of SEQ ID NO: 1 by mutation, wherein one or more amino acid residues are mutated at positions selected from the group consisting of: 37, 73, 91, 187, 352, and 432, i.e., the mutations at positions 37, 73, 91, 187, 352, and 432 may be selected from any amino acid other than the original amino acid;

[0010] or (b) the tryptophan decarboxylase has an amino acid sequence similarity of greater than 95%, preferably greater than 98%, more preferably greater than 99% to that of (a), and has the function of (a), wherein the amino acid residues at positions 37, 73, 91, 187, 352, and / or 432 corresponding to the amino acid sequence of SEQ ID NO: 1 are mutated in the same manner as in (a);

[0011] Or (c) the tryptophan decarboxylase is formed by adding or deleting 1-20, preferably 1-10, more preferably 1-5 amino acid residues at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the protein described in (a), wherein the amino acid residues at positions 37, 73, 91, 187, 352 and / or 432 corresponding to the amino acid sequence shown in SEQ ID NO: 1 are mutated in the same manner as the amino acid sequence described in (a).

[0012] Preferably, the mutation at position 37 is a mutation to proline, serine or aspartic acid, the mutation at position 73 is a mutation to isoleucine or leucine, the mutation at position 91 is a mutation to alanine or threonine, the mutation at position 187 is a mutation to valine, methionine or leucine, the mutation at position 352 is a mutation to lysine, cysteine, alanine or aspartic acid, and the mutation at position 432 is a mutation to leucine or valine.

[0013] More preferably, the mutation at position 37 is to proline, the mutation at position 73 is to isoleucine, the mutation at position 91 is to alanine, the mutation at position 187 is to valine, the mutation at position 352 is to lysine, and the mutation at position 432 is to leucine.

[0014] In a second aspect, the present invention also provides a gene encoding the above-mentioned tryptophan decarboxylase mutant.

[0015] In a third aspect, the present invention provides an expression vector comprising a gene encoding the tryptophan decarboxylase mutant and capable of expressing the tryptophan decarboxylase mutant.

[0016] In a fourth aspect, the present invention provides a recombinant genetically engineered bacterium, wherein the recombinant genetically engineered bacterium comprises the above-mentioned expression vector or a gene encoding a tryptophan decarboxylase mutant is integrated into its genome. The recombinant genetically engineered bacterium can be obtained by transferring the above-mentioned expression vector into a host cell.

[0017] In a specific embodiment, the host cell is a bacterium or a yeast; preferably, the host cell is Escherichia coli (E. coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris and Yarrowia lipolytica (Yarrowia lipolytica); more preferably, the host cell is Escherichia coli, Saccharomyces cerevisiae; most preferably, the host cell is Escherichia coli.

[0018] In a fifth aspect, the present invention provides use of the above-mentioned tryptophan decarboxylase mutant or expression vector or genetically engineered bacteria in the production of tryptamine or 5-hydroxytryptamine.

[0019] In a sixth aspect, the present invention also provides a method for producing tryptamine or 5-hydroxytryptamine, comprising the following steps: inducing protein expression in genetically engineered bacteria, then collecting the cells by centrifugation, post-treating them, and then adding them to a conversion liquid containing the raw material tryptophan or 5-hydroxytryptamine to react, and finally isolating tryptamine or 5-hydroxytryptamine from the conversion liquid.

[0020] Furthermore, the post-treatment is washing with 0.9% saline.

[0021] According to the above scheme, the conversion solution is an aqueous solution containing 30-200 g / L of tryptophan or 5-hydroxytryptophan and 0.2-0.5 g / L of pyridoxal phosphate.

[0022] According to the above scheme, the reaction temperature is 30-40°C.

[0023] According to the above scheme, the reaction process is shaken on a shaking table with a rotation speed of 100 to 300 rpm.

[0024] According to the above scheme, the reaction time is 3 to 24 hours.

[0025] According to the above scheme, the reaction system is under closed conditions.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention obtains a tryptophan decarboxylase mutant with significantly enhanced activity by mutating the original tryptophan decarboxylase sequence shown in SEQ ID NO: 1. This mutant is used to generate tryptamine and 5-hydroxytryptamine, significantly improving catalytic efficiency, reducing the amount of microbial cells used in the catalytic process, and lowering production costs. Compared to the wild-type enzyme, its catalytic yield can be increased by up to 584% and 225%, respectively, achieving yields of up to 52g / L tryptamine and 79g / L 5-hydroxytryptamine.

[0028] The initial reaction system is water. Except for the substrate tryptophan or 5-hydroxytryptophan and the coenzyme pyridoxal phosphate, no other substances are added. The obtained product has no impurities and is easy to separate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the plasmid map of pRSFDuet1-TDC.

[0030] Figure 2 The figure shows the result of TDC single mutant catalyzing the conversion of tryptophan to tryptamine.

[0031] Figure 3 The figure shows the results of TDC single mutant catalyzing the conversion of 5-hydroxytryptophan to 5-hydroxytryptamine.

[0032] Figure 4 The figure shows the results of TDC multiple mutants catalyzing the conversion of tryptophan to tryptamine.

[0033] Figure 5 The figure shows the results of TDC multiple mutants catalyzing the conversion of 5-hydroxytryptophan into 5-hydroxytryptamine. DETAILED DESCRIPTION

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0035] Example 1 Construction of wild-type tryptophan decarboxylase engineered bacteria

[0036] (1) Using the pRSFDuet-1 vector as a template, PCR amplification was performed using primers P1-F and P1-R; the pRSFDuet-1 vector is a commercial vector purchased from Novagen; the sequence of the primer P1-F is 5'-AAGCTTGCGGCCGCATAATGCTT-3', as shown in SEQ ID No. 3, and the sequence of the primer P1-R is 5'-GGTATATCTCCTTATTAAAGTTAAACAAAATTATTTCTACAGGGG-3', as shown in SEQ ID No. 4; after the PCR product was recovered, the linearized vector pRSFDuet-1 was obtained, and the size of the linearized vector fragment was 3757 bp;

[0037] (2) The artificially synthesized TDC gene after codon optimization was used as a template, and PCR amplification was performed using primers TDC-F and TDC-R. The amplified products were recovered to obtain the target TDC gene fragment with a fragment size of 1476 bp. The nucleotide sequence of the TDC gene is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1. The primer sequence TDC-F is: 5'-TTAATAAGGAGATATACCATGAACCGCATGAAAAAC-3', as shown in SEQ ID No.5, and the primer sequence TDC-R is: 5'-TTATGCGGCCGCAAGCTTTTAGCTACGATCTGCCAGATCG-3', as shown in SEQ ID No.6.

[0038] (3) The PCR reaction system is as follows: 1 μL template, 2 μL upstream and downstream primers, 25 μL PrimeSTAR Max DNA polymerase, and 20 μL sterilized double-distilled water.

[0039] (4) The PCR amplification program was as follows: 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles, and 72°C extension for 7 min.

[0040] (5) The target fragment was connected to the personalized vector pRSFDuet-1 using the ClonExpress II one-step cloning kit to obtain a recombinant plasmid, which was named pRSFDuet1-TDC. The recombinant plasmid was successfully constructed and verified to be correct by sequencing. The plasmid map is shown in Figure 2. Figure 1 shown.

[0041] (6) The pRSFDuet1-TDC plasmid was transformed into the Escherichia coli expression host strain BL21 (DE3) by electroporation and spread onto LB solid medium containing kanamycin. The LB plate was cultured at 37°C until transformants grew. Positive transformants were picked to obtain wild-type tryptophan decarboxylase engineered bacteria (WT).

[0042] Example 2 Construction of single mutant tryptophan decarboxylase engineered bacteria

[0043] Using the constructed pRSFDuet1-TDC plasmid as a template, primers were designed for plasmid amplification and mutagenesis to obtain a linearized plasmid vector with base mutations. This vector was then transformed into Escherichia coli BL21 (DE3) and repaired and circularized in vivo to obtain a plasmid with base mutations. Specifically:

[0044] (1) Using the constructed pRSFDuet1-TDC plasmid as a template, PCR amplification was performed using 6 pairs of primers. The primer sequences are shown in Table 1. Six mutant sequences were obtained. The mutations were as follows: the arginine at the 37th amino acid of TDC was mutated to proline, the valine at the 73rd amino acid was mutated to isoleucine, the serine at the 91st amino acid was mutated to alanine, the cysteine ​​at the 187th amino acid was mutated to valine, the arginine at the 352th amino acid was mutated to lysine, and the alanine at the 432th amino acid was mutated to leucine. The plasmids expressing the mutants were named pRSFDuet1-TDC(R37P), pRSFDuet1-TDC(V73I), pRSFDuet1-TDC(S91A), pRSFDuet1-TDC(C187V), pRSFDuet1-TDC(R352K), and pRSFDuet1-TDC(A432L).

[0045] Table 1. Primer sequences for mutant construction

[0046]

[0047] (2) The PCR reaction system is as follows: 1 μL template, 2 μL upstream and downstream primers, 25 μL PrimeSTAR Max DNA polymerase, and 20 μL sterilized double-distilled water.

[0048] (3) The PCR amplification program was as follows: 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min 30 s, 30 cycles, and 72°C extension for 7 min.

[0049] (4) After the reaction, take 8.5 μL of PCR product, add 0.5 μL of DpnI restriction enzyme and 1 μL of 10× CutSmart, and incubate in a 37°C water bath for 4 h.

[0050] (5) The six Dpn I-treated linearized plasmids were transformed into the Escherichia coli expression host strain BL21 (DE3) by electroporation and plated onto LB solid medium containing kanamycin. The LB plates were cultured at 37°C until transformants grew. The transformants were picked and sequenced to obtain single mutant tryptophan decarboxylase engineered bacteria.

[0051] Example 3. Determination of catalytic activity of engineered bacteria with single mutant tryptophan decarboxylase

[0052] (1) The wild-type and single mutant tryptophan decarboxylase engineered bacteria were cultured overnight in a seed culture medium containing 50 μg / mL kanamycin to obtain a seed solution. The seed culture medium (mass percentage) was 1% tryptone, 1% sodium chloride, and 0.5% yeast extract. The culture conditions of the seed solution were 37° C. and 220 rpm.

[0053] (2) The above seed solution was inoculated into a protein expression medium containing 1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 0.231% KH2PO4 and 1.254% K2HPO4 (mass percentage) at 2% at 37°C and 220 rpm.

[0054] (3) After culturing for 3 h, IPTG was added at a final concentration of 0.5 mM to induce expression. The induction expression conditions were 25°C, 220 rpm, and the induction time was 16 h.

[0055] (4) After protein induction expression was completed, the cells were collected by centrifugation at 4000 rpm for 20 min at room temperature, washed with sterile 0.9% saline, and centrifuged again.

[0056] (5) Use a certain amount of pure water (pH 7.0) to suspend the cells until the OD of the resuspension is 600 The reaction conditions for whole-cell catalysis were 37°C, 150 rpm, and 6 h.

[0057] (6) Detection of tryptamine by high performance liquid chromatography: the chromatographic column is C18 (250 mm*4.6 mm, 5 μm) or an equivalent chromatographic column, the mobile phase is 30 wt% methanol and 70 wt% ammonium formate aqueous solution (20 mmol / L, pH 4.0), the flow rate is 1 mL / min, the injection volume is 10 μL, the column oven temperature is 40°C, the tryptamine is detected at a wavelength of 220, and the content is determined by the external standard method.

[0058] (7) Detection of 5-hydroxytryptamine by high performance liquid chromatography: the chromatographic column is C18 (250 mm*4.6 mm, 5 μm) or an equivalent chromatographic column, the mobile phase is 5 wt% methanol and 95 wt% potassium dihydrogen phosphate aqueous solution (5 mmol / L, pH 3.0), the flow rate is 1 mL / min, the injection volume is 10 μL, the column oven temperature is 35°C, 5-hydroxytryptamine is detected at a wavelength of 220, and the content is determined by the external standard method.

[0059] The results are as follows Figure 2 、 Figure 3 As shown, it can be seen that compared with the wild-type TDC strain, the TDC single mutant strain catalyzed the production of tryptamine and 5-hydroxytryptamine content.

[0060] Example 4. Construction of multiple mutant tryptophan decarboxylase engineered bacteria

[0061] Based on the above-mentioned single-mutant tryptophan decarboxylase engineered bacteria, the present invention further constructs multiple-mutant tryptophan decarboxylase engineered bacteria. Specifically, the multiple-mutant tryptophan decarboxylase engineered bacteria constructed by combining the three most active single-point mutations (R37P, V73I, and C187V) are shown below. The construction method is the same as in Example 2.

[0062] Based on the existing plasmid pRSFDuet1-TDC(R37P), the double mutant plasmid pRSFDuet1-TDC(R37P / V73I) was constructed using primers V73I-F and V73I-R; the double mutant plasmid pRSFDuet1-TDC(R37P / C187V) was constructed using primers C187V-F and C187V-R; and the triple mutant plasmid pRSFDuet1-TDC(R37P / V73I / C187V) was constructed using primers V73I-F1 and C187V-R1. Based on the existing plasmid pRSFDuet1-TDC(V73I), the double mutant plasmid pRSFDuet1-TDC(V73I / C187V) was constructed using primers C187V-F and C187V-R. The sequences of primers V73I-F, V73I-R, C187V-F and C187V-R are shown in Table 1. The sequence of V73I-F1 is: 5'-ATTGAGCGCCATATTCTGCCGGGTATTACCCATTGGC-3', and the sequence of C187V-R1 is: 5'-ATTGCTGGTATAAACAACCAGCGGCAGACGACAAC-3'.

[0063] The aforementioned multiple mutants are not limited to the aforementioned site combinations. Engineered strains of tryptophan decarboxylase with multiple mutations based on other sites can also enhance enzyme activity, creating a synergistic effect. Examples include R37P / S91A, R37P / R352K, R37P / V73I / S91A, R37P / S91A / R352K, R37P / V73I / S91A / R352K, and R37P / V73I / S91A / C187V / R352K / A432L.

[0064] Example 5. Determination of catalytic activity of multiple mutant tryptophan decarboxylase engineered bacteria

[0065] The catalytic activity of each strain on substrates tryptophan and 5-hydroxytryptophan was determined using the multiple mutants constructed in Example 4, using the same method as in Example 3. Figure 4 、 Figure 5 As shown, it can be seen that compared with the wild-type TDC strain, the TDC multiple mutant strain catalyzed the production of tryptamine and 5-hydroxytryptamine at significantly increased levels, reaching 13.48 and 39.51 g / L, respectively, which can be increased by up to 584% and 225% respectively compared with the wild-type strain.

[0066] Example 6. Optimization of reaction conditions to produce tryptamine and 5-hydroxytryptamine

[0067] The reaction conditions in Example 3 were optimized by increasing the substrate tryptophan or 5-hydroxytryptophan content, the coenzyme PLP content, the catalytic reaction time, and the reaction air tightness (the decarboxylation reaction causes the substrate to lose a molecule of CO2, which escapes into the air, which increases the pH of the reaction solution and is not conducive to the enzyme catalytic process. Increasing the reaction air tightness can allow some CO2 to dissolve in water again and stabilize the pH of the reaction solution), and catalytic synthesis of tryptamine and 5-hydroxytryptamine is as follows:

[0068] The reaction conditions are different from those in Example 3: Step (1) uses triple mutant tryptophan decarboxylase engineered bacteria TDC (R37P / V73I / C187V), and Step (5) is to suspend the bacteria in a certain amount of pure water (pH 7.0) until the OD of the resuspension reaches 600 The reaction temperature was 40. A 10 mL sample was used for whole-cell catalysis. The substrate concentration of tryptophan or 5-hydroxytryptophan was 100 g / L, and the coenzyme pyridoxal phosphate was 0.5 g / L. The whole-cell catalytic reaction conditions were 37°C, 150 rpm, and a reaction time of 24 h. The reaction was performed in an Erlenmeyer flask with a solid stopper.

[0069] The catalytic results of the triple mutant TDC (R37P / V73I / C187V) strain are shown in the table below. The levels of tryptamine and 5-hydroxytryptamine were further increased to 25.21 g / L and 78.94 g / L respectively.

[0070] Table 2. Detection results after optimizing reaction conditions

[0071]

[0072]

Claims

1. A tryptophan decarboxylase mutant, characterized in that Its amino acid sequence is mutated from the sequence shown in SEQ ID NO: 1, and the mutation is a mutation at position 73 as shown in SEQ ID NO: 1, or a mutation at positions 73 and 187, or a mutation at positions 73, 37 and 187; wherein position 37 mutates to proline, position 73 mutates to isoleucine, and position 187 mutates to valine.

2. A gene encoding the tryptophan decarboxylase mutant according to claim 1.

3. An expression vector, characterized in that Comprising the gene according to claim 2, capable of expressing the tryptophan decarboxylase mutant according to claim 1.

4. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria contains the expression vector according to claim 3 or the gene according to claim 2 is integrated into the genome of the recombinant genetically engineered bacteria.

5. The recombinant genetically engineered bacterium according to claim 4, wherein the recombinant genetically engineered bacterium is obtained by transferring the expression vector according to claim 3 into a host cell, wherein the host cell is a bacterium or a yeast.

6. Use of the tryptophan decarboxylase mutant according to claim 1, the expression vector according to claim 3, or the recombinant genetically engineered bacterium according to claim 4 in the production of tryptamine or 5-hydroxytryptamine.

7. A method for producing tryptamine or 5-hydroxytryptamine, characterized in that: The method comprises the following steps: inducing protein expression in the recombinant genetically engineered bacteria according to claim 4, then collecting the cells by centrifugation, adding the cells to a conversion solution containing raw material tryptophan or 5-hydroxytryptophan for reaction, and finally separating tryptamine or 5-hydroxytryptamine from the conversion solution.

8. The method according to claim 7, wherein: The conversion solution is an aqueous solution containing 30-200 g / L of tryptophan or 5-hydroxytryptophan and 0.2-0.5 g / L of pyridoxal phosphate; The reaction temperature is 30-40° C., the reaction process is shaken culture at a rotation speed of 100-300 rpm, the reaction time is 3-24 h, and the reaction system is sealed.

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