Regulation method and application of heterologous synthesis of flavonoids

Abstract

The present application provides a method for regulating the production of heterologous flavonoids and its application. The present application realizes the significant increase of flavonoids production by regulating the genes in the production cell, which includes up-regulating the transmembrane protein thiocyanate Ygap of E. coli or down-regulating one or more target genes. The present application also discloses the optimized host cell and its application.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and pharmaceutical technology, specifically to the regulation methods and applications of heterologous synthesis of flavonoids. Background Technology

[0002] Baicalein and baicalein are flavonoids, primarily found in the traditional Chinese medicine Scutellaria baicalensis. These two active flavonoids accumulate in small amounts only in the roots of Scutellaria baicalensis and related medicinal plants. Both baicalein and baicalein are synthesized via the flavonoid biosynthesis pathway. Baicalein and baicalein possess important physiological activities such as antioxidant, antitumor, antibacterial, and cardioprotective effects. Recently, baicalein has been reported in vitro as an inhibitor of SARS-CoV-2 3Clpro, demonstrating the significant potential of traditional Chinese medicine.

[0003] Currently, the main sources of flavonoids are plant extraction and chemical synthesis. However, due to the use of toxic chemicals and extreme reaction conditions, plant extraction or chemical synthesis cannot provide a green route for large-scale production. Therefore, research on microbial synthesis of flavonoids has been extensive. Introducing complex heterogeneous pathways from plants, enzyme imbalances, and the accumulation of intermediate metabolites often result in low product titers. To address these issues, a multivariate modular approach has been employed to synthesize flavonoids by adjusting promoter strength and plasmid copy number. However, this is time-consuming and always requires substantial effort. Previous work reported the synthesis of baicalin and scutellarin in engineered yeast and *E. coli*, but the yields of baicalin and scutellarin remain at very low levels.

[0004] Therefore, there is an urgent need in the field to optimize microbial strains capable of efficiently synthesizing baicalin and baicalin or similar compounds heterologously. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for regulating the heterologous synthesis of flavonoids.

[0006] In a first aspect of the present invention, a method for synthesizing flavonoids is provided, the method comprising:

[0007] (1) Provide recombinant prokaryotic cells containing the encoding genes of the following exogenous enzymes: phenylalanine ammonia-lyase (PAL), 4-coumarate-co-A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonoid synthase I (FNSI); and upregulate Escherichia coli transmembrane protein thiocyanate Ygap in the prokaryotic cells; or downregulate target genes or combinations of target genes selected from the following groups in the prokaryotic cells: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, leuA;

[0008] (2) Using formula (I) as a substrate, synthesize flavonoids from the prokaryotic cells described above, wherein the flavonoids are apigenin compounds (such as apigenin or apigenin);

[0009]

[0010] R includes H or OH.

[0011] In one or more embodiments, (1) the prokaryotic cell further includes the encoding genes of the following exogenous enzymes: flavonoid 6-hydroxylase (F6H) and cytochrome P450 oxidoreductase (CPR); (2) the flavonoid compound is a baicalin compound (such as baicalin or scutellarin).

[0012] In one or more embodiments, the downregulation of the target gene includes: downregulating the activity of the target gene, reducing the effective duration of the target gene, downregulating the expression or stability of the target gene, etc.

[0013] In one or more embodiments, the upregulation of Escherichia coli transmembrane protein thiocyanate enzyme Ygap includes: introducing an exogenous gene encoding Escherichia coli transmembrane protein thiocyanate enzyme Ygap into the prokaryotic cell; preferably, the gene encoding Escherichia coli transmembrane protein thiocyanate enzyme Ygap is introduced into the prokaryotic cell via an expression vector.

[0014] In one or more embodiments, downregulating the target gene includes: knocking out or silencing the target gene in cells, or inhibiting the activity of the target gene; preferably, knocking out or silencing the target gene in cells includes (but is not limited to): silencing the target gene with a specific interfering molecule, knocking out the target gene by gene editing using a CRISPR system, knocking out the target gene by homologous recombination, or mutating the target gene into a loss-of-function mutation; preferably, the interfering molecule includes sRNA.

[0015] In one or more embodiments, the interfering molecule is an sRNA, with the sRNA sequence for glpC shown in SEQ ID NO:2; the sRNA sequence for leuA shown in SEQ ID NO:3; the sRNA sequence for leuC shown in SEQ ID NO:4; the sRNA sequence for leuD shown in SEQ ID NO:5; the sRNA sequence for folK shown in SEQ ID NO:6; the sRNA sequence for tktA shown in SEQ ID NO:7; the sRNA sequence for fabB shown in SEQ ID NO:8; the sRNA sequence for accC shown in SEQ ID NO:9; the sRNA sequence for accB shown in SEQ ID NO:10; the sRNA sequence for purC shown in SEQ ID NO:11; the sRNA sequence for pyrB shown in SEQ ID NO:12; or the sRNA sequence for glyA shown in SEQ ID NO:13.

[0016] In one or more embodiments, the reagent used to downregulate the target gene is called a downregulator or downregulator (including inhibitors, blockers, antagonists, etc.).

[0017] In one or more embodiments, the phenylalanine ammonia-lyase (PAL) and 4-coumaric acid coenzyme A ligase (4CL) are configured to constitute a complex (complex reactor).

[0018] In one or more embodiments, phenylalanine ammonia-lyase and 4-coumaric acid coenzyme A are brought close together through the binding of protein-protein interaction domains and their ligands to obtain a complex, or phenylalanine ammonia-lyase and 4-coumaric acid coenzyme A ligase are directly linked or linked through a linker to obtain a complex in the form of a fusion protein.

[0019] In one or more embodiments, the protein-protein interaction domain includes a PDZ domain, the ligand of which is a PDZ ligand; the phenylalanine ammonia-lyase and 4-coumaric acid coenzyme A are fused to the PDZ domain and its ligand, respectively; preferably, the phenylalanine ammonia-lyase is fused to PDZ and the 4-coumaric acid coenzyme A is fused to the PDZ ligand.

[0020] In one or more embodiments, the phenylalanine ammonia-lyase fused with PDZ further includes ER / K linker connection (PAL-ER / K-PDZ).

[0021] In one or more embodiments, the fusion of the 4-coumaric acid coenzyme A with PDZ ligand further includes the connection of (PDZlig-(GGGGS)2-4CL) with a (GGGGS)2 linker.

[0022] In one or more embodiments, the protein-protein interaction domain includes domains selected from the group consisting of: PDZ domain, SH3 domain, WW domain, LIM domain, DD domain, PH domain, EH domain, and GBD domain.

[0023] In one or more embodiments, the protein-protein interaction domain includes an SH3 domain, the ligand of which is an SH3 ligand; the phenylalanine ammonia-lyase and 4-coumaric acid coenzyme A are fused to the SH3 domain and its ligand, respectively; preferably, the phenylalanine ammonia-lyase is fused to SH3, and the 4-coumaric acid coenzyme A is fused to SH3 ligand; more preferably, when the phenylalanine ammonia-lyase is fused to SH3, it is further connected by an ER / K linker (PAL-ER / K-SH3), and when the 4-coumaric acid coenzyme A is fused to SH3 ligand, it is further connected by a (GGGGS)2 linker (SH3lig-(GGGGS)2-4CL).

[0024] In one or more embodiments, 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.

[0025] In one or more embodiments, when the 4-coumaric acid coenzyme A is fused with 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.

[0026] In one or more embodiments, 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.

[0027] In one or more embodiments, when the 4-coumaric acid coenzyme A is fused with SH3 ligand, the SH3 ligand is located at the N-terminus and the 4-coumaric acid coenzyme A is located at the C-terminus.

[0028] In one or more embodiments, (1) the prokaryotic cell further includes an exogenous gene encoding an enzyme that promotes the production of malonyl-CoA; preferably, it includes matC, matB, ACS, FabF.

[0029] In one or more embodiments, when introduced into cells, the encoding genes for PDZ ligand, 4-coumarate-coenzyme A ligase, phenylalanine ammonia-lyase, ER / K, PDZ, flavonoid synthase I, chalcone synthase, and chalcone isomerase are located in a single construct (plasmid).

[0030] In one or more embodiments, when introduced into cells, the gene encoding the flavonoid 6-hydroxylase, cytochrome P450 oxidoreductase, is located in a construct, preferably also including the 2B1 (cytochrome P450 2B1 family soluble protein) gene.

[0031] In one or more embodiments, when introduced into cells, the encoding genes for matC, matB, ACS, and FabF are located in a single construct.

[0032] In one or more embodiments, the term "promotion" is a statistically significant promotion, such as a promotion of 5% or more, 10% or more, 20% or more, 50% or more, 80% or more, 100% or more, or higher.

[0033] In one or more embodiments, when introduced into cells, the encoding genes for SH3lig, 4-coumarate-coenzyme A ligase, phenylalanine ammonia-lyase, ER / K, SH3, and chalcone synthase are located in a construct.

[0034] In one or more embodiments, when introduced into cells, the gene encoding the chalcone isomerase, flavonoid synthase I, is located in a construct.

[0035] In one or more embodiments, the prokaryotic cell is a cell having the substrate synthesis pathway of formula (I); preferably, the prokaryotic cell is an Escherichia coli cell.

[0036] In another aspect of the invention, a prokaryotic cell for synthesizing flavonoids is provided, comprising genes encoding exogenous enzymes from the following groups: phenylalanine ammonia-lyase (PAL), 4-coumarate-co-A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonoid synthase I (FNSI); and, in the prokaryotic cell, the Escherichia coli transmembrane protein thiocyanate Ygap is upregulated; or a target gene or combination of target genes selected from the following groups is downregulated in the prokaryotic cell: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, leuA; wherein the flavonoid is a apigenin compound (such as apigenin or apigenin).

[0037] In one or more embodiments, the prokaryotic cells also include the encoding genes of the following exogenous enzymes: flavonoid 6-hydroxylase (F6H) and cytochrome P450 oxidoreductase (CPR); the flavonoid compound is a baicalin compound (such as baicalin or baicalin).

[0038] In another aspect of the invention, the use of the prokaryotic cells is provided for the synthesis of flavonoids; preferably, the flavonoids include: scutellarin compounds and baicalin compounds.

[0039] In another aspect of the invention, a kit for producing flavonoids is provided, the kit comprising the aforementioned prokaryotic cells.

[0040] In one or more embodiments, the kit further includes cell culture medium.

[0041] In one or more embodiments, the kit further includes a substrate of formula (I).

[0042] In one or more embodiments, the kit contains

[0043] In another aspect of the invention, a kit is provided for establishing host cells for synthesizing flavonoids, the kit comprising: (a) a construct (e.g., an expression vector) expressing phenylalanine ammonia-lyase (PAL), 4-coumarate-co-A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonoid synthase I (FNSI); (b) a construct (e.g., an expression vector) expressing the Escherichia coli transmembrane protein thiocyanate enzyme Ygap; or, a construct (e.g., an expression vector) expressing a downregulator that downregulates a target gene or combination of target genes selected from the group consisting of: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, leuA; optionally, the kit further comprises: a construct expressing a gene encoding flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase.

[0044] In another aspect of the invention, the use of a regulatory agent in promoting the biosynthesis of flavonoids is provided, said regulatory agent being selected from: (i) Escherichia coli transmembrane protein thiocyanate enzyme Ygap or its upregulators (such as expression constructs expressing it); or (ii) downregulators that downregulate target genes or combinations of target genes selected from the group consisting of: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, leuA.

[0045] In one or more embodiments, the promotion of flavonoid biosynthesis is to promote the synthesis of flavonoids in prokaryotic cells.

[0046] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0047] Figure 1 Comparison of salicylic acid production between strain J-1 and strain J-2, which overexpresses ygaP.

[0048] Figure 2 Comparison of baicalin production between strain J-3 and strain J-4 overexpressing ygaP.

[0049] Figure 3 Comparison of baicalin production in engineered strains, control strains, and strains with downregulated target genes.

[0050] Figure 4 Schematic diagram of the plasmid structure of pET28a-ygaP.

[0051] Figure 5 A schematic diagram of the plasmid structure of PET28a-sRNA.

[0052] Figure 6 A schematic diagram of the plasmid structure of pET28a-sRNA-glpC.

[0053] Figure 7 A schematic diagram of the plasmid structure of pET28a-sRNA-leuA.

[0054] Figure 8 A schematic diagram of the plasmid structure of pET28a-sRNA-leuC.

[0055] Figure 9 A schematic diagram of the plasmid structure of pET28a-sRNA-leuD.

[0056] Figure 10 A schematic diagram of the plasmid structure of pET28a-sRNA-folK.

[0057] Figure 11 A schematic diagram of the plasmid structure of pET28a-sRNA-tktA.

[0058] Figure 12 A schematic diagram of the plasmid structure of pET28a-sRNA-fabB.

[0059] Figure 13 A schematic diagram of the plasmid structure of pET28a-sRNA-accC.

[0060] Figure 14 A schematic diagram of the plasmid structure of pET28a-sRNA-accB.

[0061] Figure 15 A schematic diagram of the plasmid structure of pET28a-sRNA-purC.

[0062] Figure 16 A schematic diagram of the plasmid structure of pET28a-sRNA-pyrB.

[0063] Figure 17 A schematic diagram of the plasmid structure of pET28a-sRNA-glyA.

[0064] Figure 18 Schematic diagram of the plasmid structure of pZZ41.

[0065] Figure 19 Schematic diagram of the plasmid structure of pZZ42.

[0066] Figure 20 Schematic diagram of the plasmid structure of pYH38.

[0067] Figure 21 A schematic diagram of the synthetic pathway for producing baicalein and scutellarin by fermentation using phenylalanine as a precursor. Detailed Implementation

[0068] Through in-depth research, the inventors have provided a novel optimization strategy for the biosynthesis of flavonoids (such as baicalin / salicylic acid compounds). By appropriately regulating genes in production cells, a significant increase in flavonoid yield was achieved. This regulation includes upregulating the E. coli transmembrane protein thiocyanate enzyme Ygap or downregulating one or more target genes (pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, or leuA). This invention also discloses the optimized host cells and their applications.

[0069] As used herein, the “flavonoids” referred to are compounds having the following core structure:

[0070]

[0071] As used herein, “capillary compounds” include capillary or variants, isomers, derivatives, precursors, salts, etc., having their parent nucleus structure, such as capillary or apigenin.

[0072] As used herein, “baicalin (or similar) compounds” include baicalin or variants, isomers, derivatives, precursors, salts, etc., having the parent nucleus structure thereon, such as baicalin or baicalin.

[0073] As used in this article, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a protein / gene and a host cell. For example, although the host cell itself may contain the corresponding gene or produce the corresponding protein, when a synthesized / recombined gene / protein is introduced into the host cell through genetic engineering methods, it is considered "exogenous" or "heterogeneous" relative to that host cell.

[0074] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the nucleic acid sequence of a target gene, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby “operationally linked” to the nucleic acid sequence.

[0075] As used herein, the term "construct" or "expression construct" refers to a recombinant DNA molecule containing the intended nucleic acid coding sequence, which may contain one or more gene expression cassettes. The "construct" is typically contained within an expression vector.

[0076] As used herein, the PAL, 4CL, CHS, CHI, and FNSI proteins are proteins that form the synthetic pathways of apigenin or chrysogenin compounds in the expression system.

[0077] As used herein, the F6H and CPR proteins are proteins that convert apigenin or sennain compounds into baicalin or scutellarin compounds in the expression system.

[0078] As used herein, the matC, matB, ACS, and / or FabF proteins are enzymes that promote malonyl-CoA production in the expression system.

[0079] The above-mentioned proteins or genes of wild type are already identified in the art and therefore can be obtained and prepared from public sources. As a preferred embodiment of the present invention, PAL is derived from Rhodotorula toruloides, having the sequence shown in GenBank accession number AAA33883.1; 4CL is derived from Petroselium crispum, having the sequence shown in GenBank accession number KF765780.1; CHS is derived from Petunia X hybrida, having the sequence shown in GenBank accession number KF765781.1; the CHI gene is derived from Medicago sativa, having the sequence shown in GenBank accession number KF765782.1; and FNS I is derived from Petroselium crispum, having the sequence shown in Swiss-Prot accession number Q7XZQ8.1.

[0080] 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 embodiment of the present invention, CPR is derived from *Arabidopsis thaliana*, which has the sequence shown in GenBank accession number NP_849472.2.

[0081] Wild-type matC, matB, ACS, and FabF proteins have also been identified in the art. As a preferred embodiment of the present invention, matC is derived from *Rhizobium leguminosarum*, with the sequence shown in GenBank accession number KF765784.1; matB is derived from *Rhizobium leguminosarum*, with the sequence shown in GenBank accession number AGZ04579.1; ACS is derived from *Escherichia coli*, with the sequence shown in GenBank accession number CP062211.1; and FabF is derived from *Escherichia coli*, with the sequence shown in GenBank accession number AP023237.1.

[0082] Baicalein and baicalein are two structurally similar and important flavonoid compounds. The molecular formula of baicalein is C1. 15 H 10 O5 has a molecular weight of 270.24, while baicalin has a molecular weight of C. 15 H10 O6 has a molecular weight of 286.24. Their structures are shown below:

[0083]

[0084] Glutinous glycoside is also a flavonoid compound. In the enzyme-mediated biosynthetic pathway, glutinous glycoside is an upstream compound (intermediate compound) of baicalin or baicalein, and its structural formula is shown below.

[0085]

[0086] The inventors of this invention are dedicated to researching optimization strategies for the biosynthesis of flavonoids (such as baicalin / stigmine compounds). Through extensive research, screening, and experimental work, the inventors have identified optimization schemes that significantly increase the yield of flavonoids, including upregulating the expression or activity of the E. coli transmembrane protein thiocyanate enzyme Ygap in host cells.

[0087] It should be understood that, once the functions of Ygap and the signaling pathways containing Ygap (preferably, also including its upstream and downstream genes) are known, various methods well-known to those skilled in the art can be used to regulate the expression or activity of Ygap or to regulate related upstream or downstream genes of Ygap. For example, various methods well-known to those skilled in the art can be used to overexpress Ygap or its upstream or downstream genes.

[0088] In this invention, upregulating the expression of the Ygap protein or its encoding gene, and its upstream or downstream proteins or their encoding genes, includes applying an upregulator of the Ygap protein or its encoding gene. The upregulator may include promoters, agonists, or activators. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein activity or protein expression, and these are statistically significant. Any substance that can increase the activity of Ygap or its signaling pathway proteins (including its upstream and downstream proteins), increase the stability of Ygap or its signaling pathway proteins, upregulate the expression of Ygap or its signaling pathway genes, increase the effective duration of action of Ygap or its signaling pathway proteins, or increase the phosphorylation / activation level of various proteins can be used in this invention as substances useful for upregulating Ygap or signaling pathways. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0089] This invention also provides a method for upregulating Ygap expression in cells, the method comprising: transferring the Ygap coding gene or an expression construct or vector containing said coding gene into cells. Alternatively, gain-of-function mutations may be performed on Ygap or its coding gene; expression of the Ygap coding gene may be promoted by expressing an enhancing promoter or a tissue-specific promoter; or, expression of the Ygap coding gene may be promoted by an enhancer. It should be understood that other methods for upregulating Ygap expression in cells should also be included in this invention.

[0090] This invention also provides another optimization scheme that is beneficial for significantly increasing the yield of flavonoids, including downregulating target proteins or target genes encoding them selected from the following group in host cells: pyrB (GenBank accession number CAD6022649.1), accC (GenBank accession number CAD6001830.1), accB (GenBank accession number CAD6001842.1), purC (GenBank accession number CAD6007197.1), glyA (GenBank accession number CAD6006651.1). The following are examples of proteins that can downregulate the target proteins or their encodings: tktA (GenBank accession number CAD6004304.1), fabB (GenBank accession number CAD6008062.1), leuD (GenBank accession number CAD6022253.1), leuC (GenBank accession number CAD6022253.1), glpC (GenBank accession number CAD6008699.1), folK (GenBank accession number CAD6022053.1), and leuA (GenBank accession number CAD6022244.1). Reagents that downregulate these target proteins or their encodings are called downregulators.

[0091] In this invention, the aforementioned target gene downregulator refers to any substance that can reduce the activity of the target protein, reduce the stability of the target protein or its encoding gene, downregulate the expression of the target protein, reduce the effective action time of the target protein, inhibit the transcription and translation of the target protein's encoding gene, or reduce the phosphorylation / activation level of the protein. These substances can all be used in this invention. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. For example, the downregulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of the target gene; or a gene editing reagent that specifically edits the target, etc.

[0092] This invention provides a method for downregulating the expression of a target gene in cells, comprising introducing an interfering molecule that interferes with the expression of the target gene into the cells. As a more specific embodiment, the interfering molecule is sRNA. Another method for downregulating the expression of a target gene in cells includes, for example, targeted mutation, gene editing, or gene recombination of the target gene to achieve downregulation. As a more specific embodiment, gene editing is performed using a CRISPR / Cas9 system to knock out or downregulate the target gene. Suitable sgRNA target sites result in higher gene editing efficiency; therefore, suitable target sites can be designed and identified before gene editing. After designing specific target sites, in vitro cell activity screening is required to obtain effective target sites for subsequent experiments. It should be understood that the methods for downregulating target genes / proteins in cells are not limited to those listed above.

[0093] In a preferred embodiment of the present invention, the inventors utilize enzyme assembly technology to ferment and produce baicalein or wild baicalein. The principle of this method is as follows: an interacting protein pair (e.g., PDZ and PDZ ligand) is fused with the enzymes PAL and 4CL in the baicalein synthesis pathway, enabling PAL and 4CL to spontaneously assemble within *E. coli*, forming a dual-enzyme complex reactor, thereby increasing the yield of the target compound. The inventors have, for the first time, discovered in a prokaryotic expression system for synthesizing baicalein / salicylic acid compounds that constructing a complex (complex reactor) of PAL and 4CL can significantly improve the yield of the expression system. Any biological materials or techniques suitable for forming an active complex of PAL and 4CL can be applied to this invention. In a preferred embodiment of the present invention, the protein-protein interaction domain may include domains selected from the group consisting of: PDZ domain, SH3 domain, WW domain, LIM domain, DD domain, PH domain, and EH domain. As a more preferred embodiment of the present invention, the protein-protein interaction domain may include domains selected from the group consisting of: PDZ domain, SH3 domain; and their corresponding ligands are PDZ ligand (PDZlig) or SH3 ligand (SH3lig).

[0094] Protein-protein interactions are primarily mediated efficiently by protein domains. Domains such as PDZ, SH3, and WW can recognize and bind to a conserved short peptide sequence of a ligand protein through one or more recognition "pockets." For example, the PDZ domain typically binds to the C-terminal 4-5 amino acid residues of a ligand protein; it can also bind to the middle sequence of the ligand protein, aggregate with itself or other domains, or bind to lipids on the membrane.

[0095] This invention may also include other methods for forming a complex of PAL and 4CL that retain the biological activity of the PAL and 4CL, such as fusing them to form a fusion protein with a suitable spatial structure; the activity of the fusion protein can be determined by experimental testing. The fusion between PAL and 4CL can be a direct connection or a connection using a linker.

[0096] As another optional embodiment of the present invention, aroG, especially its aroG, can be overexpressed in a prokaryotic expression system. fbr (the aroG gene, where G is mutated to A at position 436), and pheA, especially its pheA gene. fbr The pheA gene (the gene whose position 976 is mutated from A to C) fbr A high-yield phenylalanine prokaryotic expression system was constructed, and an exogenous baicalin or wild baicalin / sapine-like compound synthesis pathway was introduced into the prokaryotic expression system, enabling the strain to synthesize baicalin / sapine-like compounds de novo using glucose.

[0097] The prokaryotic cells described in this invention also include genes encoding exogenous enzymes that promote malonyl-CoA production; preferably, they include matC, matB, ACS, and FabF.

[0098] In this invention, the prokaryotic expression system (prokaryotic cell) used is a cell with the substrate synthesis pathway of formula (I). Commonly used prokaryotic expression systems include Escherichia coli, Bacillus subtilis, etc.; for example, it can be E. coli cells, such as E. coli BL21(DE3).

[0099] In addition to the preferred proteins listed herein (including the wild-type and mutant proteins described above), this invention also includes analogs thereof. These analogs may differ from the natural proteins in amino acid sequence, in form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, site-directed mutagenesis, or other known molecular biology techniques. Analogs also include those having residues different from natural L-amino acids (e.g., D-amino acids), and those having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the proteins of this invention are not limited to the representative proteins exemplified above.

[0100] In addition to the preferred proteins listed in this invention (including the wild-type and mutant proteins mentioned above), this invention also includes proteins that have high homology with the proteins listed (e.g., 70% or higher homology with the specific protein sequences listed; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98% or 99% homology) and have the same function as the corresponding polypeptides.

[0101] This invention lists proteins or genes from specific species. It should be understood that while proteins or genes derived from specific species are preferably studied in this invention, other proteins or genes derived from other species that are highly homologous to the said proteins or genes (e.g., having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0102] The invention also relates to providing a polynucleotide sequence encoding the protein of the invention or a conserved variant thereof. The polynucleotide of the invention may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The polynucleotide encoding the mutant mature protein of the invention includes: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences) and a non-coding sequence.

[0103] The present invention also includes a polynucleotide sequence formed by codon optimization of the sequence of the gene, for example, codon optimization according to the preferences of the host cell.

[0104] In this invention, an engineered strain capable of producing high yields of baicalin or baicalein-like compounds was constructed, comprising encoding genes for exogenous enzymes from the following group: F6H, CPR, PAL, 4CL, CHS, CHI, and FNSI; and including the encoding gene for the exogenous E. coli transmembrane protein thiocyanate enzyme Ygap; or downregulated target genes or combinations of target genes selected from the following group in the engineered strain: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, and leuA. Preferably, after the enzyme is expressed, PAL and 4CL constitute a complex (complex reactor). The engineered strain is cultured, and baicalin or baicalein-like compounds are produced using phenylalanine or tyrosine as substrates. Production using phenylalanine or tyrosine as substrates is suitable for large-scale compound production. A schematic diagram of the synthetic pathway for the production of baicalin and scutellarin from phenylalanine precursor via fermentation is shown below. Figure 21 .

[0105] In this invention, an engineered strain capable of producing high yields of baicalin-like compounds was constructed, comprising the encoding genes of exogenous enzymes from the following group: PAL, 4CL, CHS, CHI, and FNSI; and the engineered strain including the encoding gene of the exogenous E. coli transmembrane protein thiocyanate enzyme Ygap; or the target genes or combinations of target genes selected from the following group in the engineered strain being downregulated: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, and leuA. Preferably, after the enzymes are expressed, PAL and 4CL constitute a complex (complex reactor). The recombinant strain is cultured, and baicalin or scutellarin-like compounds are produced using phenylalanine or tyrosine as substrates. Production using phenylalanine or tyrosine as substrates is suitable for large-scale compound production.

[0106] In a preferred embodiment of the invention, the F6H further includes a polypeptide tag fused thereto, the polypeptide tag being selected, for example, from 8RP, Sumo, MBP, 2B1, or combinations thereof; preferably 2B1. A linker peptide may or may not be present between the polypeptide tag and the F6H, the linker peptide not affecting the biological activity of either. Linking F6H to 2B1 yields an improved F6H mutant, 2B1trF6H.

[0107] Furthermore, upstream pathways for the formation of the aforementioned substrates (phenylalanine or tyrosine) can be introduced into the engineered strains described above. These pathways may include, for example, the formation of phenylalanine or tyrosine from glucose or glycerol via glycolysis, the pentose phosphate pathway, or the shikimic acid pathway. It should be understood that schemes for forming phenylalanine or tyrosine based on such pathways are also included in this invention. Methods for enhancing the aforementioned pathways for forming phenylalanine or tyrosine using means known in the art may be included in this invention.

[0108] As a preferred approach, exogenous aroG, especially its aroG, can be further introduced into the aforementioned engineered strains using phenylalanine or tyrosine as substrates. fbr , and pheA, especially its pheA fbr Another recombinant strain was obtained, which can produce baicalin / salicylic acid compounds using glucose as a substrate. Glucose-based production is inexpensive and well-suited for large-scale compound production.

[0109] Based on the establishment of the optimized expression system as described in this invention and its use in production, those skilled in the art can systematically study a series of factors that improve the yield of baicalin, scutellarin-like compounds, or scutellarin-like compounds, including gene efficiency and suitability, gene dosage, and culture medium. Furthermore, the yield of the target compound can also be increased by scaling up production. For example, based on the yield under simple culture conditions at shake-flask scale, when further scaling up production, using a fed-batch culture medium (which can continuously provide abundant substrate), or providing good fermenter-level production conditions (such as optimized temperature control, optimized dissolved oxygen control, etc.), the yield can typically increase by 2 to 1000 times or more. These operations and optimizations should also be included in this invention. It is anticipated that the recombinant prokaryotic cells of this invention, with some optimized equipment and operating processes, will further increase the amount of the target product.

[0110] After obtaining the fermentation product, the target compound can be extracted from it using techniques known in the art. Well-known techniques such as high-performance liquid chromatography (HPLC) can be used to analyze and identify the product to confirm that the desired compound has been obtained.

[0111] The strains of this invention exhibit good stability and can be used for large-scale cultivation and production of baicalin or baicalin-like compounds / salicylic acid-like compounds in bioreactors. The preferred strains of this invention achieve very high yields of the target compounds.

[0112] Compared to traditional plant extraction methods, microbial fermentation offers advantages such as faster processing speed and less susceptibility to external factors. The yield of some compounds synthesized through microorganisms is significantly higher than that obtained from plant extraction, making it an important method for acquiring natural products. In this invention, baicalin or baicalein-like compounds / capillary compounds are produced using *Escherichia coli*, achieving a more economical and convenient manufacturing process for the target compounds.

[0113] This invention also provides a kit containing engineered strains for the production of flavonoids. Furthermore, it may include a culture medium for prokaryotic cells, substrates for synthesis such as phenylalanine, tyrosine, or glucose, and reagents for the isolation or detection of baicalin or scutellarin-like compounds. More preferably, the kit may also include instructions for use describing the method for biosynthesizing flavonoids.

[0114] This invention also provides a kit for constructing engineered strains for producing flavonoids. The kit may include a series of constructs, such as those provided in the embodiments of this invention, or other constructs containing the gene but with different gene arrangements or tandem configurations. The expression vector (expression construct) can be established using techniques familiar to those skilled in the art. Once the desired enzyme and the desired cell system for expression are known, those skilled in the art can establish the expression construct. 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 effectively expressed and active after transformation into cells. The kit may also include prokaryotic cells, a culture medium for prokaryotic cells, substrates for synthesis such as phenylalanine, tyrosine, or glucose, and reagents for the isolation or detection of baicalein or baicalein-like compounds. More preferably, the kit may also include instructions for use describing the method for biosynthesizing flavonoids.

[0115] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0116] 1. Experimental materials

[0117] Polymerase chain reaction (PCR) gel recovery kits and plasmid extraction kits were both products of Axygen (USA); PrimeSTAR Max DNA Polymerase, a high-fidelity PCR enzyme, was a product of Takara Bio Inc. (Japan); and restriction endonucleases were all products of NEB.

[0118] The standard compounds baicalein and baicalein were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Other reagents were domestically produced analytical grade or chromatographic grade reagents, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0119] 2. The strains and plasmids involved in this invention

[0120] Escherichia coli DH10B is used for gene cloning, and Escherichia coli BL21(DE3) strain is used for protein expression and the production of baicalin and scutellarin.

[0121] pCDFDuet-1, pET28a, and pACYCDuet-1 vectors are used for metabolic pathway gene assembly.

[0122] 5nm rigid linker ER / K sequence:

[0123] KAKLKEEERKQREEEERIKRLEELAKRKEEERKGT (SEQ ID NO: 14).

[0124] 3. Proteins / genes / sRNAs / domains, etc.

[0125] The wild-type proteins or genes specifically exemplified in the embodiments are all identified in the art and therefore can be obtained and prepared from public sources, as detailed in Table 1.

[0126] Table 1

[0127]

[0128]

[0129] 4. Plasmid construction

[0130] The plasmids containing a single gene were constructed, as shown in Table 2.

[0131] Table 2

[0132]

[0133]

[0134] Plasmids carrying multiple genes were constructed, as detailed in Table 3.

[0135] Table 3

[0136]

[0137]

[0138] 5. Shake-flask fermentation of baicalin and scutellarin by Escherichia coli

[0139] Preparation of fermentation strain: The constructed plasmid was transformed into Escherichia coli BL21(DE3), and after incubation at 37℃ upside down for 12h, positive clones were selected and cultured in 2mL of LB resistant medium at 37℃ and 250rpm for 10h to prepare fermentation seed strain.

[0140] 1% of the seed culture was transferred to 10 mL of MOPS medium containing 2% glucose (with the appropriate antibiotic added to the medium), and cultured at 37°C and 250 rpm until the strain OD = 0.5. 0.5 mM IPTG and 500 mg / L phenylalanine were added, and fermentation was carried out at 22°C and 250 rpm for 3 days. 1 mL of the sample was taken, and the bacterial culture was sonicated 3 times. It was extracted twice with an equal volume of ethyl acetate, centrifuged at 12000 rpm for 2 min, and the organic phase was transferred to a new tube. After evaporation at room temperature or 30°C, the organic phase was reconstituted with 200 μL of methanol (concentrated 5 times) and mixed thoroughly. After centrifugation at 12000 rpm for 2 min, the supernatant was transferred for HPLC detection.

[0141] 6. HPLC detection

[0142] Liquid chromatography detection conditions: Phase A: 0.1% formic acid solution, Phase B: acetonitrile; Separation conditions: 0-20 min 20% Phase B - 55% Phase B, 20-22 min 55% Phase B - 100% Phase B, 22-27 min 100% Phase B, 27-35 min 100% Phase B - 20% Phase B, 35-40 min 20% Phase B; Detection wavelength: 340 nm, Column temperature: 30℃.

[0143] Chromatographic column: Thermo syncronis C18 reversed-phase column (250mm × 4.6mm, 5μm).

[0144] Example 1: Production of styraxin by fermentation engineered bacteria

[0145] 1. Strains (J-1)

[0146] The plasmids pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a were transformed into BL21(DE3) to obtain engineered strain J-1, which was used to ferment salicylic acid using phenylalanine as a precursor.

[0147] 2. YGAP overexpressing strain (J-2)

[0148] The plasmids pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-T7 ygaP were transformed into BL21(DE3) to obtain engineered strain J-2, which was used to ferment styraxin using phenylalanine as a precursor.

[0149] The fermentation method is as follows: The strain was cultured overnight at 37°C on LB solid medium (spectrobacterium 80 μg / mL, chloramphenicol 34 μg / mL, kanamycin 50 μg / mL). A single clone was picked and transferred to 2 mL of LB liquid medium (spectrobacterium 80 μg / mL, chloramphenicol 34 μg / mL, kanamycin 50 μg / mL). The overnight culture was then transferred to 10 mL of new MOPS liquid resistant medium and cultured at 37°C and 250 rpm until OD600 = 0.5–0.6. The culture was then cooled to approximately 16°C in a water bath, and IPTG was added to a final concentration of 0.5 mM. Sterilized phenylalanine was then added to a final concentration of 500 mg / L, and the culture was transferred to 22°C for induction culture. The culture was continued for 72 h on a shaker at 250 rpm. Samples were taken to detect the yield of salinomycin.

[0150] The results showed that, compared with strain J-1, the ygaP overexpressing strain J-2 produced 2.99 times more apigenin. Figure 1 ).

[0151] Example 2: Production of baicalin by fermentation engineered bacteria: overexpression of ygaP

[0152] 1. Strains (J-3)

[0153] The plasmids pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a were transformed into BL21(DE3) to obtain engineered strain J-3, which was used to ferment baicalein using phenylalanine as a precursor.

[0154] 2. YGAP overexpressing strain (J-4)

[0155] The plasmids pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-T7ygaP were transformed into BL21(DE3) to obtain engineered strain J-4, which was used to ferment baicalein using phenylalanine as a precursor.

[0156] The two strains were fermented using the following method: The strains were cultured overnight at 37°C on LB solid medium (spectruminant 80 μg / mL, chloramphenicol 34 μg / mL, kanamycin 50 μg / mL). A single clone was picked and transferred to 2 mL of LB liquid medium (spectruminant 80 μg / mL, chloramphenicol 34 μg / mL, kanamycin 50 μg / mL). The overnight culture was then transferred to 10 mL of new MOPS liquid resistant medium and cultured at 37°C and 250 rpm until OD600 = 0.5–0.6. The culture was then cooled to approximately 16°C in a water bath, and IPTG was added to a final concentration of 0.5 mM. Sterilized phenylalanine at a final concentration of 500 mg / L was added, and the culture was transferred to 22°C for induction. The culture was continued for 72 h on a shaker at 250 rpm. Samples were taken to detect the baicalin yield.

[0157] The results showed that, compared with strain J-3, the baicalin production of strain J-4 overexpressing ygaP was increased by 1.59 times. Figure 2 ).

[0158] Example 3: Regulation of Baicalin Fermentation Production: Interfering with the Expression of Target Genes

[0159] 1. Engineered bacteria control

[0160] pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA plasmid were transformed into BL21(DE3) to obtain the engineered bacteria control, which was used to ferment baicalein using phenylalanine as a precursor (the sRNA sequence in this PET28a-sRNA has no target site, only an sRNA hairpin structure).

[0161] 2. Strain that inhibits glpC (glpC-)

[0162] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-glpC plasmid were transformed into BL21(DE3) to obtain the engineered bacterium glpC-, which was used to ferment baicalein using phenylalanine as a precursor.

[0163] 3. Inhibits leuA strain (leuA-)

[0164] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-leuA plasmid were transformed into BL21(DE3) to obtain the engineered strain leuA-, which was used to ferment baicalein using phenylalanine as a precursor.

[0165] 4. Inhibits leuC strain (leuC-)

[0166] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-leuC plasmid were transformed into BL21(DE3) to obtain the engineered strain leuC-, which was used to ferment baicalein using phenylalanine as a precursor.

[0167] 5. Inhibits leuD strain (leuD-)

[0168] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-leuD plasmid were transformed into BL21(DE3) to obtain the engineered strain leuD-, which was used to ferment baicalein using phenylalanine as a precursor.

[0169] 6. Inhibits folK strains (folK-)

[0170] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-folK plasmid were transformed into BL21(DE3) to obtain engineered folK-, which was used to ferment baicalein using phenylalanine as a precursor.

[0171] 7. Inhibit tktA strain (tktA-)

[0172] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-tkt plasmid were transformed into BL21(DE3) to obtain engineered strain tktA-, which was used to ferment baicalein using phenylalanine as a precursor.

[0173] 8. Inhibit fabB strain (fabB-)

[0174] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-fabB plasmid were transformed into BL21(DE3) to obtain the engineered bacterium fabB-, which was used to ferment baicalein using phenylalanine as a precursor.

[0175] 9. Inhibits accC strains (accC-)

[0176] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-accC plasmid were transformed into BL21(DE3) to obtain the engineered bacterium accC-, ​​which was used to ferment baicalein using phenylalanine as a precursor.

[0177] 10. Strain that inhibits accB (accB-)

[0178] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-accB plasmid were transformed into BL21(DE3) to obtain engineered bacteria accB-, which was used to ferment baicalein using phenylalanine as a precursor.

[0179] 11. Inhibits purC strain (purC-)

[0180] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-purC plasmid were transformed into BL21(DE3) to obtain the engineered bacterium purC-, which was used to ferment baicalein using phenylalanine as a precursor.

[0181] 12. Inhibit pyrB strain (pyrB-)

[0182] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-pyrB plasmid were transformed into BL21(DE3) to obtain the engineered strain pyrB-, which was used to ferment baicalein using phenylalanine as a precursor.

[0183] 13. Strain that inhibits glyA (glyA-)

[0184] pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER / K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF), and pET28a-sRNA-glyA plasmid were transformed into BL21(DE3) to obtain engineered bacteria glyA-, which was used to ferment baicalein using phenylalanine as a precursor.

[0185] The two strains were fermented using the following method: The strains were cultured overnight at 37°C on LB solid medium (spectruminant 80 μg / mL, chloramphenicol 34 μg / mL, kanamycin 50 μg / mL). A single clone was picked and transferred to 2 mL of LB liquid medium (spectruminant 80 μg / mL, chloramphenicol 34 μg / mL, kanamycin 50 μg / mL). The overnight culture was then transferred to 10 mL of new MOPS liquid resistant medium and cultured at 37°C and 250 rpm until OD600 = 0.5–0.6. The culture was then cooled to approximately 16°C in a water bath, and IPTG was added to a final concentration of 0.5 mM. Sterilized phenylalanine at a final concentration of 500 mg / L was added, and the culture was transferred to 22°C for induction. The culture was continued for 72 h on a shaker at 250 rpm. Samples were taken to detect the baicalin yield.

[0186] The results showed that, compared with the engineered strain control, inhibiting baicalein production by strain glpC (glpC-) increased baicalein production by 1.34 times, inhibiting strain leuA (leuA-) by 1.16 times, inhibiting strain leuC (leuC-) by 1.53 times, inhibiting strain leuD (leuD-) by 2.32 times, inhibiting strain folK (folK-) by 1.3 times, and inhibiting strain tktA (tktA-) by [the specific strain is missing from the original text]. The yield increased by 3 times, the baicalein yield inhibited by the fabB strain (fabB-) increased by 2.69 times, the baicalein yield inhibited by the accC strain (accC-) increased by 5.31 times, the baicalein yield inhibited by the accB strain (accB-) increased by 5.26 times, the baicalein yield inhibited by the purC strain (purC-) increased by 3.51 times, the baicalein yield inhibited by the pyrB strain (pyrB-) increased by 5.66 times, and the baicalein yield inhibited by the glyA strain (glyA-) increased by 3.32 times.

[0187] ( Figure 3 )

[0188] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. sequence list <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> Regulation methods and applications of heterologous synthesis of flavonoids <130> 21A206 <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 85 <212> DNA <213> Artificial Sequence <400> 1 tttctgttgg gccattgcat tgccactgat tttccaacat ataaaaagac aagcccgaac 60 agtcgtccgg gctttttttc tcgag 85 <210> 2 <211> 109 <212> DNA <213> Artificial Sequence <400> 2 gttttcgaag ctggtgtcgt tcattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggctttt tttctcgag 109 <210> 3 <211> 109 <212> DNA <213> Artificial Sequence <400> 3 gaaaataatg acttgctggc tcattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggctttt tttctcgag 109 <210> 4 <211> 109 <212> DNA <213> Artificial Sequence <400> 4 tttttcgtat aacgtcttag ccattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggctttt tttctcgag 109 <210> 5 <211> 109 <212> DNA <213> Artificial Sequence <400> 5 gtgtttgata aatttctctg ccattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggctttt tttctcgag 109 <210> 6 <211> 109 <212> DNA <213> Artificial Sequence <400> 6 tatggcaata tacgccactg tcattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggctttt tttctcgag 109 <210> 7 <211> 109 <212> DNA <213> Artificial Sequence <400> 7 ggcaagctct ttacgtgagg acattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggctttt tttctcgag 109 <210> 8 <211> 109 <212> DNA <213> Artificial Sequence <400> 8 gccagtaatc actgcacgtt tcattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggctttt tttctcgag 109 <210> 9 <211> 109 <212> DNA <213> Artificial Sequence <400> 9 ggcaataaca attttatcca gcattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggctttt tttctcgag 109 <210> 10 <211> 108 <212> DNA <213> Artificial Sequence <400> 10 ttttttaatc ttacgaatat ccattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgc cgggcttttt ttctcgag 108 <210> 11 <211> 109 <212> DNA <213> Artificial Sequence <400> 11 atacaactca gcttgctttt gcattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggcttttt tttctcgag 109 <210> 12 <211> 109 <212> DNA <213> Artificial Sequence <400> 12 tttctgatat agcggattag ccattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggcttttt tttctcgag 109 <210> 13 <211> 109 <212> DNA <213> Artificial Sequence <400> 13 aatgttcatt tcacgcttta acattttctg ttgggccatt gcattgccac tgattttcca 60 acatataaaa agacaagccc gaacagtcgt ccgggcttttt tttctcgag 109 <210> 14 <211> 36 <212> PRT <213> Artificial Sequence <400> 14 Lys Ala Lys Leu Lys Glu Glu Glu Glu Arg Lys Gln Arg Glu Glu Glu Glu 1 5 10 15 Glu Arg Ile Lys Arg Leu Glu Glu Leu Ala Lys Arg Lys Glu Glu Glu 20 25 30 Arg Lys Gly Thr 35 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <400> 15 Gly Val Lys Glu Ser Leu Val 1 5 <210> 16 <211> 57 <212> PRT <213> Artificial Sequence <400> 16 Ala Glu Tyr Val Arg Ala Leu Phe Asp Phe Asn Gly Asn Asp Glu Glu 1 5 10 15 Asp Leu Pro Phe Lys Lys Gly Asp Ile Leu Arg Ile Arg Asp Lys Pro 20 25 30 Glu Glu Gln Trp Trp Asn Ala Glu Asp Ser Glu Gly Lys Arg Gly Met 35 40 45 Ile Pro Val Pro Tyr Val Glu Lys Tyr 50 55 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <400> 17 Pro Pro Pro Ala Leu Pro Pro Lys Arg Arg Arg 1 5 10

Claims

1. A method for synthesizing flavonoids, characterized in that, The method includes: (1) Provide recombinant prokaryotic cells containing the genes encoding exogenous lower enzyme groups: phenylalanine ammonia-lyase, 4-coumarate coenzyme A ligase, chalcone synthase, chalcone isomerase and flavonoid synthase I. Furthermore, the Escherichia coli transmembrane protein thiocyanate enzyme Ygap was upregulated in the prokaryotic cells; and the target genes selected from the following group were downregulated in the prokaryotic cells: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, and leuA. The prokaryotic cells also include the genes encoding the following exogenous enzymes: flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase. The prokaryotic cells also include exogenous genes encoding enzymes that promote malonyl-CoA production, such as matC, matB, ACS, and FabF. (2) Using formula (I) as a substrate, synthesize flavonoids from the prokaryotic cells, wherein the flavonoids are baicalin or scutellarin; (I)； Where R is H; The prokaryotic cells are Escherichia coli cells.

2. The method as described in claim 1, characterized in that, The upregulation of Escherichia coli transmembrane protein thiocyanate Ygap includes introducing an exogenous gene encoding Escherichia coli transmembrane protein thiocyanate Ygap into the prokaryotic cells; the gene encoding Escherichia coli transmembrane protein thiocyanate Ygap is introduced into the prokaryotic cells via an expression vector.

3. The method as described in claim 1, characterized in that, The downregulation includes knocking out or silencing the target gene in the prokaryotic cells, or inhibiting the activity of the target gene.

4. The method as described in claim 3, characterized in that, Knocking out or silencing the target gene in the prokaryotic cells includes: silencing the target gene in the prokaryotic cells with a specific interfering molecule, knocking out the target gene by gene editing with a CRISPR system, knocking out the target gene by homologous recombination, or mutating the target gene into a loss-of-function mutation.

5. The method as described in claim 4, characterized in that, The interfering molecule is sRNA, and the sequence of the sRNA targeting glpC is shown in SEQ ID NO: 2; The sequence of the sRNA targeting leuA is shown in SEQ ID NO: 3; The sequence of the sRNA targeting leuC is shown in SEQ ID NO: 4; The sequence of the sRNA targeting leuD is shown in SEQ ID NO: 5; The sequence of the sRNA targeting folK is shown in SEQ ID NO: 6; The sRNA sequence targeting tktA is shown in SEQ ID NO: 7; The sequence of the sRNA targeting fabB is shown in SEQ ID NO: 8; The sequence of the sRNA targeting accC is shown in SEQ ID NO: 9; The sequence of the sRNA targeting accB is shown in SEQ ID NO: 10; The sRNA sequence targeting purC is shown in SEQ ID NO: 11; The sequence of the sRNA targeting pyrB is shown in SEQ ID NO: 12; or The sequence of the sRNA targeting glyA is shown in SEQ ID NO:

13.

6. The method as described in claim 1 or 2, characterized in that, The phenylalanine ammonia-lyase and 4-coumaric acid coenzyme A ligase are configured to form a complex.

7. The method as described in claim 6, characterized in that, Phenylalanine ammonia-lyase and 4-coumaric acid coenzyme A can be brought close together through the binding of protein-protein interaction domains and their ligands to obtain a complex, or phenylalanine ammonia-lyase and 4-coumaric acid coenzyme A ligase can be directly linked or linked through a linker to obtain a complex in the form of a fusion protein.

8. The method as described in claim 7, characterized in that, The protein-protein interaction domain includes a PDZ domain, whose ligand is a PDZ ligand; the phenylalanine ammonia-lyase and 4-coumaric acid coenzyme A are fused to the PDZ domain and its ligand, respectively.

9. The method as described in claim 8, characterized in that, The phenylalanine ammonia-lyase is fused with the PDZ domain, and the 4-coumaric acid coenzyme A is fused with the PDZ ligand.

10. A prokaryotic cell for synthesizing flavonoids, characterized in that, The prokaryotic cells include the encoding genes of the following exogenous enzymes: phenylalanine ammonia-lyase, 4-coumarate coenzyme A ligase, chalcone synthase, chalcone isomerase, and flavonoid synthase I; Furthermore, in the prokaryotic cells, the Escherichia coli transmembrane protein thiocyanate enzyme Ygap was upregulated; and in the prokaryotic cells, target genes selected from the following group were downregulated: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, leuA. The prokaryotic cells also include the genes encoding the following exogenous enzymes: flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase. The prokaryotic cells also include exogenous genes encoding enzymes that promote malonyl-CoA production, such as matC, matB, ACS, and FabF. The flavonoid compound is baicalin or scutellarin; The prokaryotic cells are Escherichia coli cells.

11. A reagent kit for producing flavonoids, characterized in that, The kit includes the prokaryotic cells as described in claim 10.

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