A type of flavonoid reductase and its application

Abstract

The present invention relates to a flavonoid reductase and an application thereof. Specifically, the flavonoid reductase of the present invention can catalyze the reduction of flavonoid compounds / flavonol compounds into dihydroflavonoid compounds / dihydroflavonol compounds; and catalyze the oxidation of dihydroflavonoid compounds / dihydroflavonol compounds into flavonoid compounds / flavonol compounds.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology; more specifically, the present invention relates to a flavonoid reductase and application thereof. Background Art

[0002] An important class of natural products in human food is flavonoids, which are the most common polyphenolic compounds in nature and are widely found in vegetable, fruit, grain and other plant foods. Flavonoids have inhibitory and killing effects on a variety of bacteria in vivo and in vitro experiments, and have anti-inflammatory, anti-tumor and antioxidant physiological activities on mammalian cells. They can also be used as drugs for clinical treatment of patients with cardiovascular diseases. Intestinal bacteria have long been subjected to antibacterial stress from flavonoids in the coexistence of flavonoids in the intestinal environment, and have evolved a variety of enzymes that can degrade flavonoids, so that they undergo chemical catalytic processes such as hydrolysis, dehydroxylation and reduction, and metabolize into small molecules such as phenolic acid, which not only eliminates the biological toxicity of flavonoids, but also uses the small molecules produced by their decomposition as nutrition for bacteria. Therefore, the degradation of flavonoids by intestinal bacteria directly affects the biological activity and bioavailability of flavonoids.

[0003] Flavonoids and flavonols are important members of the flavonoid class, with flavonols being the most common, accounting for about one-third of the flavonoid class, followed by flavonoids, which account for more than a quarter of the total. Among the known flavonoids, apigenin is the most typical, and its physiological and pharmacological activity mechanisms have been well studied.

[0004] At present, the enzyme gene responsible for the first key reaction of flavonoid degradation - the hydrogenation reduction of the C2-C3 double bond to dihydroflavonoids - has not been discovered and identified, and the underlying reaction mechanism is also unclear. This is an important gap in our current understanding of microbial metabolism of flavonoids.

[0005] Therefore, there is an urgent need in the art to develop an enzyme that can catalyze the reversible redox reaction of flavones / flavonols and dihydroflavones / dihydroflavonols. Summary of the invention

[0006] The object of the present invention is to provide an enzyme capable of catalyzing the reversible redox reaction of flavones / flavonols and dihydroflavones / dihydroflavonols, and the enzyme is named as flavone reductase (FLR).

[0007] Another object of the present invention is to provide amino acid sequences of flavonoid reductases from different species and their corresponding DNA sequence information.

[0008] Another object of the present invention is to provide information on substrates and corresponding products catalyzed by flavonoid reductase.

[0009] Another object of the present invention is to provide a method for constructing an in vitro catalytic system of flavone reductase and optimal catalytic conditions.

[0010] Another object of the present invention is to provide the catalytic characteristics and enzymatic parameter information of flavonoid reductase.

[0011] The first aspect of the present invention provides an isolated flavonoid reductase, wherein the flavonoid reductase is selected from:

[0012] (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; or

[0013] (b) a polypeptide derived from (a) having flavone reductase activity, which is obtained by substituting, deleting or adding one or more (e.g., 1-20, preferably 1-10, more preferably 1-5, and most preferably 1-3) amino acid residues in the amino acid sequence shown in SEQ ID NO: 1; or

[0014] (c) a polypeptide derived from (a) having at least 85% (preferably at least 90%; more preferably at least 95%, more preferably at least 98% or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having flavone reductase activity.

[0015] In another preferred embodiment, the flavonoid reductase is in a form selected from the group consisting of bacteria, crude enzyme solution, pure enzyme, crude enzyme powder, immobilized enzyme, free enzyme, fermentation broth, or a combination thereof.

[0016] In a preferred example, the flavonoid reductase activity refers to the activity of catalyzing the reduction of flavonoid compounds / flavonol compounds to dihydroflavonoid compounds / dihydroflavonol compounds; and catalyzing the oxidation of dihydroflavonoid compounds / dihydroflavonol compounds to flavonoid compounds / flavonol compounds.

[0017] In another preferred embodiment, the flavonoid reductase catalyzes the following reaction:

[0018]

[0019] Among them, R1 is H or OH, R2 is OH, R3 is H, R4 is OH, R5 is H, R6 is H, OH or -O-C1-C4 alkyl, R7 is OH, H or -O-C1-C4 alkyl, and R8 is H or OH.

[0020] In another preferred embodiment, R7 is OH, H or O-CH3.

[0021] In another preferred embodiment, the compound of formula I after substitution with R1-R8, and the compound of formula II converted from the compound of formula I are shown in the following table:

[0022]

[0023] In another preferred embodiment, when R is H, the compound of formula I is a flavonoid compound, and the compound of formula II is a dihydroflavonoid compound.

[0024] In another preferred embodiment, when R is OH, the compound of formula I is a flavonol compound, and the compound of formula II is a dihydroflavonol compound.

[0025] In another preferred embodiment, the reaction has one or more characteristics selected from the following group:

[0026] (i) the pH of the reaction system is 4-10, preferably 5-9, more preferably 6-8;

[0027] (ii) the reaction temperature is 20-50°C, preferably 25-45°C, more preferably 30-42°C, more preferably 35-40°C;

[0028] (iii) the reaction time is 10 min to 10 h, preferably 20 min to 5 h, more preferably 30 min to 1.5 h;

[0029] (iv) k of catalytic reduction reaction cat The value is 0.005s -1 -0.040s -1 , preferably, 0.012s -1 -0.020s -1 ;

[0030] (v) the Km value of the catalytic reduction reaction is 2-20 μM, preferably 4-15 μM, more preferably 6-10 μM;

[0031] (vi) k of catalytic oxidation reaction cat The value is 0.015s -1 -0.100s -1 , preferably, 0.040s -1 -0.080s -1 ;

[0032] (vii) The Km value of the catalytic oxidation reaction is 3-50 μM, preferably 6-30 μM, and more preferably 10-20 μM.

[0033] In another preferred embodiment, the flavonoid compound is selected from the following group: chrysin, apigenin, luteolin, diosgenin, or a combination thereof.

[0034] In another preferred embodiment, the flavonol compound is selected from the following group: kaempferol, quercetin, morin, myricetin, isorhamnetin, or a combination thereof.

[0035] In another preferred embodiment, the dihydroflavonoid compound is selected from the following group: naringenin, bromocriptine, eriodictyol, dihydrodioscorein, or a combination thereof.

[0036] In another preferred embodiment, the dihydroflavonol compound is selected from the following group: dihydromorin, citrus aurantium, taxol, ampelopsis, dihydroisorhamnetin, or a combination thereof.

[0037] In another preferred embodiment, when the compound of formula I is apigenin, the compound of formula II is naringenin.

[0038] In another preferred embodiment, when the compound of formula I is chrysin, the compound of formula II is chrysin.

[0039] In another preferred embodiment, when the compound of formula I is luteolin, the compound of formula II is eriodictyol.

[0040] In another preferred embodiment, when the compound of formula I is diosmetin, the compound of formula II is dihydrodiosmetin.

[0041] In another preferred embodiment, when the compound of formula I is kaempferol, the compound of formula II is citrus aurantium.

[0042] In another preferred embodiment, when the compound of formula I is quercetin, the compound of formula II is taxifolin.

[0043] In another preferred embodiment, when the compound of formula I is morin, the compound of formula II is dihydromorin.

[0044] In another preferred embodiment, when the compound of formula I is myricetin, the compound of formula II is ampelopsis.

[0045] In another preferred embodiment, when the compound of formula I is isorhamnetin, the compound of formula II is dihydroisorhamnetin.

[0046] In another preferred embodiment, the flavonoid reductase is derived from humans, animals, plants or microorganisms.

[0047] In another preferred embodiment, the flavonoid reductase is derived from a prokaryotic microorganism, more preferably an intestinal bacterium.

[0048] In another preferred embodiment, the flavonoid reductase is derived from the class Clostridium of the phylum Firmicutes.

[0049] In another preferred embodiment, the flavonoid reductase is derived from Clostridium orbiscindens (fpla), Clostridium beijerinckii (NCIMB 8052, cbe), Clostridium difficile (cdc), Clostridium ljungdahlii (clj) or Clostridium butyricum (cbut).

[0050] In another preferred embodiment, the flavonoid reductase is derived from Clostridium (Clostridium: Clostridiumorbiscindens, fpla; Clostridium beijerinckii NCIMB 8052, cbe; Clostridiumacetobutylicum ATCC 824, cac; Clostridium ljungdahlii, clju; Clostridiumcarboxidivorans, ccar), Streptomyces (Streptomyces: Streptomyces scabiei, scb), Pestalotiopsis (Pestalotiopsis: Pestalotiopsis fici, pfy), and Alternaria (Alternaria: Alternaria alternata, aalt). Fusobacterium (Fusobacterium: Fusobacteriumvarium, fva; Fusobacterium mortiferum, fmo), Brachyspira (Brachyspira: Brachyspirapilosicoli B2904, bpj; Brachyspira pilosicoli WesB, bpw).

[0051] In another preferred embodiment, the flavonoid reductase is derived from Clostridium orbiscindens (fpla), Clostridium beijerinckii (CBE), Clostridium acetobutylicum ATCC 824 (cac), Streptomyces cesscabiei (scb), Alternaria alternata (aalt), Clostridium ljungdahlii (clju), Clostridium carboxidivorans (ccar), Fusobacterium varium (fva), Fusobacterium mortiferum (fmo), Brachyspira pilosicoli WesB (Brachyspira pilosicoli WesB, bpw), Pestalotiopsis fici, pfy, Brachyspira pilosicoli B2904, bpj.

[0052] In another preferred embodiment, the sequence (c) further includes: a fusion protein formed by adding a tag sequence, a signal sequence or a secretion signal sequence to (a) or (b).

[0053] The second aspect of the present invention provides an isolated polynucleotide, which encodes the flavone reductase described in the first aspect of the present invention.

[0054] In another preferred embodiment, the polynucleotide is selected from the following group:

[0055] (a) a polynucleotide encoding the polypeptide shown in SEQ ID NO.1;

[0056] (b) a polynucleotide whose sequence is shown in any one of SEQ ID NOs. 2-4;

[0057] (c) a polynucleotide having a nucleotide sequence homology of ≥95% (preferably ≥98%, more preferably ≥99%) to the polynucleotide sequence shown in (b), and encoding the polypeptide shown in SEQ ID NO.1;

[0058] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0059] In another preferred embodiment, the polynucleotide further contains auxiliary elements selected from the following group on the flank of the ORF of flavonoid reductase: a signal peptide, a secretory peptide, a tag sequence (such as 6His), or a combination thereof.

[0060] In another preferred embodiment, the polynucleotide is selected from the following group: a DNA sequence, an RNA sequence, or a combination thereof.

[0061] In another preferred embodiment, the nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NOs: 2-4.

[0062] The third aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the second aspect of the present invention.

[0063] In another preferred embodiment, the vector includes an expression vector, a shuttle vector, and an integration vector.

[0064] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect of the present invention, or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

[0065] In another preferred embodiment, the host cell is a eukaryotic cell, including yeast cells, fungal cells, insect cells, mammalian cells, and plant cells.

[0066] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0067] The fifth aspect of the present invention provides a method for producing the flavone reductase according to the first aspect of the present invention, comprising the steps of:

[0068] Cultivating the host cell according to the fourth aspect of the present invention under conditions suitable for expression, thereby expressing flavonoid reductase; and

[0069] The flavonoid reductase is isolated.

[0070] The sixth aspect of the present invention provides an enzyme preparation, characterized in that the enzyme preparation comprises the flavonoid reductase described in the first aspect of the present invention.

[0071] In another preferred embodiment, the enzyme preparation includes an injection and / or a lyophilized preparation.

[0072] A seventh aspect of the present invention provides a method for catalyzing the redox reaction of a flavonoid compound / flavonol compound and a dihydroflavonoid compound / dihydroflavonol compound, comprising the steps of:

[0073] In the reaction system, the flavonoid reductase is contacted with a reaction substrate.

[0074] In another preferred embodiment, the flavonoid reductase is derived from humans, animals, plants or microorganisms.

[0075] In another preferred embodiment, the flavonoid reductase is derived from a prokaryotic microorganism, more preferably an intestinal bacterium.

[0076] In another preferred embodiment, the flavonoid reductase is derived from the class Clostridium of the phylum Firmicutes.

[0077] In another preferred embodiment, the flavonoid reductase is derived from Clostridium orbiscindens (fpla), Clostridium beijerinckii (NCIMB 8052, cbe), Clostridium difficile (cdc), Clostridium ljungdahlii (clj) or Clostridium butyricum (cbut).

[0078] In another preferred embodiment, the flavonoid reductase is derived from Clostridium (Clostridium: Clostridiumorbiscindens, fpla; Clostridium beijerinckii NCIMB 8052, cbe; Clostridiumacetobutylicum ATCC 824, cac; Clostridium ljungdahlii, clju; Clostridiumcarboxidivorans, ccar), Streptomyces (Streptomyces: Streptomyces scabiei, scb), Pestalotiopsis (Pestalotiopsis: Pestalotiopsis fici, pfy), and Alternaria (Alternaria: Alternaria alternata, aalt). Fusobacterium (Fusobacterium: Fusobacteriumvarium, fva; Fusobacterium mortiferum, fmo), Brachyspira (Brachyspira: Brachyspirapilosicoli B2904, bpj; Brachyspira pilosicoli WesB, bpw).

[0079] In another preferred embodiment, the flavonoid reductase is derived from Clostridium orbiscindens (fpla), Clostridium beijerinckii (CBE), Clostridium acetobutylicum ATCC 824 (cac), Streptomyces cesscabiei (scb), Alternaria alternata (aalt), Clostridium ljungdahlii (clju), Clostridium carboxidivorans (ccar), Fusobacterium varium (fva), Fusobacterium mortiferum (fmo), Brachyspira pilosicoli WesB (Brachyspira pilosicoli WesB, bpw), Pestalotiopsis fici, pfy, Brachyspira pilosicoli B2904, bpj.

[0080] In another preferred embodiment, the flavonoid reductase is a wild type or a mutant.

[0081] In another preferred embodiment, the flavonoid reductase is selected from:

[0082] (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; or

[0083] (b) a polypeptide derived from (a) having flavone reductase activity, which is obtained by substituting, deleting or adding one or more (e.g., 1-20, preferably 1-10, more preferably 1-5, and most preferably 1-3) amino acid residues in the amino acid sequence shown in SEQ ID NO: 1; or

[0084] (c) a polypeptide derived from (a) having at least 85% (preferably at least 90%; more preferably at least 95%, more preferably at least 98% or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having flavone reductase activity.

[0085] In another preferred embodiment, the amino acid sequence of the flavonoid reductase has at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence shown in SEQ ID NO.1.

[0086] In another preferred embodiment, the flavonoid reductase is in a form selected from the group consisting of resting cells, bacteria, crude enzyme solution, pure enzyme, crude enzyme powder, immobilized enzyme, free enzyme, fermentation broth, or a combination thereof.

[0087] In another preferred embodiment, in the reaction system, the concentration of the flavonoid reductase is 0.01-100 μM, preferably, 0.1-50 μM, and more preferably, 0.5-20 μM.

[0088] In another preferred embodiment, the concentration of the product of the reaction is 0.01-1 mM, preferably, 0.05-0.5 mM, and more preferably, 0.1-0.2 μM.

[0089] In another preferred embodiment, the reaction system further comprises: FMNH2 and FMN.

[0090] In another preferred embodiment, the reaction substrate is

[0091] Among them, R1 is H or OH, R2 is OH, R3 is H, R4 is OH, R5 is H, R6 is H, OH or O-C1-C4 alkyl, R7 is OH, H or O-C1-C4 alkyl, and R8 is H or OH.

[0092] In another preferred embodiment, when the reaction substrate is a compound of formula I, the reaction product is a compound of formula II.

[0093] In another preferred embodiment, when the reaction substrate is a compound of formula II, the reaction product is a compound of formula I.

[0094] In another preferred embodiment, the reaction has one or more characteristics selected from the following group:

[0095] (i) the pH of the reaction system is 4-10, preferably 5-9, more preferably 6-8;

[0096] (ii) the reaction temperature is 20-50°C, preferably 25-45°C, more preferably 30-42°C, more preferably 35-40°C;

[0097] (iii) the reaction time is 10 min to 10 h, preferably 20 min to 5 h, more preferably 30 min to 1.5 h;

[0098] (iv) k of catalytic reduction reaction cat The value is 0.005s -1 -0.040s -1 , preferably, 0.012s -1 -0.020s-1 ;

[0099] (v) the Km value of the catalytic reduction reaction is 2-20 μM, preferably 4-15 μM, more preferably 6-10 μM;

[0100] (vi) k of catalytic oxidation reaction cat The value is 0.015s -1 -0.100s -1 , preferably, 0.040s -1 -0.080s -1 ;

[0101] (vii) The Km value of the catalytic oxidation reaction is 3-50 μM, preferably 6-30 μM, and more preferably 10-20 μM.

[0102] The eighth aspect of the present invention provides a use of the flavone reductase described in the first aspect of the present invention or the host cell described in the fourth aspect of the present invention, for catalyzing the reduction of flavonoid compounds / flavonol compounds to dihydroflavonoid compounds / dihydroflavonol compounds; and / or catalyzing the oxidation of dihydroflavonoid compounds / dihydroflavonol compounds to flavonoid compounds / flavonol compounds; or for preparing a catalytic preparation that catalyzes the reduction of flavonoid compounds / flavonol compounds to dihydroflavonoid compounds / dihydroflavonol compounds; and / or catalyzes the oxidation of dihydroflavonoid compounds / dihydroflavonol compounds to flavonoid compounds / flavonol compounds.

[0103] In another preferred embodiment, the flavonoid compound / flavonol compound has a structure shown in Formula I:

[0104]

[0105] Among them, R1 is H or OH, R2 is OH, R3 is H, R4 is OH, R5 is H, R6 is H, OH or O-C1-C4 alkyl, R7 is OH, H or O-C1-C4 alkyl, and R8 is H or OH.

[0106] In another preferred embodiment, the dihydroflavonoid compound / dihydroflavonol compound has a structure shown in Formula II:

[0107]

[0108] Among them, R1 is H or OH, R2 is OH, R3 is H, R4 is OH, R5 is H, R6 is H, OH or O-C1-C4 alkyl, R7 is OH, H or O-C1-C4 alkyl, and R8 is H or OH.

[0109] Other aspects of the present invention are obvious to those skilled in the art due to the disclosure herein. It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 showed the discovery and identification of flavonoid reductase, Figure 1 a shows the degradation of flavonoids and flavonols by intestinal microorganisms into aromatic derivatives (only the parent nucleus is shown, and the side chain groups are not shown). Figure 1 b shows that isoflavone reductase IFR can catalyze the reduction of the C2-C3 double bond of isoflavones, but cannot catalyze the reduction of flavonoids. Figure 1 c shows the prediction of flavonoid reductase candidate genes by comparative genomics analysis of downstream genes in the flavonoid degradation pathway. Figure 1 d shows that A4U99_05915 was expressed in E. coli and identified as a flavone reductase gene;

[0111] Fermentation conditions: TB medium (5 ml), N2 (0.2 MPa), 5% inoculum size (v / v).

[0112] Figure 2 The establishment of an in vitro catalytic system for flavonoid reductase and the analysis of its enzymatic properties are shown. Figure 2 a shows the UV-Vis absorption spectrum of FLR enzyme solution, indicating that FMN is a possible cofactor. Figure 2 b shows the reaction system of coupling NADH to generate FMNH2, in which apigenin is reduced to naringenin by flavonoid reductase. Figure 2 c shows the liquid chromatography verification that FLR can catalyze the interconversion of apigenin and naringenin, Figure 2 d shows the verification by liquid chromatography-mass spectrometry that FLR can catalyze the mutual conversion of apigenin and naringenin. Figure 2 e shows the optimal catalytic conditions for FLR and the identification of the Michaelis-Menten curve, and the figure shows the results of three biological replicates.

[0113] Figure 3 It was shown that FLR is a type of reductase that can specifically catalyze the hydrogenation of the C2-C3 bond of the flavonoid and flavonol nucleus.

[0114] Figure 4 It shows that FLR is ubiquitous in different microorganisms. Figure 4 a shows the protein sequence and conservative amino acid residue analysis of flavonoid reductase in common Clostridium, Figure 4 b shows the evolutionary relationship of FLR proteins from different microbial sources, Figure 4 c shows the in vitro enzyme activity assay of FLR proteins from 12 different microbial hosts. Figure 4 d shows Figure 4 c Species names and gene numbers corresponding to the flavonoid reductases. DETAILED DESCRIPTION

[0115] Through extensive and in-depth research, the inventors unexpectedly screened out a flavonoid reductase that can catalyze the redox reaction of flavonoids / flavonols (compounds of formula I) and dihydroflavonoids / dihydroflavonols (compounds of formula II) through a large number of screenings. Specifically, the present invention first discovered that the flavonoid reductase of the present invention can catalyze the reduction of flavonoids / flavonols to dihydroflavonoids / dihydroflavonols; and catalyze the oxidation of dihydroflavonoids / dihydroflavonols to flavonoids / flavonols. On this basis, the present invention was completed.

[0116] Group Definition

[0117] As used herein, the term "-O-C1-C4 alkyl" refers to a group having a (C1-C4 alkyl)-O- structure, for example, CH3-O-, C2H5-O-, C3H8-O-, or the like.

[0118] Enzymes

[0119] The flavonoid reductase disclosed in the present invention can be naturally occurring, such as it can be isolated or purified from plants or microorganisms. In addition, the enzyme can also be artificially prepared, such as recombinant enzymes can be produced according to conventional genetic engineering recombination techniques. Preferably, the present invention can apply recombinant enzymes.

[0120] The flavonoid reductase includes the full-length enzyme or its biologically active fragment (or called active fragment). The amino acid sequence of the enzyme formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The meaning of the biologically active fragment of the enzyme refers to a polypeptide that can still maintain all or part of the functions of the full-length enzyme. Generally, the biologically active fragment maintains at least 50% of the activity of the full-length enzyme. Under more preferred conditions, the active fragment can maintain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the activity of the full-length enzyme. The enzyme or its biologically active fragment includes a replacement sequence of a portion of conservative amino acids, and the sequence replaced by amino acids does not affect its activity or retains part of its activity. Appropriate replacement of amino acids is a well-known technique in the art, which can be easily implemented and ensures that the biological activity of the resulting molecule is not changed. These techniques have made those skilled in the art realize that, in general, changing a single amino acid in a non-essential region of a polypeptide will not substantially change the biological activity. See Watson et al. Molecular Biology of The Gene, 4th edition, 1987, The Benjamin / Cummings Pub. Co. P224.

[0121] The present invention may also employ modified or improved flavonoid reductases, for example, enzymes modified or improved to improve their half-life, effectiveness, metabolism and / or efficacy. The modified or improved enzymes may have little in common with naturally occurring enzymes, but may function the same or substantially the same as the wild-type enzymes without causing other adverse effects. In other words, any variation that does not affect the biological activity of the enzyme may be employed in the present invention.

[0122] The present invention also includes the isolated nucleic acid encoding the biologically active fragment of the flavonoid reductase, or the complementary strand thereof. As a preferred embodiment of the present invention, the coding sequence of the enzyme can be codon optimized to improve the expression efficiency. The DNA sequence encoding the biologically active fragment of the enzyme can be synthesized artificially in the whole sequence, or can be obtained by PCR amplification. After obtaining the DNA sequence encoding the biologically active fragment of the enzyme, it is connected to a suitable expression construct (such as an expression vector) and then transferred to a suitable host cell. Finally, the desired protein is obtained by culturing the transformed host cell.

[0123] The present invention also includes an expression construct comprising a nucleic acid molecule encoding a biologically active fragment of the flavone reductase. The expression construct may include a gene expression cassette encoding the enzyme, and may also include an expression control sequence operably connected to the sequence of the nucleic acid molecule to facilitate protein expression. The design of the expression control sequence is well known in the art. In the expression control sequence, an inducible or constitutive promoter may be used according to different needs. The inducible promoter can achieve more controllable protein expression and compound production, which is conducive to industrial application.

[0124] Expression vector

[0125] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0126] The polynucleotide sequences of the present invention can be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0127] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0128] (2) Host cells cultured in a suitable culture medium;

[0129] (3) Isolate and purify proteins from culture medium or cells.

[0130] In the present invention, the polynucleotide sequence encoding the protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0131] Methods well known to those skilled in the art can be used to construct expression vectors containing the protein encoding DNA sequence of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0132] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0133] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0134] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells.

[0135] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0136] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0137] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0138] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0139] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0140] Method for catalyzing the redox reaction of flavonoids / flavonols and dihydroflavonoids / dihydroflavonols

[0141] The present invention discloses a method for heterologously producing flavonoids / flavonols or dihydroflavonoids / dihydroflavonols by using microorganisms. The method uses bioengineering technology to express relevant enzymes and catalyze the redox reaction between flavonoids / flavonols and dihydroflavonoids / dihydroflavonols in vitro.

[0142] A method for producing flavonoids / flavonols or dihydroflavonoids / dihydroflavonols is: recombinantly express the flavonoid reductase of the present invention, preferably, using host cells (prokaryotic cells or eukaryotic cells, such as commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, etc.; commonly used eukaryotic host cells include fungal cells, insect cells and mammalian cells, etc.) to recombinantly express the above enzyme. The expressed enzyme or the host cell capable of expressing the above enzyme is contacted with the substrate flavonoids / flavonols or dihydroflavonoids / dihydroflavonols, thereby catalyzing the redox reaction between the flavonoids / flavonols and the dihydroflavonoids / dihydroflavonols.

[0143] The main advantages of the present invention include:

[0144] (1) The present invention first discovered that the flavonoid reductase of the present invention can specifically catalyze the redox reaction between flavonoid compounds / flavonol compounds and dihydroflavonoid compounds / dihydroflavonol compounds.

[0145] (2) The present invention first discovered that the FMN-dependent flavonoid reduction reaction catalyzed by the flavonoid reductase of the present invention is a reversible reaction.

[0146] (3) The present invention first discovered that the flavone reductase of the present invention is a flavin-dependent oxidoreductase.

[0147] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0148] Unless otherwise specified, the reagents and materials in the examples of the present invention are all commercially available products.

[0149] Example 1 Discovery and verification of flavonoid reductase FLR

[0150] Experimental methods:

[0151] The human intestinal flora can metabolize flavonoids, among which the key enzymes responsible for the initiation of the metabolic pathways of the two main types of compounds, flavonoids and flavonols, have not yet been discovered and identified ( Figure 1a). Potential candidate genes of flavonoid reductase were predicted by comparative genomics, and the candidate genes were cloned one by one into plasmids and introduced into Escherichia coli BL21 (DE3) for expression. Anaerobic fermentation was carried out in a medium supplemented with apigenin to determine whether the target dihydroflavonoids were produced.

[0152] Experimental steps:

[0153] 1) We tested whether the reported flavonoid reductase, isoflavone reductase IFR, can catalyze the reduction reaction of flavonoids and found that IFR can only specifically catalyze the reduction of isoflavones. This indicates that the flavonoid reduction reaction is responsible for a unique reductase. Figure 1 b.

[0154] 1) We first used the position-specific iterative (PSI)-BLAST tool to find the homologous genes A4U99_05920 and A4U99_15225 of the above two genes in Clostridium orbiscindens.

[0155] 2) The two genes in 1) were input into STRING software for further analysis. We found that there were 4 and 10 genes that were adjacent or co-occurring with A4U99_05920 and A4U99_15225 in the genomic structure, respectively ( Figure 1 c) The flavonoid reductase we are looking for may exist among these 14 genes.

[0156] 3) The 14 genes in 2) were cloned into plasmids one by one and introduced into Escherichia coli BL21 (DE3) for expression. Anaerobic fermentation was performed in a medium supplemented with apigenin to determine whether the target dihydroflavonoids were produced ( Figure 1 d).

[0157] Experimental results:

[0158] The 14 predicted genes were cloned into plasmids one by one and introduced into E. coli BL21 (DE3) for expression. As expected, the E. coli strain containing the A4U99_05915 gene was able to reduce apigenin in the culture medium to naringenin ( Figure 1 d), which suggests that the product encoded by this gene is very likely to be our target protein - flavonoid reductase FLR. Here, the enzyme is named FLR. ( Figure 1 d).

[0159] Example 2 Analysis of biochemical properties of flavonoid reductase

[0160] Experimental methods:

[0161] A coupling reaction system based on flavin cofactor was constructed to realize the in vitro catalytic reaction of flavonoid reductase. The catalytic efficiency of the enzyme's flavonoid reduction reaction and dihydroflavonoid oxidation reaction was detected at different pH values, different temperatures and different substrate concentrations to analyze the enzymatic properties of FLR.

[0162] Experimental steps:

[0163] 1) According to the two characteristic absorption peaks of 380nm and 447nm appearing in the full wavelength scanning results of the enzyme extract ( Figure 2 a), It is preliminarily judged that FLR is a flavin-dependent oxidoreductase.

[0164] 2) A flavin cofactor-based coupling reaction system was constructed to analyze the enzymatic properties of FLR. Figure 2 As shown in b, the reaction system produces reduced flavin cofactor FMNH2 through NADH-dependent flavin oxidoreductase, and uses it as an electron donor for the next step of flavonoid reductase-catalyzed FMNH2 reduction of apigenin.

[0165] 3) Using the enzyme reaction system constructed in 2), the catalytic efficiency of the enzyme was detected at different pH values, different temperatures and different substrate concentrations, and the amount of product generated per unit time was determined by high performance liquid chromatography ( Figure 2 d).

[0166] Experimental results:

[0167] As expected, the results of enzyme activity assays showed that when the cofactor was FMNH2, FLR could reduce the C2-C3 double bond in apigenin to a saturated single bond, thereby generating naringenin ( Figure 2 c, d), proving that FLR is an FMN-dependent oxidoreductase. When we replace the flavin cofactor FMNH2 in the reaction system with its oxidized FMN, FLR can reversely catalyze the above reaction, oxidizing the C2-C3 saturated single bond of naringenin to a double bond, thereby regenerating apigenin ( Figure 2 c) This indicates that the FMN-dependent flavonoid reduction reaction catalyzed by FLR is a reversible reaction.

[0168] We further tested the optimal reaction conditions of FLR. The results showed that when the concentration of FLR was 0.1 μM and the concentration of substrate apigenin was 100 μM, the optimal reaction pH and temperature of FLR were 7.0 and 37 °C, respectively. Under this condition, the k of FLR-catalyzed reduction of apigenin to naringenin was cat and Km values ​​were 1.65×10-2s-1 and 8.36μM, respectively. When the concentration of FLR was 0.1μM and the concentration of substrate naringenin was 100μM, the k value of the reverse reaction, i.e., the oxidation of naringenin to apigenin, wascat and Km values ​​were 5.77×10-2s-1 and 12.27μM ( Figure 2 e).

[0169] Example 3 Universality of flavonoid reductase substrates

[0170] Experimental methods:

[0171] Under the constructed enzyme catalytic system, different types of representative flavonoid substrates were selected to test the catalytic activity of flavonoid reductase.

[0172] Experimental steps:

[0173] We selected 14 representative flavonoid compounds from the main categories of flavonoids (flavones, flavonols, isoflavones, anthocyanins and chromones) for enzyme activity tests.

[0174] Experimental results:

[0175] The results showed that FLR had catalytic activity towards these substances and showed good substrate adaptability ( Figure 3 ). Moreover, through comparison, it was found that if there are more or larger substituents on the B ring of these flavonoids, the catalytic efficiency of FLR for substrates will be reduced. For example, the ability of FLR to catalyze the reduction of chrysin, apigenin, and luteolin showed a decreasing trend, and the efficiency of FLR to catalyze the reduction of flavonoids (dioscorea odorifera) with a larger methoxyl group on the parent nucleus was significantly lower than that of other flavonoids. FLR had no activity on citrus erythritol and nobiletin ( Figure 3 ). Given that flavonols are structurally very similar to flavones (the only difference is whether the H at C3 of the C ring is replaced by OH), we also tested whether FLR can reduce flavonol substrates. The results showed that FLR has catalytic activity for several major flavonols, kaempferol, quercetin, morin, and myricetin ( Figure 3 ), and the substitution of -OH at the C3 position of the substrate did not have a significant effect on the enzyme activity, but with the increase of the substitution groups on the parent nucleus, the catalytic efficiency gradually decreased (kaempferol> morin> quercetin> myricetin) ( Figure 3 ).

[0176] Subsequently, we also tested whether FLR could catalyze the catalysis of other flavonoids, such as cyanidin and delphinidin (anthocyanidins), daidzein (isoflavones), and chromone (chromones), but no catalytic activity was observed ( Figure 3 ).

[0177] The above results indicate that FLR is a type of reductase that can specifically catalyze the hydrogenation of the C2-C3 bond of the parent nucleus of flavonoids and flavonols. Unlike flavonoid synthase and flavonol synthase, which can only specifically catalyze the oxidation of dihydroflavones / dihydroflavonols to the corresponding flavones / flavonols, FLR has a broad substrate spectrum, which enables it to simultaneously catalyze the two major categories of flavonoid compounds, flavonoids and flavonols. This is of great significance to the broad spectrum of substrates in the flavonoid metabolic pathway, ensuring that intestinal bacteria possessing this enzyme can widely degrade different types of flavonoid compounds.

[0178] Example 4 Universality of flavonoid reductase distribution

[0179] Experimental methods:

[0180] The amino acid sequence of flavonoid reductase was input into BLASTP for comparison to reveal the universality of the distribution of flavonoid reductase, and FLR proteins from different species were selected for in vitro enzyme activity assay to provide a reference for screening flavonoid reductase with better catalytic performance.

[0181] Experimental steps:

[0182] Further, the tblastn tool was used to compare the homologous genes of flavonoid reductase FLR in the NCBI database. Representative FLR proteins from common species were selected for sequence comparison and display, and a phylogenetic tree was established. Representative FLR homologous proteins were selected and purified, and their flavonoid reductase activity was determined.

[0183] Experimental results:

[0184] It was found that potential flavonoid reductase genes with highly similar sequences to the FLR encoding gene are widely present in different types of microorganisms ( Figure 4 b), the most important of which are prokaryotic microorganisms, and most of them are intestinal bacteria, and the most common are Clostridium of the Firmicutes phylum, including the common Clostridium flavone-solubilizing (FPLA), Clostridium beijerinckii (CBE), Clostridium difficile (CDC), Clostridium jondahlii (CLJ) and Clostridium butyricum (CBUT), etc. ( Figure 4 a). Twelve FLR homologous proteins from different microbial hosts were selected for in vitro enzyme activity testing ( Figure 4 d), and found that these FLR proteins all have flavonoid reductase activity ( Figure 4 c), Figure 4 d shows Figure 4 c Species names and gene numbers corresponding to the flavonoid reductases.

[0185] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for catalyzing redox reactions using flavonoid reductase, characterized in that: Includes steps: In the reaction system, the flavonoid reductase is contacted with the reaction substrate; The amino acid sequence of the flavonoid reductase is shown in SEQ ID NO: 1; The reaction substrate is wherein R1 is H or OH, R2 is OH, R3 is H, R4 is OH, R5 is H, R6 is H, OH or -O-C1-C4 alkyl, R7 is OH, H or -O-C1-C4 alkyl, and R8 is H or OH; And when the reaction substrate is a compound of formula I, the reaction product is a compound of formula II; When the reaction substrate is a compound of formula II, the reaction product is a compound of formula I.

2. The method according to claim 1, characterized in that The compounds of formula I after substitution with R1-R8, and the compounds of formula II converted from the compounds of formula I are shown in the following table:

3. The method according to claim 1, characterized in that The reaction has one or more characteristics selected from the following (i)-(vii): (i) the pH of the reaction system is 4-10; (ii) the reaction temperature is 20-50°C; (iii) The reaction time is 10 min to 10 h; (iv) k of catalytic reduction reaction cat The value is 0.005s -1 -0.040s -1 ; (v) The Km value of the catalytic reduction reaction is 2-20 μM; (vi) k of catalytic oxidation reaction cat The value is 0.015s -1 -0.100s -1 ; (vii) The Km value of the catalytic oxidation reaction is 3-50 μM.

4. The method according to claim 1, characterized in that In the reaction system, the concentration of the flavonoid reductase is 0.01-100 μM.

5. The method according to claim 1, characterized in that The concentration of the reaction product is 0.01-1 mM.

6. The method according to claim 1, characterized in that The reaction system also includes: FMNH2 or FMN.

7. A use of flavonoid reductase, characterized in that: The use of the flavonoid reductase is selected from one of the following (i)-(iv): (i) used for catalyzing the reduction of flavonoid compounds or flavonol compounds into dihydroflavonoid compounds or dihydroflavonol compounds; (ii) used for catalyzing the oxidation of dihydroflavonoids or dihydroflavonols into flavonoids or flavonols; (iii) used for preparing a catalytic preparation for catalyzing the reduction of flavonoids or flavonols to dihydroflavonoids or dihydroflavonols; (iv) used for preparing a catalytic preparation for catalyzing the oxidation of dihydroflavonoids or dihydroflavonols to flavonoids or flavonols; The amino acid sequence of the flavonoid reductase is shown in SEQ ID NO: 1; The flavonoid compound or flavonol compound has a structure shown in Formula I: The dihydroflavonoid compound or dihydroflavonol compound has a structure shown in Formula II: Among them, R1 is H or OH, R2 is OH, R3 is H, R4 is OH, R5 is H, R6 is H, OH or O-C1-C4 alkyl, R7 is OH, H or O-C1-C4 alkyl, and R8 is H or OH.

8. The use according to claim 7, characterized in that The compounds of formula I after substitution with R1-R8, and the compounds of formula II converted from the compounds of formula I are shown in the following table:

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