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WO2025050732A1 - Procédé de synthèse efficace d'un composé de protoberbérine et son utilisation - Google Patents

Procédé de synthèse efficace d'un composé de protoberbérine et son utilisation Download PDF

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WO2025050732A1
WO2025050732A1 PCT/CN2024/097774 CN2024097774W WO2025050732A1 WO 2025050732 A1 WO2025050732 A1 WO 2025050732A1 CN 2024097774 W CN2024097774 W CN 2024097774W WO 2025050732 A1 WO2025050732 A1 WO 2025050732A1
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enzyme
catalyzes
generate
dopamine
enzymes
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王勇
刘海利
张前
李建华
孙雨伟
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Center for Excellence in Molecular Plant Sciences of CAS
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of biosynthesis, and specifically relates to a method for efficiently synthesizing protoberberine compounds and an application thereof.
  • Benzylisoquinoline alkaloids are a class of natural products with various medicinal values produced by tyrosine metabolism, including protoberberine alkaloids, phthalide isoquinoline alkaloids, morphinan alkaloids, etc.
  • protoberberine compounds are widely distributed in the rhizomes of plants in the Berberidaceae, Ranunculaceae and Papaveraceae, such as golden corydaline, tetrahydroafricanine, berberine and tetrahydropalmatine.
  • the preparation methods of protoberberine include plant extraction, chemical synthesis and biosynthesis.
  • the methods of extracting bioactive ingredients from plants are limited by the following factors: 1. The planting area of plants is small; 2. The time required for plant growth is long; 3. The content of bioactive ingredients is low; 4. The loss of extraction methods is large.
  • the technology of chemical synthesis is mature, there are still many problems in the synthesis of complex natural products, including the complexity of synthesizing certain racemic compounds and the pollution of chemical synthesis pathways.
  • the present invention aims to efficiently synthesize protoberberine compounds or reticulated annonaine.
  • the first aspect of the present invention provides a nucleic acid construct for increasing the production of protoberberine compounds in cells, which comprises a coding sequence encoding the following enzymes: an enzyme that catalyzes the hydroxylation of tyrosine to produce dopa, an enzyme that catalyzes the decarboxylation of dopa to produce dopamine, an enzyme that catalyzes dopamine to produce 3,4-dihydroxyphenylacetaldehyde, an enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlaudanine, an enzyme that catalyzes norlaudanine to produce 3'hydroxycoclaurine, an enzyme that catalyzes 3'hydroxycoclaurine to produce 3'hydroxynitromethylcoclaurine, an enzyme that catalyzes 3'hydroxynitromethylcoclaurine to produce reticuline, an enzyme that catalyzes reticuline to produce golden corydaline, an enzyme that cata
  • the nucleic acid construct further comprises a coding sequence for an enzyme of a BH4 synthesis pathway and a coding sequence for an enzyme of a BH4 regeneration pathway, wherein the enzyme of the BH4 synthesis pathway is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthase and dihydropterin reductase, and the enzyme of the BH4 regeneration pathway comprises: 4 ⁇ -hydroxytetrahydropterin dehydratase and/or dihydropterin reductase,
  • the enzymes of the BH4 synthesis pathway include GTP cyclohydrolase from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthase from Rattus norvegicus, and indole reductase from Rattus norvegicus.
  • the enzymes of the BH4 regeneration pathway include: 4 ⁇ -hydroxytetrahydropterin dehydratase from Rattus norvegicus and dihydropterin reductase from Rattus norvegicus.
  • the nucleic acid construct further comprises an enzyme that catalyzes the hydroxylation of tyramine to generate dopamine.
  • the enzyme that catalyzes the hydroxylation of tyramine to generate dopamine is HpaBC or a variant thereof.
  • the nucleic acid construct further comprises a coding sequence for a purine nucleic acid kinase.
  • the nucleic acid construct further comprises a coding sequence for nuclease activity regulatory protein A and/or a coding sequence for cytochrome b5.
  • the enzyme that catalyzes the hydroxylation of tyrosine to produce DOPA is TYH or a variant thereof.
  • the enzyme that catalyzes the decarboxylation of dopa to form dopamine is DODC.
  • the enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine is MAO.
  • the enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlabdanine is NCS.
  • the enzyme that catalyzes the production of 3'-hydroxycoclaurine from norlabradanine is 6OMT.
  • the enzyme that catalyzes the production of 3'hydroxycoclaurine from 3'hydroxynitromethylcoclaurine is CNMT.
  • the enzyme that catalyzes the production of reticuline from 3'-hydroxyl nitrogen methylcoclaurine is 4'OMT.
  • the enzyme that catalyzes the production of chrysophylline from reticuline is BBE1 or a variant thereof.
  • the enzyme that catalyzes the production of tetrahydroafricanine from chrysocorrhizine is 9'OMT.
  • the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetramine is CAS.
  • the enzyme that reduces the oxidized state of cytochrome P450 is CPR2.
  • the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine eg, CAS
  • the enzyme that reduces the oxidized state of cytochrome P450 eg, CPR2
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are fused with the protein interaction domain and its ligand to form separate fusion proteins; or the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are fused to form a fusion protein.
  • the protein interaction domain comprises a domain selected from the group consisting of a PDZ domain, an SH3 domain, a WW domain, a LIM domain, a DD domain, a PH domain, an EH domain, and a GBD domain.
  • the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are respectively bound to the SH3 domain and its ligand.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused to SH3, and the enzyme that reduces the oxidized state of cytochrome P450 is fused to SH3Lig.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is connected to SH3 through a linker, and the enzyme that reduces the oxidized state of cytochrome P450 is connected to SH3Lig through a linker.
  • the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetracycline is located at the N-terminus of SH3.
  • the enzyme that reduces the oxidized state of cytochrome P450 is located at the N-terminus of SH3Lig.
  • the amino acid sequence of the linker is as shown in SEQ ID NO: 72, and the nucleic acid sequence is as shown in SEQ ID NO: 63.
  • amino acid sequence of the SH3 domain is as shown in SEQ ID NO: 73
  • nucleic acid sequence is as shown in SEQ ID NO: 64.
  • amino acid sequence of the SH3Lig is shown as SEQ ID NO: 74, and the nucleic acid sequence is shown as SEQ ID NO: 65.
  • TYH is from Rattus norvegicus.
  • the TYH variant has mutations at positions R37, R38 and W166; preferably, the mutations are R37E, R38E and W166Y.
  • the HpaBC is from E. coli.
  • the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.
  • the DODC is from Pseudomonas putida.
  • the MAO is from Micrococcus luteus.
  • the NCS is from Coptis chinensis.
  • the 6OMT is from opium poppy.
  • the CNMT is from opium poppy.
  • 4'OMT is from Coptis chinensis.
  • BBE1 is from Poppy; preferably, the BBE1 contains a truncated N-terminal transmembrane region, more preferably, the BBE1 contains a truncated N-terminal transmembrane region and is fused with a sequence of an 8RP solubility-promoting tag.
  • the BBE1 variant has a mutation at one or both of F398 and I431.
  • 9'OMT is from Coptis chinensis.
  • the CAS is from Coptis chinensis.
  • CPR2 is from Arabidopsis thaliana.
  • the sequence of TYH of Rattus norvegicus is as shown in SEQ ID NO: 1.
  • the sequence of Drosophila TYH is shown as SEQ ID NO: 2.
  • the sequence of MtrA is as shown in SEQ ID NO: 3.
  • the sequence of PTPS is as shown in SEQ ID NO: 4.
  • sequence of SPR is as shown in SEQ ID NO: 5.
  • sequence of PCD is as shown in SEQ ID NO: 6.
  • sequence of QDHPR is as shown in SEQ ID NO: 7.
  • sequence of the HpaBC variant is shown as SEQ ID NO: 8.
  • the sequence of DODC is as shown in SEQ ID NO: 9.
  • sequence of MAO is as shown in SEQ ID NO: 10.
  • sequence of NCS is as shown in SEQ ID NO: 11.
  • sequence of 6OMT is as shown in SEQ ID NO: 12.
  • the sequence of CNMT is as shown in SEQ ID NO: 13.
  • sequence of 4’OMT is as shown in SEQ ID NO: 14.
  • BBE1 or a variant thereof (1) has a sequence as shown in SEQ ID NO: 15, (2) having the sequence shown in SEQ ID NO: 15 and having one or two mutations at positions selected from F398 and I431, wherein the mutation is a substitution, deletion or addition of an amino acid residue, (3) being a truncated variant of (1) having amino acids 36-554 of SEQ ID NO: 15, (4) having a sequence that is at least 70% identical to the sequence shown in (1)-(3) and retains berberine bridge enzyme activity, or (5) having a sequence of the sequence shown in (1)-(4) fused to the N-terminus of an 8RP solubility-enhancing tag.
  • sequence of 9’OMT is as shown in SEQ ID NO: 16.
  • sequence of CAS is shown as SEQ ID NO: 17.
  • the sequence of CPR2 is as shown in SEQ ID NO: 18.
  • sequence of DeoD is shown as SEQ ID NO: 19.
  • the sequence of RraA is shown as SEQ ID NO: 20.
  • the sequence of cytochrome b5 is as shown in SEQ ID NO: 21 or 68 or 70.
  • the nucleic acid construct further comprises a coding sequence encoding one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase ( aroGfbr ) that resists feedback inhibition, chorismate translocase and prephenate dehydrogenase ( tyrAfbr ) that resists feedback inhibition, phosphoenolpyruvate synthase (ppsA), transketolase that increases erythrose-4-phosphate synthesis (tktA), and shikimate dehydrogenase (ydiB).
  • aroGfbr 3-deoxy-D-arabinoheptulose-7-phosphate synthase
  • tyrAfbr chorismate translocase and prephenate dehydrogenase
  • ppsA phosphoenolpyruvate synthase
  • tktA transketolase that increases erythrose-4-phosphate synthesis
  • aroG, tyrA, ppsA, tktA, ydiB are derived from E. coli.
  • amino acid sequence of aroG fbr is as shown in SEQ ID NO:22.
  • amino acid sequence of tyrA fbr is as shown in SEQ ID NO:23.
  • amino acid sequence of ppsA is as shown in SEQ ID NO: 24.
  • amino acid sequence of tktA is as shown in SEQ ID NO: 25.
  • amino acid sequence of ydiB is as shown in SEQ ID NO: 26.
  • the nucleic acid construct further comprises a polynucleotide that inhibits the expression or activity of pyruvate kinase, such as dsRNA, sRNA or sgRNA.
  • a polynucleotide that inhibits the expression or activity of pyruvate kinase such as dsRNA, sRNA or sgRNA.
  • the sequence of the sRNA is as shown in SEQ ID NO: 27.
  • the nucleic acid construct further comprises a polynucleotide that inhibits pheA expression, such as dsRNA, sRNA or sgRNA.
  • a polynucleotide that inhibits pheA expression such as dsRNA, sRNA or sgRNA.
  • the N20 sequence of the sgRNA is as shown in SEQ ID NO: 28.
  • the nucleic acid construct further comprises a polynucleotide that inhibits the expression of an aromatic amino acid transcriptional regulator (tyrR), such as a dsRNA, sRNA, or sgRNA.
  • an aromatic amino acid transcriptional regulator such as a dsRNA, sRNA, or sgRNA.
  • the N20 sequence of the sgRNA is as shown in SEQ ID NO: 29 and/or 30.
  • the nucleic acid construct is a vector.
  • the coding sequence of the enzyme is in one, two, three or more expression frames.
  • the coding sequence is operably linked to an expression control sequence.
  • the first aspect of the present invention also provides a host cell with improved production of protoberberine compounds, wherein the host cell:
  • an enzyme that catalyzes the hydroxylation of tyrosine to form dopa an enzyme that catalyzes the decarboxylation of dopa to form dopamine, an enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine, an enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to form norlaudanine, an enzyme that catalyzes the production of 3'hydroxycoclaurine from norlaudanine, an enzyme that catalyzes the production of 3'hydroxynitromethylcoclaurine from 3'hydroxynitromethylcoclaurine, an enzyme that catalyzes the production of reticuline from reticuline, an enzyme that catalyzes the production of chrysanthemumine from chrysanthemumine, an enzyme that catalyzes the production of tetrahydroafricanine from tetrahydr
  • the cell further expresses enzymes of the BH 4 synthesis pathway and enzymes of the BH 4 regeneration pathway, wherein the enzymes of the BH 4 synthesis pathway include one or more or all selected from the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase, and the enzymes of the BH 4 regeneration pathway include: 4 ⁇ -hydroxytetrahydropterin dehydratase and/or dihydropterin reductase.
  • the enzymes of the BH 4 synthesis pathway include GTP cyclohydrolase from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthetase from Rattus norvegicus, and indole reductase from Rattus norvegicus.
  • the enzymes of the BH 4 regeneration pathway include: 4 ⁇ -hydroxytetrahydropterin dehydratase of Rattus norvegicus and dihydropterin reductase of Rattus norvegicus.
  • the cells further express purine nucleic acid kinase (DeoD).
  • DesoD purine nucleic acid kinase
  • the cell further expresses nuclease activity regulatory protein (RraA) and/or cytochrome b5.
  • NraA nuclease activity regulatory protein
  • cytochrome b5 cytochrome b5.
  • the cell further expresses an enzyme that catalyzes the hydroxylation of tyramine to generate dopamine.
  • the enzyme that catalyzes the hydroxylation of tyramine to generate dopamine is HpaBC or a variant thereof.
  • the cell co-expresses an enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetracycline and an enzyme that reduces cytochrome P450 in an oxidized state.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are fused with the protein interaction domain and its ligand and expressed respectively; or the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are fused and expressed.
  • the protein interaction domain comprises a domain selected from the group consisting of a PDZ domain, an SH3 domain, a WW domain, a LIM domain, a DD domain, a PH domain, an EH domain, and a GBD domain.
  • the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are fused with the SH3 domain and its ligand respectively.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3, and the enzyme that reduces the oxidized state of cytochrome P450 is fused with SH3Lig.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is linked to SH3 through a ligand.
  • the enzyme of the reduced oxidized cytochrome P450 is expressed by fusion with SH3Lig through a linker.
  • the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetracycline is located at the N-terminus of SH3.
  • the enzyme that reduces the oxidized state of cytochrome P450 is located at the N-terminus of SH3Lig.
  • the enzyme that catalyzes the hydroxylation of tyrosine to produce DOPA is TYH or a variant thereof.
  • the enzyme that catalyzes the decarboxylation of dopa to form dopamine is DODC.
  • the enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine is MAO.
  • the enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlabdanine is NCS.
  • the enzyme that catalyzes the production of 3'-hydroxycoclaurine from norlabdanine is 6OMT.
  • the enzyme that catalyzes the production of 3'hydroxycoclaurine from 3'hydroxynitromethylcoclaurine is CNMT.
  • the enzyme that catalyzes the production of reticuline from 3' hydroxyl nitrogen methylcoclaurine is 4' OMT.
  • the enzyme that catalyzes the production of chrysophylline from reticuline is BBE1 or a variant thereof.
  • the enzyme that catalyzes the production of tetrahydrotetrandrine from chrysocorrhizine is 9'OMT.
  • the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetramine is CAS.
  • the enzyme that reduces the oxidized state of cytochrome P450 is CPR2.
  • the host cell is an E. coli cell.
  • TYH is from Rattus norvegicus.
  • the TYH variant has mutations at positions R37, R38 and W166; preferably, the mutations are R37E, R38E and W166Y.
  • the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.
  • the DODC is from Pseudomonas putida.
  • the MAO is from Micrococcus luteus.
  • the NCS is from Coptis chinensis.
  • the 6OMT is from opium poppy.
  • the CNMT is from opium poppy.
  • 4'OMT is from Coptis chinensis.
  • BBE1 is from Poppy somniferum; preferably, the BBE1 contains a truncated N-terminal cross Membrane region, more preferably, the BBE1 contains a truncated nitrogen-terminal transmembrane region and is fused with an 8RP solubility-promoting tag.
  • the BBE1 variant has mutations at positions F398 and I431.
  • 9'OMT is from Coptis chinensis.
  • the CAS is from Coptis chinensis.
  • CPR2 is from Arabidopsis thaliana.
  • the E. coli is a B-lineage E. coli, such as BL21(DE3).
  • the cell further expresses one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase ( aroGfbr ) that resists feedback inhibition, chorismate translocase and prephenate dehydrogenase ( tyrAfbr ) that resists feedback inhibition, phosphoenolpyruvate synthase (ppsA), transketolase that increases erythrose-4-phosphate synthesis (tktA), and shikimate dehydrogenase (ydiB).
  • aroGfbr 3-deoxy-D-arabinoheptulose-7-phosphate synthase
  • tyrAfbr chorismate translocase and prephenate dehydrogenase
  • ppsA phosphoenolpyruvate synthase
  • tktA transketolase that increases erythrose-4-phosphate synthesis
  • aroG, tyrA, ppsA, tktA, ydiB are derived from Escherichia coli.
  • amino acid sequence of aroG fbr is as shown in SEQ ID NO:22.
  • amino acid sequence of tyrA fbr is as shown in SEQ ID NO:23.
  • amino acid sequence of ppsA is as shown in SEQ ID NO: 24.
  • amino acid sequence of tktA is as shown in SEQ ID NO: 25.
  • amino acid sequence of ydiB is as shown in SEQ ID NO: 26.
  • the host cell further comprises an agent that inhibits the expression of pyruvate kinase (pykA), such as dsRNA, sRNA or sgRNA.
  • pykA pyruvate kinase
  • the sequence of the sRNA is as shown in SEQ ID NO: 27.
  • the host cell further comprises an agent that inhibits pheA expression, such as dsRNA, sRNA or sgRNA.
  • an agent that inhibits pheA expression such as dsRNA, sRNA or sgRNA.
  • the N20 sequence of the sgRNA is as shown in SEQ ID NO: 28.
  • the host cell further comprises an agent that inhibits the expression of an aromatic amino acid transcriptional regulator (tyrR), such as dsRNA, sRNA or sgRNA.
  • an aromatic amino acid transcriptional regulator such as dsRNA, sRNA or sgRNA.
  • the N20 sequence of the sgRNA is as shown in SEQ ID NO: 29 and/or 30.
  • the enzyme in the host cell as described in any embodiment of the first aspect of this document, is integrated into the host cell via chromosome.
  • the second aspect of the present invention provides a method for producing protoberberine compounds, the method comprising the following steps:
  • Tetrahydroafricanine is converted into tetrahydroberberine.
  • the method further comprises the use of an enzyme catalyzing the reduction of the oxidized state of cytochrome P450, preferably CPR2.
  • the reaction of step (1) is catalyzed by an enzyme, preferably TYH or a variant thereof.
  • step (1) further comprises catalyzing BH4 regeneration and catalyzing BH4 synthesis.
  • the enzyme catalyzing BH4 regeneration comprises: 4 ⁇ -hydroxytetrahydropterin dehydratase and/or dihydropterin reductase, and the enzyme catalyzing BH4 synthesis is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase.
  • the enzymes of the BH4 regeneration pathway comprise: 4 ⁇ -hydroxytetrahydropterin dehydratase of Rattus norvegicus and dihydropterin reductase of Rattus norvegicus.
  • the enzymes of the BH4 synthesis pathway comprise GTP cyclohydrolase from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthetase from Rattus norvegicus, and indole reductase from Rattus norvegicus.
  • reaction of step (2) is catalyzed by an enzyme, preferably DODC.
  • the method further comprises: hydroxylating tyramine to generate dopamine.
  • the hydroxylation of tyramine to generate dopamine is catalyzed by an enzyme, and the enzyme is preferably HpaBC or a variant thereof.
  • the reaction of step (3) is catalyzed by an enzyme, preferably MAO.
  • the reaction of step (4) is catalyzed by an enzyme, preferably NCS.
  • the reaction of step (5) is catalyzed by an enzyme, preferably 6OMT.
  • the reaction of step (6) is catalyzed by an enzyme, preferably CNMT.
  • the reaction of step (7) is catalyzed by an enzyme, preferably 4'OMT.
  • the reaction of step (8) is catalyzed by an enzyme, preferably BBE1 or a variant thereof.
  • reaction of step (9) is catalyzed by an enzyme, preferably 9'OMT.
  • reaction of step (10) is catalyzed by an enzyme, preferably CAS.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine e.g., CAS
  • the enzyme that reduces the oxidized state of cytochrome P450 e.g., CPR2
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine e.g., CAS
  • the enzyme that reduces the oxidized state of cytochrome P450 e.g., CPR2
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine e.g., CAS
  • the enzyme that reduces the oxidized state of cytochrome P450 e.g., CPR2
  • the protein interaction domain comprises a domain selected from the group consisting of a PDZ domain, an SH3 domain, a WW domain, a LIM domain, a DD domain, a PH domain, an EH domain, and a GBD domain.
  • the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme of the reduced oxidative state cytochrome P450 are fused with the SH3 domain and its ligand respectively.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3, and the enzyme of the reduced oxidative state cytochrome P450 is fused with SH3Lig.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3, it also includes a connection with a linker, and when the enzyme of the reduced oxidative state cytochrome P450 is fused with SH3Lig, it also includes a connection with a linker.
  • the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetracycline is located at the N-terminus of SH3.
  • the enzyme that reduces the oxidized state of cytochrome P450 is located at the N-terminus of SH3Lig.
  • the method further comprises: up-regulating the expression of purine nucleic acid kinase (DeoD).
  • the expression of purine nucleic acid kinase is up-regulated by plasmid overexpression technology.
  • the method further comprises: up-regulating the expression or activity of nuclease activity regulatory protein A (RraA) and/or cytochrome b5.
  • RraA nuclease activity regulatory protein A
  • cytochrome b5 the expression of nuclease activity regulatory protein A and/or cytochrome b5 is up-regulated by plasmid overexpression technology.
  • TYH is from Rattus norvegicus.
  • the TYH variant has mutations at positions R37, R38 and W166; preferably, the mutations are R37E, R38E and W166Y.
  • HpaBC is from Escherichia coli.
  • the HpaBC variant has mutations at positions A211 and Q212; more preferably, the mutations are A211G and Q212Y.
  • the DODC is from Pseudomonas putida.
  • the MAO is from Micrococcus luteus.
  • the NCS is from Coptis chinensis.
  • the 6OMT is from opium poppy.
  • the CNMT is from opium poppy.
  • 4'OMT is from Coptis chinensis.
  • BBE1 is from Poppy; preferably, the BBE1 contains a truncated N-terminal transmembrane region, more preferably, the BBE1 contains a truncated N-terminal transmembrane region and is fused with an 8RP solubility-enhancing tag.
  • 9'OMT is from Coptis japonica.
  • the CAS is from Coptis chinensis.
  • CPR2 is from Arabidopsis thaliana.
  • the method comprises the step of culturing the host cell as described in any embodiment herein under conditions suitable for the production of protoberberines.
  • the conditions include TB medium.
  • the culturing temperature is 20-25°C.
  • the culturing is for at least 24 hours.
  • the method further comprises the step of isolating protoberberine compounds from host cells; specifically comprising: crushing the cells, extracting with an organic solvent and vacuum drying, wherein the organic solvent is preferably n-butanol.
  • the method further comprises:
  • 3-deoxy-D-arabinoheptulose-7-phosphate synthase aroGfbr
  • tyrAfbr chorismate translocase and prephenate dehydrogenase
  • ppsA phosphoenolpyruvate synthase
  • tktA transketolase
  • shikimate dehydrogenase ydiB
  • pyruvate kinase preferably, down-regulating the expression or activity of the enzyme by RNAi or CRISPR technology;
  • chorismate translocation and prephenate dehydratase pheA
  • pheA Down-regulating the expression or activity of chorismate translocation and prephenate dehydratase
  • aromatic amino acid transcription regulator tyrR
  • the second aspect of the present invention provides a nucleic acid construct for increasing the production of reticuline in a cell, comprising a coding sequence encoding the following enzymes: an enzyme that catalyzes the hydroxylation of tyrosine to produce dopa, an enzyme that catalyzes the decarboxylation of dopa to produce dopamine, an enzyme that catalyzes dopamine to produce 3,4-dihydroxyphenylacetaldehyde, an enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlaudanine, an enzyme that catalyzes norlaudanine to produce 3'hydroxycoclaurine, an enzyme that catalyzes 3'hydroxycoclaurine to produce 3'hydroxynitromethylcoclaurine, an enzyme that catalyzes 3'hydroxynitromethylcoclaurine to produce reticuline, an enzyme that catalyzes the BH4 synthesis pathway, and an enzyme that catalyzes
  • the enzymes catalyzing the BH4 synthesis pathway include GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase.
  • the enzymes of the BH4 synthesis pathway include GTP cyclohydrolase from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthetase from Rattus norvegicus, and indole reductase from Rattus norvegicus.
  • the enzymes catalyzing the BH4 regeneration pathway include: 4 ⁇ -hydroxytetrahydropterin dehydration
  • the enzymes of the BH4 regeneration pathway include: 4a-hydroxytetrahydropterin dehydratase of Rattus norvegicus and dihydropterin reductase of Rattus norvegicus.
  • the nucleic acid construct further comprises a coding sequence for purine nucleic acid kinase (DeoD).
  • the nucleic acid construct further comprises an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine.
  • the nucleic acid construct further comprises a coding sequence encoding one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase ( aroGfbr ) that resists feedback inhibition, chorismate translocase and prephenate dehydrogenase ( tyrAfbr ) that resists feedback inhibition, phosphoenolpyruvate synthase (ppsA), transketolase that increases erythrose-4-phosphate synthesis (tktA), and shikimate dehydrogenase (ydiB).
  • aroGfbr 3-deoxy-D-arabinoheptulose-7-phosphate synthase
  • tyrAfbr chorismate translocase and prephenate dehydrogenase
  • ppsA phosphoenolpyruvate synthase
  • tktA transketolase that increases erythrose-4-phosphate synthesis
  • aroG, tyrA, ppsA, tktA, ydiB are derived from E. coli.
  • amino acid sequence of aroG fbr is as shown in SEQ ID NO:22.
  • amino acid sequence of tyrA fbr is as shown in SEQ ID NO:23.
  • amino acid sequence of ppsA is as shown in SEQ ID NO: 24.
  • amino acid sequence of tktA is as shown in SEQ ID NO: 25.
  • amino acid sequence of ydiB is as shown in SEQ ID NO: 26.
  • the nucleic acid construct further comprises a polynucleotide that inhibits the expression or activity of pyruvate kinase (pykA), such as dsRNA, sRNA or sgRNA.
  • pykA pyruvate kinase
  • the sequence of the sRNA is as shown in SEQ ID NO: 27.
  • the nucleic acid construct further comprises a polynucleotide that inhibits pheA expression or activity, such as dsRNA, sRNA or sgRNA.
  • a polynucleotide that inhibits pheA expression or activity such as dsRNA, sRNA or sgRNA.
  • the N20 sequence of the sgRNA is as shown in SEQ ID NO: 28.
  • the nucleic acid construct further comprises a polynucleotide that inhibits the expression or activity of an aromatic amino acid transcriptional regulator (tyrR), such as a dsRNA, sRNA or sgRNA.
  • an aromatic amino acid transcriptional regulator such as a dsRNA, sRNA or sgRNA.
  • the N20 sequence of the sgRNA is as shown in SEQ ID NO: 29 and/or 30.
  • the nucleic acid construct is a vector.
  • the coding sequence of the enzyme is in 1, 2, 3 or more expression frames.
  • the coding sequence is operably linked to an expression control sequence.
  • one expression frame may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more coding sequences of enzymes.
  • the second aspect of the present invention also provides a host cell with increased production of reticulated annonaine, wherein the host cell:
  • the enzymes catalyzing the BH4 regeneration pathway include: 4 ⁇ -hydroxytetrahydropterin dehydratase and/or dihydropterin reductase.
  • the enzymes catalyzing the BH4 regeneration pathway include: PCD and QDHPR from Rattus norvegicus.
  • the enzymes catalyzing the BH4 synthesis pathway include GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase.
  • the enzymes catalyzing the BH4 synthesis pathway include MtrA from Bacillus subtilis, PTPS from Rattus norvegicus, and SPR from Rattus norvegicus.
  • the cells further express purine nucleic acid kinase (DeoD).
  • DesoD purine nucleic acid kinase
  • the cell further expresses an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine.
  • the cell further expresses one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase ( aroGfbr ) that resists feedback inhibition, chorismate translocase and prephenate dehydrogenase ( tyrAfbr ) that resists feedback inhibition, phosphoenolpyruvate synthase (ppsA), transketolase that increases erythrose-4-phosphate synthesis (tktA), and shikimate dehydrogenase (ydiB).
  • aroGfbr 3-deoxy-D-arabinoheptulose-7-phosphate synthase
  • tyrAfbr chorismate translocase and prephenate dehydrogenase
  • ppsA phosphoenolpyruvate synthase
  • tktA transketolase that increases erythrose-4-phosphate synthesis
  • aroG, tyrA, ppsA, tktA, ydiB are derived from E. coli.
  • amino acid sequence of aroG fbr is as shown in SEQ ID NO:22.
  • amino acid sequence of tyrA fbr is as shown in SEQ ID NO:23.
  • amino acid sequence of ppsA is as shown in SEQ ID NO: 24.
  • amino acid sequence of tktA is as shown in SEQ ID NO: 25.
  • amino acid sequence of ydiB is as shown in SEQ ID NO: 26.
  • the host cell further comprises an agent that inhibits the expression or activity of pyruvate kinase, such as dsRNA, sRNA or sgRNA.
  • an agent that inhibits the expression or activity of pyruvate kinase such as dsRNA, sRNA or sgRNA.
  • the sequence of the sRNA is as shown in SEQ ID NO: 27.
  • the host cell further comprises an agent that inhibits pheA expression or activity, such as dsRNA, sRNA or sgRNA.
  • an agent that inhibits pheA expression or activity such as dsRNA, sRNA or sgRNA.
  • the N20 sequence of the sgRNA is as shown in SEQ ID NO: 28.
  • the host cell further comprises an agent that inhibits the expression or activity of an aromatic amino acid transcriptional regulator (tyrR), such as dsRNA, sRNA or sgRNA.
  • an aromatic amino acid transcriptional regulator such as dsRNA, sRNA or sgRNA.
  • the N20 sequence of the sgRNA is as shown in SEQ ID NO: 29 and/or 30.
  • the enzyme in the host cell as described in any embodiment of the second aspect of this invention, is integrated into the host cell via chromosome.
  • the second aspect of the present invention also provides a method for producing reticulated annonaine, the method comprising the following steps:
  • the method also includes catalyzing BH4 regeneration and catalyzing BH4 synthesis in steps (1) and/or (2).
  • the reaction of hydroxylating tyrosine to form DOPA is catalyzed by an enzyme, preferably TYH or a variant thereof.
  • the enzyme that catalyzes BH4 regeneration includes: 4 ⁇ -hydroxytetrahydropterin dehydratase and/or dihydropterin reductase.
  • the enzyme that catalyzes the BH4 regeneration pathway includes: 4 ⁇ -hydroxytetrahydropterin dehydratase of Rattus norvegicus and dihydropterin reductase of Rattus norvegicus.
  • the enzyme catalyzing BH4 synthesis is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase.
  • the enzyme catalyzing the BH4 synthesis pathway includes GTP cyclohydrolase from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthetase from Rattus norvegicus, and indole reductase from Rattus norvegicus.
  • reaction of step (2) is catalyzed by an enzyme, preferably DODC.
  • step (2) further comprises: hydroxylating tyramine to generate dopamine.
  • the hydroxylation of tyramine to generate dopamine is catalyzed by an enzyme, and the enzyme is preferably HpaBC or a variant thereof.
  • the reaction of step (3) is catalyzed by an enzyme, preferably MAO.
  • the reaction of step (4) is catalyzed by an enzyme, preferably NCS.
  • the reaction of step (5) is catalyzed by an enzyme, preferably 6OMT.
  • the reaction of step (6) is catalyzed by an enzyme, preferably CNMT.
  • the reaction of step (7) is catalyzed by an enzyme, preferably 4'OMT.
  • the TYH is from Rattus norvegicus or Drosophila, preferably, the TYH is from Rattus norvegicus; the TYH variant has mutations at R37, R38 and W166; preferably, the mutations are R37E, R38E and W166Y.
  • the HpaBC is from Escherichia coli.
  • the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.
  • the DODC is from Pseudomonas putida.
  • the MAO is from Micrococcus luteus.
  • the 6OMT is from opium poppy.
  • the CNMT is from opium poppy.
  • the 4'OMT is from Coptis chinensis.
  • the method comprises the step of culturing the host cell of the second aspect under conditions suitable for producing reticuline.
  • the conditions include TB medium.
  • the culturing temperature is 20-25°C.
  • the culturing is for at least 24 hours.
  • the method further comprises the step of isolating reticulated annonaine from host cells; specifically comprising: crushing the cells, extracting with an organic solvent and vacuum drying, wherein the organic solvent is preferably n-butanol.
  • the method further comprises:
  • 3-deoxy-D-arabinoheptulose-7-phosphate synthase aroGfbr
  • tyrAfbr chorismate translocase and prephenate dehydrogenase
  • ppsA phosphoenolpyruvate synthase
  • tktA transketolase
  • shikimate dehydrogenase ydiB
  • pyruvate kinase preferably, down-regulating the expression or activity of the enzyme by RNAi or CRISPR technology
  • chorismate translocation and prephenate dehydratase pheA
  • pheA Down-regulating the expression or activity of chorismate translocation and prephenate dehydratase
  • aromatic amino acid transcription regulator tyrR
  • FIG1 is a standard curve of reticuline drawn based on the relationship between the response of the extracted ions and the sample concentration in LC-MS/MS detection.
  • FIG2 shows the results of strain construction, tyrosine production and bacterial density.
  • FIG. 3 shows the reticuline production of strains from different sources and the regeneration and recycling pathways of tetrahydrobiopterin.
  • FIG4 is a by-product synthesis pathway inferred from the LC-MS/MS detection results.
  • FIG. 5 shows the reticuline production of strains overexpressing HpaBC-D11.
  • FIG. 6 shows the reticuline production of strains overexpressing or inhibiting different targets.
  • FIG. 7 shows the nitrogen-terminal design of tetrahydroberberine synthase (CAS) and the results of SDS-PAGE analysis.
  • FIG8 shows CAS from different sources and different co-localization designs of CAS and CPR.
  • FIG. 9 shows the in vitro activity test results of CAS from different sources and with different co-localization designs.
  • FIG. 10 shows the process and identification results of the oxidation final product produced in the in vitro activity test.
  • FIG. 11 shows the tetrahydroberberine production of strains overexpressing different effectors and cytochrome b5.
  • FIG. 12 shows the yield of protoberberine from batch fermentation of SQZ266 in a 7.5 L reactor.
  • the protoberberine compounds are a large subclass of benzylisoquinoline alkaloids well known to those skilled in the art, including berberine, hydrogenated berberine, tetrahydrocorydaline, coptisine, tetrahydrocorydaline, tetrahydroafricanine, strychnine, corydaline, golden corydaline, dehydrocorydaline, tetrahydropalmatine, corydaline and corydaline.
  • co-localization expression refers to shortening the spatial distance between the target protein expressed by fusion with the scaffold protein through the interaction of the scaffold protein.
  • the inventors obtained a chassis strain that produces high tyrosine and synthesizes tetrahydrobiopterin by integrating genes for improving tyrosine synthesis and genes for the BH4 cycle pathway into the chromosome of Escherichia coli BL21 (DE3). Moreover, the inventors also alleviated the feedback inhibition of tyramine on tyrosine hydroxylase by using a mutant HpaBC-D11 of an endogenous enzyme that catalyzes tyramine hydroxylation. In addition, by regulating targets related to tyrosine and tetrahydrobiopterin, efficient synthesis of reticulate annona was achieved.
  • the present invention provides a host cell with improved production of protoberberine compounds, which expresses the following enzymes: an enzyme that catalyzes the hydroxylation of tyrosine to produce dopa, an enzyme that catalyzes the decarboxylation of dopa to produce dopamine, an enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine, an enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlaudanine, an enzyme that catalyzes the production of 3'hydroxycoclaurine from norlaudanine, an enzyme that catalyzes the production of 3'hydroxycoclaurine from 3'hydroxynitromethylcoclaurine, an enzyme that catalyzes the production of reticuline from scutellariae, an enzyme that catalyzes the production of tetrahydroafricanine from scutellariae, an enzyme that catalyze
  • the host cell comprises a nucleic acid construct expressing the above enzyme.
  • the enzyme catalyzing tyrosine hydroxylation to generate DOPA is TYH or a variant thereof, an exemplary TYH is from Rattus norvegicus, the amino acid sequence is shown in SEQ ID NO: 1, the nucleic acid sequence is shown in SEQ ID NO: 31, and the TYH variant includes a variant having mutations at positions R37, R38 and W166, For example, R37E, R38E and W166Y, an exemplary TYH is from Drosophila, the amino acid sequence is shown in SEQ ID NO: 2, and the nucleic acid sequence is shown in SEQ ID NO: 32;
  • the enzyme that catalyzes the decarboxylation of dopa to produce dopamine is DODC
  • an exemplary DODC is from Pseudomonas putida
  • the amino acid sequence is shown in SEQ ID NO: 9
  • the nucleic acid sequence is shown in SEQ ID NO:
  • BBE1 contains a truncated nitrogen-terminal transmembrane region. In other embodiments, BBE1 contains a truncated nitrogen-terminal transmembrane region and a sequence fused with an 8RP solubility-enhancing tag; the enzyme that catalyzes the production of tetrahydroafricanine from golden corydaline is 9′OMT, an exemplary 9′OMT is from Coptis chinensis, the amino acid sequence is shown in SEQ ID NO:16, and the nucleic acid sequence is shown in SEQ ID NO:46; the enzyme that catalyzes the production of tetrahydroafricanine from tetrahydroberberine is CAS, an exemplary CAS is from Coptis chinensis, the amino acid sequence is shown in SEQ ID NO:17, and the nucleic acid sequence is shown in SEQ ID NO:47; the enzyme that reduces the oxidized state of cytochrome P450 is
  • the recycling pathway of tetrahydrobiopterin includes a synthesis pathway and a regeneration pathway.
  • the host cell further expresses enzymes of the BH4 synthesis pathway and enzymes of the BH4 regeneration pathway; the enzymes of the BH4 synthesis pathway are selected from one or more of the following: GTP cyclohydrolase (MtrA), 6-pyruvyl-tetrahydrobiopterin synthase (PTPS) and succinylcholine reductase (SPR), exemplary enzymes of the BH4 synthesis pathway include (MtrA) from Bacillus subtilis, the amino acid sequence is shown in SEQ ID NO:3, the nucleic acid sequence is shown in SEQ ID NO:33; PTPS from Rattus norvegicus, the amino acid sequence is shown in SEQ ID NO:4, the nucleic acid sequence is shown in SEQ ID NO:34; SPR from Rattus norvegicus, the amino acid sequence is shown in SEQ ID NO:5, the nucleic acid sequence is shown in SEQ ID NO:35; enzymes of the BH4 regeneration pathway
  • the inventors also found that by using a mutant HpaBC-D11 of an endogenous enzyme that catalyzes tyramine hydroxylation to relieve inhibition, efficient synthesis of reticuline can be achieved. Therefore, in some embodiments, the host cell also expresses an enzyme that catalyzes tyramine hydroxylation to produce dopamine.
  • the enzyme that catalyzes tyramine hydroxylation to produce dopamine is HpaBC or a variant thereof
  • an exemplary HpaBC is from Escherichia coli
  • the HpaBC variant includes a variant having mutations at the A211 and Q212 sites, such as A211G and Q212Y, the amino acid sequence is shown in SEQ ID NO: 8, and the nucleic acid sequence is shown in SEQ ID NO: 38.
  • the reagent for increasing tyrosine production may also include a reagent for upregulating the expression or activity of purine nucleic acid kinase (DeoD).
  • the amino acid sequence of purine nucleic acid kinase (DeoD) is shown in SEQ ID NO: 19, and the nucleic acid sequence is shown in SEQ ID NO: 49.
  • the co-localization expression of the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 can promote the oxidation of tetrahydroafricanine.
  • the cell co-localizes the expression of the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450.
  • the co-localization expression includes the expression of the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450, respectively, fused with the protein interaction domain and its ligand; or the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are fused.
  • the protein interaction domain includes a domain selected from the group consisting of a PDZ domain, an SH3 domain, a WW domain, a LIM domain, a DD domain, a PH domain, an EH domain, and a GBD domain.
  • the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidative state of cytochrome P450 are fused with the SH3 domain and its ligand for expression, respectively.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3 and expressed, and the enzyme that reduces the oxidative state of cytochrome P450 is fused with SH3Lig for expression.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3 and expressed, it also includes connecting with a linker, and when the enzyme that reduces the oxidative state of cytochrome P450 is fused with SH3Lig and expressed, it also includes connecting with a linker.
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine when the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3 and expressed, the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is located at the N-terminus of SH3. In one or more embodiments, when the reduced oxidative state cytochrome P450 enzyme is expressed in fusion with SH3Lig, the reduced oxidative state cytochrome P450 enzyme is located at the N-terminus of SH3Lig.
  • An exemplary amino acid sequence of the linker is shown in SEQ ID NO: 72, and a nucleic acid sequence is shown in SEQ ID NO: 63; an exemplary SH3 nucleic acid sequence is shown in SEQ ID NO: 64; an exemplary SH3Lig nucleic acid sequence is shown in SEQ ID NO: 65.
  • the host cell also expresses a nuclease activity regulatory protein (RraA) and/or cytochrome b5.
  • RraA nuclease activity regulatory protein
  • cytochrome b5 cytochrome b5.
  • the exemplary RraA is from Escherichia coli, and the amino acid sequence is shown in SEQ ID NO: 20.
  • the amino acid sequence is shown in SEQ ID NO: 50; an exemplary cytochrome b5 is from Chinese coptis chinensis, the amino acid sequence is shown in SEQ ID NO: 21, and the nucleic acid sequence is shown in SEQ ID NO: 51; or, an exemplary cytochrome b5 is from Phanerochaete chrysosporium, the amino acid sequence is shown in SEQ ID NO: 68, and the nucleic acid sequence is shown in SEQ ID NO: 69; or, an exemplary cytochrome b5 is from Chinese coptis chinensis, the amino acid sequence is shown in SEQ ID NO: 70, and the nucleic acid sequence is shown in SEQ ID NO: 71.
  • the enzyme is encoded by the corresponding enzyme encoding nucleic acid sequence contained in the nucleic acid construct.
  • the nucleic acid sequence can be in the form of DNA or RNA, can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.
  • the coding sequences of all enzymes may be in 1, 2, 3 or more expression frames.
  • a linker sequence is included between each coding sequence, and the linker sequence enables multiple cistrons to be expressed in a single expression frame.
  • the linker sequence is a coding sequence for a 2A peptide.
  • 2A peptides include F2A, P2A or T2A peptides.
  • Those skilled in the art can determine the type and quantity of coding sequences contained in an expression frame based on sequence length, promoter, etc.
  • the coding sequence of the enzyme is in 2 expression frames, wherein the coding sequences of TYH or its variants, BH4 , DODC, HpaBC or its variants, MAO, NCS, 6OMT, CNMT and 4'OMT are in one expression frame, and the coding sequences of BBE1, 9'OMT and CAS are in another expression frame.
  • the coding sequence of the enzyme is in three expression frames, wherein the coding sequences of TYH or its variant, BH4 , DODC, HpaBC or its variant, MAO and NCS are in one expression frame, the coding sequences of 6OMT, CNMT and 4'OMT are in one expression frame, and the coding sequences of BBE1, 9'OMT, and CAS are in one expression frame.
  • the coding sequences of TYH or its variant, BH4 , DODC, HpaBC or its variant, MAO and NCS are in one expression frame
  • the coding sequences of 6OMT, CNMT and 4'OMT are in one expression frame
  • the coding sequences of BBE1, 9'OMT, and CAS are in one expression frame.
  • nucleic acid sequence is optimized using species (e.g., E. coli) preferred codons to make the sequence more easily expressed in the species.
  • species e.g., E. coli
  • the coding sequences of the amino acid sequences shown in SEQ ID NOs: 1-26, 72, 66, 68, 70 are shown in SEQ ID NOs: 31-56, 63, 67, 69, 71, respectively.
  • the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequence and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, and more preferably at least 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • chromosome integration and low-copy overexpression are used to construct the strain.
  • the full-length nucleotide sequence of the protein or enzyme of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short.
  • a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them.
  • the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a nucleic acid construct (e.g., a vector), then transferring it into cells, and then isolating the relevant sequence from the host cells after proliferation by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
  • Nucleic acid constructs usually carry extrachromosomal elements of genes that are not part of the central metabolism of the cell, and are often in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences, linear or circular single-stranded or double-stranded DNA or RNA from any source, many of which have been joined or recombined into specific constructs that are capable of introducing the promoter fragment and DNA sequence of the selected gene product together with the appropriate 3' non-translated sequence into the cell.
  • sequences generally include one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, replication origin, transcription termination sequence, complete intron sequence containing donor and acceptor splice sites, sequence encoding leader sequence for polypeptide secretion, ribosome binding site, polyadenylation sequence, multiple linker regions and selectable marker elements for inserting nucleic acids encoding antibodies to be expressed.
  • exemplary nucleic acid constructs include pET21a and pET28a.
  • the vector may also contain sequences for integrating the coding sequence or expression cassette into the genome.
  • Plasmid overexpression technology is well known in the art, for example, after the coding sequence of a polypeptide of interest (such as RraA or cytochrome b5) is introduced into an expression vector containing a promoter and a terminator, the vector is transferred into a cell, and the cell is incubated under conditions suitable for polypeptide expression.
  • a polypeptide of interest such as RraA or cytochrome b5
  • 4 synthesized enzymes include GTP cyclohydrolase (MtrA) from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthase (PTPS) from Rattus norvegicus, and septerin reductase (SPR) from Rattus norvegicus; and/or, up-regulating the expression or activity of purine nucleic acid kinase (DeoD), for example, up-regulating the expression of purine nucleic acid kinase by plasmid overexpression technology.
  • GTP cyclohydrolase MtrA
  • PTPS 6-pyruvyl-tetrahydrobiopterin synthase
  • SPR septerin reductase
  • the method also includes an agent for increasing tyrosine production, such as expressing one or more or all of the following enzymes in the cell: 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ) that resists feedback inhibition, chorismate transposase and prephenate dehydrogenase (tyrA fbr ) that resists feedback inhibition, phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB); down-regulating the expression or activity of pyruvate kinase (pykA); preferably, down-regulating the expression or activity of the enzyme by RNAi or CRISPR technology; down-regulating the expression or activity of chorismate transposase and prephenate dehydratase (pheA);
  • aroG fbr 3-deoxy-
  • the present invention also provides a host cell with improved reticuline production, wherein the host cell expresses the following enzymes: an enzyme that catalyzes the hydroxylation of tyrosine to generate dopa, an enzyme that catalyzes the decarboxylation of dopa to generate dopamine, An enzyme that catalyzes dopamine to produce 3,4-dihydroxyphenylacetaldehyde, an enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlaudanine, an enzyme that catalyzes norlaudanine to produce 3'hydroxycoclaurine, an enzyme that catalyzes 3'hydroxycoclaurine to produce 3'hydroxynitromethylcoclaurine, an enzyme that catalyzes 3'hydroxynitromethylcoclaurine to produce reticuline, an enzyme that catalyzes the BH4 regeneration pathway, and an enzyme that catalyzes the BH4 synthesis pathway;
  • the cell also expresses purine nucleic acid kinase (DeoD). In some embodiments, the cell also expresses an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine. In some embodiments, the cell also includes an agent that increases tyrosine production and/or an agent that inhibits the expression or activity of one or more or all of the following enzymes, such as pykA, pheA, and tyrR. Preferably, in the host cell, the enzyme is integrated into the host cell by chromosome.
  • the present invention also provides a method for producing reticulate annonaine, which comprises the following steps: (1) hydroxylating tyrosine to generate dopa, (2) decarboxylating dopa to generate dopamine, (3) generating 3,4-dihydroxyphenylacetaldehyde from dopamine, (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlaudanine, (5) generating 3'-hydroxycoclaurine from norlaudanine, (6) generating 3'-hydroxycoclaurine from 3'-hydroxynitromethylcoclaurine, and (7) generating reticulate annonaine from 3'-hydroxynitromethylcoclaurine.
  • the method further comprises catalyzing BH4 regeneration and catalyzing BH4 synthesis in step (1) and/or (2).
  • RhaA endogenous ribonuclease activity regulator A
  • Escherichia coli DH10B was used as a cloning host for plasmid construction, and Escherichia coli BL21 (DE3) was used as an expression host to detect the expression of a single gene and as the original strain for fermentation.
  • the vector backbone was selected to contain four different resistance genes, namely, the ampicillin-resistant plasmid pET21a, the kanamycin-resistant plasmid pET28a, the chloramphenicol-resistant plasmid pACYC-Duet, and the spectinomycin-resistant plasmid pCDF-Duet.
  • Plasmid The detailed information of plasmid is shown in Table 1 (ori: vector backbone origin; KanR: kanamycin resistance; CmR: chloramphenicol resistance; SmR: spectinomycin resistance; AmpR: ampicillin resistance gene; PT7: T7 promoter; TT7: T7 terminator).
  • the enzyme that catalyzes the hydroxylation of L-tyrosine to generate L-DOPA is selected from the tyrosine hydroxylase from brown rats (Rattus norvegicus, tyrosine hydroxylase (RnTYH, UniProtKB: P04177)), and the amino acid mutations at three sites (R37E, R38E, W166Y) relieve feedback inhibition to increase the activity of tyrosine hydroxylase. You can also select the enzyme from Drosophila (Tyrosine 3-monooxygenase, Drosophila melanogaster (DmTYH, UniProt: P18459)). The coenzyme of this enzyme is tetrahydrobiopterin (BH 4 ).
  • BH 4 uses guanosine triphosphate (GTP) as a precursor and is BH 4 is generated by guanosine triphosphate (GTP) cyclohydrase I of Bacillus subtilis (MtrA, UniProt: P19465); 6-pyruvoyltetrahydropterin synthase of R. norvegicus (PTPS, UniProt: P27213); and Sepiapterin reductase of R. norvegicus ( SPR , UniProt: P18297).
  • GTP guanosine triphosphate
  • BH4 regeneration pathway was introduced, and BH4 regeneration was completed through rat 4 ⁇ -hydroxytetrahydropterin dehydratase (Rattus norvegicus pterin-4 alpha-carbinolamine dehydratase 1 (PCD, NCBI accession: NP_001007602)) and rat dihydropterine reductase (Rattus norvegicus quinoid dihydropteridine reductase (QDHPR, UniProtKB: P11348)).
  • PCD rat 4 ⁇ -hydroxytetrahydropterin dehydratase
  • PCD rat dihydropterine reductase
  • QDHPR rat dihydropterine reductase
  • the enzyme that catalyzes the decarboxylation of L-DOPA to generate dopamine (DOPA decarboxylase, Pseudomonas putida KT2440 (DODC, GenBank accession number: AE015451)) was selected.
  • L-tyrosine can catalyze the formation of two precursors for the synthesis of reticuline, dopamine and 3,4-DHPAA.
  • Norcoclaurine synthase 2 CjPR10A, Coptis japonica (NCS, UniprotKB: A2A1A1), which catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norcoclaurine, is selected from Coptis japonica (NCS, UniprotKB: A2A1A1).
  • the enzyme that catalyzes the production of 3'hydroxycoclaurine from norcoclaurine is selected from Papaver somniferum (6OMT, UniprotKB: Q6WUC1).
  • the enzyme that catalyzes the production of 3'hydroxycoclaurine from 3'hydroxynitromethylcoclaurine is selected from Papaver somniferum (CNMT, UniprotKB: Q7XB08).
  • the enzyme that catalyzes the conversion of 3'-hydroxy-N-methylcoclaurine to reticuline was selected from 3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase, Coptis japonica (4'OMT, UniProtKB: Q9LEL5)) from Coptis japonica.
  • reticuline can be obtained by fermentation in Escherichia coli using tyrosine as a substrate with the participation of the above enzymes.
  • the enzyme that catalyzes the conversion of reticuline to scoulerine is a mutant of berberine bridge enzyme 1 from Argemone Mexicana (AmBBE1, UniProtKB: D2SMM9), and the amino acids at two sites (F398 and I431) are mutated to obtain AmBBE1 F398W-I431F .
  • the enzyme that catalyzes the conversion of scoulerine to tetrahydroafricanine is a scoulerine 9'-oxymethyltransferase from Coptis japonica (9'OMT, GenBank: D29809).
  • the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is selected from the tetrahydroberberine synthase (Canadine synthase (CYP719), Coptis japonica (CAS, GenBank: AB026122)).
  • the enzyme that reduces the oxidized cytochrome P450 is selected from the cytochrome P450 oxidoreductase 2 (Arabidopsis thaliana P450 reductase 2 (AtCPR2, NCBI: NM_179141.2)).
  • the construction of the modified vector backbone and the construction of the sRNA-related plasmid were constructed by inverse PCR.
  • the sRNA-related plasmid was constructed by inverse PCR using pQZ135 as a template and introducing the first 24 bases of the target gene in the primers.
  • the construction of pQZ135 adopted homologous recombination.
  • the vectors pJF650 and CDF-Duet1 containing the sRNA backbone were used as templates to amplify three fragments containing overlapping bases, namely JF650, CDF1 and CDF2.
  • the primer sequences are as follows:
  • part135-CDF2 Template pCDF-Duet
  • part135-CDF2-F is shown as SEQ ID NO: 57
  • part135-CDF2-R is shown as SEQ ID NO: 58.
  • part135-JF650 Template pJF650
  • part135-JF650-F is shown in SEQ ID NO: 59
  • part135-JF650-R is shown in SEQ ID NO: 60.
  • part135-CDF1 Template pCDF-Duet
  • part135-CDF1-F is shown as SEQ ID NO: 61
  • part135-CDF1-R is shown as SEQ ID NO: 62.
  • sgRNA plasmid Construction of sgRNA plasmid. Use Benchling (https://www.benchling.com/) to find the corresponding gene in E. coli BL21 (DE3), then select the appropriate NGGPAM position and determine the corresponding 20bp guide sequence. Finally, the determined guide sequence is introduced into the pTargetF plasmid by inverse PCR.
  • the construction of the plasmid related to the overexpression target was constructed by in-fusion. After the target gene was cloned, the homologous sequence of the vector was added by overlap-PCR and constructed into the linearized vector with the help of homologous recombinase.
  • the integration process was divided into two steps: First, the sgRNA plasmid pQZ279 targeting TyrR was obtained by inverse PCR. The two ends of the editing template sequence contained homologous sequences about 500bp upstream and downstream of the knockout gene sequence. The template was obtained by designing primers for amplification and fusion through overlap extension PCR. Secondly, a new NGG was selected from the fragment integrated in the first step, and the corresponding sgRNA plasmid pQZ261 was obtained. The editing template was a fragment of the plasmid pQZ298 double-digested with XbaI and NotI, and its two sides were about 500bp away from the site integrated in the first step. 1000 bp homologous fragment.
  • the detailed editing and screening process is as follows: BL21 (DE3) cells containing pEcCas9 were obtained by chemical transformation. The single clone was expanded and cultured, and arabinose with a final concentration of 10mM was added to the culture medium to induce the expression of the ⁇ -Red system to obtain electroporated competent cells. In the subsequent electroporation process, 100 ⁇ L of electroporated competent cells was mixed with 100ng of pTargetT series plasmids or 100ng of pTargetF series plasmids and 400ng of editing template fragments. The mixture was electroporated at a voltage of 1800v in a 1mm electroporation cup.
  • the colonies with sgRNA eliminated were further inoculated into LB medium containing 5g/L glucose and cultured overnight at 37°C and 250rpm. 10 ⁇ L of the overnight culture solution was spread on LB plates containing 5g/L glucose and 10g/L sucrose, cultured overnight at 37°C, and then the colonies were picked and screened on LB plates with or without kanamycin. The colonies sensitive to kanamycin were the positive strains that eliminated pEcCas9.
  • RNA in the tissue was then extracted according to the kit instructions (TIANGEN RNA simple Total RNA kit).
  • kit instructions TIANGEN RNA simple Total RNA kit.
  • the purified RNA was reverse transcribed into cDNA using the PrimeScript RT (Takara) kit.
  • the tBLASTn tool was used to retrieve the information in the Rhizoma Coptidis transcriptome in the SRA database (SRA: SRX4550566).
  • the candidate sequences were obtained after the nitrogen and carbon end analysis of the screening results, and primers were designed based on this. PCR amplification was performed using PrimerSTAR Max DNA polymerase (Takara), and finally two CYB5 genes were obtained, which were named CcCYB5A and CcCYB5B respectively.
  • IPTG IPTG
  • the culture was shaken at 22°C, 250 rpm. After 16-20h of shaking culture, harvest the bacteria at 5000 ⁇ g, 5min, 4°C. Add 1mL bufferA to the bacteria and resuspend and mix well. Now add 1% 100mM PMSF (protease inhibitor) and mix well. Put the sample on ice and sonicate for 10min, with a working time of 15s/time and an interval of 45s/time until the transparency of the resuspension increases.
  • PMSF prote inhibitor
  • the plasmids carrying CAS and CPR were transformed into E. coli BL21 (DE3) to overexpress the target gene and test the activity of the protein.
  • the seeds were pre-cultured in LB medium at 37°C for 12 hours, and then 1% was inoculated into 10mL LB medium containing antibiotics and cultured at 37°C and 250rpm until OD 600 reached 0.6. A final concentration of 0.1mM IPTG was added, and then cultured at 22°C and 180rpm for 20 hours. After that, the bacteria were harvested at 5000 ⁇ g, 5min, 4°C.
  • the cells were resuspended in 1 mL of buffer B (100 mM Tris-HCl [pH 7.5] and 100 mM NaCl), to which 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM MgCl 2 and 5 mg/L DNase I were added and mixed.
  • the samples were placed on ice and sonicated for 10 min, with a working time of 15 s/time and an intermittent time of 45 s/time, until the transparency of the resuspension increased.
  • the supernatant was used as a crude protein extract for the subsequent in vitro reaction.
  • the in vitro enzyme activity assay was performed in a 100 ⁇ L reaction mixture containing 100 mM Tris-HCl (pH 7.5), 50 mg/L (S)-tetrahydroafricanine, 50 ⁇ L crude enzyme, 1 mM NADPH, 5 ⁇ M FAD, 5 ⁇ M FMN, 4 mM glucose-6-phosphate, 0.2 units of glucose-6-phosphate dehydrogenase and 2 ⁇ M DTT.
  • the reaction was incubated at 22°C, 180 rpm for 12 hours and then terminated with 100 ⁇ L of methanol and vigorous vortexing.
  • the strain was cultured at 22°C, 250rpm for 24h, and then 0.1mM IPTG and 30g/L glucose were added. After 12h of continuous cultivation, the production and OD 600 of strains SQZ208-209 in the culture medium were determined. After 24h of continuous cultivation, the production and OD 600 of strains SQZ219-222 and SQZ227-231 were determined. After 48h of continuous cultivation, the production and OD 600 of strains SQZ202-207 were determined.
  • the strain was cultured in TB (2% glycerol) medium at 37°C and 250 rpm for 3 h, then 0.1 mM IPTG was added and cultured at 22°C and 220 rpm for 48 h. The yield and OD 600 in the medium of strain SQZ261-266 were determined.
  • the single clone SQZ266 was inoculated into a medium containing carbenicillin (50 mg/L), chloramphenicol (34 mg/L), streptomycin (100 mg/L), The culture was then transferred into 2 mL LB liquid medium containing 50 mg/L 1% quinoline (50 mg/L) and 50 mg/L kanamycin (50 mg/L) and grown at 37°C for 12 hours. 800 ⁇ L of the culture was then transferred into a 500 mL flask containing 80 mL LB medium and cultured at 37°C, 250 rpm for 4 hours as a seed solution.
  • Batch culture was carried out in a 7.5 L reactor, and the seed solution was transferred into 4 L of fermentation basal medium at a volume ratio of 2%.
  • Each liter of basal medium consisted of 0.5 g/L L-phenylalanine, 20 g/L glucose, 12 g/L K 2 HPO 4 , 4.2 g/L KH 2 PO 4 , 2 g/L (NH 4 ) 2 SO 4 , 1.2 g/L MgSO 4 ⁇ 7H 2 O, 1.8 g/L citric acid monohydrate, 0.5 g/L yeast extract, corresponding antibiotics and 1 ⁇ trace metal solution.
  • the 1000 ⁇ trace metal mixture contained 0.03 g/L H 3 BO 3 , 1 g/L thiamine, 0.94 g/L ZnCl 2 , 0.5 g/L CoCl 2 , 0.38 g/L CuCl 2 , 1.6 g/L MnCl 2 , and 3.6 g/L FeCl 2 .
  • the feed medium contained 500 g/kg glucose, 10.7 g/L (NH 4 ) 2 SO 4 , 12 g/L MgSO 4 ⁇ 7H 2 O, 5 mL/L 1000 ⁇ trace metal solution, and antibiotics.
  • the initial culture temperature was 37°C, the air flow rate was 1 vvm, and the initial stirring rate was 400 rpm.
  • the pH value was maintained at 7 by automatically adding ammonia solution (25%, v/v).
  • OD 600 reached 19.5
  • the temperature was adjusted to 22°C.
  • the culture was immediately induced with 0.1 mM IPTG, and the OD 600 during induction was 21.
  • the duration of the entire batch culture was 58 hours.
  • tyrosine in fermentation broth The sample was diluted with an equal volume of 1N hydrochloric acid and mixed thoroughly at 37°C, 250rpm for 1h. Subsequently, the sample was centrifuged at high speed and filtered through a 0.22 ⁇ m filter membrane, and then detected by UPLC, chromatographic column: C18 (250 ⁇ 4.6mm, 5 ⁇ m), detection conditions: column temperature 30°C, flow rate 1mL/min; UV 278nm.
  • the mobile phase composition was B: deionized water (0.1% trifluoroacetic acid), C: acetonitrile (0.1% trifluoroacetic acid), mobile phase gradient setting: 0-12min, 5-15%C; 12-12.1min, 15-95%C; 12.1-15min, 95%C; 15-15.1min, 95-5%C; 15.1-20min, 5%C.
  • the fermentation broth loading volume was 5 ⁇ L.
  • the mobile phase composition was B: deionized water (0.1% formic acid), C: acetonitrile (0.1% formic acid), and the mobile phase gradient was set as follows: 0-10 min, 5-40% C; 10-16 min, 40-65% C; 16-16.1 min, 65-95% C; 16.1-19.0 min, 95% C; 19.0-19.1 min, 95-5% C; 19.1-22.0 min, 5% C.
  • the fermentation broth loading volume was 5 ⁇ L.
  • LC-MS/MS detection is used for qualitative and quantitative analysis of low-content annonaine.
  • a 20 mg/L standard solution was prepared and diluted it step by step to the following concentrations: 2.0 mg/L, 0.4 mg/L, 0.08 mg/L, 0.016 mg/L. 1 ⁇ L of the standard was loaded, and a standard curve was drawn based on the relationship between the response of the extracted ions and the sample concentration. The standard curve is shown in Figure 1.
  • Example 1 Construction of a high-yield tyrosine chassis strain
  • Tyrosine as the primary product of intracellular metabolism, participates in the synthesis of benzylisoquinoline alkaloids.
  • the inventors have integrated the existing high-yield strategy of tyrosine, including: knocking out the regulatory gene tyrR that regulates tyrosine synthesis, knocking out the gene pheA that phenylalanine synthesizes, overexpressing aroG and tyrA that release feedback inhibition, overexpressing ppsA, tktA and ydiB.
  • the present embodiment compares the situation of producing tyrosine in strains SQZ201 (plasmid, high copy overexpression) and SQZB18 (chromosome integration, low copy overexpression) of tyrosine synthesis-related genes after knocking out 2 genes.
  • SQZ201 plasmid, high copy overexpression
  • SQZB18 chromosome integration, low copy overexpression
  • the results of strain construction, yield and cell density are shown in Figure 2.
  • the results show that the tyrosine high-yield strain obtained by chromosome integration is more advantageous, and the tyrosine production 2.11g/L is more than 3 times the yield of the plasmid overexpression method.
  • Example 2 Tetrahydrobiopterin regeneration and recycling pathway improves tyrosine hydroxylase activity
  • Tyrosine hydroxylation is a key step in the metabolism of tyrosine to generate benzylisoquinoline alkaloids.
  • This example compares the effects of different sources and the regeneration and circulation pathways of tetrahydrobiopterin on the activity of tyrosine hydroxylase. A total of 4 combinations were evaluated: strains SQZ202-SQZ205 of RnTYH (BH 4 regeneration pathway), RnTYH (BH 4 circulation pathway), DmTYH (BH 4 regeneration pathway) and DmTYH (BH 4 circulation pathway).
  • the BH 4 circulation pathway was then integrated into the chromosome to construct strain SQZB19, as shown in Figure 2.
  • the results showed that the production of reticuline in strain SQZ206 using mouse tyrosine hydroxylase was only 1.08 mg/L, while the production of reticuline in strain SQZ207 using fruit fly tyrosine hydroxylase increased to 19.12 mg/L.
  • the fermentation broth was then tested by LC-MS/MS to find out why mouse tyrosine hydroxylase produced such a different production trait.
  • the LC-MS/MS test results of the fermentation broth showed that three strong response peaks appeared around the target product: m/z 300.1587[M+H] + , m/z 284.1599[M+H] + and m/z 314.1680[M+H] + (as shown in Figure 4).
  • the fragment ions it was determined that these compounds were structural analogs of reticuline and were generated by the condensation of phenylalanine or tyramine derivatives with dopamine (see Figure 4).
  • HpaBC-D11 a mutant of HpaBC that can specifically hydroxylate tyramine to generate dopamine is used to convert tyramine back into dopamine.
  • Example 3 DeoD involved in purine nucleotide metabolism contributes to the production of reticuline
  • this embodiment selected targets related to tyrosine synthesis and GTP metabolism for testing.
  • the results showed that inhibiting the activity of pyruvate kinase A (Pyruvate kinase II, E. coli, (EcPykA, UniProtKB: P21599)) and overexpressing purine nucleic acid kinase (Purine-nucleoside phosphorylase, E. coli, (EcDeoD, UniProtKB: P0ABP8)) can improve the yield of reticulate annonaine.
  • overexpression of purine nucleic acid kinase is the most effective, and the yield of reticulate annonaine can be increased to 100.13 mg/L at most, as shown in Figure 6.
  • Example 4 Scaffold connecting CAS and CPR promotes oxidation of tetrahydrotetramine
  • the inventors then evaluated the effects of three CAS from different sources and the co-localization of three different CAS and CPR on the catalytic activity of CAS, and a total of nine combinations were tested in subsequent in vitro experiments.
  • the three CAS were: one P450 with tetrahydroberberine synthase activity from Coptis chinensis and two from Corydalis yanhusuo: CjCYP719A1, CyCYP719A41 and CyCYP719A42.
  • Three different localization conditions CAS and CPR were expressed separately, CAS and CPR were fused and expressed through a linker ((GGGGS)2 flexible linker), and CAS was fused with SH3, and CPR was fused with SH3Lig.
  • Example 5 Global regulatory factors RraA and cytochrome b5 contribute to the increase of protoberberine production
  • the berberine bridge enzyme mutant and the tetrahydroberberine synthase with improved activity were integrated into the downstream synthesis pathway to form the strain SQZ261.
  • the results of shake flask culture showed that a large amount of protoberberine compounds accumulated in the fermentation broth, including 56.83 mg/L of reticulate annona, 34.79 mg/L of chrysophylline, 25.26 mg/L of tetrahydroafricanine, and 8.99 mg/L of tetrahydroberberine.
  • the accumulated reticulate annona and tetrahydroafricanine indicate that the activities of the two types of membrane proteins still need to be optimized.
  • the inventors selected two methods that can improve the activity of membrane proteins in the Escherichia coli system.
  • One is to use the effector DjlA (DnaJ-like protein A, the amino acid sequence is shown in SEQ ID NO: 66, The nucleic acid sequence is shown in SEQ ID NO: 67) and RraA (nuclease activity regulatory protein A), and the other is overexpression of cytochrome b5 (three cytochrome b5s were evaluated, namely PcCYB5, CcCYB5A and CcCYB5B, among which PcCYB5 is from Phanerochaete chrysosporium, and its amino acid sequence is shown in SEQ ID NO: 68, and its nucleic acid sequence is shown in SEQ ID NO: 69).
  • the strain SQZ266 overexpressing the effectors RraA and CcCYB5B obtained the highest tetrahydroberberine production, and the production in shake flask culture reached 16.81 mg/L, as shown in Figure 11.

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Abstract

La présente invention concerne un procédé de synthèse efficace d'un composé de protoberbérine et son utilisation. La présente invention concerne plus particulièrement une cellule hôte qui exprime les enzymes suivantes : une enzyme catalysant l'hydroxylation de la tyrosine pour produire de la dopa, une enzyme catalysant la décarboxylation de la dopa pour produire de la dopamine, une enzyme catalysant la production de 3,4-dihydroxyphénylacétaldéhyde à partir de la dopamine, une enzyme catalysant la condensation de la dopamine et du 3,4-dihydroxyphénylacétaldéhyde pour produire de la norlaudanine, une enzyme catalysant la production de 3'-hydroxycoclaurine à partir de la norlaudanine, une enzyme catalysant la production de 3'-hydroxy-N-méthylcoclaurine à partir de la 3'-hydroxycoclaurine, une enzyme catalysant la production de réticuline à partir de la 3'-hydroxy-N-méthylcoclaurine, une enzyme catalysant la production de scoulerine à partir de la réticuline, une enzyme catalysant la production de tétrahydrocolumbamine à partir de la scoulerine, une enzyme catalysant la production de tétrahydroberberine à partir de la tétrahydrocolumbamine, et une enzyme réduisant le cytochrome P450 dans un état d'oxydation. Le procédé de la présente étude permet la synthèse efficace d'un composé de protoberbérine.
PCT/CN2024/097774 2023-09-06 2024-06-06 Procédé de synthèse efficace d'un composé de protoberbérine et son utilisation Pending WO2025050732A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012039438A1 (fr) * 2010-09-22 2012-03-29 石川県公立大学法人 Procédé de production de benzylisoquinoléine-alcaloïde végétal
CN105907804A (zh) * 2016-06-04 2016-08-31 北京化工大学 一种异源生物合成香豆醇，咖啡醇及阿魏醇的方法
WO2022013407A1 (fr) * 2020-07-15 2022-01-20 Danmarks Tekniske Universitet Microbes thérapeutiques
CN114262681A (zh) * 2020-09-16 2022-04-01 中国科学院分子植物科学卓越创新中心 小檗碱生产菌株、其建立方法及其应用
CN114426929A (zh) * 2021-12-24 2022-05-03 浙江大学 一种发酵生产血根碱的酵母工程菌及其应用
WO2022109194A1 (fr) * 2020-11-19 2022-05-27 Antheia, Inc. Procédés pour améliorer la production d'alcaloïdes de type morphinane et de dérivés
CN116376796A (zh) * 2023-03-31 2023-07-04 江南大学 一种生产绿原酸的重组大肠杆菌及应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012039438A1 (fr) * 2010-09-22 2012-03-29 石川県公立大学法人 Procédé de production de benzylisoquinoléine-alcaloïde végétal
CN105907804A (zh) * 2016-06-04 2016-08-31 北京化工大学 一种异源生物合成香豆醇，咖啡醇及阿魏醇的方法
WO2022013407A1 (fr) * 2020-07-15 2022-01-20 Danmarks Tekniske Universitet Microbes thérapeutiques
CN114262681A (zh) * 2020-09-16 2022-04-01 中国科学院分子植物科学卓越创新中心 小檗碱生产菌株、其建立方法及其应用
WO2022109194A1 (fr) * 2020-11-19 2022-05-27 Antheia, Inc. Procédés pour améliorer la production d'alcaloïdes de type morphinane et de dérivés
CN114426929A (zh) * 2021-12-24 2022-05-03 浙江大学 一种发酵生产血根碱的酵母工程菌及其应用
CN116376796A (zh) * 2023-03-31 2023-07-04 江南大学 一种生产绿原酸的重组大肠杆菌及应用

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