Construction method of engineering saccharomycetes for efficient biosynthesis of chelerythrine

Abstract

The invention provides a construction method of engineering saccharomycetes for efficient biosynthesis of chelerythrine. Specifically, the invention provides an engineering bacterium for producing chelerythrine, the engineering bacterium is saccharomycetes, and the engineering bacterium contains exogenous and / or endogenous gene modules, such as BBE, SMT, TDC, TNMT, MSH, P6H, CPR and BUP1 gene modules. The invention also provides a construction method, application and a construction kit of the engineering bacterium, and a production method and a fermentation system of chelerythrine. The engineering bacteria can efficiently synthesize chelerythrine, the yield is far higher than that in the prior art, and the highest yield reaches 4.5 mg / L. Therefore, the engineering bacterium and the construction method provided by the invention can overcome the defect of long production period of chelerythrine in the field, and a new method is provided for synthesis of chelerythrine.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and metabolic engineering, and more specifically to an engineering yeast for efficiently biosynthesizing chelerythrine and a construction method thereof. Background Art

[0002] Chelerythrine belongs to the benzophenanthridine alkaloids, which has pharmacological activities such as antibacterial, anti-inflammatory, antioxidant, improving animal intestinal health, antiviral and anti-cancer, and has been widely used in agriculture and medicine.

[0003] Due to its antibacterial, anti-inflammatory, antioxidant and intestinal health-improving activities, products with chelerythrine and sanguinarine as main ingredients have been developed as feed additives for animal production (for example, patent applications with application numbers CN201310303875.4, CN200610066060.9, and CN202111276526.9). In addition, chelerythrine is toxic to a variety of tumor cells and has great potential as an anti-tumor drug (for example, patent applications with application numbers CN199880004824.4 and CN202310196314.2).

[0004] However, due to the complexity of its structure, it is difficult to fully synthesize it using chemical methods. At present, chelerythrine is mainly extracted from plants such as Papaveraceae and Rutaceae, but the content of chelerythrine in plants is relatively small (only 0.01-0.1%) and the content is unstable. The plant extraction cycle is long, and the later separation and purification is relatively difficult and easily affected by climate and geographical location. Therefore, when encountering major natural disasters and a surge in demand in a short period of time, plant extraction is difficult to meet market demand.

[0005] In addition, in terms of biosynthesis, Chinese invention patents (such as application numbers 201811423064.7 and 201811423227.1) describe the construction of engineered bacteria by heterologously expressing proopioid hydroxylase (P6H), dihydrobenzophenanthridine oxidase (DBOX), and cytochrome P450 oxidoreductase (CPR) in yeast, and the fermentation and conversion of the constructed yeast engineered bacteria using the original solution of Macleaya cordata leaves as a substrate to produce sanguinarine and chelerythrine. However, the efficiency and yield of this bioconversion are extremely low, and the yield of chelerythrine is only at the nanogram level.

[0006] In view of the above problems, the production and preparation of chelerythrine is still facing huge challenges, which greatly limits the development of its downstream products and the prospects of its application.

[0007] Therefore, researching and developing an engineered yeast that can efficiently biosynthesize chelerythrine as a production method to supplement or replace chelerythrine has important application value and significance. Summary of the invention

[0008] The purpose of the present invention is to provide an engineered yeast for efficient biosynthesis of chelerythrine and a construction method thereof. Specifically, the purpose of the present invention is to construct an engineered yeast strain for stably and efficiently producing chelerythrine, that is, to design a biosynthetic pathway of chelerythrine, to comprehensively utilize synthetic biology and metabolic engineering technology to construct and optimize a recombinant microbial cell factory for synthesizing chelerythrine, and to achieve stable and efficient synthesis of chelerythrine using (S)-carbomer.

[0009] In a first aspect of the present invention, an engineered bacterium for producing chelerythrine is provided, wherein the engineered bacterium is a yeast, and the engineered bacterium contains an exogenous and / or endogenous gene module, wherein the gene module comprises:

[0010] (a) a berberine bridge enzyme (BBE) gene module, wherein the BBE gene module contains a single copy or multiple copies of a BBE gene;

[0011] (b) a S. chrysanthemi methyltransferase (SMT) gene module, wherein the SMT gene module contains a single copy or multiple copies of the SMT gene;

[0012] (c) a tetrahydroberberine synthase (TDC) gene module, wherein the TDC gene module contains a single copy or multiple copies of a TDC gene;

[0013] (d) a tetrahydroberberine N-methyltransferase (TNMT) gene module, wherein the TNMT gene module contains a single copy or multiple copies of the TNMT gene;

[0014] (e) an N-methylaurocanine hydroxylase (MSH) gene module, wherein the MSH gene module contains a single copy or multiple copies of the MSH gene;

[0015] (f) a proopioid hydroxylase (P6H) gene module, wherein the P6H gene module contains a single copy or multiple copies of a P6H gene;

[0016] (g) a cytochrome P450 oxidoreductase (CPR) gene module, wherein the CPR gene module contains a single copy or multiple copies of a CPR gene; and

[0017] (h) a transporter benzylisoquinoline alkaloid uptake permease (BUP1) gene module, wherein the BUP1 gene module contains a single copy or multiple copies of a BUP1 gene.

[0018] In another preferred embodiment, the yeast is Saccharomyces cerevisiae.

[0019] In another preferred embodiment, the BBE gene module is used to biosynthesize (S)-corydaline using (S)-corydaline as raw material.

[0020] In another preferred embodiment, the SMT gene module is used to biosynthesize (S)-corydaline using (S)-corydaline as a raw material.

[0021] In another preferred embodiment, the TDC gene module is used to biosynthesize (S)-tetrahydroberberine using (S)-chrysoberylline as a raw material in combination with a cytochrome P450 oxidoreductase (CPR) gene module.

[0022] In another preferred embodiment, the TNMT gene module is used to biosynthesize (S)-N-methyltetrahydroberberine using (S)-tetrahydroberberine as a raw material.

[0023] In another preferred embodiment, the MSH gene module is used to biosynthesize allocryptopine using (S)-N-methyltetrahydroberberine as a raw material in combination with a cytochrome P450 oxidoreductase (CPR) gene module.

[0024] In another preferred embodiment, the P6H gene module is used to biosynthesize chelerythrine using allocryptine as a raw material in combination with a cytochrome P450 oxidoreductase (CPR) gene module.

[0025] In another preferred embodiment, the CPR gene module is used to cooperate with the tetrahydroberberine synthase (TDC) gene module, N-methylgoldopapaverine hydroxylase (MSH) gene module and / or proopioid hydroxylase (P6H) gene module to biosynthesize (S)-tetrahydroberberine, allocryptine and / or chelerythrine.

[0026] In another preferred embodiment, the BUP1 gene module is used for the transmembrane intracellular transport of the substrate (S)-carbomer.

[0027] In another preferred embodiment, the genes contained in the gene module are codon-optimized genes.

[0028] In another preferred embodiment, the BBE gene is selected from the following group: tMcoBBE, McoBBE, AmBBE, EcBBE, PsBBE, or a combination thereof.

[0029] In another preferred embodiment, the BBE gene has one or more nucleotide sequences selected from the following group:

[0030] (i) a sequence as shown in SEQ ID NO:2, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24;

[0031] (ii) a sequence with a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:2, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24;

[0032] (iii) a sequence in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 24;

[0033] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0034] In another preferred example, the nucleotide sequence of the BBE gene is selected from the following group: SEQ ID NO: 2 (tMcoBBE), SEQ ID NO: 18 (McoBBE), SEQ ID NO: 20 (AmBBE), SEQ ID NO: 22 (EcBBE), SEQ ID NO: 24 (PsBBE), or a combination thereof.

[0035] In another preferred example, the nucleotide sequence of the BBE gene is shown in SEQ ID NO:2.

[0036] In another preferred embodiment, the BBE gene has one or more amino acid sequences selected from the following group:

[0037] (i) a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23;

[0038] (ii) a sequence with a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:1, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:23;

[0039] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23;

[0040] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0041] In another preferred example, the amino acid sequence of the BBE gene is selected from the following group: SEQ ID NO: 1 (tMcoBBE), SEQ ID NO: 17 (McoBBE), SEQ ID NO: 19 (AmBBE), SEQ ID NO: 21 (EcBBE), SEQ ID NO: 23 (PsBBE), or a combination thereof.

[0042] In another preferred example, the amino acid sequence of the BBE gene is shown in SEQ ID NO:1.

[0043] In another preferred embodiment, the SMT gene is selected from the following group: tMcoBBE, McoBBE, AmBBE, EcBBE, PsBBE, or a combination thereof.

[0044] In another preferred embodiment, the SMT gene has one or more nucleotide sequences selected from the following group:

[0045] (i) a sequence as shown in SEQ ID NO:4, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:30;

[0046] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:4, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:30;

[0047] (iii) a sequence in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO:4, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:30;

[0048] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0049] In another preferred example, the nucleotide sequence of the SMT gene is selected from the following group: SEQ ID NO: 4 (TfSMT), SEQ ID NO: 26 (AmSMT), SEQ ID NO: 28 (CcSMT), SEQ ID NO: 30 (PsSMT), or a combination thereof.

[0050] In another preferred example, the nucleotide sequence of the SMT gene is shown in SEQ ID NO:4.

[0051] In another preferred embodiment, the SMT gene has an amino acid sequence selected from one or more of the following groups:

[0052] (i) a sequence as shown in SEQ ID NO:3, SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29;

[0053] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:3, SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29;

[0054] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29;

[0055] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0056] In another preferred example, the amino acid sequence of the SMT gene is selected from the following group: SEQ ID NO: 3 (TfSMT), SEQ ID NO: 25 (AmSMT), SEQ ID NO: 27 (CcSMT), SEQ ID NO: 29 (PsSMT), or a combination thereof.

[0057] In another preferred example, the amino acid sequence of the SMT gene is shown in SEQ ID NO:3.

[0058] In another preferred embodiment, the TDC gene is selected from the following group: AmTDC.

[0059] In another preferred embodiment, the TDC gene has one or more nucleotide sequences selected from the following group:

[0060] (i) the sequence shown in SEQ ID NO:6;

[0061] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO: 6;

[0062] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 6;

[0063] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0064] In another preferred example, the nucleotide sequence of the TDC gene is shown as SEQ ID NO: 6 (AmTDC).

[0065] In another preferred embodiment, the TDC gene has one or more amino acid sequences selected from the following group:

[0066] (i) the sequence shown in SEQ ID NO:5;

[0067] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:5;

[0068] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 5;

[0069] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0070] In another preferred example, the amino acid sequence of the TDC gene is shown in SEQ ID NO: 5 (AmTDC).

[0071] In another preferred embodiment, the TNMT gene is selected from the following group: EcTNMT, GfTNMT, PsTNMT, or a combination thereof.

[0072] In another preferred embodiment, the TNMT gene has one or more nucleotide sequences selected from the following group:

[0073] (i) a sequence as shown in SEQ ID NO:8, SEQ ID NO:34 or SEQ ID NO:36;

[0074] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:8, SEQ ID NO:34 or SEQ ID NO:36;

[0075] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 8, SEQ ID NO: 34 or SEQ ID NO: 36;

[0076] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0077] In another preferred example, the nucleotide sequence of the TNMT gene is selected from the following group: SEQ ID NO: 8 (EcTNMT), SEQ ID NO: 34 (GfTNMT), SEQ ID NO: 36 (PsTNMT), or a combination thereof.

[0078] In another preferred example, the nucleotide sequence of the TNMT gene is shown in SEQ ID NO:8.

[0079] In another preferred embodiment, the TNMT gene has one or more amino acid sequences selected from the following group:

[0080] (i) a sequence as shown in SEQ ID NO:7, SEQ ID NO:33 or SEQ ID NO:35;

[0081] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:7, SEQ ID NO:33 or SEQ ID NO:35;

[0082] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO:7, SEQ ID NO:33 or SEQ ID NO:35;

[0083] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0084] In another preferred embodiment, the amino acid sequence of the TNMT gene is selected from the following group: SEQ ID NO: 7 (EcTNMT), SEQ ID NO: 33 (GfTNMT), SEQ ID NO: 35 (PsTNMT), or a combination thereof.

[0085] In another preferred example, the amino acid sequence of the TNMT gene is shown in SEQ ID NO:7.

[0086] In another preferred embodiment, the MSH gene is selected from the following group: PsMSH.

[0087] In another preferred embodiment, the MSH gene has one or more nucleotide sequences selected from the following group:

[0088] (i) the sequence shown in SEQ ID NO: 10;

[0089] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO: 10;

[0090] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 10;

[0091] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0092] In another preferred example, the nucleotide sequence of the MSH gene is shown as SEQ ID NO: 10 (PsMSH).

[0093] In another preferred embodiment, the MSH gene has one or more amino acid sequences selected from the following group:

[0094] (i) the sequence shown in SEQ ID NO:9;

[0095] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:9;

[0096] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 9;

[0097] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0098] In another preferred example, the amino acid sequence of the MSH gene is shown in SEQ ID NO: 9 (PsMSH).

[0099] In another preferred example, the P6H gene is selected from the following group: EcP6H, PsP6H, McoP6H, t4EcP6H, t27EcP6H, or a combination thereof.

[0100] In another preferred embodiment, the P6H gene has one or more nucleotide sequences selected from the following group:

[0101] (i) a sequence as shown in SEQ ID NO:12, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44;

[0102] (ii) a sequence with a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:12, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44;

[0103] (iii) a sequence in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 12, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44;

[0104] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0105] In another preferred example, the nucleotide sequence of the P6H gene is selected from the following group: SEQ ID NO: 12 (EcP6H), SEQ ID NO: 38 (PsP6H), SEQ ID NO: 40 (McoP6H), SEQ ID NO: 42 (t4EcP6H), SEQ ID NO: 44 (t27EcP6H), or a combination thereof.

[0106] In another preferred example, the nucleotide sequence of the P6H gene is shown in SEQ ID NO:12.

[0107] In another preferred embodiment, the P6H gene has one or more amino acid sequences selected from the following group:

[0108] (i) a sequence as shown in SEQ ID NO:11, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43;

[0109] (ii) a sequence with a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:11, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43;

[0110] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO:11, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43;

[0111] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0112] In another preferred example, the amino acid sequence of the P6H gene is selected from the following group: SEQ ID NO:11 (EcP6H), SEQ ID NO:37 (PsP6H), SEQ ID NO:39 (McoP6H), SEQ ID NO:41 (t4EcP6H), SEQ ID NO:43 (t27EcP6H), or a combination thereof.

[0113] In another preferred example, the amino acid sequence of the P6H gene is shown in SEQ ID NO:11.

[0114] In another preferred embodiment, the CPR gene is selected from the following group: PsCPR.

[0115] In another preferred embodiment, the CPR gene has one or more nucleotide sequences selected from the following group:

[0116] (i) the sequence shown in SEQ ID NO:46;

[0117] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:46;

[0118] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 46;

[0119] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0120] In another preferred example, the nucleotide sequence of the CPR gene is shown in SEQ ID NO: 46 (PsCPR).

[0121] In another preferred embodiment, the CPR gene has one or more amino acid sequences selected from the following group:

[0122] (i) the sequence shown in SEQ ID NO:45;

[0123] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:45;

[0124] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 45;

[0125] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0126] In another preferred example, the amino acid sequence of the CPR gene is shown in SEQ ID NO: 45 (PsCPR).

[0127] In another preferred embodiment, the BUP1 gene is selected from the following group: PsBUP1.

[0128] In another preferred embodiment, the BUP1 gene has one or more nucleotide sequences selected from the following group:

[0129] (i) the sequence shown in SEQ ID NO: 14;

[0130] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO: 14;

[0131] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 14;

[0132] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0133] In another preferred example, the nucleotide sequence of the BUP1 gene is shown in SEQ ID NO: 14 (PsBUP1).

[0134] In another preferred embodiment, the BUP1 gene has one or more amino acid sequences selected from the following group:

[0135] (i) the sequence shown in SEQ ID NO: 13;

[0136] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO: 13;

[0137] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 13;

[0138] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0139] In another preferred example, the amino acid sequence of the BUP1 gene is shown in SEQ ID NO: 13 (PsBUP1).

[0140] In another preferred embodiment, the gene module further comprises:

[0141] (i) a rate-limiting enzyme gene module in the heme pathway, wherein the rate-limiting enzyme gene module contains a single copy or multiple copies of a rate-limiting enzyme gene (HEM2, HEM3 and / or HEM12);

[0142] (j) an oxidation-related gene module in the pentose phosphate pathway, wherein the oxidation-related gene module contains a single copy or multiple copies of an oxidation-related gene (GND1 and / or ZWF1);

[0143] (k) a non-oxidation-related gene module in the pentose phosphate pathway, wherein the non-oxidation-related gene module contains a single copy or multiple copies of non-oxidation-related genes (TAL1, TKL1, TKL2, XFPK and / or PTA);

[0144] (l) A product transporter gene module, wherein the product transporter gene module contains a single copy or multiple copies of a product transporter gene (ABCG10 gene).

[0145] (m) a cytochrome P450 oxidoreductase (ATR1) gene module, wherein the ATR1 gene module contains a single copy or multiple copies of the ATR1 gene;

[0146] (n) a phospholipid biosynthesis transcription factor (INO2) gene module, wherein the INO2 gene module contains a single copy or multiple copies of an INO2 gene; and / or

[0147] (o) a dihydrofolate reductase (DHFR) gene module, wherein the DHFR gene module contains a single copy or multiple copies of a DHFR gene.

[0148] In another preferred example, the gene module further includes: a product transporter (ABCG10) gene module.

[0149] In another preferred embodiment, the ABCG10 gene module is used for the transmembrane extracellular secretion of the product chelerythrine.

[0150] In another preferred embodiment, the ATR1 gene module is used to cooperate with the tetrahydroberberine synthase (TDC) gene module, N-methylgoldopapaverine hydroxylase (MSH) gene module and / or proopioid hydroxylase (P6H) gene module to biosynthesize (S)-tetrahydroberberine, allocryptine and / or chelerythrine.

[0151] In another preferred embodiment, the INO2 gene module is used to cooperate with the tetrahydroberberine synthase (TDC) gene module, N-methylgold opium poppy hydroxylase (MSH) gene module and / or protopyrine hydroxylase (P6H) gene module to biosynthesize (S)-tetrahydroberberine, allocryptine and / or chelerythrine.

[0152] In another preferred embodiment, the DHFR gene module is used to catalyze the synthesis of chelerythrine from dihydrochelerythrine.

[0153] In another preferred embodiment, the ABCG10 gene is selected from the following group: MtABCG10.

[0154] In another preferred embodiment, the ABCG10 gene has one or more nucleotide sequences selected from the following group:

[0155] (i) the sequence shown in SEQ ID NO: 16;

[0156] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO: 16;

[0157] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 16;

[0158] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0159] In another preferred example, the nucleotide sequence of the ABCG10 gene is shown in SEQ ID NO: 16 (MtABCG10).

[0160] In another preferred embodiment, the ABCG10 gene has one or more amino acid sequences selected from the following group:

[0161] (i) the sequence shown in SEQ ID NO: 15;

[0162] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO: 15;

[0163] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 15;

[0164] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0165] In another preferred example, the amino acid sequence of the ABCG10 gene is shown in SEQ ID NO: 15 (MtABCG10).

[0166] In another preferred embodiment, the ATR1 gene is selected from the following group: AtATR1.

[0167] In another preferred embodiment, the ATR1 gene has one or more nucleotide sequences selected from the following group:

[0168] (i) the sequence shown in SEQ ID NO:48;

[0169] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO: 48;

[0170] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 48;

[0171] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0172] In another preferred example, the nucleotide sequence of the ATR1 gene is shown as SEQ ID NO: 48 (AtATR1).

[0173] In another preferred embodiment, the ATR1 gene has one or more amino acid sequences selected from the following group:

[0174] (i) the sequence shown in SEQ ID NO:47;

[0175] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO: 47;

[0176] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 47;

[0177] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0178] In another preferred example, the amino acid sequence of the ATR1 gene is shown in SEQ ID NO: 47 (AtATR1).

[0179] In another preferred embodiment, the INO2 gene is selected from the following group: ScINO2.

[0180] In another preferred embodiment, the INO2 gene has one or more nucleotide sequences selected from the following group:

[0181] (i) the sequence shown in SEQ ID NO:50;

[0182] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:50;

[0183] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 50;

[0184] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0185] In another preferred example, the nucleotide sequence of the INO2 gene is shown as SEQ ID NO: 50 (ScINO2).

[0186] In another preferred embodiment, the INO2 gene has one or more amino acid sequences selected from the following group:

[0187] (i) the sequence shown in SEQ ID NO:49;

[0188] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:49;

[0189] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 49;

[0190] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0191] In another preferred example, the amino acid sequence of the INO2 gene is shown as SEQ ID NO: 49 (ScINO2).

[0192] In another preferred embodiment, the DHFR gene is selected from the following group: ScDHFR.

[0193] In another preferred embodiment, the DHFR gene has one or more nucleotide sequences selected from the following group:

[0194] (i) the sequence shown in SEQ ID NO:52;

[0195] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:52;

[0196] (iii) a sequence having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 52;

[0197] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0198] In another preferred example, the nucleotide sequence of the DHFR gene is shown in SEQ ID NO: 52 (ScDHFR).

[0199] In another preferred embodiment, the DHFR gene has one or more amino acid sequences selected from the following group:

[0200] (i) the sequence shown in SEQ ID NO:51;

[0201] (ii) a sequence having a homology of ≥95% (preferably ≥98%) to the sequence shown in SEQ ID NO:51;

[0202] (iii) a sequence in which at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and / or substituted based on the sequence shown in SEQ ID NO: 51;

[0203] (iv) a sequence complementary to any one of the sequences (i) to (iii).

[0204] In another preferred embodiment, the amino acid sequence of the DHFR gene is shown in SEQ ID NO: 51 (ScDHFR).

[0205] In another preferred embodiment, the engineered bacteria also has the following characteristics:

[0206] (Z1) The gene of feedback inhibition in the heme pathway (ROX1) was knocked out or knocked down;

[0207] (Z2) the gene for heme degradation in the heme pathway (HMX1) is knocked out or knocked down; and / or

[0208] (Z3) The gene GPP1 in the pentose phosphate pathway is knocked out or knocked down.

[0209] In another preferred embodiment, when the gene module contains ≥2 genes, some or all of the genes are expressed in tandem.

[0210] In another preferred embodiment, the multi-copy genes in each gene module are expressed in tandem.

[0211] In another preferred embodiment, the gene modules are expressed in tandem.

[0212] In another preferred example, the multi-copy genes in the BBE gene module, SMT gene module, TDC gene module, TNMT gene module, MSH gene module, P6H gene module, CPR gene module and / or BUP1 gene module are expressed in tandem.

[0213] In another preferred embodiment, the BBE gene module, SMT gene module, TDC gene module, TNMT gene module, MSH gene module, P6H gene module, CPR gene module and / or BUP1 gene module are expressed in series.

[0214] In another preferred embodiment, the multiple copies of genes in the rate-limiting enzyme gene module, oxidation-related gene module, non-oxidation-related gene module, product transporter gene module, cytochrome P450 oxidoreductase (ATR1) gene module and / or phospholipid biosynthesis transcription factor (INO2) gene module are expressed in tandem.

[0215] In another preferred embodiment, the rate-limiting enzyme gene module, oxidation-related gene module, non-oxidation-related gene module, product transporter gene module, cytochrome P450 oxidoreductase (ATR1) gene module and / or phospholipid biosynthesis transcription factor (INO2) gene module are expressed in series.

[0216] In another preferred example, the multiple copies of genes in the BBE gene module, SMT gene module, TDC gene module, TNMT gene module, MSH gene module, P6H gene module, CPR gene module, BUP1 gene module and / or product transporter (ABCG10) gene module are expressed in tandem.

[0217] In another preferred embodiment, the BBE gene module, SMT gene module, TDC gene module, TNMT gene module, MSH gene module, P6H gene module, CPR gene module, BUP1 gene module and / or product transporter (ABCG10) gene module are expressed in series.

[0218] In another preferred embodiment, each gene module further comprises a promoter element and / or a terminator element.

[0219] In another preferred embodiment, each gene in each gene module is driven by a constitutive or inducible promoter.

[0220] In another preferred embodiment, each gene in each gene module is driven by an inducible promoter.

[0221] In another preferred example, the promoter may be a promoter of any gene from yeast (preferably Saccharomyces cerevisiae).

[0222] In another preferred embodiment, the promoter is selected from the following group: TEF1 (transcription elongation factor 1) promoter, GPD (3-phosphoglyceraldehyde dehydrogenase) promoter, FBA (1,6-diphosphate fructose lyase) promoter (preferably FBA1 promoter), PGK1 promoter, ENO2 promoter, PYK2 promoter, HXT7 promoter, CDC60 promoter, GMP1 promoter, or a combination thereof.

[0223] In another preferred example, the terminator may be a terminator of any gene from yeast (preferably Saccharomyces cerevisiae).

[0224] In another preferred embodiment, the terminator is selected from the following group: TEF1 terminator, TDH1 terminator, PGK1 terminator, PGK1 terminator, PFK1 terminator, ENO2 terminator, ADH1 terminator, ADH2 terminator, CYC1 terminator, HOG1 terminator, or a combination thereof.

[0225] In another preferred embodiment, the starting strain of the engineered bacteria is Saccharomyces cerevisiae.

[0226] In another preferred embodiment, the starting strain of the engineered bacteria contains endogenous dihydrofolate reductase (DHFR) and / or endogenous phospholipid biosynthesis transcription factor (INO2).

[0227] In another preferred embodiment, the starting strain of the engineered bacteria does not contain endogenous dihydrofolate reductase (DHFR).

[0228] In another preferred embodiment, the starting strain of the engineered bacteria does not contain endogenous phospholipid biosynthesis transcription factor (INO2).

[0229] In another preferred embodiment, when the starting strain of the engineered bacteria does not contain endogenous dihydrofolate reductase (DHFR), the engineered bacteria further contains an exogenous dihydrofolate reductase (DHFR) gene module.

[0230] In another preferred embodiment, when the starting strain of the engineered bacteria does not contain endogenous phospholipid biosynthesis transcription stimulator (INO2), the engineered bacteria further contains an exogenous phospholipid biosynthesis transcription stimulator (INO2) gene module.

[0231] In another preferred embodiment, the starting strain of the engineered bacteria is selected from the following group: Saccharomyces cerevisiae W303-1A (MATαleu2-3,112trp1-1 can1-100 ura3-1 ade2-1his3-11,15), Saccharomyces cerevisiae BY4741 (MATαhis3Δ1leu2Δ0met15Δ0ura3Δ0), Saccharomyces cerevisiae CEN.PK113-11C (MATa,SUC2,MAL2-8c,his3Δ1,ura3Δ-52), Saccharomyces cerevisiae CEN.PK2-1C, or a combination thereof.

[0232] In another preferred embodiment, the starting strain of the engineered bacteria is Saccharomyces cerevisiae W303-1A (MATαleu2-3,112trp1-1 can1-100 ura3-1 ade2-1his3-11,15).

[0233] In another preferred embodiment, the chelerythrine production of the engineered bacteria is ≥1 mg / L, preferably ≥2 mg / L, more preferably ≥3 mg / L, and most preferably ≥4.5 mg / L.

[0234] In a second aspect of the present invention, there is provided a method for producing chelerythrine, the method comprising the steps of:

[0235] (1) culturing the engineered bacteria according to the first aspect of the present invention under conditions suitable for expression, and adding a substrate (S)-carbohydidine to obtain a culture product or fermentation product containing chelerythrine; and

[0236] (2) optionally isolating and / or purifying chelerythrine from the culture product or fermentation product.

[0237] In another preferred embodiment, the culture includes test tube culture, shake flask culture, and tank fermentation culture.

[0238] In another preferred embodiment, the culture temperature is 20-37°C, preferably 25-30°C, more preferably 28°C or 30°C.

[0239] In another preferred embodiment, the culture is shaking culture with a rotation speed of 200-300RPM, preferably 260RPM.

[0240] In another preferred embodiment, the culture time of the test tube culture or shake flask culture is 10-72 hours, preferably 18-48 hours, more preferably 18-24 hours or 36-48 hours.

[0241] In another preferred embodiment, the fermentation time of the tank fermentation culture is 5-20 days, preferably 7-15 days, and more preferably 10-12 days.

[0242] In another preferred embodiment, the inoculation amount of the engineered bacteria in the test tube culture or shake flask culture is OD 600 =0.1-0.5, preferably 0.2.

[0243] In another preferred embodiment, the fermentation inoculum amount of the engineered bacteria in the tank fermentation culture is OD 600 =16-45, preferably OD 600 =24-40, better OD 600 =32-40, best OD 600 =40.

[0244] In another preferred example, the culture uses SC medium and / or YPD medium.

[0245] In another preferred embodiment, the SC medium is a nutrient-deficient medium lacking both uracil and tryptophan, wherein the glucose concentration is 20 g / L.

[0246] In another preferred example, the components of the YPD medium are 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose.

[0247] In another preferred embodiment, the concentration of the substrate (S)-carbomer is 50-300 μM.

[0248] In another preferred embodiment, the conditions suitable for expression include: step-wise amplification culture (eg, three-step amplification culture).

[0249] In another preferred embodiment, the step-wise amplification culture comprises the steps of:

[0250] (S1) culturing a single colony of the engineered bacteria in SC-URA-TPR medium;

[0251] (S2) inoculating the engineered bacteria into 5×SC medium for cultivation;

[0252] (S3) centrifuging under appropriate conditions, removing the supernatant, and resuspending the precipitated cells in SC or YPD medium; and

[0253] (S4) Adding substrate (S)-carbomer to the culture medium and performing fermentation culture.

[0254] In another preferred embodiment, the culture conditions of step (S1) are: culture temperature 30°C, 260RPM in a constant temperature shaking incubator for 18-24 hours.

[0255] In another preferred embodiment, the inoculation conditions of step (S2) are: according to the inoculation concentration OD 600 =0.2 for inoculation.

[0256] In another preferred embodiment, the culture conditions of step (S2) are: culture temperature 28°C, 260RPM in a constant temperature shaking incubator for 36-48 hours.

[0257] In another preferred embodiment, the suitable conditions of step (S3) are: centrifugation at 500×g for 4 minutes.

[0258] In another preferred embodiment, the precipitated bacteria in step (S3) are resuspended to an OD of 600 It is 8-48.

[0259] In another preferred embodiment, the concentration of the substrate (S)-carbomer in step (S4) is 50-300 μM, preferably 100 μM.

[0260] In another preferred embodiment, the fermentation culture conditions of step (S4) are: fermentation for 10 days in a constant temperature shaking incubator at a culture temperature of 28°C and 260RPM.

[0261] In another preferred embodiment, the method further comprises the step of detecting chelerythrine in the culture product (eg, cell lysis solution) or the fermentation product (eg, fermentation solution).

[0262] In another preferred embodiment, the detection is performed by LC-MS measurement.

[0263] In another preferred embodiment, the detection is performed by LC-MS / MS measurement.

[0264] In the third aspect of the present invention, a method for constructing the engineered bacteria as described in the first aspect of the present invention is provided, the method comprising the steps of:

[0265] (a) constructing a BBE gene module, an SMT gene module, a TDC gene module, a TNMT gene module, an MSH gene module, a P6H gene module, a CPR gene module and a BUP1 gene module, or a vector containing the above gene modules;

[0266] (b) using the gene module or vector obtained in step (a) to transform a yeast, thereby obtaining the engineered bacteria according to the first aspect of the present invention.

[0267] In another preferred embodiment, the method further comprises the step of: (c) screening yeast transformants containing the gene module or vector in a screening medium, and picking out single colony clones for molecular identification.

[0268] In another preferred embodiment, the step (a) further comprises the sub-steps of:

[0269] (a1) According to the codon preference of yeast, the coding sequences of tMcoBBE, TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR, and BUP1 genes were synthesized respectively, and the corresponding gene modules (expression cassettes) were constructed using the overlapping PCR method.

[0270] In another preferred embodiment, the step (b) further comprises the sub-steps of:

[0271] (b1) first round of transformation: using the BBE gene module, SMT gene module, TDC gene module and CPR gene module to transform yeast, thereby obtaining a recombinant yeast after the first round of transformation; and

[0272] (b2) Second round of transformation: using the TNMT gene module, MSH gene module, P6H gene module and BUP1 gene module to transform the recombinant yeast transformed in the first round, thereby obtaining the engineered bacteria according to the first aspect of the present invention.

[0273] In another preferred embodiment, the step (b1) further comprises the step of: screening recombinant yeast containing the BBE gene module, the SMT gene module, the TDC gene module and the CPR gene module.

[0274] In another preferred embodiment, the step (b2) further comprises the step of: screening recombinant yeast containing the TNMT gene module, the MSH gene module, the P6H gene module and the BUP1 gene module.

[0275] In a fourth aspect of the present invention, there is provided a use of the engineered bacteria according to the first aspect of the present invention, wherein the engineered bacteria is used for producing chelerythrine.

[0276] In another preferred embodiment, the production is fermentation production.

[0277] In a fifth aspect of the present invention, a kit is provided, comprising:

[0278] (Z1) a first container and a berberine bridge enzyme (BBE) gene module in the first container, wherein the BBE gene module contains a single copy or multiple copies of a BBE gene;

[0279] (Z2) a second container and a S. cerasiferum methyltransferase (SMT) gene module in the second container, wherein the SMT gene module contains a single copy or multiple copies of the SMT gene;

[0280] (Z3) a third container and a tetrahydroberberine synthase (TDC) gene module located in the third container, wherein the TDC gene module contains a single copy or multiple copies of a TDC gene;

[0281] (Z4) a fourth container and a tetrahydroberberine N-methyltransferase (TNMT) gene module located in the fourth container, wherein the TNMT gene module contains a single copy or multiple copies of the TNMT gene;

[0282] (Z5) a fifth container and an N-methylpapaverine hydroxylase (MSH) gene module located in the fifth container, wherein the MSH gene module contains a single copy or multiple copies of the MSH gene;

[0283] (Z6) a sixth container and a proopioid hydroxylase (P6H) gene module located in the sixth container, wherein the P6H gene module contains a single copy or multiple copies of a P6H gene;

[0284] (Z7) a seventh container and a cytochrome P450 oxidoreductase (CPR) gene module located in the seventh container, wherein the CPR gene module contains a single copy or multiple copies of a CPR gene; and

[0285] (Z8) An eighth container and a transporter benzylisoquinoline alkaloid uptake permease (BUP1) gene module located in the eighth container, wherein the BUP1 gene module contains a single copy or multiple copies of a BUP1 gene.

[0286] In another preferred embodiment, the vector is a plasmid and / or a nucleic acid fragment.

[0287] In another preferred embodiment, the kit further comprises:

[0288] (Z9) a ninth container and a restriction endonuclease and / or a DNA ligase located in the ninth container.

[0289] In another preferred example, any one or more of the first container, the second container, the third container, the fourth container, the fifth container, the sixth container, the seventh container, the eighth container, and the ninth container may be the same or different containers.

[0290] In a sixth aspect of the present invention, a use of the kit as described in the fifth aspect of the present invention is provided, wherein the kit is used to construct the engineered bacteria as described in the first aspect of the present invention.

[0291] In a seventh aspect of the present invention, a fermentation system for producing chelerythrine is provided, the fermentation system comprising:

[0292] (S1) the engineered bacteria as described in the first aspect of the present invention; and

[0293] (S2) Substrate (S)-caryophylline.

[0294] In another preferred embodiment, the system further comprises: (S3) glucose.

[0295] In another preferred embodiment, the concentration of the substrate (S)-carbohydidine is 50-300 μM, preferably 100 μM.

[0296] In another preferred example, the concentration of glucose is 20 g / L.

[0297] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0298] Figure 1 The microbial synthesis pathway of chelerythrine is shown.

[0299] Figure 2 Shown are: A. the screening results of berberine bridge enzyme (BBE) with better catalytic activity; B. the screening results of smilax china methyltransferase (SMT) with better catalytic activity; C. the screening results of tetrahydroberberine N-methyltransferase (TNMT) with better catalytic activity; D. the screening results of protoopine hydroxylase (P6H) with better catalytic activity.

[0300] Figure 3 Validation of chelerythrine synthesis in recombinant yeast using liquid chromatography-tandem mass spectrometry is shown.

[0301] Figure 4Shown are: A. the effect of different initial inoculum amounts on the synthesis of chelerythrine; B. the effect of different culture media on the synthesis of chelerythrine.

[0302] Figure 5 The yield of chelerythrine produced by fermentation was shown, with the highest yield reaching 4.5 mg / L. DETAILED DESCRIPTION

[0303] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly discovered for the first time that the microbial synthesis pathway constructed by heterologous expression of codon-optimized exogenous berberine bridge enzyme (BBE), golden berberine methyltransferase (SMT), tetrahydroberberine synthase (TDC), tetrahydroprotoberberine N-methyltransferase (TNMT), N-methylgold opium poppy hydroxylase (MSH), protopine hydroxylase (P6H), cytochrome P450 oxidoreductase (CPR), and transporter benzylisoquinoline alkaloid uptake permease (BUP1) can be used to efficiently synthesize chelerythrine by fermentation and conversion of (S)-cholorocarpine by recombinant yeast. Furthermore, through multi-copy integration, regulation of cofactor heme, regulation of NADPH, mining of secretory transporter ABCG10, and debugging of different fermentation conditions, the maximum yield of chelerythrine reached 4.5 mg / L. The recombinant yeast strain obtained by the construction method described in the present invention can efficiently synthesize chelerythrine, achieve a yield far exceeding that of the prior art, and can solve the disadvantage of a long production cycle, thereby providing a new method for the synthesis of chelerythrine, and thus has broad application prospects in the fields of agriculture and medicine. On this basis, the present invention is completed.

[0304] the term

[0305] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0306] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0307] The terms "engineered bacteria", "genetically engineered bacteria" or "engineered yeast" are used interchangeably to refer to the specially modified recombinant yeast described in the first aspect of the present invention. The engineered bacteria of the present invention can effectively utilize the substrate (S)-carnitine to produce chelerythrine.

[0308] Biosynthesis of chelerythrine in yeast

[0309] Regarding the biosynthesis of chelerythrine in yeast, those skilled in the art know that: the enzymes required for the heterologous synthesis of chelerythrine come from plants, and Saccharomyces cerevisiae can actively express plant-derived P450 enzymes and enzymes required for the synthesis of chelerythrine; and the yeasts listed themselves contain dihydrofolate reductase (DHFR), which can catalyze the last step reaction of chelerythrine (catalyze the synthesis of chelerythrine from dihydrochelerythrine), and may also contain endogenous phospholipid biosynthesis transcription factors (INO2), which can cooperate with tetrahydroberberine synthase (TDC), N-methylgoldopapaverine hydroxylase (MSH) and / or proopioid hydroxylase (P6H) to biosynthesize (S)-tetrahydroberberine, allocryptine and / or chelerythrine; in addition, Saccharomyces cerevisiae contains an organelle - the endoplasmic reticulum, which provides a place for the catalysis involving P450 enzymes. Therefore, for Saccharomyces cerevisiae, those skilled in the art can expect that as long as the specific gene combination in the present application is introduced into the Saccharomyces cerevisiae, this type of yeast can produce high levels of chelerythrine.

[0310] Typically, the starting strain of the engineered bacteria used to produce chelerythrine in the present invention is Saccharomyces cerevisiae, preferably Saccharomyces cerevisiae containing endogenous dihydrofolate reductase (DHFR) and endogenous phospholipid biosynthesis transcription factor (INO2). Exemplary starting strains include, but are not limited to: Saccharomyces cerevisiae W303-1A (MATαleu2-3,112trp1-1can1-100 ura3-1 ade2-1his3-11,15), Saccharomyces cerevisiae BY4741 (MATαhis3Δ1leu2Δ0met15Δ0ura3Δ0), Saccharomyces cerevisiae CEN.PK113-11C (MATa,SUC2,MAL2-8c,his3Δ1,ura3Δ-52), Saccharomyces cerevisiae CEN.PK2-1C, or a combination thereof.

[0311] Those skilled in the art know that based on the aforementioned characteristics and functions of Saccharomyces cerevisiae, it can be expected that all Saccharomyces cerevisiae can achieve the technical effects of the present invention, and are not limited to the Saccharomyces cerevisiae used as illustrative examples in the embodiments.

[0312] For Saccharomyces cerevisiae containing endogenous dihydrofolate reductase (DHFR), it can be expected that they can all achieve the technical effects of the present invention; for Saccharomyces cerevisiae not containing endogenous dihydrofolate reductase (DHFR), it can be expected that the technical effects of the present invention can also be achieved after additionally introducing the dihydrofolate reductase (DHFR) gene therein.

[0313] Similarly, for Saccharomyces cerevisiae containing endogenous phospholipid biosynthesis transcription stimulator (INO2), it can be expected that they can achieve the technical effects of the present invention; for Saccharomyces cerevisiae that does not contain endogenous phospholipid biosynthesis transcription stimulator (INO2), it can be expected that the technical effects of the present invention can also be achieved after additionally introducing the phospholipid biosynthesis transcription stimulator (INO2) gene into it.

[0314] Therefore, those skilled in the art can expect that all brewing yeasts can achieve the technical effects of the present invention.

[0315] Engineering bacteria

[0316] In the present invention, an engineered bacterium for producing chelerythrine is provided, which contains an exogenous and / or endogenous gene module, wherein the gene module comprises:

[0317] (a) a berberine bridge enzyme (BBE) gene module, wherein the BBE gene module contains a single copy or multiple copies of a BBE gene;

[0318] (b) a S. chrysanthemi methyltransferase (SMT) gene module, wherein the SMT gene module contains a single copy or multiple copies of the SMT gene;

[0319] (c) a tetrahydroberberine synthase (TDC) gene module, wherein the TDC gene module contains a single copy or multiple copies of a TDC gene;

[0320] (d) a tetrahydroberberine N-methyltransferase (TNMT) gene module, wherein the TNMT gene module contains a single copy or multiple copies of the TNMT gene;

[0321] (e) an N-methylaurocanine hydroxylase (MSH) gene module, wherein the MSH gene module contains a single copy or multiple copies of the MSH gene;

[0322] (f) a proopioid hydroxylase (P6H) gene module, wherein the P6H gene module contains a single copy or multiple copies of a P6H gene;

[0323] (g) a cytochrome P450 oxidoreductase (CPR) gene module, wherein the CPR gene module contains a single copy or multiple copies of a CPR gene; and

[0324] (h) a transporter benzylisoquinoline alkaloid uptake permease (BUP1) gene module, wherein the BUP1 gene module contains a single copy or multiple copies of a BUP1 gene.

[0325] In certain preferred embodiments, the gene module may further include:

[0326] (i) a rate-limiting enzyme gene module in the heme pathway, wherein the rate-limiting enzyme gene module contains a single copy or multiple copies of a rate-limiting enzyme gene (HEM2, HEM3 and / or HEM12);

[0327] (j) an oxidation-related gene module in the pentose phosphate pathway, wherein the oxidation-related gene module contains a single copy or multiple copies of an oxidation-related gene (GND1 and / or ZWF1);

[0328] (k) a non-oxidation-related gene module in the pentose phosphate pathway, wherein the non-oxidation-related gene module contains a single copy or multiple copies of non-oxidation-related genes (TAL1, TKL1, TKL2, XFPK and / or PTA);

[0329] (l) A product transporter gene module, wherein the product transporter gene module contains a single copy or multiple copies of a product transporter gene (ABCG10 gene).

[0330] (m) a cytochrome P450 oxidoreductase (ATR1) gene module, wherein the ATR1 gene module contains a single copy or multiple copies of the ATR1 gene;

[0331] (n) a phospholipid biosynthesis transcription factor (INO2) gene module, wherein the INO2 gene module contains a single copy or multiple copies of an INO2 gene; and / or

[0332] (o) a dihydrofolate reductase (DHFR) gene module, wherein the DHFR gene module contains a single copy or multiple copies of a DHFR gene.

[0333] Furthermore, the engineered bacteria can also be transformed as follows:

[0334] (Z1) The gene of feedback inhibition in the heme pathway (ROX1) was knocked out or knocked down;

[0335] (Z2) the gene for heme degradation in the heme pathway (HMX1) is knocked out or knocked down; and / or

[0336] (Z3) The gene GPP1 in the pentose phosphate pathway is knocked out or knocked down.

[0337] The starting strain of the engineered bacteria can be selected from the following group: Saccharomyces cerevisiae W303-1A (MATαleu2-3,112trp1-1can1-100 ura3-1 ade2-1his3-11,15), Saccharomyces cerevisiae BY4741 (MATαhis3Δ1leu2Δ0met15Δ0ura3Δ0), Saccharomyces cerevisiae CEN.PK113-11C (MATa,SUC2,MAL2-8c,his3Δ1,ura3Δ-52), Saccharomyces cerevisiae CEN.PK2-1C, or a combination thereof; the starting strain may contain, or may not contain: endogenous dihydrofolate reductase (DHFR) and / or endogenous phospholipid biosynthesis transcription factor (INO2).

[0338] Through experimental verification and mechanism research of the present invention, the above-mentioned modified and starting strains can achieve the technical effect of the present invention, so that the modified engineered bacteria can effectively utilize the substrate (S)-carbohydidine to produce chelerythrine.

[0339] Gene module

[0340] The gene sequences of the gene modules provided in the present invention can be selected from any combination of one or more. Those skilled in the art can reasonably anticipate, based on the description of the present invention, that the selection and combination of various gene sequences can achieve the technical effects of the present invention.

[0341] Typically, the BBE gene module of the present invention can be selected from the following group: tMcoBBE (amino acid sequence SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 2), McoBBE (amino acid sequence SEQ ID NO: 17, nucleotide sequence SEQ ID NO: 18), AmBBE (amino acid sequence SEQ ID NO: 19, nucleotide sequence SEQ ID NO: 20), EcBBE (amino acid sequence SEQ ID NO: 21, nucleotide sequence SEQ ID NO: 22), PsBBE (amino acid sequence SEQ ID NO: 23, nucleotide sequence SEQ ID NO: 24), or a combination thereof.

[0342] The SMT gene module of the present invention can be selected from the following groups: TfSMT (amino acid sequence SEQ ID NO: 3, nucleotide sequence SEQ ID NO: 4), AmSMT (amino acid sequence SEQ ID NO: 25, nucleotide sequence SEQ ID NO: 26), CcSMT (amino acid sequence SEQ ID NO: 27, nucleotide sequence SEQ ID NO: 28), PsSMT (amino acid sequence SEQ ID NO: 29, nucleotide sequence SEQ ID NO: 30), or a combination thereof.

[0343] The TDC gene module of the present invention can be selected from the following group: AmTDC (amino acid sequence SEQ ID NO: 5, nucleotide sequence SEQ ID NO: 6).

[0344] The TNMT gene module of the present invention can be selected from the following group: EcTNMT (amino acid sequence SEQ ID NO: 7, nucleotide sequence SEQ ID NO: 8), GfTNMT (amino acid sequence SEQ ID NO: 33, nucleotide sequence SEQ ID NO: 34), PsTNMT (amino acid sequence SEQ ID NO: 35, nucleotide sequence SEQ ID NO: 36), or a combination thereof.

[0345] The MSH gene module of the present invention may be selected from the following group: PsMSH (amino acid sequence SEQ ID NO: 9, nucleotide sequence SEQ ID NO: 10).

[0346] The P6H gene module of the present invention can be selected from the following groups: EcP6H (amino acid sequence SEQ ID NO: 11, nucleotide sequence SEQ ID NO: 12), PsP6H (amino acid sequence SEQ ID NO: 37, nucleotide sequence SEQ ID NO: 38), McoP6H (amino acid sequence SEQ ID NO: 39, nucleotide sequence SEQ ID NO: 40), t4EcP6H (amino acid sequence SEQ ID NO: 41, nucleotide sequence SEQ ID NO: 42), t27EcP6H (amino acid sequence SEQ ID NO: 43, nucleotide sequence SEQ ID NO: 44), or a combination thereof.

[0347] The CPR gene module of the present invention may be selected from the following group: PsCPR (amino acid sequence SEQ ID NO: 45, nucleotide sequence SEQ ID NO: 46).

[0348] The BUP1 gene module of the present invention may be selected from the following group: PsBUP1 (amino acid sequence SEQ ID NO: 13, nucleotide sequence SEQ ID NO: 14).

[0349] In addition, the ABCG10 gene module of the present invention may be selected from the following group: MtABCG10 (amino acid sequence SEQ ID NO: 15, nucleotide sequence SEQ ID NO: 16).

[0350] The ATR1 gene module of the present invention may be selected from the following group: AtATR1 (amino acid sequence SEQ ID NO: 47, nucleotide sequence SEQ ID NO: 48).

[0351] The INO2 gene module of the present invention may be selected from the following group: ScINO2 (amino acid sequence SEQ ID NO: 49, nucleotide sequence SEQ ID NO: 50).

[0352] The DHFR gene module of the present invention may be selected from the following group: ScDHFR (amino acid sequence SEQ ID NO: 51, nucleotide sequence SEQ ID NO: 52).

[0353] Main advantages of the present invention

[0354] 1. The recombinant yeast obtained by the construction method described in the present invention can efficiently synthesize chelerythrine, with a maximum yield of 4.5 mg / L, which is the highest yield reported so far.

[0355] 2. The use of yeast to biosynthesize chelerythrine can solve the disadvantage of long production cycle and provide a new method for the synthesis of chelerythrine that is fast, effective and high-yield.

[0356] 3. The inventors have also optimized the production process of chelerythrine in many aspects, such as multi-copy integration, regulation of cofactor heme, regulation of NADPH, exploration of secretory transporter ABCG10, debugging of different fermentation conditions, etc., providing useful guidance for the production process of chelerythrine.

[0357] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0358] Example 1: Screening of berberine bridge enzyme (BBE) with better catalytic activity

[0359] The berberine bridge enzyme gene was synthesized by codon optimization, i.e., the amino acid sequences were as shown in SEQ ID NO: 1 (tMcoBBE), SEQ ID NO: 17 (McoBBE), SEQ ID NO: 19 (AmBBE), SEQ ID NO: 21 (EcBBE), SEQ ID NO: 23 (PsBBE), or the corresponding nucleotide sequences were as shown in SEQ ID NO: 2 (tMcoBBE), SEQ ID NO: 18 (McoBBE), SEQ ID NO: 20 (AmBBE), SEQ ID NO: 22 (EcBBE), SEQ ID NO: 24 (PsBBE). The present inventors obtained the expression cassette of the above berberine bridge enzyme gene by overlapping polymerase chain reaction, and constructed the expression plasmid of the berberine bridge enzyme gene by enzyme cutting and ligation.

[0360] The constructed berberine bridge enzyme gene expression plasmids were transformed into Saccharomyces cerevisiae W303-1A (Z0) (wherein the Control group represents Saccharomyces cerevisiae that was not transformed with the exogenous berberine bridge enzyme gene expression plasmid). The recombinant Saccharomyces cerevisiae containing the berberine bridge enzyme gene expression plasmid was screened, and the screened recombinant bacteria were fed with (S)-corydaline to detect the concentration of (S)-corydaline in the product.

[0361] The results are as follows Figure 2 As shown in A, according to the concentration of (S)-corydaline, berberine bridge enzymes AmBBE, EcBBE, McoBBE, tMcoBBE, and PsBBE can all achieve effective catalysis, and tMcoBBE has the highest activity.

[0362] Example 2: Screening of S. chrysanthemi methyltransferase (SMT) with better catalytic activity

[0363] The codons are optimized and synthesized to obtain the golden corydaline methyltransferase gene, which has the amino acid sequences shown in SEQ ID NO:3 (TfSMT), SEQ ID NO:25 (AmSMT), SEQ ID NO:27 (CcSMT), and SEQ ID NO:29 (PsSMT), or the corresponding nucleotide sequences shown in SEQ ID NO:4 (TfSMT), SEQ ID NO:26 (AmSMT), SEQ ID NO:28 (CcSMT), and SEQ ID NO:30 (PsSMT). The inventors obtain the expression cassette of the golden corydaline methyltransferase gene by overlapping polymerase chain reaction, and construct the expression plasmid of the golden corydaline methyltransferase gene by enzyme cutting and ligation.

[0364] The constructed golden corydaline methyltransferase gene expression plasmids were transformed into Saccharomyces cerevisiae (Z0) (wherein the Control group represents the Saccharomyces cerevisiae that was not transformed with the exogenous golden corydaline methyltransferase gene expression plasmid). The recombinant Saccharomyces cerevisiae containing the golden corydaline methyltransferase gene expression plasmid was screened, and the screened recombinant bacteria were fed with (S)-corydaline to detect the concentration of (S)-golden corydaline in the product.

[0365] The results are as follows Figure 2 As shown in B, according to the concentration of (S)-chrysocollaine, chrysocollaine methyltransferases AmSMT, CcSMT, PsSMT, and TfSMT can all achieve effective catalysis, and TfSMT has the highest activity.

[0366] Example 3: Screening of Tetrahydroberberine N-methyltransferase (TNMT) with Better Catalytic Activity

[0367] The tetrahydroberberine N-methyltransferase gene was synthesized by codon optimization, i.e., the amino acid sequences were as shown in SEQ ID NO:7 (EcTNMT), SEQ ID NO:31 (CmTNMT), SEQ ID NO:33 (GfTNMT), SEQ ID NO:35 (PsTNMT), or the corresponding nucleotide sequences were as shown in SEQ ID NO:8 (EcTNMT), SEQ ID NO:32 (CmTNMT), SEQ ID NO:34 (GfTNMT), SEQ ID NO:36 (PsTNMT). The present inventors obtained the expression cassette of the tetrahydroberberine N-methyltransferase gene by overlapping polymerase chain reaction, and constructed the expression plasmid of the tetrahydroberberine N-methyltransferase gene by enzyme cutting and ligation.

[0368] The constructed tetrahydroberberine N-methyltransferase gene expression plasmid was transformed into Saccharomyces cerevisiae (Z0) (wherein the Control / control group represents the Saccharomyces cerevisiae that was not transformed with the exogenous tetrahydroberberine N-methyltransferase gene expression plasmid). The recombinant Saccharomyces cerevisiae containing the tetrahydroberberine N-methyltransferase gene expression plasmid was screened, and the screened recombinant bacteria were fed with (S)-tetrahydroberberine to detect the concentration of (S)-N-methyltetrahydroberberine in the product.

[0369] The results are as follows Figure 2 As shown in Figure C, according to the concentration of (S)-N-methyltetrahydroberberine, tetrahydroprotoberberine N-methyltransferases EcTNMT, GfTNMT, and PsTNMT can all achieve effective catalysis, while CmTNMT cannot catalyze the production of (S)-N-methyltetrahydroberberine, and EcTNMT has the highest catalytic activity.

[0370] Example 4: Screening of protopine hydroxylase (P6H) with better catalytic activity

[0371] The original opium alkali hydroxylase gene is synthesized by codon optimization, that is, the amino acid sequence shown in SEQ ID NO: 11 (EcP6H), SEQ ID NO: 37 (PsP6H), SEQ ID NO: 39 (McoP6H), SEQ ID NO: 41 (t4EcP6H), SEQ ID NO: 43 (t27EcP6H), or the corresponding nucleotide sequence shown in SEQ ID NO: 12 (EcP6H), SEQ ID NO: 38 (PsP6H), SEQ ID NO: 40 (McoP6H), SEQ ID NO: 42 (t4EcP6H), SEQ ID NO: 44 (t27EcP6H). The inventors obtain the expression cassette of the original opium alkali hydroxylase gene by overlapping polymerase chain reaction, and construct the expression plasmid of the original opium alkali hydroxylase gene by enzyme cutting and ligation.

[0372] The constructed protopine hydroxylase gene expression plasmids were transformed into Saccharomyces cerevisiae (wherein the Control group represents Saccharomyces cerevisiae that was not transformed with the exogenous protopine hydroxylase gene expression plasmid). The recombinant Saccharomyces cerevisiae containing the protopine hydroxylase gene expression plasmid was screened, and the screened recombinant bacteria were fed with allocryptine to detect the concentration of chelerythrine in the product.

[0373] The results are as follows Figure 2 As shown in D, according to the concentration of chelerythrine, protopine hydroxylases EcP6H, t4EcP6H, t27EcP6H, McoP6H, and PsP6H can all achieve effective catalysis, and the catalytic activities of EcP6H, t4EcP6H, and PsP6H are relatively high, with EcP6H having the highest activity.

[0374] Example 5: Tetrahydroberberine synthase (TDC) and N-methylglucopapine hydroxylase with better catalytic activity (MSH), cytochrome P450 oxidoreductase (CPR), and the transporter benzylisoquinoline alkaloid uptake permease (BUP1) Filter

[0375] The present inventors also screened the tetrahydroberberine synthase gene, N-methylauropopavine hydroxylase gene, cytochrome P450 oxidoreductase gene, and transporter benzylisoquinoline alkaloid uptake permease gene that were codon optimized and synthesized.

[0376] By detecting and comparing the concentrations of (S)-tetrahydroberberine, allocryptine, and intracellular (S)-caryophyllene in the recombinant bacterial products, the following products were screened out:

[0377] The tetrahydroberberine synthase gene with better activity is AmTDC, which has an amino acid sequence as shown in SEQ ID NO:5 and a nucleotide sequence as shown in SEQ ID NO:6;

[0378] The N-methylauropopavine hydroxylase gene with better activity is PsMSH, which has an amino acid sequence as shown in SEQ ID NO:9 and a nucleotide sequence as shown in SEQ ID NO:10;

[0379] The cytochrome P450 oxidoreductase gene with better activity is PsCPR, which has an amino acid sequence as shown in SEQ ID NO:45 and a nucleotide sequence as shown in SEQ ID NO:46;

[0380] The gene of the transporter benzylisoquinoline alkaloid uptake permease with better activity is PsBUP1, which has an amino acid sequence as shown in SEQ ID NO:13 and a nucleotide sequence as shown in SEQ ID NO:14.

[0381] Example 6: Design and synthesis of gene expression cassette and homology arms

[0382] The best optimized sequences of each gene screened in Examples 1-5 (codon optimized sequences with the highest catalytic activity) are respectively used to construct gene expression cassettes. It should be understood that those skilled in the art can select one or more suitable promoter and / or terminator sequences for the construction of gene expression cassettes according to conventional selection and adjustment. Different promoter and / or terminator sequences can be used to assist the expression of the best optimized sequences of each gene above. As an example, the following gene expression cassettes are constructed in this embodiment:

[0383] The berberine bridge enzyme mutant expression cassette includes the following DNA elements: a promoter sequence (TEF1p), a berberine bridge enzyme mutant (tMcoBBE) gene sequence, and a terminator sequence (TEF1t);

[0384] The golden corydaline methyltransferase expression cassette includes the following DNA elements: a promoter sequence (PGK1p), a golden corydaline methyltransferase (TfSMT) gene sequence, and a terminator sequence (PFK1t);

[0385] The tetrahydroberberine synthase expression cassette includes the following DNA elements: a promoter sequence (ENO2p), a tetrahydroberberine synthase (AmTDC) gene sequence, and a terminator sequence (ENO2t);

[0386] The tetrahydroberberine N-methyltransferase expression cassette includes the following DNA elements: promoter sequence (PYK2p), tetrahydroberberine synthase (EcTNMT) gene sequence, terminator sequence (ADH1t);

[0387] The N-methyl auropapaveline hydroxylase expression cassette includes the following DNA elements: a promoter sequence (HXT7p), an N-methyl auropapaveline hydroxylase (PsMSH) gene sequence, and a terminator sequence (ADH2t);

[0388] The proopioline hydroxylase expression cassette includes the following DNA elements: a promoter sequence (FBA1p), a proopioline hydroxylase (EcP6H) gene sequence, and a terminator sequence (CYC1t);

[0389] The cytochrome P450 oxidoreductase expression cassette includes the following DNA elements: a promoter sequence (CDC60p), a cytochrome P450 oxidoreductase (PsPCR) gene sequence, and a terminator sequence (TDH1t);

[0390] The transporter benzylisoquinoline alkaloid uptake permease expression cassette includes the following DNA elements: a promoter sequence (GMP1p), a transporter benzylisoquinoline alkaloid uptake permease (PsBUP1) gene sequence, and a terminator sequence (HOG1t).

[0391] Among them, the promoter sequence and the terminator sequence can be obtained by PCR amplification using the Saccharomyces cerevisiae genome as a template, while the target gene sequence is obtained by PCR amplification using a synthetic gene as a template. Each expression cassette is obtained by Overlap PCR using the promoter sequence, the target gene sequence, and the terminator sequence as templates. In addition, the homology arm is obtained by PCR amplification using the yeast genome as a template.

[0392] Example 7: Obtaining chelerythrine-producing recombinant bacteria

[0393] After the gene expression cassette and homology arms are obtained, they are transformed into Saccharomyces cerevisiae in two rounds. First, the plasmid containing the Cas9 protein sequence is transformed into the starting strain (such as Saccharomyces cerevisiae W303-1A (MATαleu2-3,112trp1-1 can1-100 ura3-1 ade2-1his3-11,15), or other yeast), and screened on the SC-URA solid plate to obtain yeast containing the Cas9 protein plasmid.

[0394] First round of transformation: Transform the berberine bridge enzyme mutant (tMcoBBE) expression cassette, golden corydaline methyltransferase (TfSMT) expression cassette, tetrahydroberberine synthase (AmTDC) expression cassette, cytochrome P450 oxidoreductase (PsCPR) expression cassette, upstream and downstream homology arms and gRNA1 into yeast containing Cas9 protein plasmid, screened on SC-URA-Trp solid plates, and obtained recombinant bacteria containing target gene expression by PCR verification. Screening on SC-TRP solid plates containing 1g / L 5-FOA obtained recombinant bacteria containing target gene expression without gRNA1.

[0395] Second round of transformation: The tetrahydroprotoberberine N-methyltransferase (EcTNMT) expression cassette, N-methylgoldopapaverine hydroxylase (PsMSH) expression cassette, protopyrine hydroxylase expression cassette (PsP6H), transporter benzylisoquinoline alkaloid uptake permease (PsBUP1) expression cassette, upstream and downstream homology arms, and gRNA2 were transformed into the recombinant bacteria in which gRNA1 was removed in the first round, and the recombinant bacteria containing the expression of the target gene were screened on the solid plate of SC-URA-Trp and verified by PCR, and named Z1.

[0396] The transformation method adopts chemical transformation. For the specific operation method, please refer to the publicly published paper (Metabolic Engineering, 2020, 57: 151-161).

[0397] A single colony was selected and placed in SC-URA-TPR medium (SC medium is a nutrient-deficient medium lacking uracil and tryptophan, with a glucose concentration of 20 g / L) and cultured in a constant temperature shaking incubator at 30°C and 260 RPM for 18-24 hours. The recombinant yeast was inoculated at an OD of 1. 600 Inoculate 5×SC medium with 0.2% iodine, culture in a constant temperature shaking incubator at 28°C and 260RPM for 36-48 hours, centrifuge at 500×g for 4 minutes, and remove the supernatant. Resuspend the precipitated cells in SC medium to an OD of 600 The culture medium was added with 100 μM (S)-taurine, and the culture was incubated at 28°C and 260 RPM in a constant temperature shaking incubator for 10 days.

[0398] like Figure 3 As shown, the synthesis of chelerythrine was verified by liquid chromatography-tandem mass spectrometry, proving that the recombinant bacteria into which the above expression cassette was introduced could produce chelerythrine.

[0399] Example 8: Multi-copy integration improves the synthesis of chelerythrine

[0400] There is a rate-limiting enzyme in the synthesis pathway of chelerythrine. In order to solve the reason why the rate-limiting enzyme leads to low yield, multiple copies of the rate-limiting enzyme in the pathway are integrated. First, Z1 is used as the starting strain. After the first round of integration of multi-gene expression cassettes, the expression cassettes of the multi-genes (TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR expression cassettes) are increased by 5 copies to obtain recombinant bacteria Z2; the second round of integration of multi-gene expression cassettes increases the number of copies of the multi-gene expression cassettes (PsMSH, AtATR1, ScINO2 expression cassettes, wherein AtATR1 and ScINO2 are respectively the optimized ATR1 sequence and the optimized INO2 sequence obtained by screening using the method in the aforementioned embodiment) by 5 copies. After two rounds of multi-gene multi-copy integration, recombinant bacteria Z3 is obtained, and the yield of chelerythrine is increased by 220 times compared with Z1.

[0401] The above results indicate that multiple copies of the gene are beneficial to increasing the production of chelerythrine.

[0402] Example 9: Regulation of cofactor heme

[0403] Taking Z3 as the starting strain, the inventors obtained recombinant bacteria Z4 by enhancing the expression of rate-limiting enzymes in the heme pathway (i.e., HEM2, HEM3, and HEM12), knocking out the gene of feedback inhibition (ROX1) and the gene of heme degradation (HMX1). Compared with the recombinant bacteria Z3, the production of chelerythrine in Z4 was further increased by 70.2%.

[0404] Example 10: Regulation of NADPH

[0405] Using Z4 as the starting strain, the inventors obtained recombinant strain Z5 by strengthening the oxidation process (overexpression of GND1, ZWF1) and non-oxidation process (i.e. TAL1, TKL1, TKL2, XFPK, PTA) in the pentose phosphate pathway and knocking out the gene GPP1. Compared with the recombinant strain Z4, the production of chelerythrine was further increased by 54.7%.

[0406] Example 11: Discovery of Chelerythrine Secretion Transporter

[0407] Since chelerythrine mainly accumulates intracellularly, in order to further reduce the toxicity caused by the accumulation of intracellular chelerythrine, the inventors also explored plant-derived transporters and successfully discovered that the preferred transporter MtABCG10 obtained by screening can significantly increase the secretion of chelerythrine. Using Z5 as the starting strain, heterologous expression of MtABCG10 was obtained to obtain recombinant bacteria Z6, and the ratio of intracellular expression to extracellular expression decreased from 6:1 to 0.7:1, which increased the total production of chelerythrine by 55.6%.

[0408] Example 12: Experiments on fermentation of (S)-carbomer under different conditions

[0409] The following implementation describes in detail the effects of different initial inoculation amounts and culture media of the recombinant bacteria Z6 on the fermentation synthesis of chelerythrine.

[0410] 12.1 Effect of different initial inoculum sizes on chelerythrine synthesis

[0411] 10 ml shake flasks were filled with 1 ml of bacterial solution (SC medium dilution) with different initial inoculum volumes, the initial inoculum volumes were OD 600 The concentration of the substrate (S)-caryophyllene was 100 μM. After 10 days of fermentation, the production of chelerythrine was determined. The results are shown in Figure 4 A.

[0412] like Figure 4 As shown in A, as the initial inoculum size gradually increased from 8 to 40, the yield of chelerythrine also increased, and the increase was more significant in the 8-32 stage, while the increase in the 32-40 stage began to slow down and tended to saturation. 600 =32-40 is the optimal initial inoculum size, and the inoculum size is OD 600 =40 is the optimal initial inoculum size.

[0413] 12.2 Effects of different culture media on the synthesis of chelerythrine

[0414] Place 1 ml of different culture dilutions (SC medium or YPD medium dilution) in a 10 ml sterile test tube. The initial inoculum volume OD 600 The concentration of the substrate (S)-caryophyllene was 100 μM. After 10 days of fermentation, the production of chelerythrine was determined. The results are shown in Figure 4 B.

[0415] like Figure 4 As shown in B, the production of chelerythrine was higher when YPD medium was used than when SC medium was used. Therefore, YPD medium was a better medium.

[0416] Example 13: Experiment on production of chelerythrine by batch fermentation

[0417] The optimal recombinant strain Z6 was used to produce chelerythrine. In a 1-liter fermenter, the initial inoculation concentration was OD 600 The concentration of the feed substrate was 100 μM and the fermentation lasted for 12 days. Samples were taken every two days to measure the bacterial concentration and the production of chelerythrine.

[0418] Results Figure 5 The highest yield of chelerythrine reached 4.5 mg / L.

[0419] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0420] Sequence Listing

[0421] Berberine bridge enzyme (tMcoBBE) amino acid sequence (Organism: Macleaya cordata SEQ ID NO: 1)

[0422] MDDLLSCLTSHGVHNYTTPSSDSNSDYLRFLHVSIQNPLFENPATPKPAAIVMPRTKEELASTVRCCTRGSWTVRLRSGGHSYEGLSYTADTPFVLIDLMNMNRISIDVESETAWVESGATLGELYYA ITESTDSFGFTAGWCPTVGSGGHISGGGFGMMSRKYGLAADNVVDAILIDGNGVILDRNSMGEDVFWAIRGGGGGVWGAIYAWKIKLLPVPKKVTVFRLMKHVKIEEASNLLHKWQFVADELDDDFTL SVLGGADENEVWLMFLGLYLGPKTVAKSTIDPKFPELGLIEEEFSEMSWGESFAYLAGLKTVGELNNRFLKFDDRAFKTKVDFAKEPIPLKVINGALEILSKEPRGFVALNGLGGMMSKVSSDFTPFP HRSGTRLMVEYIIAWNKDEDSKSEEFTNWLQRFYDYMEPFVSKNPRVGYVNHIDLDLGGIDWKNKTSTSNAIEIARTWGEKYFLTNYERLVKAKTLIDPKNVFNHPQSIPPMEFDLEQNWGVNVEGIVQ

[0423] Berberine bridge enzyme (tMcoBBE) nucleotide sequence (Organism: Macleaya cordata SEQ ID NO: 2)

[0424]

[0425] TfSMT amino acid sequence (Organism: Thalictrum flavum SEQ ID NO: 3)

[0426] MALQEGVNYLSGLGLSRLICLPMALRAAIELNVFEIIFQAGPEAQLSPAEIVAKIPTKNPNAAIALDRILRMLGASSILSVTTMKDGRVYGLTEESRCLVADKNGVSVVPMLLFTSDKAVVESFYNIKDVVLEEGVIPFDRTHGMDFFAYAGKEQSVNKSFNQAMGAGSTIAFDEVF KVYKGFHDLKELVDVGGGIGTSLSNIISKYPHIKGINFELPHVIADAPNYPGVEHIAGNMFEGVPNAQNILLKWVLHDWDDERSIKILQNCWKALPEGGTVIVVEFVLPQILGNNAESFNALTPDLLMMTLNPGGKERTTTEFDGLAKAAGFAETKFFPISQGLHVMEFHKATAGVAS

[0427] TfSMT nucleotide sequence (Organism: Thalictrum flavum SEQ ID NO: 4)

[0428]

[0429] Amino acid sequence of tetrahydroberberine synthase (AmTDC) (Organism: Argemone mexicana SEQ ID NO: 5)

[0430] MEEKIMTNNSPWILTSSTTTTTTILLSLLFTIFIILRRNKSSSSKMVWPTGPKTLPIIGNMNILGGTALHVVLHNLAKTYGNVMTIWIGSWRPVIVVSDIDRAWEVLVNKSSDYSARDMPEITKLATADWKTISSSDSGPFWTNLRKGLQNVALSPQNLSSQSKFQERDIIKTIQNLKEEAKMNNGIVKPLDHLKKAMVRLISRLIYGQDFDNDEYVEEMHHTIEELIRVSGYARLAEAFYYAKYLPSHKKAVREVLQANQRVQNLVRPLLSLNSPTNTYLHFLRSQNYEDEVIIFAIFEAYLLGVDSTSSTTAWALAYLIREPNVQEKLYEELKNFTNDNDRKMVKFEDLNKLQYLQAVVKETMRMKPIAPLAIPHKACRETSLMGRKVNQGTRVMVNIYALHHNQNVWKEPYKFNPERFLQKNQDGVDGKAMEQSLLPFSAGMRICAGMELGKLQFSFALANLVNAFKWSCVSDGVFPDMSDQLGFVLLMKTPLEAGIVPRM

[0431] Nucleotide sequence of tetrahydroberberine synthase (AmTDC) (Organism: Argemone mexicana SEQ ID NO: 6)

[0432]

[0433] Tetrahydroberberine N-methyltransferase (EcTNMT) amino acid sequence (Organism: Eschscholzia californica SEQ ID NO: 7)

[0434] MGSSAGEIMGRLMKGEIEDEELKKLIRHQWDRRIEWGYKPTHEKQLAFNLDFIKGLKEMVMSGEIDTMNKETYELPTAFLEAVFGKTVKQSCCYFKDENSTIDEAEEAAHELYCERAQIKDGQTVLDIGCGQGGLVLYIAEKYKNCHVTGLTNSKAQANYIEQQAEKLELTNVDV IFADVTKFDTDKTYDRILVVETIEHMKNIQLFMKKLSTWMTEDSLLFVDHISHKTFNHNFEALDEDDWYSGFIFPKGCVTILSSSSTLLYFQDDVSALDHWVVNGMHMARSVEAWRKKLDETIEAAREILEPGLGSKEAVNQVITHIRTFCIGGYEQFSYNNGEEWMITQILFKKK

[0435] Tetrahydroberberine N-methyltransferase (EcTNMT) nucleotide sequence (Organism: Eschscholzia californica SEQ ID NO: 8)

[0436]

[0437] N-methylpapaverine hydroxylase (PsMSH) amino acid sequence (Organism: Papaver somniferum SEQ ID NO: 9)

[0438] MRTESIKTNRPMDLLLQYLQPISVALVVIALVWNYGRRNPTKKLAPEASGGRPIMGHLHLFNDGELTHRKLGAMADTYGPVFNIRFGSHKTLVVSDWEIVKECFTTNDKLFSNRPGTLGIKLMFYDADSVG YAPYGAYWRDLRKISTLKLLSNHRIDTIKHLRSSEVESCFESLYSQWGNGEKSGEFAPVRMDSWLGDLTFNVVARIVAGKKNFSANGDVGAQRYKAAMDEAMRLMRFFAFSDVIPSLSWLDNLRGLVREMKK CASEIDSIMATWVEEHRVKRNSGGNSQLEHDFIDVCLDIMEHSSLPGDDPDLVVKSTCLDMILGGSDTTTVTLTWAMSLLLNHPQVLQKAKEELETQVGKNRQVDDSDIPNLPFIQAIIKETMRLYPAGPL IERRTMEDCEVAGYQVPAGTRLLLVNVWKMQRDGNVYKGDPLEFRPDRFLTSNADVDLKGQHYELIPFGAGRRICPGVSFAVQLMHLVLARLLHEFEITTVEPETKVDMAESGGLLCYKIMPLEVLIKPRLEI

[0439] N-methylgoldopapaverine hydroxylase (PsMSH) nucleotide sequence (Organism: Papaver somniferum SEQID NO: 10)

[0440]

[0441] Amino acid sequence of protopine hydroxylase (EcP6H) (Organism: Eschscholzia californica SEQID NO:11)

[0442] MDSLMLAYLFPISVASIIAFVFLYNLFSSRTLKNKKIRTAPMATGAWPVLGHLHLFGSGELPHKMLAAMADKYGSAFRMKFGKHTTLVVSDTRIVKECFTTNDTLFSNRPSTKAFQLMTYDNESVAFTPYGSYWREIRKISTLKLLSNHRLQAIKDVRASEVNVCFKTLYDQCKNPSGSAPILIDMKKWFEEVSNNVVMRVIVGRQNFGSKIVQGEEEAIHYKKVMDELLRLASLSMFSDFAPLLGFVDIFQGNLSAMKRNAKKVDAILENWLEEHRKKKNSVAESQQDFMDVMLSIVEESKLSGHDADAVIKATCLAMIMGGTDTTAVSLTWIISLLMNNRHALKKAREELDALVGKDRQVEDSDLKNLVYMNAIVKETMRMYPLGTLLERETKEDCEIDGFHVKGGTRLLVNVWKLQRDPNVWVDPTEFRPERFLTENADIDVGGQHFELLPFGAGRRVCPGVXFALQFMHLVLARLIHGYDLNTLNEENVDLTESPEGHVNHKASPLDLILTPRLHYKLYE

[0443] Nucleotide sequence of protopine hydroxylase (EcP6H) (Organism: Eschscholzia californica SEQID NO:12)

[0444]

[0445] Transporter Benzylisoquinoline Alkaloid Uptake Permease (PsBUP1) Amino Acid Sequence (Organism: Papaversomniferum SEQ ID NO: 13)

[0446] MIIETLDILGPNQNGNSGTHTQKPIKTRNWLLIIINCALVFCGVIGGPLLMRLYYLHGGSRKWLSSFLQTAGFPVLIFPLIFLYIKPKLSTQNNDQSSSFFMEPKLFLWSAIVGIVFGVSNFMYALGLSYLPVSTSTILFATQLCFTAIFAWLIVKQKFTAFIINAVIVMTLGSILLGINTNGDRPIGVSKTQYLIG FLMTLAAAALTGLGTPFVELSFIKATRNITYPTLLQFQVILCLFGTCLNVIGMLINKDFQAIPREADMFELGKSKYYMIICLTALTWQLSGIGLVGLILYTNALFNGIYVSVLVPFTEVAAVIFFHEKFTGLKGMALALCLWGFSSYFYGEYKMMNKVGDNETHEKIEEAESEPKRLEDQQAPYSTVEQKLISEEDL

[0447] Transporter Benzylisoquinoline Alkaloid Uptake Permease (PsBUP1) Nucleotide Sequence (Organism: Papaversomniferum SEQ ID NO: 14)

[0448]

[0449] Transport protein MtABCG10 amino acid sequence (Organism: Medicago truncatula SEQ ID NO: 15)

[0450]

[0451] Transport protein MtABCG10 nucleotide sequence (Organism: Medicago truncatula SEQ ID NO: 16)

[0452]

[0453] Amino acid sequence of berberine bridge enzyme (McoBBE) (Organism: Macleaya cordata SEQ ID NO:17)

[0454] MDTKIRNLSSSLFIFISVLTCALGDDLLSCLTSHGVHNYTTPSSDSNSDYLRFLHVSIQNPLFENPATPKPAAIVMPRTKEELASTVRCCTRGSWTVRLRSGGHSYEGLSYTADTPFVLIDLMNMNRISIDVESETAWVESGATLGELYYAITESTDSFGFTAGWCPTVGSGGHISGGGFGMMSRKYGLAADNVVDAILIDGNGVILDRNSMGEDVFWAIRGGGGGVWGAIYAWKIKLLPVPKKVTVFRLMKHVKIEEASNLLHKWQFVADELDDDFTLSVLGGADENEVWLMFLGLYLGPKTVAKSTIDPKFPELGLIEEEFSEMSWGESFAYLAGLKTVGELNNRFLKFDDRAFKTKVDFAKEPIPLKVINGALEILSKEPRGFVALNGLGGMMSKVSSDFTPFPHRSGTRLMVEYIIAWNKDEDSKSEEFTNWLQRFYDYMEPFVSKNPRVGYVNHIDLDLGGIDWKNKTSTSNAIEIARTWGEKYFLTNYERLVKAKTLIDPKNVFNHPQSIPPMEFDLEQNWGVNVEGIVQ

[0455] Nucleotide sequence of berberine bridge enzyme (McoBBE) (Organism: Macleaya cordata SEQ ID NO:18)

[0456]

[0457] Amino acid sequence of berberine bridge enzyme (AmBBE) (Organism: Argemone mexicana SEQ ID NO:19)

[0458] MLTTVTMETKITKNLYSSLFIIIFFFFFSVLTCALSDDILSCLTSNGVHNYTTPSSDSNSDYLRLYHLSIQNPLFEKSTISKPSLIVLPRNKEELSNTVRCCTRGSWTIRLRSGGHSYEGLSYTADTPFVLIDLMNLNRISIDIDSETAWVESGATLGELYYAITELTDSLGFTAGWCPTVGSGGHISGGGFGMMSRKYGLAADNVEDVILIDSKGAILDRKLMGEDVFWAVRGGGGGVWGAIYAWKIKLLPVPKKVTVFRVTKNVNIEEASFLIHKWQYVADELDDDFTVSILGGANGNEVWVIFLGLHLGCKTVAKSIIDKKFPELGLIEEEFLEMNWGESFAYLSGLKTVKELNNRFLKFDDRAFKTKVDFTKETLPLEAIDGLLEILSKEPRGFIALNGFGGKMSKISNDFTPFPHRKGTKLMVEYIVAWSKDEESKSDEFFDWLRNIYDYMEMFVSKNPRVGYVNHIDLDLGGIDWSDKNSSNNAIEIARNWGEKYFLSNYERLIRAKTLIDPNNVFNHPQSIPPMMKYYNVDDEKNLKLHVDQGNVIF

[0459] Nucleotide sequence of berberine bridge enzyme (AmBBE) (Organism: Argemone mexicana SEQ ID NO:20)

[0460]

[0461] Amino acid sequence of berberine bridge enzyme (EcBBE) (Organism: Eschscholzia californica SEQ IDNO: 21)

[0462] MENKTPIFFSLSIFLSLLNCALGGNDLLSCLTFNGVRNHTVFSADSDSDFNRFLHLSIQNPLFQNSLISKPSAIILPGSKEELSNTIRCIRKGSWTIRLRSGGHSYEGLSYTSDTPFILIDLMNLNRVSIDLESETAWVESGSTLGELYYAITESSSKLGFTAGWCPTVGTGGHISGGGFGMMSRKYGLAADNVVDAILIDANGAILDRQAMGEDVFWAIRGGGGGVWGAIYAWKIKLLPVPEKVTVFRVTKNVAIDEATSLLHKWQFVAEELEEDFTLSVLGGADEKQVWLTMLGFHFGLKTVAKSTFDLLFPELGLVEEDYLEMSWGESFAYLAGLETVSQLNNRFLKFDERAFKTKVDLTKEPLPSKAFYGLLERLSKEPNGFIALNGFGGQMSKISSDFTPFPHRSGTRLMVEYIVAWNQSEQKKKTEFLDWLEKVYEFMKPFVSKNPRLGYVNHIDLDLGGIDWGNKTVVNNAIEISRSWGESYFLSNYERLIRAKTLIDPNNVFNHPQSIPPMANFDYLEKTLGSDGGEVVI

[0463] Nucleotide sequence of berberine bridge enzyme (EcBBE) (Organism: Eschscholzia californica SEQ IDNO: 22)

[0464]

[0465] Amino acid sequence of Berberine Bridge Enzyme (PsBBE) (Organism: Papaver somniferum SEQ ID NO:23)

[0466] MMCRSLTLRFFLFIVLLQTCVRGGDVNDNLLSSCLNSHGVHNFTTLSTDTNSDYFKLLHASMQNPLFAKPTVSKPSFIVMPGSKEELSSTVHCCTRESWTIRLRSGGHSYEGLSYTADTPFVIVDMMNLNRISIDVLSETAWVESGATLGELYYAIAQSTDTLGFTAGWCPTVGSGGHISGGGFGMMSRKYGLAADNVVDAILIDSNGAILDREKMGDDVFWAIRGGGGGVWGAIYAWKIKLLPVPEKLTVFRVTKNVGIEDASSLLHKWQYVADELDEDFTVSVLGGVNGNDAWLMFLGLHLGRKDAAKTIIDEKFPELGLVDKEFQEMSWGESMAFLSGLDTISELNNRFLKFDERAFKTKVDFTKVSVPLNVFRHALEMLSEQPGGFIALNGFGGKMSEISTDFTPFPHRKGTKLMFEYIIAWNQDEESKIGEFSEWLAKFYDYLEPFVSKEPRVGYVNHIDLDIGGIDWRNKSSTTNAVEIARNWGERYFSSNYERLVKAKTLIDPNNVFNHPQSIPPMMKFEEIYMLKEL

[0467] Nucleotide sequence of Berberine Bridge Enzyme (PsBBE) (Organism: Papaver somniferum SEQ ID NO:24)

[0468]

[0469] Amino acid sequence of Argemone mexicana methyltransferase (AmSMT) (Organism: Argemone mexicana SEQID NO: 25)

[0470] MDVNNGIVETSIYMSRVGKLGSYSAISMALRAAIELNLFEIISGFGPEAQVSATDLVSKMSNIINNPNAPIFLDRILRLLGVSSILSMSTRAISPLSSSNGKIMNKVERVYGLTEESRSLVPREEDGVSLLPTLMFTT SKLVVESFFNLKGVVLQKNYVPFDMTHGVSIFEYACNEPKLNKGFNEAMGFFSVIVFEEVFRVYNGFVGMKELLDVGGGTGTSISKIVSKYPSIHGINFDLSHVISVAPQYPGIENIAGDMFEEVPKAQNILLNWVLHD

[0471] Argemone mexicana nucleotide sequence (AmSMT)

[0472] ATGGATGTTAACAACGGTATTGTTGAAACTTCTATTTACATGTCTAGAGTTGGTAAATTGGGTTCTTATTCTGCTATTTCTATGGCTTTGAGAGCTGCTATTGAATTGAATTTGTTTGAAATTATTTCCGGTTTCGGTCCTGAAGCTCAAGTTTCCGCTACCGATTTGGTTTCTAAGATGTCTAATATTATTAACAACCCAAACGCTCCAATTTTTTTGGATAGAATTTTGAGATTGTTGGGTGTTTCTTCTATTTTATCTATGTCTACTAGAGCTATTTCCCCTTTGTCTTCTTCTAATGGTAAAATTATGAACAAGGTTGAAAGAGTTTACGGTTTGACAGAAGAATCAAGATCTTTGGTTCCAAGAGAAGAAGATGGTGTTTCATTGTTGCCTACTTTGATGTTTACTACTTCTAAATTGGTTGTTGAATCCTTTTTCAACTTGAAAGGTGTTGTTTTGCAAAAGAATTACGTTCCATTTGATATGACTCATGGTGTTTCTATTTTCGAATATGCTTGTAATGAACCAAAGTTGAATAAGGGTTTTAACGAAGCTATGGGTTTTTTTTCTGTTATTGTTTTCGAAGAAGTTTTCAGAGTTTACAATGGTTTTGTTGGTATGAAAGAATTGTTGGATGTTGGTGGTGGTACTGGTACTAGTATTTCTAAGATTGTTTCTAAGTACCCATCTATTCATGGTATTAATTTCGATTTGTCCCATGTTATTTCCGTTGCTCCACAATATCCTGGTATTGAAAATATTGCTGGTGATATGTTTGAAGAAGTTCCAAAAGCTCAAAATATTTTGTTGAACTGGGTTTTGCATGATTAA

[0473] Amino acid sequence of corydaline methyltransferase (CcSMT) (Organism: Coptis chinensis SEQID NO:27)

[0474] MAAQEGVNYLSGLGLSRLICLPMALRAAIELNVFEIISQAGQMLNYHHQISSQNPHENPSAAISLDRILRMLGASSILSVSTTKSGRVYGLNEESRCLVASEDKVSVVPMLLFTPDKAVVESFYNIKDVVLEEGVIPFDRTHGMDFFQYAGKEQRVNKSFNQAMGAGSTIAFDEVFEVYKGFDNLKELVDVGGGIGTSLSNIVAKYPHIRGINFELPHVIGDAPDYPGVEHVPGDMFEGVPNAQNILLKWVLHDWDDDRSIKILKNCWKALPENGTVIVIEFVLPQVLGNIAESFNALTPDLLMMALNPGGKERTTIEFDGLAKAAGFAETKFFPISQGLHVMEFHKINC

[0475] Nucleotide sequence of Corydalis aurea methyltransferase (CcSMT) (Organism: Coptis chinensis SEQ ID NO:28)

[0476]

[0477] PsSMT amino acid sequence (Organism: Papaver somniferum SEQ ID NO: 29)

[0478] MATNGEIFNTYGHNRQTATVTKITASNESSNGVCYLSETANLGKLICIPMALRAAMELNVFQLISKFGTDAKVSASEIASKMPNAKNNPEAAMYLDRILRLLGASSILSVSTTKKSINRGGDDVVVHEKLYGLTNSSCCLVPRQEDGVSLVEELLFTSDKVVVDSFFKLKCVVEEKDSVPFEVAHGAKIFEYAAT EPRMNQVFNDGMAVFSIVVFEAVFRFYDGFLDMKELLDVGGGIGTSVSKIVAKYPLIRGVNFDLPHVISVAPQYPGVEHVAGDMFEEVPKGQNMLLKWVLHDWGDERCVKLLKNCWNSLPVGGKVLIIEFVLPNELGNNAESFNALIPDLLLMALNPGGKERTISEYDDLGKAAGFIKTIPIPISNGLHVIEFHK

[0479] PsSMT nucleotide sequence (Organism: Papaver somniferum SEQ ID NO: 30)

[0480]

[0481] Tetrahydroberberine N-methyltransferase (CmTNMT) amino acid sequence (Organism: Chelidonium majus SEQ ID NO: 31)

[0482] MSKLVRHQWERRLQWGYKPTHEQQLACNIDFIKSLKYMDMSGEIDTMNEETHELPSAFLEATFGKTIKQSGCYFKDETTTLDEAEEASNELYCERAQIKDGQTILDIGCGQGGLVLYIAQKYKNCHVTGLTNSKAQM NYILMQAEKLQLSNVDVISADVTKFDTDKTYDRILVIEAIEHMKNIQLFLKKLSTWMKKDSLLFVDHISHKKFNHHFEALDEKDWYSGLIFPKGCVTILSSSTLLYFQDDVSVVDHWVVNGMHMARSMEEWRKKLDK

[0483] Tetrahydroberberine N-methyltransferase (CmTNMT) nucleotide sequence (Organism: Chelidonium majus SEQ ID NO: 32)

[0484] ATGTCTAAATTAGTTAGACATCAATGGGAAAGAAGATTGCAATGGGGTTATAAACCAACACATGAACAACAATTAGCTTGTAATATTGATTTTATAAAATCTTTGAAATATATGGATATGTCTGGTGAAATTGATACTATGAATGAAGAAACTCATGAATTGCCATCAGCTTTTCTAGAAGCTACTTTTGGTAAAACTATTAAGCAATCTGGTTGTTATTTTAAAGATGAAACAACAACTTTGGATGAAGCTGAAGAAGCATCTAATGAATTATATTGTGAAAGAGCACAAATTAAGGATGGTCAAACTATTTTAGATATAGGTTGTGGTCAAGGTGGTTTAGTGTTATATATTGCTCAAAAATATAAAAATTGTCATGTTACTGGTTTGACTAATAGTAAAGCTCAAATGAATTATATTTTGATGCAAGCTGAAAAATTGCAATTATCTAATGTTGATGTTATTTCTGCTGATGTTACTAAATTTGATACTGATAAAACTTATGATAGAATTTTGGTTATTGAAGCTATTGAACATATGAAAAATATTCAATTATTTTTGAAAAAATTAAGTACATGGATGAAAAAAGATTCTTTGTTATTTGTTGATCATATTTCTCATAAAAAGTTTAATCACCATTTTGAAGCATTAGATGAAAAAGATTGGTATTCTGGATTGATTTTTCCAAAAGGTTGTGTTACTATTTTGTCATCTTCTACTCTATTATATTTTCAAGATGATGTTTCTGTTGTTGATCATTGGGTTGTTAATGGTATGCATATGGCCAGATCTATGGAAGAATGGAGAAAAAAGTTAGATAAGTAA

[0485] Tetrahydroprotoberberine N-methyltransferase (GfTNMT) amino acid sequence (Organism: Glaucium flavum SEQ ID NO:33)

[0486] MGSSHHHHHHGTGSYITSLYKKAGWMGSNEAQVKKESIGEIMGKLMQGEIGDEELSKRIKEIFGKRLQWGYKPTHQQQLAFNLDFIKSLKEMDMSGEIDTMNEETYELPSAFLEAAFGKTIKQSGCYFKDETTTIDEAEEASHELYCERAQIKDGQTVLDIGCGQGGLVLHIAQKYKNCHVTGLTNSKAQKNYILMQAEKLQLSNVDVILADVTKHESDKTYDRILVIETIEHMKNIQLFMKKLSTWMTEDSLLFVDHICHKTFSHHFEAIDEDDWYSGFIFPKGCVTILSASALLYFQDDVTILDHWVVNGMHMARSVDAWRKKLDKNMELAREILLPGLGSKEAVNGVITHIRTFCMGGYEQFSYNNGEEWMVAQMLFKKK

[0487] Tetrahydroprotoberberine N-methyltransferase (GfTNMT) nucleotide sequence (Organism: Glaucium flavum SEQ ID NO: 34)

[0488]

[0489] Tetrahydroberberine N-methyltransferase (PsTNMT) amino acid sequence (Organism: Papaversomniferum SEQ ID NO: 35)

[0490] MGSIDEVKKESAGETLGRLLKGEIKDEELKKLIKFQFEKRLQWGYKSSHQEQLSFNLDFIKSLKKMEMSGEIETMNKETYELPSEFLEAVFGKTVKQSMCYFTHESATIDEAEAAHELYCERAQIKDGQTVLDIGCGQGGLVLYIAQKYKNCHVTGLTNSKAQVNYLLKQAEKLGLTN VDAILADVTQYESDKTYDRLLMIEAIEHMKNLQLFMKKLSTWMTKESLLFVDHVCHKTFAHFFEAVDEDDWYSGFIFPPGCATILAANSLLYFQDDVSVVDHWVVNGMHMARSVDIWRKALDKNMEAAKEILLPGLGGSHETVNGVVTHIRTFCMGGYEQFSMNNGDEWMVAQLLFKKK

[0491] Tetrahydroberberine N-methyltransferase (PsTNMT) nucleotide sequence (Organism: Papaversomniferum SEQ ID NO: 36)

[0492]

[0493] Amino acid sequence of protopine hydroxylase (PsP6H) (Organism: Papaver somniferum SEQ ID NO: 37)

[0494] MDFSSLLLLLLNTWISAYSMAALLALVLVYNLRMTKSSSSKTTSLKGKKIITRPPAVTGAWPVFGHLHLFGSGEHPHEMLSKLAEKYGPSFTMKFGKHTTLVVSDTRVVKECFTTNDTLFSNRPSTIAFDLMTYATDSIAFTPYSPYWRELRKISTLKLLSNNRLESIKQLRTSEVSVCFKELYDLTNKKNDNGAPVPIDLKRWFDEVSNNVIMRVIFGKQNFGSKIVLGEDQEAVHYKKIMDELSRLSSLTMLSDMVPLLGWLDYFKGDLRAMKRNGKELNSILQKWLEEHKSKKSSDARQDFMDVMLSISKDTQLYGHDQDTFIKATCLAMIMGGTNSTEVALTWILSLLMNNRCALHKAREEIDLLVGKDRQVEDSDVKNLTYMNAIIKETMRLYPLGFLLERDTKEDCEVSGFNIKGGTRLLINVWKLQRDPNVWTDPMEFKPERFLTENADIDVGGQHFELLPFGAGRRVCPGVSFALQFMHLVLARLIHGYDMETLNGEDVDLSVSSGGHVNIKSTPLELILTPRLHPELYDCET

[0495] Nucleotide sequence of protopine hydroxylase (PsP6H) (Organism: Papaver somniferum SEQ ID NO: 38)

[0496]

[0497] Amino acid sequence of protopine hydroxylase (McoP6H) (Organism: Macleaya cordata SEQ ID NO:39)

[0498] MAALLALVFLYNFIIIWSSSPRTTINGKKQIRKAPMAAGAWPILGHLHLFGSGELPHKMLAAMAEKYGSAFMMKFGKHTTLVVSDTRIVKECFTTNDTLFANRPSTTAFDLMTYANDSVAFTPYGPYWRELRKISTLKLLSNHRLQAIKDVRASEVNVCFRELYNLCNKQNKNDGADHVLVDMKKWFEEVSNNVVMRVIVGRQNFGSKIVRGEEEAVNYKKVMDELLRLASLSMLSDFAPLLGWLDIFQGNMSAMKRNAKKVDTILEGWLEEHRNKKKKSSSSSSSSSSSSSSSSSGENDQDFMDVMLSIIEETKLSGRDADTVIKATCLAMIMGGTDTTAVSLTWIVSLLMNNRHVLKKAREELDALVGKDRQVEDSDLKNLVYMNAIVKETMRLFPLGALLERETKEDCEVGGFQLQGGSRLLVNVWKLQRDPNVWSDPTEFRPERFLSENADIDVGGQHFELLPFGAGRRVCPGVSFALQFMHLVLARLIHGYELGTQNDEDVDLTESTEGHVNHKASPLDLILTPRLHPKLYEYEQKLISEEDL

[0499] Nucleotide sequence of protopine hydroxylase (McoP6H) (Organism: Macleaya cordata SEQ ID NO:40)

[0500]

[0501] Amino acid sequence of protopine hydroxylase (t4EcP6H) (Organism: Eschscholzia californica SEQ ID NO: 41)

[0502] MMLAYLFPISVASIIAFVFLYNLFSSRTLKNKKIRTAPMATGAWPVLGHLHLFGSGELPHKMLAAMADKYGSAFRMKFGKHTTLVVSDTRIVKECFTTNDTLFSNRPSTKAFQLMTYDNESVAFTPYGSYWREIRKISTLKLLSNHRLQAIKDVRASEVNVCFKTLYDQCKNPSGSAPILIDMKKWFEEVSNNVVMRVIVGRQNFGSKIVQGEEEAIHYKKVMDELLRLASLSMFSDFAPLLGFVDIFQGNLSAMKRNAKKVDAILENWLEEHRKKKNSVAESQQDFMDVMLSIVEESKLSGHDADAVIKATCLAMIMGGTDTTAVSLTWIISLLMNNRHALKKAREELDALVGKDRQVEDSDLKNLVYMNAIVKETMRMYPLGTLLERETKEDCEIDGFHVKGGTRLLVNVWKLQRDPNVWVDPTEFRPERFLTENADIDVGGQHFELLPFGAGRRVCPGVXFALQFMHLVLARLIHGYDLNTLNEENVDLTESPEGHVNHKASPLDLILTPRLHYKLYE

[0503] Nucleotide sequence of protopine hydroxylase (t4EcP6H) (Organism: Eschscholzia californica SEQ ID NO: 42)

[0504]

[0505] Amino acid sequence of protopine hydroxylase (t27EcP6H) (Organism: Eschscholzia californica SEQ ID NO: 43)

[0506] MSSRTLKNKKIRTAPMATGAWPVLGHLHLFGSGELPHKMLAAMADKYGSAFRMKFGKHTTLVVSDTRIVKECFTTNDTLFSNRPSTKAFQLMTYDNESVAFTPYGSYWREIRKISTLKLLSNHRLQAIKDVRASEVNVCFKTLYDQCKNPSGSAPILIDMKKWFEEVSNNVVMRVIVGRQNFGSKIVQGEEEAIHYKKVMDELLRLASLSMFSDFAPLLGFVDIFQGNLSAMKRNAKKVDAILENWLEEHRKKKNSVAESQQDFMDVMLSIVEESKLSGHDADAVIKATCLAMIMGGTDTTAVSLTWIISLLMNNRHALKKAREELDALVGKDRQVEDSDLKNLVYMNAIVKETMRMYPLGTLLERETKEDCEIDGFHVKGGTRLLVNVWKLQRDPNVWVDPTEFRPERFLTENADIDVGGQHFELLPFGAGRRVCPGVXFALQFMHLVLARLIHGYDLNTLNEENVDLTESPEGHVNHKASPLDLILTPRLHYKLYE

[0507] Nucleotide sequence of protopine hydroxylase (t27EcP6H) (Organism: Eschscholzia californica SEQ ID NO: 44)

[0508]

[0509] Cytochrome P450 Reductase (PsCPR) Amino Acid Sequence (Organism: Papaver somniferum SEQ ID NO: 45)

[0510] MGSNNLANSIESMLGISIGSEYISDPIFIMVTTVASMLIGFGFFACMKSSSSQSKPIETYKPIIDKEEEEIEVDPGKIKLTIFFGTQTGTAEGFAKALAEEIKAKYKKAVVKVVDLDDYAAEDDQYEEKLKKESLVFFMVATYGDGEPTDNAARFYKWFTQEHERGEWLQQLTYGVFGLGNRQYEHFNKIAVDVDEQLGKQGAKRIVQVGLGDDDQCIEDDFTAWRELLWTELDQLLKDEDAAPSVATPYIATVPEYRVVIHETTVAALDDKHINTANGDVAFDILHPCRTIVAQQRELHKPKSDRSCIHLEFDISGSSLTYETGDHVGVYAENCDETVEEAGKLLGQPLDLLFSIHTDKEDGSPQGSSLPPPFPGPCTLRSALARYADLLNPPRKASLIALSAHASVPSEAERLRFLSSPLGKNEYSKWVVGSQRSLLEIMAEFPSAKPPLGVFFAAVAPRLPPRYYSISSSPKFAPSRIHVTCALVYGQSPTGRVHRGVCSTWMKHAVPQDSWAPIFVRTSNFKLPADPSTPIIMVGPGTGLAPFRGFLQERMALKENGAQLGPAVLFFGCRNRNMDFIYEDELNNFVERGVISELVIAFSREGEKKEYVQHKMMEKATDVWNVISGDGYLYVCGDAKGMARDVHRTLHTIAQEQGPMESSAAEAAVKKLQVEERYLRDVW

[0511] Cytochrome P450 Reductase (PsCPR) Nucleotide Sequence (Organism: Papaver somniferum SEQ ID NO: 46)

[0512]

[0513] Cytochrome P450 Reductase (AtATR1) Amino Acid Sequence (Organism: Arabidopsis thaliana SEQ ID NO: 47)

[0514] MTSALYASDLFKQLKSIMGTDSLSDDVVLVIATTSLALVAGFVVLLWKKTTADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSIFFGTQTGTAEGFAKALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYGDGEPTDNAARFYKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKSVATPYTAVIPEYRVVTHDPRFTTQKSMESNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAPSRVHVTSALVYGPTPTGRIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVMVGPGTGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMDFIYEDELNNFVDQGVISELIMAFSREGAQKEYVQHKMMEKAAQVWDLIKEEGYLYVCGDAKGMARDVHRTLHTIVQEQEGVSSSEAEAIVKKLQTEGRYLRDVW

[0515] Cytochrome P450 Reductase (AtATR1) Nucleotide Sequence (Organism: Arabidopsis thaliana SEQ ID NO: 48)

[0516]

[0517] Phospholipid biosynthesis transcription factor (ScINO2) amino acid sequence (Organism: Saccharomyces cerevisiae SEQ ID NO: 49)

[0518] MQQATGNELLGILDLDNDIDFETAYQMLSSNFDDQMSAHIHENTFSATSPPLLTHELGIIPNVATVQPSHVETIPADNQTHHAPLHTHAHYLNHNPHQPSMGFDQALGLKLSPSSSGLLSTNESNAIEQFLDNLISQDMMSSNASMNSESHL HIRSPKKQHRYTELNQRYPETHPHSNTGELPTNTADVPTEFTTREGPHQPIGNDHYNPPPFSVPEIRIPDSDIPANIEDDPVKVRKWKHVQMEKIRRINTKEAFERLIKSVRTPPKENGKRIPKHILLTCVMNDIKSIRSANEALQHILDDS

[0519] Phospholipid biosynthesis transcription factor (ScINO2) nucleotide sequence (Organism: Saccharomyces cerevisiae SEQ ID NO: 50)

[0520] ATGCAACAAGCAACTGGGAACGAATTACTGGGTATCCTAGATCTGGATAACGATATAGACTTTGAAACTGCTTACCAAATGCTCAGCAGTAACTTCGACGACCAAATGTCTGCGCACATACATGAAAACACGTTTAGTGCAACTTCCCCTCCTCTGTTAACACACGAGCTCGGCATAATTCCTAACGTAGCAACCGTGCAACCCTCTCACGTAGAAACTATACCTGCCGATAACCAAACTCATCATGCTCCTTTGCATACTCATGCACACTATCTAAATCACAACCCTCATCAACCAAGCATGGGTTTTGATCAAGCGCTTGGTCTCAAGTTGTCTCCTTCCAGTTCGGGGTTGTTGAGCACGAATGAATCGAATGCCATTGAACAGTTTTTAGACAATCTAATATCACAGGATATGATGTCTTCCAACGCTTCCATGAACTCCGAATCACATCTACATATAAGATCACCAAAAAAGCAGCATAGGTATACCGAATTAAATCAAAGATATCCTGAAACACATCCACACAGTAACACAGGGGAGTTACCCACAAACACAGCAGATGTGCCAACTGAGTTCACCACGAGGGAAGGACCTCATCAGCCTATCGGCAATGACCACTACAACCCGCCACCGTTTTCAGTACCTGAGATACGAATCCCAGACTCTGATATTCCAGCCAATATCGAGGACGACCCTGTGAAGGTACGGAAATGGAAACACGTTCAAATGGAGAAGATACGAAGAATAAACACCAAAGAAGCCTTTGAAAGGCTCATTAAATCAGTAAGGACCCCACCGAAGGAAAACGGGAAAAGAATTCCCAAGCATATTCTTTTAACTTGTGTAATGAACGATATCAAGTCCATTAGAAGCGCAAATGAAGCACTACAGCACATACTGGATGATTCCTGA

[0521] Dihydrofolate reductase (ScDHFR) amino acid sequence (Organism: Saccharomyces cerevisiae SEQ ID NO: 51)

[0522] MAGGKIPIVGIVACLQPEMGIGFRGGLPWRLPSEMKYFRQVTSLTKDPNKKNALIMGRKTWESIPPKFRPLPNRMNVIISRSFKDDFVHDKERSIVQSNSLANAI MNLESNFKEHLERIYVIGGGEVYSQIFSITDHWLITKINPLDKNATPAMDTFLDAKKLEEVFSEQDPAQLKEFLPPKVELPETDCDQRYSLEEKGYCFEFTLYNRK

[0523] Dihydrofolate reductase (ScDHFR) nucleotide sequence (Organism: Saccharomyces cerevisiae SEQ ID NO: 52)

[0524] ATGGCTGGAGGAAAGATTCCTATTGTAGGAATTGTGGCATGTTTACAGCCGGAGATGGGGATAGGATTTCGTGGAGGTCTACCATGGAGGTTGCCCAGTGAAATGAAGTATTTCAGACAGGTCACTTCATTGACGAAAGATCCAAACAAAAAAAATGCTTTGATAATGGGAAGGAAGACATGGGAATCCATACCGCCCAAGTTTCGCCCACTGCCCAATAGAATGAATGTCATTATATCGAGAAGCTTCAAGGACGATTTTGTCCACGATAAAGAGAGATCAATAGTCCAAAGTAATTCATTGGCAAACGCAATAATGAACCTAGAAAGCAATTTTAAGGAGCATCTGGAAAGAATCTACGTGATTGGGGGTGGCGAAGTTTATAGTCAAATCTTCTCCATTACAGATCATTGGCTCATCACGAAAATAAATCCATTAGATAAAAACGCAACTCCTGCAATGGACACTTTCCTTGATGCGAAGAAATTGGAAGAAGTATTTAGCGAGCAAGATCCGGCCCAGCTGAAAGAATTTCTTCCCCCTAAAGTAGAGTTGCCCGAAACAGACTGTGATCAACGCTACTCGCTGGAAGAAAAAGGTTATTGCTTCGAATTCACTCTATACAATCGTAAATGA

Claims

1. An engineered bacterium for producing chelerythrine, characterized in that: The engineered bacteria is a yeast, and the engineered bacteria contains exogenous and / or endogenous gene modules, and the gene modules include: (a) a berberine bridge enzyme (BBE) gene module, wherein the BBE gene module contains a single copy or multiple copies of a BBE gene; (b) a S. chrysanthemi methyltransferase (SMT) gene module, wherein the SMT gene module contains a single copy or multiple copies of the SMT gene; (c) a tetrahydroberberine synthase (TDC) gene module, wherein the TDC gene module contains a single copy or multiple copies of a TDC gene; (d) a tetrahydroberberine N-methyltransferase (TNMT) gene module, wherein the TNMT gene module contains a single copy or multiple copies of the TNMT gene; (e) an N-methylaurocanine hydroxylase (MSH) gene module, wherein the MSH gene module contains a single copy or multiple copies of the MSH gene; (f) a proopioid hydroxylase (P6H) gene module, wherein the P6H gene module contains a single copy or multiple copies of a P6H gene; (g) a cytochrome P450 oxidoreductase (CPR) gene module, wherein the CPR gene module contains a single copy or multiple copies of a CPR gene; and (h) a transporter benzylisoquinoline alkaloid uptake permease (BUP1) gene module, wherein the BUP1 gene module contains a single copy or multiple copies of a BUP1 gene.

2. The engineered bacteria according to claim 1, characterized in that The gene module also includes: (i) a rate-limiting enzyme gene module in the heme pathway, wherein the rate-limiting enzyme gene module contains a single copy or multiple copies of a rate-limiting enzyme gene (HEM2, HEM3 and / or HEM12); (j) an oxidation-related gene module in the pentose phosphate pathway, wherein the oxidation-related gene module contains a single copy or multiple copies of an oxidation-related gene (GND1 and / or ZWF1); (k) a non-oxidation-related gene module in the pentose phosphate pathway, wherein the non-oxidation-related gene module contains a single copy or multiple copies of non-oxidation-related genes (TAL1, TKL1, TKL2, XFPK and / or PTA); (l) A product transporter gene module, wherein the product transporter gene module contains a single copy or multiple copies of a product transporter gene (ABCG10 gene). (m) a cytochrome P450 oxidoreductase (ATR1) gene module, wherein the ATR1 gene module contains a single copy or multiple copies of the ATR1 gene; (n) a phospholipid biosynthesis transcription factor (INO2) gene module, wherein the INO2 gene module contains a single copy or multiple copies of an INO2 gene; and / or (o) a dihydrofolate reductase (DHFR) gene module, wherein the DHFR gene module contains a single copy or multiple copies of a DHFR gene.

3. The engineered bacteria according to claim 1, characterized in that The starting strain of the engineered bacteria contains or does not contain endogenous dihydrofolate reductase (DHFR).

4. The engineered bacteria according to claim 1, characterized in that The chelerythrine production of the engineered bacteria is ≥1 mg / L, preferably ≥2 mg / L, more preferably ≥3 mg / L, and most preferably ≥4.5 mg / L.

5. A method for producing chelerythrine, characterized in that: The method comprises the steps of: (1) culturing the engineered bacteria according to any one of claims 1 to 4 under conditions suitable for expression, and adding a substrate (S)-cheerythrine to obtain a culture product or fermentation product containing chelerythrine; and (2) optionally isolating and / or purifying chelerythrine from the culture product or fermentation product.

6. A method for constructing the engineered bacteria according to any one of claims 1 to 4, characterized in that: The method comprises the steps of: (a) constructing a BBE gene module, an SMT gene module, a TDC gene module, a TNMT gene module, an MSH gene module, a P6H gene module, a CPR gene module and a BUP1 gene module, or a vector containing the above gene modules; (b) using the gene module or vector obtained in step (a) to transform a yeast, thereby obtaining the engineered bacteria according to any one of claims 1 to 4.

7. A use of the engineered bacteria according to any one of claims 1 to 4, characterized in that: The engineered bacteria is used for producing chelerythrine.

8. A kit, characterized in that: The kit comprises: (Z1) a first container and a berberine bridge enzyme (BBE) gene module in the first container, wherein the BBE gene module contains a single copy or multiple copies of a BBE gene; (Z2) a second container and a S. cerasiferum methyltransferase (SMT) gene module in the second container, wherein the SMT gene module contains a single copy or multiple copies of the SMT gene; (Z3) a third container and a tetrahydroberberine synthase (TDC) gene module located in the third container, wherein the TDC gene module contains a single copy or multiple copies of a TDC gene; (Z4) a fourth container and a tetrahydroberberine N-methyltransferase (TNMT) gene module located in the fourth container, wherein the TNMT gene module contains a single copy or multiple copies of the TNMT gene; (Z5) a fifth container and an N-methylpapaverine hydroxylase (MSH) gene module located in the fifth container, wherein the MSH gene module contains a single copy or multiple copies of the MSH gene; (Z6) a sixth container and a proopioid hydroxylase (P6H) gene module located in the sixth container, wherein the P6H gene module contains a single copy or multiple copies of a P6H gene; (Z7) a seventh container and a cytochrome P450 oxidoreductase (CPR) gene module located in the seventh container, wherein the CPR gene module contains a single copy or multiple copies of a CPR gene; and (Z8) An eighth container and a transporter benzylisoquinoline alkaloid uptake permease (BUP1) gene module located in the eighth container, wherein the BUP1 gene module contains a single copy or multiple copies of a BUP1 gene.

9. A use of the kit as claimed in claim 8, characterized in that: The kit is used to construct the engineering bacteria according to any one of claims 1 to 4.

10. A fermentation system for producing chelerythrine, characterized in that: The fermentation system comprises: (S1) The engineered bacteria according to any one of claims 1 to 4; and (S2) Substrate (S)-caryophylline.

Images