L-tryptophan 5-peroxygenase and its use in the biocatalytic synthesis of 5-hydroxytryptophan

Abstract

The application provides a bacterial-derived L-tryptophan 5-peroxygenase (TPH) and its application in the biosynthetic synthesis of 5-hydroxytryptophan (5-HTP). A brand-new L-tryptophan 5-peroxygenase gene is cloned from a specific bacterial strain. The enzyme can catalyze the highly regioselective hydroxylation reaction of the indole ring C5 of free L-tryptophan with oxygen or hydrogen peroxide as an oxidant, and one-step generates 5-hydroxytryptophan, without the need of additional tetrahydrobiopterin and its complex regeneration system. The application further constructs a recombinant expression vector and an engineering strain containing the L-tryptophan 5-peroxygenase gene, and realizes the efficient synthesis of 5-hydroxytryptophan through the whole-cell catalysis mode, the conversion rate can reach more than 92%, the regioselectivity is more than 98%, and the yield can reach 8.5 g / L.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the design of L-tryptophan 5-peroxygenase and its application in the biocatalytic synthesis of 5-hydroxytryptophan. Background Technology

[0002] 5-Hydroxytryptophan (5-HTP) is a direct precursor of serotonin, an important neurotransmitter in the human body. In the medical field, it can be used as an adjunct treatment for various diseases such as fibromyalgia, myoclonus, migraine, and cerebellar ataxia. At the same time, it is also widely used in the development of products related to mood regulation and sleep improvement in the health food field.

[0003] Currently, the main method for producing 5-hydroxytryptophan is plant extraction, which involves separating and purifying it from natural plants such as ghana seeds. This method has drawbacks such as dependence on raw materials, high extraction costs, limited yield, and susceptibility to climate and geographical influences. Early chemical synthesis routes, on the other hand, involve harsh reaction conditions, numerous byproducts, difficult separation and purification, and are prone to environmental pollution.

[0004] At the biosynthetic level, it was previously generally believed that only eukaryotic tryptophan hydroxylases could catalyze the C5-hydroxylation of the indole ring of free L-tryptophan. These eukaryotic enzymes belong to the aromatic amino acid hydroxylases family and depend on tetrahydrobiopterin (BH4) and Fe. 2+ As a cofactor, the catalytic process requires a complex cofactor recycling system, making it difficult to achieve efficient heterologous expression in prokaryotic engineered strains, which greatly limits the advancement of large-scale biological production of 5-hydroxytryptophan. For a long time, the source of bacterial 5-hydroxytryptophan biosynthesis has remained an unsolved mystery in the field. Only in recent years have related studies revealed for the first time the existence of a novel 5-hydroxytryptophan biosynthesis pathway in bacteria. However, there are currently no mature patents or application schemes related to bacterial-derived L-tryptophan 5-peroxygenase that can be used for industrial production.

[0005] Therefore, it is necessary to develop a novel bacterial L-tryptophan 5-peroxygenase. Summary of the Invention

[0006] The purpose of this invention is to provide a bacterial L-tryptophan 5-peroxygenase and its application in the biocatalytic synthesis of 5-hydroxytryptophan. This enzyme does not rely on the eukaryotic-specific tetrahydrobiopterin cofactor and can efficiently convert L-tryptophan to 5-hydroxytryptophan in simple catalytic systems or prokaryotic engineered strains, significantly reducing the biosynthesis cost of 5-hydroxytryptophan, improving production efficiency, and enabling industrial-scale production.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a second aspect of the invention, L-tryptophan 5-peroxygenase is provided, wherein the L-tryptophan 5-peroxygenase is as follows: L-tryptophan 5-peroxygenase (TPH) has the amino acid sequence shown in SEQ ID NO.1.

[0008] Furthermore, the L-tryptophan 5-peroxygenase also comprises one of the following: 1) Having an amino acid sequence with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1; 2) Having a fusion amino acid sequence obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.1; 3) An amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function.

[0009] In a second aspect of the invention, the use of the L-tryptophan 5-peroxygenase in the biocatalytic synthesis of 5-hydroxytryptophan is provided.

[0010] In a third aspect of the invention, an expression vector is provided that is capable of expressing the L-tryptophan 5-peroxygenase.

[0011] The nucleic acid molecule encoding the L-tryptophan 5-peroxygenase in the recombinant expression vector includes one of the following: 1) It has the deoxyribonucleotide sequence shown in SEQ ID NO.2; 2) A deoxyribonucleotide sequence having the amino acid sequence shown in SEQ ID NO.1; 3) Having a deoxyribonucleic acid sequence encoding L-tryptophan 5-peroxygenase activity obtained by substituting and / or deleting and / or adding one or more nucleotides to the deoxyribonucleic acid sequence shown in SEQ ID NO.2; 4) A deoxyribonucleic acid sequence having 90% or more homology with the deoxyribonucleic acid sequence defined in SEQ ID NO.2 and encoding L-tryptophan 5-peroxygenase.

[0012] In a fourth aspect of the invention, a recombinant bacterium or engineered cell line comprising the recombinant expression vector is provided.

[0013] In a fifth aspect of the present invention, a method for the biocatalytic synthesis of 5-hydroxytryptophan is provided, comprising the following steps: Step 1: Using L-tryptophan as a substrate, 5-hydroxytryptophan is generated under the catalysis of L-tryptophan 5-peroxygenase and with oxygen or hydrogen peroxide as an oxidant. Step 2: Separate and purify the reaction solution obtained in Step 1 to obtain pure 5-hydroxytryptophan.

[0014] According to an embodiment of the present invention, the method for preparing 5-hydroxytryptophan in step 1 is as follows: The recombinant engineered bacterial cells after induced expression were collected and resuspended in phosphate buffer containing L-tryptophan. In the presence of oxygen or hydrogen peroxide, L-tryptophan 5-peroxygenase catalyzed the hydroxylation reaction at the C5 position of the L-tryptophan indole ring to generate 5-hydroxytryptophan.

[0015] Furthermore, the L-tryptophan 5-peroxygenase is TPH, and its encoded gene sequence is shown in SEQ ID NO.2.

[0016] Further, the reaction system in step 1 is as follows: 50 mM sodium phosphate buffer, pH 6.5-7.5, final L-tryptophan concentration 10 g / L, final hydrogen peroxide concentration 0.1-1 mM, bacterial cell concentration OD600=30, reaction at 30-37℃ and 180 rpm for 12 hours.

[0017] Furthermore, the optimal reaction temperature of the L-tryptophan 5-peroxygenase is 30-37℃, and the optimal reaction pH is 6.5-7.5. Under the condition of adding a low concentration of hydrogen peroxide, the catalytic conversion rate of L-tryptophan can reach more than 92%, and there is almost no generation of byproducts such as 4-hydroxytryptophan and 6-hydroxytryptophan, with a site selectivity of more than 98%.

[0018] According to an embodiment of the present invention, the purification method of 5-hydroxytryptophan in step 2 is as follows: The reaction solution obtained in step 1 was analyzed by HPLC using a C18 column, with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase and a detection wavelength of 280 nm. The product was then quantitatively analyzed and purified.

[0019] Furthermore, the L-tryptophan 5-peroxygenase can be obtained by constructing a recombinant vector for expression and purification. The recombinant strain is Escherichia coli BL21(DE3), and the recombinant plasmid is pET28a-TPH.

[0020] Compared with existing related technologies, the beneficial effects of the present invention are as follows: 1. This invention overcomes the application limitations of eukaryotic enzymes: The L-tryptophan 5-peroxygenase of this invention is completely different from eukaryotic tryptophan hydroxylases, relying solely on heme as a cofactor and eliminating the need for a tetrahydrobiopterin system, thus significantly simplifying the components of the catalytic reaction and reducing production costs. This enzyme shares less than 20% amino acid sequence homology with eukaryotic tryptophan hydroxylases, exhibiting fundamental differences in protein structure, cofactor system, and catalytic mechanism.

[0021] 2. The L-tryptophan 5-peroxygenase of the present invention has high site selectivity: the enzyme has extremely high specificity for the hydroxylation of the C5 position of the indole ring of L-tryptophan, with very few byproducts. The subsequent separation and purification steps of 5-hydroxytryptophan are simple, and the product purity can easily reach more than 99.5%, meeting the quality requirements of pharmaceutical-grade raw materials.

[0022] 3. Suitable for industrial production: The recombinant E. coli engineered bacteria have good genetic stability and can be fermented at high density using inexpensive inorganic salt culture media. The whole-cell catalysis process is mild and the aqueous reaction system is green and environmentally friendly. Compared with plant extraction and chemical synthesis routes, the production cycle is shortened by more than 60% and the overall production cost is reduced by 70%.

[0023] 4. Expanding application potential: The heme-dependent monooxygenase family to which this enzyme belongs can be further expanded through protein-directed modification to synthesize other hydroxylated tryptophan derivatives, providing a new toolbox of biocatalytic elements for the synthetic biology manufacturing of related active molecules. Attached Figure Description

[0024] Figure 1 This diagram compares the bacterial and eukaryotic 5-hydroxytryptophan synthesis pathways of this invention. The bacterial pathway utilizes L-tryptophan 5-peroxygenase (TPH) to directly catalyze the conversion of L-tryptophan to 5-hydroxytryptophan, requiring only oxygen or hydrogen peroxide as an oxidant and relying on heme cofactors. The eukaryotic pathway utilizes tryptophan hydroxylase (TPH) to catalyze the conversion of L-tryptophan to 5-hydroxytryptophan, requiring tetrahydrobiopterin (BH4) and Fe2+. 2+ It serves as a cofactor and requires a complex cofactor recycling system.

[0025] Figure 2 This is an HPLC chromatogram of the whole-cell catalytic products of the recombinant strain. std represents the standard, and TPH represents the 5-hydroxytryptophan product obtained by whole-cell catalysis using L-tryptophan as a substrate. The 5-hydroxytryptophan product is singular and of high purity.

[0026] Figure 3 The results of the amino acid sequence comparison between L-tryptophan 5-peroxygenase (TPH) and eukaryotic tryptophan hydroxylase showed that the homology between the two was less than 20%. Detailed Implementation

[0027] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0028] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0030] Example 1: Discovery of L-tryptophan 5-peroxygenase I. Experimental Objective A novel L-tryptophan 5-peroxygenase gene was obtained from a specific bacterial strain to clarify the sequence characteristics of its encoded protein and its evolutionary relationship with eukaryotic tryptophan hydroxylases.

[0031] II. Experimental Methods The full-length gene sequence of L-tryptophan 5-peroxygenase was obtained from the genomic DNA of the target bacteria by PCR amplification. The amino acid sequence encoded by this gene was compared with the homology of known eukaryotic tryptophan hydroxylases, and the sequence was optimized to a codon sequence preferred by *E. coli*.

[0032] III. Experimental Results The L-tryptophan 5-peroxygenase gene was successfully cloned. The protein it encodes belongs to the heme-dependent monooxygenase family and shares less than 20% amino acid sequence homology with eukaryotic tryptophan hydroxylase. The two enzymes differ fundamentally in protein structure, cofactor system, and catalytic mechanism. The amino acid sequence of this enzyme is shown in SEQ ID NO.1: >bacteria L-tryptophan 5-peroxygenase MVDRISEQEHLDGADSKREKSYRVTTEDSLLNNSHRCDAREILSEFHKVG KHFVTHQKLQQLAVYRDELARNSAGDWMLSFLNVVLDKFDDTYSYRSYLALDLLHWQNYTENSADYIDWQLTIITMDLIRFELEALRNNGSWLYEMAPDQ RLVNMRCRRLIKCMGKVYQRMNIQWENNERDMIAICDTICSAVFLRLSDNEKLRLEYSMIPVYINHDEYIFLRILQAFETLFDWLASCLTEVIAFAKSDLKQATEELLFLSANRLNEMAAL FPLLSTLRVD GFHRFRDYTEGSSAIQSRSYKKVESLCSRP DAERFNSIAYRAVPEVSEEILCNPETIDDVFNLIPQDNLYKDDFHQAMESFGLGMKIWRQ SHYGIAVKML GASPGTGNTE GTAYLKEVRK IPIFKNI The optimized nucleotide sequence is shown in SEQ ID NO.2. If constructing a vector, an NdeI site can be introduced upstream of the 5' end of the gene and an XhoI site can be introduced downstream of the 3' end of the gene, and then ligated with the pET-28a vector that has been digested with the same enzymes.

[0033] Sequence homology alignment results are as follows Figure 3 As shown, the significant sequence differences between this bacterial enzyme and eukaryotic enzymes are clearly demonstrated.

[0034] Example 2: Construction of Recombinant Expression Vector I. Experimental Objective A recombinant expression vector capable of efficiently expressing L-tryptophan 5-peroxygenase in Escherichia coli was constructed.

[0035] II. Experimental Methods The L-tryptophan 5-peroxygenase gene (SEQ ID NO.2) was double-digested with restriction endonucleases NdeI and XhoI, then ligated with the pET-28a vector (purchased from Miaoling Biotechnology, catalog number VT0331-01) that had been digested with the same restriction enzymes. The ligation was then performed on the vector, which was transformed into competent E. coli cells. Positive clones were screened and sequenced for verification.

[0036] III. Experimental Results The recombinant expression plasmid pET28a-TPH was successfully obtained. Sequencing results confirmed that the inserted fragment sequence was completely correct and the reading frame matched the vector expression system, making it suitable for subsequent protein expression.

[0037] Example 3: Induced expression of recombinant strains I. Experimental Objective The expression of exogenous genes in recombinant strains was verified, and L-tryptophan 5-peroxygenase protein was prepared.

[0038] II. Experimental Methods The recombinant plasmid pET28a-TPH was transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37℃ and 200 rpm until... Add 0.5 mM IPTG to a final concentration, transfer to 25°C to induce expression for 16 hours. After induction, collect the bacterial cells by centrifugation, resuspend in pH 7.0 sodium phosphate buffer, sonicate, and then purify the target protein using a nickel column affinity assay. Determine the concentration and store the protein.

[0039] III. Experimental Results After IPTG induction, the recombinant strain expressed soluble enzymes accounting for more than 35% of the total bacterial protein. High-purity L-tryptophan 5-peroxygenase was obtained after purification, which can be used for subsequent enzymatic property studies. A comparison of the 5-hydroxytryptophan synthesis pathway from this bacterium with that of eukaryotic organisms is provided. Figure 1 As shown, this highlights the simplicity of the invention's approach and the advantages of the auxiliary factors.

[0040] Example 4: Determination of the enzymatic properties of L-tryptophan 5-peroxygenase I. Experimental Objective Key enzymological parameters of the enzyme, such as optimal reaction temperature, optimal reaction pH, and substrate site selectivity, were determined.

[0041] II. Experimental Methods: 1. Determination of optimal temperature: Using L-tryptophan as a substrate, enzyme activity was measured in sodium phosphate buffer at pH 7.0 at 20℃, 25℃, 30℃, 37℃, 40℃, and 45℃.

[0042] 2. Determination of optimal pH: Enzyme activity was determined in different buffer solutions with pH values ​​ranging from 5.5 to 8.5, using L-tryptophan as a substrate.

[0043] 3. Site selectivity analysis: Under the condition of adding 0.5 mM hydrogen peroxide, the hydroxylation activity of the enzyme on the C5, C4 and C6 positions of the L-tryptophan indole ring was determined.

[0044] 4. HPLC detection: The chromatographic column was C18 (5 μm, 4.6×250 mm), the mobile phase was acetonitrile-water (containing 0.1% trifluoroacetic acid), the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, and the column temperature was 30℃.

[0045] III. Experimental Results The optimal reaction temperature for this enzyme is 30-37℃, and the optimal pH is 6.5-7.5. Under conditions of low-concentration hydrogen peroxide (0.5 mM), this enzyme exhibits extremely high specificity for the C5-hydroxylation of L-tryptophan indole ring, with almost no formation of byproducts such as 4-hydroxytryptophan and 6-hydroxytryptophan, demonstrating a regioselectivity exceeding 98%. Chromatographic analysis of the products from the pure enzyme-catalyzed reaction shows a single product with a well-defined 5-hydroxytryptophan peak, confirming the high regioselectivity.

[0046] Example 5: Whole-cell catalytic synthesis of 5-hydroxytryptophan I. Experimental Objective The efficient conversion of L-tryptophan to 5-hydroxytryptophan was achieved using a whole-cell catalytic system of recombinant engineered bacteria.

[0047] II. Experimental Methods Collect the induced recombinant engineered bacterial cells, resuspend them in 50 mM pH 7.0 sodium phosphate buffer, and adjust the cell concentration to [specific value missing]. L-tryptophan at a final concentration of 10 g / L and 0.5 mM hydrogen peroxide were added to the system, and the mixture was reacted at 30°C and 180 rpm in a shaker for 12 hours. After the reaction, the supernatant was collected by centrifugation, and the yield of 5-hydroxytryptophan was quantitatively analyzed by HPLC under the same detection conditions as in Example 4.

[0048] III. Experimental Results After 12 hours of whole-cell catalysis, the conversion rate of 5-hydroxytryptophan reached 93.2%, with a site selectivity of 98.7% and a yield of 8.5 g / L. The catalytic efficiency per cell is significantly higher than that of previously reported eukaryotic enzyme recombinant systems. The HPLC chromatogram of the whole-cell catalytic product is shown below. Figure 2 As shown, apart from the main peak of 5-hydroxytryptophan, there were no obvious impurity peaks, further confirming the high efficiency and high selectivity of the reaction.

[0049] Example 6: Purification of 5-hydroxytryptophan I. Experimental Objective 5-Hydroxytryptophan was isolated and purified from the whole-cell catalytic reaction solution to obtain a high-purity product.

[0050] II. Experimental Methods The whole-cell catalytic reaction solution was centrifuged to remove bacterial cells, and the supernatant was purified by HPLC using a preparative column with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase. The fraction corresponding to 5-hydroxytryptophan was collected, and the solid pure product was obtained after freeze-drying.

[0051] III. Experimental Results The purified 5-hydroxytryptophan obtained has a purity of over 99.5% as determined by HPLC, meeting the quality requirements for pharmaceutical-grade raw materials. The entire purification process is simple and efficient, suitable for large-scale production.

[0052] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0053] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0054] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0055] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An L-tryptophan 5-peroxygenase, characterized in that, The L-tryptophan 5-peroxygenase is TPH, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The L-tryptophan 5-peroxygenase according to claim 1, characterized in that, The L-tryptophan 5-peroxygenase also includes one of the following: It has an amino acid sequence with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.

1. Having a fusion amino acid sequence obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.1; An amino acid sequence having 90% or more homology with and having the same function as the amino acid sequence shown in SEQ ID NO.

1.

3. The application of L-tryptophan 5-peroxygenase according to any one of claims 1-2 in the biocatalytic synthesis of 5-hydroxytryptophan, characterized in that, The applications include: Using L-tryptophan as a substrate, and employing the L-tryptophan 5-peroxygenase or its whole-cell catalytic system as described in any one of claims 1-2, the indole ring of L-tryptophan is hydroxylated at the C5 position in the presence of oxygen or hydrogen peroxide to generate 5-hydroxytryptophan.

4. A recombinant expression vector, characterized in that, The recombinant expression vector is capable of expressing the L-tryptophan 5-peroxygenase described in claim 1.

5. The recombinant expression vector according to claim 4, characterized in that, The nucleic acid molecule encoding the L-tryptophan 5-peroxygenase in the recombinant expression vector includes one of the following: It has the deoxyribonucleotide sequence shown in SEQ ID NO.2; A deoxyribonucleotide sequence having the amino acid sequence shown in SEQ ID NO.1; The deoxyribonucleic acid sequence encoding L-tryptophan 5-peroxygenase activity is obtained by substituting and / or deleting and / or adding one or more nucleotides to the deoxyribonucleic acid sequence shown in SEQ ID NO.

2. The deoxyribonucleic acid sequence having 90% or more homology with the deoxyribonucleic acid sequence defined in SEQ ID NO.2 and encoding L-tryptophan 5-peroxygenase.

6. A recombinant bacterial strain or engineered host cell line expressing a recombinant expression vector comprising claim 4 or 5, characterized in that, The recombinant expression vector includes one or more of the following: Escherichia coli expression vector, yeast expression vector, Bacillus subtilis expression vector, lactic acid bacteria expression vector, Streptomyces expression vector, filamentous fungus expression vector, plant expression vector, insect expression vector, or mammalian cell expression vector; the host cell includes one of the following: Escherichia coli host cell, yeast host cell, Bacillus subtilis host cell, lactic acid bacteria host cell, actinomycete host cell, filamentous fungus host cell, insect cell, or mammalian cell.

7. The use of the recombinant expression vector of claim 4 or 5 or the recombinant bacteria or engineered cell line of claim 6 in the biocatalytic synthesis of 5-hydroxytryptophan.

8. A method for the biocatalytic synthesis of 5-hydroxytryptophan, characterized in that, The method includes: using L-tryptophan as a substrate, employing the L-tryptophan 5-peroxygenase or its whole-cell catalytic system as described in claim 1, and catalyzing the indole ring C5-hydroxylation of L-tryptophan in the presence of oxygen or hydrogen peroxide to generate 5-hydroxytryptophan.

9. The method for biocatalytic synthesis of 5-hydroxytryptophan according to claim 8, characterized in that, The catalytic reaction conditions in the method include: 10 g / L L-tryptophan, 0.1-1 mM hydrogen peroxide, bacterial cell concentration OD600=30, 50 mM sodium phosphate buffer pH 6.5-7.5, reaction temperature 30-37℃, and reaction time 12 hours.

10. The method for biocatalytic synthesis of 5-hydroxytryptophan according to claim 8, characterized in that, The L-tryptophan 5-peroxygenase is a heme-dependent monooxygenase that uses oxygen or hydrogen peroxide as an oxidant to catalyze the conversion of L-tryptophan.

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