WO2020048523A1 - 合成黄芩素和野黄芩素的微生物、其制备方法及其应用 - Google Patents

合成黄芩素和野黄芩素的微生物、其制备方法及其应用 Download PDF

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WO2020048523A1
WO2020048523A1 PCT/CN2019/104658 CN2019104658W WO2020048523A1 WO 2020048523 A1 WO2020048523 A1 WO 2020048523A1 CN 2019104658 W CN2019104658 W CN 2019104658W WO 2020048523 A1 WO2020048523 A1 WO 2020048523A1
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baicalein
flavonoid
hydroxylase
cytochrome
mutant
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PCT/CN2019/104658
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French (fr)
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王勇
李建华
田晨菲
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中国科学院上海生命科学研究院
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Priority to US17/274,377 priority Critical patent/US20220033827A1/en
Priority to JP2021537458A priority patent/JP2021535757A/ja
Priority to EP19856619.2A priority patent/EP3848462A4/en
Publication of WO2020048523A1 publication Critical patent/WO2020048523A1/zh

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Definitions

  • the present invention relates to the technical field of synthetic biology and medicine, and in particular, the present invention relates to a microorganism that synthesizes baicalein and baicalein, a preparation method and application thereof.
  • Scutellaria baicalensis (Georgi) is a famous Chinese traditional medicine, which is a plant in the family Lamiaceae; Chinese herbal medicine scutellaria baicalensis is a dried root of the plant Scutellaria baicalensis.
  • Erigeron breviscapus is a dried whole grass of the gerbera breviscapus. It has a bitter cold taste, and has anti-inflammatory and analgesic effects, promoting blood circulation, removing blood stasis, and removing wind and dampness.
  • the extract of Scutellaria baicalensis and Erigeron breviscapus has been widely used in traditional Chinese medicine preparations.
  • the main raw materials of Yinhuang tablets, Shuanghuanglian oral liquid and Lancen oral liquid are extracts of Scutellaria baicalensis.
  • the main active ingredient of Chinese herbal medicine Qingkailing is baicalein. It has the functions of anti-inflammation, prevention and treatment of diarrhea, liver disease and tumors.
  • Common scutellar dosage forms include scutellarin tablets and scutellarin oral solution. Their main metabolic absorption form in the body is its aglycone wild baicalein. Therefore, baicalein and wild baicalein both have certain new drug development value.
  • Baicalin and baicalein are two important and similar flavonoids.
  • the molecular formula of baicalein is C 15 H 10 O 5 and the molecular weight is 270.24, while the molecular weight of baicalein is C 15 H 10 O 6 and the molecular weight is 286.24.
  • Their structure is shown in Figure 1.
  • baicalein and baicalein are currently mainly prepared by two methods, chemical synthesis and organic solvent extraction.
  • Organic solvent extraction is mainly used for tissue extraction of medicinal plants such as Scutellaria baicalensis, Erigeron breviscapus, Scutellaria barbata, etc.
  • a large amount of organic solvents are required, and subsequent separation processes are cumbersome and costly for industrialization.
  • This method suffers from slow plant growth and damage to medicinal resources.
  • baicalein and baicalein can also be obtained in large quantities through chemical synthesis, the raw materials will involve chemical substances such as cinnamic acid or its derivatives, oxophenols, and their applications in the fields of medicine and food are limited to a certain extent.
  • the use of toxic reagents and expensive chemical catalysts is also involved in the synthesis process.
  • Synthetic biology is based on rational design, which integrates and assembles standardized biological components to build artificial life systems with excellent performance. Once synthetic biology was born, its thinking and design profoundly affected the development of industrial microbial technology, making microbial technology play a greater role in the development and production of drugs, biofuels, and fine chemicals.
  • An object of the present invention is to provide a microorganism that synthesizes baicalein and baicalein, a preparation method thereof, and an application thereof.
  • a method for producing baicalein and baicalein comprising: (1) introducing into a host cell an expression of flavonoid 6-hydroxylase (F6H) and cytochrome P450 oxidoreductase (CPR ) Gene, and chrysin or apigenin synthesis gene; and (2) culturing the host cell in a culture system containing phenylalanine and / or tyrosine, thereby producing baicalein or baicalein.
  • F6H flavonoid 6-hydroxylase
  • CPR cytochrome P450 oxidoreductase
  • the astaxanthin or apigenin synthesis gene includes: expression of phenylalanine ammoniammy-lyase (PAL), 4-coumarate coenzyme A ligase (4-coumarate: CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavone synthase I (FNSI) genes; preferably, When introduced into host cells, the genes expressing phenylalanine ammonialyase, 4-coumaric acid coenzyme A ligase, chalcone synthase, chalcone isomerase and flavonoid synthase I existed in the same expression Vector.
  • PAL phenylalanine ammoniammy-lyase
  • 4-coumarate coenzyme A ligase (4-coumarate: CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavone synthas
  • the flavonoid 6-hydroxylase is derived from Scutcllaria baicalensis, and also includes homologues (homologous genes or polypeptides from other species);
  • the CPR is derived from Arabidopsis Arabidopsis thaliana also includes its homologues.
  • the PAL is derived from Rhodotorula toruloides and includes homologues thereof;
  • the 4CL is derived from parsley (Petroselium cristum) and includes homologues thereof;
  • the CHS is derived from petunia (hybrida) and also includes homologues thereof;
  • the CHI is derived from Medicago (sativa) and also includes homologues thereof;
  • the FNS I is derived from parsley (Petroselium crispum), including its homologues.
  • a method for producing baicalein and baicalein comprising: (1) introducing into a host cell the expression of flavonoid 6-hydroxylase (F6H) and cytochrome P450 oxidoreductase (CPR ) Gene to obtain a recombinant host cell; and (2) culturing the recombinant host cell in a culture system containing chrysin or apigenin to produce baicalein or baicalein.
  • F6H flavonoid 6-hydroxylase
  • CPR cytochrome P450 oxidoreductase
  • a method for converting chrysin or apigenin to baicalein or lutein catalyzing chrysin or apigenin with flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase, thereby Add a hydroxyl group to the structure of chrysin or apigenin to form baicalein or baicalein.
  • the flavonoid 6-hydroxylase is a mutant flavonoid 6-hydroxylase that truncates the N-terminal amino acids (1-10) to (20-30); Is a mutant flavonoid 6-hydroxylase that truncates the (2-5) to (22-28) amino acids at the N-terminus.
  • the flavonoid 6-hydroxylase is fused to a polypeptide tag
  • the polypeptide tag is selected from the group consisting of calf serum 17 hydroxylase N-terminal 8 amino acid polypeptide (8RP), and small molecule ubiquitin modification Related protein (Sumo), maltose binding protein (MBP), cytochrome P450 2B1 family soluble protein (2B1), or a combination thereof; preferably maltose binding protein or cytochrome P450 2B1 family soluble protein, or a combination thereof; preferably The polypeptide tag is located at the N-terminus.
  • the cytochrome P450 oxidoreductase is a mutant cytochrome P450 oxidoreductase that truncates the N-terminal amino acid from (1-20) to (60-85) positions; Preferably, it is a mutant cytochrome P450 oxidoreductase that truncates the (2-10) to (65-80) amino acids at the N-terminus; more preferably, it cuts (2-5) to (70) at the N-terminus -75) mutant cytochrome P450 oxidoreductase.
  • the host cells include: prokaryotic cells or eukaryotic cells; preferably, the prokaryotic cells include: E. coli cells, Bacillus subtilis cells; and the eukaryotic cells include: yeast cell.
  • a recombinant host cell which includes a foreign gene expressing flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase.
  • the recombinant host cell further includes an exogenous chrysin or apigenin synthesis gene.
  • polypeptide tag is a single copy or a sequence structure of 2-10 copies (such as 3, 4, 5, 6, 8 copies) in tandem.
  • a strain that does not have a gene for astaxanthin or apigenin in the cell it is used to produce baicalein and baicalein as a substrate for externally added chrysin or apigenin;
  • a method for preparing a host cell for producing baicalein and baicalein comprising: introducing into the host cell a gene expressing flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase To obtain a recombinant strain; preferably, the method further comprises introducing a chrysin or apigenin synthesis gene.
  • kits for producing baicalein and baicalein includes the recombinant host cell of any one of the foregoing.
  • the kit further includes: a host cell culture medium, an instruction manual, and the like.
  • a mutant flavonoid 6-hydroxylase which corresponds to the wild-type flavonoid 6-hydroxylase (F6H), and the (1-10) to (20-30) positions of the N-terminus are truncated.
  • Amino acid preferably, the N-terminal amino acid at positions (2-5) to (22-28) is truncated; preferably, it has the amino acid sequence shown in SEQ ID NO: 2.
  • a mutant cytochrome P450 oxidoreductase which corresponds to the wild-type cytochrome P450 oxidoreductase and truncates the N-terminal amino acid at positions (1-20) to (60-85);
  • the N-terminal (2-10) to (65-80) amino acids are truncated; more preferably, the N-terminal (2-5) to (70-75) amino acids are truncated; preferably, the It has the amino acid sequence shown in SEQ ID NO: 8.
  • a fusion polypeptide which includes any of the mutant flavonoid 6-hydroxylase enzymes described above, and a polypeptide tag fused thereto, the polypeptide tag is selected from: 8RP, Sumo, MBP , 2B1; preferably MBP or 2B1.
  • 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.
  • a polynucleotide which encodes: the mutant flavonoid 6-hydroxylase described above; or the mutant cytochrome P450 oxidoreductase; or the fusion polypeptide.
  • an expression construct comprising: any of the polynucleotides described above; or encoding any of the mutant flavonoid 6-hydroxylase enzymes described above or the aforementioned A polynucleotide of a fusion protein, and a polynucleotide encoding a mutant cytochrome P450 oxidoreductase as described above.
  • the expression construct further includes a promoter and a terminator operatively linked to the polynucleotide.
  • mutant flavonoid 6-hydroxylase or the fusion protein and the use of the mutant cytochrome P450 oxidoreductase for use in the structure of astaxanthin or apigenin Add a hydroxyl group to form baicalein or baicalein.
  • FIG. 1 Structural formula of baicalein and baicalein.
  • FIG. 1 Schematic diagram of plasmid pYH66 construction.
  • Figure 4 Schematic diagram of plasmid pYH57 construction.
  • FIG. 1 HPLC detection pattern of engineering strain BL21 (DE3) -pYH57-pYH66 and standard baicalein.
  • i represents BL21 (DE3) -pETDuet-1-pCDFDuet-1 fermentation broth as a blank control
  • ii represents BL21 (DE3) -pYH57-pYH66 fermentation broth supplemented with phenylalanine
  • iii represents baicalein standard.
  • FIG. 7 HPLC detection pattern of engineering strain BL21 (DE3) -pYH57-pYH66 and standard baicalein.
  • i represents BL21 (DE3) -pETDuet-1-pCDFDuet-1 fermentation broth as a blank control
  • ii represents BL21 (DE3) -pYH57-pYH66 fermentation broth supplemented with tyrosine
  • iii represents a standard of baicalein.
  • Figure 8 Mass spectrum of wild baicalein produced by engineering strain BL21 (DE3) -pYH57-pYH66.
  • A a schematic diagram of the key elements in the constructed plasmid
  • the present inventors are committed to the use of heterologous synthesis of baicalein and baicalein in microorganisms, and to increase the production of baicalein and baicalein.
  • genetic engineering methods are used to carry out heterologous metabolic pathways in host cells After transformation, engineering strains with high yield of baicalein and wild baicalein were obtained.
  • amino acid at the (1-10) to (20-30) position at the N-terminus refers to an amino acid that starts at any position from the N-terminus to 1-10 and ends at the N-terminus. Amino acids at any of positions 20-30.
  • N-terminus (2-5) to (22-28) refers to an amino acid that starts at any position 2-5 from the N-terminus and ends at the N-terminus Amino acids at any of positions 22-28.
  • N-terminal (1-20) to (60-85) refers to an amino acid that starts at any of the 1-20th position from the N-terminus and ends at the N-terminal position. Amino acids at any of positions 60-85.
  • N-terminal (2-10) to (65-80) refers to an amino acid that starts at any of the 2-10th position from the N-terminus and ends at the N-terminal position. Amino acids at positions 65-80.
  • N-terminus (2-5) to (70-75) refers to an amino acid that starts at any of the 2nd to 5th positions from the N-terminus and ends at the N-terminus. Amino acids at any of positions 70-75.
  • align or “heterologous” refers to the relationship between two or more nucleic acid or protein sequences from different sources.
  • operably linked (linked) or “operably linked (linked)” refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest, so that the transcription of the nucleic acid sequence is guided by the promoter region, so that the promoter region is “operably linked” to the nucleic acid sequence.
  • the "expression construct” refers to a recombinant DNA molecule, which contains a desired nucleic acid coding sequence, which may include one or more gene expression cassettes. Said “construct” is usually contained in an expression vector.
  • the PAL, 4CL, CHS, CHI, and FNSI proteins are proteins that form a chrysin or apigenin synthesis pathway in an expression system.
  • the F6H and CPR proteins are proteins that convert chrysin or apigenin in an expression system, and produce baicalein or baicalein.
  • PAL is derived from Rhodotorula Toruloides, which has the sequence shown in GenBank Accession No. AAA33883.1; 4CL is derived from Parsley (Petroselium Crispum), which has GenBank Accession No. KF765780.1.
  • CHS is derived from Petunia hybrida, which has the sequence shown in GenBank accession number KF765781.1; CHI gene is derived from Medicago sativa, which has the sequence shown in GenBank accession number KF765782.1 Sequence; FNS I is derived from parsley (Petroselium crispus), which has the sequence shown in Swiss-Prot accession number Q7XZQ8.1.
  • Wild-type F6H and CPR have also been identified in the art.
  • F6H is derived from Scutellaria baicalensis and has a sequence shown by GenBank accession number ASW21050.1.
  • the CPR is derived from Arabidopsis thaliana, which has the sequence shown in GenBank accession number NP_849472.2.
  • baicalein and baicalein in the process of using the host cell to produce baicalein and baicalein, the use of wild-type F6H can only produce trace products and cannot achieve large-scale production. Therefore, many proteins involved in the reaction have been modified. After a large number of screening and analysis, some preferred transformation schemes were obtained, which significantly increased the yield of baicalein and baicalein in prokaryotic expression systems such as E. coli.
  • a mutant F6H which corresponds to the wild-type F6H, and truncates the N-terminal amino acid at positions (1-10) to (20-30); preferably, N is truncated. (2-5) to (22-28) terminal amino acids; more preferably, truncated amino acids 2-25 of the N terminal.
  • a fusion protein containing the F6H or mutant F6H which includes F6H or any mutant F6H, and a polypeptide tag fused thereto, the polypeptide tag is selected from: 8RP, Sumo, MBP, 2B1, or a combination thereof; preferably MBP or 2B1.
  • the polypeptide tag and the F6H or mutant F6H may or may not include a linker peptide, and the linker peptide does not affect the biological activity of the two.
  • a mutant CPR which corresponds to the wild-type CPR, and truncates the N-terminal amino acid from (1-20) to (60-85); preferably, the N-terminal (2-10) to (65-80) amino acids; more preferably, the N-terminal amino acids (2-5) to (70-75) are truncated.
  • the present invention also includes fragments, derivatives and analogs that retain their biological activity.
  • Their protein fragments, derivatives or analogs may be several (usually 1-50, more preferably 1-20, and even more preferably 1-10, 1-5, 1-3, or 1-2) amino acid deletions, insertions and / or substitutions, and addition or deletion of one or several (e.g., 100 or less, 80 or less, 50 or less, or 20 or less) at the C-terminus and / or N-terminus It is preferably within 10 amino acids, and more preferably within 5 amino acids. For example, in the art, substitution of amino acids with similar or similar properties usually does not change the function of the protein.
  • adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein.
  • truncation to the N-terminus as described above was performed.
  • the present invention also includes their analogs.
  • the differences between these analogs and natural proteins can be differences in amino acid sequences, or differences in modified forms that do not affect the sequence, or both.
  • These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.
  • Analogs also include analogs with residues (such as D-amino acids) that are different from natural L-amino acids, and analogs with non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • the present invention also includes high homology with the protein (for example, the homology with the specific protein sequence listed is 70 % Or higher; preferably 80% or higher homology; more preferably 90% or higher homology (such as 95%, 98% or 99% homology), and has the same function as the corresponding polypeptide Proteins are also included in the present invention.
  • Proteins or genes from specific species are enumerated in the present invention. It should be understood that although the protein or gene obtained from a specific species is preferably studied in the present invention, the protein or gene obtained from other species is highly homologous to the protein or gene (eg, has more than 60%, such as 70%, 80%, 85% , 90%, 95%, or even 98% sequence identity) are also within the scope of the present invention.
  • the invention also relates to a polynucleotide sequence encoding a protein of the invention or a conservatively variant protein thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the polynucleotide encoding the mutant mature protein of the present invention includes: the coding sequence encoding only the mature protein; the coding sequence of the mature protein and various additional coding sequences; the coding sequence of the mature protein (and optional additional coding sequences); Coding sequence.
  • the invention also includes a polynucleotide sequence formed by codon optimization of the sequence of the gene, for example, codon optimization according to the preference of the host cell.
  • an engineering strain with high yield of baicalein and baicalein is also constructed, which includes exogenously introduced F6H (especially the mutant F6H or fusion protein) and CPR (especially the mutant F6H Or fusion protein).
  • F6H especially the mutant F6H or fusion protein
  • CPR especially the mutant F6H Or fusion protein
  • the recombinant strain is cultured, and chrysin or apigenin is added to the culture system to produce baicalein or baicalein.
  • baicalein or baicalein another engineering strain with high yield of baicalein or baicalein is also constructed, which includes exogenously introduced F6H (especially the mutant F6H or fusion protein) and CPR (especially the mutant type).
  • F6H or fusion protein gene, and astaxanthin or apigenin synthesis genes.
  • the astaxanthin or apigenin synthesis genes include: 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 baicalein in a bioreactor.
  • the yield of baicalein or baicalein of the preferred strain of the present invention is very high.
  • baicalein or baicalein is produced by E. coli, so that baicalein or baicalein can be produced more economically and conveniently.
  • the invention also provides a kit for producing baicalein or baicalein engineering strains.
  • it can also include E. coli culture medium, baicalein or baicalein separation or detection reagents, instructions for use, and the like.
  • AxyPrep total RNA small amount preparation kit, polymerase chain reaction (PCR) gel recovery kit, plasmid extraction kit are all American Axygen products; PrimeScript RT agent kit with gDNA Eraser (Perfect Real Time) polymerase kit, polymerization
  • the enzyme-chain reaction (PCR) high-fidelity enzyme PrimeSTAR, Max DNA and Polymerase are products of TAKARA; the restriction enzymes are all NEB products.
  • E. coli DH10B is used for gene cloning
  • E. coli BL21 (DE3) strain is used for protein expression and baicalein and baicalein production.
  • pET28a, pEDDuet-1, and pCDFDuet-1 vectors are used for gene assembly in metabolic pathways.
  • baicalein and wild baicalein were purchased from Shanghai Yuanye Biotechnology Co., Ltd.
  • Other reagents are domestic analytical or chromatographic reagents, purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Arktik Thermal Cycler (Thermo Fisher Scientific) was used for PCR; ZXGP-A2050 constant temperature incubator and ZWY-211G constant temperature culture shaker were used for constant temperature culture; 5418R high-speed refrigerated centrifuge and 5418 small centrifuge (Eppendorf) were used for centrifugation. Concentrator plus concentrator (Eppendorf) was used for vacuum concentration; OD 600 was detected by UV-1200 UV-visible spectrophotometer (Shanghai Mepida Instrument Co., Ltd.).
  • the rotary evaporation system consists of IKA RV 10digital rotary evaporator (IKA), MZ 2C NT chemical diaphragm pump, and CVC3000 vacuum controller (vacuubrand). High performance liquid chromatography used a Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific).
  • Liquid phase detection conditions A phase: 0.1% formic acid water, B phase: acetonitrile; separation conditions: 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: 30 ° C.
  • Chromatographic column Thermo syncronis C18 reversed-phase column (250 mm ⁇ 4.6 mm, 5 ⁇ m).
  • F6H The length of F6H (SbF6H) from Scutellaria baicalensis is 517aa (Genbank access no. ASW21050.1).
  • the specific sequence is as follows (SEQ ID NO: 1):
  • Modification 1 The present inventor modified the sequence of SEQ ID NO: 1, removed amino acids 2-25, and added two amino acids of MA to the N-terminus to obtain an improved F6H mutant trF6H.
  • the specific sequence is as follows (SEQ ID ID NO :2):
  • Modification 2 The inventor modified the sequence of SEQ ID NO: 1, removed the amino acids 2-25, and added the amino acid sequence of 8RP to the N-terminus to obtain an improved F6H mutant 8RPtrF6H.
  • the specific sequence is as follows (SEQ ID NO: 3):
  • Modification 3 The inventor modified the sequence of SEQ ID NO: 1, removed the amino acids 2-25, and added the Sumo amino acid sequence to the N-terminus to obtain an improved F6H mutant SumotrF6H.
  • the specific sequence is as follows (SEQ ID NO: 4):
  • Modification 4 The inventor modified the sequence of SEQ ID NO: 1, removed the amino acids 2-25, and added the amino acid sequence of MBP at the N-terminus to obtain an improved F6H mutant MBPtrF6H.
  • the specific sequence is as follows (SEQ ID NO: 5):
  • Modification 5 The inventor modified the sequence of SEQ ID NO: 1, removed the amino acids 2-25, and added the amino acid sequence of 2B1 to the N-terminus to obtain an improved F6H mutant 2B1trF6H.
  • the specific sequence is as follows (SEQ ID NO: 6):
  • the CPR (AtCPR) sequence length from Arabidopsis thaliana is 712aa (Genebank access no. NP_849472.2), as follows (SEQ ID NO: 7):
  • the inventors performed sequence modification on SEQ ID NO: 1, deleted amino acids 2-72 therein, and obtained an improved AtCPR mutant trAtCPR, as follows (SEQ ID NO: 8):
  • AtCPR was ligated to NdeI and XhoI sites by a one-step cloning method to obtain plasmid pYH45.
  • trAtCPR was ligated to the NdeI and XhoI sites by a one-step cloning method to obtain plasmid pYH46.
  • the codon-optimized F6H coding gene sequence was synthesized and ligated into the pUC19 vector to obtain pUC19-F6H.
  • F6H-F / R was used as a primer and pUC19-F6H was used as a template for PCR amplification.
  • the system was 50 ⁇ L (PrimeSTAR Max Premix, 25 ⁇ L; final concentration of dual primers 0.2 to 0.3 ⁇ M; pUC19-F6H 0.2 uL; the remaining volume was sterilized with distilled water). Make up).
  • PCR reaction conditions pre-denaturation at 98 ° C for 2 min, then denaturation at 98 ° C for 10 s, annealing at 55 ° C for 15 s, extension at 72 ° C for 20 s, 25 cycles, agarose electrophoresis detection, amplification to obtain a fragment of approximately 1.5 kb, and purification using Nco I and BamH I double digestion.
  • the digested fragment was ligated into pYH46 digested by the same enzyme, and the ligated product was transformed into E. coli DH10B competent cells.
  • the extracted plasmid was constructed and introduced into the digestion site for double digestion verification and gene sequencing to obtain the recombinant plasmid pYH59.
  • the digested fragment was ligated into pYH45 to obtain a recombinant plasmid pYH59.
  • trF6H-F / F6H-R as a primer and pUC19-trF6H as a template
  • the product obtained by PCR amplification was ligated to the NdeI and BamH I sites in pYH46 by a one-step cloning method to obtain plasmid pYH58.
  • the product was ligated to the NdeI and BamH I sites of pYH46 by a one-step cloning method to obtain pYH60.
  • a DNA fragment containing Sumo sequence was obtained using pETSumo (purchased from Invitrogen) as a template, Sumo-F / Sumo-trF6H-R as a primer, pUC19-trF6H as a template, and Sumo-trF6H-F / F6H-R as a primer extension
  • the trF6HDNA fragment was amplified.
  • the product was ligated to the NdeI and BamHI sites of pYH46 by a one-step cloning method to obtain pYH61.
  • pMAL-c5x purchased from Invitrogen
  • MBP-F / MBP-trF6H-R primers to obtain a DNA fragment containing MBP sequences.
  • pUC19-trF6H as a template
  • MBP-trF6H-F / F6H -R is a primer to obtain a DNA fragment containing trF6H.
  • the product was ligated to the NdeI and pYH46 NdeI and In the BamH I site, pYH62 was obtained.
  • the product was ligated into the NdeI and BamHI sites of pYH46 by a one-step cloning method to obtain pYH63.
  • trF6H-F / F6H-R as a primer and pUC19-trF6H as a template
  • the product obtained by PCR amplification was ligated into the NdeI and BamH I sites of pYH45 by a one-step cloning method to obtain a recombinant plasmid pYH64.
  • the primers shown in Table 2 were synthesized. Using pET28-4CL as a template and 4CL-F-NcoI / 4CL-R-BamHI as primers to obtain PCR products, pYFD40 was ligated with NcoI / BamHI double-digested pCDFDuet-1 to obtain pYH40.
  • pYH50 was obtained by ligation with pYH40 double-digested with NdeI / XhoI.
  • PCR products were obtained using pET28a-CHI as a template and T7CHI-F-XhoI / CHI-R-AvrII as primers, and ligated to pYH50 to obtain pYH51.
  • PCR products were obtained using pET28-PAL as a template and T7PAL-F-BamH I / PAL-R-Hind III as primers. After double digestion with BamH I / Hind III, the pYH51 was ligated with the same double digestion to obtain plasmid pYH55.
  • PCR products were obtained using pET28a-FNSI as a template and FNSI-HindIII-F / FNSI-NotI-R) as primers. After double digestion with Hind III / Not I, they were ligated with pYH55 with the same double digestion to obtain the final vector pYH57.
  • the construction of plasmid pYH57 is shown in Figure 4.
  • the recombinant plasmids pYH66 and pYH57 were co-transformed into competent cells of E. coli BL21 (DE3) to obtain the engineered strain BL21 (DE3) -pYH57-pYH66.
  • LB solid medium (Spectinomycin 80 ⁇ g / mL, Ampicillin 100 ⁇ g / mL) was cultured overnight at 37 ° C.
  • Pick a single clone into 2mL LB liquid medium (Spectinomycin 80 ⁇ g / mL, Ampicillin 100 ⁇ g / mL)
  • To OD 600 0.5-0.6, cool the water bath to about 16 ° C, then add the inducer IPTG to a final concentration of 1mM, add sterilized phenylalanine or tyrosine at different concentrations and transfer to 22 ° C low temperature induction culture.
  • the culture was continued for 48 h at a shaker speed of 220 r / min.
  • the BL21 (DE3) recombinant strain carrying empty plasmids pETDuet-1 and pCDFDuet-1 that had not been ligated into foreign genes was used as a blank control.
  • each of the recombinant plasmids listed in Table 2 was transformed into E. coli and cultured, and the production of each product was detected.
  • the HPLC detection pattern of the engineering strain BL21 (DE3) -pYH57-pYH66 and the standard baicalein is shown in Fig. 5.
  • the mass spectrum of baicalein produced by the engineering strain BL21 (DE3) -pYH57-pYH66 is shown in Figure 6.
  • the HPLC detection pattern of the engineering strain BL21 (DE3) -pYH57-pYH66 and the standard baicalein is shown in Fig. 7.
  • the mass spectrum of wild baicalein produced by the engineering strain BL21 (DE3) -pYH57-pYH66 is shown in Figure 8.
  • the six recombinant plasmids pYH58 ⁇ pYH66 were transformed into competent cells of E. coli BL21 (DE3) to obtain engineered strains, and named BL21 (DE3) -pYH58 ⁇ BL21 (DE3) -pYH66.
  • LB solid medium (Ampicillin 100 ⁇ g / mL) was cultured overnight at 37 ° C.
  • Pick a single clone into 2mL LB liquid medium (Ampicillin 100 ⁇ g / mL), transfer the overnight bacterial culture to a new 20mL MOPS liquid resistant medium at 37 ° C, and culture at 250r / min to OD 600 0.5-0.6
  • the temperature of the water bath was reduced to about 16 ° C, and then the inducer IPTG was added to a final concentration of 1 mM, and the culture was continued for 12 h at a shaker speed of 220 r / min and a temperature of 22 ° C.
  • reaction buffer 50 mM Tris-HCl, pH 7.4, 0.1% Trixton
  • OD 600 30.
  • 1 mL of the resuspended suspension of each recombinant bacterium was taken, 5 ⁇ L of astaxanthin (25 mM) and 2.5 ⁇ L of NADPH (100 mM) were added thereto, and the reaction was continued at 37 ° C. for 8 hours.
  • 10 ⁇ L of HCl (6M) and 1 mL of ethyl acetate were added to the reaction solution for extraction three times.
  • the residue obtained by concentrating the organic phase was dissolved in 200 ⁇ L of methanol, and 10 ⁇ L was taken for HPLC analysis.

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Abstract

提供合成黄芩素和野黄芩素的微生物、其制备方法及其应用。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。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。

Description

合成黄芩素和野黄芩素的微生物、其制备方法及其应用 技术领域
本发明涉及合成生物学及医药技术领域，具体地，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。
背景技术
黄芩(Scutcllaria Baicalensis Georgi)是中国著名的传统药物，为唇形科植物；中药黄芩是植物黄芩干燥的根，药用历史悠久，可用于风热湿热等多种疾病的治疗。灯盏花是菊科植物短葶(Erigeron breviscapus)的干燥全草，性寒味苦，具有消炎止痛、活血化瘀和祛风除湿等功效。黄芩和灯盏花提取物早已被广泛应用于中药制剂中，银黄片、双黄莲口服液和兰岑口服液等的主要原料都是黄芩提取物，中药清开灵的主要有效成分就是黄芩素，它具有消炎，防治腹泻、肝病和肿瘤的作用；常见的灯盏花剂型有灯盏花素片和灯盏花素口服液等，它们在生物体内的主要代谢吸收形式是其苷元野黄芩素。因此，黄芩素和野黄芩素均具有一定的新药开发价值。
黄芩素和野黄芩素是二个结构相似且重要的黄酮类化合物。黄芩素的分子式为C 15H 10O 5，分子量为270.24，而野黄芩素的分子量为C 15H 10O 6，分子量为286.24。它们的结构如图1所示。
与大多数植物源天然产物一样，目前黄芩素和野黄芩素的主要通过化学合成和有机溶剂萃取两种方法制备。有机溶剂萃取主要是对黄芩、灯盏花、半枝莲等药用植物进行组织抽提，在此过程中需要大量的有机溶剂，还存在随后的分离工艺繁琐，工业化造价高等问题。最主要的是该方法受到植物生长缓慢、对药用资源的破坏等难题。尽管通过化学合成也可以大量获得黄芩素和野黄芩素，但是原料会涉及肉桂酸或其衍生物、氧代苯酚等化工物质，一定程度限制了其在药物、食品领域的应用。而且在合成过程中还涉及到有毒试剂及昂贵的化学催化剂的使用等问题。
合成生物学是基于理性的设计，将标准化的生物元件进行集成与装配，从而构建性能优良的人造生命系统。合成生物学一经诞生，它的思想和设计就深刻地影响着工业微生物技术的发展，使微生物技术在药物、生物燃料、精细化学品的开发与生产过程中发挥出更加巨大的作用。
在本领域中，多种天然产物的合成元件经过组装后实现了在微生物中的异源合成。但是本发明中涉及到的黄芩素和野黄芩素这两种活性黄酮类化合物，还尚未见在微生物中成功地异源合成的报道。因此，迫切需要构建一株能够异源合成黄芩素和野黄芩素的微生物菌株。
发明内容
本发明的目的在于提供合成黄芩素和野黄芩素的微生物、其制备方法及其应用。
在本发明的第一方面，提供一种生产黄芩素和野黄芩素的方法，包括：(1)在宿主细胞中引入表达黄酮6-羟化酶(F6H)以及细胞色素P450氧化还原酶(CPR)的基因，以及白杨素或芹菜素合成基因；和(2)在含有苯丙氨酸和/或酪氨酸的培养体系中培养该宿主细胞，从而生产黄芩素或野黄芩素。
在一个优选例中，所述的白杨素或芹菜素合成基因包括：表达苯丙氨酸解氨酶(phenylalanine ammonia-lyase，PAL)、4-香豆酸辅酶A连接酶(4-coumarate：CoA ligase，4CL)、查尔酮合成酶(chalcone synthase，CHS)、查尔酮异构酶(chalcone isomerase，CHI)和黄酮合成酶I(flavone synthase I，FNSI)的基因；较佳地，在被引入宿主细胞时，所述的表达苯丙氨酸解氨酶、4-香豆酸辅酶A连接酶、查尔酮合成酶、查尔酮异构酶和黄酮合成酶I的基因存在于同一表达载体中。
在另一优选例中，所述的黄酮6-羟化酶来源于黄芩(Scutcllaria baicalensis)，也包括其同源物(来自其它物种的同源基因或多肽)；所述的CPR来源于拟南芥(Arabidopsis thaliana)，也包括其同源物。
在另一优选例中，所述的PAL来源于红景天(Rhodotorula toruloides)，也包括其同源物；所述的4CL来源于欧芹(Petroselium crispum)，也包括其同源物；所述的CHS来源于矮牵牛(Petunia X hybrida)，也包括其同源物；所述的CHI来源于苜蓿(Medicago sativa)，也包括其同源物；所述的FNS I来源于欧芹(Petroselium crispum)，也包括其同源物。
在本发明的另一方面，提供一种生产黄芩素和野黄芩素的方法，包括：(1)在宿主细胞中引入表达黄酮6-羟化酶(F6H)以及细胞色素P450氧化还原酶(CPR)的基因，获得重组的宿主细胞；和(2)在含有白杨素或芹菜素的培养体系中培养该重组的宿主细胞，从而生产黄芩素或野黄芩素。
在本发明的另一方面，提供一种将白杨素或芹菜素转化为黄芩素或野黄芩素的方法：以黄酮6-羟化酶以及细胞色素P450氧化还原酶催化白杨素或芹菜素，从而在白杨素或芹菜素的结构中加上一个羟基，形成黄芩素或野黄芩素。
在一个优选例中，所述的黄酮6-羟化酶(F6H)是截去N端第(1-10)～(20-30)位氨基酸的突变型黄酮6-羟化酶；较佳地，是截去N端第(2-5)～(22-28)位氨基酸的突变型黄酮6-羟化酶。
在另一优选例中，所述的黄酮6-羟化酶与多肽标签融合，所述的多肽标签选自：小牛血清17羟基化酶N端8氨基酸多肽(8RP)，小分子泛素修饰相关蛋白(Sumo)，麦芽糖结合蛋白(MBP)，细胞色素P450 2B1家族可溶性蛋白(2B1)，或其组合；较佳地为麦芽糖结合蛋白或细胞色素P450 2B1家族可溶性蛋白，或其组合；较佳地所述多肽标签位于N端。
在另一优选例中，所述的细胞色素P450氧化还原酶(CPR)的是截去N端第(1-20)～(60-85)位氨基酸的突变型细胞色素P450氧化还原酶；较佳地，是截去N端第(2-10)～ (65-80)位氨基酸的突变型细胞色素P450氧化还原酶；更佳地，是截去N端第(2-5)～(70-75)位氨基酸的突变型细胞色素P450氧化还原酶。
在另一优选例中，所述的宿主细胞包括：原核细胞或真核细胞；较佳地，所述的原核细胞包括：大肠杆菌细胞，枯草芽孢杆菌细胞；所述的真核细胞包括：酵母细胞。
在本发明的另一方面，提供一种重组宿主细胞，其中包括外源的表达黄酮6-羟化酶以及细胞色素P450氧化还原酶的基因。
在另一优选例中，所述的重组宿主细胞中还包括外源的白杨素或芹菜素合成基因。
在另一优选例中，所述的多肽标签为单拷贝或2～10拷贝(如3、4、5、6、8拷贝)串联的序列结构。
在本发明的另一方面，提供前面任一所述的重组宿主细胞的用途，用于生产黄芩素和野黄芩素。
在一个优选例中，对于细胞内不存在白杨素或芹菜素合成基因的菌株，用于以外源添加的白杨素或芹菜素为底物生产黄芩素和野黄芩素；对于细胞内存在白杨素或芹菜素合成基因的菌株，用于在外源添加苯丙氨酸和/或酪氨酸的培养条件下生产黄芩素和野黄芩素。
在本发明的另一方面，提供一种制备用于生产黄芩素和野黄芩素的宿主细胞的方法，包括：在宿主细胞中引入表达黄酮6-羟化酶以及细胞色素P450氧化还原酶的基因，获得重组菌株；较佳地，还包括引入白杨素或芹菜素合成基因。
在本发明的另一方面，提供一种用于生产黄芩素和野黄芩素的试剂盒所述试剂盒中包括前面任一所述的重组的宿主细胞。
在另一优选例中，所述的试剂盒中还包括：宿主细胞培养基，使用说明书等。
在本发明的另一方面，提供突变型黄酮6-羟化酶，其对应于野生型黄酮6-羟化酶(F6H)，截去N端第(1-10)～(20-30)位氨基酸；较佳地，截去N端第(2-5)～(22-28)位氨基酸；较佳地，其具有SEQ ID NO:2所示的氨基酸序列。
在本发明的另一方面，提供突变型细胞色素P450氧化还原酶，其对应于野生型细胞色素P450氧化还原酶，截去N端第(1-20)～(60-85)位氨基酸；较佳地，截去N端第(2-10)～(65-80)位氨基酸；更佳地，截去N端第(2-5)～(70-75)位氨基酸；较佳地，其具有SEQ ID NO:8所示的氨基酸序列。
在本发明的另一方面，提供融合多肽，其包括前面所述的任一突变型黄酮6-羟化酶，以及与之融合的多肽标签，所述的多肽标签选自：8RP，Sumo，MBP，2B1；较佳地为MBP或2B1。
在一个优选例中，所述的融合多肽具有选自下组的氨基酸序列：SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6。
在本发明的另一方面，提供多核苷酸，其编码：述的突变型黄酮6-羟化酶；或所述的突变型细胞色素P450氧化还原酶；或所述的融合多肽。
在本发明的另一方面，提供一种表达构建物，其包括：前面所述的任一多核苷酸；或编码前面所述的任一突变型黄酮6-羟化酶或前面所述的融合蛋白的多核苷酸，以及编码前面所述的突变型细胞色素P450氧化还原酶的多核苷酸。
在另一优选例中，所述的表达构建物中，还包括与上述的多核苷酸操作性连接的启动子和终止子。
在本发明的另一方面，提供所述的突变型黄酮6-羟化酶或所述的融合蛋白，以及突变型细胞色素P450氧化还原酶的用途，用于在白杨素或芹菜素的结构中加上一个羟基，形成黄芩素或野黄芩素。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、黄芩素和野黄芩素的结构式。
图2、黄芩素和野黄芩素的生物合成路径。
图3、质粒pYH66的构建示意图。
图4、质粒pYH57的构建示意图。
图5、工程菌株BL21(DE3)-pYH57-pYH66和标准品黄芩素的HPLC检测图谱。其中，i表示BL21(DE3)-pETDuet-1-pCDFDuet-1发酵液作为空白对照；ii表示BL21(DE3)-pYH57-pYH66添加苯丙氨酸的发酵液；iii表示黄芩素标准品。
图6、工程菌株BL21(DE3)-pYH57-pYH66产黄芩素的质谱图谱。
图7、工程菌株BL21(DE3)-pYH57-pYH66和标准品野黄芩素的HPLC检测图谱。其中，i表示BL21(DE3)-pETDuet-1-pCDFDuet-1发酵液作为空白对照；ii表示BL21(DE3)-pYH57-pYH66添加酪氨酸的发酵液；iii表示野黄芩素标准品。
图8、工程菌株BL21(DE3)-pYH57-pYH66产野黄芩素的质谱图谱。
图9、SbF6H和AtCPR突变体催化白杨素生成黄芩素。
A，所构建的质粒中关键元件的示意图；
B，重组大肠杆菌中，催化白杨素生成黄芩素的转化率；
C，HPLC分析重组大肠杆菌催化反应液。其中，Chr表示白杨素，Bai表示黄芩素。
具体实施方式
本发明人致力于利用微生物中异源合成黄芩素和野黄芩素，以及提高生物生产黄芩素和野黄芩素的产量，经过深入的研究，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。
如本文所用，所述的“N端第(1-10)～(20-30)位氨基酸”是指起始于N端起第1-10位中的任一位氨基酸，终止于N端起第20-30位中的任一位氨基酸。
如本文所用，所述的“N端第(2-5)～(22-28)位”是指起始于N端起第2-5位中的任一位氨基酸，终止于N端起第22-28位中的任一位氨基酸。
如本文所用，所述的“N端第(1-20)～(60-85)位”是指起始于N端起第1-20位中的任一位氨基酸，终止于N端起第60-85位中的任一位氨基酸。
如本文所用，所述的“N端第(2-10)～(65-80)位”是指起始于N端起第2-10位中的任一位氨基酸，终止于N端起第65-80位中的任一位氨基酸。
如本文所用，所述的“N端第(2-5)～(70-75)位”是指起始于N端起第2-5位中的任一位氨基酸，终止于N端起第70-75位中的任一位氨基酸。
如本文所用，“外源的”或“异源的”是指来自不同来源的两条或多条核酸或蛋白质序列之间的关系。
如本文所用，所述的“可操作地连接(相连)”或“操作性连接(相连)”是指两个或多个核酸区域或核酸序列的功能性的空间排列。例如：启动子区被置于相对于目的基因核酸序列的特定位置，使得核酸序列的转录受到该启动子区域的引导，从而，启动子区域被“可操作地连接”到该核酸序列上。
如本文所用，所述的“表达构建物”是指重组DNA分子，它包含预期的核酸编码序列，其可以包含一个或多个基因表达盒。所述的“构建物”通常被包含在表达载体中。
如本文所用，所述的PAL、4CL、CHS、CHI和FNSI蛋白是在表达系统中形成白杨素或芹菜素合成途径的蛋白。
如本文所用，所述的F6H和CPR蛋白是在表达系统中转化白杨素或芹菜素、生成黄芩素或野黄芩素的蛋白。
野生型的上述蛋白或基因均为本领域已经鉴定的，因此，可以从公众途径获得和制备。作为本发明的优选方式，PAL来源于红景天(Rhodotorula toruloides)，其具有GenBank登录号AAA33883.1所示的序列；4CL来源于欧芹(Petroselium crispum)，其具有GenBank登录号KF765780.1所示的序列；CHS来源于矮牵牛(Petunia X hybrida)，其具有GenBank登录号KF765781.1所示的序列；CHI基因来源于苜蓿(Medicago sativa)，其具有GenBank登录号KF765782.1所示的序列；FNS I来源于欧芹(Petroselium crispum)，其具有Swiss-Prot登录号Q7XZQ8.1所示的序列。
野生型的F6H和CPR也是本领域已经鉴定的。作为本发明的优选方式，F6H来源于黄岑(Scutellaria baicalensis)，其具有GenBank登录号ASW21050.1所示的序列。作为本发明的优选方式，CPR来自于拟南芥(Arabidopsis thaliana)，其具有GenBank登录号NP_849472.2所示的序列。
本发明人发现，利用宿主细胞生产黄芩素和野黄芩素的过程中，应用野生型的F6H，仅能产生微量的产物，无法实现规模化生产，因此对多个参与反应的蛋白进行了改造， 经过大量筛选分析，获得了一些优选的改造方案，极为显著地提高了微生物，尤其是原核表达系统如大肠杆菌中黄芩素和野黄芩素的产量。
因此，作为本发明的优选方式，提供了一种突变型F6H，其对应于野生型F6H，截去N端第(1-10)～(20-30)位氨基酸；较佳地，截去N端第(2-5)～(22-28)位氨基酸；更佳地，截去N端第2-25位氨基酸。
作为本发明的优选方式，还提供了一种含有所述F6H或突变型F6H的融合蛋白，其包括F6H或任一突变型F6H，以及与之融合的多肽标签，所述的多肽标签选自：8RP，Sumo，MBP，2B1，或它们的组合；较佳地为MBP或2B1。所述的多肽标签与所述F6H或突变型F6H之间，可以包含或不包含连接肽，所述的连接肽不影响两者的生物学活性。
作为本发明的优选方式，提供了一种突变型CPR，其对应于野生型CPR，截去N端第(1-20)～(60-85)位氨基酸；较佳地，截去N端第(2-10)～(65-80)位氨基酸；更佳地，截去N端第(2-5)～(70-75)位氨基酸。
在上述优选的蛋白(包括上述野生型的蛋白，突变型的蛋白)的基础上，本发明还包括它们保留生物活性的片段、衍生物和类似物。它们的蛋白片段、衍生物或类似物可以是若干个(通常为1-50个，更佳地1-20个，还更佳如1-10个、1-5个、1-3个、或1-2个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(例如100个以内、80个以内、50个以内、20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的功能。但是，对于上述的突变型蛋白，在其进一步的变化形式中，均进行了如上所述的针对N端的截短。
在上述优选的蛋白(包括上述野生型的蛋白，突变型的蛋白)的基础上，本发明还包括它们的类似物。这些类似物与天然蛋白的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性的蛋白。
在上述优选的蛋白(包括上述野生型的蛋白，突变型的蛋白)的基础上，本发明还包括与所述的蛋白同源性高(比如与所列举的具体蛋白序列的同源性为70％或更高；优选地同源性为80％或更高；更优选地同源性为90％或更高，如同源性95％，98％或99％)的、且具有相应多肽相同功能的蛋白也包括在本发明内。
本发明中列举了来自特定物种的蛋白或基因。应理解，虽然本发明中优选研究了获自特定物种的蛋白或基因，但是获自其它物种的与所述蛋白或基因高度同源(如具有60％以上，如70％，80％，85％、90％、95％、甚至98％序列相同性)的其它蛋白或基因也在 本发明考虑的范围之内。
发明还涉及本发明还提供了编码本发明的蛋白或其保守性变异蛋白的多核苷酸序列。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码本发明的突变体成熟蛋白的多核苷酸包括：只编码成熟蛋白的编码序列；成熟蛋白的编码序列和各种附加编码序列；成熟蛋白的编码序列(和任选的附加编码序列)以及非编码序列。
本发明还包括针对所述基因的序列，进行密码子优化后形成的多核苷酸序列，例如，根据宿主细胞的偏好进行密码子优化。
本发明中，还构建了一种高产黄芩素和野黄芩素的工程菌株，其中包括外源引入的表达F6H(特别是所述突变型F6H或融合蛋白)以及CPR(特别是所述突变型F6H或融合蛋白)的基因。培养该重组菌株，并在培养体系中加入白杨素或芹菜素，从而可生产黄芩素或野黄芩素。
本发明中，还构建了另一种高产黄芩素或野黄芩素的工程菌株，其中包括外源引入的表达F6H(特别是所述突变型F6H或融合蛋白)以及CPR(特别是所述突变型F6H或融合蛋白)的基因，以及白杨素或芹菜素合成基因。所述的白杨素或芹菜素合成基因包括：表达PAL、4CL、CHS、CHI和FNSI蛋白的基因。
本发明的菌株稳定性好，并可实现在生物反应器中规模性培养及生产黄芩素或野黄芩素。本发明优选的菌株的黄芩素或野黄芩素得率非常高。
本发明中，通过大肠杆菌生产黄芩素或野黄芩素，实现黄芩素或野黄芩素更经济、更方便的制造。
本发明还提供了用于生产黄芩素或野黄芩素工程菌株的试剂盒。此外，其中还可包括大肠杆菌培养基，黄芩素或野黄芩素分离或检测试剂，使用说明书等。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实验材料
AxyPrep总RNA小量制备试剂盒，多聚酶链式反应(PCR)胶回收试剂盒，质粒抽提试剂盒均为美国Axygen产品；PrimeScript RT reagent Kit with gDNA Eraser(Perfect Real Time)聚合酶试剂盒，聚合酶链式反应(PCR)高保真酶PrimeSTAR Max DNA Polymerase为日本宝生物公司(TAKARA)产品；限制性内切酶均为NEB产品。
大肠杆菌DH10B用于基因克隆，大肠杆菌BL21(DE3)菌株用于蛋白表达和黄芩素、野黄芩素的生产。pET28a、pEDDuet-1、pCDFDuet-1载体用于代谢途径基因装配。
标准品化合物黄芩素和野黄芩素购自上海源叶生物科技有限公司。其他试剂为国产分析纯或色谱纯试剂，购自国药集团化学试剂有限公司。
PCR使用Arktik Thermal Cycler(Thermo Fisher Scientific)；恒温培养使用ZXGP-A2050恒温培养箱和ZWY-211G恒温培养振荡器；离心使用5418R高速冷冻式离心机和5418小型离心机(Eppendorf)。真空浓缩使用Concentrator plus浓缩仪(Eppendorf)；OD 600使用UV-1200紫外可见分光光度计检测(上海美谱达仪器有限公司)。旋转蒸发系统由IKA RV 10digital旋转蒸发仪(IKA)和MZ 2C NT化学隔膜泵、CVC3000真空控制器(vacuubrand)组成。高效液相色谱使用Dionex UltiMate 3000液相色谱系统(Thermo Fisher Scientific)。
液相检测条件：A相：0.1％甲酸水，B相：乙腈；分离条件：0-20min 20％B相-55％B相，20-22min 55％B相-100％B相，22-27min 100％B相，27-35min 100％B相-20％B相，35-40min，20％B相；检测波长：340nm，柱温：30℃。色谱柱：Thermo syncronis C18反相柱(250mm×4.6mm，5μm)。
实施例1、多肽及其序列优化
1、F6H多肽序列的优化改造
来自黄岑(Scutellaria baicalensis)的F6H(SbF6H)长度为517aa(Genbank access no.ASW21050.1)，具体序列如下(SEQ ID NO:1)：
Figure PCTCN2019104658-appb-000001
改造1：本发明人针对SEQ ID NO:1进行序列改造，去除其中第2-25位氨基酸，在N端加上MA两个氨基酸，获得改进的F6H突变体trF6H，具体序列如下(SEQ ID NO:2)：
Figure PCTCN2019104658-appb-000002
改造2：本发明人针对SEQ ID NO:1进行序列改造，去除其中第2-25位氨基酸，再在N端加上8RP的氨基酸序列，获得改进的F6H突变体8RPtrF6H，具体序列如下(SEQ  ID NO:3)：
Figure PCTCN2019104658-appb-000003
改造3：本发明人针对SEQ ID NO:1进行序列改造，去除其中第2-25位氨基酸，再在N端加上Sumo的氨基酸序列，获得改进的F6H突变体SumotrF6H，具体序列如下(SEQ ID NO:4)：
Figure PCTCN2019104658-appb-000004
改造4：本发明人针对SEQ ID NO:1进行序列改造，去除其中第2-25位氨基酸，再在N端加上MBP的氨基酸序列，获得改进的F6H突变体MBPtrF6H，具体序列如下(SEQ ID NO:5)：
Figure PCTCN2019104658-appb-000005
改造5：本发明人针对SEQ ID NO:1进行序列改造，去除其中第2-25位氨基酸，再在N端加上2B1的氨基酸序列，获得改进的F6H突变体2B1trF6H，具体序列如下(SEQ ID NO:6)：
Figure PCTCN2019104658-appb-000006
Figure PCTCN2019104658-appb-000007
2、CPR的优化改造
来自拟南芥(Arabidopsis thaliana)的CPR(AtCPR)序列长度712aa(Genebank access no.NP_849472.2)，具体如下(SEQ ID NO:7)：
Figure PCTCN2019104658-appb-000008
本发明人针对SEQ ID NO:1进行序列改造，删除其中第2-72位氨基酸，获得改进的AtCPR突变体trAtCPR，具体如下(SEQ ID NO:8)：
Figure PCTCN2019104658-appb-000009
实施例2、包含新型F6H突变体的重组质粒的构建
以pETDuet-1为出发质粒，将AtCPR通过一步克隆方法连接到NdeI和XhoI位点获得质粒pYH45。
以pETDuet-1为出发质粒，将trAtCPR通过一步克隆方法连接到NdeI和XhoI位点获得质粒pYH46。
进一步，通过金斯瑞合成密码子优化的F6H的编码基因序列并连接到pUC19载体中，获得pUC19-F6H。以F6H-F/R为引物，以pUC19-F6H为模板进行PCR扩增，体系50μL(PrimeSTAR Max Premix，25μL；双引物终浓度0.2～0.3μM；pUC19-F6H 0.2uL；剩余体积用灭菌蒸馏水补足)。PCR反应条件：98℃预变性2min，然后98℃变性10s，55℃退火15s，72℃延伸20s，25个循环，琼脂糖电泳检测，扩增得到约1.5kb的片段，纯化后用Nco I和BamH I双酶切消化。消化后片段连入相同酶消化过的pYH46，连接 产物转化大肠杆菌DH10B感受态细胞，提取质粒经构建时引入酶切位点双酶切验证和基因测序，得到重组质粒pYH59。同样地，将消化后片段连入pYH45，得到重组质粒pYH59。
以trF6H-F/F6H-R为引物，pUC19-trF6H为模板，通过PCR扩增获得的产物在通过一步克隆方法连接到pYH46中的NdeI和BamH I位点获得质粒pYH58。
以pUC19-trF6H为模板，以8RP-trF6H-F/F6H-R为引物扩增后，用一步克隆方法将产物连接到pYH46的NdeI和BamH I位点中，获得pYH60。
以pETSumo(购自Invitrogen公司)为模板，以Sumo-F/Sumo-trF6H-R为引物获得含有Sumo序列的DNA片段，以pUC19-trF6H为模板，Sumo-trF6H-F/F6H-R为引物扩增获得含有trF6HDNA片段。以上述2个DNA片段为模板，以Sumo-F/F6H-R为引物进行融合PCR后，用一步克隆方法将产物连接到pYH46的NdeI和BamH I位点中，获得pYH61。
以pMAL-c5x(购自Invitrogen公司)为模板，以MBP-F/MBP-trF6H-R为引物PCR扩增获得含有MBP序列的DNA片段，以pUC19-trF6H为模板，MBP-trF6H-F/F6H-R为引物扩增获得含有trF6H的DNA片段，以上述获得的2个DNA片段为模板，MBP-F/F6H-R为引物进行融合PCR后，用一步克隆方法将产物连接到pYH46的NdeI和BamH I位点中，获得pYH62。
以pUC19-trF6H为模板，以2B1-F/F6H-R为引物扩增后，用一步克隆方法将产物连接到pYH46的NdeI和BamH I位点中，获得pYH63。
以trF6H-F/F6H-R为引物，pUC19-trF6H为模板，通过PCR扩增获得的产物再通过一步克隆方法连接到pYH45的NdeI和BamH I位点中，得到重组质粒pYH64。
以pMAL-c5x为模板，以MBP-F/MBP-trF6H-R为引物PCR扩增获得含有MBP序列的DNA片段，以pUC19-trF6H为模板，MBP-trF6H-F/F6H-R为引物扩增获得含有trF6H的DNA片段，以上述获得的2个DNA片段为模板，MBP-F/F6H-R为引物进行融合PCR后，用一步克隆方法将产物连接到pYH45的NdeI和BamH I位点中，获得pYH65。
以pUC19-2B1trF6H为模板，以2B1-F/F6H-R为引物扩增后，用一步克隆方法将产物连接到pYH45的NdeI和BamH I位点中，获得pYH66。质粒pYH66的构建示意图如图3所示。
以上构建过程所用的引物如表1。所构建的质粒中关键元件的示意图如图9A。
表1
Figure PCTCN2019104658-appb-000010
Figure PCTCN2019104658-appb-000011
实施例3、表达PAL、4CL、CHS、CHI和FNSI的重组质粒的构建
通过金斯瑞合成来源于红景天(Rhodotorula toruloides)的PAL基因、(GenBank登录号AAA33883.1)、来源于欧芹(Petroselium crispum)的4CL基因(GenBank登录号KF765780.1)、来源于矮牵牛(Petunia X hybrida)的CHS基因(GenBank登录号KF765781.1)、来源于苜蓿(Medicago sativa)的CHI基因(GenBank登录号KF765782.1)、来源于欧芹(Petroselium crispum)的FNS I基因(Swiss-Prot登录号Q7XZQ8.1)，并构建到pET28a载体上获得pET28-PAL，pET28-4CL，pET28-CHS，pET28a-CHI，pET28a-FNSI。
合成如表2的引物。以pET28-4CL为模板，4CL-F-NcoI/4CL-R-BamHI为引物获得PCR产物，经与NcoI/BamHI双酶切的pCDFDuet-1连接获得pYH40。
以pET28-CHS为模板，CHS-F-NdeI/CHS-R-XhoI为引物获得PCR产物，经与NdeI/XhoI双酶切的pYH40连接获得pYH50。
以pET28a-CHI为模板，T7CHI-F-XhoI/CHI-R-AvrII为引物获得PCR产物，连接到pYH50获得pYH51。
以pET28-PAL为模板，T7PAL-F-BamH I/PAL-R-Hind III为引物获得PCR产物，经BamH I/Hind III双酶切后与同样双酶切的pYH51连接，获得质粒pYH55。
以pET28a-FNSI为模板，FNSI-HindIII-F/FNSI-NotI-R)为引物获得PCR产物，经Hind III/Not I双酶切后与同样双酶切的pYH55连接，获得最终载体pYH57。质粒pYH57的构建示意图如图4所示。
表2
Figure PCTCN2019104658-appb-000012
Figure PCTCN2019104658-appb-000013
实施例4、黄芩素和野黄芩素合成菌株的构建及功能验证
黄芩素和野黄芩素的生物合成的示意图如图1。
将重组质粒pYH66和pYH57共转化到大肠杆菌BL21(DE3)的感受态细胞中以获得工程菌株BL21(DE3)-pYH57-pYH66。
LB固体培养基(壮观霉素80μg/mL，氨苄青霉素100μg/mL)37℃培养过夜。挑取单个克隆到2mL LB液体培养基(壮观霉素80μg/mL，氨苄青霉素100μg/mL)，转接过夜培养的菌液到新的10mL MOPS液体抗性培养基中37℃，250r/min培养至OD 600＝0.5-0.6，水浴降温至16℃左右，然后加入诱导剂IPTG至终浓度1mM，加入不同浓度经灭菌的苯丙氨酸或酪氨酸并转至22℃低温诱导培养，在摇床转速220r/min条件下继续培养48h。携带未连入外来基因的pETDuet-1和pCDFDuet-1空质粒的BL21(DE3)重组菌株作为空白对照，培养操作同上。
同时，将表2所列的各个重组质粒分别转化大肠杆菌，进行培养，检测各自的产物生成情况。
培养后，检测各个转化了重组质粒的重组菌株表达化合物的状况，如表3所示。
表3
Figure PCTCN2019104658-appb-000014
Figure PCTCN2019104658-appb-000015
经验证，本发明的各个重组菌株可以成功合成目标化合物。
工程菌株BL21(DE3)-pYH57-pYH66和标准品黄芩素的HPLC检测图谱如图5。工程菌株BL21(DE3)-pYH57-pYH66产黄芩素的质谱图谱如图6。
工程菌株BL21(DE3)-pYH57-pYH66和标准品野黄芩素的HPLC检测图谱如图7。工程菌株BL21(DE3)-pYH57-pYH66产野黄芩素的质谱图谱如图8。
实施例5、以白杨素为底物进行生产
分别将pYH58～pYH66这6个重组质粒转化到大肠杆菌BL21(DE3)的感受态细胞中以获得工程菌株，并命名为BL21(DE3)-pYH58～BL21(DE3)-pYH66。
LB固体培养基(氨苄青霉素100μg/mL)37℃培养过夜。挑取单个克隆到2mL LB液体培养基(氨苄青霉素100μg/mL)，转接过夜培养的菌液到新的20mL MOPS液体抗性培养基中37℃，250r/min培养至OD 600＝0.5-0.6，水浴降温至16℃左右，然后加入诱导剂IPTG至终浓度1mM，在摇床转速220r/min，温度22℃条件下继续培养12h。将上述发酵液并在6000rpm，4℃，10min条件下离心，去掉上清，收集菌体。用反应缓冲液(50mM Tris-HCl，pH 7.4，0.1％Trixton)重悬至OD 600＝30。取1mL各重组菌的重悬液，向其中加入5μL白杨素(25mM)和2.5μL NADPH(100mM)，继续在37℃反应8小时。待反应结束后，向反应液中加入10μL HCl(6M)和1mL乙酸乙酯进行萃取3次，浓缩有机相获得的残留物用200μL甲醇溶解后取10μL进行HPLC分析。
各个重组大肠杆菌中，催化白杨素生成黄芩素的转化率如图9B。
各个重组大肠杆菌催化反应液的HPLC分析结果如图9C。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (23)

  1. 一种生产黄芩素和野黄芩素的方法，其特征在于，包括：
    (1)在宿主细胞中引入表达黄酮6-羟化酶以及细胞色素P450氧化还原酶的基因，以及白杨素或芹菜素合成基因；
    (2)在含有苯丙氨酸和/或酪氨酸的培养体系中培养该宿主细胞，从而生产黄芩素或野黄芩素。
  2. 如权利要求1所述的方法，其特征在于，所述的白杨素或芹菜素合成基因包括：表达苯丙氨酸解氨酶、4-香豆酸辅酶A连接酶、查尔酮合成酶、查尔酮异构酶和黄酮合成酶I的基因；较佳地，在被引入宿主细胞时，所述的表达苯丙氨酸解氨酶、4-香豆酸辅酶A连接酶、查尔酮合成酶、查尔酮异构酶和黄酮合成酶I的基因存在于同一表达载体中。
  3. 一种生产黄芩素和野黄芩素的方法，其特征在于，包括：
    (1)在宿主细胞中引入表达黄酮6-羟化酶以及细胞色素P450氧化还原酶的基因，获得重组的宿主细胞；
    (2)在含有白杨素或芹菜素的培养体系中培养该重组的宿主细胞，从而生产黄芩素或野黄芩素。
  4. 一种将白杨素或芹菜素转化为黄芩素或野黄芩素的方法，其特征在于，以黄酮6-羟化酶以及细胞色素P450氧化还原酶催化白杨素或芹菜素，从而在白杨素或芹菜素的结构中加上一个羟基，生成黄芩素或野黄芩素。
  5. 如权利要求1～4任一所述的方法，其特征在于，所述的黄酮6-羟化酶是截去N端第(1-10)～(20-30)位氨基酸的突变型黄酮6-羟化酶；较佳地，是截去N端第(2-5)～(22-28)位氨基酸的突变型黄酮6-羟化酶。
  6. 如权利要求1～4任一所述的方法，其特征在于，所述的黄酮6-羟化酶与多肽标签融合，所述的多肽标签选自：小牛血清17羟基化酶N端8氨基酸多肽，小分子泛素修饰相关蛋白，麦芽糖结合蛋白，细胞色素P450 2B1家族可溶性蛋白，或其组合；较佳地为麦芽糖结合蛋白或细胞色素P450 2B1家族可溶性蛋白，或其组合；较佳地所述多肽标签位于N端。
  7. 如权利要求1～4任一所述的方法，其特征在于，所述的细胞色素P450氧化还原酶的是截去N端第(1-20)～(60-85)位氨基酸的突变型细胞色素P450氧化还原酶；较佳地，是截去N端第(2-10)～(65-80)位氨基酸的突变型细胞色素P450氧化还原酶；更 佳地，是截去N端第(2-5)～(70-75)位氨基酸的突变型细胞色素P450氧化还原酶。
  8. 如权利要求1～3任一所述的方法，其特征在于，所述的宿主细胞包括：原核细胞或真核细胞；较佳地，所述的原核细胞包括：大肠杆菌细胞，枯草芽孢杆菌细胞；所述的真核细胞包括：酵母细胞。
  9. 一种重组宿主细胞，其特征在于，其中包括外源的表达黄酮6-羟化酶以及细胞色素P450氧化还原酶的基因。
  10. 如权利要求9所述的重组宿主细胞，其特征在于，其中还包括外源的白杨素或芹菜素合成基因；较佳地，所述的白杨素或芹菜素合成基因包括：表达苯丙氨酸解氨酶、4-香豆酸辅酶A连接酶、查尔酮合成酶、查尔酮异构酶和黄酮合成酶I的基因；较佳地，所述的表达苯丙氨酸解氨酶、4-香豆酸辅酶A连接酶、查尔酮合成酶、查尔酮异构酶和黄酮合成酶I的基因于同一表达载体中被引入宿主细胞。
  11. 如权利要求9～10任一所述的重组宿主细胞，其特征在于，所述的黄酮6-羟化酶是截去N端第(1-10)～(20-30)位氨基酸的突变型黄酮6-羟化酶；较佳地，是截去N端第(2-5)～(22-28)位氨基酸的突变型黄酮6-羟化酶。
  12. 如权利要求9～10任一所述的重组宿主细胞，其特征在于，所述的黄酮6-羟化酶与多肽标签融合，所述的多肽标签选自：小牛血清17羟基化酶N端8氨基酸多肽，小分子泛素修饰相关蛋白，麦芽糖结合蛋白，细胞色素P450 2B1家族可溶性蛋白，或其组合；较佳地为麦芽糖结合蛋白或细胞色素P450 2B1家族可溶性蛋白，或其组合；较佳地所述多肽标签位于N端。
  13. 如权利要求9～10任一所述的重组宿主细胞，其特征在于，所述的细胞色素P450氧化还原酶的是截去N端第(1-20)～(60-85)位氨基酸的突变型细胞色素P450氧化还原酶；较佳地，是截去N端第(2-10)～(65-80)位氨基酸的突变型细胞色素P450氧化还原酶；更佳地，是截去N端第(2-5)～(70-75)位氨基酸的突变型细胞色素P450氧化还原酶。
  14. 权利要求9～13任一所述的重组宿主细胞的用途，用于生产黄芩素和野黄芩素。
  15. 一种制备用于生产黄芩素和野黄芩素的宿主细胞的方法，其特征在于，包括：在宿主细胞中引入表达黄酮6-羟化酶以及细胞色素P450氧化还原酶的基因，获得重组 菌株；较佳地，还包括引入白杨素或芹菜素合成基因。
  16. 一种用于生产黄芩素和野黄芩素的试剂盒，其特征在于，所述试剂盒中包括权利要求9～13任一所述的重组的宿主细胞。
  17. 突变型黄酮6-羟化酶，其对应于野生型黄酮6-羟化酶，截去N端第(1-10)～(20-30)位氨基酸；较佳地，截去N端第(2-5)～(22-28)位氨基酸；更佳地其具有SEQ ID NO:2所示的氨基酸序列。
  18. 突变型细胞色素P450氧化还原酶，其对应于野生型细胞色素P450氧化还原酶，截去N端第(1-20)～(60-85)位氨基酸；较佳地，截去N端第(2-10)～(65-80)位氨基酸；更佳地，截去N端第(2-5)～(70-75)位氨基酸；更佳地其具有SEQ ID NO:8所示的氨基酸序列。
  19. 融合多肽，其包括权利要求17所述的突变型黄酮6-羟化酶，以及与之融合的多肽标签，所述的多肽标签选自：8RP，Sumo，MBP，2B1；较佳地为MBP或2B1。
  20. 如权利要求19所述的融合多肽，其特征在于，其具有选自下组的氨基酸序列：SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6。
  21. 多核苷酸，其编码：
    权利要求17所述的突变型黄酮6-羟化酶；或
    权利要求18所述的突变型细胞色素P450氧化还原酶；或
    权利要求19或20所述的融合多肽。
  22. 一种表达构建物，其包括：
    权利要求21所述的任一多核苷酸；或
    编码权利要求17所述的任一突变型黄酮6-羟化酶或权利要求19所述的融合蛋白的多核苷酸，以及编码权利要求18所述的突变型细胞色素P450氧化还原酶的多核苷酸。
  23. 权利要求17所述的突变型黄酮6-羟化酶或权利要求19所述的融合蛋白，以及权利要求18所述的突变型细胞色素P450氧化还原酶的用途，用于在白杨素或芹菜素的结构中加上一个羟基，形成黄芩素或野黄芩素。
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