阅读说明：本技术 合成黄芩素和野黄芩素的微生物、其制备方法及其应用 (Microorganism for synthesizing baicalein and scutellarein, preparation method and application thereof ) 是由 王勇 李建华 田晨菲 于 2018-09-07 设计创作，主要内容包括：本发明涉及合成黄芩素和野黄芩素的微生物、其制备方法及其应用。本发明人通过基因工程的方法对宿主细胞的异源代谢途径进行改造,获得了高产黄芩素和野黄芩素的工程菌株。本发明还提供了利用所述工程菌株生产黄芩素和野黄芩素的工艺。(The invention relates to a microorganism for synthesizing baicalein and scutellarein, a preparation method and application thereof. The present inventors obtained high-yield baicalein and scutellarein engineering strains by modifying the heterologous metabolic pathway of host cells through a genetic engineering method. The invention also provides a process for producing baicalein and scutellarein by using the engineering strain.)

1. A method for producing baicalein and scutellarein, which is characterized by comprising the following steps:

(1) introducing genes for expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase and a chrysin or apigenin synthesis gene into a host cell;

(2) culturing the host cell in a culture system containing phenylalanine and/or tyrosine, thereby producing baicalein or scutellarein.

2. The method of claim 1, wherein the chrysin or apigenin synthesis gene comprises: genes expressing phenylalanine ammonia lyase, 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, and flavone synthase I; preferably, the genes expressing phenylalanine ammonia lyase, 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, and flavone synthase I are present in the same expression vector when introduced into the host cell.

3. A method for producing baicalein and scutellarein, which is characterized by comprising the following steps:

(1) introducing genes expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase into a host cell to obtain a recombinant host cell;

(2) culturing the recombinant host cell in a culture system containing chrysin or apigenin, thereby producing baicalein or scutellarein.

4. A method for converting chrysin or apigenin into baicalein or scutellarein is characterized in that chrysin or apigenin is catalyzed by flavone 6-hydroxylase and cytochrome P450 oxidoreductase, so that a hydroxyl group is added to the structure of the chrysin or apigenin to generate the baicalein or scutellarein.

5. The method according to any one of claims 1 to 4, wherein the flavone 6-hydroxylase is a mutant flavone 6-hydroxylase in which the amino acids at (1-10) to (20-30) th positions of the N-terminus are truncated; preferably, the mutant flavone 6-hydroxylase is one in which the amino acids at (2-5) - (22-28) of the N-terminal are truncated.

6. The method of any one of claims 1 to 4, wherein the flavone 6-hydroxylase is fused to a polypeptide tag selected from the group consisting of: calf serum 17 hydroxylase N-terminal 8 amino acid polypeptide, small ubiquitin modification related protein, maltose binding protein, cytochrome P4502B1 family soluble protein, or a combination thereof; preferably a maltose binding protein or a soluble protein of the cytochrome P4502B1 family, or a combination thereof; preferably the polypeptide tag is located at the N-terminus.

7. The method according to any one of claims 1 to 4, wherein the cytochrome P450 oxidoreductase is a mutant cytochrome P450 oxidoreductase in which the amino acids (1-20) to (60-85) of the N-terminal are cleaved; preferably, the mutant cytochrome P450 oxidoreductase is a cytochrome P450 oxidoreductase in which the (2-10) -to (65-80) -th amino acids from the N-terminus are truncated; more preferably, it is a mutant cytochrome P450 oxidoreductase in which the (2-5) - (70-75) th amino acids from the N-terminus are cleaved.

8. The method of any one of claims 1 to 3, wherein the host cell comprises: a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell comprises: coli cells, bacillus subtilis cells; the eukaryotic cell comprises: a yeast cell.

9. A recombinant host cell comprising an exogenous gene expressing a flavone 6-hydroxylase and a cytochrome P450 oxidoreductase.

10. The recombinant host cell of claim 9, further comprising an exogenous chrysin or apigenin synthesis gene; preferably, the chrysin or apigenin synthetic gene comprises: genes expressing phenylalanine ammonia lyase, 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, and flavone synthase I; preferably, the genes expressing phenylalanine ammonia lyase, 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, and flavone synthase I are introduced into the host cell in the same expression vector.

11. The recombinant host cell according to any one of claims 9 to 10, wherein the flavone 6-hydroxylase is a mutant flavone 6-hydroxylase in which amino acids (1-10) to (20-30) of the N-terminal are truncated; preferably, the mutant flavone 6-hydroxylase is one in which the amino acids at (2-5) - (22-28) of the N-terminal are truncated.

12. The recombinant host cell of any one of claims 9 to 10, wherein the flavone 6-hydroxylase is fused to a polypeptide tag selected from the group consisting of: calf serum 17 hydroxylase N-terminal 8 amino acid polypeptide, small ubiquitin modification related protein, maltose binding protein, cytochrome P4502B1 family soluble protein, or a combination thereof; preferably a maltose binding protein or a soluble protein of the cytochrome P4502B1 family, or a combination thereof; preferably the polypeptide tag is located at the N-terminus.

13. The recombinant host cell according to any one of claims 9 to 10, wherein the cytochrome P450 oxidoreductase is a mutant cytochrome P450 oxidoreductase in which the amino acids (1-20) to (60-85) of the N-terminal are truncated; preferably, the mutant cytochrome P450 oxidoreductase is a cytochrome P450 oxidoreductase in which the (2-10) -to (65-80) -th amino acids from the N-terminus are truncated; more preferably, it is a mutant cytochrome P450 oxidoreductase in which the (2-5) - (70-75) th amino acids from the N-terminus are cleaved.

14. Use of the recombinant host cell of any one of claims 9 to 13 for the production of baicalein and scutellarein.

15. A method of preparing a host cell for producing baicalein and scutellarein comprising: introducing genes expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase into host cells to obtain recombinant strains; preferably, the method also comprises introducing a chrysin or apigenin synthesis gene.

16. A kit for producing baicalein and scutellarein, comprising the recombinant host cell of any one of claims 9 to 13.

17. A mutant flavone 6-hydroxylase corresponding to the wild-type flavone 6-hydroxylase having amino acids at positions (1-10) to (20-30) of the N-terminus truncated; preferably, the amino acids (2-5) - (22-28) at the N-terminal are truncated; more preferably, it has the amino acid sequence shown in SEQ ID NO. 2.

18. A mutant cytochrome P450 oxidoreductase in which the (1-20) th to (60-85) th amino acids from the N-terminus are cleaved off in accordance with a wild-type cytochrome P450 oxidoreductase; preferably, the amino acids (2-10) - (65-80) at the N-terminal are truncated; more preferably, the amino acids at the (2-5) - (70-75) th positions of the N end are cut off; more preferably it has the amino acid sequence shown in SEQ ID NO. 8.

19. A fusion polypeptide comprising the mutant flavone 6-hydroxylase of claim 17, and a polypeptide tag fused thereto, said polypeptide tag selected from the group consisting of: 8RP, Sumo, MBP, 2B 1; preferably MBP or 2B 1.

20. The fusion polypeptide of claim 19, having an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.

21. A polynucleotide encoding:

the mutant flavone 6-hydroxylase of claim 17; or

The mutant cytochrome P450 oxidoreductase of claim 18; or

The fusion polypeptide of claim 19 or 20.

22. An expression construct, comprising:

any one of the polynucleotides of claim 21; or

A polynucleotide encoding any one of the mutant flavone 6-hydroxylase of claim 17 or the fusion protein of claim 19, and a polynucleotide encoding the mutant cytochrome P450 oxidoreductase of claim 18.

23. The mutant flavone 6-hydroxylase of claim 17 or the fusion protein of claim 19, and the use of the mutant cytochrome P450 oxidoreductase of claim 18 to add a hydroxyl group to the structure of chrysin or apigenin to form baicalein or scutellarein.

Technical Field

The invention relates to the technical field of synthetic biology and medicines, in particular to a microorganism for synthesizing baicalein and scutellarein, a preparation method and application thereof.

Background

Scutellaria Baicalensis Georgi (Scutcllaria Baicalensis Georgi) is a famous traditional medicine in China and is a labiate plant; the traditional Chinese medicine scutellaria is a dry root of plant scutellaria, has long medicinal history, and can be used for treating various diseases such as wind heat, dampness and heat. Erigeron breviscapus is dry whole plant of Erigeron breviscapus (Erigeron brevicapus) of Compositae, is cold in nature and bitter in taste, and has effects of diminishing inflammation, relieving pain, promoting blood circulation, removing blood stasis, dispelling pathogenic wind and removing dampness. The extracts of scutellaria and erigeron breviscapus have been widely applied to Chinese medicinal preparations for a long time, the main raw materials of the honeysuckle flower tablet, the scutellaria oral liquid and the like are all scutellaria extracts, and the main effective component of the Chinese medicament qingkailing is baicalein which has the functions of diminishing inflammation, preventing and treating diarrhea, liver diseases and tumors; common breviscapine preparation forms include breviscapine tablet, breviscapine oral liquid, etc., and the main metabolism absorption form of them in the organism is its aglycone scutellarin. Therefore, both the baicalein and the scutellarein have certain new drug development value.

Baicalein and scutellarein are two important flavonoid compounds with similar structures. The molecular formula of baicalein is C₁₅H₁₀O₅Molecular weight of 270.24, while scutellarein has molecular weight of C₁₅H₁₀O₆And the molecular weight is 286.24. Their structure is shown in fig. 1.

Like most natural products of plant origin, baicalein and scutellarein are currently mainly prepared by two methods, chemical synthesis and organic solvent extraction. The organic solvent extraction mainly comprises the step of carrying out tissue extraction on medicinal plants such as scutellaria baicalensis, erigeron breviscapus, sculellaria barbata and the like, a large amount of organic solvent is needed in the process, and the problems of complicated subsequent separation process, high industrial manufacturing cost and the like exist. The method is mainly difficult to solve the problems of slow plant growth, damage to medicinal resources and the like. Although baicalein and scutellarein can be obtained in large quantity by chemical synthesis, the raw materials relate to chemical substances such as cinnamic acid or derivatives thereof, oxyphenol and the like, and the application of the baicalein and scutellarein in the fields of medicines and foods is limited to a certain extent. But also the use of toxic reagents and expensive chemical catalysts during the synthesis process.

Synthetic biology is based on rational design, integrating and assembling standardized biological elements to construct a well-behaved artificial life system. Once the synthetic biology is produced, the thought and design of the synthetic biology deeply affect the development of industrial microbial technology, so that the microbial technology plays a great role in the development and production processes of medicines, biofuels and fine chemicals.

In the art, synthetic elements of various natural products are assembled to achieve heterologous synthesis in microorganisms. However, the two active flavonoid compounds of baicalein and scutellarin related in the invention have not been reported to be successfully synthesized heterologously in microorganisms. Therefore, the construction of a microbial strain capable of heterologously synthesizing baicalein and scutellarein is urgently needed.

Disclosure of Invention

The invention aims to provide a microorganism for synthesizing baicalein and scutellarein, a preparation method and application thereof.

In a first aspect of the present invention, there is provided a method for producing baicalein and scutellarein, comprising: (1) introducing genes expressing flavone 6-hydroxylase (F6H) and cytochrome P450 oxidoreductase (CPR) and a chrysin or apigenin synthesis gene into a host cell; and (2) culturing the host cell in a culture system containing phenylalanine and/or tyrosine, thereby producing baicalein or scutellarein.

In a preferred embodiment, the chrysin or apigenin synthesis gene comprises: genes expressing phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4-coumarate: CoA ligase, 4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavone synthase I (FNSI); preferably, the genes expressing phenylalanine ammonia lyase, 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, and flavone synthase I are present in the same expression vector when introduced into the host cell.

In another preferred embodiment, the flavone 6-hydroxylase is derived from scutellaria baicalensis (scutellaria baicalensis), and homologues thereof (homologous genes or polypeptides from other species); the CPR is derived from Arabidopsis thaliana (Arabidopsis thaliana), and also includes homologues thereof.

In another preferred embodiment, the PAL is derived from rhodiola rosea (rhodiola toruloides), and also includes homologues thereof; the 4CL is derived from parsley (Petroselium crispum) and also includes homologues thereof; the CHS is derived from Petunia (Petunia X hybrida) and also comprises a homologue thereof; said CHI is derived from alfalfa (medicago sativa) and includes homologues thereof; the FNS I is derived from parsley (Petroselium crispum) and includes homologues thereof.

In another aspect of the present invention, there is provided a method for producing baicalein and scutellarein, comprising: (1) introducing genes expressing flavone 6-hydroxylase (F6H) and cytochrome P450 oxidoreductase (CPR) into a host cell to obtain a recombinant host cell; and (2) culturing the recombinant host cell in a culture system containing chrysin or apigenin, thereby producing baicalein or scutellarein.

In another aspect of the present invention, there is provided a method of converting chrysin or apigenin into baicalein or scutellarein: catalyzing chrysin or apigenin by flavone 6-hydroxylase and cytochrome P450 oxidoreductase, so as to add a hydroxyl group to the structure of the chrysin or apigenin to form baicalein or scutellarein.

In a preferred embodiment, the flavone 6-hydroxylase (F6H) is a mutant flavone 6-hydroxylase with the N-terminal amino acids at positions (1-10) - (20-30) truncated; preferably, the mutant flavone 6-hydroxylase is one in which the amino acids at (2-5) - (22-28) of the N-terminal are truncated.

In another preferred embodiment, said flavone 6-hydroxylase is fused to a polypeptide tag selected from the group consisting of: calf serum 17 hydroxylase N-terminal 8 amino acid polypeptide (8RP), small ubiquitin-modifying related protein (Sumo), Maltose Binding Protein (MBP), cytochrome P4502B1 family soluble protein (2B1), or a combination thereof; preferably a maltose binding protein or a soluble protein of the cytochrome P4502B1 family, or a combination thereof; preferably the polypeptide tag is located at the N-terminus.

In another preferred embodiment, the cytochrome P450 oxidoreductase (CPR) is a mutant cytochrome P450 oxidoreductase in which the amino acids at the (1-20) to (60-85) th positions of the N-terminus are cleaved; preferably, the mutant cytochrome P450 oxidoreductase is a cytochrome P450 oxidoreductase in which the (2-10) -to (65-80) -th amino acids from the N-terminus are truncated; more preferably, it is a mutant cytochrome P450 oxidoreductase in which the (2-5) - (70-75) th amino acids from the N-terminus are cleaved.

In another preferred embodiment, the host cell comprises: a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell comprises: coli cells, bacillus subtilis cells; the eukaryotic cell comprises: a yeast cell.

In another aspect of the present invention, there is provided a recombinant host cell comprising an exogenous gene expressing a flavone 6-hydroxylase and a cytochrome P450 oxidoreductase.

In another preferred example, the recombinant host cell further comprises an exogenous chrysin or apigenin synthesis gene.

In another preferred embodiment, the polypeptide tag is a single copy or 2-10 copies (e.g. 3, 4, 5, 6, 8 copies) of a tandem sequence structure.

In another aspect of the invention there is provided the use of a recombinant host cell as described in any one of the preceding paragraphs for the production of baicalein and scutellarein.

In a preferred embodiment, the method is used for producing baicalein and scutellarein by taking exogenously added chrysin or apigenin as substrates for a strain without a chrysin or apigenin synthesis gene in cells; for the bacterial strain with chrysin or apigenin synthesis gene in the cell, the method is used for producing baicalein and scutellarein under the culture condition of exogenously adding phenylalanine and/or tyrosine.

In another aspect of the present invention, there is provided a method for preparing a host cell for producing baicalein and scutellarein, comprising: introducing genes expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase into host cells to obtain recombinant strains; preferably, the method also comprises introducing a chrysin or apigenin synthesis gene.

In another aspect of the present invention, there is provided a kit for producing baicalein and scutellarein, said kit comprising a recombinant host cell as defined in any one of the preceding.

In another preferred embodiment, the kit further comprises: host cell culture media, instructions for use, and the like.

In another aspect of the present invention, there is provided a mutant flavone 6-hydroxylase corresponding to the wild-type flavone 6-hydroxylase (F6H) having amino acids at positions (1-10) to (20-30) truncated from the N-terminus; preferably, the amino acids (2-5) - (22-28) at the N-terminal are truncated; preferably, it has the amino acid sequence shown in SEQ ID NO. 2.

In another aspect of the present invention, there is provided a mutant cytochrome P450 oxidoreductase in which the amino acids at the (1-20) - (60-85) th positions of the N-terminus are truncated in accordance with the wild-type cytochrome P450 oxidoreductase; preferably, the amino acids (2-10) - (65-80) at the N-terminal are truncated; more preferably, the amino acids at the (2-5) - (70-75) th positions of the N end are cut off; preferably, it has the amino acid sequence shown in SEQ ID NO. 8.

In another aspect of the invention, there is provided a fusion polypeptide comprising any one of the mutant flavone 6-hydroxylases described above, and a polypeptide tag fused thereto, said polypeptide tag being selected from the group consisting of: 8RP, Sumo, MBP, 2B 1; preferably MBP or 2B 1.

In a preferred embodiment, the fusion polypeptide has an amino acid sequence selected from the group consisting of: 3, 4, 5 or 6.

In another aspect of the invention, there is provided a polynucleotide encoding: the mutant flavone 6-hydroxylase described above; or said mutant cytochrome P450 oxidoreductase; or said fusion polypeptide.

In another aspect of the invention, there is provided an expression construct comprising: any of the polynucleotides described above; or a polynucleotide encoding any one of the mutant flavone 6-hydroxylase or the fusion protein, and a polynucleotide encoding the mutant cytochrome P450 oxidoreductase.

In another preferred embodiment, the expression construct further comprises a promoter and a terminator operably linked to the polynucleotide.

In another aspect of the present invention, there is provided the use of said mutant flavone 6-hydroxylase or said fusion protein, and mutant cytochrome P450 oxidoreductase, for adding a hydroxyl group to the structure of chrysin or apigenin to form baicalein or scutellarein.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, structural formulas of baicalein and scutellarein.

FIG. 2, biosynthesis pathway of baicalein and scutellarein.

FIG. 3 shows the construction of plasmid pYH 66.

FIG. 4 shows the construction of plasmid pYH 57.

FIG. 5 shows HPLC detection maps of the engineered strain BL21(DE3) -pYH57-pYH66 and baicalein as a standard. Wherein, i represents BL21(DE3) -pETDuet-1-pCDFDuet-1 fermentation broth as a blank control; ii represents a fermentation broth of BL21(DE3) -pYH57-pYH66 with phenylalanine added thereto; and iii represents a baicalein standard product.

FIG. 6 is a mass spectrum of baicalein produced by engineering strain BL21(DE3) -pYH57-pYH 66.

FIG. 7 shows HPLC detection maps of the engineered strain BL21(DE3) -pYH57-pYH66 and scutellarein as a standard. Wherein, i represents BL21(DE3) -pETDuet-1-pCDFDuet-1 fermentation broth as a blank control; ii represents a tyrosine-added fermentation broth of BL21(DE3) -pYH57-pYH 66; and iii represents a scutellarein standard substance.

FIG. 8 is a mass spectrum of scutellarein produced by the engineered strain BL21(DE3) -pYH57-pYH 66.

FIG. 9, SbF6H and the AtCPR mutants catalyze the production of baicalein from chrysin.

A, schematic representation of key elements in the constructed plasmid;

b, catalyzing the chrysin to generate the conversion rate of baicalein in the recombinant escherichia coli;

and C, analyzing the recombinant escherichia coli catalytic reaction solution by HPLC. Wherein, Chr represents chrysin, and Bai represents baicalein.

Detailed Description

The present inventors have made extensive studies to improve the yield of biologically produced scutellarin and scutellarein by heterogeneously synthesizing scutellarin and scutellarein in microorganisms, and have obtained a high-yield scutellarin and scutellarein engineering strain by modifying the heterogeneously metabolic pathway of host cells by genetic engineering methods.

As used herein, the "N-terminal amino acids (1-10) to (20-30)" refers to any amino acid from the 1-10 th position of the N-terminus and any amino acid from the 20-30 th position of the N-terminus.

As used herein, the "positions (2-5) to (22-28) of the N-terminus" means any of the amino acids at positions 2 to 5 from the N-terminus and any of the amino acids at positions 22 to 28 from the N-terminus.

As used herein, the "positions (1-20) to (60-85) of the N-terminus" refers to any of the amino acids from position 1 to 20 from the N-terminus and any of the amino acids from position 60 to 85 from the N-terminus.

As used herein, the "positions (2-10) to (65-80) of the N-terminus" means any of the amino acids from the 2-10 th position from the N-terminus and any of the amino acids from the 65-80 th position from the N-terminus.

As used herein, the "positions (2-5) to (70-75) of the N-terminus" means any of the amino acids at positions 2 to 5 from the N-terminus and any of the amino acids at positions 70 to 75 from the N-terminus.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources.

As used herein, the term "operably linked" or "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the term "expression construct" refers to a recombinant DNA molecule comprising a desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. The "construct" is typically contained in an expression vector.

As used herein, the PAL, 4CL, CHS, CHI and FNSI proteins are proteins that form the chrysin or apigenin synthesis pathway in an expression system.

As used herein, the F6H and CPR proteins are proteins that convert chrysin or apigenin, produce baicalein or scutellarein in an expression system.

Wild-type such proteins or genes are all identified in the art and, therefore, are available and prepared from public sources. In a preferred embodiment of the present invention, PAL is derived from rhodiola rosea (rhodiola toruloides) and has a sequence shown in GenBank accession AAA 33883.1; 4CL is derived from parsley (Petroselium crispum) having the sequence shown in GenBank accession KF 765780.1; the CHS is derived from Petunia (Petunia X hybrida) and has a sequence shown in GenBank accession KF 765781.1; the CHI gene is derived from alfalfa (Medicago sativa) having a sequence shown in GenBank accession KF 765782.1; FNS I is derived from parsley (Petroselium crispum) and has the sequence shown in Swiss-Prot accession Q7XZQ8.1.

Wild-type F6H and CPR have also been identified in the art. In a preferred embodiment of the present invention, F6H is derived from Scutellaria baicalensis (Scutellaria basilica) having a sequence shown in GenBank accession number ASW 21050.1. In a preferred embodiment of the present invention, the CPR is derived from Arabidopsis thaliana (Arabidopsis thaliana) having the sequence shown in GenBank accession No. NP _ 849472.2.

The inventor finds that in the process of producing baicalein and scutellarein by using host cells, wild F6H can only produce trace products and can not realize large-scale production, so that a plurality of proteins participating in reaction are modified, and a plurality of optimized modification schemes are obtained through large-scale screening analysis, and the yield of the baicalein and the scutellarein in microorganisms, particularly prokaryotic expression systems such as escherichia coli, is remarkably improved.

Accordingly, in a preferred embodiment of the present invention, there is provided a mutant F6H in which the amino acids at the (1-10) - (20-30) positions of the N-terminus are truncated, corresponding to the wild-type F6H; preferably, the amino acids (2-5) - (22-28) at the N-terminal are truncated; more preferably, the amino acids 2-25 of the N-terminus are truncated.

As a preferred embodiment of the present invention, there is also provided a fusion protein comprising F6H or mutant F6H, which comprises F6H or any mutant F6H, and a polypeptide tag fused thereto, wherein the polypeptide tag is selected from the group consisting of: 8RP, Sumo, MBP, 2B1, or a combination thereof; preferably MBP or 2B 1. The polypeptide tag may or may not comprise a linker peptide between the polypeptide tag and the F6H or mutant F6H, wherein the linker peptide does not affect the biological activity of the two.

In a preferred embodiment of the present invention, there is provided a mutant CPR in which the amino acids at positions (1-20) to (60-85) of the N-terminus are truncated in accordance with wild-type CPR; preferably, the amino acids (2-10) - (65-80) at the N-terminal are truncated; more preferably, the N-terminal amino acids at positions (2-5) to (70-75) are truncated.

On the basis of the above-mentioned preferred proteins (including the above-mentioned wild-type proteins, mutant proteins), the present invention also includes fragments, derivatives and analogs thereof which retain biological activity. The protein fragment, derivative or analogue thereof may be a deletion, insertion and/or substitution of several (usually 1 to 50, more preferably 1 to 20, still more preferably 1 to 10, 1 to 5, 1 to 3, or 1 to 2) amino acids, and addition or deletion of one or several (e.g., 100 or less, 80 or less, 50 or less, 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. However, in a further variation of the above mutant proteins, truncation to the N-terminus is performed as described above.

The present invention also includes analogs of the above-described preferred proteins (including wild-type, mutant proteins as described above) which differ from the native protein by amino acid sequence differences, by modifications which do not affect the sequence, or by both.

On the basis of the above-mentioned preferred proteins (including the above-mentioned wild-type proteins, mutant proteins), the present invention also includes proteins which have high homology with the above-mentioned proteins (for example, homology with the specified protein sequences is 70% or more; preferably homology is 80% or more; more preferably homology is 90% or more; like homology 95%, 98% or 99%) and have the same function as the corresponding polypeptides.

Proteins or genes from a particular species are listed in the present invention. It is to be understood that while proteins or genes from a particular species are preferably studied in the present invention, other proteins or genes obtained from other species that are highly homologous (e.g., have greater than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) to the proteins or genes are also within the contemplation of the present invention.

The invention also provides polynucleotide sequences encoding the proteins of the invention or conservative variant proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. Polynucleotides encoding the mutant mature proteins of the invention include: a coding sequence that encodes only the mature protein; the coding sequence for the mature protein and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature protein.

The invention also includes polynucleotide sequences formed after codon optimization of the sequence of the gene, e.g., according to the preference of the host cell.

In the invention, an engineering strain for high yield of baicalein and scutellarein is also constructed, wherein the engineering strain comprises exogenously introduced genes for expressing F6H (particularly the mutant F6H or the fusion protein) and CPR (particularly the mutant F6H or the fusion protein). Culturing the recombinant strain, and adding chrysin or apigenin into the culture system to produce baicalein or scutellarein.

In the invention, another high-yield baicalein or scutellarein engineering strain is also constructed, wherein the high-yield baicalein or scutellarein engineering strain comprises exogenously introduced genes for expressing F6H (particularly the mutant F6H or the fusion protein) and CPR (particularly the mutant F6H or the fusion protein), and a chrysin or apigenin synthesis gene. The chrysin or apigenin synthesis gene comprises: genes expressing PAL, 4CL, CHS, CHI and FNSI proteins.

The strain of the invention has good stability, and can realize large-scale culture and production of baicalein or scutellarein in a bioreactor. The yield of baicalein or scutellarein of the preferred strain of the invention is very high.

In the invention, the baicalein or the scutellarein is produced by the escherichia coli, so that the baicalein or the scutellarein can be produced more economically and conveniently.

The invention also provides a kit for producing the baicalein or scutellarein engineering strain. In addition, the kit can also comprise an Escherichia coli culture medium, a baicalein or scutellarein separation or detection reagent, an instruction for use and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Experimental Material

AxyPrep total RNA miniprep kit, Polymerase Chain Reaction (PCR) gel recovery kit, plasmid extraction kit are all American Axygen products; PrimeScript RT reagent Kit with gDNA Eraser (PerfectReal Time) polymerase Kit, Polymerase Chain Reaction (PCR) high fidelity enzyme PrimeSTAR Max DNApolymerase is a product of Nippon Baozo biology (TAKARA); all restriction enzymes are NEB products.

Coli DH10B was used for gene cloning, and E.coli BL21(DE3) strain was used for protein expression and production of baicalein and scutellarein. pET28a, pEDDuet-1, pCDFDuet-1 vectors were used for metabolic pathway gene assembly.

The standard compounds baicalein and scutellarein were purchased from Shanghai-sourced leaf Biotech, Inc. Other reagents are domestic analytical pure or chromatographic pure reagents and are purchased from national pharmaceutical group chemical reagent limited.

Arktik Thermal Cycler (Thermo Fisher Scientific) was used for PCR; the constant temperature culture uses a ZXGP-A2050 constant temperature incubator and an ZWY-211G constant temperature culture oscillator; centrifugation used a 5418R high speed refrigerated centrifuge and a 5418 mini centrifuge (Eppendorf). Vacuum concentration using a Concentrator plus Concentrator (Eppendorf); OD₆₀₀Detection was performed using a UV-1200 ultraviolet-visible spectrophotometer (Shanghai Mei spectral instruments, Ltd.). The rotary evaporation system consists of an IKARV 10digital rotary evaporator (IKA) and an MZ 2C NT chemical diaphragm pump, a CVC3000 vacuum controller (vacuubrand). High performance liquid chromatography was performed using a Dionex Ultimate 3000 liquid chromatography system (Thermo Fisher scientific).

Liquid phase detection conditions: phase A: 0.1% formic acid water, phase B: acetonitrile; separation conditions are as follows: 0-20min 20% B phase-55% B phase, 20-22min 55% B phase-100% B phase, 22-27min 100% B phase, 27-35min 100% B phase-20% B phase, 35-40min, 20% B phase; detection wavelength: 340nm, column temperature: at 30 ℃. A chromatographic column: thermo syncronis C18 reversed phase column (250 mm. times.4.6 mm, 5 μm).