WO2022148377A1 - Host cell of heterologous synthetic flavonoid compound, and use thereof - Google Patents

Host cell of heterologous synthetic flavonoid compound, and use thereof Download PDF

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WO2022148377A1
WO2022148377A1 PCT/CN2022/070316 CN2022070316W WO2022148377A1 WO 2022148377 A1 WO2022148377 A1 WO 2022148377A1 CN 2022070316 W CN2022070316 W CN 2022070316W WO 2022148377 A1 WO2022148377 A1 WO 2022148377A1
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gene
ammonia lyase
phenylalanine ammonia
pdz
coumaric acid
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Chinese (zh)
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王勇
李建华
计东尼
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中国科学院分子植物科学卓越创新中心
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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 host cells for heterologous synthesis of flavonoids and applications thereof.
  • Baicalein and scutellarin are flavonoids, which are mainly found in the traditional Chinese medicine Scutellaria baicalensis. These two active flavonoids only accumulate less in the roots of related medicinal plants such as Scutellaria baicalensis. Both baicalein and baicalein are synthesized through the flavonoid biosynthetic pathway. Baicalein and scutellarin have important physiological activities such as antioxidant, antitumor, antibacterial, and heart protection. Recently, baicalein was reported as an inhibitor of SARS-CoV-2 3Clpro in vitro, showing the great potential of traditional Chinese medicine.
  • flavonoids are extraction and chemical synthesis from plants.
  • plant extraction or chemical synthesis cannot provide a green route to mass production due to the use of toxic chemicals and extreme reaction conditions. Therefore, the research on microbial synthesis of flavonoids has been intensively carried out.
  • Low product titers typically result from enzyme imbalances and accumulation of intermediate metabolites due to complex heterogeneous pathways introduced from plants.
  • a multivariate modular approach was employed to synthesize flavonoids by modulating promoter strength and plasmid copy number.
  • it is time consuming and always requires a lot of work.
  • Previous work reported the synthesis of baicalein and scutellarin in engineered yeast and E. coli, but the yields of baicalein and scutellarin were still at very low levels.
  • the purpose of the present invention is to provide a host cell for heterologous synthesis of baicalein, scutellarin or chrysin compounds and its application.
  • a prokaryotic cell for synthesizing baicalein and scutellarin-like compounds (such as baicalein or scutellarin), which comprises an exogenous encoding gene of the following group of enzymes: flavonoid 6- Hydroxylase (F6H), Cytochrome P450 oxidoreductase (CPR), Phenylalanine ammonia lyase (PAL), 4-Coumarate CoA ligase (4CL), Chalcone synthase (CHS), Chalcone isomerase (CHI) and flavonoid synthase I (FNSI); and after the enzymes are expressed, phenylalanine ammonia lyase (PAL) and 4-coumarate coenzyme A ligase (4CL) constitute Complex (compound reactor).
  • flavonoid 6- Hydroxylase F6H
  • Cytochrome P450 oxidoreductase CPR
  • Phenylalanine ammonia lyase PAL
  • a prokaryotic cell for synthesizing chrysin-based compounds (such as chrysin or apigenin) is provided, which comprises an exogenous encoding gene of the following group of enzymes: phenylalanine ammonia lyase ( PAL), 4-coumarate coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI) and flavonoid synthase I (FNSI); and after the enzymes are expressed , phenylalanine ammonia lyase (PAL) and 4-coumarate coenzyme A ligase (4CL) constitute a complex (complex reactor).
  • PAL phenylalanine ammonia lyase
  • 4-coumarate coenzyme A ligase (4CL) constitute a complex (complex reactor).
  • the complex of phenylalanine ammonia lyase and 4-coumaric acid coenzyme A ligase comprises: phenylalanine ammonia lyase and 4-coumaric acid coenzyme A through protein-protein The binding of the interacting domains and their ligands brings them closer together, resulting in a complex.
  • the phenylalanine ammonia lyase and 4-coumarate coenzyme A ligase are directly connected or connected through a linker to obtain a complex in the form of a fusion protein.
  • the protein-protein interaction domain comprises a domain selected from the group consisting of: PDZ domain, SH3 domain, WW domain, LIM domain, DD domain, PH domain, EH domain, GBD domain.
  • the protein-protein interaction domain includes a PDZ domain, and its ligand is a PDZ ligand; the phenylalanine ammonia lyase and 4-coumarate coenzyme A are respectively associated with the PDZ Domain and its ligand are fused; preferably, the phenylalanine ammonia lyase is fused with PDZ, and the 4-coumaric acid coenzyme A is fused with PDZ ligand; more preferably, the phenylalanine ammonia lyase is fused with PDZ;
  • the aminoase is fused with PDZ, it also includes connecting with an ER/K linker (PAL-ER/K-PDZ), and when the 4-coumaric acid coenzyme A is fused with PDZ ligand, it also includes connecting with a (GGGGS) 2 linker ( PDZlig-(GGGGS) 2-4CL ).
  • the protein-protein interaction domain includes an SH3 domain, and its ligand is SH3ligand; the phenylalanine ammonia lyase and 4-coumarate coenzyme A are respectively associated with the SH3 structure Domain and its ligand are fused; preferably, the phenylalanine ammonia lyase is fused with SH3, and the 4-coumaric acid coenzyme A is fused with SH3 ligand; more preferably, the phenylalanine ammonia lyase is fused with SH3
  • the enzyme is fused with SH3, it also includes connecting with an ER/K linker (PAL-ER/K-SH3), and when the 4-coumaric acid coenzyme A is fused with SH3 ligand, it also includes connecting with a (GGGGS) 2 linker (SH3lig -(GGGGS) 2-4CL ).
  • the phenylalanine ammonia lyase when the phenylalanine ammonia lyase is fused with PDZ, the phenylalanine ammonia lyase is located at the N-terminus, and the PDZ is located at the C-terminus.
  • the PDZ ligand when the 4-coumaric acid coenzyme A is fused to the PDZ ligand, the PDZ ligand is located at the N-terminus, and the 4-coumaric acid-coenzyme A is located at the C-terminus.
  • the phenylalanine ammonia lyase when fused with SH3; the phenylalanine ammonia lyase is located at the N-terminus, and the SH3 is located at the C-terminus.
  • the SH3 ligand when the 4-coumaric acid coenzyme A is fused to the SH3 ligand, the SH3 ligand is located at the N-terminus, and the 4-coumaric acid coenzyme A is located at the C-terminus.
  • the cell also includes an exogenous gene encoding an enzyme that promotes the production of malonyl CoA; preferably, it includes matC, matB, ACS, and FabF.
  • the prokaryotic cells are Escherichia coli cells.
  • the cell also includes an exogenous gene encoding an enzyme that promotes phenylalanine synthesis; preferably, it includes: aroG, pheA; more preferably, the pheA is at position 976 composed of A gene mutated from A to C; more preferably, the aroG is a gene mutated from G to A at position 436.
  • the "promoting” is a statistically significant "promoting", such as promoting more than 5%, more than 10%, more than 20%, more than 50%, more than 80%, more than 100% or higher.
  • the application of the prokaryotic cells is provided for synthesizing baicalein or scutellarin compounds.
  • the application of the prokaryotic cells is provided for synthesizing chrysin compounds.
  • a method for synthesizing baicalein or scutellarin compounds comprising: providing the prokaryotic cells (containing F6H and CPR), and using formula (I) as a substrate to synthesize baicalein or scutellarin compounds;
  • R includes H or OH.
  • a method for synthesizing chrysin compounds comprising: providing the prokaryotic cells (which may not contain F6H and CPR), and using formula (I) as a substrate to synthesize chrysin compounds.
  • a method for synthesizing baicalein or scutellarin-like compounds or chrysin-like compounds comprising: providing the prokaryotic cells, and using glucose as a substrate to synthesize baicalein or scutellarin compounds or chrysin-like compounds.
  • the gene encoding chalcone isomerase is located in one construct (plasmid).
  • the encoding gene for flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase is located in a construct, preferably also including 2B1 (cytochrome P450 2B1 family soluble protein) gene.
  • the genes encoding matC, matB, ACS, and FabF are located in one construct.
  • the genes encoding SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, and chalcone synthase are located in one construct.
  • the gene encoding chalcone isomerase and flavonoid synthase I is located in one construct.
  • the encoding genes of matC, matB, ACS, and FabF are pheA gene (pheA fbr ) mutated from A to C at position 976, and aroG gene (aroG fbr ) mutated from G to A at position 436 ) in one construct.
  • kits for producing baicalein or scutellarin-based compounds (containing F6H and CPR in the kit) or chrysin-based compounds (without F6H and CPR in the kit), It includes said recombinant host cells.
  • kits for establishing a host cell for synthesizing baicalein or scutellarin-like compounds or chrysin-like compounds comprising: comprising PDZligand, 4-coumaric acid-CoA ligase , phenylalanine ammonia lyase, ER/K, PDZ, flavonoid synthase I, chalcone synthase, chalcone isomerase encoding gene constructs; including matC, matB, ACS, FabF encoding genes Constructs of SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, constructs encoding genes for chalcone synthase; containing chalcone isomerase , a construct encoding the gene encoding flavonoid synthase I; including the encoding genes of matC, matB,
  • the kit further includes: glucose; or, the substrate of formula (I).
  • Figure 1 Schematic diagram of the construction of plasmid pZZ41.
  • Figure 2 Schematic diagram of the construction of plasmid pZZ55.
  • Figure 3 The bar graph of the baicalein production of the non-self-assembling strain DN-1 and the self-assembling strain DN-2.
  • Figure 5 Bar graph of baicalein production of self-assembled strain DN-4 compared to control strain DN-3.
  • Figure 6 The bar graph of the scutellarin production of the non-self-assembling strain DN-1 and the self-assembling strain DN-2.
  • Figure 7 The bar graph of the baicalein production of the self-assembly engineering strain DN-6 and the non-self-assembly engineering strain DN-5.
  • Fig. 8 is a schematic diagram of the synthetic pathway for fermenting to generate baicalein and baicalein using phenylalanine as a precursor.
  • Figure 9 is a schematic diagram of the synthetic pathway of baicalein produced by fermentation with glucose as a precursor.
  • the inventors provide a novel biosynthesis optimization of baicalein or scutellarin compounds/chrysin compounds, which can realize the synthesis of baicalein or scutellarin using enzyme self-assembly technology on the basis of prokaryotes scutellarin-like compounds/chrysin-like compounds, and de novo synthesis of baicalein or scutellaria-like compounds/chrysin-like compounds using glucose.
  • the invention also discloses the optimized modified host cell and its application.
  • 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 location 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, and thus, the promoter region is "operably linked” to the nucleic acid sequence.
  • an "expression construct” refers to a recombinant DNA molecule comprising the desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes.
  • the "construct” is usually contained in an expression vector.
  • the PAL, 4CL, CHS, CHI and FNSI proteins are those that form the chrysin or apigenin synthetic pathway in an expression system.
  • the F6H and CPR proteins are proteins that convert chrysin or apigenin to produce baicalein or scutellarin-like compounds in an expression system.
  • the matC, matB, ACS and/or FabF proteins described are enzymes that promote malonyl-CoA production in an expression system.
  • aroG or a mutant thereof, pheA or a mutant thereof promotes phenylalanine synthesis in an expression system.
  • PAL is derived from Rhodiola (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 (Petunia X hybrida), which has the sequence shown in GenBank accession number KF765781.1
  • CHI gene is derived from alfalfa (Medicago sativa), which has the sequence shown in GenBank accession number KF765782.1 Sequence
  • FNS I is derived from Parsley (Petroselium crispum), which has the sequence shown in Swiss-Prot Accession No. Q7XZQ8.1.
  • Wild-type F6H and CPR have also been identified in the art.
  • F6H is derived from Scutellaria baicalensis, which has the sequence shown in GenBank accession number ASW21050.1.
  • the CPR is derived from Arabidopsis thaliana, which has the sequence shown in GenBank accession number NP_849472.2.
  • matC is derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number KF765784.1
  • matB is derived from Rhizobium leguminosarum, which has GenBank accession number AGZ04579
  • the sequence shown in .1; ACS is derived from Escherichia coli (Escherichia coli), which has the sequence shown in GenBank accession number CP062211.1
  • FabF is derived from Escherichia coli (Escherichia coli), which has GenBank accession number shown in AP023237.1 the sequence of.
  • Baicalein and scutellarin are two structurally similar and important 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 as follows:
  • scutellarin compounds such as baicalein or scutellarin
  • their precursor chrysin compounds such as chrysin or apigenin
  • chrysin compounds such as chrysin or apigenin
  • the inventors utilize enzyme assembly technology to ferment and produce baicalein or scutellarin.
  • the principle of this scheme is to use interacting protein pairs (such as PDZ and PDZ ligand) to fuse with enzymes PAL and 4CL in the baicalein synthesis pathway, so that PAL and 4CL can spontaneously assemble in E. coli to form a dual-enzyme complex reactor, thereby increasing the yield of the target compound.
  • interacting protein pairs such as PDZ and PDZ ligand
  • the inventors found for the first time in the prokaryotic expression system for synthesizing scutellaria compound/chrysin compound that PAL and 4CL are constructed into a complex (composite reactor), which can extremely effectively improve the yield of the expression system.
  • Any biological material or technical means suitable for making PAL and 4CL into an active complex can be used in the present invention.
  • the protein-protein interaction domain may comprise a domain selected from the group consisting of: PDZ domain, SH3 domain, WW domain, LIM domain, DD domain, PH domain , the EH domain.
  • the protein-protein interaction domain may comprise a domain selected from the group consisting of: PDZ domain, SH3 domain; their corresponding ligands are PDZ ligand (PDZlig) or SH3ligand(SH3lig).
  • Protein-protein interactions are efficiently mediated mainly by protein domains.
  • PDZ, SH3, WW and other domains can recognize and bind a short conserved peptide sequence of ligand proteins through one or more recognition "pockets".
  • the PDZ domain In the case of the PDZ domain, it usually binds to the C-terminal 4-5 amino acid residues of the ligand protein, and it is also capable of binding to the intermediate sequence of the ligand protein, polymerizing with itself or other domains, or binding to lipids on the membrane .
  • fusion between PAL and 4CL can be a direct connection or a linker can be used for connection.
  • the inventors overexpressed aroG, especially its aroG fbr , and pheA, especially its pheA fbr gene in a prokaryotic expression system, and constructed a prokaryotic expression system to obtain high-yield phenylalanine,
  • the exogenous baicalein or scutellarin compound/chrysin compound synthesis pathway is introduced into the prokaryotic expression system, so that the strain can utilize glucose to synthesize baicalein compound/chrysin compound de novo.
  • E. coli E. coli
  • Bacillus subtilis etc.
  • E. coli cells E. coli
  • E. coli BL21 DE3
  • the present invention also includes their analogs.
  • the differences between these analogs and the native protein may be differences in amino acid sequence, 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 a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other known molecular biology techniques.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.
  • the present invention also includes high homology with the proteins (for example, the homology with the specific protein sequences listed is 70 % or more; preferably 80% or more homology; more preferably 90% or more homology, such as 95%, 98% or 99% homology), and have the same function as the corresponding polypeptide
  • the homology with the specific protein sequences listed is 70 % or more; preferably 80% or more homology; more preferably 90% or more homology, such as 95%, 98% or 99% homology
  • the protein is also included in the present invention.
  • Proteins or genes from specific species are enumerated in the present invention. It should be understood that although proteins or genes obtained from a specific species are preferably studied in the present invention, those obtained from other species are highly homologous to said proteins or genes (eg have more than 60%, such as 70%, 80%, 85% , 90%, 95%, or even 98% sequence identity) other proteins or genes are also contemplated by the present invention.
  • the present invention also relates to the present invention also provides a polynucleotide sequence encoding the protein of the present invention or a conservative variant thereof.
  • the polynucleotides 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 the coding or non-coding strand.
  • Polynucleotides encoding the mutant mature proteins of the present invention include: coding sequences encoding only the mature protein; coding sequences for the mature protein and various additional coding sequences; coding sequences for the mature protein (and optional additional coding sequences) and non- coding sequence.
  • the present invention also includes polynucleotide sequences formed by codon optimization for the sequence of the gene, eg, codon optimization according to the preference of the host cell.
  • an engineering strain with high production of baicalein or scutellarin compounds is also constructed, which includes the encoding genes of the exogenous lower group enzymes: F6H, CPR, PAL, 4CL, CHS, CHI and FNSI; and the enzymes After being expressed, PAL and 4CL form a complex (complex reactor).
  • the recombinant strain is cultivated, and takes phenylalanine or tyrosine as a substrate to produce baicalein or scutellarin compounds.
  • the production using phenylalanine or tyrosine as a substrate is suitable for large-scale compound production.
  • an engineered strain with high production of chrysin compounds is also constructed, which includes exogenous encoding genes of the following group of enzymes: PAL, 4CL, CHS, CHI and FNSI; and after the enzymes are expressed, PAL and 4CL constitute Complex (compound reactor).
  • the recombinant strain is cultivated, and takes phenylalanine or tyrosine as a substrate to produce baicalein or scutellarin compounds.
  • the production using phenylalanine or tyrosine as a substrate is suitable for large-scale compound production.
  • the F6H further includes a polypeptide tag fused to it, for example, the polypeptide tag is selected from: 8RP, Sumo, MBP, 2B1, or a combination thereof; preferably 2B1.
  • a connecting peptide may or may not be included, and the connecting peptide does not affect the biological activity of the two. F6H was linked to 2B1 to obtain an improved F6H mutant 2B1trF6H.
  • the upstream generation pathway of the above-mentioned substrate can be further introduced, for example, including: generated by glucose or glycerol through glycolysis, pentose phosphate pathway, shikimic acid pathway Phenylalanine or tyrosine.
  • phenylalanine or tyrosine can be further introduced, for example, including: generated by glucose or glycerol through glycolysis, pentose phosphate pathway, shikimic acid pathway Phenylalanine or tyrosine.
  • protocols for the formation of phenylalanine or tyrosine based on such pathways are also encompassed by the present invention.
  • Methods of enhancing the phenylalanine or tyrosine-forming pathway by means known in the art can be included in the present invention.
  • exogenous aroG, especially its aroG fbr , and pheA, especially its pheA fbr can be further introduced into the above-mentioned engineering strain using phenylalanine or tyrosine as a substrate to obtain another A recombinant strain capable of producing baicalein compounds/chrysin compounds using glucose as a substrate.
  • the production using glucose as a substrate has low cost and is very suitable for large-scale compound production.
  • the yield of the target compound can also be increased by expanding the production scale.
  • the medium feeding scheme can be carried out (which can provide abundant substrates continuously), or the production conditions of good fermenter level (such as temperature When the optimal control of oxygen is used, the optimal control of dissolved oxygen, etc.), its output can usually be increased by 2 to 1000 times.
  • the fermentation product After the fermentation product is obtained, techniques known in the art can be used to extract the target compound from the fermentation product.
  • the product can be analytically identified using well known techniques such as high performance liquid chromatography to confirm that the desired compound has been obtained.
  • the strain of the invention has good stability, and can realize large-scale cultivation and production of baicalein or scutellarin compounds/chrysin compounds in a bioreactor.
  • the yields of the target compounds of the preferred strains of the present invention are very high.
  • microbial fermentation has the advantages of fast speed and less influence by external factors; the yield of some compounds through microbial synthesis is much higher than that of plant extraction, and has become an important means of obtaining natural products.
  • baicalein or scutellarin compound/chrysin compound through Escherichia coli, more economical and convenient manufacture of the target compound is achieved.
  • the invention also provides a kit for producing engineering strains of baicalein or scutellarin compounds.
  • the culture medium of prokaryotic cells can also be included, substrates for synthesis such as phenylalanine, tyrosine or glucose, baicalein or scutellarin-based compound separation or detection reagents.
  • the kit may further include instructions for use that describe the method for biosynthesizing xanthophylls, and the like.
  • the present invention also provides a kit for constructing the engineering strain for producing baicalein or scutellarin-like compounds/chrysin-like compounds, the kit may include a series of constructs, for example, refer to the implementation of the present invention
  • the constructs provided in the examples can also be other constructs that contain the genes but the gene arrangement or tandem manner is different.
  • Expression vectors expression constructs
  • Gene sequences can be inserted into different expression constructs (such as expression vectors), or into the same expression construct, as long as the encoded polypeptide can be efficiently expressed and active after being transferred into cells .
  • the kit can also include prokaryotic cells, culture medium of prokaryotic cells, substrates for synthesis such as phenylalanine, tyrosine or glucose, and baicalein or scutellarin compounds for separation or detection reagents. More preferably, the kit may further include instructions for use that describe the method for biosynthesizing baicalein or scutellarin.
  • PCR Polymerase chain reaction
  • gel recovery kits and plasmid extraction kits are products of American Axygen
  • PrimeSTAR Max DNA Polymerase is a product of TAKARA
  • restriction The endonucleases are all NEB products.
  • baicalein and scutellarin were purchased from Shanghai Yuanye Biotechnology Co., Ltd.
  • Other reagents were domestic analytical or chromatographic pure reagents, purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Arktik Thermal Cycler (Thermo Fisher Scientific) was used for PCR; ZXGP-A2050 incubator and ZWY-211G incubator were used for constant temperature incubation; 5418R high-speed refrigerated centrifuge and 5418 mini-centrifuge (Eppendorf) were used for centrifugation. Concentrator plus concentrator (Eppendorf) was used for vacuum concentration; OD600 was detected by UV-1200 UV-Vis spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.).
  • the rotary evaporation system consists of an IKA RV 10digital rotary evaporator (IKA), a MZ 2CNT chemical diaphragm pump, and a CVC3000 vacuum controller (vacuubrand). High performance liquid chromatography was performed using a Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific).
  • Escherichia coli DH10B was used for gene cloning, and Escherichia coli BL21(DE3) strain was used for protein expression and production of baicalein and scutellarin.
  • pCDFDuet-1 pETDuet-1
  • pACYCDuet-1 vectors were used for metabolic pathway gene assembly.
  • PAL derived from Rhodiola (Rhodotorula touloides), which has the sequence shown in GenBank accession number AAA33883.1 (RtPAL);
  • CHS derived from petunia (Petunia X hybrida), which has the sequence shown in GenBank accession number KF765781.1;
  • CHI gene derived from alfalfa (Medicago sativa), it has the sequence shown in GenBank accession number KF765782.1;
  • FNS I derived from parsley (Petroselium crispum), which has the sequence shown in Swiss-Prot accession number Q7XZQ8.1;
  • F6H derived from Scutellaria baicalensis, which has the sequence shown in GenBank accession number ASW21050.1;
  • CPR from Arabidopsis thaliana, which has the sequence shown in GenBank Accession No. NP_849472.2.
  • PDZ domain derived from mouse Mouse ⁇ -syntrophin (syn), 77-171 amino acid sequence, which has the sequence shown in GenBank accession number EDL06069.
  • matB gene derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number AGZ04579.1.
  • matC gene derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number KF765784.1.
  • ACS gene derived from Escherichia coli, which has the sequence shown in GenBank accession number CP062211.1.
  • PDZ ligand sequence is GVKESLV (SEQ ID NO: 12).
  • SH3 domain AEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWN AEDSEGKRGMIPVPYVEKY (SEQ ID NO: 13).
  • SH3lig PPPALPPKRRR (SEQ ID NO: 14).
  • NcoI restriction site was added to the N-terminus of PAL gene, and EcoRI restriction site was added to the C-terminus of PDZ sequence.
  • PAL, 5nm rigid linker ER/K, PDZ were genetically fused by Over-Lap PCR method, and pCDFDuet- 1. Connect the fusion gene PAL-ER/K-PDZ obtained after Over-Lap PCR to the NcoI and EcoRI sites of pCDFDuet-1 to obtain pCDFDuet1-T7PAL-ER/K-PDZ.
  • the PDZ ligand sequence was designed in the upstream primer, NcoI restriction site was added to the N-terminus, and BamHI restriction site was added to the C-terminus of the 4CL sequence.
  • the PDZlig-4CL fusion gene was obtained by PCR, and the fusion gene was constructed into the NcoI and BamHI of pCDFDuet-1. site, resulting in pCDFDuet1-T7PDZlig-4CL.
  • the PDZ ligand sequence was designed in the downstream primer, the NcoI restriction site was added to the N-terminus of 4CL, and the BamHI restriction site was added to the C-terminus of PDZ ligand.
  • the 4CL-PDZlig fusion gene was obtained by PCR, and the fusion gene was constructed into the NcoI and NcoI of pCDFDuet-1. BamHI site, resulting in pCDFDuet1-T7 4CL-PDZlig.
  • the pheA gene was cloned from the BL21 (DE3) genome.
  • the NcoI restriction site was added to the N-terminus of the pheA gene, and the BamHI restriction site was added to the C-terminus.
  • the 976 position of the pheA gene was mutated from A to C to obtain pheA fbr .
  • the vector backbone was selected from pETDuet-1, and the pheA fbr gene was connected to the NcoI and BamHI sites of pCDFDuet-1 to obtain pCDFDuet1-T7pheA fbr .
  • the aroG gene was cloned from the BL21 (DE3) genome.
  • the NcoI restriction site was added to the N-terminus of the aroG gene, and the BamHI restriction site was added to the C-terminus.
  • the 436 position of the aroG gene was mutated from G to A to obtain aroG fbr .
  • the vector backbone was selected from pETDuet-1, and the aroG fbr gene was connected to the NcoI and BamHI sites of pCDFDuet-1 to obtain pCDFDuet1-T7aroG fbr .
  • pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF): The matC gene and matB gene were obtained by PCR; the NcoI restriction site was added to the N-terminus of the matC gene, and the HindIII restriction site was added to the C-terminus. Linked to the NcoI and HindIII sites of pACYCDuet1 (the site has the T7 promoter that comes with the vector) to obtain T7matC. A HindIII site was added to the N-terminus of the T7matC gene site, and an Acc65I site was added to the C-terminus.
  • the T7matC gene was obtained by PCR, and the T7matC gene was linked to the HindIII and Acc65I sites of pACYCDuet1.
  • the Acc65I site was added to the N-terminus of the T7ACS gene, and the NotI site was added to the C-terminus.
  • the T7ACS gene was obtained by PCR, and the T7ACS gene was connected to the Acc65I and NotI sites of pACYCDuet1.
  • the NotI site was added to the N-terminus of the T7FabF gene, and the XbaI site was added to the C-terminus.
  • the T7FabF gene was obtained by PCR, and the T7FabF gene was connected to the NotI and XbaI sites of pACYCDuet1.
  • the pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF plasmid was obtained.
  • pZZ12 pCDFDuet1-T7-CHI-FNSI
  • NcoI site was added to the N-terminal of CHI gene, and HindIII site was added to C-terminal
  • CHI gene was obtained by PCR, and the CHI gene was connected to the NcoI and HindIII sites of pCDFDuet1.
  • a HindIII site was added to the N-terminus of the FNSI gene, and an EcoRI site was added to the C-terminus, and the FNSI gene was obtained by PCR, which was connected to the HindIII and EcoRI sites of pCDFDuet1.
  • pCDFDuet1-T7-CHI-FNSI was obtained.
  • pZZ22 pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS
  • NcoI site was added to the N-terminal of SH3lig gene
  • BamHI site was added to the C-terminal of 4CL gene.
  • Over-lap PCR fused SH3lig and 4CL to obtain the fusion gene SH3lig-4CL, which was connected to the NcoI and BamHI sites of pET28a.
  • the fusion gene PAL-ER/K was obtained by Over-Lap PCR, the BamHI site was added to the N-terminus of the PAL-ER/K gene, and the EcoRI site was added to the C-terminus of the SH3 gene.
  • PAL-ER/K-SH3 It was fused with the SH3 gene to obtain the PAL-ER/K-SH3 gene, and the PAL-ER/K-SH3 was linked to the BamHI and EcoRI sites of pET28a.
  • the EcoRI site was added to the N-terminal of the CHS gene, and the SalI site was added to the C-terminal, and the CHS was connected to the EcoRI and SalI sites of pET28a.
  • Plasmid pZZ22 Plasmid pZZ22 (pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS) was obtained.
  • NcoI site was added to the N-terminal of 4CL gene
  • BamHI site was added to the C-terminal of 4CL gene
  • pET28a was used as a vector to connect 4CL to the NcoI and BamHI sites of pET28a.
  • the BamHI site was added to the N-terminus of the PAL-ER/K gene
  • the EcoRI site was added to the C-terminus of the SH3 gene.
  • Over-Lap PCR was used to fuse the PAL-ER/K gene and the SH3 gene to obtain PAL-ER/K-SH3 gene, ligating PAL-ER/K-SH3 to the BamHI and EcoRI sites of pET28a.
  • the EcoRI site was added to the N-terminal of the CHS gene, and the SalI site was added to the C-terminal, and the CHS was connected to the EcoRI and SalI sites of pET28a.
  • Plasmid pZZ23 (pET28a-T7-4CL-PAL-ER/K-SH3-CHS) was obtained.
  • pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI): the N-terminal of the PAL-ER/K-PDZ fusion gene constructed in 4(1) was added with a BamHI enzyme cleavage site , an EcoRI restriction site was added to the C-terminal and inserted into pCDFDuet1 to obtain pCDFDuet1-T7PAL-ER/K-PDZ.
  • the gene PAL-ER/K-PDZ was constructed into the BamHI and EcoRI sites of the pCDFDuet1-T7PDZlig-4CL plasmid after PCR to obtain pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/ K-PDZ.
  • the T7FNSI-T7CHS-T7CHI gene was digested from the pYH57 plasmid using HindIII and AvrII double digestion, and connected to the HindIII and AvrII restriction sites of the above plasmid to obtain pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ -T7FNSI-T7CHS-T7CHI) plasmid.
  • pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ): Design primers in front of the T7 promoter of the pCDFDuet1-T7aroG fbr plasmid, add a BamHI restriction site, and add an EcoRI restriction site at the C-terminal, using pETDuet1-T7aroG fbr as a template, The T7aroG fbr fragment was obtained by PCR, and the fragment was ligated into the BamHI and EcoRI sites of the pCDFDuet1-T7pheA fbr plasmid to obtain the pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ) plasmid.
  • pZZ55 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr -T7aroG fbr ): using the pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ) plasmid as a template, design primers upstream of the T7 promoter to increase the AvrII plasmid, which increases at the C-terminus AvrII digestion site, PCR clone to obtain T7pheA fbr- T7aroG fbr fragment, T7pheA fbr -T7aroG fbr was ligated to the AvrII site of pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) using a one-step cloning kit to obtain
  • the detailed information of the plasmid is shown in Table 1, the cell information is shown in Table 2, and the schematic diagram of the plasmid construction is shown in Figures 1 to 2.
  • the constructed plasmid was transformed into Escherichia coli BL21 (DE3), and the positive clones were picked in 2 mL of LB-resistant medium after inverted culture at 37°C for 12 hours, and cultured at 37°C and 250 rpm for 10 hours to prepare fermented seed bacteria, fermentation engineering bacteria See Table 2 for details.
  • Amino acid, fermented at 22°C, 220rpm for 3 days sampled 1mL, sonicated the bacterial liquid for 3 times, mixed with equal volume of ethyl acetate and extracted twice, centrifuged at 12000rpm, 2min, transferred the organic phase to a new tube, and evaporated to dryness at room temperature or 30°C. Then, add 200 ⁇ L of methanol to reconstitute (concentrate 5 times) and mix well, transfer the supernatant to HPLC for 2 min at 12000 rpm.
  • phase A 0.1% formic acid water
  • phase B acetonitrile
  • separation conditions 0-20min 20% phase B-55% phase B, 20-22min 55% phase B-100% phase B, 22-27min 100% phase B, 27-35min 100% phase B-20% phase B, 35-40min, 20% phase B
  • detection wavelength 340nm
  • column temperature 30°C.
  • Chromatographic column Thermo syncronis C18 reversed-phase column (250mm ⁇ 4.6mm, 5 ⁇ m).
  • Rhodiola Rhodotorula toruloides
  • SEQ ID NO: 1 The PAL (RtPAL) length from Rhodiola (Rhodotorula toruloides) is 693aa (GenBank accession number AAA33883.1), and the specific sequence is as follows (SEQ ID NO: 1):
  • Transformation 1 The inventors carried out sequence transformation for SEQ ID NO: 1, the amino acid added a 5nm rigid linker ER/K linker at the C end to obtain an improved PAL mutant PAL-ER/K, the specific sequence is as follows (SEQ ID NO: 2):
  • Transformation 2 The inventors carried out sequence transformation for SEQ ID NO: 2, and then added the amino acid sequence of PDZ to the C-terminal to obtain an improved PAL-ER/K mutant PAL-ER/K-PDZ, the specific sequence is as follows (SEQ ID NO: 2) ID NO: 3):
  • PAL is located at positions 1 to 693 of SEQ ID NO: 3; ER/K is located at positions 694 to 729 of SEQ ID NO: 3; PDZ is located at positions 730 to 824 of SEQ ID NO: 3.
  • Transformation 3 The inventors carried out sequence transformation for SEQ ID NO: 2, and then added the amino acid sequence of SH3 at the C end to obtain an improved PAL-ER/K mutant PAL-ER/K-SH3, the specific sequence is as follows (SEQ ID NO: 2) ID NO: 4):
  • PAL is located at positions 1 to 693 of SEQ ID NO: 4; ER/K is located at positions 694 to 729 of SEQ ID NO: 3; PDZ is located at positions 730 to 786 of SEQ ID NO: 3.
  • the length of 4CL (Pc4CL) from parsley (Petroselium crispum) is 544aa (GenBank accession number KF765780.1), and the specific sequence is as follows (SEQ ID NO: 5):
  • Transformation 1 The inventors carried out sequence transformation for SEQ ID NO: 5, removed the first amino acid, and added the amino acid sequence of (GGGGS) 2 to the N-terminus to obtain an improved 4CL mutant (GGGGS) 2-4CL ,
  • the specific sequence is as follows (SEQ ID NO: 6):
  • Modification 2 The inventors carried out sequence modification according to SEQ ID NO: 6, then added the amino acid sequence of PDZlig to the N-terminus, and then added the M amino acid to the N-terminus to obtain an improved (GGGGS) 2-4CL mutant PDZlig-(GGGGS ) 2-4CL , the specific sequence is as follows (SEQ ID NO: 7):
  • PDZlig is located at positions 1-8 of SEQ ID NO: 7; (GGGGS) 2 is located at positions 9-18 of SEQ ID NO: 7; 4CL is located at positions 19-561 of SEQ ID NO: 7.
  • Modification 3 The inventors carried out sequence modification for SEQ ID NO: 5, and added the amino acid sequence of (GGGGS) 2 to the C-terminus to obtain an improved 4CL mutant 4CL-(GGGGS) 2 , the specific sequence is as follows (SEQ ID NO: 8):
  • Transformation 4 The inventors carried out sequence transformation for SEQ ID NO: 8, and added the amino acid sequence of PDZlig to the C-terminal to obtain an improved 4CL mutant 4CL-(GGGGS) 2 -PDZlig, the specific sequence is as follows (SEQ ID NO: 9 ):
  • 4CL is located at positions 1-544 of SEQ ID NO: 9; (GGGGS) 2 is located at positions 545-554 of SEQ ID NO: 10; PDZlig is located at positions 555-561 of SEQ ID NO: 9.
  • Transformation 5 The inventors carried out sequence transformation according to SEQ ID NO: 6, then added the amino acid sequence of SH3lig to the N-terminus, and then added the M amino acid to the N-terminus to obtain an improved (GGGGS) 2-4CL mutant SH3lig-(GGGGS ) 2-4CL (SH3lig-4CL for short), the specific sequence is as follows (SEQ ID NO: 10):
  • SH3lig is located at positions 1-12 of SEQ ID NO: 10; (GGGGS) 2 is located at positions 13-22 of SEQ ID NO: 10; 4CL is located at positions 23-565 of SEQ ID NO: 10.
  • Transformation 6 The inventors carried out sequence transformation according to SEQ ID NO: 8, and added the amino acid sequence of SH3lig to the C-terminal to obtain an improved 4CL mutant 4CL-(GGGGS) 2 -SH3lig, the specific sequence is as follows (SEQ ID NO: 11 ):
  • 4CL is located at positions 1-544 of SEQ ID NO: 11; (GGGGS) 2 is located at positions 545-554 of SEQ ID NO: 10; SH3lig is located at positions 555-565 of SEQ ID NO: 10.
  • Embodiment 2 fermentation engineering bacteria detect chrysin
  • the pYH57 (pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI) plasmid was transformed into BL21 (DE3) to obtain an engineered strain DN-1, which was used to ferment chrysin using phenylalanine as a precursor.
  • the pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid was transformed into BL21 (DE3) to obtain self-assembly engineering bacteria DN-0, which was used to use phenylalanine as a precursor , fermented chrysin.
  • Embodiment 3 fermentation engineering bacteria detect baicalein
  • phenylalanine is used as a precursor for fermentation, and the synthesis route is shown in Figure 8.
  • the pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid, pYH66 (pETDuet1-T72B1-trF6H-T7CPR) plasmid, pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) plasmid Co-transformed into BL21 (DE3) to obtain self-assembly engineering bacteria DN-2, which was used to ferment baicalein with phenylalanine as a precursor.
  • the pZZ23 (pET28a-T7-4CL-PAL-ER/K-SH3-CHS) plasmid, the pZZ12 (pCDFDuet1-CHI-FNSI) plasmid, and the plasmids were co-transformed into BL21 (DE3) to obtain the control engineering bacteria DN-3, which was treated with benzene.
  • Alanine is used as a precursor to ferment baicalein with phenylalanine as a precursor.
  • the pZZ22 (pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS) plasmid, the pZZ12 (pCDFDuet1-T7-CHI-FNSI) plasmid, and the plasmids were co-transformed into BL21 (DE3) to obtain the self-assembly engineering bacteria DN -4, for the fermentation of baicalein with phenylalanine as the precursor.
  • strain LB solid medium (spectinomycin 80 ⁇ g/mL, ampicillin 100 ⁇ g/mL, chloramphenicol 34 ⁇ g/mL) was cultured overnight at 37°C.
  • PAL modification 2 For the comparison of various modification schemes, the inventors preferred PAL modification 2, 4CL modification 2 or PAL modification 3, 4CL modification 5.
  • Embodiment 4 fermentation engineering bacteria detect scutellarin
  • tyrosine was used as a precursor for fermentation.
  • a non-assembled engineered bacterium DN-1 was obtained, which was used to ferment baicalein with tyrosine as a precursor.
  • the self-assembly engineering bacteria DN-2 was obtained, which was used to ferment baicalein with tyrosine as a precursor.
  • the above two strains were cultured in LB solid medium (spectinomycin 80 ⁇ g/mL, ampicillin 100 ⁇ g/mL, chloramphenicol 34 ⁇ g/mL) overnight at 37°C.
  • transfer the overnight culture into a new 10mL M9Y liquid resistance medium 37 °C, 250r/min culture to OD600 0.5-0.6, water bath cooling to about 16 °C, then add inducer IPTG to the final concentration of 0.2mM, add the final concentration of 500mg/L sterilized tyrosine and transfer to 22 Induced culture at °C low temperature, and continued to culture for 72 h at a shaking speed of 220 r/min.
  • glucose is used as a precursor for fermentation, and the synthesis route is shown in Figure 9.
  • the pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid, pYH66 (pETDuet1-T72B1trF6H-T7CPR) plasmid, pZZ55 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr- T7aroG fbr ) plasmid was co-transformed into BL21 (DE3) to obtain a self-assembled engineering bacterium DN-6 using the interaction scheme of PDZ and PDZlig, which was used to synthesize baicalein from glucose.
  • the two strains established above were cultured on LB solid medium (spectinomycin 80 ⁇ g/mL, ampicillin 100 ⁇ g/mL, chloramphenicol 34 ⁇ g/mL) overnight at 37°C.
  • transfer the overnight culture into a new 10mL M9Y liquid resistance medium 37°C, 250r/min culture to OD600 0.5-0.6, cooling down to about 16°C in water bath, then adding inducer IPTG to a final concentration of 0.2mM, and transferring to 22°C low temperature induction culture, under the condition of shaking table rotation speed 220r/min

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Abstract

Provided are a host cell of a heterologous synthetic baicalein, scutellarein or chrysin compound, and the use thereof. Provided is a new type of optimized modification for the biosynthesis of a baicalein, scutellarein or chrysin compound, which modification can realize the synthesis of the baicalein, scutellarein and chrysin compound by means of using an enzymatic self-assembly technique with prokaryotes as substrates, and realizes the de novo synthesis of baicalein compounds by means of using glucose. Further disclosed are a host cell which has been subjected to optimized modification, and the use thereof.

Description

异源合成黄酮类化合物的宿主细胞及其应用Host cells for heterologous synthesis of flavonoids and their applications 技术领域technical field
本发明涉及合成生物学及医药技术领域，具体地，本发明涉及异源合成黄酮类化合物的宿主细胞及其应用。The present invention relates to the technical field of synthetic biology and medicine, and in particular, the present invention relates to host cells for heterologous synthesis of flavonoids and applications thereof.
背景技术Background technique
黄芩素和野黄芩素是黄酮类化合物，主要存在于中药黄芩中。这两种活性黄酮仅在黄芩等相关药用植物的根中积累较少。黄芩素和黄芩素都是通过类黄酮生物合成途径合成的。黄芩素和野黄芩素具有抗氧化、抗肿瘤、抗菌、护心等重要生理活性。最近，黄芩素在体外被报道为SARS-CoV-2 3Clpro的抑制剂，显示了中药的巨大潜力。Baicalein and scutellarin are flavonoids, which are mainly found in the traditional Chinese medicine Scutellaria baicalensis. These two active flavonoids only accumulate less in the roots of related medicinal plants such as Scutellaria baicalensis. Both baicalein and baicalein are synthesized through the flavonoid biosynthetic pathway. Baicalein and scutellarin have important physiological activities such as antioxidant, antitumor, antibacterial, and heart protection. Recently, baicalein was reported as an inhibitor of SARS-CoV-2 3Clpro in vitro, showing the great potential of traditional Chinese medicine.
目前，黄酮类化合物的主要来源是从植物中提取和化学合成。然而，由于使用有毒化学物质和极端的反应条件，植物萃取或化学合成无法提供大规模生产的绿色路线。因此，微生物合成黄酮类化合物的研究已经深入开展。由于从植物引入复杂的异质途径，酶的失衡和中间代谢物的积累，通常会导致产物滴度低。为了解决这些问题，采用多变量模块化方法，通过调节启动子强度和质粒拷贝数来合成黄酮类化合物。然而，它是费时的，总是需要大量的工作。之前的工作报道了在工程酵母和大肠杆菌中实现黄芩素和野黄芩素的合成，但黄芩素和野黄芩素的产量仍处于很低水平。Currently, the main sources of flavonoids are extraction and chemical synthesis from plants. However, plant extraction or chemical synthesis cannot provide a green route to mass production due to the use of toxic chemicals and extreme reaction conditions. Therefore, the research on microbial synthesis of flavonoids has been intensively carried out. Low product titers typically result from enzyme imbalances and accumulation of intermediate metabolites due to complex heterogeneous pathways introduced from plants. To address these issues, a multivariate modular approach was employed to synthesize flavonoids by modulating promoter strength and plasmid copy number. However, it is time consuming and always requires a lot of work. Previous work reported the synthesis of baicalein and scutellarin in engineered yeast and E. coli, but the yields of baicalein and scutellarin were still at very low levels.
建立底物通道是提高酶在体内催化效率的有效途径。利用相互作用蛋白对组装多酶已被证明是提高催化效率的有效方法。脚手架也是增加产品滴度的有效策略，许多不同构象的连接子被开发用于构建各种相互作用蛋白。The establishment of substrate channels is an effective way to improve the catalytic efficiency of enzymes in vivo. The use of interacting protein pairs to assemble multiple enzymes has been shown to be an effective approach to improve catalytic efficiency. Scaffolding is also an effective strategy to increase product titers, and many linkers in different conformations have been developed for the construction of various interacting proteins.
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。但是利用生物体内酶组装策略生产黄芩素和野黄芩素这两种活性黄酮类化合物还尚未见报道；从葡萄糖合成黄芩素和野黄芩素的大肠杆菌还尚未见报道。In the art, synthetic elements of various natural products are assembled to achieve heterologous synthesis in microorganisms. However, the production of two active flavonoids, baicalein and scutellarin, has not been reported yet; the synthesis of baicalein and scutellarin from Escherichia coli has not been reported yet.
因此，本领域亟待优化能够高效地异源合成黄芩素和野黄芩素或类似化合物的微生物菌株。Therefore, there is an urgent need in the art to optimize microbial strains that can efficiently synthesize baicalein and scutellarin or similar compounds heterologously.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供异源合成黄芩素、野黄芩素类或白杨素类化合物的宿主细胞及其应用。The purpose of the present invention is to provide a host cell for heterologous synthesis of baicalein, scutellarin or chrysin compounds and its application.
在本发明的第一方面，提供一种用于合成黄芩素、野黄芩素类化合物(如黄芩素或野黄芩素)的原核细胞，其包括外源的下组酶的编码基因：黄酮6-羟化酶(F6H)，细胞色素P450氧化还原酶(CPR)，苯丙氨酸解氨酶(PAL)、4-香豆酸辅酶A连接酶(4CL)、查尔酮合成酶(CHS)、查尔酮异构酶(CHI)和黄酮合成酶I(FNSI)；且所述酶被表达后，苯丙氨酸解氨酶(PAL)和4-香豆酸辅酶A连接酶(4CL)构成复合体(复合反应器)。In the first aspect of the present invention, there is provided a prokaryotic cell for synthesizing baicalein and scutellarin-like compounds (such as baicalein or scutellarin), which comprises an exogenous encoding gene of the following group of enzymes: flavonoid 6- Hydroxylase (F6H), Cytochrome P450 oxidoreductase (CPR), Phenylalanine ammonia lyase (PAL), 4-Coumarate CoA ligase (4CL), Chalcone synthase (CHS), Chalcone isomerase (CHI) and flavonoid synthase I (FNSI); and after the enzymes are expressed, phenylalanine ammonia lyase (PAL) and 4-coumarate coenzyme A ligase (4CL) constitute Complex (compound reactor).
在本发明的另一方面，提供一种用于合成白杨素类化合物(如白杨素或芹菜素)的原核细胞，其包括外源的下组酶的编码基因：苯丙氨酸解氨酶(PAL)、4-香豆酸辅酶A连接酶(4CL)、查尔酮合成酶(CHS)、查尔酮异构酶(CHI)和黄酮合成酶I(FNSI)；且所述酶被表达后，苯丙氨酸解氨酶(PAL)和4-香豆酸辅酶A连接酶(4CL)构成复合体(复合反应器)。In another aspect of the present invention, a prokaryotic cell for synthesizing chrysin-based compounds (such as chrysin or apigenin) is provided, which comprises an exogenous encoding gene of the following group of enzymes: phenylalanine ammonia lyase ( PAL), 4-coumarate coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI) and flavonoid synthase I (FNSI); and after the enzymes are expressed , phenylalanine ammonia lyase (PAL) and 4-coumarate coenzyme A ligase (4CL) constitute a complex (complex reactor).
在一个优选例中，所述的苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶的复合体包括：苯丙氨酸解氨酶和4-香豆酸辅酶A通过蛋白-蛋白相互作用结构域及其配体的结合而靠近，获得复合体。In a preferred example, the complex of phenylalanine ammonia lyase and 4-coumaric acid coenzyme A ligase comprises: phenylalanine ammonia lyase and 4-coumaric acid coenzyme A through protein-protein The binding of the interacting domains and their ligands brings them closer together, resulting in a complex.
在另一优选例中，所述的苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶直接连接或通过连接子连接、获得融合蛋白形式的复合体。In another preferred embodiment, the phenylalanine ammonia lyase and 4-coumarate coenzyme A ligase are directly connected or connected through a linker to obtain a complex in the form of a fusion protein.
在另一优选例中，所述蛋白-蛋白相互作用结构域包括选自下组的结构域：PDZ结构域，SH3结构域，WW结构域，LIM结构域，DD结构域，PH结构域，EH结构域，GBD结构域。In another preferred embodiment, the protein-protein interaction domain comprises a domain selected from the group consisting of: PDZ domain, SH3 domain, WW domain, LIM domain, DD domain, PH domain, EH domain, GBD domain.
在另一优选例中，所述蛋白-蛋白相互作用结构域包括PDZ结构域，其配体为PDZ ligand；所述苯丙氨酸解氨酶和4-香豆酸辅酶A分别与所述PDZ结构域及其配体融合；较佳地，所述苯丙氨酸解氨酶与PDZ融合、所述4-香豆酸辅酶A与PDZ ligand融合；更佳地，所述苯丙氨酸解氨酶与PDZ融合时还包括以ER/K连接子连接(PAL-ER/K-PDZ)，所述4-香豆酸辅酶A与PDZ ligand融合时还包括以(GGGGS) 2连接子连接(PDZlig-(GGGGS) 2-4CL)。 In another preferred embodiment, the protein-protein interaction domain includes a PDZ domain, and its ligand is a PDZ ligand; the phenylalanine ammonia lyase and 4-coumarate coenzyme A are respectively associated with the PDZ Domain and its ligand are fused; preferably, the phenylalanine ammonia lyase is fused with PDZ, and the 4-coumaric acid coenzyme A is fused with PDZ ligand; more preferably, the phenylalanine ammonia lyase is fused with PDZ; When the aminoase is fused with PDZ, it also includes connecting with an ER/K linker (PAL-ER/K-PDZ), and when the 4-coumaric acid coenzyme A is fused with PDZ ligand, it also includes connecting with a (GGGGS) 2 linker ( PDZlig-(GGGGS) 2-4CL ).
在另一优选例中，所述蛋白-蛋白相互作用结构域包括SH3结构域，其配体为SH3ligand；所述苯丙氨酸解氨酶和4-香豆酸辅酶A分别与所述SH3结构域及其配体融合；较佳地，所述苯丙氨酸解氨酶与SH3融合、所述4-香豆酸辅酶A与SH3 ligand融合；更佳地，所述苯丙氨酸解氨酶与SH3融合时还包括以ER/K连接子连接(PAL-ER/K-SH3)，所述4-香豆酸辅酶A与SH3 ligand融合时还包括以(GGGGS) 2连接子连接(SH3lig-(GGGGS) 2-4CL)。 In another preferred example, the protein-protein interaction domain includes an SH3 domain, and its ligand is SH3ligand; the phenylalanine ammonia lyase and 4-coumarate coenzyme A are respectively associated with the SH3 structure Domain and its ligand are fused; preferably, the phenylalanine ammonia lyase is fused with SH3, and the 4-coumaric acid coenzyme A is fused with SH3 ligand; more preferably, the phenylalanine ammonia lyase is fused with SH3 When the enzyme is fused with SH3, it also includes connecting with an ER/K linker (PAL-ER/K-SH3), and when the 4-coumaric acid coenzyme A is fused with SH3 ligand, it also includes connecting with a (GGGGS) 2 linker (SH3lig -(GGGGS) 2-4CL ).
在另一优选例中，所述苯丙氨酸解氨酶与PDZ融合时，所述苯丙氨酸解氨酶位于N端，所述PDZ位于C端。In another preferred embodiment, when the phenylalanine ammonia lyase is fused with PDZ, the phenylalanine ammonia lyase is located at the N-terminus, and the PDZ is located at the C-terminus.
在另一优选例中，所述4-香豆酸辅酶A与PDZ ligand融合时，所述PDZ ligand位于N端，所述4-香豆酸辅酶A位于C端。In another preferred embodiment, when the 4-coumaric acid coenzyme A is fused to the PDZ ligand, the PDZ ligand is located at the N-terminus, and the 4-coumaric acid-coenzyme A is located at the C-terminus.
在另一优选例中，所述苯丙氨酸解氨酶与SH3融合时；所述苯丙氨酸解氨酶位于N端，所述SH3位于C端。In another preferred embodiment, when the phenylalanine ammonia lyase is fused with SH3; the phenylalanine ammonia lyase is located at the N-terminus, and the SH3 is located at the C-terminus.
在另一优选例中，所述4-香豆酸辅酶A与SH3 ligand融合时，所述SH3 ligand位于N端，所述4-香豆酸辅酶A位于C端。In another preferred example, when the 4-coumaric acid coenzyme A is fused to the SH3 ligand, the SH3 ligand is located at the N-terminus, and the 4-coumaric acid coenzyme A is located at the C-terminus.
在另一优选例中，所述细胞中还包括外源的促进丙二酰CoA生成的酶的编码基因；较佳地，包括matC，matB，ACS，FabF。In another preferred embodiment, the cell also includes an exogenous gene encoding an enzyme that promotes the production of malonyl CoA; preferably, it includes matC, matB, ACS, and FabF.
在另一优选例中，所述的原核细胞为大肠杆菌细胞。In another preferred embodiment, the prokaryotic cells are Escherichia coli cells.
在另一优选例中，所述细胞中还包括外源的促进苯丙氨酸合成的酶的编码基因；较佳地，包括：aroG，pheA；更佳地，所述pheA为第976位由A突变为C的基因；更佳地，所述aroG为第436位由G突变为A的基因。In another preferred embodiment, the cell also includes an exogenous gene encoding an enzyme that promotes phenylalanine synthesis; preferably, it includes: aroG, pheA; more preferably, the pheA is at position 976 composed of A gene mutated from A to C; more preferably, the aroG is a gene mutated from G to A at position 436.
在另一优选例中，所述“促进”为统计学意义的“促进”，例如促进5％以上，10％以上，20％以上，50％以上，80％以上，100％以上或更高。In another preferred example, the "promoting" is a statistically significant "promoting", such as promoting more than 5%, more than 10%, more than 20%, more than 50%, more than 80%, more than 100% or higher.
在本发明的另一方面，提供所述的原核细胞的应用，用于合成黄芩素或野黄芩素类化合物。In another aspect of the present invention, the application of the prokaryotic cells is provided for synthesizing baicalein or scutellarin compounds.
在本发明的另一方面，提供所述的原核细胞的应用，用于合成白杨素类化合物。In another aspect of the present invention, the application of the prokaryotic cells is provided for synthesizing chrysin compounds.
在本发明的另一方面，提供一种合成黄芩素或野黄芩素类化合物的方法，包括：提供所述的原核细胞(含有F6H和CPR)，以式(I)为底物，合成黄芩素或野黄芩素类化合物；In another aspect of the present invention, a method for synthesizing baicalein or scutellarin compounds is provided, comprising: providing the prokaryotic cells (containing F6H and CPR), and using formula (I) as a substrate to synthesize baicalein or scutellarin compounds;
Figure PCTCN2022070316-appb-000001
Figure PCTCN2022070316-appb-000001
其中，R包括H或OH。wherein R includes H or OH.
在本发明的另一方面，提供一种合成白杨素类化合物的方法，包括：提供所述的原核细胞(可不含有F6H和CPR)，以式(I)为底物，合成白杨素类化合物。In another aspect of the present invention, a method for synthesizing chrysin compounds is provided, comprising: providing the prokaryotic cells (which may not contain F6H and CPR), and using formula (I) as a substrate to synthesize chrysin compounds.
在本发明的另一方面，提供一种合成黄芩素或野黄芩素类化合物或白杨素类化合物的方法，包括：提供所述的原核细胞，以葡萄糖为底物，合成黄芩素或野黄芩素类化合物或白杨素类化合物。In another aspect of the present invention, there is provided a method for synthesizing baicalein or scutellarin-like compounds or chrysin-like compounds, comprising: providing the prokaryotic cells, and using glucose as a substrate to synthesize baicalein or scutellarin compounds or chrysin-like compounds.
在一个优选例中，在引入细胞时，所述PDZligand、4-香豆酸辅酶A连接酶、苯丙氨酸解氨酶、ER/K、PDZ、黄酮合成酶I、查尔酮合成酶、查尔酮异构酶的编码基因位于一个构建体(质粒)中。In a preferred example, when introduced into cells, the PDZligand, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, PDZ, flavonoid synthase I, chalcone synthase, The gene encoding chalcone isomerase is located in one construct (plasmid).
在另一优选例中，所述黄酮6-羟化酶，细胞色素P450氧化还原酶的编码基因位于一个构建体中，较佳地还包括2B1(细胞色素P450 2B1家族可溶性蛋白)基因。In another preferred embodiment, the encoding gene for flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase is located in a construct, preferably also including 2B1 (cytochrome P450 2B1 family soluble protein) gene.
在另一优选例中，所述matC，matB，ACS，FabF的编码基因位于一个构建体中。In another preferred embodiment, the genes encoding matC, matB, ACS, and FabF are located in one construct.
在另一优选例中，所述SH3lig，4-香豆酸辅酶A连接酶，苯丙氨酸解氨酶，ER/K，SH3，查尔酮合成酶的编码基因位于一个构建体中。In another preferred embodiment, the genes encoding SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, and chalcone synthase are located in one construct.
在另一优选例中，所述查尔酮异构酶，黄酮合成酶I的编码基因位于一个构建体中。In another preferred embodiment, the gene encoding chalcone isomerase and flavonoid synthase I is located in one construct.
在另一优选例中，所述matC、matB、ACS、FabF的编码基因，第976位由A突变为C的pheA基因(pheA fbr)，第436位由G突变为A的aroG基因(aroG fbr)位于一个构建体中。 In another preferred embodiment, the encoding genes of matC, matB, ACS, and FabF are pheA gene (pheA fbr ) mutated from A to C at position 976, and aroG gene (aroG fbr ) mutated from G to A at position 436 ) in one construct.
在本发明的另一方面，提供一种用于生产黄芩素或野黄芩素类化合物(试剂盒中 含有F6H和CPR)或白杨素类化合物(试剂盒中可不含有F6H和CPR)的试剂盒，其包括所述的重组的宿主细胞。In another aspect of the present invention, there is provided a kit for producing baicalein or scutellarin-based compounds (containing F6H and CPR in the kit) or chrysin-based compounds (without F6H and CPR in the kit), It includes said recombinant host cells.
在本发明的另一方面，提供一种用于建立合成黄芩素或野黄芩素类化合物或白杨素类化合物的宿主细胞的试剂盒，其包括：包含PDZligand、4-香豆酸辅酶A连接酶、苯丙氨酸解氨酶、ER/K、PDZ、黄酮合成酶I、查尔酮合成酶、查尔酮异构酶的编码基因的构建体；包含matC，matB，ACS，FabF的编码基因的构建体；包含SH3lig，4-香豆酸辅酶A连接酶，苯丙氨酸解氨酶，ER/K，SH3，查尔酮合成酶的编码基因的构建体；包含查尔酮异构酶，黄酮合成酶I的编码基因的构建体；包含matC、matB、ACS、FabF的编码基因，第976位由A突变为C的pheA基因(pheA fbr)，第436位由G突变为A的aroG基因(aroG fbr)的构建体；可选地，还包含黄酮6-羟化酶，细胞色素P450氧化还原酶的编码基因的构建体；较佳地所述构建体还包含2B1基因。 In another aspect of the present invention, there is provided a kit for establishing a host cell for synthesizing baicalein or scutellarin-like compounds or chrysin-like compounds, comprising: comprising PDZligand, 4-coumaric acid-CoA ligase , phenylalanine ammonia lyase, ER/K, PDZ, flavonoid synthase I, chalcone synthase, chalcone isomerase encoding gene constructs; including matC, matB, ACS, FabF encoding genes Constructs of SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, constructs encoding genes for chalcone synthase; containing chalcone isomerase , a construct encoding the gene encoding flavonoid synthase I; including the encoding genes of matC, matB, ACS, FabF, the pheA gene (pheA fbr ) mutated from A to C at position 976, and aroG mutated from G to A at position 436 The construct of the gene (aroG fbr ); optionally, the construct further comprising the gene encoding flavone 6-hydroxylase, cytochrome P450 oxidoreductase; preferably, the construct further comprises the 2B1 gene.
在一个优选例中，所述的试剂盒中还包括：葡萄糖；或，式(I)底物。In a preferred embodiment, the kit further includes: glucose; or, the substrate of formula (I).
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。Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.
附图说明Description of drawings
图1、质粒pZZ41的构建示意图。Figure 1. Schematic diagram of the construction of plasmid pZZ41.
图2、质粒pZZ55的构建示意图。Figure 2. Schematic diagram of the construction of plasmid pZZ55.
图3、非自组装菌株DN-1、自组装菌株DN-2的黄芩素产量的柱形图。Figure 3. The bar graph of the baicalein production of the non-self-assembling strain DN-1 and the self-assembling strain DN-2.
图4、非自组装菌株DN-1、自组装菌株DN-2产生的黄芩素的HPLC检测图谱。Figure 4. HPLC detection pattern of baicalein produced by non-self-assembling strain DN-1 and self-assembling strain DN-2.
图5、对照菌株DN-3相比，自组装菌株DN-4的黄芩素产量的柱形图。Figure 5. Bar graph of baicalein production of self-assembled strain DN-4 compared to control strain DN-3.
图6、非自组装菌株DN-1、自组装菌株DN-2的野黄芩素产量的柱形图。Figure 6. The bar graph of the scutellarin production of the non-self-assembling strain DN-1 and the self-assembling strain DN-2.
图7、自组装工程菌株DN-6、非自组装工程菌株DN-5的黄芩素产量的柱形图。Figure 7. The bar graph of the baicalein production of the self-assembly engineering strain DN-6 and the non-self-assembly engineering strain DN-5.
图8、以苯丙氨酸为前体，进行发酵生成黄芩素、野黄岑素的合成途径示意图。Fig. 8 is a schematic diagram of the synthetic pathway for fermenting to generate baicalein and baicalein using phenylalanine as a precursor.
图9、以葡萄糖为前体，进行发酵生成黄芩素的合成途径示意图。Figure 9 is a schematic diagram of the synthetic pathway of baicalein produced by fermentation with glucose as a precursor.
图10、非自组装的菌株JH-0相比与自组装菌株DN-0的白杨素产量比较。Figure 10. Comparison of chrysin production between non-self-assembled strain JH-0 and self-assembled strain DN-0.
具体实施方式Detailed ways
本发明人经过深入的研究，提供了新型的黄芩素或野黄芩素类化合物/白杨素类化合物生物合成的优化改造，可实现以原核生物为底盘利用酶自组装技术合成黄芩素或野黄芩素类化合物/白杨素类化合物，以及实现利用葡萄糖从头合成黄芩素或野黄芩类化合物/白杨素类化合物。本发明也揭示了优化改造后的宿主细胞及其应用。After in-depth research, the inventors provide a novel biosynthesis optimization of baicalein or scutellarin compounds/chrysin compounds, which can realize the synthesis of baicalein or scutellarin using enzyme self-assembly technology on the basis of prokaryotes scutellarin-like compounds/chrysin-like compounds, and de novo synthesis of baicalein or scutellaria-like compounds/chrysin-like compounds using glucose. The invention also discloses the optimized modified host cell and its application.
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。As used herein, "foreign" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources.
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。As used herein, "operably linked (linked)" or "operably linked (linked)" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, the promoter region is placed at a specific location 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, and thus, the promoter region is "operably linked" to the nucleic acid sequence.
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。As used herein, an "expression construct" refers to a recombinant DNA molecule comprising the desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. The "construct" is usually contained in an expression vector.
如本文所用，所述的PAL、4CL、CHS、CHI和FNSI蛋白是在表达***中形成白杨素或芹菜素合成途径的蛋白。As used herein, the PAL, 4CL, CHS, CHI and FNSI proteins are those that form the chrysin or apigenin synthetic pathway in an expression system.
如本文所用，所述的F6H和CPR蛋白是在表达***中转化白杨素或芹菜素、生成黄芩素或野黄芩素类化合物的蛋白。As used herein, the F6H and CPR proteins are proteins that convert chrysin or apigenin to produce baicalein or scutellarin-like compounds in an expression system.
如本文所用，所述的matC、matB、ACS和/或FabF蛋白在表达***中促进丙二酰CoA生成的酶。As used herein, the matC, matB, ACS and/or FabF proteins described are enzymes that promote malonyl-CoA production in an expression system.
如本文所用，所述的aroG或其突变体，pheA或其突变体在表达***中促进苯丙氨酸合成。As used herein, aroG or a mutant thereof, pheA or a mutant thereof, promotes phenylalanine synthesis in an expression system.
野生型的上述蛋白或基因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。The wild-type protein or gene described above has been identified in the art, and therefore, can be obtained and prepared from public sources. As a preferred mode of the present invention, PAL is derived from Rhodiola (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. The sequence shown; CHS is derived from petunia (Petunia X hybrida), which has the sequence shown in GenBank accession number KF765781.1; CHI gene is derived from alfalfa (Medicago sativa), which has the sequence shown in GenBank accession number KF765782.1 Sequence; FNS I is derived from Parsley (Petroselium crispum), which has the sequence shown in Swiss-Prot Accession No. Q7XZQ8.1.
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。Wild-type F6H and CPR have also been identified in the art. As a preferred embodiment of the present invention, F6H is derived from Scutellaria baicalensis, which has the sequence shown in GenBank accession number ASW21050.1. As a preferred mode of the present invention, the CPR is derived from Arabidopsis thaliana, which has the sequence shown in GenBank accession number NP_849472.2.
野生型的matC、matB、ACS、FabF蛋白也是本领域已经鉴定的。作为本发明的优选方式，matC来源于豆科根瘤菌(Rhizobium leguminosarum)，其具有GenBank登录号KF765784.1所示的序列；matB来源于豆科根瘤菌(Rhizobium leguminosarum)，其具有GenBank登录号AGZ04579.1所示的序列；ACS来源于大肠杆菌(Escherichia coli)，其具有GenBank登录号CP062211.1所示的序列；FabF来源于大肠杆菌(Escherichia coli)，其具有GenBank登录号AP023237.1所示的序列。Wild-type matC, matB, ACS, FabF proteins have also been identified in the art. As a preferred mode of the present invention, matC is derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number KF765784.1; matB is derived from Rhizobium leguminosarum, which has GenBank accession number AGZ04579 The sequence shown in .1; ACS is derived from Escherichia coli (Escherichia coli), which has the sequence shown in GenBank accession number CP062211.1; FabF is derived from Escherichia coli (Escherichia coli), which has GenBank accession number shown in AP023237.1 the sequence of.
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。它们的结构如下所示： Baicalein and scutellarin are two structurally similar and important 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 as follows:
Figure PCTCN2022070316-appb-000002
Figure PCTCN2022070316-appb-000002
本发明人发现，利用宿主细胞生产黄芩素、野黄岑素类化合物(如黄芩素或野黄芩素)或其前体白杨素类化合物(如白杨素或芹菜素)的过程中，仍然存在产物含量不够高的情形，因此对多个参与反应的蛋白进行了分析，经过大量筛选和实验，获得了一种优选的改造方案，极为显著地提高了微生物，尤其是原核表达***(原核细胞)如大肠杆菌中化合物的产量。The inventors found that in the process of using host cells to produce baicalein, scutellarin compounds (such as baicalein or scutellarin) or their precursor chrysin compounds (such as chrysin or apigenin), there are still products When the content is not high enough, a number of proteins involved in the reaction were analyzed, and after a lot of screening and experiments, a preferred transformation scheme was obtained, which significantly improved microorganisms, especially prokaryotic expression systems (prokaryotic cells) such as Yields of compounds in E. coli.
作为本发明的改造方案的一个方面，本发明人利用酶组装技术发酵生产黄芩素或野黄芩素。该方案的原理为：利用相互作用的蛋白对(例如PDZ和PDZ ligand)与黄芩素合成途径中的酶PAL和4CL进行融合，使PAL和4CL能够在大肠杆菌体内进行自发组装，形成双酶复合反应器，从而提目标化合物的产量。As an aspect of the modification scheme of the present invention, the inventors utilize enzyme assembly technology to ferment and produce baicalein or scutellarin. The principle of this scheme is to use interacting protein pairs (such as PDZ and PDZ ligand) to fuse with enzymes PAL and 4CL in the baicalein synthesis pathway, so that PAL and 4CL can spontaneously assemble in E. coli to form a dual-enzyme complex reactor, thereby increasing the yield of the target compound.
本发明人首次在合成黄岑类化合物/白杨素类化合物的原核表达***中发现将PAL与4CL构建成复合体(复合反应器)，可极为有效地提高表达***的产量。适用于使得PAL与4CL构成有活性的复合体的任何生物材料或技术手段可被应用于本发明中。作为本发明的优选方式，所述的蛋白-蛋白相互作用结构域可包括选自下组的结构域：PDZ结构域，SH3结构域，WW结构域，LIM结构域，DD结构域，PH结构域，EH结构域。作为本发明的更优选的方式，所述的蛋白-蛋白相互作用结构域可包括选自下组的结构域：PDZ结构域，SH3结构域；它们的相应的配体为PDZ ligand(PDZlig)或SH3 ligand(SH3lig)。The inventors found for the first time in the prokaryotic expression system for synthesizing scutellaria compound/chrysin compound that PAL and 4CL are constructed into a complex (composite reactor), which can extremely effectively improve the yield of the expression system. Any biological material or technical means suitable for making PAL and 4CL into an active complex can be used in the present invention. As a preferred mode of the present invention, the protein-protein interaction domain may comprise a domain selected from the group consisting of: PDZ domain, SH3 domain, WW domain, LIM domain, DD domain, PH domain , the EH domain. As a more preferred mode of the present invention, the protein-protein interaction domain may comprise a domain selected from the group consisting of: PDZ domain, SH3 domain; their corresponding ligands are PDZ ligand (PDZlig) or SH3ligand(SH3lig).
蛋白质-蛋白质相互作用主要由蛋白质结构域来高效介导。PDZ、SH3、WW等结构域可通过一个或多个识别“口袋”来识别和结合配体蛋白的一段保守的短肽序列。就PDZ结构域而言，它通常结合配体蛋白C末端4-5个氨基酸残基，其也能够结合配体蛋白的中间序列，与自身或其他结构域聚合，或与膜上的脂类结合。Protein-protein interactions are efficiently mediated mainly by protein domains. PDZ, SH3, WW and other domains can recognize and bind a short conserved peptide sequence of ligand proteins through one or more recognition "pockets". In the case of the PDZ domain, it usually binds to the C-terminal 4-5 amino acid residues of the ligand protein, and it is also capable of binding to the intermediate sequence of the ligand protein, polymerizing with itself or other domains, or binding to lipids on the membrane .
本发明中，也可包括其它的将PAL与4CL构成复合体、且能保留所述PAL与4CL的生物学活性的方法，例如将它们进行融合，构成具有适合的空间结构的融合蛋白；可通过实验测试来确定融合蛋白的活性。PAL与4CL之间的融合可以是直接连接，也可以利用连接子(Linker)来进行连接。In the present invention, other methods for forming a complex between PAL and 4CL and retaining the biological activity of the PAL and 4CL can also be included, such as fusing them to form a fusion protein with a suitable spatial structure; Experimental tests to determine the activity of fusion proteins. The fusion between PAL and 4CL can be a direct connection or a linker can be used for connection.
作为本发明的改造方案的另一个方面，本发明人在原核表达***中过表达aroG、特别是其aroG fbr，以及pheA、特别是其pheA fbr基因，构建获得高产苯丙氨酸原核表达***，在该原核表达***中引入外源的黄芩素或野黄芩素类化合物/白杨素类化合物合成途径，使该菌株能够利用葡萄糖从头合成黄芩素化合物/白杨素类化合物。 As another aspect of the transformation scheme of the present invention, the inventors overexpressed aroG, especially its aroG fbr , and pheA, especially its pheA fbr gene in a prokaryotic expression system, and constructed a prokaryotic expression system to obtain high-yield phenylalanine, The exogenous baicalein or scutellarin compound/chrysin compound synthesis pathway is introduced into the prokaryotic expression system, so that the strain can utilize glucose to synthesize baicalein compound/chrysin compound de novo.
常用的原核表达***包括大肠杆菌、枯草杆菌等；例如可为大肠杆菌细胞(E.coli)，如大肠杆菌BL21(DE3)。Commonly used prokaryotic expression systems include E. coli, Bacillus subtilis, etc.; for example, E. coli cells (E. coli), such as E. coli BL21 (DE3).
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。On the basis of the above-mentioned preferred proteins (including the above-mentioned wild-type proteins and mutant proteins), the present invention also includes their analogs. The differences between these analogs and the native protein may be differences in amino acid sequence, 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 a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.
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。On the basis of the above-mentioned preferred proteins (including the above-mentioned wild-type proteins and mutant proteins), the present invention also includes high homology with the proteins (for example, the homology with the specific protein sequences listed is 70 % or more; preferably 80% or more homology; more preferably 90% or more homology, such as 95%, 98% or 99% homology), and have the same function as the corresponding polypeptide The protein is also included in the present invention.
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。Proteins or genes from specific species are enumerated in the present invention. It should be understood that although proteins or genes obtained from a specific species are preferably studied in the present invention, those obtained from other species are highly homologous to said proteins or genes (eg have more than 60%, such as 70%, 80%, 85% , 90%, 95%, or even 98% sequence identity) other proteins or genes are also contemplated by the present invention.
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。The present invention also relates to the present invention also provides a polynucleotide sequence encoding the protein of the present invention or a conservative variant thereof. The polynucleotides 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 the coding or non-coding strand. Polynucleotides encoding the mutant mature proteins of the present invention include: coding sequences encoding only the mature protein; coding sequences for the mature protein and various additional coding sequences; coding sequences for the mature protein (and optional additional coding sequences) and non- coding sequence.
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。The present invention also includes polynucleotide sequences formed by codon optimization for the sequence of the gene, eg, codon optimization according to the preference of the host cell.
本发明中，还构建了高产黄芩素或野黄芩素类化合物的工程菌株，其中包括外源的下组酶的编码基因：F6H，CPR，PAL、4CL、CHS、CHI和FNSI；且所述酶被表达后，PAL和4CL构成复合体(复合反应器)。培养该重组菌株，并以苯丙氨酸或酪氨酸为底物，生产黄芩素或野黄芩素类化合物。以苯丙氨酸或酪氨酸为底物的生产，适合于规模化的化合物生产。In the present invention, an engineering strain with high production of baicalein or scutellarin compounds is also constructed, which includes the encoding genes of the exogenous lower group enzymes: F6H, CPR, PAL, 4CL, CHS, CHI and FNSI; and the enzymes After being expressed, PAL and 4CL form a complex (complex reactor). The recombinant strain is cultivated, and takes phenylalanine or tyrosine as a substrate to produce baicalein or scutellarin compounds. The production using phenylalanine or tyrosine as a substrate is suitable for large-scale compound production.
本发明中，还构建了高产白杨素类化合物的工程菌株，其中包括外源的下组酶的编码基因：PAL、4CL、CHS、CHI和FNSI；且所述酶被表达后，PAL和4CL构成复合体(复合反应器)。培养该重组菌株，并以苯丙氨酸或酪氨酸为底物，生产黄芩素或野黄芩素类化合物。以苯丙氨酸或酪氨酸为底物的生产，适合于规模化的化合物生产。In the present invention, an engineered strain with high production of chrysin compounds is also constructed, which includes exogenous encoding genes of the following group of enzymes: PAL, 4CL, CHS, CHI and FNSI; and after the enzymes are expressed, PAL and 4CL constitute Complex (compound reactor). The recombinant strain is cultivated, and takes phenylalanine or tyrosine as a substrate to produce baicalein or scutellarin compounds. The production using phenylalanine or tyrosine as a substrate is suitable for large-scale compound production.
为本发明的优选方式，所述F6H还包括与之融合的多肽标签，所述的多肽标签例如选自：8RP，Sumo，MBP，2B1，或它们的组合；较佳地为2B1。所述的多肽标 签与所述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。F6H于2B1连接，可获得一种改进的F6H突变体2B1trF6H。In a preferred mode of the present invention, the F6H further includes a polypeptide tag fused to it, for example, the polypeptide tag is selected from: 8RP, Sumo, MBP, 2B1, or a combination thereof; preferably 2B1. Between the polypeptide tag and the F6H, a connecting peptide may or may not be included, and the connecting peptide does not affect the biological activity of the two. F6H was linked to 2B1 to obtain an improved F6H mutant 2B1trF6H.
还可以在上述的工程菌株中，进一步引入上述底物(苯丙氨酸或酪氨酸)的上游生成途径，例如包括：由葡萄糖或甘油通过糖酵解、磷酸戊糖途径、莽草酸途径生成苯丙氨酸或酪氨酸。应理解，基于此类途径来形成苯丙氨酸或酪氨酸的方案也包含在本发明中。通过本领域已知手段来加强所述形成苯丙氨酸或酪氨酸途径的方法可包含在本发明中。Also in the above-mentioned engineering strain, the upstream generation pathway of the above-mentioned substrate (phenylalanine or tyrosine) can be further introduced, for example, including: generated by glucose or glycerol through glycolysis, pentose phosphate pathway, shikimic acid pathway Phenylalanine or tyrosine. It should be understood that protocols for the formation of phenylalanine or tyrosine based on such pathways are also encompassed by the present invention. Methods of enhancing the phenylalanine or tyrosine-forming pathway by means known in the art can be included in the present invention.
作为一种优选方式，可以在上述以苯丙氨酸或酪氨酸为底物的工程菌株中，进一步引入外源的aroG、特别是其aroG fbr，以及pheA、特别是其pheA fbr，获得另一种重组菌株，该菌株能够以葡萄糖为底物，生产黄芩素类化合物/白杨素类化合物。以葡萄糖为底物的生产，成本低廉，非常适合于规模化的化合物生产。 As a preferred method, exogenous aroG, especially its aroG fbr , and pheA, especially its pheA fbr can be further introduced into the above-mentioned engineering strain using phenylalanine or tyrosine as a substrate to obtain another A recombinant strain capable of producing baicalein compounds/chrysin compounds using glucose as a substrate. The production using glucose as a substrate has low cost and is very suitable for large-scale compound production.
在建立如本发明优化的表达***以及利用其进行生产的基础上，本领域人员还可***研提高黄芩素、野黄芩素类化合物或白杨素类化合物产量的一系列因素，包括基因的效率和适宜性、基因剂量和培养基。此外，也可通过扩大生产规模来提高目标化合物的产量。例如，在摇瓶规模、简单培养条件下的产量基础上，当进一步扩大生产规模、进行培养基流加方案(可以源源不断提供充沛的底物)或给予良好的发酵罐水平生产条件(如温度的优化控制、溶氧的优化控制等)时，其产量通常可增加2～1000倍。这些操作和优化方式也应被包含在本发明中。可以预期，本发明的重组原核细胞，在一些优化的设备和操作工艺中，目标产物的量会发生长足的增长。On the basis of establishing the optimized expression system as in the present invention and utilizing it for production, those in the art can also systematically study a series of factors that increase the yield of baicalein, scutellarin-like compounds or chrysin-like compounds, including gene efficiency and Suitability, gene dosage and media. In addition, the yield of the target compound can also be increased by expanding the production scale. For example, on the basis of the yield under the shake flask scale and simple culture conditions, when the production scale is further expanded, the medium feeding scheme can be carried out (which can provide abundant substrates continuously), or the production conditions of good fermenter level (such as temperature When the optimal control of oxygen is used, the optimal control of dissolved oxygen, etc.), its output can usually be increased by 2 to 1000 times. These operations and optimizations should also be included in the present invention. It can be expected that, in the recombinant prokaryotic cells of the present invention, the amount of the target product will increase significantly in some optimized equipment and operating techniques.
在获得了发酵产物后，从发酵产物中提取目标化合物可以采用本领域已知的技术。可以采用一些公知技术如高效液相色谱来对产物进行分析鉴定，以确定获得了所需的化合物。After the fermentation product is obtained, techniques known in the art can be used to extract the target compound from the fermentation product. The product can be analytically identified using well known techniques such as high performance liquid chromatography to confirm that the desired compound has been obtained.
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类化合物/白杨素类化合物。本发明优选的菌株的目标化合物得率非常高。The strain of the invention has good stability, and can realize large-scale cultivation and production of baicalein or scutellarin compounds/chrysin compounds in a bioreactor. The yields of the target compounds of the preferred strains of the present invention are very high.
相对于传统的植物提取手段，微生物发酵具有速度快、受外界因素影响较小等优势；部分化合物通过微生物合成的产量远高于植物提取，已经成为天然产物获得的一种重要手段。本发明中，通过大肠杆菌生产黄芩素或野黄芩素类化合物/白杨素类化合物，实现目标化合物更经济、更方便的制造。Compared with traditional plant extraction methods, microbial fermentation has the advantages of fast speed and less influence by external factors; the yield of some compounds through microbial synthesis is much higher than that of plant extraction, and has become an important means of obtaining natural products. In the present invention, by producing baicalein or scutellarin compound/chrysin compound through Escherichia coli, more economical and convenient manufacture of the target compound is achieved.
本发明还提供了用于生产黄芩素或野黄芩素类化合物工程菌株的试剂盒。此外，其中还可包括原核细胞的培养基，用于合成的底物如苯丙氨酸、酪氨酸或葡萄糖，黄芩素或野黄芩素类化合物分离或检测试剂。更佳地，所述试剂盒中还可包括说明进行生物合成黄层素的方法的使用说明书等。The invention also provides a kit for producing engineering strains of baicalein or scutellarin compounds. In addition, the culture medium of prokaryotic cells can also be included, substrates for synthesis such as phenylalanine, tyrosine or glucose, baicalein or scutellarin-based compound separation or detection reagents. More preferably, the kit may further include instructions for use that describe the method for biosynthesizing xanthophylls, and the like.
本发明还提供了用于构建所述生产黄芩素或野黄芩素类化合物/白杨素类化合物，工程菌株的试剂盒，所述试剂盒中可包括一系列构建体，例如可参考本发明的实施例中所提供的构建体，也可以为含有所述基因但基因排列或串联方式不同的其它构 建体。表达载体(表达构建物)的建立可以采用本领域技术人员熟悉的技术。在得知了所需选择的酶以及所需表达的细胞体系之后，本领域技术人员可以进行表达构建物的建立。基因序列可以被***到不同的表达构建物(如表达载体)中，也可以被***到同一表达构建物中，只要在转入到细胞后其编码的多肽能够被有效地表达和发挥活性即可。所述试剂盒中还可包括原核细胞，原核细胞的培养基，用于合成的底物如苯丙氨酸、酪氨酸或葡萄糖，黄芩素或野黄芩素类化合物分离或检测试剂。更佳地，所述试剂盒中还可包括说明进行生物合成黄芩素或野黄芩素的方法的使用说明书等。The present invention also provides a kit for constructing the engineering strain for producing baicalein or scutellarin-like compounds/chrysin-like compounds, the kit may include a series of constructs, for example, refer to the implementation of the present invention The constructs provided in the examples can also be other constructs that contain the genes but the gene arrangement or tandem manner is different. Expression vectors (expression constructs) can be established using techniques familiar to those skilled in the art. After knowing the desired enzyme of choice and the desired cell system for expression, those skilled in the art can establish expression constructs. Gene sequences can be inserted into different expression constructs (such as expression vectors), or into the same expression construct, as long as the encoded polypeptide can be efficiently expressed and active after being transferred into cells . The kit can also include prokaryotic cells, culture medium of prokaryotic cells, substrates for synthesis such as phenylalanine, tyrosine or glucose, and baicalein or scutellarin compounds for separation or detection reagents. More preferably, the kit may further include instructions for use that describe the method for biosynthesizing baicalein or scutellarin.
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。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.
1.实验材料1. Experimental materials
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；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。Polymerase chain reaction (PCR) gel recovery kits and plasmid extraction kits are products of American Axygen; polymerase chain reaction (PCR) high-fidelity enzyme PrimeSTAR Max DNA Polymerase is a product of TAKARA; restriction The endonucleases are all NEB products.
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。The standard compounds baicalein and scutellarin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Other reagents were domestic analytical or chromatographic pure reagents, purchased from Sinopharm Chemical Reagent Co., Ltd.
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)；OD600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***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***(Thermo Fisher Scientific)。Arktik Thermal Cycler (Thermo Fisher Scientific) was used for PCR; ZXGP-A2050 incubator and ZWY-211G incubator were used for constant temperature incubation; 5418R high-speed refrigerated centrifuge and 5418 mini-centrifuge (Eppendorf) were used for centrifugation. Concentrator plus concentrator (Eppendorf) was used for vacuum concentration; OD600 was detected by UV-1200 UV-Vis spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.). The rotary evaporation system consists of an IKA RV 10digital rotary evaporator (IKA), a MZ 2CNT chemical diaphragm pump, and a CVC3000 vacuum controller (vacuubrand). High performance liquid chromatography was performed using a Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific).
2、本发明所涉及的菌株及质粒2. Strain and plasmid involved in the present invention
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。Escherichia coli DH10B was used for gene cloning, and Escherichia coli BL21(DE3) strain was used for protein expression and production of baicalein and scutellarin.
pCDFDuet-1、pETDuet-1、pACYCDuet-1载体用于代谢途径基因装配。pCDFDuet-1, pETDuet-1, pACYCDuet-1 vectors were used for metabolic pathway gene assembly.
5nm rigid linker ER/K序列：KAKLKEEEERKQREEEERIKRLEELAKR KEEERKGT。5nm rigid linker ER/K sequence: KAKLKEEEERKQREEEERIKRLEELAKR KEEERKGT.
3、酶的选择3. Enzyme selection
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，具体如下：The above-mentioned wild-type proteins or genes have been identified in the art, and therefore, can be obtained and prepared from public sources, as follows:
PAL：来源于红景天(Rhodotorula toruloides)，其具有GenBank登录号AAA33883.1所示的序列(RtPAL)；PAL: derived from Rhodiola (Rhodotorula touloides), which has the sequence shown in GenBank accession number AAA33883.1 (RtPAL);
4CL：来源于欧芹(Petroselium crispum)，其具有GenBank登录号KF765780.1所示的序列(Pc4CL)；4CL: derived from parsley (Petroselium crispum), which has the sequence shown in GenBank accession number KF765780.1 (Pc4CL);
CHS：来源于矮牵牛(Petunia X hybrida)，其具有GenBank登录号KF765781.1所示的序列；CHS: derived from petunia (Petunia X hybrida), which has the sequence shown in GenBank accession number KF765781.1;
CHI基因：来源于苜蓿(Medicago sativa)，其具有GenBank登录号KF765782.1所示的序列；CHI gene: derived from alfalfa (Medicago sativa), it has the sequence shown in GenBank accession number KF765782.1;
FNS I：来源于欧芹(Petroselium crispum)，其具有Swiss-Prot登录号Q7XZQ8.1所示的序列；FNS I: derived from parsley (Petroselium crispum), which has the sequence shown in Swiss-Prot accession number Q7XZQ8.1;
F6H：来源于黄岑(Scutellaria baicalensis)，其具有GenBank登录号ASW21050.1所示的序列；F6H: derived from Scutellaria baicalensis, which has the sequence shown in GenBank accession number ASW21050.1;
CPR：来自于拟南芥(Arabidopsis thaliana)，其具有GenBank登录号NP_849472.2所示的序列。CPR: from Arabidopsis thaliana, which has the sequence shown in GenBank Accession No. NP_849472.2.
PDZ结构域：来自于小鼠Mouseα-syntrophin(syn)，77-171氨基酸序列，其具有GenBank登录号EDL06069所示的序列。PDZ domain: derived from mouse Mouseα-syntrophin (syn), 77-171 amino acid sequence, which has the sequence shown in GenBank accession number EDL06069.
matB基因：来源于豆科根瘤菌(Rhizobium leguminosarum)，其具有GenBank登录号AGZ04579.1所示的序列。matB gene: derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number AGZ04579.1.
matC基因：来源于豆科根瘤菌(Rhizobium leguminosarum)，其具有GenBank登录号KF765784.1所示的序列。matC gene: derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number KF765784.1.
ACS基因：来源于大肠杆菌(Escherichia coli)，其具有GenBank登录号CP062211.1所示的序列。ACS gene: derived from Escherichia coli, which has the sequence shown in GenBank accession number CP062211.1.
PDZ ligand：序列为GVKESLV(SEQ ID NO:12)。PDZ ligand: sequence is GVKESLV (SEQ ID NO: 12).
SH3结构域：AEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWN AEDSEGKRGMIPVPYVEKY(SEQ ID NO:13)。SH3 domain: AEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWN AEDSEGKRGMIPVPYVEKY (SEQ ID NO: 13).
SH3lig：PPPALPPKRRR(SEQ ID NO:14)。SH3lig: PPPALPPKRRR (SEQ ID NO: 14).
4、质粒的构建4. Plasmid construction
构建含单个基因的质粒Construction of plasmids containing a single gene
(1)PAL和PDZ的融合蛋白质粒的构建(1) Construction of fusion protein particles of PAL and PDZ
首先在PAL基因N端增加NcoI酶切位点，PDZ序列C端增加EcoRI酶切位点，利用Over-Lap PCR方法将PAL，5nm rigid linker ER/K，PDZ进行基因融合，载体骨架选择pCDFDuet-1，将Over-Lap PCR后得到的融合基因PAL-ER/K-PDZ连接到 pCDFDuet-1的NcoI和EcoRI位点，得到pCDFDuet1-T7PAL-ER/K-PDZ。Firstly, NcoI restriction site was added to the N-terminus of PAL gene, and EcoRI restriction site was added to the C-terminus of PDZ sequence. PAL, 5nm rigid linker ER/K, PDZ were genetically fused by Over-Lap PCR method, and pCDFDuet- 1. Connect the fusion gene PAL-ER/K-PDZ obtained after Over-Lap PCR to the NcoI and EcoRI sites of pCDFDuet-1 to obtain pCDFDuet1-T7PAL-ER/K-PDZ.
(2)4CL和PDZ ligand的融合蛋白质粒的构建(2) Construction of fusion protein particles of 4CL and PDZ ligand
将PDZ ligand序列设计在上游引物中，N端增加NcoI酶切位点，4CL序列C端增加BamHI酶切位点，PCR得到PDZlig-4CL融合基因，将融合基因构建到pCDFDuet-1的NcoI和BamHI位点，得到pCDFDuet1-T7PDZlig-4CL。The PDZ ligand sequence was designed in the upstream primer, NcoI restriction site was added to the N-terminus, and BamHI restriction site was added to the C-terminus of the 4CL sequence. The PDZlig-4CL fusion gene was obtained by PCR, and the fusion gene was constructed into the NcoI and BamHI of pCDFDuet-1. site, resulting in pCDFDuet1-T7PDZlig-4CL.
(3)4CL和PDZ ligand的融合蛋白质粒的构建(3) Construction of fusion protein particles of 4CL and PDZ ligand
将PDZ ligand序列设计在下游引物中，4CL N端增加NcoI酶切位点，PDZ ligand C端增加BamHI酶切位点，PCR得到4CL-PDZlig融合基因，将融合基因构建到pCDFDuet-1的NcoI和BamHI位点，得到pCDFDuet1-T7 4CL-PDZlig。The PDZ ligand sequence was designed in the downstream primer, the NcoI restriction site was added to the N-terminus of 4CL, and the BamHI restriction site was added to the C-terminus of PDZ ligand. The 4CL-PDZlig fusion gene was obtained by PCR, and the fusion gene was constructed into the NcoI and NcoI of pCDFDuet-1. BamHI site, resulting in pCDFDuet1-T7 4CL-PDZlig.
(4)pheA基因从BL21(DE3)基因组克隆得到，在pheA基因N端增加NcoI酶切位点，C端增加BamHI酶切位点。利用PCR引物，将pheA基因的976位由A突变为C得到pheA fbr，载体骨架选择pETDuet-1，将pheA fbr基因连接到pCDFDuet-1的NcoI和BamHI位点，得到pCDFDuet1-T7pheA fbr。 (4) The pheA gene was cloned from the BL21 (DE3) genome. The NcoI restriction site was added to the N-terminus of the pheA gene, and the BamHI restriction site was added to the C-terminus. Using PCR primers, the 976 position of the pheA gene was mutated from A to C to obtain pheA fbr . The vector backbone was selected from pETDuet-1, and the pheA fbr gene was connected to the NcoI and BamHI sites of pCDFDuet-1 to obtain pCDFDuet1-T7pheA fbr .
(5)aroG基因从BL21(DE3)基因组克隆得到，在aroG基因N端增加NcoI酶切位点，C端增加BamHI酶切位点。利用PCR引物，将aroG基因的436位由G突变为A得到aroG fbr，载体骨架选择pETDuet-1，将aroG fbr基因连接到pCDFDuet-1的NcoI和BamHI位点，得到pCDFDuet1-T7aroG fbr。 (5) The aroG gene was cloned from the BL21 (DE3) genome. The NcoI restriction site was added to the N-terminus of the aroG gene, and the BamHI restriction site was added to the C-terminus. Using PCR primers, the 436 position of the aroG gene was mutated from G to A to obtain aroG fbr . The vector backbone was selected from pETDuet-1, and the aroG fbr gene was connected to the NcoI and BamHI sites of pCDFDuet-1 to obtain pCDFDuet1-T7aroG fbr .
5、构建携带多基因的质粒5. Construction of plasmids carrying multiple genes
pYH57(pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI)的建立：参见引用文献1：Production of plant-specific flavones baicalein and scutellarein in an engineered E.coli from available phenylalanine and tyrosine。Establishment of pYH57 (pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI): see Citation 1: Production of plant-specific flavones baicalein and scutellarein in an engineered E. coli from available phenylalanine and tyrosine.
pYH66(pETDuet1-T72B1trF6H-T7CPR)的建立：参见引用文献1：Production of plant-specific flavones baicalein and scutellarein in an engineered E.coli from available phenylalanine and tyrosine。Establishment of pYH66 (pETDuet1-T72B1trF6H-T7CPR): see Citation 1: Production of plant-specific flavones baicalein and scutellarein in an engineered E. coli from available phenylalanine and tyrosine.
pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)的建立：利用PCR得到matC基因和matB基因；在matC基因N端加上NcoI酶切位点，C端加上HindIII酶切位点，将matC基因连到pACYCDuet1的NcoI和HindIII位点(该位点前具有载体自带的T7启动子)，获得T7matC。在T7matC基因位点N端加上HindIII位点，C端加上Acc65I位点，利用PCR得到T7matC基因，将T7matC基因连到pACYCDuet1的HindIII和Acc65I位点。在T7ACS基因N端加上Acc65I位点，C端加上NotI位点，利用PCR得到T7ACS基因，将T7ACS基因连接到pACYCDuet1的Acc65I和NotI位点。在T7FabF基因的N端加上NotI位点，在C端加上XbaI位点，PCR得到T7FabF基因，将T7FabF基因连接到pACYCDuet1的NotI和XbaI位点。得到pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF质粒。Establishment of pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF): The matC gene and matB gene were obtained by PCR; the NcoI restriction site was added to the N-terminus of the matC gene, and the HindIII restriction site was added to the C-terminus. Linked to the NcoI and HindIII sites of pACYCDuet1 (the site has the T7 promoter that comes with the vector) to obtain T7matC. A HindIII site was added to the N-terminus of the T7matC gene site, and an Acc65I site was added to the C-terminus. The T7matC gene was obtained by PCR, and the T7matC gene was linked to the HindIII and Acc65I sites of pACYCDuet1. The Acc65I site was added to the N-terminus of the T7ACS gene, and the NotI site was added to the C-terminus. The T7ACS gene was obtained by PCR, and the T7ACS gene was connected to the Acc65I and NotI sites of pACYCDuet1. The NotI site was added to the N-terminus of the T7FabF gene, and the XbaI site was added to the C-terminus. The T7FabF gene was obtained by PCR, and the T7FabF gene was connected to the NotI and XbaI sites of pACYCDuet1. The pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF plasmid was obtained.
pZZ12(pCDFDuet1-T7-CHI-FNSI)的建立：在CHI基因N端加上NcoI位点，C端加上HindIII位点，PCR得到CHI基因，将CHI基因连接到pCDFDuet1的NcoI和HindIII位点。在FNSI基因N端加上HindIII位点，C端加上EcoRI位点，PCR得到FNSI基因，连接到pCDFDuet1的HindIII和EcoRI位点。得到pCDFDuet1-T7-CHI-FNSI。The establishment of pZZ12 (pCDFDuet1-T7-CHI-FNSI): NcoI site was added to the N-terminal of CHI gene, and HindIII site was added to C-terminal, CHI gene was obtained by PCR, and the CHI gene was connected to the NcoI and HindIII sites of pCDFDuet1. A HindIII site was added to the N-terminus of the FNSI gene, and an EcoRI site was added to the C-terminus, and the FNSI gene was obtained by PCR, which was connected to the HindIII and EcoRI sites of pCDFDuet1. pCDFDuet1-T7-CHI-FNSI was obtained.
pZZ22(pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS)的建立：在SH3lig基因N端加上NcoI位点，4CL基因C端加上BamHI位点，以pET28a为载体，利用Over-lap PCR将SH3lig和4CL融合得到融合基因SH3lig-4CL，将SH3lig-4CL连接到pET28a的NcoI和BamHI位点。Over-Lap PCR得到融合基因PAL-ER/K，在PAL-ER/K基因N端加上BamHI位点，SH3基因C端加上EcoRI位点，利用Over-lap PCR将PAL-ER/K基因和SH3基因融合，得到PAL-ER/K-SH3基因，将PAL-ER/K-SH3连接到pET28a的BamHI和EcoRI位点。在CHS基因N端加上EcoRI位点，C端加上SalI位点，将CHS连接到pET28a的EcoRI和SalI位点。得到质粒pZZ22(pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS)。The establishment of pZZ22 (pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS): NcoI site was added to the N-terminal of SH3lig gene, and BamHI site was added to the C-terminal of 4CL gene. Over-lap PCR fused SH3lig and 4CL to obtain the fusion gene SH3lig-4CL, which was connected to the NcoI and BamHI sites of pET28a. The fusion gene PAL-ER/K was obtained by Over-Lap PCR, the BamHI site was added to the N-terminus of the PAL-ER/K gene, and the EcoRI site was added to the C-terminus of the SH3 gene. It was fused with the SH3 gene to obtain the PAL-ER/K-SH3 gene, and the PAL-ER/K-SH3 was linked to the BamHI and EcoRI sites of pET28a. The EcoRI site was added to the N-terminal of the CHS gene, and the SalI site was added to the C-terminal, and the CHS was connected to the EcoRI and SalI sites of pET28a. Plasmid pZZ22 (pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS) was obtained.
pZZ23(pET28a-T7-4CL-PAL-ER/K-SH3-CHS)的建立：在4CL基因N端加上NcoI位点，4CL基因C端加上BamHI位点，以pET28a为载体，将4CL连接到pET28a的NcoI和BamHI位点。在PAL-ER/K基因N端加上BamHI位点，SH3基因C端加上EcoRI位点，利用Over-Lap PCR将PAL-ER/K基因和SH3基因融合，得到PAL-ER/K-SH3基因，将PAL-ER/K-SH3连接到pET28a的BamHI和EcoRI位点。在CHS基因N端加上EcoRI位点，C端加上SalI位点，将CHS连接到pET28a的EcoRI和SalI位点。得到质粒pZZ23(pET28a-T7-4CL-PAL-ER/K-SH3-CHS)。Establishment of pZZ23 (pET28a-T7-4CL-PAL-ER/K-SH3-CHS): NcoI site was added to the N-terminal of 4CL gene, BamHI site was added to the C-terminal of 4CL gene, and pET28a was used as a vector to connect 4CL to the NcoI and BamHI sites of pET28a. The BamHI site was added to the N-terminus of the PAL-ER/K gene, and the EcoRI site was added to the C-terminus of the SH3 gene. Over-Lap PCR was used to fuse the PAL-ER/K gene and the SH3 gene to obtain PAL-ER/K-SH3 gene, ligating PAL-ER/K-SH3 to the BamHI and EcoRI sites of pET28a. The EcoRI site was added to the N-terminal of the CHS gene, and the SalI site was added to the C-terminal, and the CHS was connected to the EcoRI and SalI sites of pET28a. Plasmid pZZ23 (pET28a-T7-4CL-PAL-ER/K-SH3-CHS) was obtained.
pZZ41(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI)的建立：将4(1)中构建的PAL-ER/K-PDZ融合基因N端增加BamHI酶切位点，C端增加EcoRI酶切位点，***到pCDFDuet1中，获得pCDFDuet1-T7PAL-ER/K-PDZ。以pCDFDuet1-T7PAL-ER/K-PDZ为模板，PCR后将基因PAL-ER/K-PDZ构建到pCDFDuet1-T7PDZlig-4CL质粒的BamHI和EcoRI位点，得到pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ。将T7FNSI-T7CHS-T7CHI基因利用HindIII和AvrII双酶切从pYH57质粒上酶切下来，连接到上述质粒的HindIII和AvrII酶切位点得到pZZ41(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI)质粒。Establishment of pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI): the N-terminal of the PAL-ER/K-PDZ fusion gene constructed in 4(1) was added with a BamHI enzyme cleavage site , an EcoRI restriction site was added to the C-terminal and inserted into pCDFDuet1 to obtain pCDFDuet1-T7PAL-ER/K-PDZ. Using pCDFDuet1-T7PAL-ER/K-PDZ as a template, the gene PAL-ER/K-PDZ was constructed into the BamHI and EcoRI sites of the pCDFDuet1-T7PDZlig-4CL plasmid after PCR to obtain pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/ K-PDZ. The T7FNSI-T7CHS-T7CHI gene was digested from the pYH57 plasmid using HindIII and AvrII double digestion, and connected to the HindIII and AvrII restriction sites of the above plasmid to obtain pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ -T7FNSI-T7CHS-T7CHI) plasmid.
pZZ52(pETDuet1-T7pheA fbr-T7aroG fbr)的建立：在pCDFDuet1-T7aroG fbr质粒T7启动子前设计引物，增加BamHI酶切位点，C端增加EcoRI酶切位点，以pETDuet1-T7aroG fbr为模板，PCR得到T7aroG fbr片段，将该片段连接到pCDFDuet1-T7pheA fbr质粒的BamHI和EcoRI位点，得到pZZ52(pETDuet1-T7pheA fbr-T7aroG fbr)质粒。 Establishment of pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ): Design primers in front of the T7 promoter of the pCDFDuet1-T7aroG fbr plasmid, add a BamHI restriction site, and add an EcoRI restriction site at the C-terminal, using pETDuet1-T7aroG fbr as a template, The T7aroG fbr fragment was obtained by PCR, and the fragment was ligated into the BamHI and EcoRI sites of the pCDFDuet1-T7pheA fbr plasmid to obtain the pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ) plasmid.
pZZ55(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr-T7aroG fbr)的建立：以pZZ52(pETDuet1-T7pheA fbr-T7aroG fbr)质粒为模板，T7启动子上游设计引物增加AvrII质粒，在C端增加AvrII酶切位点，PCR克隆得到T7pheA fbr-T7aroG fbr片段，利用一步克隆试剂盒将T7pheA fbr-T7aroG fbr连接到pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)的AvrII位点，得到pZZ55(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr-T7aroG fbr)质粒。 Establishment of pZZ55 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr -T7aroG fbr ): using the pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ) plasmid as a template, design primers upstream of the T7 promoter to increase the AvrII plasmid, which increases at the C-terminus AvrII digestion site, PCR clone to obtain T7pheA fbr- T7aroG fbr fragment, T7pheA fbr -T7aroG fbr was ligated to the AvrII site of pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) using a one-step cloning kit to obtain pZZ55 (pACYCDuet1 -T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr -T7aroG fbr ) plasmid.
质粒详细信息见表1，细胞信息见表2，质粒构建示意图见图1～图2。The detailed information of the plasmid is shown in Table 1, the cell information is shown in Table 2, and the schematic diagram of the plasmid construction is shown in Figures 1 to 2.
表1Table 1
Figure PCTCN2022070316-appb-000003
Figure PCTCN2022070316-appb-000003
表2Table 2
Figure PCTCN2022070316-appb-000004
Figure PCTCN2022070316-appb-000004
Figure PCTCN2022070316-appb-000005
Figure PCTCN2022070316-appb-000005
6、大肠杆菌摇瓶发酵黄芩素和野黄芩素6. Escherichia coli shake flask fermentation of baicalein and scutellarin
发酵菌株的准备：构建好的质粒转化大肠杆菌BL21(DE3)，37℃倒置培养12h后挑阳性克隆于2mL LB抗性的培养基中，37℃，250rpm培养10h制备发酵种子菌，发酵工程菌的详细信息见表2。Preparation of fermentation strains: The constructed plasmid was transformed into Escherichia coli BL21 (DE3), and the positive clones were picked in 2 mL of LB-resistant medium after inverted culture at 37°C for 12 hours, and cultured at 37°C and 250 rpm for 10 hours to prepare fermented seed bacteria, fermentation engineering bacteria See Table 2 for details.
种子液1％转接至10mL含2％葡萄糖的M9培养基中(培养基中加入相应的抗生素)，37℃，250rpm培养至菌株OD＝0.5，加入0.1mM的IPTG及500mg/L的苯丙氨酸，22℃，220rpm发酵3天，取样1mL，菌液超声破碎3次，等体积乙酸乙酯混匀萃取两次，12000rpm，2min离心转移有机相至新管，室温或30℃旋转蒸干后，加200μL甲醇复溶(浓缩5倍)充分混匀，12000rpm，2min后转移上清HPLC检测。1% of the seed solution was transferred to 10 mL of M9 medium containing 2% glucose (corresponding antibiotics were added to the medium), cultured at 37° C., 250 rpm until the strain OD=0.5, and 0.1 mM IPTG and 500 mg/L phenylpropane were added. Amino acid, fermented at 22°C, 220rpm for 3 days, sampled 1mL, sonicated the bacterial liquid for 3 times, mixed with equal volume of ethyl acetate and extracted twice, centrifuged at 12000rpm, 2min, transferred the organic phase to a new tube, and evaporated to dryness at room temperature or 30°C. Then, add 200 μL of methanol to reconstitute (concentrate 5 times) and mix well, transfer the supernatant to HPLC for 2 min at 12000 rpm.
7、HPLC检测7. HPLC detection
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℃。Liquid phase detection conditions: phase A: 0.1% formic acid water, phase B: acetonitrile; separation conditions: 0-20min 20% phase B-55% phase B, 20-22min 55% phase B-100% phase B, 22-27min 100% phase B, 27-35min 100% phase B-20% phase B, 35-40min, 20% phase B; detection wavelength: 340nm, column temperature: 30°C.
色谱柱：Thermo syncronis C18反相柱(250mm×4.6mm，5μm)。Chromatographic column: Thermo syncronis C18 reversed-phase column (250mm×4.6mm, 5μm).
实施例Example
实施例1、蛋白的优化改造Example 1. Optimization of protein
1、PAL蛋白序列的优化改造1. Optimization of PAL protein sequence
来自红景天(Rhodotorula toruloides)的PAL(RtPAL)长度为693aa(GenBank登录号AAA33883.1)，具体序列如下(SEQ ID NO：1)：The PAL (RtPAL) length from Rhodiola (Rhodotorula toruloides) is 693aa (GenBank accession number AAA33883.1), and the specific sequence is as follows (SEQ ID NO: 1):
Figure PCTCN2022070316-appb-000006
Figure PCTCN2022070316-appb-000006
改造1：本发明人针对SEQ ID NO：1进行序列改造，氨基酸，在C端加上5nm rigid linker ER/K连接子，获得改进的PAL突变体PAL-ER/K，具体序列如下(SEQ  ID NO：2)：Transformation 1: The inventors carried out sequence transformation for SEQ ID NO: 1, the amino acid added a 5nm rigid linker ER/K linker at the C end to obtain an improved PAL mutant PAL-ER/K, the specific sequence is as follows (SEQ ID NO: 2):
Figure PCTCN2022070316-appb-000007
Figure PCTCN2022070316-appb-000007
改造2：本发明人针对SEQ ID NO：2进行序列改造，再在C端加上PDZ的氨基酸序列，获得改进的PAL-ER/K突变体PAL-ER/K-PDZ，具体序列如下(SEQ ID NO：3)：Transformation 2: The inventors carried out sequence transformation for SEQ ID NO: 2, and then added the amino acid sequence of PDZ to the C-terminal to obtain an improved PAL-ER/K mutant PAL-ER/K-PDZ, the specific sequence is as follows (SEQ ID NO: 2) ID NO: 3):
Figure PCTCN2022070316-appb-000008
Figure PCTCN2022070316-appb-000008
其中，PAL位于SEQ ID NO：3的第1～693位；ER/K位于SEQ ID NO：3的第694～729位；PDZ位于SEQ ID NO：3的第730～824位。Wherein, PAL is located at positions 1 to 693 of SEQ ID NO: 3; ER/K is located at positions 694 to 729 of SEQ ID NO: 3; PDZ is located at positions 730 to 824 of SEQ ID NO: 3.
改造3：本发明人针对SEQ ID NO：2进行序列改造，再在C端加上SH3的氨基酸序列，获得改进的PAL-ER/K突变体PAL-ER/K-SH3，具体序列如下(SEQ ID NO：4)：Transformation 3: The inventors carried out sequence transformation for SEQ ID NO: 2, and then added the amino acid sequence of SH3 at the C end to obtain an improved PAL-ER/K mutant PAL-ER/K-SH3, the specific sequence is as follows (SEQ ID NO: 2) ID NO: 4):
Figure PCTCN2022070316-appb-000009
Figure PCTCN2022070316-appb-000009
其中，PAL位于SEQ ID NO：4的第1～693位；ER/K位于SEQ ID NO：3的第694～729位；PDZ位于SEQ ID NO：3的第730～786位。Wherein, PAL is located at positions 1 to 693 of SEQ ID NO: 4; ER/K is located at positions 694 to 729 of SEQ ID NO: 3; PDZ is located at positions 730 to 786 of SEQ ID NO: 3.
2、4CL蛋白序列的优化改造2. Optimization of 4CL protein sequence
来自欧芹(Petroselium crispum)的4CL(Pc4CL)长度为544aa(GenBank登录号KF765780.1)，具体序列如下(SEQ ID NO：5)：The length of 4CL (Pc4CL) from parsley (Petroselium crispum) is 544aa (GenBank accession number KF765780.1), and the specific sequence is as follows (SEQ ID NO: 5):
Figure PCTCN2022070316-appb-000010
Figure PCTCN2022070316-appb-000010
改造1：本发明人针对SEQ ID NO：5进行序列改造，去除其中第1位氨基酸，再在N端加上(GGGGS) 2的氨基酸序列，获得改进的4CL突变体(GGGGS) 2-4CL，具体序列如下(SEQ ID NO：6)： Transformation 1: The inventors carried out sequence transformation for SEQ ID NO: 5, removed the first amino acid, and added the amino acid sequence of (GGGGS) 2 to the N-terminus to obtain an improved 4CL mutant (GGGGS) 2-4CL , The specific sequence is as follows (SEQ ID NO: 6):
Figure PCTCN2022070316-appb-000011
Figure PCTCN2022070316-appb-000011
改造2：本发明人针对SEQ ID NO：6进行序列改造，再在N端加上PDZlig的氨基酸序列，再N端加上M氨基酸，获得改进的(GGGGS) 2-4CL突变体PDZlig-(GGGGS) 2-4CL，具体序列如下(SEQ ID NO：7)： Modification 2: The inventors carried out sequence modification according to SEQ ID NO: 6, then added the amino acid sequence of PDZlig to the N-terminus, and then added the M amino acid to the N-terminus to obtain an improved (GGGGS) 2-4CL mutant PDZlig-(GGGGS ) 2-4CL , the specific sequence is as follows (SEQ ID NO: 7):
Figure PCTCN2022070316-appb-000012
Figure PCTCN2022070316-appb-000012
其中，PDZlig位于SEQ ID NO：7的第1～8位；(GGGGS) 2位于SEQ ID NO：7的第9～18位；4CL位于SEQ ID NO：7的第19～561位。 Wherein, PDZlig is located at positions 1-8 of SEQ ID NO: 7; (GGGGS) 2 is located at positions 9-18 of SEQ ID NO: 7; 4CL is located at positions 19-561 of SEQ ID NO: 7.
改造3：本发明人针对SEQ ID NO：5进行序列改造，在C端加上(GGGGS) 2的氨基酸序列，获得改进的4CL突变体4CL-(GGGGS) 2，具体序列如下(SEQ ID NO：8)： Modification 3: The inventors carried out sequence modification for SEQ ID NO: 5, and added the amino acid sequence of (GGGGS) 2 to the C-terminus to obtain an improved 4CL mutant 4CL-(GGGGS) 2 , the specific sequence is as follows (SEQ ID NO: 8):
Figure PCTCN2022070316-appb-000013
Figure PCTCN2022070316-appb-000013
Figure PCTCN2022070316-appb-000014
Figure PCTCN2022070316-appb-000014
改造4：本发明人针对SEQ ID NO：8进行序列改造，在C端加上PDZlig的氨基酸序列，获得改进的4CL突变体4CL-(GGGGS) 2-PDZlig，具体序列如下(SEQ ID NO：9)： Transformation 4: The inventors carried out sequence transformation for SEQ ID NO: 8, and added the amino acid sequence of PDZlig to the C-terminal to obtain an improved 4CL mutant 4CL-(GGGGS) 2 -PDZlig, the specific sequence is as follows (SEQ ID NO: 9 ):
Figure PCTCN2022070316-appb-000015
Figure PCTCN2022070316-appb-000015
其中，4CL位于SEQ ID NO：9的第1～544位；(GGGGS) 2位于SEQ ID NO：10的第545～554位；PDZlig位于SEQ ID NO：9的第555～561位。 Wherein, 4CL is located at positions 1-544 of SEQ ID NO: 9; (GGGGS) 2 is located at positions 545-554 of SEQ ID NO: 10; PDZlig is located at positions 555-561 of SEQ ID NO: 9.
改造5：本发明人针对SEQ ID NO：6进行序列改造，再在N端加上SH3lig的氨基酸序列，再N端加上M氨基酸，获得改进的(GGGGS) 2-4CL突变体SH3lig-(GGGGS) 2-4CL(简称SH3lig-4CL)，具体序列如下(SEQ ID NO：10)： Transformation 5: The inventors carried out sequence transformation according to SEQ ID NO: 6, then added the amino acid sequence of SH3lig to the N-terminus, and then added the M amino acid to the N-terminus to obtain an improved (GGGGS) 2-4CL mutant SH3lig-(GGGGS ) 2-4CL (SH3lig-4CL for short), the specific sequence is as follows (SEQ ID NO: 10):
Figure PCTCN2022070316-appb-000016
Figure PCTCN2022070316-appb-000016
其中，SH3lig位于SEQ ID NO：10的第1～12位；(GGGGS) 2位于SEQ ID NO：10的第13～22位；4CL位于SEQ ID NO：10的第23～565位。 Wherein, SH3lig is located at positions 1-12 of SEQ ID NO: 10; (GGGGS) 2 is located at positions 13-22 of SEQ ID NO: 10; 4CL is located at positions 23-565 of SEQ ID NO: 10.
改造6：本发明人针对SEQ ID NO：8进行序列改造，在C端加上SH3lig的氨基酸序列，获得改进的4CL突变体4CL-(GGGGS) 2-SH3lig，具体序列如下(SEQ ID NO：11)： Transformation 6: The inventors carried out sequence transformation according to SEQ ID NO: 8, and added the amino acid sequence of SH3lig to the C-terminal to obtain an improved 4CL mutant 4CL-(GGGGS) 2 -SH3lig, the specific sequence is as follows (SEQ ID NO: 11 ):
Figure PCTCN2022070316-appb-000017
Figure PCTCN2022070316-appb-000017
Figure PCTCN2022070316-appb-000018
Figure PCTCN2022070316-appb-000018
其中，4CL位于SEQ ID NO：11的第1～544位；(GGGGS) 2位于SEQ ID NO：10的第545～554位；SH3lig位于SEQ ID NO：10的第555～565位。 Wherein, 4CL is located at positions 1-544 of SEQ ID NO: 11; (GGGGS) 2 is located at positions 545-554 of SEQ ID NO: 10; SH3lig is located at positions 555-565 of SEQ ID NO: 10.
实施例2、发酵工程菌检测白杨素 Embodiment 2, fermentation engineering bacteria detect chrysin
1、非自组装菌株(JH-0)1. Non-self-assembling strain (JH-0)
将pYH57(pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI)质粒转化入BL21(DE3)，得到工程菌DN-1，用于以苯丙氨酸为前体，发酵白杨素。The pYH57 (pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI) plasmid was transformed into BL21 (DE3) to obtain an engineered strain DN-1, which was used to ferment chrysin using phenylalanine as a precursor.
2、4CL-改造2、PAL-改造2菌株(DN-0)2. 4CL-transformation 2, PAL-transformation 2 strain (DN-0)
将pZZ41(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI)质粒转化入BL21(DE3)，得到自组装工程菌DN-0，用于以苯丙氨酸为前体，发酵白杨素。The pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid was transformed into BL21 (DE3) to obtain self-assembly engineering bacteria DN-0, which was used to use phenylalanine as a precursor , fermented chrysin.
发酵方法如下：菌株LB固体培养基(壮观霉素80μg/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)，转接过夜培养的菌液到新的10mL M9Y液体抗性培养基中37℃，250r/min培养至OD600＝0.5～0.6，水浴降温至16℃左右，然后加入诱导剂IPTG至终浓度0.2mM，加入终浓度为500mg/L经灭菌的苯丙氨酸并转至22℃低温诱导培养，在摇床转速220r/min条件下继续培养72h。取样检测黄芩素产量。The fermentation method was as follows: the strain LB solid medium (spectinomycin 80 μg/mL) was cultured at 37°C overnight. Pick a single clone into 2mL LB liquid medium (spectinomycin 80μg/mL), transfer the bacterial liquid cultured overnight to a new 10mL M9Y liquid resistance medium at 37℃, 250r/min to OD600=0.5～0.6 , the water bath was cooled to about 16 ℃, then the inducer IPTG was added to a final concentration of 0.2 mM, and the final concentration of 500 mg/L of sterilized phenylalanine was added, and then transferred to 22 ℃ for low temperature induction culture, and the shaking speed was 220 r/min. Continue to culture for 72h under conditions. Sampling to detect baicalein yield.
结果显示，与非自组装的菌株JH-0相比，自组装菌株DN-0的白杨素产量，提高到1.6倍(图10)。The results showed that, compared with the non-self-assembled strain JH-0, the chrysin production of the self-assembled strain DN-0 was increased by 1.6 times (Fig. 10).
实施例3、发酵工程菌检测黄芩素 Embodiment 3, fermentation engineering bacteria detect baicalein
本实施例中，以苯丙氨酸为前体，进行发酵，合成途径如图8。In this example, phenylalanine is used as a precursor for fermentation, and the synthesis route is shown in Figure 8.
1、非自组装菌株(DN-1)1. Non-self-assembling strain (DN-1)
将pYH57(pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI)质粒，pYH66(pETDuet1-T72B1-trF6H-T7CPR)质粒，pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)质粒共同转化入BL21(DE3)，得到工程菌DN-1，用于以苯丙氨酸为前体，发酵黄芩素。The pYH57 (pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI) plasmid, pYH66 (pETDuet1-T72B1-trF6H-T7CPR) plasmid, pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) plasmid were co-transformed into BL21 (DE3), The engineering bacteria DN-1 is obtained, which is used for fermenting baicalein with phenylalanine as a precursor.
2、4CL-改造2、PAL-改造2菌株(DN-2)2. 4CL-transformation 2, PAL-transformation 2 strain (DN-2)
将pZZ41(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI)质粒，pYH66(pETDuet1-T72B1-trF6H-T7CPR)质粒，pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)质粒共同转化入BL21(DE3)，得到自组装工程菌DN-2，用于以苯丙氨酸为前体发酵黄芩素。The pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid, pYH66 (pETDuet1-T72B1-trF6H-T7CPR) plasmid, pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) plasmid Co-transformed into BL21 (DE3) to obtain self-assembly engineering bacteria DN-2, which was used to ferment baicalein with phenylalanine as a precursor.
3、4CL无改造、PAL-改造3菌株(DN-3，作为对照菌株)3. 4CL unmodified, PAL-modified 3 strains (DN-3, as a control strain)
将pZZ23(pET28a-T7-4CL-PAL-ER/K-SH3-CHS)质粒，pZZ12(pCDFDuet1-CHI-FNSI)质粒，质粒共同转化入BL21(DE3)，得到对照工程菌DN-3，以苯丙氨酸为 前体，用于以苯丙氨酸为前体发酵黄芩素。The pZZ23 (pET28a-T7-4CL-PAL-ER/K-SH3-CHS) plasmid, the pZZ12 (pCDFDuet1-CHI-FNSI) plasmid, and the plasmids were co-transformed into BL21 (DE3) to obtain the control engineering bacteria DN-3, which was treated with benzene. Alanine is used as a precursor to ferment baicalein with phenylalanine as a precursor.
4、4CL-改造5、PAL-改造3菌株(DN-4)4. 4CL-transformation 5, PAL-transformation 3 strain (DN-4)
将pZZ22(pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS)质粒，pZZ12(pCDFDuet1-T7-CHI-FNSI)质粒，质粒共同转化入BL21(DE3)，得到自组装工程菌DN-4，用于以苯丙氨酸为前体发酵黄芩素。The pZZ22 (pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS) plasmid, the pZZ12 (pCDFDuet1-T7-CHI-FNSI) plasmid, and the plasmids were co-transformed into BL21 (DE3) to obtain the self-assembly engineering bacteria DN -4, for the fermentation of baicalein with phenylalanine as the precursor.
上述四种菌株进行发酵，发酵方法如下：菌株LB固体培养基(壮观霉素80μg/mL，氨苄青霉素100μg/mL，氯霉素34μg/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，氯霉素34μg/mL)，转接过夜培养的菌液到新的10mL M9Y液体抗性培养基中37℃，250r/min培养至OD600＝0.5～0.6，水浴降温至16℃左右，然后加入诱导剂IPTG至终浓度0.2mM，加入终浓度为500mg/L经灭菌的苯丙氨酸并转至22℃低温诱导培养，在摇床转速220r/min条件下继续培养72h。取样检测黄芩素产量。The above four strains were fermented, and the fermentation method was as follows: strain LB solid medium (spectinomycin 80 μg/mL, ampicillin 100 μg/mL, chloramphenicol 34 μg/mL) was cultured overnight at 37°C. Pick a single clone into 2mL LB liquid medium (spectinomycin 80μg/mL, ampicillin 100μg/mL, chloramphenicol 34μg/mL), transfer the overnight culture into a new 10mL M9Y liquid resistance medium 37 ℃, 250r/min culture to OD600 = 0.5 ~ 0.6, water bath cooling to about 16 ℃, then add inducer IPTG to a final concentration of 0.2mM, add a final concentration of 500mg/L sterilized phenylalanine and transfer to Induced culture at 22°C and continued to culture for 72h at a shaker speed of 220r/min. Sampling to detect baicalein yield.
结果显示，与非自组装的菌株DN-1相比，自组装菌株DN-2的黄芩素产量，提高6.6倍(图3)。HPLC检测图谱如图4。The results showed that compared with the non-self-assembled strain DN-1, the baicalein production of the self-assembled strain DN-2 was increased by 6.6 times (Fig. 3). The HPLC detection spectrum is shown in Figure 4.
结果显示，与对照菌株DN-3相比，自组装菌株DN-4的黄芩素产量，提高2.5倍(图5)。The results showed that compared with the control strain DN-3, the baicalein production of the self-assembled strain DN-4 was increased by 2.5 times (Fig. 5).
根据上述结果，说明本发明的改造方案能够显著提高黄芩素产量，如表3。According to the above results, it is shown that the transformation scheme of the present invention can significantly improve the yield of baicalein, as shown in Table 3.
表3table 3
Figure PCTCN2022070316-appb-000019
Figure PCTCN2022070316-appb-000019
对于多种改造方案进行比较，本发明人优选PAL改造2、4CL改造2或PAL改造3、4CL改造5。For the comparison of various modification schemes, the inventors preferred PAL modification 2, 4CL modification 2 or PAL modification 3, 4CL modification 5.
实施例4、发酵工程菌检测野黄芩素 Embodiment 4, fermentation engineering bacteria detect scutellarin
本实施例中，以酪氨酸为前体，进行发酵。In this example, tyrosine was used as a precursor for fermentation.
如前一实施例获得非组装的工程菌DN-1，用于以酪氨酸为前体发酵黄芩素。As in the previous example, a non-assembled engineered bacterium DN-1 was obtained, which was used to ferment baicalein with tyrosine as a precursor.
如前一实施例得到自组装工程菌DN-2，用于以酪氨酸为前体发酵黄芩素。As in the previous example, the self-assembly engineering bacteria DN-2 was obtained, which was used to ferment baicalein with tyrosine as a precursor.
上述两种菌株，在LB固体培养基(壮观霉素80μg/mL，氨苄青霉素100μg/mL，氯霉素34μg/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，氯霉素34μg/mL)，转接过夜培养的菌液到新的10mL M9Y液体抗性培养基中37℃，250r/min培养至OD600＝0.5-0.6，水浴降温至16℃左右，然后加入诱导剂IPTG至终浓度0.2mM，加入终浓度为500mg/L经灭菌的酪氨酸并转至22℃低温诱导培养，在摇床转速220r/min条件下继续培养72h。The above two strains were cultured in LB solid medium (spectinomycin 80 μg/mL, ampicillin 100 μg/mL, chloramphenicol 34 μg/mL) overnight at 37°C. Pick a single clone into 2mL LB liquid medium (spectinomycin 80μg/mL, ampicillin 100μg/mL, chloramphenicol 34μg/mL), transfer the overnight culture into a new 10mL M9Y liquid resistance medium 37 ℃, 250r/min culture to OD600=0.5-0.6, water bath cooling to about 16 ℃, then add inducer IPTG to the final concentration of 0.2mM, add the final concentration of 500mg/L sterilized tyrosine and transfer to 22 Induced culture at ℃ low temperature, and continued to culture for 72 h at a shaking speed of 220 r/min.
取样检测生长情况，野黄芩素产量。与菌株DN-1相比，自组装菌株DN-2的野 黄芩素产量，提高1.43倍(图6)。Sampling was used to detect growth and scutellarin yield. Compared with strain DN-1, the scutellarin production of self-assembled strain DN-2 was increased by 1.43 times (Fig. 6).
实施例5、从葡萄糖合成黄芩素Example 5. Synthesis of baicalein from glucose
本实施例中，以葡萄糖为前体，进行发酵，合成途径如图9。In this example, glucose is used as a precursor for fermentation, and the synthesis route is shown in Figure 9.
1、非自组装菌株(DN-5)1. Non-self-assembling strain (DN-5)
将pYH57(pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI)质粒，pYH66(pETDuet1-T72B1trF6H-T7CPR)质粒，pZZ55(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr-T7aroG fbr)质粒共同转化入BL21(DE3)，得到非自组装工程菌DN-5，用于从葡萄糖合成黄芩素。 The pYH57 (pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI) plasmid, pYH66 (pETDuet1-T72B1trF6H-T7CPR) plasmid, pZZ55 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr -T7aroG fbr ) plasmid were co-transformed into BL21 (DE3), the non-self-assembling engineering bacteria DN-5 was obtained for the synthesis of baicalein from glucose.
2、4CL改造2，PAL改造2菌株(DN-5)2. 4CL transformation 2, PAL transformation 2 strain (DN-5)
将pZZ41(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI)质粒，pYH66(pETDuet1-T72B1trF6H-T7CPR)质粒，pZZ55(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr-T7aroG fbr)质粒共同转化入BL21(DE3)，得到采用PDZ和PDZlig互作方案的自组装工程菌DN-6，用于从葡萄糖合成黄芩素。 The pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid, pYH66 (pETDuet1-T72B1trF6H-T7CPR) plasmid, pZZ55 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr- T7aroG fbr ) plasmid was co-transformed into BL21 (DE3) to obtain a self-assembled engineering bacterium DN-6 using the interaction scheme of PDZ and PDZlig, which was used to synthesize baicalein from glucose.
上述建立的两种菌株，LB固体培养基(壮观霉素80μg/mL，氨苄青霉素100μg/mL，氯霉素34μg/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，氯霉素34μg/mL)，转接过夜培养的菌液到新的10mL M9Y液体抗性培养基中37℃，250r/min培养至OD600＝0.5-0.6，水浴降温至16℃左右，然后加入诱导剂IPTG至终浓度0.2mM，并转至22℃低温诱导培养，在摇床转速220r/min条件下继续培养72h。The two strains established above were cultured on LB solid medium (spectinomycin 80 μg/mL, ampicillin 100 μg/mL, chloramphenicol 34 μg/mL) overnight at 37°C. Pick a single clone into 2mL LB liquid medium (spectinomycin 80μg/mL, ampicillin 100μg/mL, chloramphenicol 34μg/mL), transfer the overnight culture into a new 10mL M9Y liquid resistance medium 37°C, 250r/min culture to OD600=0.5-0.6, cooling down to about 16°C in water bath, then adding inducer IPTG to a final concentration of 0.2mM, and transferring to 22°C low temperature induction culture, under the condition of shaking table rotation speed 220r/min Continue to cultivate for 72h.
结果显示，非自组装工程菌DN-5能够从葡萄糖中合成黄芩素，自组装工程菌株DN-6能够从葡萄糖中合成黄芩素，自组装工程菌株DN-6比非自组装工程菌株DN-5提高了111.7％(图7)。The results show that the non-self-assembly engineering strain DN-5 can synthesize baicalein from glucose, and the self-assembly engineering strain DN-6 can synthesize baicalein from glucose. An increase of 111.7% (Figure 7).
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。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (20)

  1. 一种用于合成黄芩素、野黄芩素类化合物的原核细胞，其包括外源的下组酶的编码基因：黄酮6-羟化酶，细胞色素P450氧化还原酶，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；且所述酶被表达后，苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶构成复合体。A prokaryotic cell for synthesizing baicalein and scutellarin compounds, which comprises exogenous encoding genes of the following group of enzymes: flavonoid 6-hydroxylase, cytochrome P450 oxidoreductase, phenylalanine ammonia lyase , 4-coumaric acid coenzyme A ligase, chalcone synthase, chalcone isomerase and flavone synthase I; and after the enzymes are expressed, phenylalanine ammonia lyase and 4-coumaric acid Coenzyme A ligase forms a complex.
  2. 一种用于合成白杨素类化合物的原核细胞，其包括外源的下组酶的编码基因：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；且所述酶被表达后，苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶构成复合体。A prokaryotic cell for synthesizing chrysin compounds, which comprises the coding genes of exogenous lower group enzymes: phenylalanine ammonia lyase, 4-coumaric acid coenzyme A ligase, chalcone synthase, chalcone synthase, ketone isomerase and flavonoid synthase I; and after the enzymes are expressed, phenylalanine ammonia lyase and 4-coumarate coenzyme A ligase form a complex.
  3. 如权利要求1或2所述的原核细胞，其特征在于，所述的苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶的复合体包括：The prokaryotic cell of claim 1 or 2, wherein the complex of the phenylalanine ammonia lyase and 4-coumaric acid coenzyme A ligase comprises:
    苯丙氨酸解氨酶和4-香豆酸辅酶A通过蛋白-蛋白相互作用结构域及其配体的结合而靠近，获得复合体；或Phenylalanine ammonia lyase and 4-coumarate-CoA come into close proximity through the binding of the protein-protein interaction domain and its ligand to obtain a complex; or
    苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶直接连接或通过连接子连接、获得融合蛋白形式的复合体。Phenylalanine ammonia lyase and 4-coumarate coenzyme A ligase are directly connected or connected through a linker to obtain a complex in the form of a fusion protein.
  4. 如权利要求3所述的原核细胞，其特征在于，所述蛋白-蛋白相互作用结构域包括选自下组的结构域：PDZ结构域，SH3结构域，WW结构域，LIM结构域，DD结构域，PH结构域，EH结构域，GBD结构域。The prokaryotic cell of claim 3, wherein the protein-protein interaction domain comprises a domain selected from the group consisting of PDZ domain, SH3 domain, WW domain, LIM domain, DD structure domain, PH domain, EH domain, GBD domain.
  5. 如权利要求3所述的原核细胞，其特征在于，所述蛋白-蛋白相互作用结构域包括PDZ结构域，其配体为PDZ ligand；所述苯丙氨酸解氨酶和4-香豆酸辅酶A分别与所述PDZ结构域及其配体融合；较佳地，所述苯丙氨酸解氨酶与PDZ融合、所述4-香豆酸辅酶A与PDZ ligand融合；更佳地，所述苯丙氨酸解氨酶与PDZ融合时还包括以ER/K连接子连接，所述4-香豆酸辅酶A与PDZ ligand融合时还包括以(GGGGS)2连接子连接。The prokaryotic cell of claim 3, wherein the protein-protein interaction domain comprises a PDZ domain whose ligand is PDZ ligand; the phenylalanine ammonia lyase and 4-coumaric acid Coenzyme A is fused with the PDZ structural domain and its ligand respectively; preferably, the phenylalanine ammonia lyase is fused with PDZ, and the 4-coumaric acid coenzyme A is fused with PDZ ligand; more preferably, When the phenylalanine ammonia lyase is fused with PDZ, it also includes connecting with an ER/K linker, and when the 4-coumaric acid coenzyme A is fused with PDZ ligand, it also includes connecting with a (GGGGS)2 linker.
  6. 如权利要求3所述的原核细胞，其特征在于，所述蛋白-蛋白相互作用结构域包括SH3结构域，其配体为SH3 ligand；所述苯丙氨酸解氨酶和4-香豆酸辅酶A分别与所述SH3结构域及其配体融合；较佳地，所述苯丙氨酸解氨酶与SH3融合、所述4-香豆酸辅酶A与SH3 ligand融合；更佳地，所述苯丙氨酸解氨酶与SH3融合时还包括以ER/K连接子连接，所述4-香豆酸辅酶A与SH3 ligand融合时还包括以(GGGGS)2连接子连接。The prokaryotic cell of claim 3, wherein the protein-protein interaction domain comprises an SH3 domain whose ligand is SH3 ligand; the phenylalanine ammonia lyase and 4-coumaric acid Coenzyme A is respectively fused with the SH3 structural domain and its ligand; preferably, the phenylalanine ammonia lyase is fused with SH3, and the 4-coumaric acid coenzyme A is fused with SH3 ligand; more preferably, When the phenylalanine ammonia lyase is fused with SH3, it also includes connecting with an ER/K linker, and when the 4-coumaric acid coenzyme A is fused with SH3 ligand, it also includes connecting with a (GGGGS)2 linker.
  7. 如权利要求5或6所述的原核细胞，其特征在于，所述苯丙氨酸解氨酶与PDZ融合时，所述苯丙氨酸解氨酶位于N端，所述PDZ位于C端；或The prokaryotic cell according to claim 5 or 6, wherein when the phenylalanine ammonia lyase is fused with PDZ, the phenylalanine ammonia lyase is located at the N-terminal, and the PDZ is located at the C-terminal; or
    所述4-香豆酸辅酶A与PDZ ligand融合时，所述PDZ ligand位于N端，所述4-香豆酸辅酶A位于C端；或When the 4-coumaric acid coenzyme A is fused to the PDZ ligand, the PDZ ligand is located at the N-terminal, and the 4-coumaric acid-CoA is located at the C-terminal; or
    所述苯丙氨酸解氨酶与SH3融合时；所述苯丙氨酸解氨酶位于N端，所述SH3位于C端；或When the phenylalanine ammonia lyase is fused with SH3; the phenylalanine ammonia lyase is located at the N-terminal, and the SH3 is located at the C-terminal; or
    所述4-香豆酸辅酶A与SH3 ligand融合时，所述SH3 ligand位于N端，所述4-香豆酸辅酶A位于C端。When the 4-coumaric acid coenzyme A is fused with the SH3 ligand, the SH3 ligand is located at the N-terminus, and the 4-coumaric acid coenzyme A is located at the C-terminus.
  8. 如权利要求1所述的原核细胞，其特征在于，所述细胞中还包括外源的促进丙二酰CoA生成的酶的编码基因；较佳地，包括matC，matB，ACS，FabF。The prokaryotic cell according to claim 1, characterized in that, the cell further comprises a gene encoding an exogenous enzyme that promotes the production of malonyl CoA; preferably, it comprises matC, matB, ACS, and FabF.
  9. 如权利要求1所述的原核细胞，其特征在于，所述的原核细胞为大肠杆菌细胞。The prokaryotic cell of claim 1, wherein the prokaryotic cell is an Escherichia coli cell.
  10. 如权利要求1～9任一所述的原核细胞，其特征在于，所述细胞中还包括外源的促进苯丙氨酸合成的酶的编码基因；较佳地，包括：aroG，pheA；更佳地，所述pheA为第976位由A突变为C的基因；更佳地，所述aroG为第436位由G突变为A的基因。The prokaryotic cell according to any one of claims 1 to 9, wherein the cell further comprises an exogenous gene encoding an enzyme that promotes phenylalanine synthesis; preferably, it comprises: aroG, pheA; more Preferably, the pheA is a gene whose position 976 is mutated from A to C; more preferably, the aroG is a gene whose position 436 is mutated from G to A.
  11. 权利要求1、3～10任一所述的原核细胞的应用，用于合成黄芩素或野黄芩素类化合物。The application of the prokaryotic cells according to any one of claims 1, 3 to 10, for synthesizing baicalein or scutellarin compounds.
  12. 权利要求2、3～10任一所述的原核细胞的应用，用于合成白杨素类化合物。The application of the prokaryotic cell according to any one of claims 2, 3 to 10, for synthesizing chrysin compounds.
  13. 一种合成黄芩素或野黄芩素类化合物的方法，包括：提供权利要求1、3～10任一所述的原核细胞，以式(I)为底物，合成黄芩素或野黄芩素类化合物；A method for synthesizing baicalein or scutellarin compounds, comprising: providing the prokaryotic cells according to any one of claims 1, 3 to 10, and using formula (I) as a substrate to synthesize baicalein or scutellarin compounds ;
    Figure PCTCN2022070316-appb-100001
    Figure PCTCN2022070316-appb-100001
    其中，R包括H或OH。wherein R includes H or OH.
  14. 一种合成白杨素类化合物的方法，包括：提供权利要求2、3～10任一所述的原核细胞，以式(I)为底物，合成白杨素类化合物；A method for synthesizing chrysin compounds, comprising: providing the prokaryotic cell according to any one of claims 2, 3 to 10, and using formula (I) as a substrate to synthesize chrysin compounds;
    Figure PCTCN2022070316-appb-100002
    Figure PCTCN2022070316-appb-100002
    其中，R包括H或OH。wherein R includes H or OH.
  15. 一种合成黄芩素类化合物或白杨素类化合物的方法，包括：提供权利要求10所述的原核细胞，以葡萄糖为底物，合成黄芩素类化合物或白杨素类化合物。A method for synthesizing baicalein compounds or chrysin compounds, comprising: providing the prokaryotic cell according to claim 10, and using glucose as a substrate to synthesize baicalein compounds or chrysin compounds.
  16. 如权利要求13～15任一所述的方法，其特征在于，在引入细胞时，The method according to any one of claims 13 to 15, wherein when introducing into cells,
    所述PDZligand、4-香豆酸辅酶A连接酶、苯丙氨酸解氨酶、ER/K、PDZ、黄酮合成酶I、查尔酮合成酶、查尔酮异构酶的编码基因位于一个构建体中；The coding genes of described PDZligand, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, PDZ, flavonoid synthase I, chalcone synthase, chalcone isomerase are located in a in the construct;
    所述黄酮6-羟化酶，细胞色素P450氧化还原酶的编码基因位于一个构建体中，较佳地还包括2B1基因；The flavonoid 6-hydroxylase, the coding gene of cytochrome P450 oxidoreductase is located in a construct, and preferably also includes the 2B1 gene;
    所述matC，matB，ACS，FabF的编码基因位于一个构建体中；The encoding genes of described matC, matB, ACS, FabF are located in a construct;
    所述SH3lig，4-香豆酸辅酶A连接酶，苯丙氨酸解氨酶，ER/K，SH3，查尔酮合 成酶的编码基因位于一个构建体中；Described SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, the coding gene of chalcone synthase is located in a construct;
    所述查尔酮异构酶，黄酮合成酶I的编码基因位于一个构建体中；或The chalcone isomerase, the gene encoding flavonoid synthase I is in a construct; or
    所述matC、matB、ACS、FabF的编码基因，第976位由A突变为C的pheA基因，第436位由G突变为A的aroG基因位于一个构建体中。The encoding genes of matC, matB, ACS, and FabF, the pheA gene mutated from A to C at position 976, and the aroG gene mutated from G to A at position 436 are located in one construct.
  17. 一种试剂盒，其包括权利要求1～10任一所述的重组的宿主细胞。A kit comprising the recombinant host cell of any one of claims 1-10.
  18. 一种试剂盒，其包括：包含PDZligand、4-香豆酸辅酶A连接酶、苯丙氨酸解氨酶、ER/K、PDZ、黄酮合成酶I、查尔酮合成酶、查尔酮异构酶的编码基因的构建体；A kit comprising: comprising PDZligand, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, PDZ, flavonoid synthase I, chalcone synthase, chalcone iso The construct of the gene encoding the enzyme;
    包含黄酮6-羟化酶，细胞色素P450氧化还原酶的编码基因的构建体；较佳地所述构建体还包含2B1基因；A construct comprising a gene encoding flavone 6-hydroxylase and cytochrome P450 oxidoreductase; preferably, the construct further comprises a 2B1 gene;
    包含matC，matB，ACS，FabF的编码基因的构建体；Constructs encoding genes comprising matC, matB, ACS, FabF;
    包含SH3lig，4-香豆酸辅酶A连接酶，苯丙氨酸解氨酶，ER/K，SH3，查尔酮合成酶的编码基因的构建体；A construct comprising genes encoding SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, chalcone synthase;
    包含查尔酮异构酶，黄酮合成酶I的编码基因的构建体；A construct comprising a gene encoding chalcone isomerase, flavonoid synthase I;
    包含matC、matB、ACS、FabF的编码基因，第976位由A突变为C的pheA基因，第436位由G突变为A的aroG基因的构建体。Constructs comprising the coding genes of matC, matB, ACS, FabF, the pheA gene mutated from A to C at position 976, and the aroG gene mutated from G to A at position 436.
  19. 一种试剂盒，其包括：包含PDZligand、4-香豆酸辅酶A连接酶、苯丙氨酸解氨酶、ER/K、PDZ、黄酮合成酶I、查尔酮合成酶、查尔酮异构酶的编码基因的构建体；A kit comprising: comprising PDZligand, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, PDZ, flavonoid synthase I, chalcone synthase, chalcone iso The construct of the gene encoding the enzyme;
    包含matC，matB，ACS，FabF的编码基因的构建体；Constructs encoding genes comprising matC, matB, ACS, FabF;
    包含SH3lig，4-香豆酸辅酶A连接酶，苯丙氨酸解氨酶，ER/K，SH3，查尔酮合成酶的编码基因的构建体；A construct comprising genes encoding SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, chalcone synthase;
    包含查尔酮异构酶，黄酮合成酶I的编码基因的构建体；A construct comprising a gene encoding chalcone isomerase, flavonoid synthase I;
    包含matC、matB、ACS、FabF的编码基因，第976位由A突变为C的pheA基因，第436位由G突变为A的aroG基因的构建体。Constructs comprising the coding genes of matC, matB, ACS, and FabF, the pheA gene mutated from A to C at position 976, and the aroG gene mutated from G to A at position 436.
  20. 如权利要求17～19任一所述的试剂盒，其特征在于，其中还包括：葡萄糖；或，式(I)底物；The kit according to any one of claims 17 to 19, further comprising: glucose; or, a substrate of formula (I);
    Figure PCTCN2022070316-appb-100003
    Figure PCTCN2022070316-appb-100003
    其中，R包括H或OH。wherein R includes H or OH.
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