WO2022148377A1 - Cellule hôte d'un composé flavonoïde synthétique hétérologue, et son utilisation - Google Patents

Cellule hôte d'un composé flavonoïde synthétique hétérologue, et son utilisation Download PDF

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WO2022148377A1
WO2022148377A1 PCT/CN2022/070316 CN2022070316W WO2022148377A1 WO 2022148377 A1 WO2022148377 A1 WO 2022148377A1 CN 2022070316 W CN2022070316 W CN 2022070316W WO 2022148377 A1 WO2022148377 A1 WO 2022148377A1
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gene
ammonia lyase
phenylalanine ammonia
pdz
coumaric acid
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王勇
李建华
计东尼
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中国科学院分子植物科学卓越创新中心
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Definitions

  • the present invention relates to the technical field of synthetic biology and medicine, and in particular, the present invention relates to host cells for heterologous synthesis of flavonoids and applications thereof.
  • Baicalein and scutellarin are flavonoids, which are mainly found in the traditional Chinese medicine Scutellaria baicalensis. These two active flavonoids only accumulate less in the roots of related medicinal plants such as Scutellaria baicalensis. Both baicalein and baicalein are synthesized through the flavonoid biosynthetic pathway. Baicalein and scutellarin have important physiological activities such as antioxidant, antitumor, antibacterial, and heart protection. Recently, baicalein was reported as an inhibitor of SARS-CoV-2 3Clpro in vitro, showing the great potential of traditional Chinese medicine.
  • flavonoids are extraction and chemical synthesis from plants.
  • plant extraction or chemical synthesis cannot provide a green route to mass production due to the use of toxic chemicals and extreme reaction conditions. Therefore, the research on microbial synthesis of flavonoids has been intensively carried out.
  • Low product titers typically result from enzyme imbalances and accumulation of intermediate metabolites due to complex heterogeneous pathways introduced from plants.
  • a multivariate modular approach was employed to synthesize flavonoids by modulating promoter strength and plasmid copy number.
  • it is time consuming and always requires a lot of work.
  • Previous work reported the synthesis of baicalein and scutellarin in engineered yeast and E. coli, but the yields of baicalein and scutellarin were still at very low levels.
  • the purpose of the present invention is to provide a host cell for heterologous synthesis of baicalein, scutellarin or chrysin compounds and its application.
  • a prokaryotic cell for synthesizing baicalein and scutellarin-like compounds (such as baicalein or scutellarin), which comprises an exogenous encoding gene of the following group of enzymes: flavonoid 6- Hydroxylase (F6H), Cytochrome P450 oxidoreductase (CPR), Phenylalanine ammonia lyase (PAL), 4-Coumarate CoA ligase (4CL), Chalcone synthase (CHS), Chalcone isomerase (CHI) and flavonoid synthase I (FNSI); and after the enzymes are expressed, phenylalanine ammonia lyase (PAL) and 4-coumarate coenzyme A ligase (4CL) constitute Complex (compound reactor).
  • flavonoid 6- Hydroxylase F6H
  • Cytochrome P450 oxidoreductase CPR
  • Phenylalanine ammonia lyase PAL
  • a prokaryotic cell for synthesizing chrysin-based compounds (such as chrysin or apigenin) is provided, which comprises an exogenous encoding gene of the following group of enzymes: phenylalanine ammonia lyase ( PAL), 4-coumarate coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI) and flavonoid synthase I (FNSI); and after the enzymes are expressed , phenylalanine ammonia lyase (PAL) and 4-coumarate coenzyme A ligase (4CL) constitute a complex (complex reactor).
  • PAL phenylalanine ammonia lyase
  • 4-coumarate coenzyme A ligase (4CL) constitute a complex (complex reactor).
  • the complex of phenylalanine ammonia lyase and 4-coumaric acid coenzyme A ligase comprises: phenylalanine ammonia lyase and 4-coumaric acid coenzyme A through protein-protein The binding of the interacting domains and their ligands brings them closer together, resulting in a complex.
  • the phenylalanine ammonia lyase and 4-coumarate coenzyme A ligase are directly connected or connected through a linker to obtain a complex in the form of a fusion protein.
  • the protein-protein interaction domain comprises a domain selected from the group consisting of: PDZ domain, SH3 domain, WW domain, LIM domain, DD domain, PH domain, EH domain, GBD domain.
  • the protein-protein interaction domain includes a PDZ domain, and its ligand is a PDZ ligand; the phenylalanine ammonia lyase and 4-coumarate coenzyme A are respectively associated with the PDZ Domain and its ligand are fused; preferably, the phenylalanine ammonia lyase is fused with PDZ, and the 4-coumaric acid coenzyme A is fused with PDZ ligand; more preferably, the phenylalanine ammonia lyase is fused with PDZ;
  • the aminoase is fused with PDZ, it also includes connecting with an ER/K linker (PAL-ER/K-PDZ), and when the 4-coumaric acid coenzyme A is fused with PDZ ligand, it also includes connecting with a (GGGGS) 2 linker ( PDZlig-(GGGGS) 2-4CL ).
  • the protein-protein interaction domain includes an SH3 domain, and its ligand is SH3ligand; the phenylalanine ammonia lyase and 4-coumarate coenzyme A are respectively associated with the SH3 structure Domain and its ligand are fused; preferably, the phenylalanine ammonia lyase is fused with SH3, and the 4-coumaric acid coenzyme A is fused with SH3 ligand; more preferably, the phenylalanine ammonia lyase is fused with SH3
  • the enzyme is fused with SH3, it also includes connecting with an ER/K linker (PAL-ER/K-SH3), and when the 4-coumaric acid coenzyme A is fused with SH3 ligand, it also includes connecting with a (GGGGS) 2 linker (SH3lig -(GGGGS) 2-4CL ).
  • the phenylalanine ammonia lyase when the phenylalanine ammonia lyase is fused with PDZ, the phenylalanine ammonia lyase is located at the N-terminus, and the PDZ is located at the C-terminus.
  • the PDZ ligand when the 4-coumaric acid coenzyme A is fused to the PDZ ligand, the PDZ ligand is located at the N-terminus, and the 4-coumaric acid-coenzyme A is located at the C-terminus.
  • the phenylalanine ammonia lyase when fused with SH3; the phenylalanine ammonia lyase is located at the N-terminus, and the SH3 is located at the C-terminus.
  • the SH3 ligand when the 4-coumaric acid coenzyme A is fused to the SH3 ligand, the SH3 ligand is located at the N-terminus, and the 4-coumaric acid coenzyme A is located at the C-terminus.
  • the cell also includes an exogenous gene encoding an enzyme that promotes the production of malonyl CoA; preferably, it includes matC, matB, ACS, and FabF.
  • the prokaryotic cells are Escherichia coli cells.
  • the cell also includes an exogenous gene encoding an enzyme that promotes phenylalanine synthesis; preferably, it includes: aroG, pheA; more preferably, the pheA is at position 976 composed of A gene mutated from A to C; more preferably, the aroG is a gene mutated from G to A at position 436.
  • the "promoting” is a statistically significant "promoting", such as promoting more than 5%, more than 10%, more than 20%, more than 50%, more than 80%, more than 100% or higher.
  • the application of the prokaryotic cells is provided for synthesizing baicalein or scutellarin compounds.
  • the application of the prokaryotic cells is provided for synthesizing chrysin compounds.
  • a method for synthesizing baicalein or scutellarin compounds comprising: providing the prokaryotic cells (containing F6H and CPR), and using formula (I) as a substrate to synthesize baicalein or scutellarin compounds;
  • R includes H or OH.
  • a method for synthesizing chrysin compounds comprising: providing the prokaryotic cells (which may not contain F6H and CPR), and using formula (I) as a substrate to synthesize chrysin compounds.
  • a method for synthesizing baicalein or scutellarin-like compounds or chrysin-like compounds comprising: providing the prokaryotic cells, and using glucose as a substrate to synthesize baicalein or scutellarin compounds or chrysin-like compounds.
  • the gene encoding chalcone isomerase is located in one construct (plasmid).
  • the encoding gene for flavonoid 6-hydroxylase and cytochrome P450 oxidoreductase is located in a construct, preferably also including 2B1 (cytochrome P450 2B1 family soluble protein) gene.
  • the genes encoding matC, matB, ACS, and FabF are located in one construct.
  • the genes encoding SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, and chalcone synthase are located in one construct.
  • the gene encoding chalcone isomerase and flavonoid synthase I is located in one construct.
  • the encoding genes of matC, matB, ACS, and FabF are pheA gene (pheA fbr ) mutated from A to C at position 976, and aroG gene (aroG fbr ) mutated from G to A at position 436 ) in one construct.
  • kits for producing baicalein or scutellarin-based compounds (containing F6H and CPR in the kit) or chrysin-based compounds (without F6H and CPR in the kit), It includes said recombinant host cells.
  • kits for establishing a host cell for synthesizing baicalein or scutellarin-like compounds or chrysin-like compounds comprising: comprising PDZligand, 4-coumaric acid-CoA ligase , phenylalanine ammonia lyase, ER/K, PDZ, flavonoid synthase I, chalcone synthase, chalcone isomerase encoding gene constructs; including matC, matB, ACS, FabF encoding genes Constructs of SH3lig, 4-coumaric acid coenzyme A ligase, phenylalanine ammonia lyase, ER/K, SH3, constructs encoding genes for chalcone synthase; containing chalcone isomerase , a construct encoding the gene encoding flavonoid synthase I; including the encoding genes of matC, matB,
  • the kit further includes: glucose; or, the substrate of formula (I).
  • Figure 1 Schematic diagram of the construction of plasmid pZZ41.
  • Figure 2 Schematic diagram of the construction of plasmid pZZ55.
  • Figure 3 The bar graph of the baicalein production of the non-self-assembling strain DN-1 and the self-assembling strain DN-2.
  • Figure 5 Bar graph of baicalein production of self-assembled strain DN-4 compared to control strain DN-3.
  • Figure 6 The bar graph of the scutellarin production of the non-self-assembling strain DN-1 and the self-assembling strain DN-2.
  • Figure 7 The bar graph of the baicalein production of the self-assembly engineering strain DN-6 and the non-self-assembly engineering strain DN-5.
  • Fig. 8 is a schematic diagram of the synthetic pathway for fermenting to generate baicalein and baicalein using phenylalanine as a precursor.
  • Figure 9 is a schematic diagram of the synthetic pathway of baicalein produced by fermentation with glucose as a precursor.
  • the inventors provide a novel biosynthesis optimization of baicalein or scutellarin compounds/chrysin compounds, which can realize the synthesis of baicalein or scutellarin using enzyme self-assembly technology on the basis of prokaryotes scutellarin-like compounds/chrysin-like compounds, and de novo synthesis of baicalein or scutellaria-like compounds/chrysin-like compounds using glucose.
  • the invention also discloses the optimized modified host cell and its application.
  • align or “heterologous” refers to the relationship between two or more nucleic acid or protein sequences from different sources.
  • operably linked (linked) or “operably linked (linked)” refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific location relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, and thus, the promoter region is "operably linked” to the nucleic acid sequence.
  • an "expression construct” refers to a recombinant DNA molecule comprising the desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes.
  • the "construct” is usually contained in an expression vector.
  • the PAL, 4CL, CHS, CHI and FNSI proteins are those that form the chrysin or apigenin synthetic pathway in an expression system.
  • the F6H and CPR proteins are proteins that convert chrysin or apigenin to produce baicalein or scutellarin-like compounds in an expression system.
  • the matC, matB, ACS and/or FabF proteins described are enzymes that promote malonyl-CoA production in an expression system.
  • aroG or a mutant thereof, pheA or a mutant thereof promotes phenylalanine synthesis in an expression system.
  • PAL is derived from Rhodiola (Rhodotorula toruloides), which has the sequence shown in GenBank Accession No. AAA33883.1; 4CL is derived from Parsley (Petroselium crispum), which has GenBank Accession No. KF765780.1.
  • CHS is derived from petunia (Petunia X hybrida), which has the sequence shown in GenBank accession number KF765781.1
  • CHI gene is derived from alfalfa (Medicago sativa), which has the sequence shown in GenBank accession number KF765782.1 Sequence
  • FNS I is derived from Parsley (Petroselium crispum), which has the sequence shown in Swiss-Prot Accession No. Q7XZQ8.1.
  • Wild-type F6H and CPR have also been identified in the art.
  • F6H is derived from Scutellaria baicalensis, which has the sequence shown in GenBank accession number ASW21050.1.
  • the CPR is derived from Arabidopsis thaliana, which has the sequence shown in GenBank accession number NP_849472.2.
  • matC is derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number KF765784.1
  • matB is derived from Rhizobium leguminosarum, which has GenBank accession number AGZ04579
  • the sequence shown in .1; ACS is derived from Escherichia coli (Escherichia coli), which has the sequence shown in GenBank accession number CP062211.1
  • FabF is derived from Escherichia coli (Escherichia coli), which has GenBank accession number shown in AP023237.1 the sequence of.
  • Baicalein and scutellarin are two structurally similar and important flavonoids.
  • the molecular formula of baicalein is C 15 H 10 O 5 and the molecular weight is 270.24, while the molecular weight of baicalein is C 15 H 10 O 6 and the molecular weight is 286.24.
  • Their structure is as follows:
  • scutellarin compounds such as baicalein or scutellarin
  • their precursor chrysin compounds such as chrysin or apigenin
  • chrysin compounds such as chrysin or apigenin
  • the inventors utilize enzyme assembly technology to ferment and produce baicalein or scutellarin.
  • the principle of this scheme is to use interacting protein pairs (such as PDZ and PDZ ligand) to fuse with enzymes PAL and 4CL in the baicalein synthesis pathway, so that PAL and 4CL can spontaneously assemble in E. coli to form a dual-enzyme complex reactor, thereby increasing the yield of the target compound.
  • interacting protein pairs such as PDZ and PDZ ligand
  • the inventors found for the first time in the prokaryotic expression system for synthesizing scutellaria compound/chrysin compound that PAL and 4CL are constructed into a complex (composite reactor), which can extremely effectively improve the yield of the expression system.
  • Any biological material or technical means suitable for making PAL and 4CL into an active complex can be used in the present invention.
  • the protein-protein interaction domain may comprise a domain selected from the group consisting of: PDZ domain, SH3 domain, WW domain, LIM domain, DD domain, PH domain , the EH domain.
  • the protein-protein interaction domain may comprise a domain selected from the group consisting of: PDZ domain, SH3 domain; their corresponding ligands are PDZ ligand (PDZlig) or SH3ligand(SH3lig).
  • Protein-protein interactions are efficiently mediated mainly by protein domains.
  • PDZ, SH3, WW and other domains can recognize and bind a short conserved peptide sequence of ligand proteins through one or more recognition "pockets".
  • the PDZ domain In the case of the PDZ domain, it usually binds to the C-terminal 4-5 amino acid residues of the ligand protein, and it is also capable of binding to the intermediate sequence of the ligand protein, polymerizing with itself or other domains, or binding to lipids on the membrane .
  • fusion between PAL and 4CL can be a direct connection or a linker can be used for connection.
  • the inventors overexpressed aroG, especially its aroG fbr , and pheA, especially its pheA fbr gene in a prokaryotic expression system, and constructed a prokaryotic expression system to obtain high-yield phenylalanine,
  • the exogenous baicalein or scutellarin compound/chrysin compound synthesis pathway is introduced into the prokaryotic expression system, so that the strain can utilize glucose to synthesize baicalein compound/chrysin compound de novo.
  • E. coli E. coli
  • Bacillus subtilis etc.
  • E. coli cells E. coli
  • E. coli BL21 DE3
  • the present invention also includes their analogs.
  • the differences between these analogs and the native protein may be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both.
  • These proteins include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other known molecular biology techniques.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.
  • the present invention also includes high homology with the proteins (for example, the homology with the specific protein sequences listed is 70 % or more; preferably 80% or more homology; more preferably 90% or more homology, such as 95%, 98% or 99% homology), and have the same function as the corresponding polypeptide
  • the homology with the specific protein sequences listed is 70 % or more; preferably 80% or more homology; more preferably 90% or more homology, such as 95%, 98% or 99% homology
  • the protein is also included in the present invention.
  • Proteins or genes from specific species are enumerated in the present invention. It should be understood that although proteins or genes obtained from a specific species are preferably studied in the present invention, those obtained from other species are highly homologous to said proteins or genes (eg have more than 60%, such as 70%, 80%, 85% , 90%, 95%, or even 98% sequence identity) other proteins or genes are also contemplated by the present invention.
  • the present invention also relates to the present invention also provides a polynucleotide sequence encoding the protein of the present invention or a conservative variant thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • Polynucleotides encoding the mutant mature proteins of the present invention include: coding sequences encoding only the mature protein; coding sequences for the mature protein and various additional coding sequences; coding sequences for the mature protein (and optional additional coding sequences) and non- coding sequence.
  • the present invention also includes polynucleotide sequences formed by codon optimization for the sequence of the gene, eg, codon optimization according to the preference of the host cell.
  • an engineering strain with high production of baicalein or scutellarin compounds is also constructed, which includes the encoding genes of the exogenous lower group enzymes: F6H, CPR, PAL, 4CL, CHS, CHI and FNSI; and the enzymes After being expressed, PAL and 4CL form a complex (complex reactor).
  • the recombinant strain is cultivated, and takes phenylalanine or tyrosine as a substrate to produce baicalein or scutellarin compounds.
  • the production using phenylalanine or tyrosine as a substrate is suitable for large-scale compound production.
  • an engineered strain with high production of chrysin compounds is also constructed, which includes exogenous encoding genes of the following group of enzymes: PAL, 4CL, CHS, CHI and FNSI; and after the enzymes are expressed, PAL and 4CL constitute Complex (compound reactor).
  • the recombinant strain is cultivated, and takes phenylalanine or tyrosine as a substrate to produce baicalein or scutellarin compounds.
  • the production using phenylalanine or tyrosine as a substrate is suitable for large-scale compound production.
  • the F6H further includes a polypeptide tag fused to it, for example, the polypeptide tag is selected from: 8RP, Sumo, MBP, 2B1, or a combination thereof; preferably 2B1.
  • a connecting peptide may or may not be included, and the connecting peptide does not affect the biological activity of the two. F6H was linked to 2B1 to obtain an improved F6H mutant 2B1trF6H.
  • the upstream generation pathway of the above-mentioned substrate can be further introduced, for example, including: generated by glucose or glycerol through glycolysis, pentose phosphate pathway, shikimic acid pathway Phenylalanine or tyrosine.
  • phenylalanine or tyrosine can be further introduced, for example, including: generated by glucose or glycerol through glycolysis, pentose phosphate pathway, shikimic acid pathway Phenylalanine or tyrosine.
  • protocols for the formation of phenylalanine or tyrosine based on such pathways are also encompassed by the present invention.
  • Methods of enhancing the phenylalanine or tyrosine-forming pathway by means known in the art can be included in the present invention.
  • exogenous aroG, especially its aroG fbr , and pheA, especially its pheA fbr can be further introduced into the above-mentioned engineering strain using phenylalanine or tyrosine as a substrate to obtain another A recombinant strain capable of producing baicalein compounds/chrysin compounds using glucose as a substrate.
  • the production using glucose as a substrate has low cost and is very suitable for large-scale compound production.
  • the yield of the target compound can also be increased by expanding the production scale.
  • the medium feeding scheme can be carried out (which can provide abundant substrates continuously), or the production conditions of good fermenter level (such as temperature When the optimal control of oxygen is used, the optimal control of dissolved oxygen, etc.), its output can usually be increased by 2 to 1000 times.
  • the fermentation product After the fermentation product is obtained, techniques known in the art can be used to extract the target compound from the fermentation product.
  • the product can be analytically identified using well known techniques such as high performance liquid chromatography to confirm that the desired compound has been obtained.
  • the strain of the invention has good stability, and can realize large-scale cultivation and production of baicalein or scutellarin compounds/chrysin compounds in a bioreactor.
  • the yields of the target compounds of the preferred strains of the present invention are very high.
  • microbial fermentation has the advantages of fast speed and less influence by external factors; the yield of some compounds through microbial synthesis is much higher than that of plant extraction, and has become an important means of obtaining natural products.
  • baicalein or scutellarin compound/chrysin compound through Escherichia coli, more economical and convenient manufacture of the target compound is achieved.
  • the invention also provides a kit for producing engineering strains of baicalein or scutellarin compounds.
  • the culture medium of prokaryotic cells can also be included, substrates for synthesis such as phenylalanine, tyrosine or glucose, baicalein or scutellarin-based compound separation or detection reagents.
  • the kit may further include instructions for use that describe the method for biosynthesizing xanthophylls, and the like.
  • the present invention also provides a kit for constructing the engineering strain for producing baicalein or scutellarin-like compounds/chrysin-like compounds, the kit may include a series of constructs, for example, refer to the implementation of the present invention
  • the constructs provided in the examples can also be other constructs that contain the genes but the gene arrangement or tandem manner is different.
  • Expression vectors expression constructs
  • Gene sequences can be inserted into different expression constructs (such as expression vectors), or into the same expression construct, as long as the encoded polypeptide can be efficiently expressed and active after being transferred into cells .
  • the kit can also include prokaryotic cells, culture medium of prokaryotic cells, substrates for synthesis such as phenylalanine, tyrosine or glucose, and baicalein or scutellarin compounds for separation or detection reagents. More preferably, the kit may further include instructions for use that describe the method for biosynthesizing baicalein or scutellarin.
  • PCR Polymerase chain reaction
  • gel recovery kits and plasmid extraction kits are products of American Axygen
  • PrimeSTAR Max DNA Polymerase is a product of TAKARA
  • restriction The endonucleases are all NEB products.
  • baicalein and scutellarin were purchased from Shanghai Yuanye Biotechnology Co., Ltd.
  • Other reagents were domestic analytical or chromatographic pure reagents, purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Arktik Thermal Cycler (Thermo Fisher Scientific) was used for PCR; ZXGP-A2050 incubator and ZWY-211G incubator were used for constant temperature incubation; 5418R high-speed refrigerated centrifuge and 5418 mini-centrifuge (Eppendorf) were used for centrifugation. Concentrator plus concentrator (Eppendorf) was used for vacuum concentration; OD600 was detected by UV-1200 UV-Vis spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.).
  • the rotary evaporation system consists of an IKA RV 10digital rotary evaporator (IKA), a MZ 2CNT chemical diaphragm pump, and a CVC3000 vacuum controller (vacuubrand). High performance liquid chromatography was performed using a Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific).
  • Escherichia coli DH10B was used for gene cloning, and Escherichia coli BL21(DE3) strain was used for protein expression and production of baicalein and scutellarin.
  • pCDFDuet-1 pETDuet-1
  • pACYCDuet-1 vectors were used for metabolic pathway gene assembly.
  • PAL derived from Rhodiola (Rhodotorula touloides), which has the sequence shown in GenBank accession number AAA33883.1 (RtPAL);
  • CHS derived from petunia (Petunia X hybrida), which has the sequence shown in GenBank accession number KF765781.1;
  • CHI gene derived from alfalfa (Medicago sativa), it has the sequence shown in GenBank accession number KF765782.1;
  • FNS I derived from parsley (Petroselium crispum), which has the sequence shown in Swiss-Prot accession number Q7XZQ8.1;
  • F6H derived from Scutellaria baicalensis, which has the sequence shown in GenBank accession number ASW21050.1;
  • CPR from Arabidopsis thaliana, which has the sequence shown in GenBank Accession No. NP_849472.2.
  • PDZ domain derived from mouse Mouse ⁇ -syntrophin (syn), 77-171 amino acid sequence, which has the sequence shown in GenBank accession number EDL06069.
  • matB gene derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number AGZ04579.1.
  • matC gene derived from Rhizobium leguminosarum, which has the sequence shown in GenBank accession number KF765784.1.
  • ACS gene derived from Escherichia coli, which has the sequence shown in GenBank accession number CP062211.1.
  • PDZ ligand sequence is GVKESLV (SEQ ID NO: 12).
  • SH3 domain AEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWN AEDSEGKRGMIPVPYVEKY (SEQ ID NO: 13).
  • SH3lig PPPALPPKRRR (SEQ ID NO: 14).
  • NcoI restriction site was added to the N-terminus of PAL gene, and EcoRI restriction site was added to the C-terminus of PDZ sequence.
  • PAL, 5nm rigid linker ER/K, PDZ were genetically fused by Over-Lap PCR method, and pCDFDuet- 1. Connect the fusion gene PAL-ER/K-PDZ obtained after Over-Lap PCR to the NcoI and EcoRI sites of pCDFDuet-1 to obtain pCDFDuet1-T7PAL-ER/K-PDZ.
  • the PDZ ligand sequence was designed in the upstream primer, NcoI restriction site was added to the N-terminus, and BamHI restriction site was added to the C-terminus of the 4CL sequence.
  • the PDZlig-4CL fusion gene was obtained by PCR, and the fusion gene was constructed into the NcoI and BamHI of pCDFDuet-1. site, resulting in pCDFDuet1-T7PDZlig-4CL.
  • the PDZ ligand sequence was designed in the downstream primer, the NcoI restriction site was added to the N-terminus of 4CL, and the BamHI restriction site was added to the C-terminus of PDZ ligand.
  • the 4CL-PDZlig fusion gene was obtained by PCR, and the fusion gene was constructed into the NcoI and NcoI of pCDFDuet-1. BamHI site, resulting in pCDFDuet1-T7 4CL-PDZlig.
  • the pheA gene was cloned from the BL21 (DE3) genome.
  • the NcoI restriction site was added to the N-terminus of the pheA gene, and the BamHI restriction site was added to the C-terminus.
  • the 976 position of the pheA gene was mutated from A to C to obtain pheA fbr .
  • the vector backbone was selected from pETDuet-1, and the pheA fbr gene was connected to the NcoI and BamHI sites of pCDFDuet-1 to obtain pCDFDuet1-T7pheA fbr .
  • the aroG gene was cloned from the BL21 (DE3) genome.
  • the NcoI restriction site was added to the N-terminus of the aroG gene, and the BamHI restriction site was added to the C-terminus.
  • the 436 position of the aroG gene was mutated from G to A to obtain aroG fbr .
  • the vector backbone was selected from pETDuet-1, and the aroG fbr gene was connected to the NcoI and BamHI sites of pCDFDuet-1 to obtain pCDFDuet1-T7aroG fbr .
  • pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF): The matC gene and matB gene were obtained by PCR; the NcoI restriction site was added to the N-terminus of the matC gene, and the HindIII restriction site was added to the C-terminus. Linked to the NcoI and HindIII sites of pACYCDuet1 (the site has the T7 promoter that comes with the vector) to obtain T7matC. A HindIII site was added to the N-terminus of the T7matC gene site, and an Acc65I site was added to the C-terminus.
  • the T7matC gene was obtained by PCR, and the T7matC gene was linked to the HindIII and Acc65I sites of pACYCDuet1.
  • the Acc65I site was added to the N-terminus of the T7ACS gene, and the NotI site was added to the C-terminus.
  • the T7ACS gene was obtained by PCR, and the T7ACS gene was connected to the Acc65I and NotI sites of pACYCDuet1.
  • the NotI site was added to the N-terminus of the T7FabF gene, and the XbaI site was added to the C-terminus.
  • the T7FabF gene was obtained by PCR, and the T7FabF gene was connected to the NotI and XbaI sites of pACYCDuet1.
  • the pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF plasmid was obtained.
  • pZZ12 pCDFDuet1-T7-CHI-FNSI
  • NcoI site was added to the N-terminal of CHI gene, and HindIII site was added to C-terminal
  • CHI gene was obtained by PCR, and the CHI gene was connected to the NcoI and HindIII sites of pCDFDuet1.
  • a HindIII site was added to the N-terminus of the FNSI gene, and an EcoRI site was added to the C-terminus, and the FNSI gene was obtained by PCR, which was connected to the HindIII and EcoRI sites of pCDFDuet1.
  • pCDFDuet1-T7-CHI-FNSI was obtained.
  • pZZ22 pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS
  • NcoI site was added to the N-terminal of SH3lig gene
  • BamHI site was added to the C-terminal of 4CL gene.
  • Over-lap PCR fused SH3lig and 4CL to obtain the fusion gene SH3lig-4CL, which was connected to the NcoI and BamHI sites of pET28a.
  • the fusion gene PAL-ER/K was obtained by Over-Lap PCR, the BamHI site was added to the N-terminus of the PAL-ER/K gene, and the EcoRI site was added to the C-terminus of the SH3 gene.
  • PAL-ER/K-SH3 It was fused with the SH3 gene to obtain the PAL-ER/K-SH3 gene, and the PAL-ER/K-SH3 was linked to the BamHI and EcoRI sites of pET28a.
  • the EcoRI site was added to the N-terminal of the CHS gene, and the SalI site was added to the C-terminal, and the CHS was connected to the EcoRI and SalI sites of pET28a.
  • Plasmid pZZ22 Plasmid pZZ22 (pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS) was obtained.
  • NcoI site was added to the N-terminal of 4CL gene
  • BamHI site was added to the C-terminal of 4CL gene
  • pET28a was used as a vector to connect 4CL to the NcoI and BamHI sites of pET28a.
  • the BamHI site was added to the N-terminus of the PAL-ER/K gene
  • the EcoRI site was added to the C-terminus of the SH3 gene.
  • Over-Lap PCR was used to fuse the PAL-ER/K gene and the SH3 gene to obtain PAL-ER/K-SH3 gene, ligating PAL-ER/K-SH3 to the BamHI and EcoRI sites of pET28a.
  • the EcoRI site was added to the N-terminal of the CHS gene, and the SalI site was added to the C-terminal, and the CHS was connected to the EcoRI and SalI sites of pET28a.
  • Plasmid pZZ23 (pET28a-T7-4CL-PAL-ER/K-SH3-CHS) was obtained.
  • pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI): the N-terminal of the PAL-ER/K-PDZ fusion gene constructed in 4(1) was added with a BamHI enzyme cleavage site , an EcoRI restriction site was added to the C-terminal and inserted into pCDFDuet1 to obtain pCDFDuet1-T7PAL-ER/K-PDZ.
  • the gene PAL-ER/K-PDZ was constructed into the BamHI and EcoRI sites of the pCDFDuet1-T7PDZlig-4CL plasmid after PCR to obtain pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/ K-PDZ.
  • the T7FNSI-T7CHS-T7CHI gene was digested from the pYH57 plasmid using HindIII and AvrII double digestion, and connected to the HindIII and AvrII restriction sites of the above plasmid to obtain pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ -T7FNSI-T7CHS-T7CHI) plasmid.
  • pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ): Design primers in front of the T7 promoter of the pCDFDuet1-T7aroG fbr plasmid, add a BamHI restriction site, and add an EcoRI restriction site at the C-terminal, using pETDuet1-T7aroG fbr as a template, The T7aroG fbr fragment was obtained by PCR, and the fragment was ligated into the BamHI and EcoRI sites of the pCDFDuet1-T7pheA fbr plasmid to obtain the pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ) plasmid.
  • pZZ55 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr -T7aroG fbr ): using the pZZ52 (pETDuet1-T7pheA fbr -T7aroG fbr ) plasmid as a template, design primers upstream of the T7 promoter to increase the AvrII plasmid, which increases at the C-terminus AvrII digestion site, PCR clone to obtain T7pheA fbr- T7aroG fbr fragment, T7pheA fbr -T7aroG fbr was ligated to the AvrII site of pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) using a one-step cloning kit to obtain
  • the detailed information of the plasmid is shown in Table 1, the cell information is shown in Table 2, and the schematic diagram of the plasmid construction is shown in Figures 1 to 2.
  • the constructed plasmid was transformed into Escherichia coli BL21 (DE3), and the positive clones were picked in 2 mL of LB-resistant medium after inverted culture at 37°C for 12 hours, and cultured at 37°C and 250 rpm for 10 hours to prepare fermented seed bacteria, fermentation engineering bacteria See Table 2 for details.
  • Amino acid, fermented at 22°C, 220rpm for 3 days sampled 1mL, sonicated the bacterial liquid for 3 times, mixed with equal volume of ethyl acetate and extracted twice, centrifuged at 12000rpm, 2min, transferred the organic phase to a new tube, and evaporated to dryness at room temperature or 30°C. Then, add 200 ⁇ L of methanol to reconstitute (concentrate 5 times) and mix well, transfer the supernatant to HPLC for 2 min at 12000 rpm.
  • phase A 0.1% formic acid water
  • phase B acetonitrile
  • separation conditions 0-20min 20% phase B-55% phase B, 20-22min 55% phase B-100% phase B, 22-27min 100% phase B, 27-35min 100% phase B-20% phase B, 35-40min, 20% phase B
  • detection wavelength 340nm
  • column temperature 30°C.
  • Chromatographic column Thermo syncronis C18 reversed-phase column (250mm ⁇ 4.6mm, 5 ⁇ m).
  • Rhodiola Rhodotorula toruloides
  • SEQ ID NO: 1 The PAL (RtPAL) length from Rhodiola (Rhodotorula toruloides) is 693aa (GenBank accession number AAA33883.1), and the specific sequence is as follows (SEQ ID NO: 1):
  • Transformation 1 The inventors carried out sequence transformation for SEQ ID NO: 1, the amino acid added a 5nm rigid linker ER/K linker at the C end to obtain an improved PAL mutant PAL-ER/K, the specific sequence is as follows (SEQ ID NO: 2):
  • Transformation 2 The inventors carried out sequence transformation for SEQ ID NO: 2, and then added the amino acid sequence of PDZ to the C-terminal to obtain an improved PAL-ER/K mutant PAL-ER/K-PDZ, the specific sequence is as follows (SEQ ID NO: 2) ID NO: 3):
  • PAL is located at positions 1 to 693 of SEQ ID NO: 3; ER/K is located at positions 694 to 729 of SEQ ID NO: 3; PDZ is located at positions 730 to 824 of SEQ ID NO: 3.
  • Transformation 3 The inventors carried out sequence transformation for SEQ ID NO: 2, and then added the amino acid sequence of SH3 at the C end to obtain an improved PAL-ER/K mutant PAL-ER/K-SH3, the specific sequence is as follows (SEQ ID NO: 2) ID NO: 4):
  • PAL is located at positions 1 to 693 of SEQ ID NO: 4; ER/K is located at positions 694 to 729 of SEQ ID NO: 3; PDZ is located at positions 730 to 786 of SEQ ID NO: 3.
  • the length of 4CL (Pc4CL) from parsley (Petroselium crispum) is 544aa (GenBank accession number KF765780.1), and the specific sequence is as follows (SEQ ID NO: 5):
  • Transformation 1 The inventors carried out sequence transformation for SEQ ID NO: 5, removed the first amino acid, and added the amino acid sequence of (GGGGS) 2 to the N-terminus to obtain an improved 4CL mutant (GGGGS) 2-4CL ,
  • the specific sequence is as follows (SEQ ID NO: 6):
  • Modification 2 The inventors carried out sequence modification according to SEQ ID NO: 6, then added the amino acid sequence of PDZlig to the N-terminus, and then added the M amino acid to the N-terminus to obtain an improved (GGGGS) 2-4CL mutant PDZlig-(GGGGS ) 2-4CL , the specific sequence is as follows (SEQ ID NO: 7):
  • PDZlig is located at positions 1-8 of SEQ ID NO: 7; (GGGGS) 2 is located at positions 9-18 of SEQ ID NO: 7; 4CL is located at positions 19-561 of SEQ ID NO: 7.
  • Modification 3 The inventors carried out sequence modification for SEQ ID NO: 5, and added the amino acid sequence of (GGGGS) 2 to the C-terminus to obtain an improved 4CL mutant 4CL-(GGGGS) 2 , the specific sequence is as follows (SEQ ID NO: 8):
  • Transformation 4 The inventors carried out sequence transformation for SEQ ID NO: 8, and added the amino acid sequence of PDZlig to the C-terminal to obtain an improved 4CL mutant 4CL-(GGGGS) 2 -PDZlig, the specific sequence is as follows (SEQ ID NO: 9 ):
  • 4CL is located at positions 1-544 of SEQ ID NO: 9; (GGGGS) 2 is located at positions 545-554 of SEQ ID NO: 10; PDZlig is located at positions 555-561 of SEQ ID NO: 9.
  • Transformation 5 The inventors carried out sequence transformation according to SEQ ID NO: 6, then added the amino acid sequence of SH3lig to the N-terminus, and then added the M amino acid to the N-terminus to obtain an improved (GGGGS) 2-4CL mutant SH3lig-(GGGGS ) 2-4CL (SH3lig-4CL for short), the specific sequence is as follows (SEQ ID NO: 10):
  • SH3lig is located at positions 1-12 of SEQ ID NO: 10; (GGGGS) 2 is located at positions 13-22 of SEQ ID NO: 10; 4CL is located at positions 23-565 of SEQ ID NO: 10.
  • Transformation 6 The inventors carried out sequence transformation according to SEQ ID NO: 8, and added the amino acid sequence of SH3lig to the C-terminal to obtain an improved 4CL mutant 4CL-(GGGGS) 2 -SH3lig, the specific sequence is as follows (SEQ ID NO: 11 ):
  • 4CL is located at positions 1-544 of SEQ ID NO: 11; (GGGGS) 2 is located at positions 545-554 of SEQ ID NO: 10; SH3lig is located at positions 555-565 of SEQ ID NO: 10.
  • Embodiment 2 fermentation engineering bacteria detect chrysin
  • the pYH57 (pCDFDuet1-T74CL-T7PAL-T7FNSI-T7CHS-T7CHI) plasmid was transformed into BL21 (DE3) to obtain an engineered strain DN-1, which was used to ferment chrysin using phenylalanine as a precursor.
  • the pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid was transformed into BL21 (DE3) to obtain self-assembly engineering bacteria DN-0, which was used to use phenylalanine as a precursor , fermented chrysin.
  • Embodiment 3 fermentation engineering bacteria detect baicalein
  • phenylalanine is used as a precursor for fermentation, and the synthesis route is shown in Figure 8.
  • the pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid, pYH66 (pETDuet1-T72B1-trF6H-T7CPR) plasmid, pYH38 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF) plasmid Co-transformed into BL21 (DE3) to obtain self-assembly engineering bacteria DN-2, which was used to ferment baicalein with phenylalanine as a precursor.
  • the pZZ23 (pET28a-T7-4CL-PAL-ER/K-SH3-CHS) plasmid, the pZZ12 (pCDFDuet1-CHI-FNSI) plasmid, and the plasmids were co-transformed into BL21 (DE3) to obtain the control engineering bacteria DN-3, which was treated with benzene.
  • Alanine is used as a precursor to ferment baicalein with phenylalanine as a precursor.
  • the pZZ22 (pET28a-SH3lig-T7-4CL-PAL-ER/K-SH3-CHS) plasmid, the pZZ12 (pCDFDuet1-T7-CHI-FNSI) plasmid, and the plasmids were co-transformed into BL21 (DE3) to obtain the self-assembly engineering bacteria DN -4, for the fermentation of baicalein with phenylalanine as the precursor.
  • strain LB solid medium (spectinomycin 80 ⁇ g/mL, ampicillin 100 ⁇ g/mL, chloramphenicol 34 ⁇ g/mL) was cultured overnight at 37°C.
  • PAL modification 2 For the comparison of various modification schemes, the inventors preferred PAL modification 2, 4CL modification 2 or PAL modification 3, 4CL modification 5.
  • Embodiment 4 fermentation engineering bacteria detect scutellarin
  • tyrosine was used as a precursor for fermentation.
  • a non-assembled engineered bacterium DN-1 was obtained, which was used to ferment baicalein with tyrosine as a precursor.
  • the self-assembly engineering bacteria DN-2 was obtained, which was used to ferment baicalein with tyrosine as a precursor.
  • the above two strains were cultured in LB solid medium (spectinomycin 80 ⁇ g/mL, ampicillin 100 ⁇ g/mL, chloramphenicol 34 ⁇ g/mL) overnight at 37°C.
  • transfer the overnight culture into a new 10mL M9Y liquid resistance medium 37 °C, 250r/min culture to OD600 0.5-0.6, water bath cooling to about 16 °C, then add inducer IPTG to the final concentration of 0.2mM, add the final concentration of 500mg/L sterilized tyrosine and transfer to 22 Induced culture at °C low temperature, and continued to culture for 72 h at a shaking speed of 220 r/min.
  • glucose is used as a precursor for fermentation, and the synthesis route is shown in Figure 9.
  • the pZZ41 (pCDFDuet1-T7PDZlig-4CL-T7PAL-ER/K-PDZ-T7FNSI-T7CHS-T7CHI) plasmid, pYH66 (pETDuet1-T72B1trF6H-T7CPR) plasmid, pZZ55 (pACYCDuet1-T7matC-T7matB-T7ACS-T7FabF-T7pheA fbr- T7aroG fbr ) plasmid was co-transformed into BL21 (DE3) to obtain a self-assembled engineering bacterium DN-6 using the interaction scheme of PDZ and PDZlig, which was used to synthesize baicalein from glucose.
  • the two strains established above were cultured on LB solid medium (spectinomycin 80 ⁇ g/mL, ampicillin 100 ⁇ g/mL, chloramphenicol 34 ⁇ g/mL) overnight at 37°C.
  • transfer the overnight culture into a new 10mL M9Y liquid resistance medium 37°C, 250r/min culture to OD600 0.5-0.6, cooling down to about 16°C in water bath, then adding inducer IPTG to a final concentration of 0.2mM, and transferring to 22°C low temperature induction culture, under the condition of shaking table rotation speed 220r/min

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Abstract

L'invention concerne une cellule hôte d'un composé synthétique hétérologue de baicaleine, scutellaréine ou chrysine, et son utilisation. L'invention concerne un nouveau type de modification optimisée pour la biosynthèse d'un composé de baicaléine, de scutellaréine ou de chrysine, ladite modification pouvant réaliser la synthèse du composé de baicaléine, de scutellaréine et de chrysine en utilisant une technique d'auto-assemblage enzymatique avec des procaryotes comme substrats, et réaliser la synthèse de novo des composés de baicaléine en utilisant du glucose. L'invention concerne également une cellule hôte ayant subi une modification optimisée, et son utilisation.
PCT/CN2022/070316 2021-01-05 2022-01-05 Cellule hôte d'un composé flavonoïde synthétique hétérologue, et son utilisation WO2022148377A1 (fr)

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WO2013067173A1 (fr) * 2011-11-02 2013-05-10 The Board Of Trustees Of The Leland Stanford Junior University Contrôle systématique d'interaction protéique à l'aide de lieur modulaire er/k
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US20200181677A1 (en) * 2017-06-05 2020-06-11 Regents Of The University Of Minnesota Screening system and methods for identifying enzyme substrates and modulators of enzyme activity
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US20200181677A1 (en) * 2017-06-05 2020-06-11 Regents Of The University Of Minnesota Screening system and methods for identifying enzyme substrates and modulators of enzyme activity
CN107723317A (zh) * 2017-11-29 2018-02-23 华东理工大学 一种在大肠杆菌中生产衣康酸的方法
CN111440734A (zh) * 2020-04-07 2020-07-24 华东理工大学 生产黄芩素类化合物的基因工程酵母菌、其构建方法及应用

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XIAN KUN, ET AL.: "Construction of Eukaryotic Expression Vectors of FNSII-1 and CHS-2 of Scutellaria Baicalensis Georgi", SHANXI NONGYE DAXUE XUEBAO (ZIRAN KEXUE BAN) - SHANXI UNIVERSITY OF AGRICULTURE. JOURNAL (NATURAL SCIENCE EDITION), SHANXI NONGYE DAXUE, CN, vol. 40, no. 1, 31 December 2020 (2020-12-31), CN , pages 11 - 16, XP055949430, ISSN: 1671-8151, DOI: 10.13842/j.cnki.issn1671-8151.201908021 *

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