WO2023138679A1 - 异源合成黄酮类化合物的调控方法与应用 - Google Patents

异源合成黄酮类化合物的调控方法与应用 Download PDF

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WO2023138679A1
WO2023138679A1 PCT/CN2023/073308 CN2023073308W WO2023138679A1 WO 2023138679 A1 WO2023138679 A1 WO 2023138679A1 CN 2023073308 W CN2023073308 W CN 2023073308W WO 2023138679 A1 WO2023138679 A1 WO 2023138679A1
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srna
target gene
sequence
flavonoids
protein
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French (fr)
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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 invention relates to the technical fields of synthetic biology and medicine, in particular, the invention relates to a regulation method and application of heterologous synthesis of flavonoids.
  • Baicalein and scutellarein are flavonoids mainly found in the traditional Chinese medicine Scutellaria baicalensis. These two active flavonoids only accumulated 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 scutellarein 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 the main sources of flavonoids are extraction and chemical synthesis from plants.
  • plant extraction or chemical synthesis cannot provide a green route for large-scale production due to the use of toxic chemicals and extreme reaction conditions. Therefore, research on microbial synthesis of flavonoids has been carried out in depth.
  • Low product titers are often caused by the introduction of complex heterogeneous pathways from plants, enzyme imbalances and accumulation of intermediate metabolites.
  • 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 scutellarein in engineered yeast and Escherichia coli, but the yields of baicalein and scutellarein were still at a very low level.
  • the object of the present invention is to provide a regulation method and application of heterologous synthesis of flavonoids.
  • a method for synthesizing flavonoids comprising:
  • Recombinant prokaryotic cells which include exogenous coding genes of the following group of enzymes: phenylalanine ammonia lyase (PAL), 4-coumaric acid coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI) and flavone synthase I (FNSI); and, up-regulate Escherichia coli transmembrane protein rhodanase Ygap in the prokaryotic cells; or down-regulate target genes or target genes selected from the following group in the prokaryotic cells Combinations: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, leuA;
  • PAL phenylalanine ammonia lyase
  • 4CL 4-coumaric acid coenzyme A ligase
  • CHS
  • flavonoids are chrysin compounds (such as chrysin or apigenin);
  • R includes H or OH.
  • the prokaryotic cells also include exogenous genes encoding the following enzymes: flavone 6-hydroxylase (F6H), cytochrome P450 oxidoreductase (CPR); (2), the flavonoids are baicalein compounds (such as baicalein or scutellarein).
  • flavone 6-hydroxylase F6H
  • CPR cytochrome P450 oxidoreductase
  • the flavonoids are baicalein compounds (such as baicalein or scutellarein).
  • the down-regulation of the target gene includes: down-regulation of the activity of the target gene, reduction of the effective time of the target gene, down-regulation of the expression or stability of the target gene, and the like.
  • the up-regulation of Escherichia coli transmembrane protein rhodanase Ygap includes: introducing an exogenous Escherichia coli transmembrane protein rhodanase Ygap encoding gene into the prokaryotic cell; preferably, the Escherichia coli transmembrane protein rhodanase Ygap encoding gene is introduced into the prokaryotic cell through an expression vector.
  • the down-regulation of the target gene includes: knocking out or silencing the target gene in the cell, or inhibiting the activity of the target gene; preferably, knocking out or silencing the target gene in the cell includes (but not limited to): silencing the target gene with a specific interfering molecule, performing gene editing with a CRISPR system to knock out the target gene, knocking out the target gene by homologous recombination, or subjecting the target gene to a loss-of-function mutation; preferably, the interfering molecule includes sRNA.
  • the interfering molecule is sRNA
  • the sequence of the sRNA against glpC is shown in SEQ ID NO: 2
  • the sequence of the sRNA against leuA is shown in SEQ ID NO: 3
  • the sequence of the sRNA against leuC is shown in SEQ ID NO: 4
  • the sequence of the sRNA against leuD is shown in SEQ ID NO: 5
  • the sequence of the sRNA against folK is shown in SEQ ID NO: 6
  • the sequence of the RNA is shown in SEQ ID NO: 7
  • the sequence of the sRNA against fabB is shown in SEQ ID NO: 8
  • the sequence of the sRNA against accC is shown in SEQ ID NO: 9
  • the sequence of the sRNA against accB is shown in SEQ ID NO: 10
  • the sequence of the sRNA against purC is shown in SEQ ID NO: 11
  • the sequence of sRNA is shown in SEQ ID NO: 13.
  • the reagents for down-regulating target genes are called down-modulating agents or down-regulating agents (including inhibitors, blockers, antagonists, etc.).
  • the phenylalanine ammonia lyase (PAL) and 4-coumarate-CoA ligase (4CL) are configured to form a complex (complex reactor).
  • the phenylalanine ammonia lyase and the 4-coumaric acid-CoA ligase are approached to obtain a complex through the binding of the protein-protein interaction domain and its ligand, or the phenylalanine ammonia-lyase and the 4-coumaric acid-CoA ligase are connected directly or through a linker to obtain a complex in the form of a fusion protein.
  • the protein-protein interaction domain includes a PDZ domain, and its ligand is PDZ ligand; the phenylalanine ammonia lyase and 4-coumaric acid coenzyme A are respectively fused to the PDZ domain and its ligand; preferably, the phenylalanine ammonia lyase is fused to PDZ, and the 4-coumaric acid coenzyme A is fused to PDZ ligand.
  • phenylalanine ammonia lyase when fused to PDZ, it also includes linking with an ER/K linker (PAL-ER/K-PDZ).
  • the 4-coumaric acid-CoA when fused to PDZ ligand, it also includes linking (GGGGS) 2 linker (PDZlig-(GGGGS) 2 -4CL).
  • 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 an SH3 domain, and its ligand is SH3 ligand; the phenylalanine ammonia lyase and 4-coumaric acid coenzyme A are respectively fused with the SH3 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 an ER/K linker connection (PAL-ER /K-SH3), when the 4-coumaric acid coenzyme A is fused with SH3 ligand, it also includes (GGGGS) 2 linker connection (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-CoA is fused with the PDZ ligand, the PDZ ligand is located at the N-terminus, and the 4-coumaric-acid-CoA is located at the C-terminus.
  • the phenylalanine ammonia lyase 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.
  • the SH3 ligand when the 4-coumaric acid coenzyme A is fused with the SH3 ligand, the SH3 ligand is located at the N terminal, and the 4-coumaric acid coenzyme A is located at the C terminal.
  • the prokaryotic cells also include exogenous genes encoding enzymes that promote malonyl-CoA production; preferably, matC, matB, ACS, and FabF are included.
  • the coding genes of PDZ ligand, 4-coumarate-CoA ligase, phenylalanine ammonia lyase, ER/K, PDZ, flavone synthase I, chalcone synthase, and chalcone isomerase are located in a construct (plasmid).
  • the gene encoding flavone 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 "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 genes encoding SH3lig, 4-coumarate-CoA ligase, phenylalanine ammonia lyase, ER/K, SH3, and chalcone synthase are located in one construct.
  • the gene encoding chalcone isomerase, flavone synthase I is located in one construct when introduced into cells.
  • the prokaryotic cell is a cell having a substrate synthesis pathway of formula (I); preferably, the prokaryotic cell is an Escherichia coli cell.
  • a prokaryotic cell for the synthesis of flavonoids which includes exogenous coding genes of the following group of enzymes: phenylalanine ammonia lyase (PAL), 4-coumaric acid coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI) and flavone synthase I (FNSI); and, the Escherichia coli transmembrane protein rhodanase Ygap is up-regulated in the prokaryotic cell;
  • the target gene or target gene combination is down-regulated: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, leuA;
  • the flavonoids are chrysin compounds (such as chrysin or apigenin).
  • the prokaryotic cells also include exogenous genes encoding the following enzymes: flavone 6-hydroxylase (F6H), cytochrome P450 oxidoreductase (CPR); the flavonoids are baicalein compounds (such as baicalein or scutellarein).
  • F6H flavone 6-hydroxylase
  • CPR cytochrome P450 oxidoreductase
  • the flavonoids are baicalein compounds (such as baicalein or scutellarein).
  • the application of the prokaryotic cells is provided for synthesizing flavonoids; preferably, the flavonoids include: chrysin compounds and baicalein compounds.
  • a kit for producing flavonoids includes said prokaryotic cells.
  • the kit further includes a cell culture medium.
  • the kit further includes a substrate of formula (I).
  • kits for establishing a host cell for the synthesis of flavonoids comprising: (a) constructs (such as expression vectors) expressing phenylalanine ammonia lyase (PAL), 4-coumaric acid-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI) and flavone synthase I (FNSI); (b) constructs (such as expression vectors) expressing E.
  • constructs such as expression vectors
  • PAL phenylalanine ammonia lyase
  • 4CL 4-coumaric acid-CoA ligase
  • CHS chalcone synthase
  • CHI chalcone isomerase
  • FNSI flavone synthase I
  • the promoting the biosynthesis of flavonoids is promoting the synthesis of flavonoids in prokaryotic cells.
  • Figure 1 Comparison of chrysin production between strain J-1 and strain J-2 overexpressing ygaP.
  • Fig. 4 Schematic diagram of the plasmid structure of pET28a-ygaP.
  • Fig. 5 Schematic diagram of the plasmid structure of PET28a-sRNA.
  • Fig. 7 Schematic diagram of the plasmid structure of pET28a-sRNA-leuA.
  • Fig. 8 Schematic diagram of the plasmid structure of pET28a-sRNA-leuC.
  • Fig. 9 Schematic diagram of the plasmid structure of pET28a-sRNA-leuD.
  • Fig. 10 Schematic diagram of the plasmid structure of pET28a-sRNA-folK.
  • FIG. 12 Schematic diagram of the plasmid structure of pET28a-sRNA-fabB.
  • Figure 13 Schematic diagram of the plasmid structure of pET28a-sRNA-accC.
  • Figure 14 Schematic diagram of the plasmid structure of pET28a-sRNA-accB.
  • Figure 15 Schematic diagram of the plasmid structure of pET28a-sRNA-purC.
  • Figure 16 Schematic diagram of the plasmid structure of pET28a-sRNA-pyrB.
  • Figure 17 Schematic diagram of the plasmid structure of pET28a-sRNA-glyA.
  • FIG. 1 Schematic diagram of the plasmid structure of pZZ41.
  • FIG. 19 Schematic diagram of the plasmid structure of pZZ42.
  • Figure 21 Schematic diagram of the synthesis pathway of baicalein and scutellarein produced by fermentation using phenylalanine as a precursor.
  • the present inventors provided a novel optimal transformation strategy for the biosynthesis of flavonoids (such as baicalein compounds/chrysin compounds). Through appropriate gene regulation in the production cells, a significant increase in the production of flavonoids was achieved.
  • flavonoids such as baicalein compounds/chrysin compounds.
  • the invention also discloses the optimized host cell and its application.
  • chrysin (like) compound includes chrysin or variants with its core structure, Isomers, derivatives, precursors, salts, etc. include, for example, chrysin or apigenin.
  • the "baicalein (like) compound” includes baicalein or variants, isomers, derivatives, precursors, salts and the like having its core structure, such as baicalein or scutellarein.
  • exogenous or heterologous 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.
  • synthetically/recombinantly established genes/proteins are “foreign” or “heterologous” with respect to the host cell when introduced into the host cell by genetic engineering methods, although the host cell itself may contain the corresponding gene or produce the corresponding protein.
  • operably linked (linked) or “operably linked (linked)” refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, thus, the promoter region is "operably linked” to the nucleic acid sequence.
  • construct refers to a recombinant DNA molecule comprising the desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. Said “construct (construct)” is usually contained in an expression vector.
  • the PAL, 4CL, CHS, CHI and FNSI proteins are proteins that form the synthesis pathway of chrysinoids or apigeninoids in the expression system.
  • the F6H and CPR proteins are proteins that convert chrysin-like compounds or apigenin-like compounds into baicalein-like compounds or scutellarein-like compounds in an expression system.
  • PAL is derived from Rhodiola rosea (Rhodotorula toruloides), which has the sequence shown in GenBank accession number AAA33883.1; 4CL is derived from parsley (Petroselium crispum), which has the sequence shown in GenBank accession number KF765780.1; CHS is derived from petunia (Petunia X hybrida), which has GenBank accession number KF765 The sequence shown in 781.1; CHI gene is derived from alfalfa (Medicago sativa), which has the sequence shown in GenBank accession number KF765782.1; FNS I is derived from parsley (Petroselium crispum), which has the sequence shown in Swiss-Prot accession number Q7XZQ8.1.
  • Wild-type F6H and CPR have also been identified in the art.
  • F6H is derived from Scutellaria baicalensis, which has the sequence shown in GenBank accession number ASW21050.1.
  • CPR comes from Arabidopsis thaliana, which has the sequence shown in GenBank accession number NP_849472.2.
  • matC is derived from leguminous rhizobium (Rhizobium leguminosarum), which has GenBank registration The sequence shown in No.
  • KF765784.1; matB is derived from Rhizobium leguminosarum (Rhizobium leguminosarum), which has the sequence shown in GenBank accession number AGZ04579.1; 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 The sequence shown in AP023237.1.
  • Baicalein and scutellarein 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 scutellarein is C 15 H 10 O 6 and the molecular weight is 286.24.
  • Their structure is as follows:
  • Chrysin is also a flavonoid compound.
  • chrysin is an upstream compound (intermediate compound) of baicalein or scutellarein, and its structural formula is shown below.
  • the inventors of the present invention are committed to studying the optimal modification strategy for the biosynthesis of flavonoids (such as baicalein compounds/chrysin compounds). After a lot of research, screening and experimental work, the inventors have determined an optimized solution that is beneficial to greatly increase the production of flavonoids, including up-regulating the expression or activity of Escherichia coli transmembrane protein rhodanase Ygap in host cells.
  • flavonoids such as baicalein compounds/chrysin compounds
  • Ygap preferably, also including its upstream genes and downstream genes
  • various methods well known to those skilled in the art can be used to regulate the expression or activity of the Ygap or regulate the relevant upstream genes or downstream genes of Ygap.
  • Ygap or its upstream or downstream genes can be overexpressed by various methods well known to those skilled in the art.
  • up-regulating the expression of the Ygap protein or its coding gene, its upstream or downstream protein or its coding gene includes using an up-regulator of the Ygap protein or its coding gene.
  • the up-regulators may include enhancers, agonists, activators.
  • the "up-regulation” and “promotion” include “up-regulation” and “promotion” of protein activity or “up-regulation” and “promotion” of protein expression, and they are “up-regulation” and "promotion” with statistical significance.
  • any material that can improve the activity of Ygap or its signaling pathway protein (including its upstream and downstream proteins), improve the stability of Ygap or its signaling pathway protein, up-regulate the expression of Ygap or its signaling pathway protein, increase the effective time of Ygap or its signaling pathway protein, increase the phosphorylation/activation level of each protein
  • these substances can be used in the present invention as substances useful for upregulating Ygap or its signaling pathway.
  • They can be chemical compounds, small chemical molecules, biological molecules.
  • the biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.
  • the present invention also provides a method for up-regulating the expression of Ygap in cells, the method comprising: transferring the coding gene of Ygap or the expression construct or vector containing the coding gene into the cells.
  • Ygap or its coding gene can also be subjected to gain-of-function mutations; an expression-enhanced promoter or a tissue-specific promoter can be used to promote the expression of the Ygap coding gene; or, an enhancer can be used to promote the expression of the Ygap coding gene. It should be understood that other methods for up-regulating the expression of Ygap in cells should also be included in the present invention.
  • the present invention also provides another optimization scheme that is conducive to greatly increasing the yield of flavonoids, including down-regulating the target protein selected from the following group or the target gene encoding it in the host cell: pyrB (GenBank accession number CAD6022649.1), accC (GenBank accession number CAD6001830.1), accB (GenBank accession number CAD6001842.1), purC (GenBank accession number CAD60071 97.1), glyA (GenBank Accession No. CAD6006651.1), tktA (GenBank Accession No. CAD6004304.1), fabB (GenBank Accession No. CAD6008062.1), leuD (GenBank Accession No. CAD6022253.1), leuC (GenBank Accession No.
  • the down-regulator of the target gene refers to any substance that can reduce the activity of the target protein, reduce the stability of the target protein or its coding gene, down-regulate the expression of the target protein, reduce the effective time of the target protein, inhibit the transcription and translation of the coding gene of the target protein, or reduce the phosphorylation/activation level of the protein.
  • These substances can be used in the present invention. They can be chemical compounds, small chemical molecules, biological molecules.
  • the biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.
  • the down-regulator is: an interfering RNA molecule or an antisense nucleotide that specifically interferes with the expression of a target gene; or a gene editing reagent that specifically edits a target, and so on.
  • the present invention provides a method for down-regulating the expression of the target gene in a cell, comprising introducing into the cell an interfering molecule that interferes with the expression of the target gene.
  • the interfering molecule is sRNA.
  • Another method for down-regulating the expression of a target gene in a cell includes: performing targeted mutation, gene editing or gene recombination on the target gene, so as to achieve down-regulation.
  • the CRISPR/Cas9 system is used for gene editing, thereby knocking out or down-regulating target genes.
  • Appropriate sgRNA target sites will lead to higher gene editing efficiency, so before proceeding with gene editing, suitable target sites can be designed and found. 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 methods for down-regulating target genes/target proteins in cells are not limited to those listed above.
  • the inventors use enzyme assembly technology to produce baicalein or scutellarein by fermentation.
  • the principle of the scheme is: use interacting protein pairs (such as PDZ and PDZ ligand) to fuse with the enzymes PAL and 4CL in the baicalein synthesis pathway, so that PAL and 4CL can be spontaneously assembled in E. coli to form a dual-enzyme complex reactor, thereby increasing the yield of the target compound.
  • PAL and 4CL can be spontaneously assembled in E. coli to form a dual-enzyme complex reactor, thereby increasing the yield of the target compound.
  • the inventors found for the first time that constructing a complex (composite reactor) between PAL and 4CL can effectively increase the output of the expression system.
  • 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, EH domain.
  • the protein-protein interaction domain may include a domain selected from the following group: PDZ domain, SH3 domain; their corresponding ligands are PDZ ligand (PDZlig) or SH3 ligand (SH3lig).
  • Protein-protein interactions are primarily mediated efficiently by protein domains.
  • PDZ, SH3, WW and other structural domains can recognize and bind a conserved short peptide sequence of ligand protein through one or more recognition "pockets".
  • the PDZ domain usually binds to the C-terminal 4-5 amino acid residues of the ligand protein, which can also bind to the middle sequence of the ligand protein, polymerize with itself or other domains, or bind to lipids on the membrane.
  • PAL and 4CL other methods of forming a complex of PAL and 4CL and retaining the biological activity of PAL and 4CL may also be included, 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 linker (Linker) can be used for connection.
  • aroG especially its aroG fbr (the aroG gene whose position 436 is mutated from G to A), and pheA, especially its pheA fbr gene (the pheA gene (pheA fbr ) whose position 976 is mutated from A to C)
  • aroG especially its aroG fbr (the aroG gene whose position 436 is mutated from G to A)
  • pheA especially its pheA fbr gene (the pheA gene (pheA fbr ) whose position 976 is mutated from A to C
  • aroG especially its aroG fbr (the aroG gene whose position 436 is mutated from G to A)
  • pheA especially its pheA fbr gene (the pheA gene (pheA fbr ) whose position 976 is mutated from A to C)
  • pheA especially its phe
  • the prokaryotic cells of the present invention also include exogenous genes encoding enzymes that promote malonyl-CoA production; preferably, matC, matB, ACS, and FabF are included.
  • the prokaryotic expression system used is a cell with a synthetic pathway for the substrate of formula (I).
  • Commonly used prokaryotic expression systems include Escherichia coli, Bacillus subtilis, etc.; for example, Escherichia coli cells (E. coli), such as Escherichia coli BL21 (DE3).
  • the present invention also includes their analogs.
  • the difference between these analogs and the natural protein may be the difference in amino acid sequence, or the difference in the modified form 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 by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, ⁇ , ⁇ -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 proteins with high homology (such as 70% or higher homology with the specific protein sequence listed; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98% or 99% homology) and proteins with the same function as corresponding polypeptides are also included in the present invention.
  • proteins with high homology such as 70% or higher homology with the specific protein sequence listed; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98% or 99% homology
  • proteins with the same function as corresponding polypeptides are also included in the present invention.
  • Proteins or genes from specific species are listed in the present invention. It should be understood that although the present invention preferably studies Proteins or genes obtained from a specific species, but other proteins or genes obtained from other species that are highly homologous to said protein or gene (such as having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of the present invention.
  • the invention also relates to the invention also provides a polynucleotide sequence encoding the protein of the invention or its conservative variant protein.
  • a polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.
  • the polynucleotide encoding the mutant mature protein of the present invention includes: the coding sequence encoding only the mature protein; the coding sequence of the mature protein and various additional coding sequences; the coding sequence of the mature protein (and optional additional coding sequences) and non-coding sequences.
  • the present invention also includes the polynucleotide sequence formed by codon-optimizing the sequence of the gene, for example, codon-optimizing according to the preference of the host cell.
  • an engineering strain with high yield of baicalein or scutellarein compounds which includes the coding genes of the following exogenous enzymes: F6H, CPR, PAL, 4CL, CHS, CHI and FNSI; and, the engineering strain includes the coding gene of the exogenous Escherichia coli transmembrane protein rhodanase Ygap; or the target gene or target gene combination selected from the following group in the engineering strain is down-regulated: pyrB, accC, accB, purC, glyA, tktA, fabB, leuD, leuC, glpC, folK, leuA.
  • PAL and 4CL form a complex (complex reactor).
  • the engineering strain is cultivated, and phenylalanine or tyrosine is used as a substrate to produce baicalein or scutellarein compounds. Production with phenylalanine or tyrosine as a substrate is suitable for large-scale compound production.
  • the schematic diagram of the synthesis pathway of baicalein and scutellarein produced by fermentation using phenylalanine as a precursor is shown in Figure 21.
  • an engineering strain with high yield of chrysin compounds which includes exogenous coding genes of the following enzymes: PAL, 4CL, CHS, CHI and FNSI; and, the engineering strain includes the coding gene of exogenous Escherichia coli transmembrane protein rhodanase Ygap; or the target gene or target gene combination selected from the following group in the engineering strain is down-regulated: pyrB, accC, accB, purC, glyA, tktA, fabB , leuD, leuC, glpC, folK, leuA.
  • PAL and 4CL form a complex (complex reactor).
  • the recombinant strain is cultivated, and phenylalanine or tyrosine is used as a substrate to produce baicalein or scutellarein compounds. Production with phenylalanine or tyrosine as a substrate is suitable for large-scale compound production.
  • the F6H further includes a polypeptide tag fused thereto, 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 between the polypeptide tag and the F6H, and the connecting peptide does not affect the biological activity of the two.
  • F6H is linked to 2B1, and an improved F6H mutant 2B1trF6H can be obtained.
  • exogenous aroG especially its aroG fbr
  • pheA especially its pheA fbr
  • exogenous aroG can be further introduced into the above-mentioned engineering strains using phenylalanine or tyrosine as a substrate to obtain another recombinant strain, which can use glucose as a substrate to produce baicalein compounds/chrysin compounds.
  • the production with glucose as the substrate has low cost and is very suitable for large-scale compound production.
  • the yield of the target compound can also be increased by scaling up the production.
  • the medium feeding scheme which can continuously provide abundant substrates
  • good fermentor level production conditions such as optimal control of temperature, optimal control of dissolved oxygen, etc.
  • the production can usually be increased by 2 to 1000 times or more.
  • 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 analyzed and identified using known techniques such as high performance liquid chromatography to confirm that the desired compound is obtained.
  • the strain of the invention has good stability, and can realize large-scale cultivation and production of baicalein or scutellarein/chrysin compounds in a bioreactor.
  • the yield of the target compound of the preferred strain of the present invention is very high.
  • Escherichia coli produces baicalein or scutellarein/chrysin compounds to achieve more economical and convenient manufacture of the target compound.
  • the invention also provides a kit comprising engineering strains for producing flavonoids.
  • it may also include the culture medium of prokaryotic cells, substrates for synthesis such as phenylalanine, tyrosine or glucose, baicalein or scutellarein compounds separation or detection reagents.
  • the kit may also include an instruction manual explaining the method for biosynthesizing flavonoids and the like.
  • the present invention also provides a kit for constructing the engineering strain for producing flavonoids.
  • the kit may include a series of constructs, for example, reference may be made to the constructs provided in the examples of the present invention, or other constructs containing the genes but with different gene arrangements or tandem methods.
  • 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 effectively expressed and active after being transferred into cells.
  • the kit may also include prokaryotic cells, culture medium of prokaryotic cells, substrates for synthesis such as phenylalanine, tyrosine or glucose, baicalein or scutellarein compounds separation or detection reagents. More preferably, the kit may also include an instruction manual explaining the method for biosynthesizing flavonoids and the like.
  • PCR Polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • PrimeSTAR Max DNA Polymerase is a product of TAKARA
  • restriction enzymes are all products of NEB.
  • baicalein and scutellarein were purchased from Shanghai Yuanye Biotechnology Co., Ltd.
  • Other reagents were domestic analytically pure or chromatographically pure reagents, which were purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Escherichia coli DH10B was used for gene cloning, and Escherichia coli BL21 (DE3) strain was used for protein expression and production of baicalein and wild scutellarein.
  • pCDFDuet-1, pET28a, pACYCDuet-1 vectors were used for metabolic pathway gene assembly.
  • a plasmid carrying multiple genes was constructed, as shown in Table 3.
  • Preparation of fermentation strains transform the constructed plasmid into Escherichia coli BL21(DE3), invert culture at 37°C for 12 hours, pick positive clones in 2 mL of LB-resistant medium, culture at 37°C, 250 rpm for 10 hours to prepare fermentation seed bacteria.
  • Liquid phase detection conditions A phase: 0.1% formic acid water, B phase: acetonitrile; separation conditions: 0-20min 20% B phase-55% B phase, 20-22min 55% B phase-100% B phase, 22-27min 100% B phase, 27-35min 100% B phase-20% B phase, 35-40min, 20% B phase; detection wavelength: 340nm, column temperature: 30°C.
  • Chromatographic column Thermo syncronis C18 reversed-phase column (250mm ⁇ 4.6mm, 5 ⁇ m).
  • Embodiment 1 ferment engineering bacteria to produce chrysin
  • Transform pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a plasmids into BL21(DE3) to obtain engineering bacteria J-1 for using phenylalanine as a precursor , fermented chrysin.
  • Transform pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-T7 ygaP plasmids into BL21 (DE3) to obtain engineering bacteria J-2 for Using phenylalanine as a precursor, ferment chrysin.
  • Example 2 production of baicalein by fermentation engineering bacteria: overexpression of ygaP
  • pZZ42 pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR
  • pYH38 pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF
  • pET28a plasmids were transformed into BL21(DE3), Obtain engineering bacteria J-3, which is used to ferment baicalein with phenylalanine as precursor.
  • Transform pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-T7ygaP plasmids into BL2 1(DE3), the engineering strain J-4 was obtained, which was used to ferment baicalein with phenylalanine as the precursor.
  • strain LB solid medium (spectinomycin 80 ⁇ g/mL, chloramphenicol 34 ⁇ g/mL, kanamycin 50 ⁇ g/mL) was cultured overnight at 37°C.
  • the pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-glpC plasmids were transformed into BL 21(DE3), the engineering strain glpC- was obtained, which was used to ferment baicalein with phenylalanine as precursor.
  • the pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-leuC plasmids were transformed into BL 21(DE3), obtained the engineering strain leuC-, which was used to ferment baicalein with phenylalanine as the precursor.
  • the pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-leuD plasmids were transformed into BL 21(DE3), the engineering strain leuD- was obtained, which was used to ferment baicalein with phenylalanine as the precursor.
  • the pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-folK plasmids were transformed into BL 21(DE3), obtained engineering bacteria folK-, which was used to ferment baicalein with phenylalanine as precursor.
  • the pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-tkt plasmids were transformed into BL 21(DE3), the engineering strain tktA- was obtained, which was used to ferment baicalein with phenylalanine as precursor.
  • the pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-fabB plasmids were transformed into BL 21(DE3), obtained engineering bacteria fabB-, used to ferment baicalein with phenylalanine as precursor.
  • the pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-accC plasmids were transformed into BL 21(DE3), to obtain engineering bacteria accC-, which is used to ferment baicalein with phenylalanine as precursor.
  • Transform pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-accB plasmids into BL 21(DE3), obtained engineering bacteria accB-, used to ferment baicalein with phenylalanine as precursor.
  • the pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-pyrB plasmids were transformed into BL 21(DE3), obtained engineering bacteria pyrB-, which was used to ferment baicalein with phenylalanine as precursor.
  • Transform pZZ42 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR), pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) and pET28a-sRNA-glyA plasmids into BL 21(DE3), the engineering strain glyA- was obtained, which was used to ferment baicalein with phenylalanine as precursor.
  • strain LB solid medium (spectinomycin 80 ⁇ g/mL, chloramphenicol 34 ⁇ g/mL, kanamycin 50 ⁇ g/mL) was cultured overnight at 37°C.

Abstract

提供了一种合成黄酮类化合物的方法，包括提供重组的原核细胞，在该原核细胞中上调大肠杆菌跨膜蛋白硫氰酸酶Ygap或下调选自下组的靶基因或靶基因组合：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA。还提供了用于合成黄酮类化合物的原核细胞及其应用，以及试剂盒和调控试剂的应用。实现了黄酮类化合物产量的显著性提高。

Description

异源合成黄酮类化合物的调控方法与应用 技术领域
本发明涉及合成生物学及医药技术领域，具体地，本发明涉及异源合成黄酮类化合物的的调控方法与应用。
背景技术
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。
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。
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。
发明内容
本发明的目的在于提供异源合成黄酮类化合物的的调控方法与应用。
在本发明的第一方面，提供一种合成黄酮类化合物的方法，所述方法包括：
(1)提供重组的原核细胞，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，在该原核细胞中上调大肠杆菌跨膜蛋白硫氰酸酶Ygap；或在该原核细胞中下调选自下组的靶基因或靶基因组合：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA；
(2)以式(I)为底物，以所述的原核细胞合成黄酮类化合物，所述黄酮类化合物为白杨素类化合物(如白杨素或芹菜素)；
其中，R包括H或OH。
在一个或多个实施方式中，(1)中，所述原核细胞中还包括外源的以下酶的编码基因：黄酮6-羟化酶(F6H)，细胞色素P450氧化还原酶(CPR)；(2)中，所述黄酮类化合物为黄芩素类化合物(如黄芩素或野黄芩素)。
在一个或多个实施方式中，所述下调靶基因包括：下调靶基因的活性，减少靶基因有效作用时间、下调靶基因的表达或稳定性等。
在一个或多个实施方式中，所述上调大肠杆菌跨膜蛋白硫氰酸酶Ygap包括：在该原核细胞中引入外源的大肠杆菌跨膜蛋白硫氰酸酶Ygap的编码基因；较佳地，所述大肠杆菌跨膜蛋白硫氰酸酶Ygap的编码基因通过表达载体被引入到该原核细胞中。
在一个或多个实施方式中，所述下调靶基因包括：在细胞中敲除或沉默所述靶基因，或抑制所述靶基因的活性；较佳地，在细胞中敲除或沉默所述靶基因包括(但不限于)：以特异性的干扰分子来沉默所述靶基因，以CRISPR系统进行基因编辑从而敲除所述靶基因，以同源重组的方法敲除所述靶基因，或将所述靶基因进行功能丧失性突变；较佳地，所述干扰分子包括sRNA。
在一个或多个实施方式中，所述干扰分子为sRNA，针对glpC的sRNA的序列如SEQ ID NO：2所示；针对leuA的sRNA的序列如SEQ ID NO：3所示；针对leuC的sRNA的序列如SEQ ID NO：4所示；针对leuD的sRNA的序列如SEQ ID NO：5所示；针对folK的sRNA的序列如SEQ ID NO：6所示；针对tktA的sRNA的序列如SEQ ID NO：7所示；针对fabB的sRNA的序列如SEQ ID NO：8所示；针对accC的sRNA的序列如SEQ ID NO：9所示；针对accB的sRNA的序列如SEQ ID NO：10所示；针对purC的sRNA的序列如SEQ ID NO：11所示；针对pyrB的sRNA的序列如SEQ ID NO：12所示；或针对glyA的sRNA的序列如SEQ ID NO：13所示。
在一个或多个实施方式中，用于下调靶基因的试剂称为下调试剂或下调剂(包括抑制剂，阻滞剂，拮抗剂等)。
在一个或多个实施方式中，所述苯丙氨酸解氨酶(PAL)和4-香豆酸辅酶A连接酶(4CL)设置为构成复合体(复合反应器)。
在一个或多个实施方式中，使得苯丙氨酸解氨酶和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以获得复合体，或使得苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶直接连接或通过连接子连接以获得融合蛋白形式的复合体。
在一个或多个实施方式中，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。
在一个或多个实施方式中，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)。
在一个或多个实施方式中，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)。
在一个或多个实施方式中，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。
在一个或多个实施方式中，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(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)。
在一个或多个实施方式中，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。
在一个或多个实施方式中，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。
在一个或多个实施方式中，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。
在一个或多个实施方式中，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。
在一个或多个实施方式中，(1)中，所述原核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。
在一个或多个实施方式中，在引入细胞时，所述PDZ ligand、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。
在一个或多个实施方式中，在引入细胞时，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。
在一个或多个实施方式中，在引入细胞时，所述matC，matB，ACS，FabF的编码基因位于一个构建体中。
在一个或多个实施方式中，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。
在一个或多个实施方式中，在引入细胞时，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。
在一个或多个实施方式中，在引入细胞时，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。
在一个或多个实施方式中，所述的原核细胞为具有式(I)底物合成途径的细胞；较佳地，所述的原核细胞为大肠杆菌细胞。
在本发明的另一方面，提供一种用于合成黄酮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(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，该原核细胞中大肠杆菌跨膜蛋白硫氰酸酶Ygap被上调；或该原核细胞中选自下组的靶基因或靶基因组合被下调：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA；所述黄酮类化合物为白杨素类化合物(如白杨素或芹菜素)。
在一个或多个实施方式中，所述原核细胞中还包括外源的以下酶的编码基因：黄酮6-羟化酶(F6H)，细胞色素P450氧化还原酶(CPR)；所述黄酮类化合物为黄芩素类化合物(如黄芩素或野黄芩素)。
在本发明的另一方面，提供所述的原核细胞的应用，用于合成黄酮类化合物；较佳地，所述的黄酮类化合物包括：白杨素类化合物，黄芩素类化合物。
在本发明的另一方面，提供一种用于生产黄酮类化合物的试剂盒，所述试剂盒中包括所述的原核细胞。
在一个或多个实施方式中，所述试剂盒中还包括细胞培养基。
在一个或多个实施方式中，所述试剂盒中还包括式(I)底物。
在一个或多个实施方式中，所述试剂盒中
在本发明的另一方面，提供一种用于建立合成黄酮类化合物的宿主细胞的试剂盒，所述试剂盒中包括：(a)表达苯丙氨酸解氨酶(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)的构建体(如表达载体)；(b)表达大肠杆菌跨膜蛋白硫氰酸酶Ygap的构建体(如表达载体)；或，表达下调剂的构建体(如表达载体)，所述下调剂下调选自下组的靶基因或靶基因组合：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA；可选地，所述试剂盒中还包括：表达黄酮6-羟化酶，细胞色素P450氧化还原酶的编码基因的构建体。
在本发明的另一方面，提供调控试剂在促进黄酮类化合物的生物合成中的应用，所述的调控试剂选自：(i)大肠杆菌跨膜蛋白硫氰酸酶Ygap或其上调剂(如表达其的表达构建体)；或(ii)下调剂，所述下调剂下调选自下组的靶基因或靶基因组合：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA。
在一个或多个实施方式中，所述的促进黄酮类化合物的生物合成为促进黄酮类化合物在原核细胞中的合成。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、菌株J-1以及过表达ygaP的菌株J-2的白杨素产量比较。
图2、菌株J-3以及过表达ygaP菌株J-4的黄芩素产量比较。
图3、工程菌control以及多种靶基因受到下调调控的菌株的黄芩素的产量的比较。
图4、pET28a-ygaP的质粒结构示意图。
图5、PET28a-sRNA的质粒结构示意图。
图6、pET28a-sRNA-glpC的质粒结构示意图。
图7、pET28a-sRNA-leuA的质粒结构示意图。
图8、pET28a-sRNA-leuC的质粒结构示意图。
图9、pET28a-sRNA-leuD的质粒结构示意图。
图10、pET28a-sRNA-folK的质粒结构示意图。
图11、pET28a-sRNA-tktA的质粒结构示意图。
图12、pET28a-sRNA-fabB的质粒结构示意图。
图13、pET28a-sRNA-accC的质粒结构示意图。
图14、pET28a-sRNA-accB的质粒结构示意图。
图15、pET28a-sRNA-purC的质粒结构示意图。
图16、pET28a-sRNA-pyrB的质粒结构示意图。
图17、pET28a-sRNA-glyA的质粒结构示意图。
图18、pZZ41的质粒结构示意图。
图19、pZZ42的质粒结构示意图。
图20、pYH38的质粒结构示意图。
图21、以苯丙氨酸为前体，进行发酵生成黄芩素、野黄芩素的合成途径示意图。
具体实施方式
本发明人经过深入的研究，提供了新型的黄酮类化合物(如黄芩素类化合物/白杨素类化合物)生物合成的优化改造策略，通过在生产细胞中进行适当的基因调控，实现了黄酮类化合物产量的显著性提高，所述的调控包括上调大肠杆菌跨膜蛋白硫氰酸酶Ygap或下调一种或多种靶基因(pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK或leuA)。本发明也揭示了优化改造后的宿主细胞及其应用。
如本文所用，所述“黄酮类化合物”具有以下母核结构的化合物：
如本文所用，所述“白杨素(类)化合物”包括白杨素或具有其母核结构的变体、 异构体、衍生物、前体、盐等，例如包括白杨素或芹菜素。
如本文所用，所述“黄芩素(类)化合物”包括黄芩素或具有其母核结构的变体、异构体、衍生物、前体、盐等，例如包括黄芩素或野黄芩素。
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，或者是指蛋白质/基因与宿主细胞之间的关系。例如，尽管宿主细胞自身也可能包含相应的基因或产生相应的蛋白质，合成/重组建立的基因/蛋白质当通过基因工程方法引入到宿主细胞中时，其相应于该宿主细胞为“外源的”或“异源的”。
如本文所用，所述的“可操作地连接(相连)”或“操作性连接(相连)”是指两个或多个核酸区域或核酸序列的功能性的空间排列。例如：启动子区被置于相对于目的基因核酸序列的特定位置，使得核酸序列的转录受到该启动子区域的引导，从而，启动子区域被“可操作地连接”到该核酸序列上。
如本文所用，所述的“构建体(构建物)”或“表达构建体(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“构建体(构建物)”通常被包含在表达载体中。
如本文所用，所述的PAL、4CL、CHS、CHI和FNSI蛋白是在表达系统中形成白杨素类化合物或芹菜素类化合物合成途径的蛋白。
如本文所用，所述的F6H和CPR蛋白是在表达系统中转化白杨素类化合物或芹菜素类化合物、生成黄芩素类化合物或野黄芩素类化合物的蛋白。
如本文所用，所述的matC、matB、ACS和/或FabF蛋白在表达系统中促进丙二酰CoA生成的酶。
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。
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来源于黄芩(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。
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。
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C15H10O5，分子量为270.24，而野黄芩素的分子量为C15H10O6，分子量为286.24。它们的结构如下所示：
白杨素也是一种黄酮类化合物，在酶参与的生物合成途径中，白杨素是黄芩素或野黄芩素的上游化合物(中间化合物)，其结构式如下所示。
本发明人致力于研究黄酮类化合物(如黄芩素类化合物/白杨素类化合物)生物合成的优化改造策略。经过大量研究筛选和实验工作，本发明人确定了有利于大幅度提高黄酮类化合物产量的优化方案，包括在宿主细胞中上调大肠杆菌跨膜蛋白硫氰酸酶Ygap的表达或活性。
应理解，在得知了所述Ygap以及包含Ygap的信号通路(较佳地，也包括其上游基因和下游基因)的功能后，可以采用本领域人员熟知的多种方法来调节所述的Ygap的表达或活性或调节Ygap的相关上游基因或下游基因。比如可以采用本领域人员熟知的多种方法来过表达Ygap或其上游基因或下游基因。
本发明中，上调所述的Ygap蛋白或其编码基因、其上游或下游蛋白或其编码基因的表达包括应用Ygap蛋白或其编码基因的上调剂。所述上调剂可包括促进剂、激动剂、激活剂。所述的“上调”、“促进”包括了蛋白活性的“上调”、“促进”或蛋白表达的“上调”、“促进”，且它们为具有统计学意义的“上调”、“促进”。任何可提高Ygap或含有其的信号通路蛋白(包括其上游和下游蛋白)的活性、提高Ygap或包含其的号通路蛋白的稳定性、上调Ygap或包含其的信号通路基因的表达、增加Ygap或包含其的信号通路蛋白有效作用时间的物质、增加各个蛋白的磷酸化/激活水平，这些物质均可用于本发明，作为对于上调Ygap或信号通路有用的物质。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是蛋白水平的。
本发明还提供了一种上调细胞中Ygap表达的方法，所述的方法包括：将Ygap的编码基因或含有所述编码基因的表达构建物或载体转入细胞中。此外，也可对Ygap或其编码基因进行功能获得性突变；以表达增强型启动子或组织特异性启动子促进Ygap的编码基因的表达；或，以增强子促进Ygap的编码基因的表达。应理解，其它的上调细胞中Ygap表达的方法也应被包含在本发明中。
本发明还提供了另一有利于大幅度提高黄酮类化合物产量的优化方案，包括在宿主细胞中下调选自下组的靶蛋白或编码其的靶基因：pyrB(GenBank登录号CAD6022649.1)、accC(GenBank登录号CAD6001830.1)、accB(GenBank登录号CAD6001842.1)、purC(GenBank登录号CAD6007197.1)、glyA(GenBank登录号CAD6006651.1)、tktA(GenBank登录号CAD6004304.1)、fabB(GenBank登录号CAD6008062.1)、leuD(GenBank登录号CAD6022253.1)、leuC(GenBank登录号CAD6022253.1)、glpC(GenBank登录号CAD6008699.1)、folK(GenBank登录号CAD6022053.1)、leuA(GenBank登录号CAD6022244.1)。对此类靶蛋白或其编码具有下调作用的试剂称为下调剂。
本发明中，上述靶基因的下调剂是指任何可降低靶蛋白的活性、降低靶蛋白或其编码基因的稳定性、下调靶蛋白的表达、减少靶蛋白有效作用时间、抑制靶蛋白的编码基因的转录和翻译的物质、或降低蛋白的磷酸化/激活水平，这些物质均可用于本发明。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是蛋白水平的。例如，所述的下调剂是：特异性干扰靶基因表达的干扰RNA分子或反义核苷酸；或是特异性编辑靶的基因编辑试剂，等等。
本发明提供了下调细胞中所述靶基因的表达的方法，包括将干扰靶基因表达的干扰分子引入到细胞中。作为一种更为具体的实施例方式，所述的干扰分子为sRNA。另一种下调细胞中靶基因的表达的方法例如包括：对靶基因进行靶向性地突变、基因编辑或基因重组，从而实现下调。作为一种更为具体的实施例方式，采用CRISPR/Cas9系统进行基因编辑，从而敲除或下调靶基因。合适的sgRNA靶位点，会带来更高的基因编辑效率，所以在着手进行基因编辑前，可以设计并找到合适的靶位点。在设计特异性靶位点后，还需要进行体外细胞活性筛选，以获得有效的靶位点用于后续实验。应理解，下调细胞中靶基因/靶蛋白的方法不限于以上所列举的。
作为本发明的一种优选方式，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。本发明人首次在合成黄芩类化合物/白杨素类化合物的原核表达系统中发现将PAL与4CL构建成复合体(复合反应器)，可极为有效地提高表达系统的产量。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)。
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。
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。
作为本发明的另一种可选的实施方式，可在原核表达系统中过表达aroG、特别是其aroGfbr(第436位由G突变为A的aroG基因)，以及pheA、特别是其pheAfbr基因(第976位由A突变为C的pheA基因(pheAfbr))，构建获得高产苯丙氨酸原核表达系统，在该原核表达系统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。
本发明所述原核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。
本发明中，所用的原核表达系统(原核细胞)是具有式(I)底物合成途径的细胞。常用的原核表达系统包括大肠杆菌、枯草杆菌等；例如可为大肠杆菌细胞(E.coli)，如大肠杆菌BL21(DE3)。
在本发明中所列举的优选的蛋白(包括上述野生型的蛋白，突变型的蛋白)的基础上，本发明还包括它们的类似物。这些类似物与天然蛋白的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性的蛋白。
在本发明中所列举的优选的蛋白(包括上述野生型的蛋白，突变型的蛋白)的基础上，本发明还包括与所述的蛋白同源性高(比如与所列举的具体蛋白序列的同源性为70％或更高；优选地同源性为80％或更高；更优选地同源性为90％或更高，如同源性95％，98％或99％)的、且具有相应多肽相同功能的蛋白也包括在本发明内。
本发明中列举了来自特定物种的蛋白或基因。应理解，虽然本发明中优选研究了 获自特定物种的蛋白或基因，但是获自其它物种的与所述蛋白或基因高度同源(如具有60％以上，如70％，80％，85％、90％、95％、甚至98％序列相同性)的其它蛋白或基因也在本发明考虑的范围之内。
发明还涉及本发明还提供了编码本发明的蛋白或其保守性变异蛋白的多核苷酸序列。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码本发明的突变体成熟蛋白的多核苷酸包括：只编码成熟蛋白的编码序列；成熟蛋白的编码序列和各种附加编码序列；成熟蛋白的编码序列(和任选的附加编码序列)以及非编码序列。
本发明还包括针对所述基因的序列，进行密码子优化后形成的多核苷酸序列，例如，根据宿主细胞的偏好进行密码子优化。
本发明中，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，该工程菌株中包括外源的大肠杆菌跨膜蛋白硫氰酸酶Ygap的编码基因；或该工程菌株中选自下组的靶基因或靶基因组合被下调：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA。较佳地，所述酶被表达后，PAL和4CL构成复合体(复合反应器)。培养该工程菌株，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。以苯丙氨酸为前体，进行发酵生成黄芩素、野黄芩素的合成途径示意图如图21。
本发明中，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，该工程菌株中包括外源的大肠杆菌跨膜蛋白硫氰酸酶Ygap的编码基因；或该工程菌株中选自下组的靶基因或靶基因组合被下调：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA。较佳地，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。
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。
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。
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aroGfbr，以及pheA、特别是其pheAfbr，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。
在建立如本发明优化的表达系统以及利用其进行生产的基础上，本领域人员还可系统研提高黄芩素、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倍或更多倍数。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进一步的增长。
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。
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。
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。
本发明还提供了包含用于生产黄酮类化合物的工程菌株的试剂盒。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酮类化合物的方法的使用说明书等。
本发明还提供了用于构建所述生产黄酮类化合物的工程菌株的试剂盒，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。基因序列可以被插入到不同的表达构建物(如表达载体)中，也可以被插入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酮类化合物的方法的使用说明书等。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社中所述的条件，或按照制造厂商所建议的条件。
1、实验材料
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。
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。
2、本发明所涉及的菌株及质粒
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。
pCDFDuet-1、pET28a、pACYCDuet-1载体用于代谢途径基因装配。
5nm rigid linker ER/K序列：
3、蛋白/基因/sRNA/结构域等
实施例中具体举例的野生型的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，具体如表1。
表1

4、质粒的构建
构建含单个基因的质粒，具体如表2。
表2

构建携带多基因的质粒，具体如表3。
表3

5、大肠杆菌摇瓶发酵白杨素和黄芩素
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。
种子液1％转接至10mL含2％葡萄糖的MOPS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0.5mM的IPTG及500mg/L的苯丙氨酸，22℃，250rpm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。
6、HPLC检测
液相检测条件：A相：0.1％甲酸水，B相：乙腈；分离条件：0-20min 20％B相-55％B相，20-22min 55％B相-100％B相，22-27min 100％B相，27-35min 100％B相-20％B相，35-40min，20％B相；检测波长：340nm，柱温：30℃。
色谱柱：Thermo syncronis C18反相柱(250mm×4.6mm，5μm)。
实施例1、发酵工程菌生产白杨素
1、菌株(J-1)
将pZZ41(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a质粒转化入BL21(DE3)，得到工程菌J-1，用于以苯丙氨酸为前体，发酵白杨素。
2、过表达ygaP菌株(J-2)
将pZZ41(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-T7 ygaP质粒转化入BL21(DE3)，得到工程菌J-2，用于以苯丙氨酸为前体，发酵白杨素。
发酵方法如下：菌株LB固体培养基(壮观霉素80μg/mL，氯霉素34μg/mL，卡那霉素50μg/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，氯霉素34μg/mL，卡那霉素50μg/mL)，转接过夜培养的菌液到新的10mL MOPS液体抗性培养基中37℃，250r/min培养至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0.5mM，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250r/min条件下继续培养72h。取样检测白杨素产量。
结果显示，与菌株J-1相比，过表达ygaP菌株J-2的白杨素产量，提高到2.99倍(图1)。
实施例2、发酵工程菌生产黄芩素：过表达ygaP
1、菌株(J-3)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a质粒转化入BL21(DE3)，得到工程菌J-3，用于以苯丙氨酸为前体，发酵黄芩素。
2、过表达ygaP菌株(J-4)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-T7ygaP质粒转化入BL21(DE3)，得到工程菌J-4，用于以苯丙氨酸为前体，发酵黄芩素。
上述两种菌株进行发酵，发酵方法如下：菌株LB固体培养基(壮观霉素80μg/mL，氯霉素34μg/mL，卡那霉素50μg/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，氯霉素34μg/mL，卡那霉素50μg/mL)，转接过夜培养的菌液到新的10mL MOPS液体抗性培养基中37℃，250r/min培养至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0.5mM，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250r/min条件下继续培养72h。取样检测黄芩素产量。
结果显示，与菌株J-3相比，过表达ygaP菌株J-4的黄芩素产量提高到1.59倍(图2)。
实施例3、黄芩素发酵生产调控：干扰靶基因的表达
1、工程菌control
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA质粒转化入BL21(DE3)，得到工程菌control，用于以苯丙氨酸为前体，发酵黄芩素(该PET28a-sRNA中的sRNA序列无靶点，仅存在sRNA发卡结构)。
2、抑制glpC菌株(glpC-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-glpC质粒转化入BL21(DE3)，得到工程菌glpC-，用于以苯丙氨酸为前体，发酵黄芩素。
3、抑制leuA菌株(leuA-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-leuA质粒转化入BL21(DE3)，得到工程菌leuA-，用于以苯丙氨酸为前体，发酵黄芩素。
4、抑制leuC菌株(leuC-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-leuC质粒转化入BL21(DE3)，得到工程菌leuC-，用于以苯丙氨酸为前体，发酵黄芩素。
5、抑制leuD菌株(leuD-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-leuD质粒转化入BL21(DE3)，得到工程菌leuD-，用于以苯丙氨酸为前体，发酵黄芩素。
6、抑制folK菌株(folK-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-folK质粒转化入BL21(DE3)，得到工程菌folK-，用于以苯丙氨酸为前体，发酵黄芩素。
7、抑制tktA菌株(tktA-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-tkt质粒转化入BL21(DE3)，得到工程菌tktA-，用于以苯丙氨酸为前体，发酵黄芩素。
8、抑制fabB菌株(fabB-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-fabB质粒转化入BL21(DE3)，得到工程菌fabB-，用于以苯丙氨酸为前体，发酵黄芩素。
9、抑制accC菌株(accC-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-accC质粒转化入BL21(DE3)，得到工程菌accC-，用于以苯丙氨酸为前体，发酵黄芩素。
10、抑制accB菌株(accB-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-accB质粒转化入BL21(DE3)，得到工程菌accB-，用于以苯丙氨酸为前体，发酵黄芩素。
11、抑制purC菌株(purC-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-purC质粒转化入BL21(DE3)，得到工程菌purC-，用于以苯丙氨酸为前体，发酵黄芩素。
12、抑制pyrB菌株(pyrB-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-pyrB质粒转化入BL21(DE3)，得到工程菌pyrB-，用于以苯丙氨酸为前体，发酵黄芩素。
13、抑制glyA菌株(glyA-)
将pZZ42(pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI-T72B1trF6H-T7CPR)、pYH38(pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF)和pET28a-sRNA-glyA质粒转化入BL21(DE3)，得到工程菌glyA-，用于以苯丙氨酸为前体，发酵黄芩素。
上述两种菌株进行发酵，发酵方法如下：菌株LB固体培养基(壮观霉素80μg/mL，氯霉素34μg/mL，卡那霉素50μg/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，氯霉素34μg/mL，卡那霉素50μg/mL)，转接过夜培养的菌液到新的10mL MOPS液体抗性培养基中37℃，250r/min培养至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0.5mM，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250r/min条件下继续培养72h。取样检测黄芩素产量。
结果显示，与工程菌株control相比，抑制glpC菌株(glpC-)的黄芩素产量提高到1.34倍，抑制leuA菌株(leuA-)的黄芩素产量提高到1.16倍，抑制leuC菌株(leuC-)的黄芩素产量提高到1.53倍，抑制leuD菌株(leuD-)的黄芩素产量提高到2.32倍，抑制folK菌株(folK-)的黄芩素产量提高到1.3倍，抑制tktA菌株(tktA-)的黄芩素产量提高到3倍，抑制fabB菌株(fabB-)的黄芩素产量提高到2.69倍，抑制accC菌株(accC-)的黄芩素产量提高到5.31倍，抑制accB菌株(accB-)的黄芩素产量提高到5.26倍，抑制purC菌株(purC-)的黄芩素产量提高到3.51倍，抑制pyrB菌株(pyrB-)的黄芩素产量提高到5.66倍，抑制glyA菌株(glyA-)的黄芩素产量提高到3.32倍。(图3)
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。同时，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。

Claims (13)

  1. 一种合成黄酮类化合物的方法，其特征在于，所述方法包括：
    (1)提供重组的原核细胞，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，在该原核细胞中上调大肠杆菌跨膜蛋白硫氰酸酶Ygap；或在该原核细胞中下调选自下组的靶基因或靶基因组合：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA；
    (2)以式(I)为底物，以权利要求1的原核细胞合成黄酮类化合物，所述黄酮类化合物为白杨素类化合物；
    其中，R包括H或OH。
  2. 如权利要求1所述的方法，其特征在于，(1)中，所述原核细胞中还包括外源的以下酶的编码基因：黄酮6-羟化酶，细胞色素P450氧化还原酶；
    (2)中，所述黄酮类化合物为黄芩素类化合物。
  3. 如权利要求1或2所述的方法，其特征在于，所述上调大肠杆菌跨膜蛋白硫氰酸酶Ygap包括：在该原核细胞中引入外源的大肠杆菌跨膜蛋白硫氰酸酶Ygap的编码基因；较佳地，所述大肠杆菌跨膜蛋白硫氰酸酶Ygap的编码基因通过表达载体被引入到该原核细胞中；或
    所述下调靶基因包括：在细胞中敲除或沉默所述靶基因，或抑制所述靶基因的活性；较佳地，在细胞中敲除或沉默所述靶基因包括：以特异性的干扰分子来沉默所述靶基因，以CRISPR系统进行基因编辑从而敲除所述靶基因，以同源重组的方法敲除所述靶基因，或将所述靶基因进行功能丧失性突变；较佳地，所述干扰分子包括sRNA。
  4. 如权利要求3所述的方法，其特征在于，所述干扰分子为sRNA，针对glpC的sRNA的序列如SEQ ID NO：2所示；
    针对leuA的sRNA的序列如SEQ ID NO：3所示；
    针对leuC的sRNA的序列如SEQ ID NO：4所示；
    针对leuD的sRNA的序列如SEQ ID NO：5所示；
    针对folK的sRNA的序列如SEQ ID NO：6所示；
    针对tktA的sRNA的序列如SEQ ID NO：7所示；
    针对fabB的sRNA的序列如SEQ ID NO：8所示；
    针对accC的sRNA的序列如SEQ ID NO：9所示；
    针对accB的sRNA的序列如SEQ ID NO：10所示；
    针对purC的sRNA的序列如SEQ ID NO：11所示；
    针对pyrB的sRNA的序列如SEQ ID NO：12所示；或
    针对glyA的sRNA的序列如SEQ ID NO：13所示。
  5. 如权利要求1或2所述的方法，其特征在于，所述苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶设置为构成复合体；
    较佳地，使得苯丙氨酸解氨酶和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以获得复合体，或使得苯丙氨酸解氨酶和4-香豆酸辅酶A连接酶直接连接或通过连接子连接以获得融合蛋白形式的复合体；
    较佳地，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。
  6. 如权利要求1或2所述的方法，其特征在于，(1)中，所述原核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。
  7. 如权利要求1或2所述的方法，其特征在于，所述的原核细胞为具有式(I)底物合成途径的细胞；较佳地，所述的原核细胞为大肠杆菌细胞。
  8. 一种用于合成黄酮类化合物的原核细胞，其特征在于，所述原核细胞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，该原核细胞中大肠杆菌跨膜蛋白硫氰酸酶Ygap被上调；或该原核细胞中选自下组的靶基因或靶基因组合被下调：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA；
    所述黄酮类化合物为白杨素类化合物。
  9. 如权利要求8所述的原核细胞，其特征在于，所述原核细胞中还包括外源的以下酶的编码基因：黄酮6-羟化酶，细胞色素P450氧化还原酶；所述黄酮类化合物为黄芩素类化合物。
  10. 权利要求8或9所述的原核细胞的应用，用于合成黄酮类化合物；较佳地， 所述的黄酮类化合物包括：白杨素类化合物，黄芩素类化合物。
  11. 一种用于生产黄酮类化合物的试剂盒，其特征在于，所述试剂盒中包括权利要求8或9所述的原核细胞。
  12. 一种用于建立合成黄酮类化合物的宿主细胞的试剂盒，其特征在于，所述试剂盒中包括：
    (a)表达苯丙氨酸解氨酶、4-香豆酸辅酶A连接酶、查尔酮合成酶、查尔酮异构酶和黄酮合成酶I的构建体；
    (b)表达大肠杆菌跨膜蛋白硫氰酸酶Ygap的构建体；或，表达下调剂的构建体，所述下调剂下调选自下组的靶基因或靶基因组合：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA；
    可选地，所述试剂盒中还包括：表达黄酮6-羟化酶，细胞色素P450氧化还原酶的编码基因的构建体。
  13. 调控试剂在促进黄酮类化合物的生物合成中的应用，所述的调控试剂选自：
    (i)大肠杆菌跨膜蛋白硫氰酸酶Ygap或其上调剂；或
    (ii)下调剂，所述下调剂下调选自下组的靶基因或靶基因组合：pyrB、accC、accB、purC、glyA、tktA、fabB、leuD、leuC、glpC、folK、leuA。
PCT/CN2023/073308 2022-01-20 2023-01-20 异源合成黄酮类化合物的调控方法与应用 WO2023138679A1 (zh)

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