Biosynthesis method of 6-hydroxy-L-tryptophan

Abstract

The invention provides a biosynthesis method of 6-hydroxy-L-tryptophan, on the basis that a novel tryptophan 6-site hydroxylase protein family enzyme is obtained through screening, a cell factory is constructed for biotransformation, and green synthesis of 6-hydroxy-L-tryptophan is achieved. The technical problems of limited types of biocatalysts, insufficient chemical synthesis region selectivity and the like in the prior art are effectively solved. The amino acid sequence of the tryptophan 6-site hydroxylase is shown as SEQ ID NO: 1. The invention provides a protein sequence for catalyzing a new family of tryptophan 6-site hydroxylase, and 6-hydroxy-L-tryptophan can be directly and efficiently synthesized through a cell factory. The method has the advantages of mild reaction conditions, environmental friendliness, high regioselectivity, good product purity, flexible process route and the like, and a brand new technical scheme is provided for industrial production of 6-hydroxy-L-tryptophan.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis technology of functional macromolecules, and specifically relates to a biosynthesis method of 6-hydroxy-L-tryptophan. Background Technology

[0002] 6-Hydroxy-L-tryptophan is a rare amino acid with significant biological activity. Its core biological activity is as an effective inhibitor of tyrosinase. Literature studies have shown that 6-hydroxy-L-tryptophan is effective against tyrosinase. Agaricus bisporus This inhibitor exhibits competitive inhibition of tyrosinase, with an IC50 value of 0.23 mM, significantly higher than that of hydroquinone (IC50 9.82 mM), a known tyrosinase inhibitor. Furthermore, its inhibitory activity is stereospecific; the activity of 6-hydroxy-L-tryptophan is 8.1 times that of 6-hydroxy-D-tryptophan and 78 times that of 5-hydroxy-L-tryptophan, indicating that the position of the hydroxyl group on the indole ring and the chirality of the amino acid are crucial to its activity.

[0003] Based on its potent tyrosinase inhibitory activity, 6-hydroxy-L-tryptophan has a clear potential application in inhibiting melanin production. Tyrosinase is a key rate-limiting enzyme in the melanin biosynthesis pathway, therefore this compound could serve as a pharmaceutical and cosmetic ingredient for developing novel skin-whitening agents or treating hyperpigmentation-related diseases. Due to safety concerns with some existing tyrosinase inhibitors (such as hydroquinone and rhododendronol), there is a significant need to find efficient and safe alternatives, and 6-hydroxy-L-tryptophan is a promising candidate compound.

[0004] 6-Hydroxy-L-tryptophan is rarely reported in natural resources, and the number of related research papers is limited, classifying it as a rare compound. This highlights the necessity and value of developing efficient and sustainable methods for its preparation. Chemical synthesis of 6-hydroxy-L-tryptophan typically relies on the assistance of directing groups, involves cumbersome reaction steps and demanding conditions, and is prone to generating byproducts, resulting in low overall efficiency and a significant environmental burden. In biosynthesis, most reported catalytic systems belong to the tryptophan dioxygenase family. These enzymes can catalyze the hydroxylation of tryptophan, providing a feasible route for the green biomanufacturing of 6-hydroxy-L-tryptophan. However, from the perspective of catalytic tool diversity and process optimization, discovering novel hydroxylases with different catalytic properties, higher efficiency, or better adaptability remains of significant research and application value. Summary of the Invention

[0005] This invention provides a method for the biosynthesis of 6-hydroxy-L-tryptophan. Based on the screening and obtaining of a novel tryptophan 6-hydroxylase protein family enzyme, a cell factory is constructed for biotransformation, realizing the green synthesis of 6-hydroxy-L-tryptophan. This effectively solves the technical problems of limited types of biocatalysts and insufficient regioselectivity in chemical synthesis in the prior art.

[0006] This invention first provides a tryptophan 6-hydroxylase, the amino acid sequence of which is SEQ ID NO:1; MNNTSNLSLEKLSFTLKLIPISEILLSDEFYSGTWKTVAQWCVDYLCNPHPELGRSGVVCPHSQVSMKKETFWITEIKTKGRTEKEIKEDIVSLAHLFYEQEPRNGNEVQFKTIVSV WDDLYPEEYISNLHSEMKPIFLRMGLMLGEFFSSCEKTGLRNPIFYPLCSPVPLLVVRDMLEFDIAFLSDSEEYVSEYINKYGERGVLAINNVLNNNKKIHLSDKQVSILKSYLMHK; The nucleotide sequence of the gene encoding the tryptophan 6-hydroxylase is SEQ ID NO:2; (SEQ ID NO: 2).

[0007] The present invention also provides a recombinant engineered strain carrying a nucleic acid fragment encoding the above-mentioned tryptophan 6-hydroxylase; Furthermore, the recombinant engineered strain also contains a nucleic acid fragment encoding tryptophan synthase; This invention also provides an application of the aforementioned tryptophan 6-hydroxylase, which is used to catalyze the L-tryptophan 6-hydroxylation reaction; In another aspect, the present invention provides a method for the 6-hydroxylation of L-tryptophan, wherein the method uses a tryptophan 6-hydroxylase with the amino acid sequence SEQ ID NO:1 to catalyze a substrate to generate 6-hydroxy-L-tryptophan; The substrate is indole or -L-tryptophan; Furthermore, the tryptophan 6-hydroxylase with the amino acid sequence SEQ ID NO:1 is wholly or partially replaced with an enzyme with the amino acid sequence SEQ ID NO:3 or SEQ ID NO:4.

[0008] The amino acid sequence of Xk970 is SEQ ID NO:3, and the amino acid sequence of Rr100 is SEQ ID NO:4.

[0009] MNTNPTLLVNKPYCAPKLIPLSDVLSSYELNTSALKVIAQWCVDYLCNPHPELGRSGVVCPYSPTSMKKETFWFTEIKTKGRTEEEIKQDIISLSYLFHEQEPRTGEATQFKTIVSIWD DIYSEEDIARLHYEMKPIFLHEGLMLGEFFSSCEKRGLRNSNFYPLRSPVPLLVVREMLEFDIAFLSDSIEYIKEYIHKYGDRGLNTIKNMLNNNKRIGLNDEQIAVLKSYLMHQ (SEQ ID NO:3); MPIQRRVGPRSGTVWVDLFGETSEYAREASAHLLRTWASGYLTEPNPELGRSGPVCPFVRPSIGKQLFGAAFVHGSGIDAGALGTIVEDQFDLYATLSREDDRDRSLKTLVTVLPDLTG FEVIDVVHAEFKSRFVEHGFMLGQFYPGCTQPGLWNHDFHPLDAPLPMLVARNMMTTDFPFLVGRQEWLCAYFKKFAPALPSTLRLTIAKRMKYDGDAVDAITAHHELTGNEPAR (SEQ ID NO:4).

[0010] This invention provides a new family of protein sequences that catalyze the 6-hydroxylation of tryptophan, enabling the direct and efficient synthesis of 6-hydroxy-L-tryptophan via cell factories. This invention offers multiple advantages, including mild reaction conditions, environmental friendliness, high regioselectivity, high product purity, and flexible process routes, providing a novel technical solution for the industrial production of 6-hydroxy-L-tryptophan. Attached Figure Description

[0011] Figure 1 Schematic diagram of the synthesis of 6-hydroxy-L-tryptophan. Figure 2 : LC-MS results of cell factories fed with indole Figure 3 Figure: LC-MS results of cell factories fed with L-tryptophan. Detailed Implementation

[0012] This invention realizes the cell factory fermentation synthesis of 6-hydroxy-L-tryptophan starting from indole. Using indole as a substrate, the tryptophan synthase gene and the tryptophan 6-hydroxylase (Xn269, SEQ ID NO: 1) gene are constructed into the expression vector pRSFduet and transformed into Escherichia coli. The expression of tryptophan synthase and tryptophan 6-hydroxylase is achieved by expansion culture at 37°C and induction culture at 18°C. Furthermore, the synthesis of 6-hydroxy-L-tryptophan is achieved by feeding indole at 30°C.

[0013] The tryptophan 6-hydroxylase (Xn269, amino acid sequence SEQ ID NO:1) can be replaced by other provided sequences, such as SEQ ID NO:3 or SEQ ID NO:4, and can also be its homologous protease.

[0014] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0015] Example 1: Synthesis of 6-hydroxy-L-tryptophan using indole as a substrate via a cell factory 1) Construction of engineered strains The tryptophan 6-hydroxylase gene (Xn269, SEQ ID NO: 1) and a tryptophan synthase derived from *E. coli* were constructed into the expression vector pRSFDuet. The tryptophan 6-hydroxylase gene was located between the polyclonal restriction sites NcoI and BamHI, and the tryptophan synthase gene was located between the polyclonal restriction sites NdeI and XhoI. The nucleotide sequence of the Xn269 protein gene is SEQ ID NO: 2.

[0016] Tryptophan synthase can be any conventional tryptophan synthase. The tryptophan synthase used in this embodiment consists of two subunits with amino acid sequences SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The corresponding nucleotide sequence of the encoding gene is SEQ ID NO: 7, which contains the coding sequences for the two subunits of tryptophan synthase.

[0017] MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKDPEFQAQFNDLLKNYAGRPTALTKCQNITAGTNTTLYLKREDLLHGGAHKTNQVLGQALLAKRMGKTEIIAETGAGQHGVASALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGSYETAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILEREGRLPDAVIACVGGGSNAIGMFADFINETNVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQIEESYSISAGLDFPSVGPQHAYLNSTGRADYVSITDDEALEAFKTLCLHEGIIPALESSHALAHALKMMRENPDKEQLLVVNLSGRGDKDIFTVHDILKARGEI(SEQ ID NO:5) MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRS(SEQ ID NO:6)

[0018] The constructed expression vector was further transformed into Escherichia coli, thereby realizing the construction of an engineered strain for the synthesis of 6-hydroxy-L-tryptophan.

[0019] 2) Fermentation and transformation of strains An engineered *E. coli* strain expressing Xn269 (tryptophan 6-hydroxylase) and tryptophan synthase was activated on LB agar plates. Single colonies were picked and inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to prepare a seed culture. The seed culture was transferred at a 1% (v / v) inoculation rate to a 2 L Erlenmeyer flask containing 500 mL of M9CA medium, and kanamycin was added to a final concentration of 50 μg / mL. The culture was then incubated at 37°C and 220 rpm until OD500 was reached. 600 When the pH was 0.8–1.0, 0.2 mM IPTG was added, and expression was induced at 18 °C and 150 r / min for 18–22 h. Subsequently, the temperature was adjusted to 30 °C, and the culture was continued at 200 rpm for 24 h, during which 1 mM indole was added as a substrate for transformation.

[0020] 3) Product detection and analysis Take 100 μL of fermentation broth, add 200 μL of methanol, vortex for 10 min, centrifuge at high speed (10 min), and collect the supernatant for analysis. Analyze using a 1260 quadrupole-time-of-flight high-resolution liquid chromatography-mass spectrometry (LC-QTOF-MS) system.

[0021] The results are as follows Figure 2 As shown, a new product was observed to be generated, with a conversion rate of approximately 46%. High-resolution mass spectrometry identification showed that the molecular weight of the product was consistent with that of 6-hydroxy-L-tryptophan (theoretical value of [M+H]⁺: 221.0926, measured value: 221.0922), confirming it as the target compound 6-hydroxy-L-tryptophan.

[0022] Example 2: Synthesis of 6-hydroxy-L-tryptophan using tryptophan as a substrate via a cell factory 1) Fermentation and transformation of strains The tryptophan 6-hydroxylase gene (e.g., Xn269, SEQ ID NO: 1) was constructed into the expression vector pET28a. The tryptophan 6-hydroxylase gene is located between the multiple cloning restriction sites NdeI and BamHI. The constructed expression vector was further transformed into Escherichia coli, thereby realizing the construction of an engineered strain for the synthesis of 6-hydroxy-L-tryptophan.

[0023] 2) Fermentation and transformation of strains An engineered *E. coli* strain expressing Xn269 (tryptophan 6-hydroxylase) was activated on LB agar plates. Single colonies were picked and inoculated into LB liquid medium, and cultured overnight at 37°C and 220 rpm to prepare a seed culture. A 1% (v / v) inoculum was transferred to a 2 L Erlenmeyer flask containing 500 mL of M9CA medium (supplemented with 1% glycerol), and kanamycin was added to a final concentration of 50 μg / mL. The culture was then incubated at 37°C and 220 rpm until OD500 was reached. 600 When the pH was 0.8–1.0, 0.2 mM IPTG was added, and expression was induced at 18℃ and 150 r / min for 18–22 h. Subsequently, the temperature was adjusted to 30℃, and the culture was continued at 200 rpm for 24 h, during which 1 mM L-tryptophan was added as a substrate for transformation.

[0024] 3) Product detection and analysis Take 100 μL of fermentation broth, add 200 μL of methanol, vortex for 10 min, centrifuge at high speed (10 min), and collect the supernatant for analysis. Analyze using an LC-QTOF-MS system.

[0025] The results are as follows Figure 3 As shown, a new product was generated with a conversion rate of approximately 69%. High-resolution mass spectrometry results showed that its molecular weight was consistent with that of 6-hydroxy-L-tryptophan (theoretical value of [M+H]⁺: 221.0926, measured value: 221.0922), confirming it as the target product 6-hydroxy-L-tryptophan.

Claims

1. A tryptophan 6-hydroxylase, characterized in that, The amino acid sequence of the tryptophan 6-hydroxylase is SEQ ID NO:

1.

2. The tryptophan 6-hydroxylase of claim 1, wherein, The nucleotide sequence of the gene encoding the tryptophan 6-hydroxylase is SEQ ID NO:

2.

3. A recombinant engineered bacterial strain, characterized in that, The recombinant engineered strain carries a nucleic acid fragment encoding the tryptophan 6-hydroxylase of claim 1.

4. The application of the tryptophan 6-hydroxylase according to claim 1 in catalyzing the L-tryptophan 6-hydroxylation reaction.

5. A method for L-tryptophan 6-hydroxylation reaction, characterized in that, The method uses the tryptophan 6-hydroxylase of claim 1 to catalyze the substrate to generate 6-hydroxy-L-tryptophan.

6. The method as described in claim 5, characterized in that, The substrate is indole or -L-tryptophan.

7. The method as described in claim 5, characterized in that, The tryptophan 6-hydroxylase may be entirely or partially replaced with an enzyme whose amino acid sequence is SEQ ID NO:3 or SEQ ID NO:4.

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