Method for efficiently synthesizing protoberberine compound and application of protoberberine compound

Abstract

The invention relates to a method for efficiently synthesizing protoberberine compounds and application of the protoberberine compounds. The invention provides a host cell, and the host cell expresses the following enzymes: an enzyme for catalyzing tyrosine hydroxylation to generate dopa, an enzyme for catalyzing dopamine decarboxylation to generate dopamine, an enzyme for catalyzing dopamine to generate 3, 4-dihydroxyphenylacetaldehyde, and an enzyme for catalyzing dopamine and 3, 4-dihydroxyphenylacetaldehyde to condense to generate norlaudanalkali. The enzyme is used for catalyzing norladanine to generate 3 '-hydroxy combined spiceamine, the enzyme is used for catalyzing the 3'-hydroxy combined spiceamine to generate 3 '-hydroxy nitrogen methyl combined spiceamine, the enzyme is used for catalyzing the 3'-hydroxy nitrogen methyl combined spiceamine to generate reticulatonine, the enzyme is used for catalyzing the reticulatonine to generate golden corydalis alkali, and the enzyme is used for catalyzing the golden corydalis alkali to generate tetrahydrocolumbamine. The enzyme is used for catalyzing tetrahydrocotetrandrine to generate tetrahydroberberine, and the enzyme is used for reducing cytochrome P450 in an oxidation state. According to the invention, efficient synthesis of the protoberberine compound is realized.

Description

Technical Field

[0001] The invention belongs to the field of biosynthesis, and specifically relates to a method for efficiently synthesizing protoberberine compounds and an application thereof. Background Art

[0002] Benzylisoquinoline alkaloids (BIAs) are a class of natural products with various medicinal values ​​produced by tyrosine metabolism, including protoberberine alkaloids, phthalide isoquinoline alkaloids, morphinan alkaloids, etc. Among them, some protoberberine compounds are widely distributed in the rhizomes of plants in the Berberidaceae, Ranunculaceae and Papaveraceae, such as golden purpurine, tetrahydroafricanine, berberine and tetrahydropalmatine. In addition to being used as classic drugs for anti-inflammatory, antibacterial and analgesic purposes, recent studies have shown that these compounds may also play an important role in the treatment of tumors, diabetes and cardiovascular diseases. At present, the preparation methods of protoberberine include plant extraction, chemical synthesis and biosynthesis. However, 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. At the same time, although 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. Therefore, it is necessary to find a new preparation method to make up for the shortcomings of plant extraction and chemical synthesis and increase the supply of protoberberine compounds. Synthetic biology, especially with Escherichia coli and Saccharomyces cerevisiae as the chassis, has successfully achieved the synthesis of a variety of complex and high-value-added natural products, such as flavonoids, opioids and paclitaxel.

[0003] A series of progress has been made in the synthesis of protoberberine compounds in the microbial chassis. Among them, the Smolke research team at Stanford University, in 2008, used norlabradanine as a precursor, and through mining and adapting elements, achieved the synthesis of tetrahydroberberine in brewer's yeast. The subsequent work published in 2015 increased the yield of tetrahydroberberine by about 60 times to 1.8 mg / L. However, the method of feeding norlabradanine greatly increased the cost and limited its industrial application. Subsequently, in the work in 2018, the de novo synthesis of noscapine was achieved through the modification of chassis strains and protein engineering, but the yield could only reach the microgram level.

[0004] In addition to the work on the Saccharomyces cerevisiae chassis, there have been some advances in Escherichia coli, but most of them are the work on the synthesis of the intermediate reticuline. Japanese researchers Nakagawa and others, by modifying the chassis bacteria, selecting appropriate functional elements, and optimizing culture conditions, finally achieved the synthesis of 126 mg / L of reticuline using glucose as a carbon source. Recently, Liu Jihua's team from China Pharmaceutical University used dopamine as a precursor in Escherichia coli, and improved the catalytic activity of the enzyme by truncating the nitrogen end of the berberine bridge enzyme from California poppy and fusing it with a hydrophilic tag. On this basis, they introduced a continuously methylated enzyme and a P450 enzyme to achieve the synthesis of tetrahydropalmatine and corydaline, but the yield of tetrahydropalmatine was only 1.19 mg / L. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention aims to efficiently synthesize protoberberine compounds or reticulated annonaine.

[0006] 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 catalyzes golden corydaline to produce tetrahydroafrican pine, an enzyme that catalyzes tetrahydroafrican pine to produce tetrahydroberberine, and an enzyme that reduces oxidized cytochrome P450.

[0007] In one or more embodiments, 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 synthetase and dihydropterin reductase, and the enzyme of the BH4 regeneration pathway comprises: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase,

[0008] Preferably, 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.

[0009] Preferably, the enzymes of the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase from Rattus norvegicus and dihydropterin reductase from Rattus norvegicus.

[0010] In one or more embodiments, the nucleic acid construct further comprises an enzyme that catalyzes the hydroxylation of tyramine to generate dopamine. Preferably, the enzyme that catalyzes the hydroxylation of tyramine to generate dopamine is HpaBC or a variant thereof.

[0011] In one or more embodiments, the nucleic acid construct further comprises a coding sequence for a purine nucleic acid kinase.

[0012] In one or more embodiments, the nucleic acid construct further comprises a coding sequence for nuclease activity regulatory protein A and / or a coding sequence for cytochrome b5.

[0013] In one or more embodiments, the enzyme that catalyzes the hydroxylation of tyrosine to produce DOPA is TYH or a variant thereof.

[0014] In one or more embodiments, the enzyme that catalyzes the decarboxylation of dopa to form dopamine is DODC.

[0015] In one or more embodiments, the enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine is MAO.

[0016] In one or more embodiments, the enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlabdanine is NCS.

[0017] In one or more embodiments, the enzyme that catalyzes the production of 3'-hydroxycoclaurine from norlabdanine is 6OMT.

[0018] In one or more embodiments, the enzyme that catalyzes the production of 3'-hydroxycoclaurine from 3'-hydroxynitromethylcoclaurine is CNMT.

[0019] In one or more embodiments, the enzyme that catalyzes the production of reticuline from 3'hydroxyl nitrogenmethylcoclaurine is 4'OMT.

[0020] In one or more embodiments, the enzyme that catalyzes the production of chrysophylline from reticuline is BBE1 or a variant thereof.

[0021] In one or more embodiments, the enzyme that catalyzes the production of tetrahydrotetrandrine from chrysocorrhizine is 9'OMT.

[0022] In one or more embodiments, the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetramine is CAS.

[0023] In one or more embodiments, the enzyme that reduces the oxidized state of cytochrome P450 is CPR2.

[0024] In one or more embodiments, in the nucleic acid construct, the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine (eg, CAS) and the enzyme that reduces the oxidized state of cytochrome P450 (eg, CPR2) form a complex.

[0025] In one or more embodiments, 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.

[0026] In one or more embodiments, 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.

[0027] In one or more embodiments, the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig. Preferably, 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. Preferably, 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. Preferably, 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.

[0028] In one or more embodiments, the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetracycline is located at the N-terminus of SH3.

[0029] In one or more embodiments, the enzyme that reduces the oxidized state of cytochrome P450 is located at the N-terminus of SH3Lig.

[0030] In one or more embodiments, the amino acid sequence of the linker is shown in SEQ ID NO:72, and the nucleic acid sequence is shown in SEQ ID NO:63.

[0031] In one or more embodiments, the amino acid sequence of the SH3 domain is as shown in SEQ ID NO:73, and the nucleic acid sequence is as shown in SEQ ID NO:64.

[0032] In one or more embodiments, the amino acid sequence of the SH3Lig is shown in SEQ ID NO:74, and the nucleic acid sequence is shown in SEQ ID NO:65.

[0033] In one or more embodiments, TYH is from Rattus norvegicus.

[0034] In one or more embodiments, the TYH variant has mutations at positions R37, R38 and W166; preferably, the mutations are R37E, R38E and W166Y.

[0035] In one or more embodiments, the HpaBC is from E. coli.

[0036] In one or more embodiments, the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.

[0037] In one or more embodiments, the DODC is from Pseudomonas putida.

[0038] In one or more embodiments, the MAO is from Micrococcus luteus.

[0039] In one or more embodiments, the NCS is from Coptis chinensis.

[0040] In one or more embodiments, the 6OMT is from opium poppy.

[0041] In one or more embodiments, the CNMT is from opium poppy.

[0042] In one or more embodiments, the 4'OMT is from Coptis japonica.

[0043] In one or more embodiments, 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-enhancing tag.

[0044] In one or more embodiments, the BBE1 variant has a mutation at one or both of F398 and I431.

[0045] In one or more embodiments, 9'OMT is from Coptis japonica.

[0046] In one or more embodiments, the CAS is from Coptis chinensis.

[0047] In one or more embodiments, CPR2 is from Arabidopsis thaliana.

[0048] In one or more embodiments, the sequence of TYH of Rattus norvegicus is shown as SEQ ID NO:1.

[0049] In one or more embodiments, the sequence of Drosophila TYH is shown in SEQ ID NO:2.

[0050] In one or more embodiments, the sequence of MtrA is shown in SEQ ID NO:3.

[0051] In one or more embodiments, the sequence of PTPS is shown as SEQ ID NO:4.

[0052] In one or more embodiments, the sequence of the SPR is shown in SEQ ID NO:5.

[0053] In one or more embodiments, the sequence of PCD is as shown in SEQ ID NO:6.

[0054] In one or more embodiments, the sequence of QDHPR is as shown in SEQ ID NO:7.

[0055] In one or more embodiments, the sequence of the HpaBC variant is shown as SEQ ID NO:8.

[0056] In one or more embodiments, the sequence of DODC is as shown in SEQ ID NO:9.

[0057] In one or more embodiments, the sequence of MAO is shown in SEQ ID NO:10.

[0058] In one or more embodiments, the sequence of NCS is shown as SEQ ID NO:11.

[0059] In one or more embodiments, the sequence of 6OMT is as shown in SEQ ID NO:12.

[0060] In one or more embodiments, the sequence of CNMT is as shown in SEQ ID NO:13.

[0061] In one or more embodiments, the sequence of 4'OMT is as shown in SEQ ID NO:14.

[0062] In one or more embodiments, BBE1 or its variant: (1) has the sequence shown in SEQ ID NO:15, (2) has the sequence shown in SEQ ID NO:15 and has 1 or 2 mutations at positions selected from F398 and I431, wherein the mutation is a substitution, deletion or addition of an amino acid residue, (3) is a truncated variant of (1), having amino acids 36-554 of SEQ ID NO:15, (4) has a sequence that is at least 70% identical to the sequence shown in (1)-(3) and retains berberine bridge enzyme activity, or (5) has a sequence of the sequence shown in (1)-(4) fused to the N-terminus of an 8RP solubility-enhancing tag.

[0063] In one or more embodiments, the sequence of 9'OMT is as shown in SEQ ID NO:16.

[0064] In one or more embodiments, the sequence of CAS is as shown in SEQ ID NO:17.

[0065] In one or more embodiments, the sequence of CPR2 is as shown in SEQ ID NO:18.

[0066] In one or more embodiments, the sequence of DeoD is shown in SEQ ID NO:19.

[0067] In one or more embodiments, the sequence of RraA is shown in SEQ ID NO:20.

[0068] In one or more embodiments, the sequence of cytochrome b5 is shown as SEQ ID NO: 21 or 68 or 70.

[0069] In one or more embodiments, 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 (aroG fbr ), chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB).

[0070] In one or more embodiments, aroG, tyrA, ppsA, tktA, ydiB are derived from E. coli.

[0071] In one or more embodiments, aroG fbr The amino acid sequence is shown in SEQ ID NO:22.

[0072] In one or more embodiments, tyrA fbr The amino acid sequence is shown in SEQ ID NO:23.

[0073] In one or more embodiments, the amino acid sequence of ppsA is as shown in SEQ ID NO:24.

[0074] In one or more embodiments, the amino acid sequence of tktA is as shown in SEQ ID NO:25.

[0075] In one or more embodiments, the amino acid sequence of ydiB is as shown in SEQ ID NO:26.

[0076] In one or more embodiments, the nucleic acid construct further comprises a polynucleotide that inhibits the expression or activity of pyruvate kinase, such as dsRNA, sRNA or sgRNA. In one or more embodiments, the sequence of sRNA is as shown in SEQ ID NO:27.

[0077] In one or more embodiments, the nucleic acid construct further comprises a polynucleotide that inhibits pheA expression, such as dsRNA, sRNA or sgRNA. In one or more embodiments, the N20 sequence of the sgRNA is as shown in SEQ ID NO:28.

[0078] In one or more embodiments, 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. In one or more embodiments, the N20 sequence of the sgRNA is as shown in SEQ ID NO: 29 and / or 30.

[0079] In one or more embodiments, the nucleic acid construct is a vector. Preferably, the coding sequence of the enzyme is in one, two, three or more expression frames. Preferably, the coding sequence is operably linked to an expression control sequence.

[0080] The first aspect of the present invention also provides a host cell with improved production of protoberberine compounds, wherein the host cell:

[0081] (1) expressing the following enzymes: 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'-hydroxycoclaurine from 3'-hydroxycoclaurine, an enzyme that catalyzes the production of 3'-hydroxynitromethylcoclaurine from reticuline, an enzyme that catalyzes the production of chrysanthemum from reticuline, an enzyme that catalyzes the production of tetrahydroafricanine from chrysanthemum, an enzyme that catalyzes the production of tetrahydroafricanine from tetrahydroberberine, and an enzyme that reduces the oxidized state of cytochrome P450, or

[0082] (2) comprising the nucleic acid construct according to any embodiment of the first aspect of the present invention,

[0083] In one or more embodiments, the cell also expresses enzymes of the BH4 synthesis pathway and enzymes of the BH4 regeneration pathway, wherein the enzymes of the BH4 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 BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase. Preferably, 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. Preferably, the enzymes of the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase of Rattus norvegicus and dihydropterin reductase of Rattus norvegicus.

[0084] In one or more embodiments, the cells further express purine nucleic acid kinase (DeoD).

[0085] In one or more embodiments, the cell further expresses nuclease activity regulatory protein (RraA) and / or cytochrome b5.

[0086] In one or more embodiments, the cell further expresses an enzyme that catalyzes the hydroxylation of tyramine to generate dopamine. Preferably, the enzyme that catalyzes the hydroxylation of tyramine to generate dopamine is HpaBC or a variant thereof.

[0087] In one or more embodiments, 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.

[0088] In one or more embodiments, the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are expressed by fusion with the protein interaction domain and its ligand, respectively; or the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are expressed by fusion.

[0089] In one or more embodiments, 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.

[0090] In one or more embodiments, the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig. Preferably, 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 for expression, respectively. Preferably, the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3 and expressed, and the enzyme that reduces the oxidized state of cytochrome P450 is fused with SH3Lig. Preferably, the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3 through a linker, and the enzyme that reduces the oxidized state of cytochrome P450 is fused with SH3Lig through a linker.

[0091] In one or more embodiments, the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetracycline is located at the N-terminus of SH3.

[0092] In one or more embodiments, the enzyme that reduces the oxidized state of cytochrome P450 is located at the N-terminus of SH3Lig.

[0093] In one or more embodiments, the enzyme that catalyzes the hydroxylation of tyrosine to produce DOPA is TYH or a variant thereof.

[0094] In one or more embodiments, the enzyme that catalyzes the decarboxylation of dopa to form dopamine is DODC.

[0095] In one or more embodiments, the enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine is MAO.

[0096] In one or more embodiments, the enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlabdanine is NCS.

[0097] In one or more embodiments, the enzyme that catalyzes the production of 3'-hydroxycoclaurine from norlabdanine is 6OMT.

[0098] In one or more embodiments, the enzyme that catalyzes the production of 3'hydroxycoclaurine from 3'hydroxynitromethylcoclaurine is CNMT.

[0099] In one or more embodiments, the enzyme that catalyzes the production of reticuline from 3' hydroxyl nitrogenmethylcoclaurine is 4' OMT.

[0100] In one or more embodiments, the enzyme that catalyzes the production of chrysophylline from reticuline is BBE1 or a variant thereof.

[0101] In one or more embodiments, the enzyme that catalyzes the production of tetrahydrotetrandrine from chrysocorrhizine is 9'OMT.

[0102] In one or more embodiments, the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetramine is CAS.

[0103] In one or more embodiments, the enzyme that reduces the oxidized state of cytochrome P450 is CPR2.

[0104] In one or more embodiments, the host cell is an E. coli cell.

[0105] In one or more embodiments, TYH is from Rattus norvegicus.

[0106] In one or more embodiments, the TYH variant has mutations at positions R37, R38 and W166; preferably, the mutations are R37E, R38E and W166Y.

[0107] In one or more embodiments, the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.

[0108] In one or more embodiments, the DODC is from Pseudomonas putida.

[0109] In one or more embodiments, the MAO is from Micrococcus luteus.

[0110] In one or more embodiments, the NCS is from Coptis chinensis.

[0111] In one or more embodiments, the 6OMT is from opium poppy.

[0112] In one or more embodiments, the CNMT is from opium poppy.

[0113] In one or more embodiments, the 4'OMT is from Coptis japonica.

[0114] In one or more embodiments, 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.

[0115] In one or more embodiments, the BBE1 variant has mutations at positions F398 and I431.

[0116] In one or more embodiments, 9'OMT is from Coptis japonica.

[0117] In one or more embodiments, the CAS is from Coptis chinensis.

[0118] In one or more embodiments, CPR2 is from Arabidopsis thaliana.

[0119] In one or more embodiments, the E. coli is a B-lineage E. coli, such as BL21(DE3).

[0120] In one or more embodiments, the cell further expresses one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ), chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB).

[0121] In one or more embodiments, aroG, tyrA, ppsA, tktA, ydiB are derived from E. coli.

[0122] In one or more embodiments, aroG fbr The amino acid sequence is shown in SEQ ID NO:22.

[0123] In one or more embodiments, tyrA fbr The amino acid sequence is shown in SEQ ID NO:23.

[0124] In one or more embodiments, the amino acid sequence of ppsA is as shown in SEQ ID NO:24.

[0125] In one or more embodiments, the amino acid sequence of tktA is as shown in SEQ ID NO:25.

[0126] In one or more embodiments, the amino acid sequence of ydiB is as shown in SEQ ID NO:26.

[0127] In one or more embodiments, the host cell also includes an agent that inhibits the expression of pyruvate kinase (pykA), such as dsRNA, sRNA or sgRNA. In one or more embodiments, the sequence of sRNA is as shown in SEQ ID NO:27.

[0128] In one or more embodiments, the host cell further comprises an agent that inhibits pheA expression, such as dsRNA, sRNA or sgRNA. In one or more embodiments, the N20 sequence of the sgRNA is as shown in SEQ ID NO:28.

[0129] In one or more embodiments, 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. In one or more embodiments, the N20 sequence of the sgRNA is as shown in SEQ ID NO: 29 and / or 30.

[0130] In one or more embodiments, in the host cell as described in any embodiment of the first aspect of this invention, the enzyme is integrated into the host cell via chromosome.

[0131] A second aspect of the present invention provides a method for producing a protoberberine compound, the method comprising the following steps:

[0132] (1) Hydroxylation of tyrosine to generate DOPA,

[0133] (2) decarboxylating dopa to generate dopamine,

[0134] (3) Dopamine generates 3,4-dihydroxyphenylacetaldehyde,

[0135] (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlabdanine,

[0136] (5) converting norlabradanine into 3'-hydroxycoclaurine,

[0137] (6) converting 3'-hydroxy-coclaurine into 3'-hydroxy-nitro-methyl-coclaurine,

[0138] (7) 3'-hydroxyl nitrogen methylcoclaurine is converted into reticulate annonaine,

[0139] (8) Reticulated annonaine generates golden corydaline,

[0140] (9) generating tetrahydrotetramine from golden corydaline, and

[0141] (10) Tetrahydroafricanine is converted into tetrahydroberberine.

[0142] In one or more embodiments, the method further comprises the use of an enzyme catalyzing the reduction of the oxidized state of cytochrome P450, preferably CPR2.

[0143] In one or more embodiments, the reaction of step (1) is catalyzed by an enzyme, preferably TYH or a variant thereof.

[0144] In one or more embodiments, step (1) further comprises catalyzing BH4 regeneration and catalyzing BH4 synthesis. The enzyme catalyzing BH4 regeneration comprises: 4α-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. Preferably, the enzymes of the BH4 regeneration pathway comprise: 4α-hydroxytetrahydropterin dehydratase of Rattus norvegicus and dihydropterin reductase of Rattus norvegicus. Preferably, 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.

[0145] In one or more embodiments, the reaction of step (2) is catalyzed by an enzyme, preferably DODC.

[0146] In one or more embodiments, the method further comprises: hydroxylating tyramine to generate dopamine. Preferably, the hydroxylation of tyramine to generate dopamine is catalyzed by an enzyme, and the enzyme is preferably HpaBC or a variant thereof.

[0147] In one or more embodiments, the reaction of step (3) is catalyzed by an enzyme, preferably MAO.

[0148] In one or more embodiments, the reaction of step (4) is catalyzed by an enzyme, preferably NCS.

[0149] In one or more embodiments, the reaction of step (5) is catalyzed by an enzyme, preferably 6OMT.

[0150] In one or more embodiments, the reaction of step (6) is catalyzed by an enzyme, preferably CNMT.

[0151] In one or more embodiments, the reaction of step (7) is catalyzed by an enzyme, preferably 4'OMT.

[0152] In one or more embodiments, the reaction of step (8) is catalyzed by an enzyme, preferably BBE1 or a variant thereof.

[0153] In one or more embodiments, the reaction of step (9) is catalyzed by an enzyme, preferably 9'OMT.

[0154] In one or more embodiments, the reaction of step (10) is catalyzed by an enzyme, preferably CAS.

[0155] In one or more embodiments, the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine (e.g., CAS) and the enzyme that reduces the oxidized state of cytochrome P450 (e.g., CPR2) are fused to the protein interaction domain and its ligand to form separate fusion proteins; or the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine (e.g., CAS) and the enzyme that reduces the oxidized state of cytochrome P450 (e.g., CPR2) are fused to form a fusion protein.

[0156] In one or more embodiments, 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.

[0157] In one or more embodiments, the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig. Preferably, 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. Preferably, 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. Preferably, when 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.

[0158] In one or more embodiments, the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetracycline is located at the N-terminus of SH3.

[0159] In one or more embodiments, the enzyme that reduces the oxidized state of cytochrome P450 is located at the N-terminus of SH3Lig.

[0160] In one or more embodiments, the method further comprises: up-regulating the expression of purine nucleic acid kinase (DeoD). Preferably, the expression of purine nucleic acid kinase is up-regulated by plasmid overexpression technology.

[0161] In one or more embodiments, the method further comprises: up-regulating the expression or activity of nuclease activity regulatory protein A (RraA) and / or cytochrome b5. Preferably, the expression of nuclease activity regulatory protein A and / or cytochrome b5 is up-regulated by plasmid overexpression technology.

[0162] In one or more embodiments, TYH is from Rattus norvegicus.

[0163] In one or more embodiments, the TYH variant has mutations at positions R37, R38 and W166; preferably, the mutations are R37E, R38E and W166Y.

[0164] In one or more embodiments, HpaBC is from Escherichia coli. Preferably, the HpaBC variant has mutations at positions A211 and Q212; more preferably, the mutations are A211G and Q212Y.

[0165] In one or more embodiments, the DODC is from Pseudomonas putida.

[0166] In one or more embodiments, the MAO is from Micrococcus luteus.

[0167] In one or more embodiments, the NCS is from Coptis chinensis.

[0168] In one or more embodiments, the 6OMT is from opium poppy.

[0169] In one or more embodiments, the CNMT is from opium poppy.

[0170] In one or more embodiments, the 4'OMT is from Coptis japonica.

[0171] In one or more embodiments, 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.

[0172] In one or more embodiments, 9'OMT is from Coptis japonica.

[0173] In one or more embodiments, the CAS is from Coptis chinensis.

[0174] In one or more embodiments, CPR2 is from Arabidopsis thaliana.

[0175] In one or more embodiments, the method comprises the step of culturing the host cell as described in any embodiment herein under conditions suitable for the production of protoberberines.

[0176] In one or more embodiments, the conditions include TB medium.

[0177] In one or more embodiments, the culturing temperature is 20-25°C.

[0178] In one or more embodiments, the culturing is for at least 24 hours.

[0179] In one or more embodiments, 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.

[0180] In one or more embodiments, the method further comprises:

[0181] One or more or all of the following enzymes are expressed in the cell: 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ), chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB);

[0182] 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;

[0183] Down-regulating the expression or activity of chorismate translocation and prephenate dehydratase (pheA); preferably, down-regulating the expression or activity of the enzyme by RNAi or CRISPR technology; and / or

[0184] Down-regulate the expression or activity of aromatic amino acid transcription regulator (tyrR); preferably, down-regulate the expression or activity of the enzyme by RNAi or CRISPR technology.

[0185] 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 BH4 regeneration pathway.

[0186] In one or more embodiments, the enzymes catalyzing the BH4 synthesis pathway include GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase. Preferably, 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.

[0187] In one or more embodiments, the enzymes catalyzing the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase. Preferably, the enzymes of the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase of Rattus norvegicus and dihydropterin reductase of Rattus norvegicus. In one or more embodiments, the nucleic acid construct further comprises a coding sequence for purine nucleic acid kinase (DeoD).

[0188] In one or more embodiments, the nucleic acid construct further comprises an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine.

[0189] In one or more embodiments, 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 (aroG fbr ), chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB).

[0190] In one or more embodiments, aroG, tyrA, ppsA, tktA, ydiB are derived from E. coli.

[0191] In one or more embodiments, aroG fbr The amino acid sequence is shown in SEQ ID NO:22.

[0192] In one or more embodiments, tyrA fbr The amino acid sequence is shown in SEQ ID NO:23.

[0193] In one or more embodiments, the amino acid sequence of ppsA is as shown in SEQ ID NO:24.

[0194] In one or more embodiments, the amino acid sequence of tktA is as shown in SEQ ID NO:25.

[0195] In one or more embodiments, the amino acid sequence of ydiB is as shown in SEQ ID NO:26.

[0196] In one or more embodiments, the nucleic acid construct further comprises a polynucleotide that inhibits the expression or activity of pyruvate kinase (pykA), such as dsRNA, sRNA or sgRNA. In one or more embodiments, the sequence of sRNA is as shown in SEQ ID NO:27.

[0197] In one or more embodiments, the nucleic acid construct further comprises a polynucleotide that inhibits pheA expression or activity, such as dsRNA, sRNA or sgRNA. In one or more embodiments, the N20 sequence of the sgRNA is shown in SEQ ID NO: 28.

[0198] In one or more embodiments, 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. In one or more embodiments, the N20 sequence of the sgRNA is as shown in SEQ ID NO: 29 and / or 30.

[0199] In one or more embodiments, the nucleic acid construct is a vector. Preferably, the coding sequence of the enzyme is in 1, 2, 3 or more expression frames. Preferably, the coding sequence is operably linked to an expression control sequence. Typically, one expression frame may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more coding sequences of enzymes.

[0200] The second aspect of the present invention also provides a host cell with increased production of reticulated annonaine, wherein the host cell:

[0201] (1) Expression of the following enzymes: an enzyme catalyzing the hydroxylation of tyrosine to generate dopa, an enzyme catalyzing the decarboxylation of dopa to generate dopamine, an enzyme catalyzing the generation of 3,4-dihydroxyphenylacetaldehyde from dopamine, an enzyme catalyzing the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlaudanine, an enzyme catalyzing the generation of 3'-hydroxycoclaurine from norlaudanine, an enzyme catalyzing the generation of 3'-hydroxycoclaurine from 3'-hydroxynitromethylcoclaurine, an enzyme catalyzing the generation of reticuline from 3'-hydroxynitromethylcoclaurine, an enzyme catalyzing the BH4 regeneration pathway, an enzyme catalyzing the BH4 synthesis pathway,

[0202] (2) A nucleic acid construct comprising any one of the embodiments of the second aspect of this invention.

[0203] In one or more embodiments, the enzymes catalyzing the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase. Preferably, the enzymes catalyzing the BH4 regeneration pathway include: PCD and QDHPR from Rattus norvegicus.

[0204] In one or more embodiments, the enzymes catalyzing the BH4 synthesis pathway include GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase. Preferably, the enzymes catalyzing the BH4 synthesis pathway include MtrA from Bacillus subtilis, PTPS from Rattus norvegicus, and SPR from Rattus norvegicus.

[0205] In one or more embodiments, the cells further express purine nucleic acid kinase (DeoD).

[0206] In one or more embodiments, the cell further expresses an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine.

[0207] In one or more embodiments, the cell further expresses one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ), chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB).

[0208] In one or more embodiments, aroG, tyrA, ppsA, tktA, ydiB are derived from E. coli.

[0209] In one or more embodiments, aroG fbr The amino acid sequence is shown in SEQ ID NO:22.

[0210] In one or more embodiments, tyrA fbr The amino acid sequence is shown in SEQ ID NO:23.

[0211] In one or more embodiments, the amino acid sequence of ppsA is as shown in SEQ ID NO:24.

[0212] In one or more embodiments, the amino acid sequence of tktA is as shown in SEQ ID NO:25.

[0213] In one or more embodiments, the amino acid sequence of ydiB is as shown in SEQ ID NO:26.

[0214] In one or more embodiments, the host cell further comprises an agent that inhibits the expression or activity of pyruvate kinase, such as dsRNA, sRNA or sgRNA. In one or more embodiments, the sequence of sRNA is as shown in SEQ ID NO:27.

[0215] In one or more embodiments, the host cell further comprises an agent that inhibits pheA expression or activity, such as dsRNA, sRNA or sgRNA. In one or more embodiments, the N20 sequence of the sgRNA is as shown in SEQ ID NO:28.

[0216] In one or more embodiments, 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. In one or more embodiments, the N20 sequence of the sgRNA is as shown in SEQ ID NO: 29 and / or 30.

[0217] In one or more embodiments, in the host cell as described in any embodiment of the second aspect of this invention, the enzyme is integrated into the host cell via chromosome.

[0218] The second aspect of the present invention also provides a method for producing reticulated annonaine, the method comprising the following steps:

[0219] (1) Hydroxylation of tyrosine to generate DOPA,

[0220] (2) decarboxylating dopa to generate dopamine,

[0221] (3) Dopamine generates 3,4-dihydroxyphenylacetaldehyde,

[0222] (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlabdanine,

[0223] (5) converting norlabradanine into 3'-hydroxycoclaurine,

[0224] (6) converting 3'-hydroxy-coclaurine into 3'-hydroxy-nitro-methyl-coclaurine,

[0225] (7) 3'-hydroxyl nitrogen methylcoclaurine is converted into reticulate annonaine,

[0226] The method also includes catalyzing BH4 regeneration and catalyzing BH4 synthesis in steps (1) and / or (2).

[0227] In one or more embodiments, the reaction of hydroxylating tyrosine to form DOPA is catalyzed by an enzyme, preferably TYH or a variant thereof.

[0228] In one or more embodiments, the enzymes catalyzing BH4 regeneration include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase. Preferably, the enzymes catalyzing the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase of Rattus norvegicus and dihydropterin reductase of Rattus norvegicus.

[0229] In one or more embodiments, the enzyme catalyzing BH4 synthesis is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase. Preferably, 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.

[0230] In one or more embodiments, the reaction of step (2) is catalyzed by an enzyme, preferably DODC.

[0231] In one or more embodiments, step (2) further comprises: hydroxylating tyramine to generate dopamine. Preferably, the hydroxylation of tyramine to generate dopamine is catalyzed by an enzyme, and the enzyme is preferably HpaBC or a variant thereof.

[0232] In one or more embodiments, the reaction of step (3) is catalyzed by an enzyme, preferably MAO.

[0233] In one or more embodiments, the reaction of step (4) is catalyzed by an enzyme, preferably NCS.

[0234] In one or more embodiments, the reaction of step (5) is catalyzed by an enzyme, preferably 6OMT.

[0235] In one or more embodiments, the reaction of step (6) is catalyzed by an enzyme, preferably CNMT.

[0236] In one or more embodiments, the reaction of step (7) is catalyzed by an enzyme, preferably 4'OMT.

[0237] In one or more embodiments, 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.

[0238] In one or more embodiments, the HpaBC is from Escherichia coli.

[0239] In one or more embodiments, the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.

[0240] In one or more embodiments, the DODC is from Pseudomonas putida.

[0241] In one or more embodiments, the MAO is from Micrococcus luteus.

[0242] In one or more embodiments, the NCS is from Coptis chinensis.

[0243] In one or more embodiments, the 6OMT is from opium poppy.

[0244] In one or more embodiments, the CNMT is from opium poppy.

[0245] In one or more embodiments, the 4'OMT is from Coptis chinensis.

[0246] In one or more embodiments, the method comprises the step of culturing the host cell of the second aspect under conditions suitable for producing reticuline.

[0247] In one or more embodiments, the conditions include TB medium.

[0248] In one or more embodiments, the culturing temperature is 20-25°C.

[0249] In one or more embodiments, the culturing is for at least 24 hours.

[0250] In one or more embodiments, the method further comprises the step of isolating reticulated annonaine from the host cells; specifically comprising: crushing the cells, extracting with an organic solvent and vacuum drying, wherein the organic solvent is preferably n-butanol.

[0251] In one or more embodiments, the method further comprises:

[0252] One or more or all of the following enzymes are expressed in the cell: 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ), chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB);

[0253] 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,

[0254] Down-regulating the expression or activity of chorismate translocation and prephenate dehydratase (pheA); preferably, down-regulating the expression or activity of the enzyme by RNAi or CRISPR technology; and / or

[0255] Down-regulate the expression or activity of aromatic amino acid transcription regulator (tyrR); preferably, down-regulate the expression or activity of the enzyme by RNAi or CRISPR technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0256] Figure 1 The standard curve of reticuline was drawn based on the relationship between the response of the extracted ions and the sample concentration in LC-MS / MS detection.

[0257] Figure 2 The results of strain construction, tyrosine production and bacterial density are shown in Figure 2.

[0258] Figure 3 Reticuline production by strains from different sources and regeneration and recycling pathways of tetrahydrobiopterin.

[0259] Figure 4 The synthetic pathway of the by-products was inferred based on the LC-MS / MS detection results.

[0260] Figure 5 is the reticuline production of the strain overexpressing HpaBC-D11.

[0261] Figure 6 Reticuline production in strains overexpressing or suppressing different targets.

[0262] Figure 7 Design of the nitrogen terminus of tetrahydroberberine synthase (CAS) and the results of SDS-PAGE analysis.

[0263] Figure 8 Designed for CAS from different sources and different co-localizations of CAS and CPR.

[0264] Fig. 9 In vitro activity test results of CAS designed for different sources and different co-localization.

[0265] Fig.10 The process and identification results of the oxidation end product in the in vitro activity test.

[0266] Fig.11 Tetrahydroberberine production in strains overexpressing different effectors and cytochrome b5.

[0267] Fig.12 The yield of protoberberine from batch fermentation of SQZ266 in a 7.5 L reactor. DETAILED DESCRIPTION

[0268] In the present invention, 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.

[0269] In this article, 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.

[0270] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0271] 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 enzyme that endogenously catalyzes tyramine hydroxylation. In addition, by regulating targets related to tyrosine and tetrahydrobiopterin, efficient synthesis of reticulate annona was achieved. Subsequently, the functional expression of two types of membrane proteins (BBE and CAS) was optimized by introducing a strategy of mutants and scaffold proteins. Finally, the global regulatory factors RraA and cytochrome b5 were introduced to achieve efficient synthesis of protoberberine compounds.

[0272] Therefore, the present invention provides a host cell with improved production of protoberberine compounds, wherein the host cell expresses the following enzymes: an enzyme that catalyzes tyrosine hydroxylation to produce dopa, an enzyme that catalyzes dopa decarboxylation 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 chrysanthemumine, an enzyme that catalyzes chrysanthemumine to produce tetrahydroafricanine, an enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine, and an enzyme that reduces oxidized cytochrome P450.

[0273] Typically, the host cell comprises a nucleic acid construct expressing the above enzyme. Among them, the enzyme that catalyzes the hydroxylation of tyrosine to produce dopa is TYH or its variants, 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, TYH variants include variants with mutations at R37, R38 and W166, such as R37E, R38E and W166Y, an exemplary TYH is from Drosophila, the amino acid sequence is shown in SEQ ID NO:2, 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:39; the enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine is MAO, an exemplary MAO is from Micrococcus luteus, the amino acid sequence is shown in SEQ ID NO:10, the nucleic acid sequence is shown in SEQ ID NO:40; the enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlabradanine is NCS, an exemplary NCS is from Japanese Coptis chinensis, the amino acid sequence is shown in SEQ ID NO:11, and the nucleic acid sequence is shown in SEQ ID NO:41; the enzyme that catalyzes norlabradanine to produce 3'hydroxycoclaurine is 6OMT, an exemplary 6OMT is from opium poppy, the amino acid sequence is shown in SEQ ID NO:12, and the nucleic acid sequence is shown in SEQ ID NO:42; the enzyme that catalyzes 3'hydroxycoclaurine to produce 3'hydroxynitromethylcoclaurine is CNMT, an exemplary CNMT is from opium poppy, the amino acid sequence is shown in SEQ ID NO:13, and the nucleic acid sequence is shown in SEQ ID NO:43; the enzyme that catalyzes 3'hydroxynitromethylcoclaurine to produce reticulate annona is 4'OMT, an exemplary 4'OMT is from Japanese Coptis chinensis, the amino acid sequence is shown in SEQ ID NO:14, and the nucleic acid sequence is shown in SEQ ID NO:44; the enzyme that catalyzes reticulate annonaine to produce chrysanthemine is BBE1 or a variant thereof, an exemplary BBE1 is from Papaver somniferum, the amino acid sequence is shown in SEQ ID NO:15, the nucleic acid sequence is shown in SEQ ID NO:45, BBE1 variants include variants with mutations at F398 and I431, such as F398W and I431F, in some embodiments, 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-promoting tag; the enzyme that catalyzes chrysanthemine to produce tetrahydroafricanine is 9'OMT, an exemplary 9'OMT is from Coptis chinensis, the amino acid sequence is shown in SEQ ID NO:16, the nucleic acid sequence is shown in SEQ ID NO:46;The enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is CAS, and an exemplary CAS is from Coptis chinensis, and 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 CPR2, and an exemplary CPR2 is from Arabidopsis thaliana, and the amino acid sequence is shown in SEQ ID NO: 18, and the nucleic acid sequence is shown in SEQ ID NO: 48. ;

[0274] The inventors have found that the recycling pathway of tetrahydrobiopterin can improve the activity of tyrosine hydroxylase, and the production of reticulate annonaine is significantly increased. The recycling pathway of tetrahydrobiopterin includes a synthesis pathway and a regeneration pathway. Therefore, in some embodiments, the host cell also 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), and exemplary enzymes of the BH4 synthesis pathway include (MtrA) from Bacillus subtilis, the amino acid sequence is shown in SEQ ID NO:3, and 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, and 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, and the nucleic acid sequence is shown in SEQ ID NO:35; enzymes of the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase (PCD) and / or dihydropterin reductase (QDHPR), exemplary enzymes of the BH4 regeneration pathway include: PCD from Rattus norvegicus, the amino acid sequence is shown in SEQ ID NO:6, and the nucleic acid sequence is shown in SEQ ID NO:36; QDHPR from Rattus norvegicus, the amino acid sequence is shown in SEQ ID NO:7, and the nucleic acid sequence is shown in SEQ ID NO:37.

[0275] 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 generate dopamine. The enzyme that catalyzes tyramine hydroxylation to generate dopamine is HpaBC or a variant thereof, an exemplary HpaBC is from Escherichia coli, and 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.

[0276] Further, the inventors have also found that overexpression of purine nucleic acid kinase can improve the production of reticuline. Therefore, the reagent for increasing tyrosine production may also include a reagent for upregulating the expression or activity of purine nucleic acid kinase (DeoD). Preferably, 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.

[0277] The inventors also found that 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. In other embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig. Preferably, 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 for expression, respectively. Preferably, the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused with SH3 and expressed, and the enzyme that reduces the oxidized state of cytochrome P450 is fused with SH3Lig for expression. Preferably, when 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 oxidized state of cytochrome P450 is fused with SH3Lig and expressed, it also includes connecting with a linker. In one or more embodiments, 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 enzyme of the reduced oxidative state cytochrome P450 is expressed in fusion with SH3Lig, the enzyme of the reduced oxidative state cytochrome P450 is located at the N-terminus of SH3Lig. The amino acid sequence of an exemplary linker is shown in SEQ ID NO:72, and the nucleic acid sequence is shown in SEQ ID NO:63; the nucleic acid sequence of an exemplary SH3 is shown in SEQ ID NO:64; and the nucleic acid sequence of an exemplary SH3Lig is shown in SEQ ID NO:65.

[0278] The inventors also found that the introduction of highly active berberine bridge enzyme mutants and global regulatory factors achieved efficient synthesis of protoberberine compounds. In other embodiments, the host cell also expresses nuclease activity regulatory protein (RraA) and / or cytochrome b5, exemplary RraA from Escherichia coli, amino acid sequence as shown in SEQ ID NO: 20, nucleic acid sequence as shown in SEQ ID NO: 50; exemplary cytochrome b5 from Chinese coptis, amino acid sequence as shown in SEQ ID NO: 21, nucleic acid sequence as shown in SEQ ID NO: 51; or, exemplary cytochrome b5 from chrysosporium original hairy fungus, amino acid sequence as shown in SEQ ID NO: 68, nucleic acid sequence as shown in SEQ ID NO: 69; or, exemplary cytochrome b5 from Chinese coptis, amino acid sequence as shown in SEQ ID NO: 70, nucleic acid sequence as shown in SEQ ID NO: 71.

[0279] 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.

[0280] The coding sequences of all enzymes may be in 1, 2, 3 or more expression frames. In embodiments where multiple coding sequences are in the same expression frame, a connecting sequence is included between each coding sequence, and the connecting sequence enables multiple cistrons to be expressed in a single expression frame. In one or more embodiments, the connecting 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. In some embodiments, 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. In other embodiments, the coding sequence of the enzyme is in three expression frames, wherein the coding sequences of TYH or its variants, BH4, DODC, HpaBC or its variants, 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. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be produced, all of which encode the antibodies or antigen-binding fragments thereof of the present invention. Therefore, in the case where a specific amino acid sequence has been identified, those skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. For example, a species (e.g., E. coli) preferred codon is used to optimize the nucleic acid sequence to make the sequence easier to express in the species. In an exemplary embodiment, the coding sequences of the amino acid sequences shown in SEQ ID NOs: 1-26, 72, 66, 68, 70 are shown in, for example, SEQ ID NOs: 31-56, 63, 67, 69, 71, respectively. Therefore, 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. In the present invention, "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%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0281] The inventors found that different overexpression methods also affect the tyrosine production of the strain, and the tyrosine strain obtained by chromosome integration has a higher production. Therefore, in some embodiments, chromosome integration and low-copy overexpression are used to construct the strain.

[0282] 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. Usually, a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them.

[0283] Once the relevant sequence is obtained, 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. At present, 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.

[0284] 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 into the cell together with the appropriate 3' non-translated sequence.

[0285] Examples of nucleic acid constructs include expression vectors and recombinant vectors. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors generally contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) 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.

[0286] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0287] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media (e.g., LB or TB supplemented with glycerol). Culture is carried out under conditions suitable for the growth of the host cells (e.g., 37° C.). When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction), and the cells are cultured for a period of time (e.g., 22° C., more than 96 hours).

[0288] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0289] Host cell of the present invention can be present in fungi or bacteria family and grow in wide temperature, pH value and solvent tolerance range of microbial host, including but not limited to bacterial cell, fungal cell, such as yeast cell, mammalian cell, insect cell and plant cell.For example, it is expected that any bacterium, yeast and filamentous fungi can be suitable hosts for expressing nucleic acid molecules of the present invention.In one or more embodiments, the host cell is Escherichia coli, such as B system Escherichia coli, including BL21 (DE3).In exemplary embodiments, the recombinant Escherichia coli system of the present invention artificial construction improves the output of reticulate annonaine produced by tyrosine through fermentation engineering.

[0290] In addition, the host cell also includes an agent for increasing tyrosine production, such as expressing an enzyme that increases tyrosine production and an enzyme that further catalyzes a reaction on reticulate annona (e.g., berberine bridge enzyme). Exemplarily, the enzyme that increases tyrosine production includes, but is not limited to, 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ), chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB). These enzymes are preferably derived from Escherichia coli, but homologous sequences or variants of these enzymes with similar functions in various species are also easily obtained by those skilled in the art. Preferably, aroG fbr The amino acid sequence is shown in SEQ ID NO: 22, and the nucleic acid sequence is shown in SEQ ID NO: 52; tyrA fbr The amino acid sequence of tktA is shown in SEQ ID NO: 23, and the nucleic acid sequence thereof is shown in SEQ ID NO: 53; the amino acid sequence of ppsA is shown in SEQ ID NO: 24, and the nucleic acid sequence thereof is shown in SEQ ID NO: 54; the amino acid sequence of tktA is shown in SEQ ID NO: 25, and the nucleic acid sequence thereof is shown in SEQ ID NO: 55; the amino acid sequence of ydiB is shown in SEQ ID NO: 26, and the nucleic acid sequence thereof is shown in SEQ ID NO: 56. Expression can be expressed episomally by an expression vector or integrated into the genome (e.g., by CRISPR).

[0291] The reagent for improving tyrosine production may also include a reagent for lowering the expression or activity of one or more of the following enzymes: pyruvate kinase (pykA), chorismate transposition and prephenate dehydratase (pheA), aromatic amino acid transcription regulator (tyrR). Those skilled in the art know methods for lowering gene expression or activity in cells, such as by RNAi or CRISPRi technology, and corresponding reagents, such as dsRNA, sRNA or sgRNA. SgRNA is paired with the homologous sequence of the target gene through the N20 sequence, guiding the Cas9 bound thereto to recognize the target sequence for cutting, forming a DNA double-strand break. Those skilled in the art know methods for raising gene expression or activity in cells, such as methods for introducing recombinant plasmids and CRISPRa technology.

[0292] In addition, the present invention also provides a method for producing a protoberberine compound, comprising the following steps: (1) hydroxylating tyrosine to generate dopa (for example, using TYH or a variant thereof), (2) decarboxylating dopa to generate dopamine (for example, using DODC catalysis), (3) generating 3,4-dihydroxyphenylacetaldehyde from dopamine (for example, using MAO catalysis), (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlaudanine (for example, using NCS catalysis), (5) generating 3'-hydroxycoclaurine from norlaudanine (for example, using 6OMT catalysis), (6) 3'hydroxycoclaurine is converted to 3'hydroxynitromethylcoclaurine (e.g., catalyzed by CNMT), (7) 3'hydroxynitromethylcoclaurine is converted to reticuline (e.g., catalyzed by 4'OMT), (8) reticuline is converted to chrysanthemumine (e.g., catalyzed by BBE1 or a variant thereof), (9) chrysanthemumine is converted to tetrahydroafricanine (e.g., catalyzed by 9'OMT), and (10) tetrahydroafricanine is converted to tetrahydroberberine (e.g., catalyzed by CAS).

[0293] Based on the nucleic acid constructs and cells of the present invention, in order to increase the production of protoberberine, the method further includes an enzyme that hydroxylates tyramine to generate dopamine, such as HpaBC and its variants; and / or, increasing the expression level of tetrahydroberberine synthase, such as truncating the transmembrane region at the nitrogen end of tetrahydroberberine synthase and truncating and fusing a solubility tag such as 8RP; and / or, increasing the activity of tetrahydroberberine synthase, such as overexpressing effectors such as RraA and / or cytochrome b5, exemplarily, upregulating the expression or activity of RraA and / or cytochrome b5 by plasmid overexpression technology; and / or, reducing the enzyme of cytochrome P450 in an oxidized state, such as CPR2. 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.

[0294] In order to increase the content of reticulate annonaine, the method also includes an agent for increasing the production of reticulate annonaine, such as expressing an enzyme in the recycling pathway of tetrahydrobiopterin, such as an enzyme that catalyzes the regeneration of BH4 and an enzyme that catalyzes the synthesis of BH4. Exemplarily, the enzyme that catalyzes the regeneration of BH4 includes: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase, and the enzyme that catalyzes the synthesis of BH4 is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthase and indole reductase. Preferably, the enzyme that catalyzes the regeneration of BH4 includes: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase. The enzymes include: 4α-hydroxytetrahydropterin dehydratase (PCD) from Rattus norvegicus and dihydropterin reductase (QDHPR) from Rattus norvegicus, preferably, the enzymes catalyzing BH4 synthesis include GTP cyclohydrolase (MtrA) from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthetase (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.

[0295] In order to increase the content of substrate tyrosine, the method further comprises an agent for increasing tyrosine production, for example, expressing one or more or all of the following enzymes in the cell: 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ), chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), a transketolase that increases erythrose-4-phosphate synthesis (tktA), and shikimate dehydrogenase (ydiB); downregulating the expression or activity of pyruvate kinase (pykA); preferably, downregulating the expression or activity of the enzyme by RNAi or CRISPR technology; downregulating the expression or activity of chorismate translocation and prephenate dehydratase (pheA); preferably, downregulating the expression or activity of the enzyme by RNAi or CRISPR technology; and downregulating the expression or activity of aromatic amino acid transcription regulator (tyrR); preferably, downregulating the expression or activity of the enzyme by RNAi or CRISPR technology.

[0296] Therefore, in some embodiments, the present invention also provides a host cell with improved production of reticuline, wherein the host cell 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 3'hydroxynitromethylcoclaurine, an enzyme that catalyzes the BH4 regeneration pathway, and an enzyme that catalyzes the BH4 synthesis pathway; (2) comprising the nucleic acid construct described in the above embodiments. In some embodiments, 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 further comprises an agent for increasing tyrosine production and / or an agent for inhibiting the expression or activity of one or more or all of the following enzymes, such as pykA, pheA, tyrR. Preferably, in the host cell, the enzyme is integrated into the host cell via chromosome.

[0297] In addition, 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).

[0298] The present invention has the following beneficial effects:

[0299] 1) They discovered the regulatory effect of tyramine on tyrosine hydroxylase in Rattus norvegicus and, for the first time, relieved the feedback inhibition using a mutant of endogenous 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) in Escherichia coli;

[0300] 2) It was first discovered that the gene purine nucleoside phosphorylase (DeoD) related to purine synthesis promotes the synthesis of reticuline;

[0301] 3) The endogenous ribonuclease activity regulator A (RraA) was discovered for the first time. As a global regulatory factor, it helps to increase the production of protoberberine products;

[0302] 4) For the first time, it was achieved to synthesize protoberberine products from scratch in Escherichia coli using simple carbon sources such as glucose, and the total yield was the highest level available at 371.42 mg / L (reticulated annona 205.68 mg / L, tetrahydroberberine 165.74 mg / L), reaching the highest yield in existing microbial systems.

[0303] The present invention will be further described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and reagents used in the examples are, unless otherwise stated, conventional methods and reagents in the art.

[0304] Example

[0305] Materials and Methods

[0306] 1. Strains and plasmids involved in the embodiments of the present invention

[0307] 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.

[0308] 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).

[0309] See Table 2 for detailed information on strains (“Δ” for knockout, “::” for insertion, and “ori” for chassis strains)

[0310] Table 1: Plasmids involved in the examples

[0311]

[0312] Table 2: Strains involved in the examples

[0313]

[0314] 2. Enzyme selection

[0315] 2.1 Enzyme genes related to reticulate annona synthesis

[0316] The enzyme catalyzes the hydroxylation of L-tyrosine to generate L-DOPA (L-DOPA), and selects tyrosine hydroxylase (RnTYH, UniProtKB: P04177) from brown rats, and removes feedback inhibition by amino acid mutations at three sites (R37E, R38E, W166Y) to increase the activity of tyrosine hydroxylase. Tyrosine 3-monooxygenase (Drosophila melanogaster (DmTYH, UniProt: P18459)) from fruit flies can also be selected. The coenzyme of this enzyme is tetrahydrobiopterin (BH4). BH4 uses guanosine triphosphate (GTP) as a precursor and is catalyzed 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). In order to ensure the supply of BH4, the BH4 regeneration pathway was introduced, and the regeneration of BH4 was completed through rat 4α-hydroxytetrahydropterin dehydratase (Rattusnorvegicus pterin-4alpha-carbinolamine dehydratase 1 (PCD, NCBI accession: NP_001007602)) and rat dihydropterin reductase (Rattusnorvegicus quinoid dihydropteridinereductase (QDHPR, UniProtKB: P11348)).

[0317] The enzyme that catalyzes the decarboxylation of L-DOPA to generate dopamine (DOPA decarboxylase, Pseudomonas putida KT2440 (DODC, GenBank accession number: AE015451)) is selected. The enzyme that catalyzes dopamine to generate 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) is selected from Micrococcus luteus (Monoamine oxidase, Micrococcus luteus (MAO, UniProtKB: C5CB11)). At this point, L-tyrosine can catalyze the formation of two precursors for the synthesis of reticuline, dopamine and 3,4-DHPAA.

[0318] Norcoclaurine synthase 2, CjPR10A, Coptis japonica (NCS, UniprotKB: A2A1A1), which catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norcoclaurine, is from Coptis japonica (NCS, UniprotKB: A2A1A1). The enzyme that catalyzes the production of 3'-hydroxycoclaurine from norcoclaurine is selected from Norcoclaurine 6-O-methyltransferase, Papaver somniferum (6OMT, UniprotKB: Q6WUC1). The enzyme that catalyzes the production of 3'-hydroxycoclaurine from 3'-hydroxynitromethylcoclaurine is selected from Coclaurine N-methyltransferase, Papaver somniferum (CNMT, UniprotKB: Q7XB08). The enzyme that catalyzes the production of reticuline from 3'-hydroxy-N-methylcoclaurine is 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. Thus, reticuline can be obtained by fermentation in Escherichia coli using tyrosine as a substrate with the participation of the above enzymes.

[0319] 2.2 Enzyme genes related to the synthesis of protoberberine compounds

[0320] The enzyme catalyzing the production of chrysanthemine from reticulate annonaine was selected from a mutant of berberine bridge enzyme 1 from Argemone Mexicana (AmBBE1, UniProtKB: D2SMM9), and the amino acids at two sites (F398 and I431) were mutated to obtain AmBBE1. F398W-I431F . The enzyme that catalyzes the conversion of scoulerine to tetrahydroscoulerine is selected from scoulerine 9'-oxymethyltransferase (S)-scoulerine 9-O-methyltransferase, Coptis japonica (9'OMT, GenBank: D29809) from Japanese Coptis. The enzyme that catalyzes the conversion of tetrahydroscoulerine to tetrahydroberberine is selected from tetrahydroberberine synthase (Canadine synthase (CYP719), Coptis japonica (CAS, GenBank: AB026122) from Japanese Coptis. The enzyme that reduces the oxidized cytochrome P450 is selected from cytochrome P450 oxidoreductase 2 (Arabidopsis thaliana P450 reductase 2 (AtCPR2, NCBI: NM_179141.2) from Arabidopsis.

[0321] 3. Plasmid construction

[0322] 3.1 The construction of the modified vector backbone and the construction of the sRNA-related plasmids were constructed by inverse PCR. The sRNA-related plasmids were 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. First, by PCR, 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:

[0323] part135-CDF2: Template pCDF-Duet

[0324] part135-CDF2-F is shown in SEQ ID NO:57, and part135-CDF2-R is shown in SEQ ID NO:58.

[0325] part135-JF650: Template pJF650

[0326] part135-JF650-F is shown in SEQ ID NO:59, and part135-JF650-R is shown in SEQ ID NO:60.

[0327] part135-CDF1: Template pCDF-Duet

[0328] part135-CDF1-F is shown in SEQ ID NO:61, and part135-CDF1-R is shown in SEQ ID NO:62.

[0329] Construction of sgRNA plasmid. https: / / www.benchling.com / ) Find the corresponding gene in Escherichia 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 reverse PCR.

[0330] 3.3 Construction of plasmids related to overexpression targets was constructed by in-fusion. After cloning the target gene, the homologous sequence of the vector was added by overlap-PCR and constructed into the linearized vector with the help of homologous recombinase.

[0331] 3.4 The construction of plasmids involved in the synthesis of tyrosine, reticuline, tetrahydroberberine and regulating the synthesis of tetrahydroberberine in vivo was carried out by the "biobrick" method (see Knight T.Idempotent vector design for standard assembly of biobricks [J]. 2003.). Restriction sites were added at both ends of a single functional gene, and the genes were connected in series in sequence by restriction ligation with the help of a combination of isotailing enzymes XbaI and SpeI.

[0332] 3.5 Plasmid for in vitro test of tetrahydroberberine synthase. The scaffold of tetrahydroberberine synthase was designed and constructed by extended overlap extension PCR (POE-PCR) to obtain plasmid pQZ344. Tetrahydroberberine synthase from different sources was replaced. The specific construction method is as follows: Plasmids containing CyCYP719A41 and CyCYP719A42 and pQZ344 were used as templates, and DNA fragments and vector backbones were amplified by high-fidelity PCR. Both contained overlapping fragments of about 40 bp at the 3' and 5' ends. In the subsequent PCR system, the DNA fragments containing overlapping bases and the linearized vector were amplified as templates to produce a DNA polymer product. 10 μL of PCR product was directly transformed into DH10B and positive clones were screened.

[0333] 4 Chromosome Editing Process

[0334] In order to integrate the gene related to high tyrosine production into the chromosome, the integration process was divided into two steps: First, the sgRNA plasmid pQZ279 targeting TyrR was obtained by inverse PCR. The editing template sequence contained homologous sequences of about 500 bp upstream and downstream of the knockout gene sequence at both ends. The template was obtained after primers were designed for amplification and fusion by 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 there were about 1000 bp homologous fragments on both sides of the site integrated in the first step.

[0335] In order to integrate the BH4 recycling pathway, we chose to construct a plasmid pQZ266 containing both the editing template and sgRNA. The construction method was divided into the construction of the sgRNA plasmid (pQZ265) targeting the pheA site and the connection of the homologous fragment to the vector (the fragment of overlapPCR was recombined into the linearized plasmid pQZ265 double-digested with XbaI and HindIII).

[0336] 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 λ-Red system to obtain electroporated competent cells. In the subsequent electroporation process, 100μL of electroporated competent cells were 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. After the electroporation, 1mL of fresh LB medium was added immediately, and the cells were revived at 37°C and 250rpm for 2 hours. The revived culture medium was spread on LB plates with kanamycin and spectinomycin resistance and cultured overnight at 37°C. The transformants were then verified by colony PCR and DNA sequencing.

[0337] To eliminate the sgRNA series plasmid, positive single clones were inoculated into 2 mL of LB medium containing rhamnose (10 mM) and kanamycin (50 μg / mL). The culture was cultured overnight at 37 ° C, 250 rpm, and then diluted and plated on LB plates with kanamycin (50 μg / mL). Randomly selected clones were verified on LB plates with kanamycin (50 μg / mL) and LB plates with double resistance to spectinomycin (50 μg / mL) and kanamycin (50 μg / mL). Colonies sensitive to spectinomycin were considered to have eliminated the sgRNA plasmid.

[0338] In order to eliminate pEcCas9, the colonies with sgRNA eliminated were further inoculated into LB medium containing 5g / L glucose and cultured overnight at 37°C and 250rpm. 10μL 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.

[0339] 5. Amplification of cytochrome b5 gene from cDNA of Coptis chinensis

[0340] Liquid nitrogen was used to precool a sterile mortar, and then the Rhizoma Coptidis roots cut into small pieces were placed in the mortar and liquid nitrogen was added to fully grind them. Total RNA in the tissue was then extracted according to the kit instructions (TIANGEN RNA simple Total RNA kit). In order to clone the gene, the purified RNA was reverse transcribed into cDNA using the PrimeScript RT (Takara) kit. Using AaCYB5 from Artemisia annua as the query sequence, the tBLASTn tool was used to retrieve the information in the Rhizoma Coptidis transcriptome (SRA: SRX4550566) in the SRA database. The candidate sequences were obtained after the nitrogen and carbon end analysis of the screening results, and primers were designed accordingly. PCR amplification was performed using PrimerSTAR Max DNA polymerase (Takara), and finally two CYB5 genes were obtained, which were named CcCYB5A and CcCYB5B, respectively.

[0341] Expression and SDS-PAGE detection of 6CAS

[0342] The expression vector containing a single gene (full-length CAS, truncated transmembrane region trCAS, truncated and fused 8RP solubility tag 8RP-trCAS) was transformed into BL21 (DE3), and after culturing at 37°C for 12 hours, a single clone was picked from the plate and placed in 2 mL LB resistance medium, and shaken at 37°C, 250 rpm for 12 hours as seed liquid. 1% of the seed liquid was inoculated in 10 mL LB resistance medium and incubated at 37°C, 250 rpm until OD 600=0.6, after cooling down sufficiently, induce with 0.1mM IPTG, 22℃, 250rpm, shake culture for 16-20h, 5000×g, 5min, 4℃ to harvest bacteria. 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, sonicate for 10min, working time 15s / time, intermittent time 45s / time, until the transparency of the resuspension increases. Then the sample is centrifuged at low temperature and high speed (6000×g, 4℃) for 10min, take 100μL of the supernatant and place it on ice, record it as the soluble protein sample. Add 25μL of 5×SDS loading buffer to the soluble sample and mix well, boil in boiling water for 10min, centrifuge at 12000rpm, 3min. The sample loading volume is as follows: marker 5μL, supernatant sample 5μL. After electrophoresis at 100v for 20min and 160v for 60min, Coomassie brilliant blue staining was used to observe gene expression.

[0343] In vitro activity test of 7CAS

[0344] 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 10 mL LB medium containing antibiotics and cultured at 37°C, 250 rpm until OD 600 When the concentration reached 0.6, IPTG was added to a final concentration of 0.1 mM, and then cultured at 22°C, 180 rpm for 20 hours. Afterwards, the bacteria were harvested at 5000×g, 5 min, 4°C.

[0345] After discarding the supernatant, 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 MgCl2 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 μL reaction mixture containing 100 mM Tris-HCl (pH 7.5), 50 mg / L (S)-tetrahydroafricanine, 50 μL crude enzyme, 1 mM NADPH, 5 μM FAD, 5 μM FMN, 4 mM glucose-6-phosphate, 0.2 units of glucose-6-phosphate dehydrogenase and 2 μM DTT. The reaction was incubated at 22°C, 180 rpm for 12 hours and then terminated with 100 μL of methanol and vigorous vortexing.

[0346] 8 Bacterial shake flask culture conditions

[0347] All shake flask culture experiments were conducted in 100 mL round-bottomed thick test tubes, and the culture volume was controlled at 10 mL to ensure dissolved oxygen in the culture medium. 1% of the overnight culture seed solution was uniformly inoculated into TB culture medium.

[0348] For the tyrosine-producing strain, after culturing at 22°C and 250 rpm for 12 h, 0.1 mM IPTG and 30 g / L glucose were added, and the bacterial concentration OD was measured after further culturing for 48 h. 600 and tyrosine production.

[0349] To detect the production of reticuline, the strain was cultured at 22°C, 250 rpm for 24 h, and then 0.1 mM IPTG and 30 g / L glucose were added. After 12 h of continuous culture, the production and OD of the culture medium of strain SQZ208-209 were measured. 600 After 24 hours of continuous cultivation, the yield and OD of strains SQZ219-222 and SQZ227-231 were measured. 600 After 48 hours of continuous cultivation, the yield and OD of strain SQZ202-207 were measured. 600 .

[0350] To determine the production of protoberberine, the strains were cultured in TB (2% glycerol) medium at 37°C and 250 rpm for 3 h, and then 0.1 mM IPTG was added. After further culture at 22°C and 220 rpm for 48 h, the yield and OD in the medium of strain SQZ261-266 were determined. 600 .

[0351] 9 Fed-batch culture conditions

[0352] The single clone SQZ266 was inoculated into 2 mL LB liquid medium containing carbenicillin (50 mg / L), chloramphenicol (34 mg / L), streptomycin (50 mg / L) and kanamycin (50 mg / L) and grown for 12 hours at 37° C. Then 800 μL of the culture was 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.

[0353] Batch culture was carried out in a 7.5L reactor, and the seed solution was transferred into 4L of fermentation basal medium at a volume ratio of 2%. Each liter of basal medium consisted of 0.5g / L L-phenylalanine, 20g / L glucose, 12g / L K2HPO4, 4.2g / L KH2PO4, 2g / L (NH4)2SO4, 1.2g / L MgSO4·7H2O, 1.8g / L citric acid monohydrate, 0.5g / L yeast extract, corresponding antibiotics and 1× trace metal solution. The 1000× trace metal mixed solution contained 0.03g / L H3BO3, 1g / L thiamine, 0.94g / L ZnCl2, 0.5g / L CoCl2, 0.38g / LCuCl2, 1.6g / L MnCl2, and 3.6g / L FeCl2. The feed medium contained 500 g / kg glucose, 10.7 g / L (NH4)2SO4, 12 g / L MgSO4·7H2O, 5 mL / L 1000× trace metal solution and antibiotics.

[0354] 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 automatic addition of ammonia solution (25%, v / v). When OD 600 When the temperature reached 19.5, the temperature was adjusted to 22°C. After the temperature stabilized at 22°C, 0.1 mM IPTG was used to induce the culture. The OD 600 The duration of the entire batch culture was 58 hours.

[0355] 10 Metabolite detection

[0356] Detection of 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 0.22μm filter membrane, and then detected by UPLC, chromatographic column: C18 (250×4.6mm, 5μm), 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 5μL.

[0357] To detect alkaloid compounds in fermentation broth, add an equal volume of n-butanol to the sample and mix and extract twice. Centrifuge at 12000rpm for 5min and transfer the organic phase to a new tube. After rotary evaporation at 30℃, add 100μL methanol (concentrated 5 times) and mix thoroughly. Transfer the supernatant to UPLC and UPLC-QTOF / MS for detection at 12000rpm for 3min. UPLC detection, chromatographic column: C18 (250×4.6mm, 5μm), detection conditions: column temperature 30℃, flow rate 1mL / min; UV 235&280nm. 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 μL.

[0358] LC-MS / MS detection is used for qualitative and quantitative analysis of low-content annonaine. In order to accurately quantify the annonaine in the fermentation broth, we prepared a 20 mg / L standard solution 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 μL of the standard solution 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. Figure 1. Instrument: Agilent LC1260&1290-MS / QTOF6545, chromatographic column: Agilent infinityLab poroshell 120EC-C18 (3×150mm, 2.7μm), detection conditions: column temperature 30℃, flow rate 0.3mL / min, UV 235&280nm. Mobile phase setting: A: (0.1% formic acid) water; B: (0.1% formic acid) acetonitrile, mobile phase gradient: 5% B (0-0.8min); 5-98% B (0.8-12min); 98% B (12-15min); 98-5% B (15-15.5min). Sample volume: 1.0μL. Other parameter settings: positive ion mode, ion source: GasTemp = 300 ° C, flow rate: 6 L / min; pressure: 30 psig; Sheath GasTemp = 320 ° C; flow rate: 11 L / min; pressure: 30 psig. Fragmentor: 135 V; Skimmer: 65 V; Oct1RFVpp: 750 V; Vcap: 3500 V; Nozzle Voltage: 1000 V; Scan (m / z): 100-1100; 2nd level mass spectrometry collision energy: 0 eV, 10 eV, 20 eV, 40 eV.

[0359] Example 1: Construction of a high-yield tyrosine chassis strain

[0360] Tyrosine, as the primary product of intracellular metabolism, participates in the synthesis of benzylisoquinoline alkaloids. In order to ensure that the strain can use glucose to produce the target product, the inventors integrated the existing strategies for high tyrosine production, including: knocking out the regulatory gene tyrR that regulates tyrosine synthesis, knocking out the gene pheA that regulates phenylalanine synthesis, overexpressing aroG and tyrA that relieve feedback inhibition, and overexpressing ppsA, tktA, and ydiB. This example compares the production of tyrosine by strains SQZ201 (plasmid, high copy overexpression) and SQZB18 (chromosomal integration, low copy overexpression) that overexpress tyrosine synthesis-related genes after knocking out two genes. The results of strain construction, yield, and bacterial density are shown in Figure 2 The results showed that the tyrosine high-yield strain obtained by chromosome integration was more advantageous, producing 2.11 g / L tyrosine, which was more than three times the yield of the plasmid overexpression method.

[0361] Example 2: Tetrahydrobiopterin regeneration and recycling pathway improves tyrosine hydroxylase activity

[0362] Tyrosine hydroxylation is a key step in the metabolism of tyrosine to generate benzylisoquinoline alkaloids. This example compares the effects of different sources and regeneration and recycling pathways of tetrahydrobiopterin on the activity of tyrosine hydroxylase. A total of four combinations were evaluated: strains SQZ202-SQZ205 of RnTYH (BH4 regeneration pathway), RnTYH (BH4 recycling pathway), DmTYH (BH4 regeneration pathway) and DmTYH (BH4 recycling pathway). The results showed that the BH4 recycling pathway can improve the activity of hydroxylase, and the mouse tyrosine hydroxylase and the BH4 recycling pathway have the best effect together, producing 9.26 mg / L of reticuline, such as Figure 3 The BH4 recycling 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. In contrast, 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] + (like Figure 4 By comparing the fragment ions, it was determined that these compounds were structural analogs of reticulate annonaine and were generated by the condensation of phenylalanine or tyramine derivatives with dopamine (results see Figure 4 ). Excluding the influence of phenylalanine from the culture medium, the production of tyramine derivatives becomes a prominent issue. Drawing on previous research hypotheses on the inhibitory effect of dopa decarboxylase on mouse tyrosine hydroxylase, and the fact that tyrosine hydroxylase is susceptible to feedback inhibition by substrates and products, the inventors associated the formation of tyramine with the sudden decrease in tyrosine hydroxylase activity, and speculated that tyramine is likely to feedback inhibit the activity of tyrosine hydroxylase, resulting in extremely low production of reticuline.

[0363] Since tyramine is produced from tyrosine by catalysis of dopa decarboxylase, the flow originally used for synthesizing dopamine is diverted. In this example, a known enzyme HpaBC-D11 (a mutant of HpaBC) that can specifically hydroxylate tyramine to generate dopamine is used to convert tyramine back into dopamine. In strains with increased expression of HpaBC-D11, the production of reticuline in SQZ208 (overexpressing tyrosine hydroxylase from Rattus norvegicus) and SQZ209 (overexpressing tyrosine hydroxylase from Drosophila) reached 82.31 mg / L and 44.10 mg / L, respectively (see the results). Figure 5 ).

[0364] Example 3: DeoD involved in purine nucleotide metabolism contributes to the production of reticuline

[0365] In order to improve the production of reticulate annonaine, this example 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-nucleosidephosphorylase, E. coli, (EcDeoD, UniProtKB: P0ABP8)) can improve the production of reticulate annonaine. Among them, overexpression of purine nucleic acid kinase is the most effective, and the highest production of reticulate annonaine can be increased to 100.13 mg / L. Figure 6 shown.

[0366] Example 4: Scaffold connecting CAS and CPR promotes oxidation of tetrahydrotetramine

[0367] In order to achieve efficient synthesis of protoberberine, the inventors optimized two types of membrane proteins involved in the protoberberine synthesis pathway. First, PsBBE1 (see CN202010976017.6 "Berberine production strain, its establishment method and its application") was replaced with the mutant AmBBE1 with improved activity. F398W-I431F Subsequently, because the expression level of CAS was low, the inventors optimized the expression of the enzyme. After truncating the transmembrane region at the N-terminus and truncating and fusing the 8RP solubility tag, the expression of the enzyme was tested. The SDS-PAGE results showed that the expression of the protein with truncated N-terminal transmembrane sequence was significantly improved, such as Figure 7 .

[0368] The inventors then evaluated the effects of three CAS from different sources and three different co-localization situations of CAS and CPR on the catalytic activity of CAS. 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 situations: CAS and CPR expressed separately, CAS and CPR fused via linker ((GGGGS)2 flexible linker), CAS fused with SH3, and CPR fused with SH3Lig. The construction strategy is shown in Figure 8 Finally, only after the co-expression of CjCYP719A1 from Japanese Coptis chinensis and CPR, the generation of oxidation products could be detected. Moreover, in the reaction system where CjCYP719A1 was fused with SH3 and AtCPR2 was fused with SH3Lig, the substrates were completely converted, of which 32.66% of the substrates were converted into berberine and 67.34% of the substrates were converted into the oxidized form of terunine, such as Fig. 9 The identification of the oxidized form of the product is shown in Fig.10 .

[0369] Example 5: Global regulatory factors RraA and cytochrome b5 contribute to the increase of protoberberine production

[0370] The berberine bridge enzyme mutant and the tetrahydroberberine synthase with improved activity were integrated into the downstream synthesis pathway to form 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 annonaine, 34.79 mg / L of golden violaceous alkaloids, 25.26 mg / L of tetrahydroafricanine, and 8.99 mg / L of tetrahydroberberine. The accumulated reticulate annonaine and tetrahydroafricanine indicated that the activity of the two types of membrane proteins still needed to be optimized. In order to further improve the activity of membrane proteins, the inventors selected two methods that can improve the activity of membrane proteins in the E. 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 to overexpress cytochrome b5 (three cytochrome b5s were evaluated, namely PcCYB5, CcCYB5A and CcCYB5B, among which PcCYB5 is from Phanerochaete chrysosporium, its amino acid sequence is shown in SEQ ID NO:68, and its nucleic acid sequence is shown in SEQ ID NO:69). From the production of tetrahydroberberine, the strain SQZ266 that overexpressed the effector RraA and CcCYB5B obtained the highest tetrahydroberberine yield, and the yield of shake flask culture reached 16.81 mg / L, as shown in Figure 6. Fig.11 We then carried out batch fermentation culture of the SQZ266 strain in a 7.5L reactor, where the production of reticulate annona quickly accumulated to 205.68 mg / L within 25-42 h, tetrahydroafricanine accumulated to a maximum of 21.57 mg / L at 42 h, and tetrahydroberberine reached a maximum production of 165.74 mg / L at 58 h, as shown in Figure 2. Fig.12 shown.

Claims

1. A nucleic acid construct for increasing the production of protoberberine compounds in cells, characterized in that: The nucleic acid construct comprises a coding sequence encoding the following enzymes: an enzyme that catalyzes tyrosine hydroxylation to generate dopa, an enzyme that catalyzes dopa decarboxylation to generate dopamine, an enzyme that catalyzes dopamine to generate 3,4-dihydroxyphenylacetaldehyde, an enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlaudanine, an enzyme that catalyzes norlaudanine to generate 3'hydroxycoclaurine, an enzyme that catalyzes 3'hydroxycoclaurine to generate 3'hydroxynitromethylcoclaurine, an enzyme that catalyzes 3'hydroxynitromethylcoclaurine to generate reticuline, an enzyme that catalyzes reticuline to generate chrysanthemumine, an enzyme that catalyzes chrysanthemumine to generate tetrahydroafricanine, an enzyme that catalyzes tetrahydroafricanine to generate tetrahydroberberine, and an enzyme that reduces oxidized cytochrome P450. Preferably, In the nucleic acid construct, the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 form a complex, The enzyme that catalyzes tetrahydroafricanine to generate tetrahydroberberine and the enzyme that reduces the oxidized cytochrome P450 are fused with the protein interaction domain and its ligand to form separate fusion proteins; or the enzyme that catalyzes tetrahydroafricanine to generate tetrahydroberberine and the enzyme that reduces the oxidized 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, a GBD domain, The protein interaction domain includes an SH3 domain, whose ligand is SH3Lig, Preferably, The enzyme that catalyzes tyrosine hydroxylation to generate DOPA is TYH or a variant thereof, The enzyme that catalyzes the decarboxylation of dopa to generate dopamine is DODC. The enzyme that catalyzes dopamine to produce 3,4-dihydroxyphenylacetaldehyde is MAO. The enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlabdanine is NCS. The enzyme that catalyzes norlabradanine to generate 3'-hydroxycoclaurine is 6OMT, The enzyme that catalyzes 3'-hydroxycoclaurine to generate 3'-hydroxynitromethylcoclaurine is CNMT, The enzyme that catalyzes 3'hydroxyl nitrogen methylcoclaurine to generate reticuline is 4'OMT, The enzyme that catalyzes reticuline to produce chrysophylline is BBE1 or a variant thereof, The enzyme that catalyzes the production of tetrahydrotetrandrine from chrysocorrhizine is 9'OMT, The enzyme that catalyzes tetrahydrotetramine to produce tetrahydroberberine is CAS, The enzyme that reduces the oxidized state of cytochrome P450 is CPR2, More preferably, The TYH is from Rattus norvegicus or Drosophila; preferably, the 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 DODC is derived from Pseudomonas putida, The MAO is derived from Micrococcus luteus, The NCS is derived from Japanese Coptis chinensis, The 6OMT comes from opium poppy, The CNMT is derived from opium poppy, The 4'OMT is derived from Coptis chinensis, The BBE1 is from Poppyseed, The 9'OMT is derived from Japanese Coptis chinensis, The CAS is derived from Coptis chinensis, The CPR2 is from Arabidopsis thaliana.

2. The nucleic acid construct according to claim 1, wherein The nucleic acid construct further comprises: (1) Enzymes that catalyze the hydroxylation of tyramine to produce dopamine, Preferably, the enzyme catalyzing the hydroxylation of tyramine to generate dopamine is HpaBC or a variant thereof. More preferably, the HpaBC variant has mutations at positions A211 and Q212, for example, the mutations are A211G and Q212Y; and / or (2) coding sequences of enzymes in the BH4 synthesis pathway and coding sequences of enzymes in the BH4 regeneration pathway, wherein the enzymes in the BH4 synthesis pathway are selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthase and dihydropterin reductase, and the enzymes in the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase, Preferably, 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. Preferably, the enzymes of the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase from Rattus norvegicus and dihydropterin reductase from Rattus norvegicus; and / or (3) the coding sequence of purine nucleic acid kinase, Preferably, the purine nucleic acid kinase is derived from Escherichia coli; and / or (4) the coding sequence of nuclease activity regulatory protein A and / or cytochrome b5, Preferably, the nuclease activity regulating protein A is derived from Escherichia coli, Preferably, the cytochrome b5 is derived from Coptis chinensis; and / or (5) a coding sequence for one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase that is resistant to feedback inhibition, chorismate translocase and prephenate dehydrogenase that are resistant to feedback inhibition, phosphoenolpyruvate synthase, a transketolase that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase, Preferably, the feedback-inhibition-resistant 3-deoxy-D-arabinoheptulose-7-phosphate synthase, the feedback-inhibition-resistant chorismate translocase and prephenate dehydrogenase, the phosphoenolpyruvate synthase, the transketolase that increases erythrose-4-phosphate synthesis, and the shikimate dehydrogenase are derived from Escherichia coli; and / or (6) a polynucleotide that inhibits the expression of pyruvate kinase; and / or (7) polynucleotides that inhibit chorismate translocation and prephenate dehydratase expression; and / or (8) a polynucleotide that inhibits the expression of an aromatic amino acid transcriptional regulator, Preferably, the nucleic acid construct is a vector. Preferably, the coding sequence of the enzyme is in 1, 2, 3 or more expression frames.

3. The nucleic acid construct according to claim 1 or 2, wherein: The BBE1 or its variant: (1) has the sequence shown in SEQ ID NO: 15, (2) has the sequence shown in SEQ ID NO: 15 and has a mutation at a position selected from F398 and I431, wherein the mutation is a substitution, deletion or addition of an amino acid residue, (3) is a truncated variant of (1), having amino acids 36-554 of SEQ ID NO: 15, (4) has a sequence that is at least 70% identical to the sequence shown in (1)-(3) and retains the berberine bridge enzyme activity, or, (5) the sequence shown in (1)-(4) is fused to the N-terminus of the sequence with an 8RP solubility tag.

4. A host cell, characterized in that The host cell: (1) expressing the following enzymes: 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'-hydroxycoclaurine from 3'-hydroxycoclaurine, an enzyme that catalyzes the production of 3'-hydroxynitromethylcoclaurine from reticuline, an enzyme that catalyzes the production of chrysanthemum from reticuline, an enzyme that catalyzes the production of tetrahydroafricanine from chrysanthemum, an enzyme that catalyzes the production of tetrahydroafricanine from tetrahydroberberine, and an enzyme that reduces the oxidized state of cytochrome P450, or (2) comprising the nucleic acid construct according to any one of claims 1 to 3, Preferably, the host cell also has one or more characteristics selected from the following: The host cell also expresses enzymes of the BH4 synthesis pathway and enzymes of the BH4 regeneration pathway, wherein the enzymes of the BH4 synthesis pathway include one or more or all selected from the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and dihydropterin reductase, and the enzymes of the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase, The host cell also expresses purine nucleic acid kinase, The host cell also expresses nuclease activity regulatory protein A and / or cytochrome b5, The host cell also expresses an enzyme that catalyzes the hydroxylation of tyramine to generate dopamine. The host cell co-expresses an enzyme that catalyzes tetrahydroafricanine to generate tetrahydroberberine and an enzyme that reduces oxidized cytochrome P450, More preferably, the host cell further has one or more characteristics selected from the following: The enzyme that catalyzes tyrosine hydroxylation to generate DOPA is TYH or a variant thereof, The enzyme that catalyzes the decarboxylation of dopa to generate dopamine is DODC. The enzyme that catalyzes the hydroxylation of tyramine to generate dopamine is HpaBC or a variant thereof, The enzyme that catalyzes dopamine to produce 3,4-dihydroxyphenylacetaldehyde is MAO. The enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlabdanine is NCS. The enzyme that catalyzes norlabradanine to generate 3'-hydroxycoclaurine is 6OMT, The enzyme that catalyzes 3'-hydroxycoclaurine to generate 3'-hydroxynitromethylcoclaurine is CNMT, The enzyme that catalyzes 3'hydroxyl nitrogen methylcoclaurine to generate reticuline is 4'OMT, The enzyme that catalyzes reticuline to produce chrysophylline is BBE1 or a variant thereof, The enzyme that catalyzes the production of tetrahydrotetrandrine from chrysocorrhizine is 9'OMT, The enzyme that catalyzes tetrahydrotetramine to produce tetrahydroberberine is CAS, The enzyme that reduces the oxidized state of cytochrome P450 is CPR2, The Escherichia coli is a B-type Escherichia coli, such as BL21 (DE3).

5. A method for producing protoberberine compounds, characterized in that: The method comprises the following steps: (1) Hydroxylation of tyrosine to generate DOPA, (2) decarboxylating dopa to generate dopamine, (3) Dopamine generates 3,4-dihydroxyphenylacetaldehyde, (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlabdanine, (5) converting norlabradanine into 3'-hydroxycoclaurine, (6) converting 3'-hydroxy-coclaurine into 3'-hydroxy-nitro-methyl-coclaurine, (7) 3'-hydroxyl nitrogen methylcoclaurine is converted into reticulate annonaine, (8) Reticulated annonaine generates golden corydaline, (9) generating tetrahydrotetramine from golden corydaline, and (10) converting tetrahydroafricanine into tetrahydroberberine, Preferably, the method further has one or more features selected from the following: The reaction in step (1) is catalyzed by an enzyme, preferably TYH or a variant thereof, The reaction in step (2) is catalyzed by an enzyme, preferably DODC. The reaction in step (3) is catalyzed by an enzyme, preferably MAO. The reaction in step (4) is catalyzed by an enzyme, preferably NCS. The reaction in 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 in step (8) is catalyzed by an enzyme, preferably BBE1 or a variant thereof, The reaction in step (9) is catalyzed by an enzyme, preferably 9'OMT, The reaction in step (10) is catalyzed by an enzyme, preferably CAS, The enzyme that catalyzes tetrahydroafricanine to generate tetrahydroberberine and the enzyme that reduces the oxidized cytochrome P450 are fused with the protein interaction domain and its ligand to form separate fusion proteins; or the enzyme that catalyzes tetrahydroafricanine to generate tetrahydroberberine and the enzyme that reduces the oxidized cytochrome P450 are fused to form a fusion protein, More preferably, the method further has one or more features selected from the following: Step (1) further comprises catalyzing BH4 regeneration and catalyzing BH4 synthesis; preferably, the enzyme catalyzing BH4 regeneration comprises: 4α-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 dihydropterin reductase, Step (2) further comprises: hydroxylating tyramine to generate dopamine; preferably, using HpaBC or a variant thereof to catalyze the hydroxylation of tyramine to generate dopamine, The method further comprises: upregulating the expression or activity of purine nucleic acid kinase, The method further comprises: up-regulating the expression or activity of nuclease activity regulatory protein and / or cytochrome b5.

6. The method according to claim 5, characterized in that The method comprises the steps of culturing the host cell of claim 4 under conditions suitable for producing protoberberine compounds, Preferably, the method further has one or more features selected from the following: The conditions include TB medium, The culture temperature is 20-25°C, The culturing is for at least 24 hours, The method also includes the step of isolating the protoberberine compounds from the host cells; specifically, the steps include: crushing the cells, extracting with an organic solvent, and vacuum drying, wherein the organic solvent is preferably n-butanol.

7. A nucleic acid construct for increasing the production of reticuline in a cell, characterized in that: The nucleic acid construct comprises a coding sequence encoding the following enzymes: an enzyme catalyzing tyrosine hydroxylation to generate dopa, an enzyme catalyzing dopa decarboxylation to generate dopamine, an enzyme catalyzing dopamine to generate 3,4-dihydroxyphenylacetaldehyde, an enzyme catalyzing dopamine and 3,4-dihydroxyphenylacetaldehyde condensation to generate norlaudanine, an enzyme catalyzing norlaudanine to generate 3'hydroxycoclaurine, an enzyme catalyzing 3'hydroxycoclaurine to generate 3'hydroxynitromethylcoclaurine, an enzyme catalyzing 3'hydroxynitromethylcoclaurine to generate reticuline, an enzyme catalyzing a BH4 synthesis pathway, and an enzyme catalyzing a BH4 regeneration pathway. Preferably, the nucleic acid construct further has one or more characteristics selected from the following: The enzymes catalyzing the BH4 synthesis pathway include GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and bisopterin reductase, The enzymes catalyzing the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase, The nucleic acid construct further comprises a coding sequence for a purine nucleic acid kinase, The nucleic acid construct further comprises an enzyme that catalyzes the hydroxylation of tyramine to generate 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 that resists feedback inhibition, chorismate mutase and prephenate dehydrogenase that resists feedback inhibition, phosphoenolpyruvate synthase, a transketolase that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase, preferably, 3-deoxy-D-arabinoheptulose-7-phosphate synthase that resists feedback inhibition, chorismate mutase and prephenate dehydrogenase that resists feedback inhibition, phosphoenolpyruvate synthase, a transketolase that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase are derived from Escherichia coli, A polynucleotide that inhibits the expression of pyruvate kinase, A polynucleotide that inhibits chorismate translocation and prephenate dehydratase expression, A polynucleotide that inhibits the expression of aromatic amino acid transcriptional regulators, 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.

8. A host cell with improved reticuline production, characterized in that: The host cell: (1) Expression of the following enzymes: an enzyme catalyzing the hydroxylation of tyrosine to generate dopa, an enzyme catalyzing the decarboxylation of dopa to generate dopamine, an enzyme catalyzing the generation of 3,4-dihydroxyphenylacetaldehyde from dopamine, an enzyme catalyzing the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlaudanine, an enzyme catalyzing the generation of 3'-hydroxycoclaurine from norlaudanine, an enzyme catalyzing the generation of 3'-hydroxycoclaurine from 3'-hydroxynitromethylcoclaurine, an enzyme catalyzing the generation of reticuline from 3'-hydroxynitromethylcoclaurine, an enzyme catalyzing the BH4 regeneration pathway, an enzyme catalyzing the BH4 synthesis pathway, (2) comprising the nucleic acid construct according to claim 7, Preferably, the host cell also has one or more characteristics selected from the following: The enzymes catalyzing the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase, The enzymes catalyzing the BH4 synthesis pathway include GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and bisopterin reductase, The host cell also expresses purine nucleic acid kinase, The host cell also expresses an enzyme that catalyzes the hydroxylation of tyramine to generate dopamine. The host cell further expresses one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase that resists feedback inhibition, chorismate mutase and prephenate dehydrogenase that resists feedback inhibition, phosphoenolpyruvate synthase, a transketolase that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase, preferably, 3-deoxy-D-arabinoheptulose-7-phosphate synthase that resists feedback inhibition, chorismate mutase and prephenate dehydrogenase that resists feedback inhibition, phosphoenolpyruvate synthase, a transketolase that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase are derived from Escherichia coli, The host cell further comprises an agent that inhibits the expression of pyruvate kinase, The host cell further comprises an agent that inhibits chorismate translocation and prephenate dehydratase expression, The host cell also contains an agent that inhibits expression of the aromatic amino acid transcriptional regulator. The enzyme is integrated into the host cell via chromosome.

9. A method for producing reticulated annonaine, characterized in that: The method comprises the following steps: (1) Hydroxylation of tyrosine to generate DOPA, (2) decarboxylating dopa to generate dopamine, (3) Dopamine generates 3,4-dihydroxyphenylacetaldehyde, (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlabdanine, (5) converting norlabradanine into 3'-hydroxycoclaurine, (6) converting 3'-hydroxy-coclaurine into 3'-hydroxy-nitro-methyl-coclaurine, (7) 3'-hydroxyl nitrogen methylcoclaurine is converted into reticulate annonaine, The method further comprises catalyzing BH4 regeneration and catalyzing BH4 synthesis in step (1) and / or (2), Preferably, the method further has one or more features selected from the following: The reaction of hydroxylating tyrosine to form DOPA is catalyzed by an enzyme, preferably TYH or a variant thereof, Enzymes that catalyze the regeneration of BH4 include: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase, The enzyme catalyzing BH4 synthesis is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthetase and indole reductase, The reaction in step (2) is catalyzed by an enzyme, preferably DODC. Step (2) further comprises: hydroxylating tyramine to generate dopamine. Preferably, the hydroxylation of tyramine to generate dopamine is catalyzed by an enzyme, and the enzyme is preferably HpaBC or a variant thereof. The reaction in step (3) is catalyzed by an enzyme, preferably MAO. The reaction in step (4) is catalyzed by an enzyme, preferably NCS. The reaction in 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, More preferably, the method further has one or more features selected from the following: The TYH is from Rattus norvegicus or Drosophila, preferably, the TYH is from Rattus norvegicus, and 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 derived from Pseudomonas putida, The MAO is derived from Micrococcus luteus, The NCS is derived from Japanese Coptis chinensis, The 6OMT comes from opium poppy, The CNMT is derived from opium poppy The 4'OMT is from Coptis chinensis.

10. The method according to claim 9, characterized in that The method comprises the steps of culturing the host cell according to claim 8 under conditions suitable for producing reticuline, Preferably, the method further has one or more features selected from the following: The conditions include TB medium, The culture temperature is 20-25°C, The culturing is for at least 24 hours, The method further comprises the step of isolating and obtaining 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.

Images