Baicalein- and Scutellarein- Synthesizing Microorganism, Preparation Method and Applications Thereof

Abstract

Provided are a baicalein- and scutellarein-synthesizing microorganism, a preparation method for same, and applications thereof. By modifying a heterologous metabolic pathway of a host cell per a genetic engineering method, acquired is an engineered strain providing a high yield of baicalein and scutellarein. Also provided is a process for utilizing the engineered strain to produce baicalein and scutellarein.

Claims

1. A method of producing baicalein and scutellarein, comprising: (1) introducing genes expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase, as well as genes for synthesizing chrysin or apigenin, into a host cell; (2) culturing the host cell in a culture system containing phenylalanine and/or tyrosine to produce baicalein or scutellarein.

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

3. A method of producing baicalein and scutellarein, comprising: (1) introducing genes expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase into a host cell to obtain a recombinant strain; (2) culturing the recombinant host cell in a culture system containing chrysin or apigenin to produce baicalein or scutellarein.

4. A method for converting chrysin or apigenin into baicalein or scutellarein, comprising: catalyzing chrysin or apigenin by flavone 6-hydroxylase and cytochrome P450 oxidoreductase, thereby adding a hydroxyl group to the structure of chrysin or apigenin to form baicalein or scutellarein.

5. The method according to claim 1, wherein, the flavone 6-hydroxylase is a mutant flavone 6-hydroxylase with the N-terminal amino acids (1-10) to (20-30) truncated; preferably, it is a mutant flavone 6-hydroxylase with the N-terminal amino acids (2-5) to (22-28) truncated.

6. The method according to claim 1, wherein, the flavone 6-hydroxylase is fused with a peptide tag, the peptide tag is selected from N-terminal 8 amino acid peptide of bovine calf serum 17 hydroxylase, small ubiquitin-related modifier, maltose binding protein, 2B1 family soluble protein of cytochrome P450, or a combination thereof, preferably, the peptide tag is maltose binding protein or 2B1 family soluble protein of cytochrome P450, or a combination thereof; preferably, the peptide tag is located at the N-terminal.

7. The method according to claim 1, wherein, the cytochrome P450 oxidoreductase is a mutant cytochrome P450 oxidoreductase with the N-terminal amino acids (1-20) to (60-85) truncated; preferably, it is a mutant cytochrome P450 oxidoreductase with the N-terminal amino acids (2-10) to (65-80) truncated; more preferably, it is a mutant cytochrome P450 oxidoreductase with the N-terminal amino acids (2-5) to (70-75) truncated.

8. The method according to claim 1, wherein, the host cell comprises: prokaryotic cell or eukaryotic cell; preferably, the prokaryotic cell comprises: Escherichia coli cell, Bacillus subtilis cell; the eukaryotic cell comprises yeast cell.

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

10. The recombinant host cell according to claim 9, wherein, the recombinant host cell also comprises exogenous genes for synthesizing chrysin or apigenin; preferably, the genes for synthesizing chrysin or apigenin 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 claim 9, wherein, the flavone 6-hydroxylase is a mutant flavone 6-hydroxylase with the N-terminal amino acids (1-10) to (20-30) truncated; preferably, it is a mutant flavone 6-hydroxylase with the N-terminal amino acids (2-5) to (22-28) truncated.

12. The recombinant host cell according to claim 9, wherein, the flavone 6-hydroxylase is fused with a peptide tag, the peptide tag is selected from N-terminal 8 amino acid peptide of bovine calf serum 17 hydroxylase, small ubiquitin-related modifier, maltose binding protein, 2B1 family soluble protein of cytochrome P450, or a combination thereof, preferably, the peptide tag is maltose binding protein or 2B1 family soluble protein of cytochrome P450, or a combination thereof; preferably, the peptide tag is located at the N-terminal.

13. The recombinant host cell according to claim 9, wherein, the cytochrome P450 oxidoreductase is a mutant cytochrome P450 oxidoreductase with the N-terminal amino acids (1-20) to (60-85) truncated; preferably, it is a mutant cytochrome P450 oxidoreductase with the N-terminal amino acids (2-10) to (65-80) truncated; more preferably, it is a mutant cytochrome P450 oxidoreductase with the N-terminal amino acids (2-5) to (70-75) truncated.

14. (canceled)

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 the host cell to obtain a recombinant strain; preferably, the method also comprises: introducing genes for synthesizing chrysin or apigenin.

16. A kit for the production of baicalein and scutellarein, wherein the kit comprises the recombinant host cell according to claim 9.

17. A mutant flavonoid 6-hydroxylase, which corresponds to the wild-type flavonoid 6-hydroxylase but the N-terminal amino acids (1-10) to (20-30) are truncated; preferably, the N-terminal amino acids (2-5) to (22-28) are truncated; more preferably, the mutant flavonoid 6-hydroxylase has the amino acid sequence shown in SEQ ID NO: 2.

18. A mutant cytochrome P450 oxidoreductase, which corresponds to the wild-type cytochrome P450 oxidoreductase but the N-terminal amino acids (1-20) to (60-85) are truncated; preferably, the N-terminal amino acids (2-10) to (65-80) are truncated; more preferably, the N-terminal amino acids (2-5) to (70-75) are truncated; more preferably, the mutant cytochrome P450 oxidoreductase has the amino acid sequence shown in SEQ ID NO: 8.

19. A fusion polypeptide comprising the mutant flavone 6-hydroxylase according to claim 17 fused with a peptide tag, the peptide tag is selected from the group consisting of: 8RP, Sumo, MBP, 2B1; preferably, the peptide tag is MBP or 2B1.

20. The fusion polypeptide according to claim 19, wherein, the fusion polypeptide has an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

21. A polynucleotide, encoding: a mutant flavone 6-hydroxylase having N-terminal amino acids (1-10) to (20-30) or (2-5) to (22-28) truncated from wild-type flavonoid 6-hydroxylase; wherein preferably the mutant flavonoid 6-hydroxylase has the amino acid sequence of SEQ ID NO: 2; or a mutant cytochrome P450 oxidoreductase having N-terminal amino acids (1-20) to (60-85), or (2-10) to (65-80), or (2-5) to (70-75) truncated from wild-type cytochrome P450 oxidoreductase; wherein preferably the mutant cytochrome P450 oxidoreductase has the amino acid sequence of SEQ ID NO: 8; or a fusion polypeptide comprising a mutant flavone 6-hydroxylase fused with a peptide tag, the peptide tag being selected from the group consisting of 8RP, Sumo, MBP, and 2B1; wherein preferably the peptide tag is MBP or 2B1.

22. An expression construct, comprising: any polynucleotide according to claim 21.

23. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1. Structural formula of baicalein and scutellarein.

[0036] FIG. 2. Schematic of biosynthesis pathway of baicalein and scutellarein.

[0037] FIG. 3. Schematic of plasmid pYH66.

[0038] FIG. 4. Schematic of plasmid pYH57.

[0039] FIG. 5. HPLC results of engineering strain BL21(DE3)-pYH57-pYH66 and baicalein standard. i: BL21(DE3)-pETDuet-1-pCDFDuet-1 fermentation broth, as blank control; ii: BL21(DE3)-pYH57-pYH66 fermentation broth added with phenylalanine; baicalein standard.

[0040] FIG. 6. Mass spectrum results of baicalein produced by engineering strain BL21(DE3)-pYH57-pYH66.

[0041] FIG. 7. HPLC results of engineering strain BL21(DE3)-pYH57-pYH66 and scutellarein standard. i: BL21(DE3)-pETDuet-1-pCDFDuet-1 fermentation broth, as blank control; ii: BL21(DE3)-pYH57-pYH66 fermentation broth added with tyrosine; iii: scutellarein standard.

[0042] FIG. 8. Mass spectrum results of scutellarein produced by engineering strain BL21(DE3)-pYH57-pYH66.

[0043] FIG. 9. Production of baicalein from chrysin catalyzed by SbF6H and AtCPR mutants.

[0044] A. Schematic of the key elements in the constructed plasmid;

[0045] B. The conversion rates of baicalein from chrysin in recombinant E. coli;

[0046] C. HPLC results of the catalytic reaction solution of recombinant E. coli. Chr: chrysin; Bai: baicalein.

DETAILED DESCRIPTION

[0047] The inventor is committed to the heterologous synthesis of baicalein and scutellarein from microorganisms, and to improving biological production of baicalein and scutellarein. After in-depth study, engineering strains with high yield of baicalein and scutellarein is obtained by modifying the heterologous metabolic pathway of host cells through genetic engineering.

[0048] As used herein, “N-terminal amino acids (1-10) to (20-30)” refers to a sequence starting from any amino acid in N-terminal amino acids 1-10 and ending at any amino acid in N-terminal amino acids 20-30.

[0049] As used herein, “N-terminal amino acids (2-5) to (22-28)” refers to a sequence starting from any amino acid in N-terminal amino acids 2-5 and ending at any amino acid in N-terminal amino acids 22-28.

[0050] As used herein, “N-terminal amino acids (1-20) to (60-85)” refers to a sequence starting from any amino acid in N-terminal amino acids 1-20 and ending at any amino acid in N-terminal amino acids 60-85.

[0051] As used herein, “N-terminal amino acids (2-10) to (65-80)” refers to a sequence starting from any amino acid in N-terminal amino acids 2-10 and ending at any amino acid in N-terminal amino acids 65-80.

[0052] As used herein, “N-terminal amino acids (2-5) to (70-75)” refers to a sequence starting from any amino acid in N-terminal amino acids 2-5 and ending at any amino acid in N-terminal amino acids 70-75.

[0053] As used herein, “exogenous” or “heterologous” refers to two or more nucleic acid or protein sequences from different sources.

[0054] As used herein, “operably linked (to)” or “operably connected (to)” is intended to mean a functional spatial arrangement between two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is “operatively linked” to the nucleic acid sequence of a target gene when the promoter region is placed at a specific position relative to the nucleic acid sequence so that the transcription of the nucleic acid sequence is guided by the promoter region.

[0055] As used herein, the “expression construct” refers to a recombinant DNA molecule that contains the desired nucleic acid coding sequence. An expression construct may contain one or more gene expression cassettes. The “construct” is usually contained in an expression vector.

[0056] As used herein, the PAL, 4CL, CHS, CHI and FNSI proteins are proteins that constitute the biosynthesis pathway of chrysin or apigenin in the expression system.

[0057] As used herein, the F6H and CPR proteins are the proteins that convert chrysin or apigenin into baicalein or scutellarein in the expression system.

[0058] Wild types of the above proteins or genes have been identified in the art, so they can be available and prepared from the public. As a preferable embodiment of the disclosure, PAL is derived from Rhodotorula toruloides, with the sequence shown in GenBank accession number AAA33883.1; 4CL is derived from Petroselium crispum, with the sequence shown in GenBank accession number KF765780.1; CHS is derived from Petunia X hybrida, with the sequence shown in GenBank accession number KF765781.1; CHI is derived from Medicago sativa, with the sequence shown in GenBank accession number KF765782.1; FNS I is derived from Petroselium crispum, with the sequence shown in Swiss-Prot accession number Q7XZQ8.1.

[0059] Wild types of F6H and CPR have also been identified in the art. As a preferable embodiment of the disclosure, F6H is derived from Scutellaria baicalensis, with the sequence shown in GenBank accession number ASW21050.1. As a preferable embodiment of the disclosure, CPR is derived from Arabidopsis thaliana, with the sequence shown in GenBank accession number NP_849472.2.

[0060] The inventor found that when using host cells to produce baicalein and scutellarein, the wild-type F6H can only produce a small amount of products, which cannot achieve large-scale production. Through modification of multiple proteins involved in the reaction and a large number of screening and analysis, optimized modification schemes were obtained, which greatly improved the yield of baicalein and scutellarein of microorganisms, especially prokaryotic expression systems such as E. coli.

[0061] Therefore, a preferable embodiment of the present disclosure provides a mutant F6H that corresponds to the wild-type F6H with N-terminal amino acids (1-10) to (20-30) truncated; preferably, it is a mutant F6H with N-terminal amino acids (2-5) to (22-28) truncated; more preferably, it is a mutant F6H with N-terminal amino acids 2 to 25 truncated.

[0062] In a preferable embodiment of the disclosure, a fusion protein containing F6H or mutant F6H is provided, which includes F6H or any mutant F6H, and a peptide tag fused therewith, wherein the peptide tag is selected from the group consisting of 8RP, Sumo, MBP, 2B1, or a combination of them; preferably is MBP or 2B1. The peptide tag and the F6H or mutant F6H may or may not contain a linker peptide, and the linker peptide does not affect their biological activities.

[0063] In a preferable embodiment of the present disclosure, a mutant CPR is provided, which corresponds to the wild-type CPR with N-terminal amino acids (1-20) to (60-85) truncated; preferably, it is a mutant CPR with N-terminal amino acids (2-10) to (65-80) truncated; more preferably, it is a mutant CPR with N-terminal amino acids (2-5) to (70-75) truncated.

[0064] In addition to the above preferable proteins (including the above wild-type proteins and mutant proteins), the disclosure also includes their bioactive fragments, derivatives and analogues. Their fragments, derivatives or analogues may comprise deletion, insertion and/or substitution of several (usually 1-50, more preferably 1-20, yet more preferably 1-10, 1-5, 1-3, or 1-2) amino acids, as well as addition or deletion of one or more (for example, less than 100, 80, 50, 20, more preferably less than 10, yet more preferably less than 5) amino acids at C-terminal and/or N-terminal. For example, substitution with amino acids of comparable or similar properties usually does not change protein function in the art. As another example, addition of deletion of one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of a protein either. However, for further variation of the above mutant protein, the N-terminal was truncated as described above.

[0065] In addition to the above preferable proteins (including the above wild-type proteins and mutant proteins), the disclosure also includes their analogues. The differences between analogs and the original protein may be the difference in amino acid sequences, and may also be the difference in the forms of modifications that will not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as generating random mutagenesis by irradiation or exposure to mutagens, and can also be obtained by directed mutagenesis or other known molecular biology techniques. Analogs mentioned herein also include analogs with residue(s) different from natural L-amino acid (e.g., D-amino acids), as well as analogs with a non-naturally occurred or synthetic amino acid (such as β, γ-amino acids). It should be understood that the proteins of the present disclosure are not limited to the representative proteins described above.

[0066] In addition to the above preferable proteins (including the above wild-type proteins and mutant proteins), the disclosure also includes the protein with high homology (for example, having 70% or higher, preferable 80% or higher, more preferable 90% or higher (such as 95%, 98% or 99%) homology with the sequence of the particular described protein) and having the same function as the corresponding protein.

[0067] The disclosure describes proteins or genes from specific species. It should be understood that although the proteins or genes obtained from a specific species are preferably studied in the present disclosure, other proteins or genes obtained from other species and having high homology (such as having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) with the proteins or genes also fall within the scope of the present disclosure.

[0068] The disclosure also provides a polynucleotide sequence encoding the protein of the disclosure or a conserved variant thereof. The polynucleotide sequences herein can be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. The DNA may be coding strand or non-coding strand. The polynucleotide encoding the mutant mature protein of the disclosure includes: the coding sequence only encoding the mature protein; the coding sequence encoding the mature protein and a various additional coding sequence; the coding sequence encoding the mature protein (and an optional additional coding sequence) and a noncoding sequence.

[0069] The disclosure also includes the codon-optimized polynucleotide sequence of the gene sequence, for example, the codon-optimized according to the codon bias of the host cell.

[0070] In the disclosure, an engineering strain with high yield of baicalein and scutellarein is also constructed, which includes exogenous genes expressing F6H (especially the mutant F6H or fusion protein) and CPR (especially the mutant CPR or fusion protein). Baicalein or scutellarein can be produced by culturing the recombinant strain and adding chrysin or apigenin into the culture system.

[0071] In the disclosure, another engineering strain with high yield of baicalein and scutellarein is constructed, which includes exogenous genes expressing F6H (especially the mutant F6H or fusion protein) and CPR (especially the mutant CPR or fusion protein), as well as genes for synthesizing chrysin or apigenin. The genes for synthesizing chrysin or apigenin comprise genes expressing PAL, 4CL, CHS, CHI and FNSI proteins.

[0072] By use of the strain according to the disclosure, which has great stability, large-scale cultivation and production of baicalein or scutellarein in a bioreactor can be realized. The yield of baicalein or scutellarein of the optimized strain of the disclosure is very high.

[0073] In the disclosure, more economical and convenient manufacture of baicalein or scutellarein can be conducted by production of baicalein or scutellarein from E. coli.

[0074] The disclosure also provides a kit for producing baicalein or scutellarein engineering strains. In addition, it can also include culture medium for E. coli, separation or detection reagent for baicalein or scutellarein, instruction for use, etc.

[0075] The disclosure is further illustrated by the specific examples described below. It should be understood that these examples are merely illustrative, and do not limit the scope of the present disclosure. The experimental methods without specifying the specific conditions in the following examples generally used the conventional conditions, such as those described in J. Sambrook, Molecular Cloning: A Laboratory Manual (3rd ed. Science Press, 2002) or followed the manufacturer's recommendation.

[0076] Experimental Materials

[0077] AxyPrep Total RNA Miniprep Kit, PCR Gel Extraction Kit, Plasmid Extraction Kit are from Axygen; PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time), PrimeSTAR Max DNA Polymerase are from Takara, and restriction enzymes are from NEB.

[0078] E. coli DH10B was used for gene cloning, E. coli BL21 (DE3) was used for protein expression and baicalein and scutellarein production. pET28a, pEDDuet-1 and pCDFDuet-1 vectors were used for assembling of genes in metabolic pathway.

[0079] Baicalein and scutellarein standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Other reagents are analytical grade reagent or chromatographic grade reagent, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0080] PCR was conducted on Arktik Thermal Cycler (Thermo Fisher Scientific); ZXGP-A2050 Incubator and ZWY-211G Constant Temperature Oscillator were used for culture; high-speed freezing Centrifuge 5418R and Centrifuge 5418 (Eppendorf) were used for centrifugation. Vacuum concentration was performed with Concentrator Plus (Eppendorf); OD.sub.600 was detected using UV-1200 Ultraviolet/Visible Spectrophotometer (Shanghai Mapada Instrument Co., Ltd.). Rotary evaporation system consists of IKA RV 10 Digital Rotary Evaporator (IKA), MZ 2C NT Chemical Diaphragm Pump and CVC3000 vacuum controller (Vacuubrand). Dionex UltiMate 3000 Liquid Chromatography System (Thermo Fisher Scientific) was used for HPLC.

[0081] Liquid phase detection conditions: A phase: 0.1% formic acid solution, B phase: acetonitrile; separation conditions: 0-20 min, 20% B phase-55% B phase, 20-22 min, 55% B phase-100% B phase, 22-27 min, 100% B phase-20% B phase, 27-35 min, 100% B phase-20% B phase, 35-40 min, 20% B phase; detection wavelength: 340 nm, column temperature: 30° C. The chromatographic column was Thermo syncronis C18 RP column (250 mm*4.6 mm, 5 μm).

Example 1. Polypeptide and its Sequence Optimization

[0082] 1. Optimization of F6H Polypeptide Sequence

[0083] The sequence of Scutellaria baicalensis F6H (SbF6H, 517aa, Genbank access No. ASW21050.1) is:

TABLE-US-00001 MELSSVIYGAIALLSLFYCYLHFSKPKKSSLNAPPEAGGARFITGHLHLM DGRSASDKLPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTH DTAVMARPRLIADDYLSYDGASLGFSPYGPYWREIRKLVTTELLSARRIE LQRATRVREITQFTGELYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRM VVGKRFYGGDDSETTKRWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFE KRTRDTAYELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSLVQANPTLQ LHYDADTIIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELD EQVGRERRVEESDISNLPYLQAVVKETMRLYPPAPFAGVRAFSEDCTVGG YHIQKGTFLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELM PFGGGRRMCPGSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMK ATPLDAILTPRLSPTLY*

[0084] Modification 1: the modified F6H mutant trF6H was constituted by removing the amino acids 2-25 of SEQ ID NO: 1 and adding two amino acids MA to the N-terminal. The sequence of trF6H is as follows (SEQ ID NO: 2):

TABLE-US-00002 MAMPKKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGLLADQHG PIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYLSYDGAS LGFSPYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGELYKLWE EKKDGSGRVLVDMKQWLGNLSLNLVSRMVVGKRFYGGDDSETTKRWRGVM REFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAMWLAEYR KREYSGDDKEQCFMALMLSLVQANPTLQLHYDADTIIKATCQVLISAASD TTTVILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISNLPYLQA VVKETMRLYPPAPFAGVRAFSEDCTVGGYHIQKGTFLIVNLWKLHRDPRV WSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSNLGMHMVHFV LANILQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTLY*

[0085] Modification 2: the modified F6H mutant 8RPtrF6H was constituted by removing the amino acids 2-25 of SEQ ID NO: 1 and adding amino acids of 8RP to the N-terminal. The sequence of 8RPtrF6H is as follows (SEQ ID NO: 3):

TABLE-US-00003 MALLLAVFMPKKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGL LADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYL SYDGASLGFSPYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGE LYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRMVVGKRFYGGDDSETTK RWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAM WLAEYRKREYSGDDKEQCFMALMLSLVQANPTLQLHYDADTIIKATCQVL ISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISN LPYLQAVVKETMRLYPPAPFAGVRAFSEDCTVGGYHIQKGTFLIVNLWKL HRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSNLGM HMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTL Y*

[0086] Modification 3: the modified F6H mutant SumotrF6H was constituted by removing the amino acids 2-25 of SEQ ID NO: 1 and adding amino acids of Sumo to the N-terminal. The sequence of SumotrF6H is as follows (SEQ ID NO: 4):

TABLE-US-00004 MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLME AFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGMP KKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGLLADQHGPIFT IRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYLSYDGASLGFS PYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGELYKLWEEKKD GSGRVLVDMKQWLGNLSLNLVSRMVVGKRFYGGDDSETTKRWRGVMREFF QLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAMWLAEYRKREY SGDDKEQCFMALMLSLVQANPTLQLHYDADTIIKATCQVLISAASDTTTV ILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISNLPYLQAVVKE TMRLYPPAPFAGVRAFSEDCTVGGYHIQKGTFLIVNLWKLHRDPRVWSDD ALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSNLGMHMVHFVLANI LQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTLY*

[0087] Modification 4: the modified F6H mutant MBPtrF6H was constituted by removing the amino acids 2-25 of SEQ ID NO: 1 and adding amino acids of MBP to the N-terminal. The sequence of MBPtrF6H is as follows (SEQ ID NO: 5):

TABLE-US-00005 MAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFP QVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVR YNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALM FNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDL IKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLP TFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKP LGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVI NAASGRQTVDEALKDAQTMPKKSSLNAPPEAGGARFITGHLHLMDGRSAS DKLPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMA RPRLIADDYLSYDGASLGFSPYGPYVVREIRKLVTTELLSARRIELQRAT RVREITQFTGELYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRMVVGKR FYGGDDSETTKRWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRD TAYELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSLVQANPTLQLHYDA DTIIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGR ERRVEESDISNLPYLQAVVKETMRLYPPAPFAGVRAFSEDCTVGGYHIQK GTFLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGG RRMCPGSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLD AILTPRLSPTLY*

[0088] Modification 5: the modified F6H mutant 2B1trF6H was constituted by removing the amino acids 2-25 of SEQ ID NO: 1 and adding amino acids of 2B1 to the N-terminal. The sequence of 2B 1trF6H is as follows (SEO ID NO: 6):

TABLE-US-00006 MAKKTSSKGKLPPGPSMPKKSSLNAPPEAGGARFITGHLHLMDGRSASDK LPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARP RLIADDYLSYDGASLGFSPYGPYVVREIRKLVTTELLSARRIELQRATRV REITQFTGELYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRMVVGKRFY GGDDSETTKRWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTA YELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSLVQANPTLQLHYDADT IIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGRER RVEESDISNLPYLQAVVKETMRLYPPAPFAGVRAFSEDCTVGGYHIQKGT FLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRR MCPGSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLDAI LTPRLSPTLY*

[0089] 2. Modification of CPR

[0090] The sequence of Arabidopsis thaliana CPR (AtCPR, 712aa, Genebank access No. NP_849472.2) is as follows (SEQ ID NO: 7):

TABLE-US-00007 MSSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENR QFAMIVTTSIAVLIGCIVMLVWRRSGSGNSKRVEPLKPLVIKPREEEIDD GRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDYAADDDE YEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKYG VFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDDFTAW REALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDINMAN GNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTYETGD HVGVLCDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFPP CNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKVDE YSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKI AETRIHVTCALVYEKMPTGRIHKGVCSTWMKNAVPYEKSENCSSAPIFVR QSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFF GCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKAS DIWNMISQGAYLYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVKN LQTSGRYLRDVW*

[0091] The modified AtCPR mutant trAtCPR was constituted by removing the amino acids 2-72 of SEQ ID NO: 1. The sequence of trAtCPR is as follows (SEO ID NO: 8):

TABLE-US-00008 MRRSGSGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGFAK ALGEEAKARYEKTRFKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDG EPTDNAARFYKWFTEGNDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDD ILVEQGAQRLVQVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVA TPYTAAVLEYRVSIHDSEDAKFNDINMANGNGYTVFDAQHPYKANVAVKR ELHTPESDRSCIHLEFDIAGSGLTYETGDHVGVLCDNLSETVDEALRLLD MSPDTYFSLHAEKEDGTPISSSLPPPFPPCNLRTALTRYACLLSSPKKSA LVALAAHASDPTEAERLKHLASPAGKVDEYSKWVVESQRSLLEVMAEFPS AKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRI HKGVCSTWMKNAVPYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPGT GLAPFRGFLQERLALVESGVELGPSVLFFGCRNRRMDFIYEEELQRFVES GALAELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYLYVCGDAKGM ARDVHRSLHTIAQEQGSMDSTKAEGFVKNLQTSGRYLRDVW*

Example 2. Construction of Recombinant Plasmid Containing Novel F6H Mutant

[0092] Based on pETDuet-1, plasmid pYH45 was constituted by linking AtCPR into NdeI and XhoI sites by one-step cloning method.

[0093] Based on pETDuet-1, plasmid pYH46 was constituted by linking trAtCPR into NdeI and XhoI sites by one-step cloning method.

[0094] Furthermore, pUC19-F6H was constituted by linking the codon optimized coding sequence of F6H (synthesized by GenScript) into pUC19. PCR was conducted using F6H-F/R as primers and pUC19-F6H as templates. The PCR system was 50 μL (Primestar Max Premix, 25 μL; final concentration 0.2-0.3 μM of the two primers; pUC19-F6H, 0.2 μL; the remaining volume was supplemented with sterilized distilled water). PCR reaction procedure is: pre-denaturation at 98° C. for 2 min, denaturation at 98° C. for 10 s, annealing at 55° C. for 15 s, extension at 72° C. for 20 s, 25 cycles. The amplified fragment of about 1.5 kb was detected by agarose electrophoresis, purified and digested with Nco I and BamH I. The digested fragment was ligated into pYH46 digested by the same enzymes, and the ligated product was transformed into competent cells of E. coli DH10B. The plasmid was extracted. The recombinant plasmid pYH59 was verified by double digestion (on restriction sites introduced during plasmid construction) and gene sequencing. Similarly, the digested fragment was ligated into pYH45 to obtain the recombinant plasmid pYH59.

[0095] PCR was conducted using trF6H-F/F6H-R as primers and pUC19-trF6H as templates. Plasmid pYH58 was obtained by ligating the amplified fragment into NdeI and BamH I of pYH46 by one-step cloning method.

[0096] PCR was conducted using 8RP-trF6H-F/F6H-R as primers and pUC19-trF6H as templates. Plasmid pYH60 was obtained by ligating the amplified fragment into NdeI and BamH I of pYH46 by one-step cloning method.

[0097] DNA fragment containing Sumo sequence was amplified using pETSumo (Invitrogen) as templates and Sumo-F/Sumo-trF6H-R as primers. DNA fragment containing trF6H was amplified using pUC19-trF6H as templates and Sumo-trF6H-F/F6H-R as primers. PCR amplification was conducted using Sumo-F/F6H-R as primers and the above two DNA fragments as templates. Plasmid pYH61 was obtained by ligating the amplified fragment into NdeI and BamH I of pYH46 by one-step cloning method.

[0098] DNA fragments containing MBP sequence was amplified using pMAL-c5x (Invitrogen) as templates and MBP-F/MBP-trF6H-R as primers. DNA fragments containing trF6H was amplified using pUC19-trF6H as templates and MBP-trF6H-F/F6H-R as primers. Then PCR amplification was conducted using MBP-F/F6H-R as primers and the above two DNA fragments as templates. Plasmid pYH62 was obtained by ligating the amplified fragment into NdeI and BamH I of pYH46 by one-step cloning method.

[0099] PCR was conducted using 2B1-F/F6H-R as primers and pUC19-trF6H as templates. Plasmid pYH63 was obtained by ligating the amplified fragment into NdeI and BamH I of pYH46 by one-step cloning method.

[0100] PCR was conducted using trF6H-F/F6H-R as primers and pUC19-trF6H as templates. Plasmid pYH64 was obtained by ligating the amplified fragment into NdeI and BamH I of pYH45 by one-step cloning method.

[0101] DNA fragments containing MBP sequence was amplified using pMAL-c5x as templates and MBP-F/MBP-trF6H-R as primers. DNA fragments containing trF6H was amplified using pUC19-trF6H as templates and MBP-trF6H-F/F6H-R as primers. Then PCR amplification was conducted using MBP-F/F6H-R as primers and the above two DNA fragments as templates. Plasmid pYH65 was obtained by ligating the amplified fragment into NdeI and BamH I of pYH45 by one-step cloning method.

[0102] PCR was conducted using 2B1-F/F6H-R as primers and pUC19-2B1trF6H as templates. Plasmid pYH66 was obtained by ligating the amplified fragment into NdeI and BamH I of pYH45 by one-step cloning method. Schematic of plasmid pYH66 is shown in FIG. 3.

[0103] The primers used in the above constructions are shown in Table 1. Schematic of the key elements in the constructed plasmid is shown in FIG. 9A.

TABLE-US-00009 TABLE 1 Primers Sequences F6H-F TATACCATGGAACTGAGCAGTGTGA (SEQ ID NO: 9) F6H-R CTCGAATTCGGATCCACTAGTTTAATATAAAGTCGG (SEQ ID NO: 10) trF6H-F CTTTAAGAAGGAGATATACCATGGCGATGCCGAAGAAAAGCTC (SEQ ID NO: 11) 8RP-trF6H-F CTTTAAGAAGGAGATATACCATGGCTCTGTTATTAGCAGTTTTTAT GCCGAAGAAAAGCTCTT (SEQ ID NO: 12) MBP-F CTTTAAGAAGGAGATATACCATGGCTAAAATCGAAGAAG (SEQ ID NO: 13) MBP-trF6H-F CTGAAAGACGCGCAGACTATGCCGAAGAAAAGCTC (SEQ ID NO: 14) MBP-trF6H-R GAGCTTTTCTTCGGCATAGTCTGCGCGTCTTTCAG (SEQ ID NO: 15) 2B1-F CTTTAAGAAGGAGATATACCATGGCTAAGAAAACGAGCTCTAAA GGGAAGCTCCCACCAGGACCTAGCATGCCGAAGAAAAGCTCTT (SEQ ID NO: 16) Sumo-F CTTTAAGAAGGAGATATACCATGGCGGACTCAGAAGTCAATCTT (SEQ ID NO: 17) Sumo-trF6H-F GAGAACAGATTGGTGGTATGCCGAAGAAAAGCTCTT (SEQ ID NO: 18) Sumo-trF6H-R AAGAGCTTTTCTTCGGCATACCACCAATCTGTTCTC (SEQ ID NO: 19)

Example 3. Construction of Recombinant Plasmids Expressing PAL, 4CL, CHS, CHI and FNSI

[0104] Rhodotorula toruloides PAL (GenBankAccess No. AAA33883.1), Petroselium crispum 4CL (GenBank Access No. KF765780.1), Petunia X hybrid CHS (GenBankAccess No. KF765781.1), Medicago sativa CHI gene (GenBankAccess No. KF765782.1), Petroselium crispum FNS I gene (Swiss-ProtAccess No. Q7XZQ8.1) were synthesized by GenScript and constructed into pET28a, forming plasmids pET28-PAL, pET28-4CL, pET28-CHS, pET28a-CHI, and pET28a-FNSI, respectively.

[0105] The primers in Table 2 were synthesized. PCR amplification was conducted using pET28-4CL as templates and 4CL-F-NcoI/4CL-R-BamHI as primers. pYH40 was constructed by ligation of the amplified products with NcoI/BamHI digested pCDFDuet-1.

[0106] PCR amplification was conducted using pET28-CHS as templates and CHS-F-NdeI/CHS-R-XhoI as primers. pYH50 was constructed by ligation of the amplified products with NdeI/XhoI digested pYH40.

[0107] PCR amplification was conducted using pET28a-CHI as templates and T7CHI-F-XhoI/CHI-R-AvrII as primers. Then pYH51 was constructed by ligation of the amplified products with pYH50.

[0108] PCR amplification was conducted using pET28-PAL as templates and T7PAL-F-BamH I/PAL-R-Hind III as primers. The amplified products were digested by BamH I and Hind III, and ligated with pYH51 digested by the same enzymes to form plasmid pYH55.

[0109] PCR amplification was conducted using pET28a-FNSI as templates and FNSI-HindIII-F/FNSI-NotI-R as primers. The amplified products were digested by Hind III and Not I, and ligated with pYH55 digested by the same enzymes to form plasmid pYH57. Schematic of plasmid pYH57 is shown in FIG. 4.

TABLE-US-00010 TABLE 2 Primers Sequences 4CL-F-NcoI TATACCATGGGTGACTGCGTTGCCCCG (SEQ ID NO: 20) 4CL-R-BamHI CGGGATCCTTACTTCGGCAGGTCGCCGCTC (SEQ ID NO: 21) T7PAL-F-BamHI CGGGATCCCTTATGCGACTCCTGCATTAG (SEQ ID NO: 22) PAL-R-HindIII GCCCAAGCTTTTATGCCAGCATCTTC (SEQ ID NO: 23) CHS-F-NdeI AGATATACATATGGTTACGGTGGAAGAATAC (SEQ ID NO: 24) CHS-R-XhoI CCGCTCGAGTTAGGTAGCCACACTATGCAG (SEQ ID NO: 25) T7CHI-F-XhoI CCGCTCGAGCTAGAAATAATTTTGTTTAAC (SEQ ID NO: 26) CHI-R-AvrII GAGCCTAGGTTAGTTACCGATTTTAAAG (SEQ ID NO: 27) FNSI-HindIII-F GAAGATGCTGGCATAAAAGCTTCGATCCCGCGAAATTA (SEQ ID NO: 28) FNSI-NotI-R CGACTTAAGCATTATGCGGCCGCCTACGCCAGGTTTTC (SEQ ID NO: 29)

Example 4. Construction and Functional Verification of Baicalein and Scutellarein Synthesizing Strains

[0110] The biosynthesis process of baicalein and scutellarein is shown in FIG. 1.

[0111] The engineering strain BL21(DE3)-pYH57-pYH66 was obtained by co-transformation of the recombinant plasmids pYH66 and pYH57 into E. coli BL21 (DE3) competent cells.

[0112] The cells were cultured in LB solid medium (containing 80 μg/ml spectinomycin, 100 μg/ml ampicillin) overnight at 37° C. Single colony was transferred to a 2 mL LB liquid medium (containing 80 μg/ml spectinomycin, and 100 μg/ml ampicillin) and incubated overnight. The bacterial fluid was transferred to a new 10 ml MOPS liquid medium with antibiotics and incubated at 37° C. and 250 r/min until OD.sub.600 reached 0.5-0.6. The culture was cooled down to 16° C. in a water bath. Then inducer IPTG was added at a final concentration of 1 mM, and different concentrations of sterilized phenylalanine or tyrosine was added. The mixture was placed at 22° C. for low temperature induction, and cultured for 48 h at 220 r/min. The BL21 (DE3) recombinant strain containing empty plasmid pETDuet-1 and pCDFDuet-1 without foreign gene(s) was used as blank control, and the culture procedure was the same as above.

[0113] At the same time, the recombinant plasmids listed in Table 2 were transformed into E. coli and cultured to detect the production of their products.

[0114] After culture, the expression of compounds in each recombinant strain harboring recombinant plasmid was detected, which are shown in Table 3.

TABLE-US-00011 TABLE 3 Plasmids Features Uses pYH40 expressing 4CL protein Synthesis of pinocembrin and pYH50 expressing 4CL and CHS proteins naringenin pYH51 expressing 4CL, CHS and CHI proteins pYH55 expressing PAL, 4CL, CHS and CHI proteins pYH57 expressing PAL, 4CL, CHS, CHI and FNSI Synthesis of chrysin from   proteins phenylalanine Synthesis of apigenin from   tyrosine pYH58 expressing trF6H and trCPR proteins Synthesis of baicalein from pYH59 expressing F6H and CPR proteins chrysin pYH60 expressing 8RPF6H and trCPR proteins Synthesis of scutellarein from pYH61 expressing SumotrF6H and trCPR proteins apigenin pYH62 expressing MBPtrF6H and trCPR proteins pYH63 expressing 2B1trF6H and trCPR proteins pYH64 expressing trF6H and CPR proteins pYH65 expressing MBPtrF6H and CPR proteins pYH66 expressing 2B1trF6H and CPR proteins

[0115] It was verified that each recombinant strain of the disclosure can successfully synthesize the target compound.

[0116] HPLC results of engineering strain BL21(DE3)-pYH57-pYH66 and baicalein standard are shown in FIG. 5. Mass spectrum results of baicalein produced by engineering strain BL21(DE3)-pYH57-pYH66 are shown in FIG. 6.

[0117] HPLC results of engineering strain BL21(DE3)-pYH57-pYH66 and scutellarein standard are shown in FIG. 7. Mass spectrum results of scutellarein produced by engineering strain BL21(DE3)-pYH57-pYH66 are shown in FIG. 8.

Example 5: Production with Chrysin as Substrate

[0118] Six recombinant plasmids (pYH58 to pYH66) were transformed into competent cells of E. coli BL21 (DE3) to obtain the engineering strains BL21(DE3)-pYH58 to BL21(DE3)-pYH66, respectively.

[0119] The cells were cultured in LB solid medium (containing 100 μg/ml ampicillin) overnight at 37° C. Single colony was transferred to a 2 mL LB liquid medium (containing 100 μg/ml ampicillin) and incubated overnight. The bacterial fluid was transferred to a new 20 ml MOPS liquid medium with antibiotics and incubated at 37° C. and 250 r/min until OD.sub.600 reached 0.5-0.6. The culture was cooled down to 16° C. in a water bath. Then inducer IPTG was added at a final concentration of 1 mM. The mixture was cultured for 12 h at 22° C. and 220 r/min. After centrifugation at 6000 rpm, 4° C. for 10 min, the supernatant was removed, and the bacteria were collected and re-suspended in a reaction buffer (50 mM Tris-HCl, pH 7.4, 0.1% Trixton) until OD.sub.600 reached 30.5 μL chrysin (25 mM) and 2.5 μL NADPH (100 mM) were added to 1 mL of the suspension of the recombinant bacteria, and the reaction was continued at 37° C. for 8 hours. After completion of the reaction, the solution was extracted for 3 times by 10 μL HCl (6 M) and 1 mL ethyl acetate. The organic phase was concentrated, and the resulting residue was dissolved with 200 μL methanol, wherein 10 μL was used for HPLC analysis.

[0120] The conversion rates of baicalein from chrysin in each recombinant E. coli were shown in FIG. 9B.

[0121] HPLC results of the catalytic reaction solution of each recombinant E. coli were shown in FIG. 9C.

[0122] Each reference provided herein is incorporated by reference to the same extent as if each reference was individually incorporated by reference. In addition, it should be understood that based on the above teaching content of the disclosure, those skilled in the art can practice various changes or modifications to the disclosure, and these equivalent forms also fall within the scope of the appended claims.