Use of flavin-dependent halogenases

Abstract

This invention relates to the application of flavin-dependent halogenases, specifically disclosing the application of the following proteins, expression vectors containing nucleotide sequences encoding the proteins, or host cells capable of expressing the following proteins in catalyzing the bromination of natural polyphenolic compounds. The proteins are: (a) proteins as described in any amino acid sequence of SEQ ID No. 1-3, or (b) proteins having the function of the protein described in (a) formed by substitution, deletion, or addition of one or more amino acid residues of any of the amino acids described in SEQ ID No. 1-3. They exhibit broad activity towards natural polyphenolic compounds and excellent catalytic bromination activity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. Specifically, this invention relates to the application of proteins with flavin-dependent halogenase activity, expression vectors containing genes encoding these proteins, and genetically engineered bacteria capable of expressing these proteins in the catalytic bromination of natural polyphenolic compounds. Background Technology

[0002] Halides play a vital role in medicine and agriculture, and are essential intermediates in many synthetic routes. Introducing halogen substituents into organic compounds significantly impacts their bioactivity. Traditional chemical halogenation methods suffer from various drawbacks, such as the generation of toxic byproducts, lack of regioselectivity, or specific substrate requirements. Enzymatic halogenation has become a promising synthetic method in synthetic chemistry. Halogenating enzymes, as biocatalysts, can be used for the modification of molecular structures, the late-stage functionalization of complex molecules, and can also be combined with chemical catalysts.

[0003] Among different types of halogenases, flavin-dependent halogenases have received widespread attention and research as promising halogenases. However, to date, research reports on flavin-dependent halogenases mainly focus on tryptophan halogenases, which primarily undergo chlorination reactions. There are no research reports on brominases that catalyze polyphenolic compounds.

[0004] Natural polyphenols are secondary metabolites of plants, abundant in fruits, vegetables, and grains. Brominated polyphenols can have various functions, depending on their structure and application. For example, research suggests that 4'-bromo-resveratrol may be helpful in targeted therapy of gastric cancer stem cells. Some brominated polyphenols can be used as broad-spectrum antibacterial agents, killing many types of bacteria, fungi, and viruses; these compounds are widely used in water treatment, disinfection, medical, and agricultural fields. Brominated polyphenols can also be used as flame retardants, improving the combustion performance of combustible materials; these compounds are widely used in electronics, construction, and automotive industries. Some brominated polyphenols are used as pharmaceuticals to treat certain diseases, such as cancer and heart disease. Brominated polyphenols can be used as preservatives, extending the shelf life of food, wood, and textiles; they can also be used to prepare polymers, resins, coatings, and dyes for industrial manufacturing. Furthermore, brominated polyphenols can serve as intermediates, introducing other groups through cross-coupling reactions to synthesize other valuable compounds. However, to date, no brominases capable of catalyzing polyphenol compounds have been reported.

[0005] Therefore, exploring high-performance flavin-dependent brominases for catalyzing the bromination of polyphenolic compounds is of great significance for expanding research in this field. Summary of the Invention

[0006] The purpose of this invention is to provide an application of a flavin-dependent halogenase in the catalytic bromination of natural polyphenolic compounds. It exhibits broad activity towards natural polyphenolic compounds and excellent catalytic bromination activity.

[0007] To achieve the above technical objectives, the inventors conducted extensive experiments and relentless exploration, identifying flavin-dependent halogenases from many different bacterial strains. Through candidate gene cloning, heterologous expression, and catalytic activity analysis, three flavin-dependent halogenase genes with catalytic activity for the bromination of natural polyphenolic compounds were ultimately identified and screened. Based on this, the following technical solution was finally obtained:

[0008] This invention provides the use of the following proteins or expression vectors containing nucleotide sequences encoding the proteins or host cells capable of expressing the following proteins in catalyzing the bromination of natural polyphenolic compounds, wherein the proteins are: (a) proteins as described in any amino acid sequence of SEQ ID No. 1-3, or (b) proteins having the function of the protein described in (a) formed by substitution, deletion or addition of one or more amino acid residues of any of the amino acids described in SEQ ID No. 1-3.

[0009] According to the above scheme, the host cells include, but are not limited to, Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus oryzae.

[0010] According to the above scheme, the natural polyphenolic compounds include stilbene compounds, flavonoids, lignans, and other natural polyphenolic compounds.

[0011] According to the above scheme, the natural polyphenolic compounds include, but are not limited to, sesamin, paclitaxel, resveratrol, quercetin, naringenin, epicatechin, apigenin, and daidzein.

[0012] According to the above scheme, when the natural polyphenolic compound is trans-resveratrol, the protein (AmFDH5) shown in SEQ ID No. 1 catalyzes the bromination of trans-resveratrol to generate 4-bromoresveratrol and 3'-bromoresveratrol, and the protein (LaFDH2) shown in SEQ ID No. 2 or the protein (LaFDH4) shown in SEQ ID No. 3 catalyzes the bromination of resveratrol to generate 4-bromoresveratrol.

[0013] When the catalytic bromination substrate is sesamin, paclitaxel, resveratrol, quercetin, naringenin, epicatechin, apigenin, or daidzein, the catalytic bromination reaction products are brominated sesamin, brominated paclitaxel, brominated resveratrol, brominated quercetin, brominated naringenin, brominated epicatechin, brominated apigenin, or brominated daidzein.

[0014] According to the above scheme, a tag sequence is added to the end of the amino acid sequence of the protein shown in (a) or (b), and the protein has the function of the protein described in (a). The tag can specifically be a His tag, MBP tag, sumo tag, etc.

[0015] According to the above scheme, the application method is as follows: in the presence of flavin and flavin reductase, natural polyphenols are catalyzed to bromine natural polyphenols.

[0016] The advancements and advantages of this invention are as follows:

[0017] This invention is the first to discover, clone, and identify three flavin-dependent halogenase genes derived from bacteria such as *Alteromonas macleodii* and *Lacimicrobium alkaliphilum*, which possess catalytic activity for the bromination of natural polyphenolic compounds. The flavin-dependent halogenases provided by this invention have a broad substrate spectrum, excellent bromination catalytic activity, and promising prospects for industrial applications. Attached Figure Description

[0018] Figure 1 Structural formulas of natural polyphenol compounds;

[0019] Figure 2 AmFDH5 catalyzes the trans-resveratrol reaction;

[0020] Figure 3 LaFDH2 / LaFDH5 catalyzes the trans-resveratrol reaction;

[0021] Figure 4 HPLC was used to detect the resveratrol catalysis spectra of halogenases AmFDH5, LaFDH2, and LaFDH4. Detailed Implementation

[0022] Through extensive and in-depth research, the inventors unexpectedly discovered three flavin-dependent brominase genes with high bromination activity and a broad substrate spectrum for natural polyphenolic compounds. This discovery provides a foundation for developing superior flavin-dependent halogenases for the production of brominated polyphenols. Based on this, the present invention was completed.

[0023] The following description further illustrates the principles and features of the present invention. The listed embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention. Furthermore, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0024] Example 1: Construction of heterologous expression vector plasmid in Escherichia coli

[0025] Through extensive experimentation and relentless exploration, the inventors of this invention have identified flavin-dependent halogenases from various bacterial strains. Through candidate gene cloning, heterologous expression, and catalytic activity analysis, three flavin-dependent halogenase genes with polyphenol bromination activity were ultimately identified and screened, originating from bacteria such as *Alteromonas macleodii* and *Lacimicrobium alkaliphilum*. These identified protease genes were ligated into the pET28a vector to obtain recombinant plasmids. The constructed recombinant plasmids were transformed into *E. coli* DH5α competent cells and plated on solid LB agar plates containing 50 μg / mL kanamycin. Single colonies were picked for culture and plasmid extraction to obtain the recombinant plasmids. Colony PCR was used to verify the successful construction of the recombinant plasmids. Three flavin-dependent halogenases with catalytic activity for the bromination of polyphenolic compounds were successfully expressed, namely: the amino acid sequence of AmFDH5 is shown in SEQ ID No.1, the amino acid sequence of LaFDH2 is shown in SEQ ID No.2, and the amino acid sequence of LaFDH4 is shown in SEQ ID No.3.

[0026] Simultaneously, a tagged recombinant plasmid was constructed. Specifically, this invention constructed a recombinant plasmid with a histidine tag.

[0027] In view of the teachings of this invention and the prior art, those skilled in the art should also understand that the proteins described above with catalytic polyphenol compound bromination activity should further include variants of the proteins shown in any of the amino acid sequences of SEQ ID No. 1-3, with one or more (usually 1-30, preferably 1-10, more preferably 1-6, most preferably 1-3) amino acid deletions, insertions, and / or substitutions, having the same or similar functions as SEQ ID No. 1-3, and with one or more (usually up to 20, preferably up to 10, more preferably up to 6) amino acids added to the C-terminus and / or N-terminus. For example, adding one or more amino acids to the C-terminus and / or N-terminus, for example, with a tag added for ease of separation, generally does not change the function of the resulting protein.

[0028] Example 2: Catalytic activity experiment of Fl-Hals

[0029] Halogenases AmFDH5, LaFDH2, and LaFDH4 have no bromination activity on tryptophan substrates.

[0030] Halogenases AmFDH5, LaFDH2, and LaFDH4 require flavin reductases in the reaction, such as the flavin reductase RebF reported in the prior art, which provides FADH2 for the bromination of natural polyphenolic compounds. Cell lysis supernatant (cell OD) is used. 600 (20), in the presence of flavin and flavin reductase RebF, catalyzed the reaction of 0.25 mM substrate for 10 h at 25 °C and pH 7.4, followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Based on the mass spectrometry and HPLC results, the catalytic bromination activity was analyzed, and the substrate conversion rate was calculated. The bromination activities of AmFDH5, LaFDH2, and LaFDH4 for common representative natural polyphenols—sesamin, leucopicrin, trans-resveratrol, quercetin, naringenin, epicatechin, apigenin, and daidzein—are shown in Table 1. The structural formulas of common representative natural polyphenols are shown in [Table 1]. Figure 1 .

[0031] Table 1

[0032] AmFDH5 LaFDH2 LaFDH4 trans-resveratrol 100％ 100％ 100％ Sesamin 27％ 24％ 50％ Paclitaxel 100％ 100％ 100％ Quercetin 23％ 33％ 40％ Naringin 42％ 20％ 32％ Catechin 42％ 22％ 22％ Celery 30％ 23％ 64％ daidzein 12％ 8％ 30％

[0033] HPLC analysis of resveratrol catalyzed by halogenases AmFDH5, LaFDH2, and LaFDH4 is shown in the following chromatograms. Figure 4 As shown, nuclear magnetic resonance characterization of the resveratrol bromination products confirmed that AmFDH5 catalyzes the bromination of trans-resveratrol to produce 4-bromoresveratrol and 3'-bromoresveratrol; LaFDH2 or LaFDH4 catalyzes the bromination of trans-resveratrol to produce 4-bromoresveratrol, as detailed below.

[0034] The flavin-dependent halogenases AmFDH5, LaFDH2, and LaFDH4 of the present invention can selectively brominate trans-resveratrol at different positions.

[0035] As shown in SEQ ID No. 1, AmFDH5 catalyzes the bromination of trans-resveratrol to produce 4-bromoresveratrol and 3'-bromoresveratrol. Further, pure 4-bromoresveratrol and 3'-bromoresveratrol were prepared by separation, and their structures were identified as 4-bromoresveratrol and 3'-bromoresveratrol by NMR analysis. The reaction formula is as follows. Figure 2 As shown.

[0036] For example, LaFDH2 as shown in SEQ ID No. 2 or LaFDH4 as shown in SEQ ID No. 3 catalyzes the bromination of trans-resveratrol to produce 4-bromoresveratrol, and NMR analysis identifies its structure as 4-bromoresveratrol. The reaction formula is as follows. Figure 3 As shown.

[0037] The flavin-dependent halogenases AmFDH5, LaFDH2, and LaFDH4 of this invention exhibit bromination activity against common and representative natural polyphenol compounds tested. Liquid chromatography-mass spectrometry analysis confirmed the formation of corresponding bromination products after catalyzing different substrates.

[0038] The activities of His-tagged and His-free halogenases AmFDH5, LaFDH2, and LaFDH4 on resveratrol were measured. The results showed that the addition of the tag did not affect the catalytic bromination activity of the untagged halogenases, and the catalytic bromination activity did not fluctuate significantly compared to the untagged halogenases. Specifically, the His-tagged halogenases AmFDH5, LaFDH2, and LaFDH4 maintained approximately 90%, 95%, and 90% of the activity of the untagged halogenases, respectively, for the catalytic bromination of resveratrol.

[0039] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of the following protein or an expression vector containing a nucleotide sequence encoding the protein or a host cell capable of expressing the following protein in catalyzing the bromination of natural polyphenolic compounds, wherein the protein is: (a) a protein as described in any amino acid sequence of SEQ ID No. 1-3, or a protein shown in (a) with a tag sequence added to the end of its amino acid sequence and having the function of the protein described in (a), wherein the natural polyphenolic compound is sesamin, paclitaxel, trans-resveratrol, quercetin, naringenin, epicatechin, or apigenin, wherein the application is: catalyzing the bromination of natural polyphenolic compounds to synthesize brominated natural polyphenolic compounds in the presence of flavin and flavin reductase.

2. Use according to claim 1, characterized in that: When the natural polyphenolic compound is trans-resveratrol, the protein shown in SEQ ID No. 1 catalyzes the bromination of trans-resveratrol to generate 4-bromoresveratrol and 3'-bromoresveratrol, and the protein shown in SEQ ID No. 2 or SEQ ID No. 3 catalyzes the bromination of trans-resveratrol to generate 4-bromoresveratrol.

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