Method for efficiently synthesizing protoberberine compound and use thereof

Abstract

A method for efficiently synthesizing a protoberberine compound and the use thereof. A host cell, which expresses the following enzymes: an enzyme that catalyzes the hydroxylation of tyrosine to produce dopa, an enzyme that catalyzes the decarboxylation of dopa to produce dopamine, an enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine, an enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlaudanine, an enzyme that catalyzes the production of 3'-hydroxycoclaurine from norlaudanine, an enzyme that catalyzes the production of 3'-hydroxy-N-methylcoclaurine from 3'-hydroxycoclaurine, an enzyme that catalyzes the production of reticuline from 3'-hydroxy-N-methylcoclaurine, an enzyme that catalyzes the production of scoulerine from reticuline, an enzyme that catalyzes the production of tetrahydrocolumbamine from scoulerine, an enzyme that catalyzes the production of tetrahydroberberine from tetrahydrocolumbamine, and an enzyme that reduces cytochrome P450 in an oxidation state. The method realizes the efficient synthesis of a protoberberine compound.

Description

High-efficiency synthesis method of protoberberine compounds and its application Technical Field

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

[0002] Benzylisoquinoline alkaloids (BIAs) are a class of natural products derived from tyrosine metabolism with diverse medicinal properties. They include protoberberine alkaloids, phthalide isoquinoline alkaloids, and morphinan alkaloids. Among these, some protoberberine compounds, such as chrysoberberine, tetrahydroafricanine, berberine, and tetrahydropalmatine, are widely distributed in the rhizomes of plants from the Berberidaceae, Ranunculaceae, and Papaveraceae families. In addition to their classic anti-inflammatory, antibacterial, and analgesic properties, recent studies have shown that these compounds may also play an important role in the treatment of tumors, diabetes, and cardiovascular diseases. Currently, protoberberine is prepared through plant extraction, chemical synthesis, and biosynthesis. However, the extraction of bioactive components from plants is limited by: 1. limited plant cultivation area; 2. long plant growth time; 3. low bioactive component content; and 4. significant losses during extraction. Meanwhile, while chemical synthesis technology is mature, numerous challenges remain when synthesizing complex natural products, including the complexity of synthesizing certain racemic compounds and the polluting nature of chemical synthesis pathways. Therefore, a new preparation method is needed to address the shortcomings of plant extraction and chemical synthesis and increase the supply of protoberberine compounds. Synthetic biology, particularly using Escherichia coli and Saccharomyces cerevisiae as a platform, has successfully synthesized a variety of complex and high-value-added natural products, such as flavonoids, opioids, and taxanes.

[0003] A series of progress has been made in the synthesis of protoberberine compounds in 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 work subsequently 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 also been some progress in Escherichia coli, but most of them are the work on the synthesis of the intermediate reticuline. Japanese researchers Nakagawa and others finally achieved the synthesis of 126 mg / L of reticuline using glucose as a carbon source by modifying the chassis bacteria, selecting appropriate functional elements, and optimizing culture conditions. Recently, Liu Jihua's team from China Pharmaceutical University used dopamine as a precursor in Escherichia coli. By truncating the nitrogen end of the berberine bridge enzyme from California poppy and fusing it with a hydrophilic tag, they improved the catalytic activity of the enzyme. On this basis, they introduced a continuously methylating enzyme and a P450 enzyme to achieve the synthesis of tetrahydrobamipine and corydaline. However, the yield of tetrahydrobamipine was only 1.19 mg / L.

[0005] Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention aims to efficiently synthesize protoberberine compounds or reticulated annonine.

[0007] In a first aspect, the present invention provides a nucleic acid construct for increasing the production of protoberberine compounds in cells, which comprises coding sequences 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 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 chrysoprine, an enzyme that catalyzes the production of tetrahydroafricanine from tetrahydroafricanine, an enzyme that catalyzes the production of tetrahydroberberine from tetrahydroafricanine, and an enzyme that reduces the oxidized state of cytochrome P450.

[0008] In one or more embodiments, the nucleic acid construct further comprises a coding sequence for an enzyme of the BH4 synthesis pathway and a coding sequence for an enzyme of the BH4 regeneration pathway, wherein the enzyme of the BH4 synthesis pathway is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthase, and dihydropterin reductase, and the enzyme of the BH4 regeneration pathway comprises: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase,

[0009] Preferably, the enzymes of the BH4 synthesis pathway include GTP cyclohydrolase from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthase from Rattus norvegicus, and bisopterin reductase from Rattus norvegicus.

[0010] Preferably, the enzymes of the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase from Rattus norvegicus and dihydropterin reductase from Rattus norvegicus.

[0011] In one or more embodiments, the nucleic acid construct further comprises an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine. Preferably, the enzyme that catalyzes the hydroxylation of tyramine to produce dopamine is HpaBC or a variant thereof.

[0012] In one or more embodiments, the nucleic acid construct further comprises a coding sequence for a purine nucleic acid kinase.

[0013] 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.

[0014] In one or more embodiments, the enzyme that catalyzes the hydroxylation of tyrosine to produce DOPA is TYH or a variant thereof.

[0015] In one or more embodiments, the enzyme that catalyzes the decarboxylation of dopa to produce dopamine is DODC.

[0016] In one or more embodiments, the enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine is MAO.

[0017] In one or more embodiments, the enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlabdanine is NCS.

[0018] In one or more embodiments, the enzyme that catalyzes the production of 3'-hydroxycoclaurine from norlabdanine is 6OMT.

[0019] In one or more embodiments, the enzyme that catalyzes the production of 3'-hydroxycoclaurine from 3'-hydroxynitromethylcoclaurine is CNMT.

[0020] In one or more embodiments, the enzyme that catalyzes the conversion of 3'-hydroxylaminomethylcoclaurine to reticuline is 4'OMT.

[0021] In one or more embodiments, the enzyme that catalyzes the production of chrysanthemine from reticuline is BBE1 or a variant thereof.

[0022] In one or more embodiments, the enzyme that catalyzes the production of tetrahydrotetrandrine from chrysocorydaline is 9'OMT.

[0023] In one or more embodiments, the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetramine is CAS.

[0024] In one or more embodiments, the enzyme that reduces the oxidized state of cytochrome P450 is CPR2.

[0025] In one or more embodiments, in the nucleic acid construct, the enzyme that catalyzes the conversion of tetrahydrotetracycline to tetrahydroberberine (eg, CAS) and the enzyme that reduces the oxidative state of cytochrome P450 (eg, CPR2) form a complex.

[0026] 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.

[0027] 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.

[0028] In one or more embodiments, the protein interaction domain includes an SH3 domain, and its ligand is SH3Lig. Preferably, the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine and the enzyme that reduces the oxidative state of cytochrome P450 are bound to the SH3 domain and its ligand, respectively. Preferably, the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine is fused to SH3, and the enzyme that reduces the oxidative state of cytochrome P450 is fused to SH3Lig. Preferably, the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine is connected to SH3 via a linker, and the enzyme that reduces the oxidative state of cytochrome P450 is connected to SH3Lig via a linker.

[0029] In one or more embodiments, the enzyme that catalyzes the conversion of tetrahydrotetramine to tetrahydroberberine is located at the N-terminus of SH3.

[0030] In one or more embodiments, the enzyme that reduces the oxidative state of cytochrome P450 is located at the N-terminus of SH3Lig.

[0031] 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.

[0032] In one or more embodiments, the amino acid sequence of the SH3 domain is shown in SEQ ID NO: 73, and the nucleic acid sequence is shown in SEQ ID NO: 64.

[0033] 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.

[0034] In one or more embodiments, TYH is from Rattus norvegicus.

[0035] In one or more embodiments, the TYH variant has mutations at positions R37, R38, and W166; preferably, the mutations are R37E, R38E, and W166Y.

[0036] In one or more embodiments, the HpaBC is from E. coli.

[0037] In one or more embodiments, the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.

[0038] In one or more embodiments, the DODC is from Pseudomonas putida.

[0039] In one or more embodiments, the MAO is from Micrococcus luteus.

[0040] In one or more embodiments, the NCS is from Coptis chinensis.

[0041] In one or more embodiments, the 6OMT is from opium poppy.

[0042] In one or more embodiments, the CNMT is from opium poppy.

[0043] In one or more embodiments, the 4'OMT is from Coptis chinensis.

[0044] 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-promoting tag.

[0045] In one or more embodiments, the BBE1 variant has a mutation at one or both of F398 and I431.

[0046] In one or more embodiments, 9'OMT is from Coptis chinensis.

[0047] In one or more embodiments, the CAS is from Coptis chinensis.

[0048] In one or more embodiments, CPR2 is from Arabidopsis thaliana.

[0049] In one or more embodiments, the sequence of Rattus norvegicus TYH is shown in SEQ ID NO:1.

[0050] In one or more embodiments, the sequence of Drosophila TYH is shown in SEQ ID NO:2.

[0051] In one or more embodiments, the sequence of MtrA is shown in SEQ ID NO:3.

[0052] In one or more embodiments, the sequence of PTPS is shown in SEQ ID NO:4.

[0053] In one or more embodiments, the sequence of the SPR is shown in SEQ ID NO:5.

[0054] In one or more embodiments, the sequence of the PCD is shown in SEQ ID NO:6.

[0055] In one or more embodiments, the sequence of QDHPR is shown in SEQ ID NO:7.

[0056] In one or more embodiments, the sequence of the HpaBC variant is shown in SEQ ID NO:8.

[0057] In one or more embodiments, the sequence of DODC is shown in SEQ ID NO:9.

[0058] In one or more embodiments, the sequence of MAO is shown in SEQ ID NO:10.

[0059] In one or more embodiments, the sequence of NCS is shown in SEQ ID NO:11.

[0060] In one or more embodiments, the sequence of 6OMT is shown in SEQ ID NO:12.

[0061] In one or more embodiments, the sequence of CNMT is as shown in SEQ ID NO:13.

[0062] In one or more embodiments, the sequence of 4'OMT is as shown in SEQ ID NO:14.

[0063] 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 one or two mutations at positions selected from F398 and I431, wherein the mutation is a substitution, deletion or addition of an amino acid residue, (3) 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 tag.

[0064] In one or more embodiments, the sequence of 9'OMT is as shown in SEQ ID NO:16.

[0065] In one or more embodiments, the sequence of CAS is shown in SEQ ID NO:17.

[0066] In one or more embodiments, the sequence of CPR2 is as shown in SEQ ID NO:18.

[0067] In one or more embodiments, the sequence of DeoD is shown in SEQ ID NO:19.

[0068] In one or more embodiments, the sequence of RraA is shown in SEQ ID NO:20.

[0069] In one or more embodiments, the sequence of cytochrome b5 is as shown in SEQ ID NO: 21 or 68 or 70.

[0070] 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 ), feedback-resistant chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB).

[0071] In one or more embodiments, aroG, tyrA, ppsA, tktA, ydiB are derived from Escherichia coli.

[0072] In one or more embodiments, aroG fbr The amino acid sequence is shown in SEQ ID NO: 22.

[0073] In one or more embodiments, tyrA fbr The amino acid sequence is shown in SEQ ID NO: 23.

[0074] In one or more embodiments, the amino acid sequence of ppsA is as shown in SEQ ID NO:24.

[0075] In one or more embodiments, the amino acid sequence of tktA is as shown in SEQ ID NO:25.

[0076] In one or more embodiments, the amino acid sequence of ydiB is as shown in SEQ ID NO:26.

[0077] 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 the sRNA is as shown in SEQ ID NO: 27.

[0078] 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.

[0079] In one or more embodiments, the nucleic acid construct further comprises a polynucleotide that inhibits 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.

[0080] 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.

[0081] The first aspect of the present invention further provides a host cell with increased production of protoberberine compounds, wherein the host cell:

[0082] (1) expressing 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'-hydroxynitromethylcoclaurine from 3'-hydroxynitromethylcoclaurine from reticuline, an enzyme that catalyzes the production of chrysanthemine from reticuline, an enzyme that catalyzes the production of tetrahydroafricanine from tetrahydroafricanine, an enzyme that catalyzes the production of tetrahydroberberine from tetrahydroafricanine, and an enzyme that reduces the oxidized state of cytochrome P450, or

[0083] (2) comprising the nucleic acid construct according to any embodiment of the first aspect of this invention,

[0084] In one or more embodiments, the cell further 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-pyruvoyl-tetrahydrobiopterin synthase, 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-pyruvoyl-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.

[0085] In one or more embodiments, the cells further express purine nucleic acid kinase (DeoD).

[0086] In one or more embodiments, the cell further expresses nuclease activity regulator protein (RraA) and / or cytochrome b5.

[0087] In one or more embodiments, the cell further expresses an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine. Preferably, the enzyme that catalyzes the hydroxylation of tyramine to produce dopamine is HpaBC or a variant thereof.

[0088] In one or more embodiments, the cell co-expresses an enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetramine and an enzyme that reduces the oxidized state of cytochrome P450.

[0089] 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 a protein interaction domain and its ligand and expressed respectively; or the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine and the enzyme that reduces the oxidized state of cytochrome P450 are fused and expressed.

[0090] 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.

[0091] 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 oxidative state of 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 that reduces the oxidative 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 oxidative state of cytochrome P450 is fused with SH3Lig through a linker.

[0092] In one or more embodiments, the enzyme that catalyzes the conversion of tetrahydrotetramine to tetrahydroberberine is located at the N-terminus of SH3.

[0093] In one or more embodiments, the enzyme that reduces the oxidative state of cytochrome P450 is located at the N-terminus of SH3Lig.

[0094] In one or more embodiments, the enzyme that catalyzes the hydroxylation of tyrosine to produce DOPA is TYH or a variant thereof.

[0095] In one or more embodiments, the enzyme that catalyzes the decarboxylation of dopa to dopamine is DODC.

[0096] In one or more embodiments, the enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine is MAO.

[0097] In one or more embodiments, the enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlabdanine is NCS.

[0098] In one or more embodiments, the enzyme that catalyzes the production of 3'-hydroxycoclaurine from norlabdanine is 6OMT.

[0099] In one or more embodiments, the enzyme that catalyzes the production of 3'-hydroxycoclaurine to 3'-hydroxynitromethylcoclaurine is CNMT.

[0100] In one or more embodiments, the enzyme that catalyzes the production of reticuline from 3'hydroxynitromethylcoclaurine is 4'OMT.

[0101] In one or more embodiments, the enzyme that catalyzes the production of reticuline from chrysocortinine is BBE1 or a variant thereof.

[0102] In one or more embodiments, the enzyme that catalyzes the production of tetrahydrotetrandrine from chrysocorydaline is 9'OMT.

[0103] In one or more embodiments, the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetramine is CAS.

[0104] In one or more embodiments, the enzyme that reduces the oxidized state of cytochrome P450 is CPR2.

[0105] In one or more embodiments, the host cell is an E. coli cell.

[0106] In one or more embodiments, TYH is from Rattus norvegicus.

[0107] In one or more embodiments, the TYH variant has mutations at positions R37, R38, and W166; preferably, the mutations are R37E, R38E, and W166Y.

[0108] In one or more embodiments, the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.

[0109] In one or more embodiments, the DODC is from Pseudomonas putida.

[0110] In one or more embodiments, the MAO is from Micrococcus luteus.

[0111] In one or more embodiments, the NCS is from Coptis chinensis.

[0112] In one or more embodiments, the 6OMT is from opium poppy.

[0113] In one or more embodiments, the CNMT is from opium poppy.

[0114] In one or more embodiments, the 4'OMT is from Coptis chinensis.

[0115] 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.

[0116] In one or more embodiments, the BBE1 variant has mutations at positions F398 and I431.

[0117] In one or more embodiments, 9'OMT is from Coptis chinensis.

[0118] In one or more embodiments, the CAS is from Coptis chinensis.

[0119] In one or more embodiments, CPR2 is from Arabidopsis thaliana.

[0120] In one or more embodiments, the E. coli is a B-lineage E. coli, such as BL21(DE3).

[0121] 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 ), feedback-resistant chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB).

[0122] In one or more embodiments, aroG, tyrA, ppsA, tktA, ydiB are derived from Escherichia coli.

[0123] In one or more embodiments, aroG fbr The amino acid sequence is shown in SEQ ID NO: 22.

[0124] In one or more embodiments, tyrA fbr The amino acid sequence is shown in SEQ ID NO: 23.

[0125] In one or more embodiments, the amino acid sequence of ppsA is as shown in SEQ ID NO:24.

[0126] In one or more embodiments, the amino acid sequence of tktA is as shown in SEQ ID NO:25.

[0127] In one or more embodiments, the amino acid sequence of ydiB is as shown in SEQ ID NO:26.

[0128] In one or more embodiments, the host cell further comprises an agent that inhibits the expression of pyruvate kinase (pykA), such as dsRNA, sRNA or sgRNA. In one or more embodiments, the sequence of the sRNA is as shown in SEQ ID NO: 27.

[0129] 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.

[0130] In one or more embodiments, the host cell further comprises an agent that inhibits 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.

[0131] In one or more embodiments, in the host cell according to any embodiment of the first aspect of this invention, the enzyme is integrated into the host cell via chromosome.

[0132] A second aspect of the present invention provides a method for producing protoberberine compounds, the method comprising the following steps:

[0133] (1) Hydroxylation of tyrosine to generate dopa,

[0134] (2) decarboxylating dopa to generate dopamine,

[0135] (3) Dopamine generates 3,4-dihydroxyphenylacetaldehyde,

[0136] (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlabdanine,

[0137] (5) converting norlabdanine into 3'-hydroxycoclaurine,

[0138] (6) 3'-hydroxy-coclaurine is converted into 3'-hydroxy-nitromethyl-coclaurine,

[0139] (7) 3'-hydroxyl nitrogen methyl-coclaurine is converted into reticulate annonine,

[0140] (8) Reticulated annonine generates golden corydaline,

[0141] (9) generating tetrahydroafricanine from golden corydaline, and

[0142] (10) Tetrahydroafricanine is converted into tetrahydroberberine.

[0143] In one or more embodiments, the method further comprises using an enzyme catalyzing the reduction of an oxidative state of cytochrome P450, preferably CPR2.

[0144] In one or more embodiments, the reaction of step (1) is catalyzed by an enzyme, preferably TYH or a variant thereof.

[0145] 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 synthase, and indole reductase. Preferably, the enzymes of the BH4 regeneration pathway comprise: 4α-hydroxytetrahydropterin dehydratase from Rattus norvegicus and dihydropterin reductase from Rattus norvegicus. Preferably, the enzymes of the BH4 synthesis pathway comprise GTP cyclohydrolase from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthase from Rattus norvegicus, and indole reductase from Rattus norvegicus.

[0146] In one or more embodiments, the reaction of step (2) is catalyzed by an enzyme, preferably DODC.

[0147] 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.

[0148] In one or more embodiments, the reaction of step (3) is catalyzed by an enzyme, preferably MAO.

[0149] In one or more embodiments, the reaction of step (4) is catalyzed by an enzyme, preferably NCS.

[0150] In one or more embodiments, the reaction of step (5) is catalyzed by an enzyme, preferably 6OMT.

[0151] In one or more embodiments, the reaction of step (6) is catalyzed by an enzyme, preferably CNMT.

[0152] In one or more embodiments, the reaction of step (7) is catalyzed by an enzyme, preferably 4'OMT.

[0153] In one or more embodiments, the reaction of step (8) is catalyzed by an enzyme, preferably BBE1 or a variant thereof.

[0154] In one or more embodiments, the reaction of step (9) is catalyzed by an enzyme, preferably 9'OMT.

[0155] In one or more embodiments, the reaction of step (10) is catalyzed by an enzyme, preferably CAS.

[0156] In one or more embodiments, the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine (e.g., CAS) and the enzyme that reduces the oxidized state of cytochrome P450 (e.g., CPR2) are fused to a protein interaction domain and its ligand to form separate fusion proteins; or the enzyme that catalyzes the conversion of tetrahydroafricanine to 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.

[0157] 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.

[0158] 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-oxidation cytochrome P450 are fused to the SH3 domain and its ligand, respectively. Preferably, the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused to SH3, and the enzyme of the reduced-oxidation cytochrome P450 is fused to SH3Lig. Preferably, when the enzyme that catalyzes tetrahydroafricanine to produce tetrahydroberberine is fused to SH3, it is further connected with a linker, and when the enzyme of the reduced-oxidation cytochrome P450 is fused to SH3Lig, it is further connected with a linker.

[0159] In one or more embodiments, the enzyme that catalyzes the production of tetrahydroberberine from tetrahydrotetramine is located at the N-terminus of SH3.

[0160] In one or more embodiments, the enzyme that reduces the oxidative state of cytochrome P450 is located at the N-terminus of SH3Lig.

[0161] In one or more embodiments, the method further comprises: upregulating the expression of purine nucleic acid kinase (DeoD). Preferably, the expression of purine nucleic acid kinase is upregulated by plasmid overexpression technology.

[0162] In one or more embodiments, the method further comprises: upregulating 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 upregulated by plasmid overexpression technology.

[0163] In one or more embodiments, TYH is from Rattus norvegicus.

[0164] In one or more embodiments, the TYH variant has mutations at positions R37, R38, and W166; preferably, the mutations are R37E, R38E, and W166Y.

[0165] In one or more embodiments, the HpaBC is from Escherichia coli. Preferably, the HpaBC variant has mutations at positions A211 and Q212; more preferably, the mutations are A211G and Q212Y.

[0166] In one or more embodiments, the DODC is from Pseudomonas putida.

[0167] In one or more embodiments, the MAO is from Micrococcus luteus.

[0168] In one or more embodiments, the NCS is from Coptis chinensis.

[0169] In one or more embodiments, the 6OMT is from opium poppy.

[0170] In one or more embodiments, the CNMT is from opium poppy.

[0171] In one or more embodiments, the 4'OMT is from Coptis chinensis.

[0172] 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.

[0173] In one or more embodiments, 9'OMT is from Coptis chinensis.

[0174] In one or more embodiments, the CAS is from Coptis chinensis.

[0175] In one or more embodiments, CPR2 is from Arabidopsis thaliana.

[0176] In one or more embodiments, the method includes the step of culturing the host cell according to any embodiment herein under conditions suitable for the production of a protoberberine compound.

[0177] In one or more embodiments, the conditions comprise TB medium.

[0178] In one or more embodiments, the culturing temperature is 20-25°C.

[0179] In one or more embodiments, the culturing is for at least 24 hours.

[0180] In one or more embodiments, the method further comprises the step of isolating the protoberberine compounds 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.

[0181] In one or more embodiments, the method further comprises:

[0182] One or more or all of the following enzymes are expressed in the cell: 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ), feedback-resistant chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB);

[0183] 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;

[0184] Down-regulating the expression or activity of chorismate translocation and prephenate dehydratase (pheA); preferably, down-regulating the expression or activity of said enzymes by RNAi or CRISPR technology; and / or

[0185] Downregulating the expression or activity of the aromatic amino acid transcription regulator (tyrR); preferably, downregulating the expression or activity of the enzyme by RNAi or CRISPR technology.

[0186] A second aspect of the present invention provides a nucleic acid construct for increasing the production of reticuline in a cell, comprising coding sequences 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 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 synthesis pathway, and an enzyme that catalyzes the BH4 regeneration pathway.

[0187] 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 GTP cyclohydrolase from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthetase from Rattus norvegicus, and indole reductase from Rattus norvegicus.

[0188] 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: 4α-hydroxytetrahydropterin dehydratase from Rattus norvegicus and dihydropterin reductase from Rattus norvegicus. In one or more embodiments, the nucleic acid construct further comprises a coding sequence for purine nucleic acid kinase (DeoD).

[0189] In one or more embodiments, the nucleic acid construct further comprises an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine.

[0190] 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 ), feedback-resistant chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB).

[0191] In one or more embodiments, aroG, tyrA, ppsA, tktA, ydiB are derived from Escherichia coli.

[0192] In one or more embodiments, aroG fbr The amino acid sequence is shown in SEQ ID NO: 22.

[0193] In one or more embodiments, tyrA fbr The amino acid sequence is shown in SEQ ID NO: 23.

[0194] In one or more embodiments, the amino acid sequence of ppsA is as shown in SEQ ID NO:24.

[0195] In one or more embodiments, the amino acid sequence of tktA is as shown in SEQ ID NO:25.

[0196] In one or more embodiments, the amino acid sequence of ydiB is as shown in SEQ ID NO:26.

[0197] 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 the sRNA is as shown in SEQ ID NO: 27.

[0198] 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 as shown in SEQ ID NO: 28.

[0199] 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.

[0200] 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. Typically, one expression frame may contain the coding sequence of one, two, three, four, five, six, seven, eight, nine, ten or more enzymes.

[0201] The second aspect of the present invention further provides a host cell with increased reticuline production, wherein the host cell:

[0202] (1) Expression of 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, an enzyme that catalyzes the BH4 synthesis pathway,

[0203] (2) A nucleic acid construct comprising any one of the embodiments of the second aspect of this invention.

[0204] In one or more embodiments, the enzymes catalyzing the BH4 regeneration pathway include: α - hydroxytetrahydropterin dehydratase and / or dihydropterin reductase. Preferably, the enzymes catalyzing the BH4 regeneration pathway include: PCD and QDHPR from Rattus norvegicus.

[0205] In one or more embodiments, the enzymes catalyzing the BH4 synthesis pathway include GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthase, 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.

[0206] In one or more embodiments, the cells further express purine nucleic acid kinase (DeoD).

[0207] In one or more embodiments, the cell further expresses an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine.

[0208] 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 ), feedback-resistant chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB).

[0209] In one or more embodiments, aroG, tyrA, ppsA, tktA, ydiB are derived from Escherichia coli.

[0210] In one or more embodiments, aroG fbr The amino acid sequence is shown in SEQ ID NO: 22.

[0211] In one or more embodiments, tyrA fbr The amino acid sequence is shown in SEQ ID NO: 23.

[0212] In one or more embodiments, the amino acid sequence of ppsA is as shown in SEQ ID NO:24.

[0213] In one or more embodiments, the amino acid sequence of tktA is as shown in SEQ ID NO:25.

[0214] In one or more embodiments, the amino acid sequence of ydiB is as shown in SEQ ID NO:26.

[0215] 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 the sRNA is as shown in SEQ ID NO: 27.

[0216] 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.

[0217] 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.

[0218] In one or more embodiments, in the host cell according to any embodiment of the second aspect of this invention, the enzyme is integrated into the host cell via chromosome.

[0219] The second aspect of the present invention also provides a method for producing reticulated annonaine, which comprises the following steps:

[0220] (1) Hydroxylation of tyrosine to generate dopa,

[0221] (2) decarboxylating dopa to generate dopamine,

[0222] (3) Dopamine generates 3,4-dihydroxyphenylacetaldehyde,

[0223] (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlabdanine,

[0224] (5) converting norlabdanine into 3'-hydroxycoclaurine,

[0225] (6) 3'-hydroxy-coclaurine is converted into 3'-hydroxy-nitromethyl-coclaurine,

[0226] (7) 3'-hydroxyl nitrogen methyl-coclaurine is converted into reticulate annonine,

[0227] The method further comprises catalyzing BH4 regeneration and catalyzing BH4 synthesis in steps (1) and / or (2).

[0228] In one or more embodiments, the reaction of hydroxylating tyrosine to form DOPA is catalyzed by an enzyme, preferably TYH or a variant thereof.

[0229] In one or more embodiments, the enzyme that catalyzes the regeneration of BH4 comprises: α -hydroxytetrahydropterin dehydratase and / or dihydropterin reductase. Preferably, the enzymes catalyzing the BH4 regeneration pathway include: 4α-hydroxytetrahydropterin dehydratase from Rattus norvegicus and dihydropterin reductase from Rattus norvegicus.

[0230] In one or more embodiments, the enzyme catalyzing BH4 synthesis is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvoyl-tetrahydrobiopterin synthetase, and indole reductase. Preferably, the enzyme catalyzing the BH4 synthesis pathway includes GTP cyclohydrolase from Bacillus subtilis, 6-pyruvoyl-tetrahydrobiopterin synthetase from Rattus norvegicus, and indole reductase from Rattus norvegicus.

[0231] In one or more embodiments, the reaction of step (2) is catalyzed by an enzyme, preferably DODC.

[0232] 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.

[0233] In one or more embodiments, the reaction of step (3) is catalyzed by an enzyme, preferably MAO.

[0234] In one or more embodiments, the reaction of step (4) is catalyzed by an enzyme, preferably NCS.

[0235] In one or more embodiments, the reaction of step (5) is catalyzed by an enzyme, preferably 6OMT.

[0236] In one or more embodiments, the reaction of step (6) is catalyzed by an enzyme, preferably CNMT.

[0237] In one or more embodiments, the reaction of step (7) is catalyzed by an enzyme, preferably 4'OMT.

[0238] 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.

[0239] In one or more embodiments, the HpaBC is from Escherichia coli.

[0240] In one or more embodiments, the HpaBC variant has mutations at positions A211 and Q212; preferably, the mutations are A211G and Q212Y.

[0241] In one or more embodiments, the DODC is from Pseudomonas putida.

[0242] In one or more embodiments, the MAO is from Micrococcus luteus.

[0243] In one or more embodiments, the NCS is from Coptis chinensis.

[0244] In one or more embodiments, the 6OMT is from opium poppy.

[0245] In one or more embodiments, the CNMT is from opium poppy.

[0246] In one or more embodiments, the 4'OMT is from Coptis chinensis.

[0247] 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.

[0248] In one or more embodiments, the conditions comprise TB medium.

[0249] In one or more embodiments, the culturing temperature is 20-25°C.

[0250] In one or more embodiments, the culturing is for at least 24 hours.

[0251] In one or more embodiments, the method further comprises the step of isolating and obtaining reticuline from host cells; specifically comprising: disrupting the cells, extracting with an organic solvent, and vacuum drying, wherein the organic solvent is preferably n-butanol.

[0252] In one or more embodiments, the method further comprises:

[0253] One or more or all of the following enzymes are expressed in the cell: 3-deoxy-D-arabinoheptulose-7-phosphate synthase (aroG fbr ), feedback-resistant chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase (tktA) that increases erythrose-4-phosphate synthesis, and shikimate dehydrogenase (ydiB);

[0254] 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,

[0255] Down-regulating the expression or activity of chorismate translocation and prephenate dehydratase (pheA); preferably, down-regulating the expression or activity of said enzymes by RNAi or CRISPR technology; and / or

[0256] Downregulating the expression or activity of the aromatic amino acid transcription regulator (tyrR); preferably, downregulating the expression or activity of the enzyme by RNAi or CRISPR technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0257] FIG1 is a standard curve of reticuline drawn based on the relationship between the response of the extracted ions and the sample concentration in LC-MS / MS detection.

[0258] FIG2 shows the results of strain construction, tyrosine production and bacterial density.

[0259] FIG3 shows the reticuline production of strains from different sources and the regeneration and recycling pathways of tetrahydrobiopterin.

[0260] Figure 4 shows the synthetic pathway of the by-products inferred from the LC-MS / MS detection results.

[0261] FIG5 shows the reticuline production of the strain overexpressing HpaBC-D11.

[0262] FIG6 shows the reticuline production of strains overexpressing or inhibiting different targets.

[0263] FIG7 shows the nitrogen-terminal design of tetrahydroberberine synthase (CAS) and the results of SDS-PAGE analysis.

[0264] FIG8 shows CAS from different sources and different co-localization designs of CAS and CPR.

[0265] FIG9 shows the in vitro activity test results of CAS from different sources and with different co-localization designs.

[0266] FIG10 shows the process and identification results of the oxidized end product produced in the in vitro activity test.

[0267] FIG11 shows the tetrahydroberberine production of strains overexpressing different effectors and cytochrome b5.

[0268] FIG12 shows the protoberberine yield of SQZ266 in a 7.5 L batch fermentation reactor. DETAILED DESCRIPTION

[0269] 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, tetrahydroberberine, coptisine, tetrahydrocorydaline, tetrahydroafricanine, swiss corydaline, corydaline, golden corydaline, dehydrocorydaline, tetrahydropalmatine, corydaline and corydaline, etc.

[0270] 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.

[0271] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0272] The inventors transformed the chromosomes of Escherichia coli BL21 (DE3) by integrating genes that improve tyrosine synthesis and genes involved in the BH4 cycle pathway into the strain, thereby obtaining a chassis strain that produces high levels of tyrosine and synthesizes tetrahydrobiopterin. Furthermore, the inventors alleviated the feedback inhibition of tyramine on tyrosine hydroxylase by using a mutant of the enzyme HpaBC-D11 that endogenously catalyzes tyramine hydroxylation. Furthermore, by regulating targets related to tyrosine and tetrahydrobiopterin, the efficient synthesis of reticuline was achieved. Subsequently, by introducing mutants and scaffold proteins, the functional expression of two types of membrane proteins (BBE and CAS) was optimized. Finally, by introducing the global regulatory factors RraA and cytochrome b5, the efficient synthesis of protoberberine compounds was achieved.

[0273] Therefore, the present invention provides a host cell with improved production of protoberberine compounds, which expresses the following enzymes: an enzyme that catalyzes the hydroxylation of tyrosine to produce dopa, an enzyme that catalyzes the decarboxylation of dopa to produce dopamine, an enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine, an enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlaudanine, an enzyme that catalyzes the production of 3'-hydroxycoclaurine from norlaudanine, an enzyme that catalyzes the production of 3'-hydroxynitromethylcoclaurine from 3'-hydroxynitromethylcoclaurine from reticuline, an enzyme that catalyzes the production of chrysanthemumine from reticuline, an enzyme that catalyzes the production of tetrahydroafricanine from tetrahydroafricanine, an enzyme that catalyzes the production of tetrahydroberberine from tetrahydroafricanine, and an enzyme that reduces the oxidized state of cytochrome P450.

[0274] Typically, the host cell comprises a nucleic acid construct expressing the above enzymes. The enzyme that catalyzes the hydroxylation of tyrosine to produce dopa is TYH or a variant thereof, an exemplary TYH being from Rattus norvegicus, with an amino acid sequence as shown in SEQ ID NO: 1 and a nucleic acid sequence as shown in SEQ ID NO: 31. TYH variants include variants having mutations at R37, R38, and W166, such as R37E, R38E, and W166Y, an exemplary TYH being from Drosophila, with an amino acid sequence as shown in SEQ ID NO: 2 and a nucleic acid sequence as shown in SEQ ID NO: 32; the enzyme that catalyzes the decarboxylation of dopa to produce dopamine is DODC, an exemplary DODC being from Pseudomonas putida, with an amino acid sequence as shown in SEQ ID NO: 9 and a nucleic acid sequence as shown in SEQ ID NO: 39; the enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde from dopamine is MAO, an exemplary MAO being from Micrococcus luteus, with an amino acid sequence as shown in SEQ ID NO: 10 and a nucleic acid sequence as shown in SEQ ID NO: 11. NO: 40; the enzyme that catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlaudanine 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 norlaudanine 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 reticuline 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 reticuline to produce chrysanthemine is BBE1 or a variant thereof, an exemplary BBE1 is from Papaver somniferum, has an amino acid sequence as shown in SEQ ID NO:15, and a nucleic acid sequence as shown in SEQ ID NO:45. BBE1 variants include variants having mutations at positions F398 and I431, such as F398W and I431F. In some embodiments, BBE1 contains a truncated nitrogen-terminal transmembrane region, and in other embodiments, BBE1 contains a truncated nitrogen-terminal transmembrane region fused to an 8RP solubility tag; the enzyme that catalyzes chrysanthemine to produce tetrahydroafricanine is 9'OMT, an exemplary 9'OMT is from Coptis chinensis, has an amino acid sequence as shown in SEQ ID NO:16, and a nucleic acid sequence as shown in SEQ ID NO:46;The enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine is CAS. An exemplary CAS is from Coptis chinensis, with an amino acid sequence as shown in SEQ ID NO: 17 and a nucleic acid sequence as shown in SEQ ID NO: 47. The enzyme that reduces the oxidized state of cytochrome P450 is CPR2. An exemplary CPR2 is from Arabidopsis thaliana, with an amino acid sequence as shown in SEQ ID NO: 18 and a nucleic acid sequence as shown in SEQ ID NO: 48.

[0275] The inventors have found that the recycling pathway of tetrahydrobiopterin can improve the activity of tyrosine hydroxylase and significantly increase the production of reticuline. 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). Exemplary enzymes of the BH4 synthesis pathway include (MtrA) from Bacillus subtilis, with an amino acid sequence as shown in SEQ ID NO: 3 and a nucleic acid sequence as shown in SEQ ID NO: 33; PTPS from Rattus norvegicus, with an amino acid sequence as shown in SEQ ID NO: 4 and a nucleic acid sequence as shown in SEQ ID NO: 34; SPR from Rattus norvegicus, with an amino acid sequence as shown in SEQ ID NO: 5 and a nucleic acid sequence as shown in SEQ ID NO: 6. NO: 35; enzymes of the BH4 regeneration pathway include: 4a-hydroxytetrahydropterin dehydratase (PCD) and / or dihydropterin reductase (QDHPR), exemplary enzymes of the BH4 regeneration pathway include: PCD from Rattus norvegicus, the amino acid sequence of which is shown in SEQ ID NO: 6, and the nucleic acid sequence of which is shown in SEQ ID NO: 36; QDHPR from Rattus norvegicus, the amino acid sequence of which is shown in SEQ ID NO: 7, and the nucleic acid sequence of which is shown in SEQ ID NO: 37.

[0276] The inventors have also discovered that efficient reticuline synthesis can be achieved by alleviating inhibition using a mutant of the endogenous enzyme catalyzing tyramine hydroxylation, HpaBC-D11. Therefore, in some embodiments, the host cell also expresses an enzyme that catalyzes the hydroxylation of tyramine to produce dopamine. The enzyme that catalyzes the hydroxylation of tyramine to produce dopamine is HpaBC or a variant thereof. Exemplary HpaBC is from Escherichia coli. HpaBC variants include those with mutations at positions A211 and Q212, such as A211G and Q212Y. The amino acid sequence is set forth in SEQ ID NO: 8, and the nucleic acid sequence is set forth in SEQ ID NO: 38.

[0277] Furthermore, the inventors have discovered that overexpressing purine nucleic acid kinase can improve reticuline production. Therefore, agents for increasing tyrosine production may also include agents that upregulate 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.

[0278] The inventors have also discovered that co-localized expression of an enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine and an enzyme that reduces oxidized cytochrome P450 can promote the oxidation of tetrahydroafricanine. In other embodiments, the cells co-localize the expression of an enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine and an enzyme that reduces oxidized cytochrome P450. In some embodiments, the co-localized expression includes fusion expression of an enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine and an enzyme that reduces oxidized cytochrome P450, respectively, with a protein interaction domain and its ligand; or fusion expression of an enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine and an enzyme that reduces oxidized cytochrome P450. 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 the conversion of tetrahydroafricanine to tetrahydroberberine and the enzyme that reduces the oxidative state of cytochrome P450 are fused with the SH3 domain and its ligand, respectively. Preferably, the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine is fused with SH3 and expressed, and the enzyme that reduces the oxidative state of cytochrome P450 is fused with SH3Lig. Preferably, when the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine is fused with SH3 and expressed, it is further connected with a linker, and when the enzyme that reduces the oxidative state of cytochrome P450 is fused with SH3Lig and expressed, it is further connected with a linker. In one or more embodiments, when the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine is fused with SH3 and expressed, the enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine is located at the N-terminus of SH3. In one or more embodiments, when the cytochrome P450 enzyme that reduces the oxidative state is expressed as a fusion with SH3Lig, the cytochrome P450 enzyme that reduces the oxidative state is located at the N-terminus of SH3Lig. An exemplary linker amino acid sequence is shown in SEQ ID NO: 72, and a nucleic acid sequence is shown in SEQ ID NO: 63; an exemplary SH3 nucleic acid sequence is shown in SEQ ID NO: 64; and an exemplary SH3Lig nucleic acid sequence is shown in SEQ ID NO: 65.

[0279] The inventors also discovered 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 a nuclease activity regulatory protein (RraA) and / or cytochrome b5, an exemplary RraA from Escherichia coli, with an amino acid sequence as shown in SEQ ID NO: 20 and a nucleic acid sequence as shown in SEQ ID NO: 50; an exemplary cytochrome b5 from Chinese coptis chinensis, with an amino acid sequence as shown in SEQ ID NO: 21 and a nucleic acid sequence as shown in SEQ ID NO: 51; or an exemplary cytochrome b5 from Phanerochaete chrysosporium, with an amino acid sequence as shown in SEQ ID NO: 68 and a nucleic acid sequence as shown in SEQ ID NO: 69; or an exemplary cytochrome b5 from Chinese coptis chinensis, with an amino acid sequence as shown in SEQ ID NO: 70 and a nucleic acid sequence as shown in SEQ ID NO: 71.

[0280] The enzymes are encoded by the corresponding enzyme encoding nucleic acid sequences contained in the nucleic acid constructs. The nucleic acid sequences can be in the form of DNA or RNA, can be single-stranded or double-stranded, and can be coding strands or non-coding strands.

[0281] The coding sequences of all enzymes may be in one, two, three or more expression frames. In embodiments where multiple coding sequences are in the same expression frame, a linker sequence is included between each coding sequence, and the linker sequence enables the expression of multiple cistrons in a single expression frame. In one or more embodiments, the linker sequence is a coding sequence for a 2A peptide. 2A peptides include F2A, P2A or T2A peptides. Those skilled in the art can determine the type and quantity of coding sequences contained in an expression frame based on sequence length, promoter, etc. In some embodiments, the coding sequences of the enzymes are in two 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 sequences for the enzymes are present in three expression frames, wherein the coding sequences for TYH or a variant thereof, BH4, DODC, HpaBC or a variant thereof, MAO, and NCS are present in one expression frame, the coding sequences for 6OMT, CNMT, and 4'OMT are present in one expression frame, and the coding sequences for BBE1, 9'OMT, and CAS are present in one expression frame. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a vast number of nucleic acids can be produced, all of which encode the antibodies or antigen-binding fragments thereof of the present invention. Therefore, once a specific amino acid sequence has been identified, one skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons without altering the amino acid sequence of the encoded protein. For example, a nucleic acid sequence can be optimized using codons preferred by a species (e.g., E. coli) to facilitate expression in that species. In exemplary embodiments, the coding sequences for the amino acid sequences set forth in SEQ ID NOs: 1-26, 72, 66, 68, and 70 are, for example, set forth in SEQ ID NOs: 31-56, 63, 67, 69, and 71, respectively. Therefore, the present invention also relates to polynucleotides that hybridize to the above-mentioned polynucleotide sequences 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 hybridize to 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) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0282] The inventors have found that different overexpression methods can also affect the tyrosine production of the strain, and the tyrosine strain obtained by chromosomal integration has a higher yield. Therefore, in some embodiments, chromosomal integration and low-copy overexpression are used to construct the strain.

[0283] The full-length nucleotide sequence of the protein or enzyme of the present invention, or a fragment thereof, can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. One feasible method is to synthesize the relevant sequence by synthetic methods, especially when the fragment length is relatively short. Typically, very long fragments can be obtained by synthesizing multiple small fragments and then ligating them.

[0284] Once the relevant sequence is obtained, it can be obtained in large quantities using 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 propagated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form. Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. This 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 sequences of the present invention through chemical synthesis.

[0285] 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 a specific construct that is capable of introducing the promoter fragment and DNA sequence of the selected gene product together with the appropriate 3' non-translated sequence into the cell.

[0286] 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 typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multi-linker region and a selectable marker element for inserting a nucleic acid encoding an antibody to be expressed. Exemplary nucleic acid constructs include pET21a and pET28a. The vector may also contain a sequence for integrating the coding sequence or expression cassette into the genome.

[0287] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0288] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media (e.g., LB or TB supplemented with glycerol). Culture is performed under conditions suitable for host cell growth (e.g., 37° C.). After the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (e.g., temperature conversion or chemical induction), and the cells are cultured for a further period of time (e.g., 22° C., for more than 96 hours).

[0289] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing 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, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0290] The host cell of the present invention can be a microbial host present in a fungus or bacteria family and grown in a wide temperature, pH value and solvent tolerance range, including but not limited to bacterial cells, fungal cells, such as yeast cells, mammalian cells, insect cells and plant cells. For example, it is expected that any bacterium, yeast and filamentous fungi can be a suitable host 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 an exemplary embodiment, the recombinant Escherichia coli system of the present invention has improved the output of reticulate annonine produced by tyrosine through fermentation engineering.

[0291] In addition, the host cell also includes a reagent for increasing tyrosine production, such as expressing an enzyme that increases tyrosine production and an enzyme that further catalyzes the reaction of reticuline (such as 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 ), feedback-resistant 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 achieved by episomal expression via an expression vector or by integrating the sequence into the genome (e.g., via CRISPR).

[0292] The reagents for improving tyrosine production may also include reagents 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.

[0293] In addition, the present invention also provides a method for producing protoberberine compounds, 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 dopamine to generate 3,4-dihydroxyphenylacetaldehyde (for example, using MAO catalysis), (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to generate norlaudanine (for example, using NCS catalysis), (5) generating norlaudanine to generate 3'-hydroxycoclaurine (for example, using 6OMT catalysis), (6) 3'hydroxycoclaurine to 3'hydroxynitromethylcoclaurine (e.g., using CNMT catalysis), (7) 3'hydroxynitromethylcoclaurine to reticuline (e.g., using 4'OMT catalysis), (8) reticuline to chrysanthemumine (e.g., using BBE1 or a variant thereof catalysis), (9) chrysanthemumine to tetrahydroafricanine (e.g., using 9'OMT catalysis), and (10) tetrahydroafricanine to tetrahydroberberine (e.g., using CAS catalysis).

[0294] Based on the nucleic acid constructs and cells herein, 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, illustratively, upregulating the expression or activity of RraA and / or cytochrome b5 by plasmid overexpression technology; and / or, reducing the oxidized state of cytochrome P450 enzymes, such as CPR2. Plasmid overexpression technology is well known in the art. For example, the coding sequence of a polypeptide of interest (e.g., RraA or cytochrome b5) is introduced into an expression vector containing a promoter and a terminator, and the vector is then transferred into cells, and the cells are incubated under conditions suitable for polypeptide expression.

[0295] In order to increase the content of reticuline, the method further includes an agent for increasing the production of reticuline, such as expressing an enzyme in the recycling pathway of tetrahydrobiopterin, such as an enzyme that catalyzes BH4 regeneration and an enzyme that catalyzes BH4 synthesis. Exemplarily, the enzyme that catalyzes BH4 regeneration includes: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase, and the enzyme that catalyzes BH4 synthesis is selected from one or more of the following: GTP cyclohydrolase, 6-pyruvyl-tetrahydrobiopterin synthase and indole reductase. Preferably, the enzyme that catalyzes BH4 regeneration includes: 4α-hydroxytetrahydropterin dehydratase and / or dihydropterin reductase. Enzymes include: 4α-hydroxytetrahydropterin dehydratase (PCD) from Rattus norvegicus and dihydropterin reductase (QDHPR) from Rattus norvegicus. Preferably, enzymes catalyzing BH4 synthesis include GTP cyclohydrolase (MtrA) from Bacillus subtilis, 6-pyruvyl-tetrahydrobiopterin synthase (PTPS) from Rattus norvegicus, and succinyl pterin reductase (SPR) from Rattus norvegicus; and / or, upregulating the expression or activity of purine nucleic acid kinase (DeoD), for example, by upregulating the expression of purine nucleic acid kinase through plasmid overexpression technology.

[0296] 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 ), feedback-resistant chorismate translocation and prephenate dehydrogenase (tyrA fbr ), phosphoenolpyruvate synthase (ppsA), transketolase that increases erythrose-4-phosphate synthesis (tktA), and shikimate dehydrogenase (ydiB); down-regulating the expression or activity of pyruvate kinase (pykA); preferably, down-regulating the expression or activity of the enzyme by RNAi or CRISPR technology; down-regulating the expression or activity of chorismate translocation and prephenate dehydratase (pheA); preferably, down-regulating the expression or activity of the enzyme by RNAi or CRISPR technology; and down-regulating the expression or activity of aromatic amino acid transcription regulator (tyrR); preferably, down-regulating the expression or activity of the enzyme by RNAi or CRISPR technology.

[0297] Therefore, in some embodiments, the present invention also provides a host cell with improved reticuline production, wherein the host cell expresses the following enzymes: an enzyme that catalyzes the hydroxylation of tyrosine to 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 that increases tyrosine production and / or an agent that inhibits the expression or activity of one or more or all of the following enzymes, such as pykA, pheA, tyrR. Preferably, in the host cell, the enzyme is integrated into the host cell via chromosome.

[0298] In addition, the present invention also provides a method for producing reticulate annonaine, which comprises the following steps: (1) hydroxylating tyrosine to produce dopa, (2) decarboxylating dopa to produce dopamine, (3) producing dopamine to produce 3,4-dihydroxyphenylacetaldehyde, (4) condensing dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norlaudanine, (5) producing norlaudanine to produce 3'-hydroxycoclaurine, (6) producing 3'-hydroxycoclaurine to produce 3'-hydroxynitromethylcoclaurine, and (7) producing reticulate annonaine to produce 3'-hydroxynitromethylcoclaurine. The method further comprises catalyzing BH4 regeneration and catalyzing BH4 synthesis in steps (1) and / or (2).

[0299] The present invention has the following beneficial effects:

[0300] 1) They discovered the regulatory effect of tyramine on tyrosine hydroxylase in Rattus norvegicus and, for the first time, relieved feedback inhibition using a mutant of endogenous 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) from Escherichia coli.

[0301] 2) It was discovered for the first time that the gene purine nucleoside phosphorylase (DeoD), which is related to purine synthesis, promotes the synthesis of reticuline;

[0302] 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;

[0303] 4) For the first time, the de novo synthesis of protoberberine products was achieved in Escherichia coli using simple carbon sources such as glucose, with a total yield of 371.42 mg / L (205.68 mg / L of reticuline and 165.74 mg / L of tetrahydroberberine), the highest level available, reaching the highest yield in existing microbial systems.

[0304] 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.

[0305] Example

[0306] Materials and Methods

[0307] 1. Strains and plasmids involved in the embodiments of the present invention

[0308] Escherichia coli DH10B was used as a cloning host for plasmid construction, while Escherichia coli BL21(DE3) was used as an expression host for detecting single gene expression and as a primary strain for fermentation. Vector backbones were selected, containing four different resistance genes: ampicillin-resistant plasmid pET21a, kanamycin-resistant plasmid pET28a, chloramphenicol-resistant plasmid pACYC-Duet, and spectinomycin-resistant plasmid pCDF-Duet.

[0309] The detailed information of the 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).

[0310] See Table 2 for strain details ("Δ" knockout, "::" insertion, "ori" chassis strains)

[0311] Table 1: Plasmids involved in the examples

[0312] Table 2: Strains involved in the examples

[0313] 2. Enzyme selection

[0314] 2.1 Enzyme genes related to reticuline synthesis

[0315] Tyrosine hydroxylase (RnTYH, UniProtKB: P04177) from Rattus norvegicus was selected for the enzyme that catalyzes the hydroxylation of L-tyrosine to produce L-DOPA. Amino acid mutations at three sites (R37E, R38E, and W166Y) relieved feedback inhibition and enhanced tyrosine hydroxylase activity. Tyrosine 3-monooxygenase (Drosophila melanogaster, DmTYH, UniProt: P18459) from Drosophila melanogaster was also selected. This enzyme's coenzyme is tetrahydrobiopterin (BH4). BH4 uses guanosine triphosphate (GTP) as a precursor and is catalyzed by guanosine triphosphate (GTP) cyclohydrase I (Bacillus subtilis (MtrA, UniProt: P19465)), 6-pyruvoyltetrahydropterin synthase (R. norvegicus (PTPS, UniProt: P27213)), and sepiapterin reductase (R. norvegicus (SPR, UniProt: P18297)) from Rattus norvegicus. To ensure the supply of BH4, a BH4 regeneration pathway was introduced, which completed the regeneration of BH4 through rat 4α-hydroxytetrahydropterin dehydratase (Rattus norvegicus pterin-4 alpha-carbinolamine dehydratase 1 (PCD, NCBI accession: NP_001007602)) and rat dihydropterin reductase (Rattus norvegicus quinoid dihydropteridine reductase (QDHPR, UniProtKB: P11348)).

[0316] The enzyme that catalyzes the decarboxylation of L-DOPA to produce dopamine is DOPA decarboxylase (Pseudomonas putida KT2440 (DODC, GenBank accession number: AE015451)). The enzyme that catalyzes the conversion of dopamine to 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) is monoamine oxidase (Micrococcus luteus (MAO, UniProtKB: C5CB11)). Thus, L-tyrosine can catalyze the formation of dopamine and 3,4-DHPAA, two precursors for reticuline synthesis.

[0317] Norcoclaurine synthase 2, CjPR10A, Coptis japonica (NCS, UniprotKB: A2A1A1), catalyzes the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to produce norcoclaurine. Norcoclaurine 6-O-methyltransferase, Papaver somniferum (6OMT, UniprotKB: Q6WUC1), catalyzes the conversion of norcoclaurine to 3'-hydroxycoclaurine. Coclaurine N-methyltransferase, Papaver somniferum (CNMT, UniprotKB: Q7XB08), catalyzes the conversion of 3'-hydroxycoclaurine to 3'-hydroxynitromethylcoclaurine. The enzyme that catalyzes the conversion of 3'-hydroxy-N-methylcoclaurine to reticuline is 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 these enzymes.

[0318] 2.2 Enzyme genes related to the synthesis of protoberberine compounds

[0319] A mutant of berberine bridge enzyme 1 (AmBBE1, UniProtKB: D2SMM9) from Argemone Mexicana was selected to catalyze the conversion of reticuline to scoulerine. Two amino acid positions (F398 and I431) were mutated to obtain AmBBE1F398W-I431F. A mutant of (S)-scoulerine 9-O-methyltransferase (9'OMT, Coptis japonica, GenBank: D29809) was selected to catalyze the conversion of scoulerine to tetrahydroafricanine. The enzyme that catalyzes the conversion of tetrahydroafricanine to tetrahydroberberine is selected from tetrahydroberberine synthase (CYP719) from Coptis japonica (CAS, GenBank: AB026122). The enzyme that reduces oxidized cytochrome P450 is selected from cytochrome P450 oxidoreductase 2 (AtCPR2, NCBI: NM_179141.2) from Arabidopsis thaliana.

[0320] 3. Plasmid construction

[0321] 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 plasmid was constructed by inverse PCR using pQZ135 as a template and introducing the first 24 bases of the target gene into the primers. The construction of pQZ135 used homologous recombination. First, by PCR, three fragments containing overlapping bases were amplified using the vectors pJF650 and CDF-Duet1 containing the sRNA backbone as templates, respectively. The primer sequences are as follows:

[0322] part135-CDF2: Template pCDF-Duet

[0323] Part135-CDF2-F is shown in SEQ ID NO: 57, and part135-CDF2-R is shown in SEQ ID NO: 58.

[0324] part135-JF650: Template pJF650

[0325] Part135-JF650-F is shown in SEQ ID NO: 59, and part135-JF650-R is shown in SEQ ID NO: 60.

[0326] part135-CDF1: Template pCDF-Duet

[0327] Part135-CDF1-F is shown in SEQ ID NO: 61, and part135-CDF1-R is shown in SEQ ID NO: 62.

[0328] Construction of sgRNA plasmids: Use Benchling (https: / / www.benchling.com / ) to locate the corresponding gene in E. coli BL21(DE3). Then, select the appropriate NGGPAM position and determine the corresponding 20bp guide sequence. Finally, the determined guide sequence is introduced into the pTargetF plasmid via inverse PCR.

[0329] 3.3 Construction of target overexpression plasmids was performed by in-fusion. After cloning the target gene, the homologous sequence of the vector was added by overlap-PCR and then constructed into the linearized vector using homologous recombinase.

[0330] 3.4 Plasmids involved in the synthesis of tyrosine, reticuline, and tetrahydroberberine, as well as those regulating the in vivo synthesis of tetrahydroberberine, were constructed using the "biobrick" approach (see Knight T. Idempotent vector design for standard assembly of biobricks [J], 2003). Restriction sites were added to both ends of individual functional genes, and the genes were sequentially linked through enzyme ligation using a combination of the isoenzymes XbaI and SpeI.

[0331] 3.5 Plasmids for in vitro testing of tetrahydroberberine synthase: A tetrahydroberberine synthase scaffold was designed and constructed by extended overlap extension PCR (POE-PCR), obtaining plasmid pQZ344. Tetrahydroberberine synthases from different sources were replaced with the following specific construction method: plasmids containing CyCYP719A41 and CyCYP719A42, and pQZ344, were used as templates, respectively. DNA fragments and vector backbones were amplified by high-fidelity PCR. Both contained approximately 40 bp overlapping fragments at the 3' and 5' ends. In the subsequent PCR system, the DNA fragments containing overlapping bases and the linearized vector served as templates for mutual amplification to produce a DNA multimer product. 10 μL of the PCR product was directly transformed into DH10B cells, and positive clones were screened.

[0332] 4 Chromosome Editing Process

[0333] To integrate the gene associated with high tyrosine production into the chromosome, the integration process was divided into two steps. First, the TyrR-targeting sgRNA plasmid pQZ279 was generated by inverse PCR. The editing template sequence contained approximately 500 bp of homologous sequences upstream and downstream of the knockout gene sequence. Primers were designed for each of these sequences and then fused via overlap extension PCR to obtain the template. Second, a new NGG residue was selected from the fragment integrated in the first step, and the corresponding sgRNA plasmid pQZ261 was obtained. The editing template was a fragment obtained by double-digesting plasmid pQZ298 with XbaI and NotI, flanked by approximately 1000 bp of homologous fragments to the site of integration in the first step.

[0334] In order to integrate the BH4 recycling pathway, we chose to construct the 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).

[0335] The detailed editing and screening process is as follows: BL21(DE3) cells harboring pEcCas9 were obtained by chemical transformation. Single clones were expanded and cultured, and arabinose was added to the culture medium at a final concentration of 10 mM to induce expression of the λ-Red system, resulting in electrocompetent cells. During electroporation, 100 μL of the electrocompetent cells was mixed with 100 ng of either a pTargetT series plasmid or a pTargetF series plasmid and 400 ng of the editing template fragment. The mixture was electroporated in a 1 mm cuvette at 1800 V. Immediately after electroporation, 1 mL of fresh LB medium was added and the cells were revived at 37°C, 250 rpm, for 2 hours. The revived culture was then plated on LB plates containing kanamycin and spectinomycin resistance and incubated overnight at 37°C. Transformants were then verified by colony PCR and DNA sequencing.

[0336] 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 onto LB plates containing kanamycin (50 μg / mL). Randomly selected clones were verified on LB plates containing kanamycin (50 μg / mL) and LB plates containing both spectinomycin (50 μg / mL) and kanamycin (50 μg / mL). Colonies sensitive to spectinomycin were considered to have eliminated the sgRNA plasmid.

[0337] To eliminate pEcCas9, colonies with sgRNA eliminated were further inoculated into LB medium containing 5 g / L glucose and cultured overnight at 37°C, 250 rpm. 10 μL of the overnight culture was spread onto LB plates containing 5 g / L glucose and 10 g / L sucrose and cultured overnight at 37°C. Colonies were then selected and screened on LB plates with or without kanamycin. Colonies sensitive to kanamycin were identified as positive strains for pEcCas9 elimination.

[0338] 5. Amplification of cytochrome b5 gene from cDNA of Coptis chinensis

[0339] Liquid nitrogen was pre-cooled in a sterile mortar, and then the root of Coptis chinensis, cut into small pieces, was placed in the mortar and thoroughly ground with liquid nitrogen. Total RNA was then extracted from the tissue according to the kit instructions (TIANGEN RNA simple Total RNA kit). To clone the gene, the purified RNA was reverse transcribed into cDNA using the PrimeScript RT kit (Takara). Using AaCYB5 from Artemisia annua as the query sequence, the tBLASTn tool was used to search the transcriptome of Coptis chinensis in the SRA database (SRA: SRX4550566). The screening results were analyzed for nitrogen and carbon termini to obtain candidate sequences. Primers were designed based on these sequences, and PCR amplification was performed using PrimerSTAR Max DNA Polymerase (Takara). Ultimately, two CYB5 genes were obtained and named CcCYB5A and CcCYB5B.

[0340] 6. CAS expression and SDS-PAGE detection

[0341] The expression vector containing a single gene (full-length CAS, truncated transmembrane region trCAS, truncated 8RP-trCAS fused with 8RP solubility tag) was transformed into BL21 (DE3) and cultured at 37°C for 12 hours. A single colony was picked from the plate and placed in 2 mL LB resistance medium and shaken at 37°C, 250 rpm for 12 hours as a seed solution. 1% of the seed solution was inoculated into 10 mL LB resistance medium and shaken at 37°C, 250 rpm until the OD reached 0. 600=0.6. After sufficient cooling, induce with 0.1 mM IPTG and culture with shaking at 22°C, 250 rpm for 16-20 hours. Harvest the cells at 5000 × g for 5 minutes at 4°C. Resuspend the cells in 1 mL of buffer A and mix thoroughly. Now add 1% 100 mM PMSF (protease inhibitor) and mix thoroughly. Place the sample on ice and sonicate for 10 minutes, with a working interval of 15 seconds and a rest interval of 45 seconds, until the resuspension becomes transparent. The sample is then centrifuged at low temperature (6000 × g, 4°C) for 10 minutes. Remove 100 μL of the supernatant and place on ice to record it as the soluble protein sample. Add 25 μL of 5× SDS loading buffer to the soluble sample, mix thoroughly, boil in boiling water for 10 minutes, and centrifuge at 12000 rpm for 3 minutes. Load the sample in the following amounts: 5 μL marker and 5 μL supernatant. After electrophoresis at 100V for 20min and 160V for 60min, the cells were stained with Coomassie Brilliant Blue to observe gene expression.

[0342] 7 In vitro activity test of CAS

[0343] 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 of LB medium containing antibiotics and cultured at 37°C and 250 rpm until the OD 600 When the nitric oxide concentration reached 0.6, IPTG was added to a final concentration of 0.1 mM, and the cells were then cultured at 22°C, 180 rpm for 20 hours. Afterwards, the cells were harvested at 5000 × g for 5 min at 4°C.

[0344] 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) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM MgCl2, and 5 mg / L DNase I. The sample was placed on ice and sonicated for 10 min, with a 15-second interval and a 45-second pause, until the suspension became transparent. The supernatant was used as the crude protein extract for the subsequent in vitro reaction. In vitro enzyme activity was determined in a 100 μL reaction mixture containing 100 mM Tris-HCl (pH 7.5), 50 mg / L (S)-tetrahydroafricanine, 50 μL of 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.

[0345] 8 Bacterial shake flask culture conditions

[0346] All shake flask experiments were performed in 100 mL round-bottomed test tubes, with the culture volume kept at 10 mL to ensure dissolved oxygen in the culture medium. From the overnight culture, 1% of the seed solution was uniformly inoculated into TB medium.

[0347] For tyrosine-producing strains, after culturing at 22°C and 250 rpm for 12 h, 0.1 mM IPTG and 30 g / L glucose were added, and the culture was continued for 48 h before sampling and measuring the bacterial concentration OD. 600 and tyrosine production.

[0348] To detect the production of reticuline, the strain was cultured at 22°C and 250 rpm for 24 h, and then 0.1 mM IPTG and 30 g / L glucose were added. After further culture for 12 h, 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 determined. 600 .

[0349] To determine the production of protoberberine, the strains were cultured in TB (2% glycerol) medium at 37°C and 250 rpm for 3 h. 0.1 mM IPTG was then added and cultured at 22°C and 220 rpm for 48 h. The yield and OD of the culture medium of strain SQZ261-266 were measured. 600 .

[0350] 9 Fed-batch culture conditions

[0351] A single clone of SQZ266 was inoculated into 2 mL of LB liquid medium containing carbenicillin (50 mg / L), chloramphenicol (34 mg / L), streptomycin (50 mg / L), and kanamycin (50 mg / L) and grown at 37°C for 12 hours. 800 μL of the culture was then transferred to a 500 mL flask containing 80 mL of LB medium and incubated at 37°C, 250 rpm for 4 hours as a seed solution.

[0352] Batch cultures were carried out in a 7.5-L reactor, with the seed solution transferred to 4 L of fermentation basal medium at a 2% volume ratio. Each liter of basal medium consisted of 0.5 g / L L-phenylalanine, 20 g / L glucose, 12 g / L K₂HPO₄, 4.2 g / L KH₂PO₄, 2 g / L (NH₄)₂SO₄, 1.2 g / L MgSO₄·7H₂O, 1.8 g / L citric acid monohydrate, 0.5 g / L yeast extract, the corresponding antibiotics, and 1× trace metal solution. The 1000× trace metal solution contained 0.03 g / L H₃BO₃, 1 g / L thiamine, 0.94 g / L ZnCl₂, 0.5 g / L CoCl₂, 0.38 g / L CuCl₂, 1.6 g / L MnCl₂, and 3.6 g / 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.

[0353] The initial culture temperature was 37°C, the air flow rate was 1 vvm, and the initial stirring rate was 400 rpm. The pH value was maintained at 7 by automatically adding ammonia solution (25%, v / v). 600 When the temperature reaches 19.5, adjust the temperature to 22 ° C. After the temperature stabilizes at 22 ° C, immediately induce the culture with 0.1mM IPTG. The OD at the time of induction is 600 The total batch culture time was 58 hours.

[0354] 10 Metabolite detection

[0355] Detection of tyrosine in fermentation broth. Samples were diluted with an equal volume of 1N hydrochloric acid and thoroughly mixed at 37°C, 250 rpm, for 1 hour. Subsequently, the samples were centrifuged at high speed and filtered through a 0.22 μm filter membrane. UPLC analysis was performed using a C18 column (250 × 4.6 mm, 5 μm). Detection conditions were: column temperature 30°C, flow rate 1 mL / min, UV spectroscopy 278 nm. The mobile phase composition was: B: deionized water (0.1% trifluoroacetic acid), C: acetonitrile (0.1% trifluoroacetic acid). The mobile phase gradient was: 0-12 min, 5-15% C; 12-12.1 min, 15-95% C; 12.1-15 min, 95% C; 15-15.1 min, 95-5% C; 15.1-20 min, 5% C. The fermentation broth sample volume was 5 μL.

[0356] To detect alkaloids in the fermentation broth, the sample was extracted twice with an equal volume of n-butanol. The organic phase was centrifuged at 12,000 rpm for 5 minutes, transferred to a fresh tube, and rotary evaporated to dryness at 30°C. The sample was then reconstituted with 100 μL of methanol (concentrated 5x), mixed thoroughly, and centrifuged at 12,000 rpm for 3 minutes. The supernatant was transferred to UPLC and UPLC-QTOF / MS for analysis. UPLC analysis was performed using a C18 column (250 × 4.6 mm, 5 μm). Detection conditions were: column temperature 30°C, flow rate 1 mL / min, and UV spectroscopy at 235 and 280 nm. The mobile phase composition was B: deionized water (0.1% formic acid), C: acetonitrile (0.1% formic acid), with a mobile phase gradient setting of: 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 sample volume was 5 μL.

[0357] LC-MS / MS detection was used for the qualitative and quantitative analysis of low-level reticuline. To accurately quantify reticuline in the fermentation broth, a 20 mg / L standard solution was prepared and serially diluted to the following concentrations: 2.0 mg / L, 0.4 mg / L, 0.08 mg / L, and 0.016 mg / L. 1 μL of the standard solution was loaded onto the sample. A calibration curve was constructed based on the relationship between the extracted ion response and sample concentration. The calibration curve is shown in Figure 1. The instrumentation was an Agilent LC1260 & 1290-MS / QTOF6545, using an Agilent InfinityLab Poroshell 120EC-C18 column (3×150 mm, 2.7 μm). Detection conditions were: column temperature 30°C, flow rate 0.3 mL / min, and UV spectroscopy at 235 & 280 nm. Mobile phase settings: A: (0.1% formic acid) water; B: (0.1% formic acid) acetonitrile, mobile phase gradient: 5% B (0-0.8 min); 5-98% B (0.8-12 min); 98% B (12-15 min); 98-5% B (15-15.5 min). Sample load: 1.0 μL. Other parameters: Positive ion mode, ion source: Gas Temp = 300°C, flow rate: 6 L / min; pressure: 30 psig; Sheath Gas Temp = 320°C, flow rate: 11 L / min; pressure: 30 psig. Fragmentor: 135V; Skimmer: 65V; Oct1RFVpp: 750V; Vcap: 3500V; Nozzle Voltage: 1000V; Scan (m / z): 100-1100; Level 2 mass spectrometry collision energy: 0eV, 10eV, 20eV, 40eV.

[0358] Example 1: Construction of a high-yield tyrosine chassis strain

[0359] Tyrosine, as the primary product of intracellular metabolism, participates in the synthesis of benzylisoquinoline alkaloids. In order to ensure that bacterial strains can utilize glucose to generate target products, the inventors have integrated the strategy of existing tyrosine high yield, including: knocking out the regulatory gene tyrR for regulating tyrosine synthesis, knocking out the gene pheA for phenylalanine synthesis, overexpressing aroG and tyrA that release feedback inhibition, overexpressing ppsA, tktA and ydiB. After the present embodiment compares knocking out 2 genes, overexpressing the bacterial strain SQZ201 (plasmid, high copy overexpression) and SQZB18 (chromosome integration, low copy overexpression) of tyrosine synthesis-related genes to produce tyrosine, the results of bacterial strain construction and output and cell density are shown in Figure 2, and the result shows that the tyrosine high-yield bacterial strain obtained by the chromosome integration mode has more advantages, and producing tyrosine 2.11g / L is more than 3 times of the plasmid overexpression mode output.

[0360] Example 2: Tetrahydrobiopterin regeneration and recycling pathway improves tyrosine hydroxylase activity

[0361] Tyrosine hydroxylation is a key step in the metabolism of tyrosine to produce benzylisoquinoline alkaloids. This example compares the effects of different sources and regeneration and recycling pathways of tetrahydrobiopterin on tyrosine hydroxylase activity. Four combinations were evaluated: strains SQZ202-SQZ205 containing 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 improved hydroxylase activity, with the mouse tyrosine hydroxylase and BH4 recycling pathway combined to produce the best results, producing 9.26 mg / L of reticuline, as shown in 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 Drosophila tyrosine hydroxylase increased to 19.12 mg / L. The fermentation broth was then tested by LC-MS / MS to find out the reason for the different production characteristics of mouse tyrosine hydroxylase. The LC-MS / MS analysis of the fermentation broth showed that three strong peaks appeared around the target product: m / z 300.1587 [M+H] + 、m / z 284.1599[M+H] + and m / z 314.1680 [M+H] +(As shown in Figure 4). By comparing the fragment ions, it was determined that these compounds are structural analogs of reticuline, and are generated by the condensation of derivatives of phenylalanine or tyramine with dopamine (see Figure 4 for the results). Excluding the influence of phenylalanine from the culture medium, the generation of tyramine derivatives becomes a prominent problem. 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 a 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.

[0362] Because tyramine is produced from tyrosine by dopa decarboxylase, diverting the flow originally used for dopamine synthesis, this example used HpaBC-D11 (a mutant of HpaBC), a known enzyme that specifically hydroxylates tyramine to produce dopamine, to convert tyramine back into dopamine. In strains with increased HpaBC-D11 expression, reticuline production reached 82.31 mg / L and 44.10 mg / L in strains SQZ208 (overexpressing tyrosine hydroxylase from Rattus norvegicus) and SQZ209 (overexpressing tyrosine hydroxylase from Drosophila melanogaster), respectively (see Figure 5 ).

[0363] Example 3: DeoD involved in purine nucleotide metabolism contributes to the production of reticuline

[0364] To improve the production of reticuline, 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-nucleoside phosphorylase, E. coli, (EcDeoD, UniProtKB: P0ABP8)) can improve the production of reticuline. Among them, overexpressing purine nucleic acid kinase is the most effective, increasing the production of reticuline to 100.13 mg / L, as shown in Figure 6.

[0365] Example 4: Scaffold connecting CAS and CPR promotes oxidation of tetrahydrotetramine

[0366] 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 AmBBE1F398W-I431F with improved activity. Subsequently, because the expression level of CAS was low, the inventors immediately optimized the expression of the enzyme. After truncating the transmembrane region at the nitrogen end and truncating and fusing the 8RP solubility tag, the expression of the enzyme was tested. The SDS-PAGE results showed that the expression level of the protein with truncated nitrogen-terminal transmembrane sequence was significantly improved, as shown in Figure 7.

[0367] The inventors subsequently evaluated the effects of three different CAS sources and three different colocalization scenarios with CPRs on CAS catalytic activity. A total of nine combinations were tested in subsequent in vitro experiments. The three CASs were: one P450 with tetrahydroberberine synthase activity from Coptis chinensis (Coptis japonica) and two from Corydalis yanhusuo: CjCYP719A1, CyCYP719A41, and CyCYP719A42. Three different localization scenarios were tested: expression of the CAS and CPR alone, fusion expression of the CAS and CPR via a linker ((GGGGS)2 flexible linker), and fusion expression of the CAS with SH3 and the CPR with SH3Lig. The construction strategy is shown in Figure 8. Ultimately, only the coexpression of CjCYP719A1 from Coptis chinensis with CPRs resulted in detectable production of oxidation products. In the reaction systems where CjCYP719A1 was fused to SH3 and AtCPR2 was fused to SH3Lig, the substrates were completely converted, with 32.66% of the substrate converted to berberine and 67.34% to the oxidized form of fangfang rine, as shown in Figure 9. The identification results of the oxidized form are shown in Figure 10.

[0368] Example 5: Global regulatory factors RraA and cytochrome b5 contribute to the increase of protoberberine production

[0369] Strain SQZ261 was constructed by integrating a berberine bridge enzyme mutant and a tetrahydroberberine synthase with improved activity into the downstream biosynthesis pathway. Shake flask culture results showed that the fermentation broth accumulated a significant amount of protoberberine compounds, including 56.83 mg / L of reticuline, 34.79 mg / L of chrysanthemine, 25.26 mg / L of tetrahydroafricanine, and 8.99 mg / L of tetrahydroberberine. The accumulation of reticuline and tetrahydroafricanine indicated that the activities of these two membrane proteins needed to be optimized. To further improve membrane protein activity, the inventors selected two methods for improving membrane protein activity in E. coli: one was the use of the effector factors DjlA (DnaJ-like protein A, amino acid sequence shown in SEQ ID NO: 66, nucleic acid sequence shown in SEQ ID NO: 67) and RraA (nuclease activity regulatory protein A), and the other was the overexpression of cytochrome b5 (three cytochrome b5s were evaluated: PcCYB5, CcCYB5A, and CcCYB5B. PcCYB5 is from Phanerochaete chrysosporium, amino acid sequence shown in SEQ ID NO: 68, nucleic acid sequence shown in SEQ ID NO: 69). In terms of tetrahydroberberine production, strain SQZ266, which overexpressed the effector factors RraA and CcCYB5B, achieved the highest tetrahydroberberine production, reaching 16.81 mg / L in shake flask culture, as shown in Figure 11. We then carried out batch fermentation culture of the SQZ266 strain in a 7.5 L reactor, where the production of reticuline rapidly 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 12.

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) Encoding sequences of one or more or all of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase that resists feedback inhibition, chorismate translocation and prephenate dehydrogenase that resists feedback inhibition, phosphoenolpyruvate synthase, Increase transketolase and shikimate dehydrogenase in the synthesis of erythrose-4-phosphate, 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'-hydroxynitromethylcoclaurine, an enzyme that catalyzes the production of reticuline from scutellarine, an enzyme that catalyzes the production of tetrahydroafricanine from scutellarine, 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) expressing 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'-hydroxycoclaurine to 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: 4a-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.

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