CN106318920B - Flavone-6-hydroxylase and its application in the synthesis of scutellarin - Google Patents

Abstract

The present invention provides flavones -6- hydroxylase and its applications in scutellarin synthesis, and specifically, the present invention provides a kind of flavones -6- hydroxylases, it can be catalyzed apiolin 6 hydroxylatings, and can get scutellarin.In addition, utilizing glycosyltransferase and glycosyl donor, the method that scutellarin is made in scutellarin the present invention also provides a kind of.

Description

Flavone-6-hydroxylase and its application in the synthesis of scutellarin

technical field

The invention relates to the field of biotechnology, in particular, the invention relates to flavone-6-hydroxylase and its application in the synthesis of scutellarin.

Background technique

Erigeron breviscapus (Vant.) Hand-Mazz is the dry whole herb of Compositae plant Erigeron breviscapus (Vant.) Hand-Mazz. It is cold in nature, slightly bitter, sweet and warm. Active, anti-inflammatory and analgesic effects. Erigeron breviscapus medicines have remarkable clinical application effects, and are listed as nationally developed Chinese herbal medicine varieties and clinically necessary emergency medicines for the treatment of cardiovascular and cerebrovascular diseases by traditional Chinese medicine. In addition to being mainly used for diseases of the cardiovascular and cerebrovascular systems in clinical practice, Erigeron Injection also has good curative effects in the treatment of diabetes, nephropathy, cervical vertigo, and senile diseases. Yunnan Institute of Materia Medica and other units have conducted chemical and pharmacological research on scutellaria breviscapine, and isolated and identified a variety of chemical components from scutellaria breviscapine, the main active components include scutellarin and a small amount of scutellarin (the content of scutellarin accounted for more than 90%).

The natural synthesis pathway of scutellarin is still unclear. In 2013, Anna Berim and David R. Gang discovered a catalyzed 7-position formazan in basil (Ocimum basilicum L.) and mint (Mentha piperita L.). There are no P450 enzymes (CYP82D) that sylate flavonoids to 6-hydroxylation, but none of them have been reported to be functional in scutellarin synthesis. At present, the F6H in the synthesis of scutellarin is still unclear.

At present, there is no relevant report on flavone 6-hydroxylase derived from Scutellaria scutellariae, so there is an urgent need in this field to develop a method for naturally synthesizing scutellarein and scutellarin.

Contents of the invention

The object of the present invention is to provide a method for naturally synthesizing scutellarein and scutellarin.

The first aspect of the present invention provides a flavone-6-hydroxylase for catalyzing the 6-hydroxylation of apigenin, wherein the flavone-6-hydroxylase is selected from the group consisting of:

(a) a protein having the amino acid sequence shown in SEQ ID NOs.:1;

(b) passing the protein of the amino acid sequence shown in SEQ ID NOs.: 1 through one or more amino acid residues (preferably, 1-50, more preferably, 1-30, more preferably, 1-10 One, optimally, 1-6) substitutions, deletions or additions formed, or formed after adding a signal peptide sequence, and have a derivative protein that catalyzes the 6-hydroxylation activity of apigenin;

(c) a derivative protein containing the protein sequence described in (a) or (b) in its sequence;

(d) the homology of the amino acid sequence and the amino acid sequence shown in SEQ ID NOs.: 1 is ≥ 65% (preferably ≥ 80%, more preferably ≥ 90%), and has the activity of catalyzing the 6-hydroxylation of apigenin Derivative protein.

In another preferred example, the sequence (c) is a fusion protein formed by adding a tag sequence, signal sequence or secretion signal sequence to (a) or (b).

In another preferred example, the flavone-6-hydroxylase is from Compositae, preferably from Breviscapita.

In another preferred example, the amino acid sequence shown in SEQ ID NOs.: 1 can be fused with a compound, wherein the compound is a compound that prolongs the half-life of the protein.

In another preferred example, the compound includes polyethylene glycol.

In another preferred example, the protein is the protein with the amino acid sequence shown in SEQ ID NOs.:1.

A second aspect of the present invention provides an isolated nucleotide selected from the group consisting of:

(a) a nucleotide sequence encoding the protein shown in SEQ ID NOs.:1;

(b) a nucleotide sequence as shown in SEQ ID NOs.:2;

(c) a nucleotide sequence with a homology of ≥70% (preferably ≥80%, more preferably ≥90%) to the sequence shown in SEQ ID NOs.:2;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1-10) at the 5' end and/or 3' end of the nucleotide sequence shown in SEQ ID NOs.:2 Nucleotide sequences formed from nucleotides;

(e) A nucleotide sequence that is complementary (preferably fully complementary) to any of the nucleotide sequences described in (a)-(d).

In another preferred example, the nucleotide sequence is shown in SEQ ID NOs.:2.

In another preferred example, the polynucleotide whose sequence is shown in SEQ ID NOs.:2 encodes the polypeptide whose amino acid sequence is shown in SEQ ID NOs.:1.

The third aspect of the present invention provides a vector containing the polynucleotide described in the second aspect of the present invention.

In another preferred embodiment, the vector is selected from the group consisting of expression vectors, shuttle vectors, integration vectors, or combinations thereof.

In another preferred embodiment, the vector is selected from the group consisting of bacterial plasmids, phages, yeast plasmids, plant cell viruses, animal cell viruses, retroviruses, or combinations thereof.

In another preferred embodiment, the vectors include vectors expressed in yeast, such as YCp series vectors, YEp series vectors, YIp series vectors, pCS series vectors, pRS series vectors.

The fourth aspect of the present invention provides a host cell containing the vector of the third aspect of the present invention, or the polynucleotide of the second aspect of the present invention integrated in its genome.

In another preferred example, the host cells are prokaryotic cells or eukaryotic cells.

In another preferred embodiment, the host cell is selected from the group consisting of bacteria, yeast, higher plants, insects or mammalian cells.

In another preferred embodiment, the host cells are lower eukaryotic cells, such as yeast cells.

In another preferred embodiment, the host cell is a higher eukaryotic cell, such as a mammalian cell.

In another preferred embodiment, the host cell is a prokaryotic cell, such as a bacterial cell, preferably Escherichia coli.

In another preferred embodiment, the host cell is selected from the group consisting of Saccharomyces cerevisiae, Escherichia coli, or a combination thereof.

In another preferred example, the host cell is a Saccharomyces cerevisiae cell.

The fifth aspect of the present invention provides a method for preparing flavone-6-hydroxylase, the method comprising:

(a) cultivating the host cell under conditions suitable for expression;

(b) isolating said flavone-6-hydroxylase from the culture.

The sixth aspect of the present invention provides a use of flavone-6-hydroxylase or its derivative protein or the carrier described in the third aspect of the present invention or the host cell described in the fourth aspect of the present invention, for catalyzing the 6-position of apigenin Hydroxylation.

In another preferred example, the flavone-6-hydroxylase is selected from the group consisting of proteins with amino acid sequences shown in any one of SEQ ID NO.: 1, 3, 4, and 5.

In another preferred example, the source of the flavone-6-hydroxylase is selected from one or more plants in the following group: scutellaria breviscapita, artichoke, purple basil, mint.

The seventh aspect of the present invention provides a method for catalyzing the 6-hydroxylation of apigenin, comprising steps:

In the presence of flavone-6-hydroxylase or its derivative protein, the catalytic reaction of hydroxylation at the 6-position of apigenin is carried out.

In another preferred example, the flavone-6-hydroxylase is selected from the group consisting of proteins with amino acid sequences shown in any one of SEQ ID NO.: 1, 3, 4, and 5.

In another preferred example, the source of the flavone-6-hydroxylase is selected from one or more plants in the following group: scutellaria breviscapita, artichoke, purple basil, mint.

The eighth aspect of the present invention provides a method for preparing scutellarein, comprising the steps of:

In the presence of cytochrome reductase (CPR), utilize flavone-6-hydroxylase to catalyze apigenin, thereby obtaining scutellarein;

In another preferred example, the method further includes the step of preparing flavone-6-hydroxylase.

In another preferred example, the preparation steps of the flavone-6-hydroxylase are as follows:

(a) introducing the nucleotide sequence shown in any one of SEQ ID NO.: 2, 6, 7, 8 or a recombinant expression vector containing the nucleotide sequence into a host cell, and culturing the host cell; and

(b) isolating said flavone-6-hydroxylase from the culture.

The ninth aspect of the present invention provides a method for preparing scutellarin, comprising the steps of:

(i) in the presence of cytochrome reductase (CPR), utilize flavone-6-hydroxylase to catalyze apigenin, thereby obtaining scutellarein;

and

(ii) performing a glycosylation reaction on the scutellarein obtained in step (i), thereby obtaining scutellarin;

In another preferred example, the step (ii) is carried out in the presence of a glycosyltransferase and a glycosyl donor.

In another preferred example, the glycosyltransferase is selected from the group consisting of flavone-7-glycosyltransferase F7GAT, flavone-7-glycosyltransferase UGT73C6, flavone-7-glycosyltransferase UGT73B1, flavone-7-glycosyltransferase UGT73B1, flavone-7-glycosyltransferase 7-glycosyltransferase UBGAT, or a combination thereof.

In another preferred example, the source of the glycosyltransferase is selected from one or more plants in the following group: Breviscapus breviscapita, Arabidopsis thaliana, Scutellaria baicalensis, or a combination thereof.

In another preferred embodiment, the glycosyl donor is selected from the group consisting of UDP-glucuronic acid, UDP-glucose, or a combination thereof.

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 in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Fig. 1 is an evolution analysis diagram of EbF6H, CcsF6H, ObF6H and MpF6H.

Fig. 2 is a PCR electrophoresis diagram of genes encoding EbF6H, CcsF6H, ObF6H and MpF6H.

Fig. 3 is the electrophoresis diagram of SpeI digestion of recombinant plasmids Y22-ATR2-EbF6H, Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2-MpF6H.

Fig. 4 is a schematic diagram of the plasmid map of the recombinant plasmid Y22-ATR2-EbF6H.

Fig. 5 is a schematic diagram of the plasmid map of the recombinant plasmid Y22-ATR2-CcsF6H.

Fig. 6 is a schematic diagram of the plasmid map of the recombinant plasmid Y22-ATR2-ObF6H.

Fig. 7 is a schematic diagram of the plasmid map of the recombinant plasmid Y22-ATR2-MpF6H.

Fig. 8 is an HPLC-MS analysis chart of synthesis of scutellarein by EbF6H catalyzing the hydroxylation reaction at the 6-position of apigenin.

Fig. 9 is an HPLC-MS analysis diagram of scutellarein synthesized by EbF6H catalyzing the hydroxylation reaction at the 6-position of apigenin, and then scutellarein undergoes glycosylation modification and UDP-glucuronic acid to produce scutellarin.

Fig. 10 is an HPLC-MS analysis diagram of synthesis of scutellarein by CcsF6H catalyzing the 6-hydroxylation reaction of apigenin.

Figure 11 is an HPLC-MS analysis chart of scutellarein synthesized by CcsF6H catalyzing the hydroxylation reaction at the 6-position of apigenin, and then scutellarein was glycosylated and added with UDP-glucuronic acid to produce scutellarin.

Fig. 12 is an HPLC-MS analysis chart of synthesis of scutellarein by ObF6H catalyzing the hydroxylation reaction at the 6-position of apigenin.

Figure 13 is the HPLC-MS analysis diagram of ObF6H catalyzing the hydroxylation reaction at the 6-position of apigenin to synthesize scutellarein, and then scutellarein is modified by glycosylation and added with UDP-glucuronic acid to produce scutellarin.

Fig. 14 is an HPLC-MS analysis diagram of synthesis of scutellarein by MpF6H-catalyzed hydroxylation reaction at the 6-position of apigenin.

Fig. 15 is an HPLC-MS analysis diagram of scutellarein synthesized by MpF6H catalyzing the 6-hydroxylation reaction of apigenin, and then scutellarein was glycosylated and added with UDP-glucuronic acid to produce scutellarin.

Detailed ways

After extensive and in-depth research, the inventors have discovered for the first time that flavone-6-hydroxylase derived from breviscapine, artichoke, basil violet, or mint can catalyze the hydroxylation reaction at the 6-position of apigenin, thereby obtaining Scutellarein. In addition, the inventors also found that in the presence of glycosyltransferases (such as UDP-glucuronate synthase (UDPGDH)) and glycosyl donors (such as UDP-glucuronic acid), the C7 position of scutellarein Scutellarin can be obtained through glycosylation reaction. On this basis, the present inventors have completed the present invention.

flavone-6-hydroxylase

As used herein, the terms "protein of the present invention", "polypeptide of the present invention", "flavone-6-hydroxylase", "enzyme of the present invention" are used interchangeably, and all refer to the C6 hydroxylase of catalyzed apigenin . In the present invention, the flavone-6-hydroxylase is the protein shown in SEQID NO.: 1, 3, 4, 5 or its derivative protein, and the flavone-6-hydroxylase is derived from Breviscapus breviscapus, Artichoke, purple basil, and mint are named as EbF6H, CcsF6H, ObF6H, and MpF6H respectively, and through homologous sequence alignment, it is found that the protein sequence shown in SEQ ID NO: 1 is the same as that shown in SEQ ID NO: 3 The protein similarity is 69%. The homology comparison results of the flavone-6-hydroxylase derived from breviscapella and flavone-6-hydroxylase from other species are shown in Figure 1.

As used herein, the term "isolated" means that the material is separated from its original environment (in the case of a native material, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state . Accordingly, the term "isolated flavone-6-hydroxylase" as used herein means that the protein is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify the flavone-6-hydroxylase of the present invention using standard protein purification techniques. A substantially pure protein yields a single band on a non-reducing polyacrylamide gel. However, in view of the teaching of the present invention and the prior art, those skilled in the art should also understand that "flavone-6-hydroxylase" should also include variant forms of the protein, and the variant forms have the same properties as the "flavone-6-hydroxylase of the present invention". 6-hydroxylase" has the same or similar function, but its amino acid sequence has a small amount of difference with the amino acid sequence shown in SEQ ID NO: 1, 3, 4 or 5. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and more preferably as 1-8, 1-6) amino acid deletions, insertions and/or substitutions, and addition of one or more (usually within 20, preferably within 10, more at the C-terminal and/or N-terminal Preferably within 6) amino acids. For example, those skilled in the art are well aware that substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of flavone-6-hydroxylase proteins.

Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and "flavone-6-hydroxylase of the present invention" under high or low stringency conditions "The coding DNA hybridizes to the protein encoded by the DNA. The present invention also includes other polypeptides, such as fusion proteins comprising the "flavone-6-hydroxylase of the present invention" or fragments thereof. Besides the almost full-length polypeptide, the present invention should also include active fragments of the "flavone-6-hydroxylase of the present invention". Usually, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least About 80 contiguous amino acids, optimally at least about 100 contiguous amino acids.

The present invention also provides analogs of "flavone-6-hydroxylase". The difference between these analogs and the natural "flavone-6-hydroxylase of the present invention" may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins listed above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modification also includes glycosylation. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the conservative variant polypeptide of "flavone-6-hydroxylase" means that compared with the amino acid sequence shown in SEQ ID NO: 1, 3, 4 or 5, there are at most 20, preferably at most 10 , more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide, but the conservative variant polypeptide still has the same amino acid sequence as SEQ ID NO: 1, 3, 4 Or the same or similar activity of the protein shown in 5, that is, the activity of catalyzing the 6-hydroxylation of apigenin.

Therefore, in view of the teachings of the present invention and the prior art, those skilled in the art can generate mutants with conservative variations by making amino acid substitutions, for example, as shown in the following table.

Therefore, as used herein, "comprising", "having" or "comprising" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of Consists of ", "essentially composed of" and "consisting of" belong to the sub-concepts of "contain", "have" or "include".

The protein of the present invention can be recombinant protein, natural protein, synthetic protein, preferably recombinant protein. The proteins of the present invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the proteins of the invention may be glycosylated, or may be non-glycosylated. The proteins of the invention may or may not include an initial methionine residue.

Those skilled in the art will understand that the "flavone-6-hydroxylase" of the present invention also includes fragments, derivatives and analogs of the "flavone-6-hydroxylase". As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the "flavone-6-hydroxylase" of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). These fragments, derivatives and analogs are within the purview of those skilled in the art as defined herein.

In view of the prior art in the art and the teaching of the present invention, it is not difficult for those skilled in the art to obtain the active fragment of the flavone-6-hydroxylase of the present invention. Therefore, any biologically active fragment of "flavone-6-hydroxylase" can be used in the present invention. In this paper, the biologically active fragment of "flavone-6-hydroxylase" refers to the fragment of "flavone-6-hydroxylase", but it can still maintain all or all of the full-length "flavone-6-hydroxylase". Some functions. Usually, the biologically active fragment maintains at least 50% of the activity of the full-length "flavone-6-hydroxylase". Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length "flavone-6-hydroxylase".

Based on the teaching of the present invention and the prior art, those skilled in the art can also understand that the flavone-6-hydroxylase of the present invention can be made into other utilization forms such as immobilized enzyme.

The present invention also provides a polynucleotide sequence encoding the "flavone-6-hydroxylase" or its conservative variant polypeptide.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 2, 6, 7 or 8 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a protein encoded with SEQ ID NO.: 1, 3, 4 or 5, but not identical to SEQ ID NO.: 2, 6, 7 or 8 Nucleic acid sequences that differ from the coding region sequences shown.

The polynucleotide encoding the mature polypeptide shown in SEQ ID NO.: 1, 3, 4 or 5 includes: the coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide ( and optionally additional coding sequences) and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 1, 3, 4 or 5.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding "flavone-6-hydroxylase".

The "flavone-6-hydroxylase" nucleotide full-length sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods. In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In a preferred embodiment, the "flavone-6-hydroxylase" is: (a) a protein having the amino acid sequence shown in SEQ ID NO.: 1, 3, 4 or 5; NO.: The amino acid sequence shown in 1, 3, 4 or 5 is formed by substitution, deletion or addition of one or several amino acid residues and has the function of "flavone-6-hydroxylase" derived from (a) protein; or (c) through one or several amino acid sequences shown in SEQ ID NO.: 1, 3, 4 or 5, preferably 1-50, more preferably 1-30, and preferably 1-10, Most preferably, a derivative protein formed by deletion or addition of 1-6 amino acid residues and having (a) the protein function; or (d) the amino acid sequence shown in SEQ ID NO.: 1, 3, 4 or 5 C-terminal and/or N-terminal addition or deletion of one or several, preferably 1-50, more preferably 1-30, but also preferably 1-10, most preferably 1-6 amino acid residues and A derivative protein having the function of the protein described in (a).

Correspondingly, the coding gene of the "flavone-6-hydroxylase" is:

(a) the coding nucleotide sequence of the protein whose amino acid sequence is shown in SEQ ID NO: 1, 3, 4 or 5; or

(b) The amino acid sequence shown in SEQ ID NO.: 1, 3, 4 or 5 is formed by substitution, deletion or addition of one or several amino acid residues and has an amino acid sequence such as SEQ ID NO.: 1, 3 , nucleotide sequence encoding a derivative protein of the protein function shown in 4 or 5; or

(c) one or several amino acid sequences represented by SEQ ID NO.: 1, 3, 4 or 5, preferably 1-50, more preferably 1-30, preferably 1-10, most preferably 1 - the coding sequence of a derivative protein formed by deletion or addition of 6 amino acid residues and having the function of the protein described in (a); or

(d) Add or delete one or several, preferably 1-50, more preferably 1-30, at the C-terminal and/or N-terminal of the amino acid sequence shown in SEQ ID NO: 1, 3, 4 or 5, and also It is preferably 1-10, most preferably 1-6 amino acid residues and has the coding sequence of the derivative protein described in (a).

In a further preferred embodiment, the gene encoding "flavone-6-hydroxylase" is: (i) a polynucleotide having a sequence shown in SEQ ID NO.: 2, 6, 7, 8; or (ii ) has a polynucleotide complementary to the sequence shown in SEQ ID NO.: 2, 6, 7, 8.

Cytochrome reductase (CPR)

Cytochrome reductase (CPR) is the main electron donor for the function of P450 enzymes. In the present invention, the enzyme that catalyzes the 6-hydroxylation of apigenin (ie, flavone-6-hydroxylase) is a P450 enzyme that can work together with CPR to catalyze apigenin to obtain scutellarein.

Expression vector

The present invention also relates to an expression vector comprising the coding sequence of the present invention, and a host cell produced by genetic engineering with the expression vector or the "flavone-6-hydroxylase" coding sequence of the present invention, and the polypeptide of the present invention produced by recombinant technology Methods.

The polynucleotide sequence of the present invention can be used to express or produce recombinant "flavone-6-hydroxylase" by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally speaking, there are the following steps:

1. Use the polynucleotide (or its variant) encoding "flavone-6-hydroxylase" of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

2. Host cells cultured in a suitable medium;

3. Isolate and purify protein from culture medium or cells.

In the present invention, the coding polynucleotide sequence of "flavone-6-hydroxylase" can be inserted into a recombinant expression vector or genome. The term "recombinant expression vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Those skilled in the art can use well-known methods to construct expression vectors containing "flavone-6-hydroxylase" coding DNA sequences and suitable transcription/translation control signals, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo restructuring techniques, etc. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescence protein (GFP), or kanamycin or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell described herein includes the host cell containing the expression vector or the "flavone-6-hydroxylase" coding sequence of the present invention integrated in the genome. The host cell or bacterial strain of the present invention can efficiently express flavone-6-hydroxylase with apigenin C6 hydroxylation performance, and the host cell of the present invention can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as yeast cell. In specific embodiments, the strains include, but are not limited to: Saccharomyces cerevisiae or Escherichia coli. In a preferred embodiment, the strain is Saccharomyces cerevisiae.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. When the host is Saccharomyces cerevisiae, lithium acetate transformation can be used

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

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

In view of the teachings of the present invention and the prior art, those of ordinary skill in the art will understand that the flavone-6-hydroxylase and its coding sequence, expression vector, and host cell of the present invention can be used to catalyze the C6 hydroxylation of apigenin.

The preparation method of scutellarin

The present invention also provides a preparation method of scutellarin, comprising the steps of:

(i) in the presence of cytochrome reductase (CPR), utilize flavone-6-hydroxylase to catalyze apigenin, thereby obtaining scutellarein;

and

(ii) performing a glycosylation reaction on the scutellarein obtained in step (i), thereby obtaining scutellarin;

The main advantages of the present invention include:

(1) The present invention finds for the first time that flavone-6-hydroxylase can catalyze the hydroxylation of C6 of apigenin, thereby obtaining scutellarein.

(2) The present invention provides for the first time flavone-6-hydroxylase derived from scutellaria breviscapus, and finds that the flavone-6-hydroxylase derived from scutellaria breviscapus can catalyze the C6 of apigenin to carry out the catalytic reaction of hydroxylation, thereby obtaining wild scutellaria baicalensis In addition, the present invention also reveals for the first time that flavonoid-6-hydroxylase from four plant sources of artichoke, purple basil and mint also has the function of catalyzing the hydroxylation of C6 of apigenin.

(3) The present invention provides a new approach for preparing scutellarin for the first time, catalyzing apigenin into scutellarein by flavone-6-hydroxylase, and then in the presence of glycosyltransferase and glycosyl donor, the The obtained scutellarein is further converted into scutellarin.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise specified, the materials used in the examples are all commercially available products.

The gene sequence SEQ ID NO: 6 used in the present invention was synthesized by Suzhou Synbio Biotechnology Co., Ltd. The rest of the gene sequences were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the primers were synthesized by Tianjin Institute of Industrial Biotechnology.

In the present invention, four plant-derived polypeptides with flavonoid 6-hydroxylase functions from scutellaria breviscapita, artichoke, purple basil, and mint are respectively named as EbF6H, CcsF6H, ObF6H, and MpF6H, and are respectively SEQ ID NO: 1. A polypeptide composed of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

Example 1 Obtaining of EbF6H Encoding Nucleotides

According to the nucleotide sequence information (SEQ ID NO.: 2) provided by the present invention, it is obtained by amplifying or isolating the RNA reverse transcriptome of Breviscapus breviscapus. Specifically, RNA can be extracted from plants using conventional methods or using the Easy spin plus plant RNA rapid extraction kit (purchased from Beijing Aidelai Biotechnology Co., Ltd.), and then the obtained RNA is used as a template to synthesize cDNA using First Strand The kit (Thermo, USA) reverse-transcribed the cDNA obtained according to the operation steps on the kit, and used it as a template, using primers EbF6H-F1 (SEQ ID NO: 9) and EbF6H-R1 (SEQ ID NO: 10) using conventional The PCR method specifically amplifies the coding nucleotide of EbF6H. The PCR amplified fragments were subjected to agarose gel electrophoresis, and the results are shown in Figure 2.

The acquisition of the coding nucleotide of embodiment 2 CcsF6H

Using the amino acid sequence of EbF6H of Erigeron breviscapus to compare with other Asteraceae species, it is found that there is an amino acid sequence with about 69% similarity with EbF6H in Artichoke, another Asteraceae species whose genome has been sequenced, and its Genbank number is KVH90146.1. After identification, it was deduced that this gene also has a similar function of flavonoid 6-hydroxylation, and it was named CcsF6H.

According to the amino acid sequence of SEQ ID NO: 3, the nucleotide sequence codon is optimized for the target host cell (Saccharomyces cerevisiae) and artificially synthesized to obtain the sequence shown in SEQ ID NO: 6. Using CcsF6H-F1 (SEQ ID NO: 11) and CcsF6H-R1 (SEQ ID NO: 12) as primers, a conventional PCR method was used to specifically amplify the coding nucleotide of CcsF6H. The PCR amplified fragments were subjected to agarose gel electrophoresis, and the results are shown in Figure 2.

Obtaining of the coding nucleotide of embodiment 3 ObF6H and MpF6H

According to the information of sequence SEQ ID NO: 7 and SEQ ID NO: 8, the coding nucleotides of the genes ObF6H (Genbank number AGF30364.1) and MpF6H (Genbank number AGF30366.1) were artificially synthesized, or the amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 5, the nucleotide sequence codon is optimized for the target host cell (Saccharomyces cerevisiae) and artificially synthesized to obtain the sequences shown in SEQ ID NO.: 7 and 8. Using ObF6H-F1 (SEQ ID NO: 13) and ObF6H-R1 (SEQ ID NO: 14), MpF6H-F1 (SEQ ID NO: 15) and MpF6H-R1 (SEQ ID NO: 16) as primers respectively, using conventional The PCR method specifically amplifies the coding nucleotides of ObF6H and MpF6H. The PCR amplified fragments were subjected to agarose gel electrophoresis, and the results are shown in Figure 2. The evolutionary relationship of F6H from four plant sources is shown in Fig. 1.

Example 4 Construction of EbF6H, CcsF6H, ObF6H, and MpF6H expression vectors using the Golden Gate cloning method

According to the differences in the host cells expressing EbF6H, CcsF6H, ObF6H, and MpF6H, the shuttle plasmid vector was selected for YCPlacz-22 cloning construction.

4.1 Amplification and purification of gene fragments

Using EbF6H-F1 (SEQ ID NO: 12) and EbF6H-R1 (SEQ ID NO: 13) as primers and using the cDNA containing the EbF6H coding nucleotide sequence as a template to perform conventional PCR to obtain the coding nucleotide of EbF6H, and The resulting amplified fragments were gel-cut and purified;

CcsF6H-F1 (SEQ ID NO: 14) and CcsF6H-R1 (SEQ ID NO: 15), ObF6H-F1 (SEQ ID NO: 16) and ObF6H-R1 (SEQ ID NO: 17), MpF6H-F1 (SEQ ID NO: 17), respectively ID NO: 18) and MpF6H-R1 (SEQ ID NO: 19) as primers, with the artificially synthesized codon-optimized coding nucleotide sequences of CcsF6H, ObF6H, and MpF6H as templates, conventional PCR was performed to obtain The coding nucleotide sequence fragment (Fig. 2) of the CcsF6H, ObF6H, MpF6H of construction, and the amplified fragment of gained is carried out gel cutting purification;

Primers ATR2-F (SEQ ID NO: 20) and ATR2-R ( SEQ ID NO: 21), using Arabidopsis thaliana cDNA as a template to carry out conventional PCR to obtain the encoding nucleotide of ATR2, and the resulting amplified fragment was gel-cut and purified;

The nucleotides of the bidirectional promoter PGK1+TDH3 were artificially synthesized according to the information of the sequence SEQ ID NO:10. Using this as a template, use primers PGK1+TDH-F (SEQ ID NO: 22) and PGK1+TDH-R (SEQ ID NO: 23) to specifically amplify the nucleus of the bidirectional promoter PGK1+TDH3 using conventional PCR methods nucleotides, and the resulting amplified fragments were gel-cut and purified.

Nucleotides of the empty vector YCplac22 (GenBank number: X75455.1) were artificially synthesized according to the sequence of SEQ ID NO: 11. Using this as a template, primers Y22-F (SEQ ID NO: 24) and Y22-R (SEQ ID NO: 25), nucleotides of the vector YCplac22 were used, and the resulting amplified fragment was gel-cut and purified.

4.2 Using Golden gate cloning technology to construct recombinant vectors Y22-ATR2-EbF6H, Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2-MpF6H containing genes EbF6H, CcsF6H, ObF6H, MpF6H and ATR2 respectively, Golden gate connection The system and reaction conditions are as follows:

The reaction system is as follows:

Vector backbone (terminator element) (200ng)

Equimolar amounts of promoter element and vector backbone

Equimolar amount of gene 1 and vector backbone

Equimolar amount of gene 2 and vector backbone

10XNEB T4buffer (NEB) 1.5ul

100XBSA*(NEB)0.15ul

BsaI (NEB) 1ul

NEB T4Ligase (NEB) 1ul

ddH2O make up to 15ul

Total 15ul

The reaction conditions are as follows:

The obtained product was directly transformed into DH5α Escherichia coli (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), cultivated for 12-16 hours, picked a single colony for verification, and analyzed by agarose gel electrophoresis to find the correct clone (Figure 2). The resulting correct recombinant vectors were named Y22-ATR2-EbF6H, Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2-MpF6H, respectively. The results of enzyme digestion are shown in Figure 3, and their physical maps are shown in Figure 4 and Figure 5 respectively , Figure 6, Figure 7 shown.

Example 5 Construction of recombinant vector Y33-UDPGDH-F7GAT using the Golden Gate clone construction method

Utilizing the Golden gate cloning construction technique described in 4.2 of Example 4, the glycosyltransferase gene F7GAT derived from scutellaria breviscapus was combined with the UDP-glucuronide synthase gene UDPGDH derived from Streptococcus pyogenes and the artificially synthesized bidirectional promoter ADH1 +TDH3 was constructed on the synthetic vector YCplac33 (synthetic Genbank number: X75456.1) to obtain the recombinant vector Y33-UDPGDH-F7GAT.

Example 6 Construction of apigenin-producing Saccharomyces cerevisiae host bacteria

Apigenin synthesis related genes PAL (phenylalanine deaminase gene), C4H (cinnamic acid hydroxylase gene), 4CL (coumaric acid coenzyme A synthase gene), CHS (chalcone synthesis gene) Enzyme gene), CHI (chalcone isomerase gene), FSII (flavone synthase II gene) were integrated into conventional Saccharomyces cerevisiae wild strain W303 (Thomas, B.J.and Rothstein R (1989) Elevated recombination rates inscriptionally active DNA. Cell, 56(4):619-663.), a histidine auxotrophic apigenin-producing Saccharomyces cerevisiae host strain SC1 was obtained.

Example 7 Construction of scutellarein-producing Saccharomyces cerevisiae and scutellarin-producing Saccharomyces cerevisiae

7.1 Construction of Scutellarein-producing Saccharomyces cerevisiae

The recombinant vectors Y22-ATR2-EbF6H, Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, and Y22-ATR2-MpF6H described in 4.2 of Example 4 were transformed into apigenin-producing Saccharomyces cerevisiae host strain SC1 using conventional lithium acetate transformation methods. , pick the clones that can grow on the CM medium deficient in histidine and tryptophan, numbered SC223, SC251, SC114, SC137;

7.2 Construction of scutellarin-producing Saccharomyces cerevisiae

The recombinant vectors Y22-ATR2-EbF6H, Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2-MpF6H and Y33-UDPGDH-F7GAT in 4.2 of Example 4 were co-transformed into the implementation In the Saccharomyces cerevisiae host strain SC1 producing apigenin in Example 6, the clones that can grow on the CM medium deficient in histidine, tryptophan, and uracil were selected, and the numbers were SC225, SC254, SC125, SC145.

Example 8 Fermentative production of scutellarein and scutellarin

8.1 Saccharomyces cerevisiae engineered strain seed liquid culture

Select CM medium (purchased from Suo Laibao Biotechnology Co., Ltd.) (formulation: YNB w/o AA (0.67%), glucose (2g/L), Dropout powder (0.083%), wherein the Dropout powder contains (mg/ L): threonine 150, tyrosine 30, valine 150, lysine 30, glutamic acid 100, serine 150, aspartic acid 100, methionine 20, phenylalanine 50, iso Leucine 30, arginine 20; other nutrients (mg/L): adenine 50, uracil 50, histidine 100, leucine 100, tryptophan 100 (amino acid)), liquid medium pH 5 .6. Add 1.5% agar powder (purchased from Suleibao Biotechnology Co., Ltd.) to the solid medium to adjust the pH to 6.5. Pick single clones on the plate and place them in test tubes containing 4mL of sterilized medium, and carry out overnight treatment on S. nourish.

8.2. Fermentation production

The seed solution was inoculated into an Erlenmeyer flask containing 50 mL of sterilized medium at an inoculation ratio of 1:50, and fermented at 30°C and 200 rpm for 5 days.

The LC-MS identification of embodiment 9 reaction product

9.1 After the fermentation is over, take 900uL sample, add an equal volume of anhydrous methanol, and use an ultrasonic cleaner to perform ultrasonication for 30min. Centrifuge at 12000rpm for 10min, and the supernatant is analyzed by high performance liquid phase.

9.2 HPLC, LC-MS detection conditions

HPLC analysis:

Instrument: Shimadzu HPLC 1200

Chromatographic column: Kinetex H15-168747 (4.6×250mm), ultraviolet detector, detection wavelength 335nm.

Mobile phase: A phase is 0.1% formic acid; B phase is acetonitrile; C phase is methanol

Initial concentration A: 75% B: 22% C: 5%

Flow rate: 1mL/min

Column temperature: 30°C

Detector: PDA detector

Gradient elution program: (concentration is the percentage of phase B)

MS analysis

Mass spectrometer: Bruker-micrOTOF-II:

ESI ion source, positive ion mode

Nucleoplasmic ratio (m/z): 50-1000

Nitrogen flow rate: 6.0 L/min

Temperature: 180°C

Atomizer pressure: 1bar

Probe voltage: 14.5KV.

9.3 Identification of EbF6H activity

For the fermentation product of strain SC223, the HPLC test results show that EbF6H can catalyze apigenin to generate a new product, and the peak time is 8.5 minutes, which is consistent with the standard scutellarein (Figure 8A), and the mass spectrometry results show (Figure 8B) the molecular weight of the new product It is (m/z, 287[M+H] ⁺ ), which is consistent with the molecular weight of the standard scutellarein. It is determined that the substance is scutellarein, and the shake flask fermentation yield is 16.4mg/L.

In addition, the new product was catalyzed by glycosyltransferase and UDP-glucuronic acid to generate a new product, and the peak time was 4.5 minutes, which was consistent with the standard scutellarin (Figure 9A). The mass spectrometry results showed (Figure 9B) that the new product The molecular weight of the product is (m/z, 463[M+H] ⁺ ), which is consistent with the molecular weight of the scutellarin standard product. It is confirmed that the substance is scutellarin, and the shake flask fermentation yield is 22.7mg/L.

9.4 Identification of CcsF6H activity

For the fermentation product of strain SC251, the HPLC test results show that CcsF6H can catalyze apigenin to generate a new product, and the peak time is 8.5min, which is consistent with the standard scutellarein (Figure 10A), and the mass spectrometry results show (Figure 10B) that the new product The molecular weight is (m/z, 287[M+H] ⁺ ), which is consistent with the molecular weight of the standard scutellarein. It is determined that the substance is scutellarein, and the shake flask fermentation yield is 15.2mg/L.

In addition, the new product was catalyzed by glycosyltransferase and UDP-glucuronic acid to form a new product, and the peak time was 4.5 minutes, which was consistent with the standard scutellarin (Figure 11A). The mass spectrometry results showed (Figure 11B) that the new product The molecular weight of the product is (m/z, 463[M+H] ⁺ ), which is consistent with the molecular weight of the scutellarin standard product. It is confirmed that the substance is scutellarin, and the shake flask fermentation yield is 22mg/L.

9.5 Identification of ObF6H activity

The results of HPLC detection of the fermentation product of strain SC114 show that ObF6H can catalyze a small amount of apigenin to form a new product, and the peak time is 8.5 minutes, which is consistent with the standard product of scutellarein (Figure 12A). Mass spectrometry results show (Figure 12B) that the new product The molecular weight of the product is (m/z, 287[M+H] ⁺ ), which is consistent with the molecular weight of the standard scutellarein. It is confirmed that the substance is scutellarein, and the shake flask fermentation yield is 0.5 mg/L.

In addition, the new product was catalyzed by glycosyltransferase and UDP-glucuronic acid to generate a new product, and the peak time was 4.5 minutes, which was consistent with the standard scutellarin (Figure 13A), and the mass spectrometry results showed (Figure 13B) that the new product The molecular weight of the product is (m/z, 463[M+H] ⁺ ), which is consistent with the molecular weight of the standard scutellarin. It is confirmed that the substance is scutellarin, and the shake flask fermentation yield is 3.5mg/L.

9.6 MpF6H activity identification

The results of HPLC detection of the fermentation product of strain SC137 show that MpF6H can catalyze a small amount of apigenin to generate a new product, and the peak time is 8.5 minutes, which is consistent with the standard product of scutellarein (Figure 14A), and the mass spectrometry results show (Figure 14B) that the new product The molecular weight of the product is (m/z, 287[M+H] ⁺ ), which is consistent with the molecular weight of the standard scutellarein. It is confirmed that the substance is scutellarein, and the shake flask fermentation yield is 0.5 mg/L.

In addition, the new product was catalyzed by glycosyltransferase and UDP-glucuronic acid to generate a new product, and the peak time was 4.5 minutes, which was consistent with the standard scutellarin (Figure 15A). The mass spectrometry results showed (Figure 15B) that the new product The molecular weight of the product is (m/z, 463[M+H] ⁺ ), which is consistent with the molecular weight of the standard scutellarin. It is confirmed that the substance is scutellarin, and the shake flask fermentation yield is 3.5mg/L.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 appended claims of the present application.

Claims (9)

1. a flavone-6-hydroxylase for catalyzing the 6-hydroxylation of apigenin, characterized in that, the flavone-6-hydroxylase is A protein having the amino acid sequence shown in SEQ ID NOs.:1. 2. An isolated nucleotide, characterized in that, the nucleotide is (a) a nucleotide sequence encoding the protein shown in SEQ ID NOs.:1; (b) a nucleotide sequence as shown in SEQ ID NOs.:2; (e) A nucleotide sequence complementary to the nucleotide sequence described in any one of (a)-(b). 3. A vector, characterized in that the vector contains the polynucleotide according to claim 2. 4. A host cell, characterized in that the host cell contains the vector according to claim 3, or integrates the polynucleotide according to claim 2 into its genome. 5. a preparation method of flavone-6-hydroxylase as claimed in claim 1, is characterized in that, described method comprises: (a) cultivating the host cell under conditions suitable for expression; (b) isolating said flavone-6-hydroxylase from the culture. 6. a kind of flavone-6-hydroxylase or its derivative protein or the purposes of the described carrier of claim 3 or the described host cell of claim 4, it is characterized in that, for catalyzing the 6 hydroxylation of apigenin, described The flavone-6-hydroxylase is selected from the group consisting of proteins with the amino acid sequence shown in SEQ ID NO.:1. 7. A method for catalyzing the 6-hydroxylation of apigenin, characterized in that, comprising steps: In the presence of flavone-6-hydroxylase or its derivative protein, carry out the catalytic reaction of apigenin 6-hydroxylation, the flavone-6-hydroxylase is selected from the following group: the amino acid sequence shown in SEQ ID NO.:1 protein. 8. A method for preparing scutellarein, characterized in that, comprising steps: In the presence of cytochrome reductase (CPR), utilize flavone-6-hydroxylase to catalyze apigenin, thereby obtaining scutellarein; Wherein, the flavone-6-hydroxylase is selected from the group consisting of proteins with the amino acid sequence shown in SEQ ID NO.:1. 9. A method for preparing scutellarin, comprising the steps of: (i) in the presence of cytochrome reductase (CPR), utilize flavone-6-hydroxylase to catalyze apigenin, thereby obtaining scutellarein; and (ii) performing a glycosylation reaction on the scutellarein obtained in step (i), thereby obtaining scutellarin; Wherein, the flavone-6-hydroxylase is selected from the group consisting of proteins with the amino acid sequence shown in SEQ ID NO.:1.