Recombinant bacterium and application thereof in production of bisbenzylisoquinoline alkaloid

Abstract

The invention discloses a recombinant bacterium and an application of the recombinant bacterium in production of bisbenzylisoquinoline alkaloid. According to the recombinant bacterium constructed by the invention, the synthetic route of the bisbenzylisoquinoline alkaloid is divided into four modules for research, and the de novo synthesis of the bisbenzylisoquinoline alkaloid is realized through multiple strategies; wherein the original strain is an IMX581 yeast strain. Experiments prove that the engineering strain constructed by the invention can be used for efficiently producing the bisbenzylisoquinoline alkaloids, such as (R, S)-actinodaphne, Bisnoraromoline, Bisnoramegine, isotetrandrine, Obaberine and the like, and can be used for efficiently producing the bisbenzylisoquinoline alkaloids, such as the (R, S)-actinodaphne, the Bisnoramegine, the isotetrandrine and the like. The method has an important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a recombinant bacterium and application thereof in production of bisbenzylisoquinoline alkaloids. BACKGROUND

[0002] Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites with important research and medicinal value, including antitussive and anticancer noscapine, analgesic morphine and codeine, etc. At present, BIA compounds are mainly obtained from plant extraction or chemical synthesis, but due to the low content of plant secondary metabolites and multiple stereoisomers of compounds, extraction and purification and chemical synthesis have great limitations, and biosynthesis in a microbial heterologous host has great potential to overcome these production obstacles. Production of BIA compounds in Saccharomyces cerevisiae as a host has attracted widespread attention, however, it is still a great challenge to recombine a long heterologous pathway in yeast, and multiple optimizations including identification of missing key enzymes, repositioning or cofactor engineering to improve the efficiency of rate-limiting enzymes are needed to improve the yield.

[0003] Isotetrandrine is a compound isolated from Stephania sinica Diels with antiviral and anti-inflammatory activities, and construction of an isotetrandrine-producing engineering strain has important application value. SUMMARY

[0004] The present application aims to provide a recombinant bacterium for improving the yield of bisbenzylisoquinoline alkaloids (such as (R, S)-obaberine, Bisnoraromoline, Bisnorobamegine, Isotetrandrine or Obaberine) or producing bisbenzylisoquinoline alkaloids (such as (R, S)-obaberine, Bisnoraromoline, Bisnorobamegine, Isotetrandrine or Obaberine).

[0005] The present application first protects a recombinant bacterium. The recombinant bacterium can contain or express tyrosine hydroxylase and / or dopa decarboxylase in vivo.

[0006] In the above-mentioned recombinant bacterium, the tyrosine hydroxylase and dopa decarboxylase contained or expressed in vivo can be introduced into a starting bacterium by introducing a nucleic acid encoding tyrosine hydroxylase and a nucleic acid encoding dopa decarboxylase into the starting bacterium.

[0007] In the above-mentioned recombinant bacterium, the introduction of the nucleic acid encoding tyrosine hydroxylase into the starting bacterium can be achieved by introducing an expression cassette containing the nucleic acid encoding tyrosine hydroxylase into the starting bacterium. The expression cassette containing the nucleic acid encoding tyrosine hydroxylase can include a promoter, the nucleic acid encoding tyrosine hydroxylase and a terminator.

[0008] In the aforementioned recombinant bacteria, the introduction of the dopa decarboxylase-encoding nucleic acid into the starting bacteria can be described as introducing an expression cassette containing the dopa decarboxylase-encoding nucleic acid into the starting bacteria. The expression cassette containing the dopa decarboxylase-encoding nucleic acid may include a promoter, a dopa decarboxylase-encoding nucleic acid, and a terminator.

[0009] In the aforementioned recombinant bacteria, the promoter may be TDH3p, CCW12p, or TEF1p. The terminator may be IDP1t, TPS1t, or ENO2t.

[0010] In the recombinant bacteria mentioned above, the expression cassette is introduced into the originating bacteria in the form of a plasmid and / or integrated into the chromosome.

[0011] Specifically, the recombinant bacteria can be a strain obtained by integrating an expression cassette containing tyrosine hydroxylase encoding nucleic acid and an expression cassette containing dopa decarboxylase encoding nucleic acid into the XI-5 site of the originating bacteria using a genome editing method.

[0012] In the above-mentioned recombinant bacteria, the tyrosine hydroxylase may be BvCYP76AD5 (genbank number AJD87473.1), BvCYP76AD1* (genbank number AET43289.1), AtC3H (genbank number NP_001030991.2), ZmC3H (genbank number ACG46606.1), PaHpaB (genbank number WP_003104533.1), PaHpaC (genbank number WP_058166060.1), or SeHpaC (genbank number WP_001195556.1), preferably BvCYP76AD5.

[0013] In the above recombinant bacteria, the dopa decarboxylase may specifically be DODC (genbank number DAA64376.1).

[0014] The recombinant bacteria described above may also contain or express norcodonine synthase (NCS or NCS enzyme) or N-terminally truncated norcodonine synthase.

[0015] In the above recombinant bacteria, the NCS enzyme can be CjNCS (genbank number BAF45338.2) derived from Coptis japonica.

[0016] In the above recombinant bacteria, the N-terminally truncated NCS enzyme can specifically be CjNCSN_Δ35, CjNCSN_Δ24, or CjNCSN_Δ29, that is, the N-terminus of the amino acid sequence of CjNCS is truncated by 35, 24, or 29 amino acids, respectively.

[0017] In the above-mentioned recombinant bacteria, the presence or expression of norcodonine synthase or N-terminal truncated NCS enzyme can be achieved by introducing the coding nucleic acid of NCS enzyme or the coding nucleic acid of N-terminal truncated NCS enzyme into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0018] In the aforementioned recombinant bacteria, the introduction of the coding nucleic acid of the NCS enzyme or the N-terminally truncated coding nucleic acid of the NCS enzyme into the starting bacteria or any of the aforementioned recombinant bacteria can be achieved by introducing an expression cassette containing the coding nucleic acid of the NCS enzyme or the N-terminally truncated coding nucleic acid of the NCS enzyme into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette containing the coding nucleic acid of the NCS enzyme or the N-terminally truncated coding nucleic acid of the NCS enzyme may include a promoter, the coding nucleic acid of the NCS enzyme or the N-terminally truncated coding nucleic acid of the NCS enzyme, and a terminator.

[0019] In the above recombinant bacteria, the promoter may be TEF1p. The terminator may be TDH2t.

[0020] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the originating bacteria or any of the above-described recombinant bacteria in the form of a plasmid and / or integrated into the chromosome.

[0021] In the recombinant bacteria described above, the presence or expression of NCS enzyme or N-terminal truncated NCS enzyme in the organism can increase the copy number of the encoding nucleic acid of NCS enzyme or N-terminal truncated NCS enzyme in the originating bacteria or any of the recombinant bacteria described above.

[0022] In the above recombinant bacteria, the copy number of the nucleic acid encoding the N-terminally truncated NCS enzyme can be 3, 2 or 1, preferably 3.

[0023] The recombinant bacteria described above may also contain or express tyrosine metabolic flux-related proteins.

[0024] In the aforementioned recombinant bacteria, the tyrosine metabolic flux-related protein may be EcAROL (genbank number NP_414922.1) or ARO4* (a mutant of the yeast endogenous gene ARO4) from Escherichia coli. K229L ARO7* (a mutant of the yeast endogenous gene ARO7) G141S MtPDH1 (genbank number KM507076.1), ARO1 (yeast endogenous gene), ARO2 (yeast endogenous gene), and / or ARO3 (yeast endogenous gene) derived from alfalfa.

[0025] In the above-mentioned recombinant bacteria, the body may also contain or express tyrosine metabolic flux-associated proteins, which can be achieved by introducing the encoding nucleic acid of tyrosine metabolic flux-associated proteins into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0026] In the aforementioned recombinant bacteria, the introduction of the nucleic acid encoding the tyrosine metabolic flux-associated protein into the starting bacteria or any of the aforementioned recombinant bacteria may be described as introducing an expression cassette encoding the nucleic acid of the tyrosine metabolic flux-associated protein into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the nucleic acid encoding the tyrosine metabolic flux-associated protein, and a terminator.

[0027] In the aforementioned recombinant bacteria, the promoter may be TPI1p, PGK1p, TEF1p, TDH3p, or tHXT7p. The terminator may be pYX212t, ADH1t, FBA1t, CYC1t, or TDH2t.

[0028] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria in the form of plasmids and / or integrated into the chromosome.

[0029] In vivo, proteins associated with the synthesis of 4-hydroxyphenylethanol and / or 4-hydroxyphenylacetic acid can also be knocked out or downregulated in any of the recombinant bacteria described above.

[0030] In the above recombinant bacteria, the proteins associated with the synthetic activity of 4-hydroxyphenylethanol and / or 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, YDR541C, AAD3, GRE2 and / or HFD1.

[0031] In the above recombinant bacteria, the proteins associated with the synthetic activity of 4-hydroxyphenylethanol and / or 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, GRE2, and HFD1.

[0032] The recombinant bacteria described above may also contain or express aromatic amino acid decarboxylases.

[0033] In the above recombinant bacteria, the aromatic amino acid decarboxylase may be PcAAS from Petroselinum crispum, RrAAS from Rhodiola Salidroside, or the mutant PsAAAD* from P. somniferum, preferably PsAAAD*.

[0034] In the above-mentioned recombinant bacteria, the presence or expression of aromatic amino acid decarboxylase in the body can be achieved by introducing the encoding nucleic acid of aromatic amino acid decarboxylase into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0035] In the recombinant bacteria described above, the introduction of the nucleic acid encoding the aromatic amino acid decarboxylase into the starting bacteria or any of the aforementioned recombinant bacteria can be achieved by introducing an expression cassette containing the nucleic acid encoding the aromatic amino acid decarboxylase into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the nucleic acid encoding the aromatic amino acid decarboxylase, and a terminator.

[0036] The promoter may be FBA1p. The terminator may be TPS1t-ADH1t.

[0037] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria in the form of plasmids and / or integrated into the chromosome.

[0038] The recombinant bacteria described above may also contain or express prephenate transaminase (PAT) and arogenate dehydratase (ADH). The PAT may be AtPAT (NP_565529.1), BvPAT1 (KMS97510.1), or BvPAT2 (KMT14107.1). The ADH may be BvADHα or MtncADH.

[0039] In the above-mentioned recombinant bacteria, the presence or expression of PAT and ADH in vivo can be achieved by introducing the coding nucleic acid of PAT and the coding nucleic acid of ADH into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0040] In the aforementioned recombinant bacteria, the introduction of the PAT-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria may be described as introducing an expression cassette containing the PAT-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the PAT-coding nucleic acid, and a terminator.

[0041] In the aforementioned recombinant bacteria, the introduction of ADH-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can be described as introducing an expression cassette containing ADH-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, ADH-coding nucleic acid, and a terminator. The promoter may be TDH3p, TEF2p, or FBA1p. The terminator may be TDH2t or pYX212t.

[0042] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria in the form of plasmids and / or integrated into the chromosome.

[0043] Preferably, any of the recombinant bacteria described above may also contain or express AtPAT and MtncADH.

[0044] The recombinant bacteria also contain or express methyltransferase 6OMT, isomerase, P450 reduction chaperone, and / or dimerase.

[0045] In the above recombinant bacteria, the methyltransferase 6OMT can be Ps6OMT (AAQ01669.1) derived from P. somniferum.

[0046] In the above recombinant bacteria, the isomerase may be PrDCS from Papaver rhoeas and / or PbDCR from Papaverbracteatum.

[0047] In the above-mentioned recombinant bacteria, the P450 reduction chaperone may be the P450 reduction chaperone NCP1.

[0048] In the above recombinant bacteria, the dimerase may be CYP80Q4 derived from Stephania tetrandra.

[0049] In the above-mentioned recombinant bacteria, the presence or expression of isomerase and / or dimerase can increase the copy number of PrDCS, PbDCR and CYP80Q4 or the copy number of the encoding nucleic acids of PrDCS, PbDCR and CYP80Q4 in the originating bacteria or any of the above-mentioned recombinant bacteria.

[0050] In the aforementioned recombinant bacteria, the copy number of the encoded nucleic acid of PrDCS, PbDCR, and CYP80Q4 can be more than one, specifically up to three.

[0051] In the above-mentioned recombinant bacteria, the presence or expression of methyltransferase 6OMT in the body can be achieved by introducing the coding nucleic acid of Ps6OMT into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0052] In the aforementioned recombinant bacteria, the introduction of the Ps6OMT-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can be achieved by introducing an expression cassette containing the Ps6OMT-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the Ps6OMT-encoding nucleic acid, and a terminator.

[0053] In the aforementioned recombinant bacteria, the presence or expression of an isomerase can be achieved by introducing the PrDCS-encoding nucleic acid (nucleotide sequence as shown in SEQ ID NO: 58) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the PrDCS-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can be achieved by introducing an expression cassette containing the PrDCS-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the PrDCS-encoding nucleic acid, and a terminator.

[0054] In the aforementioned recombinant bacteria, the presence or expression of an isomerase can be achieved by introducing the PbDCR-encoding nucleic acid (nucleotide sequence as shown in SEQ ID NO: 62) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the PbDCR-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the PbDCR-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the PbDCR-encoding nucleic acid, and a terminator.

[0055] In the aforementioned recombinant bacteria, the presence or expression of a P450 reduction chaperone can be achieved by introducing the NCP1-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the NCP1-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the NCP1-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the NCP1-coding nucleic acid, and a terminator.

[0056] In the aforementioned recombinant bacteria, the presence or expression of CYP80Q4 in vivo can be achieved by introducing the coding nucleic acid of CYP80Q4 into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the coding nucleic acid of CYP80Q4 into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the coding nucleic acid of CYP80Q4 into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the coding nucleic acid of CYP80Q4, and a terminator.

[0057] The promoter can be either promoter GPD1p or promoter CCW12p.

[0058] The terminator can be terminator IDP1t, terminator IDP1t, or terminator CYC1t.

[0059] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria in the form of plasmids and / or integrated into the chromosome.

[0060] The recombinant bacteria also contain or express P450 cyclase.

[0061] In the above recombinant bacteria, the P450 cyclase may be a P450 cyclase CYP82BC1, CYP82BC2, CYP82BC3 or CYP82BC4 derived from Stephania tetrandra.

[0062] In the aforementioned recombinant bacteria, the presence or expression of CYP82BC1 in vivo can be achieved by introducing the coding nucleic acid of CYP82BC1 (nucleotide sequence as shown in SEQ ID NO: 65) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the coding nucleic acid of CYP82BC1 into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the coding nucleic acid of CYP82BC1 into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the coding nucleic acid of CYP82BC1, and a terminator.

[0063] In the aforementioned recombinant bacteria, the presence or expression of CYP82BC2 in vivo can be achieved by introducing the coding nucleic acid of CYP82BC2 (nucleotide sequence as shown in SEQ ID NO: 68) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the coding nucleic acid of CYP82BC2 into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the coding nucleic acid of CYP82BC2 into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the coding nucleic acid of CYP82BC2, and a terminator.

[0064] In the aforementioned recombinant bacteria, the presence or expression of CYP82BC3 in vivo can be achieved by introducing the CYP82BC3-encoding nucleic acid (nucleotide sequence as shown in SEQ ID NO: 69) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the CYP82BC3-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the CYP82BC3-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the CYP82BC3-encoding nucleic acid, and a terminator.

[0065] In the aforementioned recombinant bacteria, the presence or expression of CYP82BC4 in vivo can be achieved by introducing the coding nucleic acid of CYP82BC4 (nucleotide sequence as shown in SEQ ID NO: 70) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the coding nucleic acid of CYP82BC4 into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the coding nucleic acid of CYP82BC4 into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the coding nucleic acid of CYP82BC4, and a terminator.

[0066] The promoter may be the CCW12p promoter.

[0067] The terminator can be terminator PGI1t.

[0068] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria in the form of plasmids and / or integrated into the chromosome.

[0069] The recombinant bacteria mentioned above also contain or express methyltransferases CNMT6, T5, T7 and OMT10.

[0070] In the aforementioned recombinant bacteria, the presence or expression of CNMT6 in vivo can be achieved by introducing the coding nucleic acid of CNMT6 (nucleotide sequence as shown in SEQ ID NO:73) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the coding nucleic acid of CNMT6 into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the coding nucleic acid of CNMT6 into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the coding nucleic acid of CNMT6, and a terminator.

[0071] In the aforementioned recombinant bacteria, the presence or expression of T5 in the in vivo can be achieved by introducing the T5-coding nucleic acid (nucleotide sequence as shown in SEQ ID NO: 74) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the T5-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the T5-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the T5-coding nucleic acid, and a terminator.

[0072] In the aforementioned recombinant bacteria, the presence or expression of T7 in the in vivo can be achieved by introducing the T7-coding nucleic acid (nucleotide sequence as shown in SEQ ID NO: 75) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the T7-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the T7-coding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the T7-coding nucleic acid, and a terminator.

[0073] In the aforementioned recombinant bacteria, the presence or expression of OMT10 in vivo can be achieved by introducing the coding nucleic acid of OMT10 (nucleotide sequence as shown in SEQ ID NO:76) into the starting bacteria or any of the aforementioned recombinant bacteria. The introduction of the coding nucleic acid of OMT10 into the starting bacteria or any of the aforementioned recombinant bacteria can also be achieved by introducing an expression cassette containing the coding nucleic acid of OMT10 into the starting bacteria or any of the aforementioned recombinant bacteria. The expression cassette may include a promoter, the coding nucleic acid of OMT10, and a terminator.

[0074] The promoter may be promoter PDC1p, promoter GPM1p, promoter TPI1p or promoter PGK1p.

[0075] The terminator can be ENO2t, IDP1t, ENO2t, ADH1t, or FBA1t.

[0076] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria in the form of plasmids and / or integrated into the chromosome.

[0077] The starting strain may be *Saccharomyces cerevisiae*. Specifically, the *Saccharomyces cerevisiae* strain may be IMX581.

[0078] Any of the recombinant bacteria mentioned above is a species of the genus *Saccharomyces*.

[0079] Any of the recombinant bacteria mentioned above may be Saccharomyces cerevisiae.

[0080] The application of any of the recombinant bacteria described above in the production of dibenzylisoquinoline alkaloids or in increasing the yield of dibenzylisoquinoline alkaloids is also within the scope of protection of this invention.

[0081] In the above applications, the bisbenzylisoquinoline alkaloids may be (R,S)-bisnoraromoline, bisnorobamegine, isotetracycline, and / or obaberine.

[0082] This invention also protects a method for producing dibenzylisoquinoline alkaloids, which may include the following steps: fermenting and culturing any of the recombinant bacteria described above, collecting the fermentation products, and obtaining dibenzylisoquinoline alkaloids therefrom.

[0083] In the above method, the bisbenzylisoquinoline alkaloid may be (R,S)-bisnoraromoline, bisnorobamegine, isotetracycline, and / or obaberine.

[0084] The structural formulas of Bisnoraromoline, Bisnorobamegine, and Obaberine can be seen above. Figure 1 .

[0085] The inventors of this invention have constructed a recombinant strain for producing dibenzylisoquinoline alkaloids (such as isotetracycline and obaberine) through extensive experiments. The synthetic pathway of dibenzylisoquinoline alkaloids was divided into four modules for study, and de novo synthesis of dibenzylisoquinoline alkaloids was achieved through various strategies; the starting strain was the IMX581 yeast strain. Experiments have demonstrated that the engineered strain constructed in this invention can efficiently produce (R,S)-bisnoraromoline, bisnorobamegine, isotetracycline, obaberine, and other dibenzylisoquinoline alkaloids. This invention has significant application value. Attached Figure Description

[0086] Figure 1 This is a biosynthetic pathway diagram of isotetracycline.

[0087] Figure 2 The pathways and modification strategies for the de novo production of (S)-norcodine (Module I) in Saccharomyces cerevisiae, and the detection results of steps (v)-(viii) in step one.

[0088] Figure 3 The results are the detection results of steps (ii) and (iii) in step one of the embodiments.

[0089] Figure 4 The result is the detection result of step (vii) in the first step of the example.

[0090] Figure 5 The result is the detection result in step (viii) of the example.

[0091] Figure 6The results of the detection of (S)-COC, 1,2-dehydroCOC and (R,S)-lindoldhamine (Module II) were obtained.

[0092] Figure 7 The results are for the detection of (R,S)-bisnoraromoline, cyclized bisbenzylisoquinoline alkaloids, and / or bisnorobamegine.

[0093] Figure 8 The results are for the detection of the yield of the dibenzylisoquinoline alkaloid isotetracycline (Module IV).

[0094] Figure 9 The results show the detection of the bisbenzylisoquinoline alkaloids isotetracycline and obaberine (module IV). Among them, a is the detection result with a mass-to-charge ratio of 623.0, b is the secondary mass spectrometry of obaberine and isotetracycline synthesized in strains XJ06326 and XJ06328, ① is obaberine, and ② isotetracycline. Detailed Implementation

[0095] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0096] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0097] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0098] The primer names and their nucleotide sequences involved in the following examples are shown in Table 1.

[0099] Table 1

[0100]

[0101]

[0102]

[0103] The strain names and their genotypes involved in the following examples are shown in Table 2.

[0104] Table 2

[0105]

[0106]

[0107]

[0108] The IMX581 yeast strain (described in the following literature: Mans R et al. CRISPR / Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res 2015, 15, fov004.) was purchased from the euroscarf website, the specific link is http: / / www.euroscarf.de / plasmid_details.php?accno=Y40593.

[0109] The LC-MS / MS was performed using a Shimadzu Nexera UHPLC system, with a Sciex QTRAP 6500+ mass spectrometer detector and a Luna Omega 1.6μm polar C18 column. The column was a Phenomenex. The mobile phase consisted of mobile phase A (0.1% formic acid solution, solvent: water) and mobile phase B (0.1% formic acid solution, solvent: acetonitrile), with a flow rate of 0.4 mL / min. The mass spectrometry conditions were: Curtain Gas (50); Collision Gas (Medium); IonSpray Voltage: -4500V (4-HP acid and Tyrosol), 4500V (Dopamine, (S)-NOR and (S)-RET); Temperature (450℃). The mobile phase gradient was: 0–0.1 min, 10% B; 0.1–5 min, 10% B–45% B; 5–5.5 min, 45% B–85% B; 5.5–7 min, 90% B; 7–7.01 min, 90% B–10% B; 7.01–9.5 min, 10% B. Quantitative detection is performed using Q3 MI mode or MS2 mode (parameters: Product of 623.0 Da; 100Da-650Da; CE 10V-47V; DP 130V; EP 10V; CXP 17V).

[0110] In the following examples, plasmid pMEL10 is a product of the EUROSCARF collection, acc.no. P30779. Plasmid pMEL10 carries the SNR52 promoter and gRNA backbone.

[0111] Example

[0112] The inventors of this invention constructed a recombinant strain that produces dibenzylisoquinoline alkaloids (such as (R,S)-bisnoraromoline, bisnorobamegine, isotetracycline, and obaberine) through extensive experiments. The synthetic pathway of dibenzylisoquinoline alkaloids was divided into four modules for study, and de novo synthesis of dibenzylisoquinoline alkaloids was achieved through various strategies. The starting strain was the IMX581 yeast strain, which is a Saccharomyces cerevisiae strain that integrates the Cas9 gene under the TEF1 promoter, with the genotype MATa ura3-52 can1Δ::cas9-natNT2 TRP1 LEU2HIS3,CEN.PK113-5D. In the following text, the IMX581 yeast strain will be abbreviated as IMX581.

[0113] Biosynthetic pathway diagrams of isotetracycline and obaberine are shown below. Figure 1 .

[0114] I. De novo production of (S)-norcoclaurine (S)-NOR from brewer's yeast (Module I)

[0115] For the approach and modification strategy of Module I, see Figure 2 a.

[0116] (I) Construction of the XJ001 strain for dopamine production

[0117] IMX581 was spread onto YPD solid plates and incubated upside down at 30°C for 48-72 h to obtain newly activated IMX581 yeast colonies. Then, the beet (Beta vulgaris) gene fragments BvCYP76AD1* (genbank number AET43289.1; encoding tyrosine hydroxylase) and DODC (genbank number DAA64376.1; encoding dopa decarboxylase) were integrated into the XI-5 site of IMX581 using CRISPR-Cas9 genome editing.

[0118] 1. Construction of plasmid pMEL10-XI-5

[0119] gRNA plasmids were constructed using the Gibson assembly method. The specific steps are as follows:

[0120] (1) Using plasmid pMEL10 as a template, PCR amplification was performed using primer pairs consisting of Vector backbone-F and Vector backbone-R to obtain linearized plasmid pMEL10.

[0121] (2) XI-5-gRNA-F and XI-5-gRNA-R were artificially synthesized, then mixed in equal amounts, treated at 95℃ for 10 min, and cooled to room temperature to obtain annealed short nucleic acid sequences, which include a 20bp sequence homologous to the SNR52 promoter, a 20bp gRNA sequence, and a 20bp sequence homologous to the gRNA backbone.

[0122] (3) The annealed short nucleic acid sequence and the linearized plasmid pMEL10 were assembled according to the standard Gibson assembly to obtain plasmid pMEL10-XI-5.

[0123] 2. Preparation of repair fragments (Donor DNA)

[0124] A. The repair fragment XI-5 us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p consists of, from 5' to 3', the upstream homologous arm XI-5 us (nucleotide sequence as shown in SEQ ID NO:1), the terminator IDP1t (nucleotide sequence as shown in SEQ ID NO:2), the gene fragment BvCYP76AD1*, the promoter TDH3p (nucleotide sequence as shown in SEQ ID NO:3), and the promoter CCW12p (nucleotide sequence as shown in SEQ ID NO:4). The specific steps for preparing the repair fragment XI-5us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p using the overlap method are as follows:

[0125] (1) Using yeast genome or synthesized gene as templates, upstream homologous arm XI-5 us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p and promoter CCW12p were amplified.

[0126] (2) The upstream homologous arm XI-5 us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p and promoter CCW12p were connected using the overlap system to obtain the template.

[0127] (2-1) Preparation of the overlap system. The overlap system consisted of 25 μL of 5*Prime STAR buffer (5 μL), dNTPs (2.5 mM each) (2 μL), upstream homologous arm XI-5 us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p, promoter CCW12p, 0.5 μL of polymerase, and ddH2O. In the overlap system, the concentration of upstream homologous arm XI-5 us was 0.03 pmol, and the molar ratio of upstream homologous arm XI-5 us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p, and promoter CCW12p was 1:3:5:3:1.

[0128] (2-2) Take the overlap system and react it to obtain the product.

[0129] The reaction conditions were: denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 2 min, for 15 cycles; and a final extension at 70℃ for 10 min.

[0130] (2-3) Take 2 μl of the product obtained in step (2-2) and use it as a template. Perform PCR amplification using primers consisting of XI-5 us-F and CCW12p-DODC-R1 to obtain the repair fragment XI-5 us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p.

[0131] The reaction conditions were: denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 4 min, for 35 cycles; and a final extension at 70℃ for 10 min.

[0132] B. The repair fragment CCW12p-DODC-TPS1t-XI-5 ds consists of the promoter CCW12p, the gene fragment DODC, the terminator TPS1t (nucleotide sequence shown in SEQ ID NO:5), and the downstream homologous arm XI-5 ds (nucleotide sequence shown in SEQ ID NO:6) from 5' to 3'. The specific procedures for preparing the repair fragment CCW12p-DODC-TPS1t-XI-5 ds using the overlap method are as follows:

[0133] (1) Using yeast genome or synthesized gene as templates, promoter CCW12p, gene fragment DODC, terminator TPS1t and downstream homologous arm XI-5 ds were amplified.

[0134] (2) The promoter CCW12p, gene fragment DODC, terminator TPS1t and downstream homologous arm XI-5ds were connected using the overlap system to obtain the template.

[0135] (2-1) Preparation of the overlap system. The overlap system consisted of 25 μL of 5*Prime STARbuffer, 2 μL of dNTPs (2.5 mM each), promoter CCW12p, gene fragment DODC, terminator TPS1t, downstream homologous arm XI-5 ds, 0.5 μL of Polymerase, and ddH2O. In the overlap system, the concentration of promoter CCW12p was 0.03 pmol, and the molar ratio of promoter CCW12p, gene fragment DODC, terminator TPS1t, and downstream homologous arm XI-5 ds was 1:3:3:1.

[0136] (2-2) Take the overlap system and react it to obtain the product.

[0137] The reaction conditions were: denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 2 min, for 15 cycles; and a final extension at 70℃ for 10 min.

[0138] (2-3) Take 2 μl of the product obtained in step (2-2) and use it as a template. Perform PCR amplification using primers consisting of CCW12p1-F and XI-5 ds-R to obtain the repair fragment CCW12p-DODC-TPS1t-XI-5 ds.

[0139] The reaction conditions were: denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 4 min, for 35 cycles; and a final extension at 70℃ for 10 min.

[0140] 3. Obtaining positive clones

[0141] (1) The plasmid pMEL10-XI-5 obtained in step 1, the repair fragment XI-5us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p obtained in step 2, and the repair fragment CCW12p-DODC-TPS1t-XI-5 ds) were added to IMX581 yeast competent cells in accordance with Table 3. After mixing, the cells were treated at 42℃ for 30-60 min. The cells were then centrifuged at 10000g for 1 min at room temperature. The cells were collected and resuspended in 100μL of sterile water to obtain the resuspension.

[0142] Table 3

[0143]

[0144] Note: The repaired fragment needs to be denatured in a boiling water bath for 5 minutes, and then placed on ice for 5 minutes.

[0145] (2) Place the resuspended liquid droplet in the center of the SD-Ura plate and spread it evenly with a spreader until all the bacterial solution is completely absorbed. Then, place it in a 30℃ incubator and invert it for 2-3 days.

[0146] SD-Ura plates: Add distilled water to 8g Ura minus medium and 15g agar powder, then bring the volume to 1L with distilled water and sterilize at 121℃ for 15min; finally, add glucose filtered through a 0.22μm filter membrane to a final concentration of 2%, pour into sterile plates and cool.

[0147] (2) Single clones were picked from the SD-Ura plates and colony PCR was performed. Specifically, the picked single clones were placed in 20 mmol / L NaOH solution and lysed at 99℃ for 15 min, followed by centrifugation for 2 min, and the supernatant was collected. The supernatant was used as a template and PCR amplification was performed using primer pair a (composed of XI-5-vF and IDP1t-rvR), primer pair b (composed of DODC-TPS1t-F and XI-5-vR), and primer pair c (composed of AtC3H-TDH3p-rF and CCW12p-DODC-R1) to obtain PCR amplification products.

[0148] The reaction conditions were: denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 1 min, for 30 cycles; and a final extension at 70℃ for 10 min.

[0149] (3) Perform agarose gel electrophoresis on the PCR amplification products respectively, and then make the following judgment: if the PCR amplification product obtained by a certain single clone using primer pair a contains a DNA fragment of 1056bp, the PCR amplification product obtained using primer pair b contains a DNA fragment of 919bp, and the PCR amplification product obtained using primer pair c contains a DNA fragment of 1384bp, then the single clone is a positive clone.

[0150] 4. Randomly select 3 positive clones and streak them on a 5-FOA plate. Incubate at 30℃ for 2-3 days. The resulting colonies are the strains that have lost the gRNA plasmid. The obtained strain is named XJ001 yeast strain (hereinafter referred to as XJ001). The genotype of XJ001 yeast strain is IMX581(XI-5::IDP1t-BvCYP76A1*-TDH3p+CCW12p-DODC-TPS1t).

[0151] 5-FOA plates: Add distilled water to 8g yeast SC medium and 20g agar powder, then bring the volume to 1L with distilled water and sterilize at 121℃ for 20min; finally, when the temperature of the medium drops to about 40℃, add 5-FOA stock solution (obtained by dissolving 5-fluoroorotic acid (5-FOA) powder in dimethyl sulfoxide (DMSO)) in a sterile laminar flow hood to make the final concentration of 5-FOA 0.1%, pour into sterile petri dishes and cool.

[0152] (II) Screening of norepinephrine synthase (NCS enzyme)

[0153] The roles of three N-terminally truncated CjNCS sequences derived from *Coptis japonica* (genbank number BAF45338.2) (CjNCSN_Δ24, CjNCSN_Δ29, and CjNCSN_Δ35, i.e., the N-terminus of the CjNCS sequence truncated by 24, 29, and 35 amino acids, respectively) in XJ001 were compared. The NCS enzyme efficiency comparison experiments were conducted in XJ001.

[0154] The expression cassette XI-3 us-TEF1p-CjNCS_Δ24-TDH2t-XI-3 ds consists of, from 5' to 3', the upstream homologous arm XI-3 us (nucleotide sequence as shown in SEQ ID NO:7), the promoter TEF1p (nucleotide sequence as shown in SEQ ID NO:8), the gene encoding CjNCS_Δ24, the terminator TDH2t (nucleotide sequence as shown in SEQ ID NO:9), and the downstream homologous arm XI-3 ds (nucleotide sequence as shown in SEQ ID NO:10).

[0155] 1. Construction of plasmid pMEL10-XI-3

[0156] Following the method in step (I), replace XI-5-gRNA-F with XI-3-gRNA-F and XI-5-gRNA-R with XI-3-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-XI-3.

[0157] 2. The plasmid pMEL10-XI-3 and the expression cassette XI-3 us-TEF1p-CjNCS_Δ24-TDH2t-XI-3 ds were transformed into XJ001 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the expression cassette XI-3 us-TEF1p-CjNCS_Δ24-TDH2t-XI-3 ds was integrated into the XI-3 site of XJ001. The resulting strain was named XJ003 yeast strain (hereinafter referred to as XJ003), and the genotype of XJ003 yeast strain was XJ001 (XI-3::TEF1p-CjNCS_Δ24-TDH2t).

[0158] 3. Replace the expression cassette XI-3 us-TEF1p-CjNCS_Δ24-TDH2t-XI-3 ds in step 2 with the expression cassette XI-3 us-TEF1p-CjNCS_Δ29-TDH2t-XI-3 ds, keeping all other steps unchanged. Integrate the expression cassette XI-3 us-TEF1p-CjNCS_Δ29-TDH2t-XI-3 ds into the XI-3 site of XJ001. The resulting strain is named yeast strain XJ006 (hereinafter referred to as XJ006), and the genotype of yeast strain XJ006 is XJ001 (XI-3::TEF1p-CjNCS_Δ29-TDH2t).

[0159] The expression cassette XI-3 us-TEF1p-CjNCS_Δ29-TDH2t-XI-3 ds consists of the upstream homologous arm XI-3 us, the promoter TEF1p, the coding gene for CjNCS_Δ29, the terminator TDH2t, and the downstream homologous arm XI-3 ds, from 5' to 3'.

[0160] 4. Replace the expression cassette XI-3 us-TEF1p-CjNCS_Δ24-TDH2t-XI-3 ds in step 2 with the expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3 ds, keeping all other steps unchanged. Integrate the expression cassette XI-3 us-TEF1p-CjNCS_Δ35-TDH2t-XI-3 ds into the XI-3 site of XJ001. The resulting strain is named XJ007 yeast strain (hereinafter referred to as XJ007), and the genotype of XJ007 yeast strain is XJ001 (XI-3::TEF1p-CjNCS_Δ35-TDH2t).

[0161] The expression cassette XI-3 us-TEF1p-CjNCS_Δ35-TDH2t-XI-3 ds consists of the upstream homologous arm XI-3 us, the promoter TEF1p, the coding gene for CjNCS_Δ35, the terminator TDH2t, and the downstream homologous arm XI-3 ds, from 5' to 3'.

[0162] 5. Three single clones from each of XJ003, XJ006, and XJ007 were selected for parallel experiments. 1 ml of Delft medium (solutes and concentrations: 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) was added to a 14 mL shaker tube. The single clone was then inoculated and cultured at 30°C and 220 rpm for 18-24 h to obtain the bacterial culture. The bacterial culture was then transferred to a 125 mL shaker flask containing 20 mL of Delft medium for further culture and incubated at 30°C and 220 rpm for 72 h to obtain the fermentation broth. The OD of the fermentation broth was measured. 600nm .

[0163] 7. Detection of bacterial metabolic components

[0164] Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and perform LC-MS / MS detection.

[0165] Test results are shown Figure 3 The results show that the engineered bacteria XJ007, which contains the gene encoding CjNCS_Δ35, produced the highest content of (S)-norcodine.

[0166] (III) Screening for Tyrosine Hydroxylase Genes

[0167] Efficiency comparisons were performed using CYP76AD1* and CYP76AD5 from beet (Beta vulgaris), C3H from Arabidopsis thaliana and maize (Zea mays), and HpaB and HpaC from Pseudomonas aeruginosa and Salmonella enterica. Tyrosine hydroxylase efficiency comparisons were conducted using IMX581.

[0168] 1. Construction of plasmid pMEL10-XI-5

[0169] Same as step 1 in step one.

[0170] 2. Strain construction

[0171] The overlapping method is used to connect the fragments to form a complete gene expression cassette. The gene expression cassette consists of a promoter, the nucleic acid sequence of the candidate gene, and a terminator. The candidate genes are: CYP76AD1* (genbank number AET43289.1), DODC (genbank number DAA64376.1), CYP76AD5 (genbank number AJD87473.1), AtC3H (genbank number NP_001030991.2), ZmC3H (genbank number ACG46606.1), PaHpaB (genbank number WP_003104533.1), PaHpaC (genbank number WP_058166060.1), and SeHpaC (genbank number WP_001195556.1); the promoters are TDH3p, CCW12p, or TEF1p; the terminators are TPS1t, IDP1t, or ENO2t (nucleotide sequences are shown in SEQ ID NO:11). Details are as follows:

[0172] (1) The upstream homologous arm XI-5 us, terminator IDP1t, CYP76AD1*, promoter TDH3p, and promoter CCW12p were ligated using the overlap method to obtain the gene expression cassette fragment XI-5 us-IDP1t-CYP76AD1*-TDH3p-CCW12p (named fragment 1). The promoter TDH3p, promoter CCW12p, DODC, terminator TPS1t, and downstream homologous arm XI-5 ds were ligated using the overlap method to obtain the gene expression cassette fragment TDH3p-CCW12p-DODC-TPS1t-XI-5 ds (named fragment 2). The plasmid pMEL10-XI-5, fragment 1, and fragment 2 obtained in step 1 were transformed into IMX581 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the integrated genes CYP76AD1* and DODC into the XI-5 site of IMX581, resulting in strain XJ001.

[0173] (1) Following the method in step (1) above, replace CYP76AD1* with CYP76AD5, and keep all other steps unchanged to obtain strain XJ013 integrated with CYP76AD5, abbreviated as XJ013.

[0174] (2) Following the method in step (1) above, replace CYP76AD1* with AtC3H, and keep all other steps unchanged to obtain strain XJ011 integrated with AtC3H, abbreviated as XJ011.

[0175] (3) Following the method in step (1) above, replace CYP76AD1* with ZmC3H, and keep all other steps unchanged to obtain strain XJ012 integrated with ZmC3H, abbreviated as XJ012.

[0176] (4) Following the method in step (1) above, replace CYP76AD1* with PaHpaB-PaHpaC (i.e., the fusion gene of PaHpaB and PaHpaC), and keep all other steps unchanged to obtain strain XJ014 integrating PaHpaB-PaHpaC, abbreviated as XJ014.

[0177] (5) Following the method in step (1) above, replace CYP76AD1* with PaHpaB-SeHpaC (i.e., the fusion gene of PaHpaB and SeHpaC), and keep all other steps unchanged to obtain strain XJ015 integrating PaHpaB-SeHpaC, abbreviated as XJ015.

[0178] (6) Following the method in step (1) above, replace CYP76AD1* with PaHpaC-PaHpaB (i.e., the fusion gene of PaHpaC and PaHpaB), and keep all other steps unchanged to obtain strain XJ016 integrating PaHpaC-PaHpaB, abbreviated as XJ016.

[0179] (7) Following the method in step (1) above, replace CYP76AD1* with SeHpaC-PaHpaB (i.e., the fusion gene of SeHpaC and PaHpaB), and keep all other steps unchanged to obtain strain XJ017 integrating SeHpaC-PaHpaB, abbreviated as XJ017.

[0180] (8) Using the overlap method, connect the upstream homologous arm XI-5 us, promoter TEF1p, PaHpaC, terminator ENO2t, and terminator IDP1t to obtain XI-5 us-TEF1p-PaHpaC-ENO2t-IDP1t (named segment 3). Using the overlap method, connect the terminator ENO2t, terminator IDP1t, PaHpaB, promoter TDH3p, and promoter CCW12p to obtain ENO2t-IDP1t-PaHpaB-TDH3p-CCW12p (named segment 4); Using the overlap method, connect the promoter TDH3p, promoter CCW12p, DODC, terminator TPS1t, and downstream homologous arm XI-5 ds to obtain TDH3p-CCW12p-DODC-TPS1t-XI-5 ds (named segment 5). The plasmid pMEL10-XI-5, fragments 3, 4, and 5 obtained in step 1 were transformed into IMX581 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the XI-5 site of IMX581, resulting in strain XJ018 integrating PaHpaC and PaHpaB, abbreviated as XJ018.

[0181] (9) Following the method in step (9) above, replace PaHpaC with SeHpaC, and keep all other steps unchanged to obtain strain XJ019 integrating PaHpaB and SeHpaC, abbreviated as XJ019.

[0182] 3. Select three single clones from each of the strains obtained in step 2 for parallel experiments. Add 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) to a 14 mL shaker tube, then inoculate with a single clone and incubate at 30°C and 220 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a shaker flask (125 mL) containing 20 mL of Delft medium for expansion culture, and incubate at 30°C and 220 rpm for 72 h to obtain the fermentation broth. Measure the OD of the fermentation broth. 600nm .

[0183] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and perform LC-MS / MS detection.

[0184] Test results are shown Figure 3 The right-middle figure shows the strains from left to right: XJ001, XJ013, XJ011, XJ012, XJ014, XJ015, XJ016, XJ017, XJ018, and XJ019. The results indicate that strain XJ013 has the highest dopamine content. Therefore, the CYP76AD5 enzyme contained in this strain is a highly efficient tyrosine hydroxylase screened in this experiment.

[0185] (iv) Construction of XJ053 strain for producing (S)-NOR

[0186] 1. Construction of strain XJ023

[0187] Starting with IMX581, strain XJ023 was constructed by increasing tyrosine metabolic flux, specifically by integrating tyrosine metabolic flux-related target genes into the IMX581 chromosome. The gene expression cassette consists of a promoter, the nucleic acid sequence of the tyrosine metabolic flux-related target gene, and a terminator. The tyrosine metabolic flux-related target genes are EcAROL (genbank ID NP_414922.1) and ARO4* (a mutant of the yeast endogenous gene ARO4) from *E. coli*. K229L ARO7* (a mutant of the yeast endogenous gene ARO7) G141S The yeast-derived MtPDH1 (genbank number KM507076.1), ARO1 (endogenous yeast gene), ARO2 (endogenous yeast gene), and ARO3 (endogenous yeast gene) are used. The promoters are TPI1p (nucleotide sequence as shown in SEQ ID NO:13), PGK1p (nucleotide sequence as shown in SEQ ID NO:16), TEF1p, TDH3p, or tHXT7p (nucleotide sequence as shown in SEQ ID NO:19). The terminators are pYX212t (nucleotide sequence as shown in SEQ ID NO:14), ADH1t (nucleotide sequence as shown in SEQ ID NO:15), FBA1t (nucleotide sequence as shown in SEQ ID NO:18), CYC1t (nucleotide sequence as shown in SEQ ID NO:17), or TDH2t. The specific steps are as follows:

[0188] (1) Construction of plasmid pMEL10-X-2

[0189] Following the method in step (I), replace XI-5-gRNA-F with X-2-gRNA-F and XI-5-gRNA-R with X-2-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-X-2.

[0190] (2) Construction of plasmid pMEL10-X-4

[0191] Following the method in step (I), replace XI-5-gRNA-F with X-4-gRNA-F and XI-5-gRNA-R with X-4-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-X-4.

[0192] (3) The plasmid pMEL10-X-2, the expression cassette X-2 us-TPI1p-EcaroL-pYX212t-ADH1t (composed of the upstream homologous arm X-2 us (nucleotide sequence as shown in SEQ ID NO:12), the promoter TPI1p (nucleotide sequence as shown in SEQ ID NO:13), the EcAROL nucleic acid sequence, the terminator pYX212t (nucleotide sequence as shown in SEQ ID NO:14) and the terminator ADH1t (nucleotide sequence as shown in SEQ ID NO:15), and the expression cassette were combined.

[0193] pYX212t-ADH1t-ARO7*-PGK1p-TEF1p (composed of the nucleic acid sequences of terminator pYX212t, terminator ADH1t, and ARO7*, promoter PGK1p (nucleotide sequence as shown in SEQ ID NO:16), and promoter TEF1p), expression cassette PGK1p-TEF1p-ARO4*-CYC1t-FBA1t (composed of the nucleic acid sequences of promoter PGK1p, promoter TEF1p, and ARO4*, terminator CYC1t (nucleotide sequence as shown in SEQ ID NO:17), and terminator FBA1t (nucleotide sequence as shown in SEQ ID NO:18)), and expression cassette CYC1t-FBA1t-MtPDH1-tHXT7p-X-2 ds (composed of the nucleic acid sequences of terminator CYC1t, terminator FBA1t, and MtPDH1, promoter tHXT7p (nucleotide sequence as shown in SEQ ID NO:18)). The plasmid pMEL10-X-4 and the expression cassette X-4 us-CYC1t-ARO1-TPI1p-TDH3p (composed of the upstream homologous arm X-4 us (nucleotide sequence as shown in SEQ ID NO:19)) were transformed into IMX581 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30°C for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid, i.e., integrated into the X-2 site of IMX581, yielding intermediate yeast. The plasmid pMEL10-X-4 and the expression cassette X-4 us-CYC1t-ARO1-TPI1p-TDH3p (composed of the upstream homologous arm X-4 us (nucleotide sequence as shown in SEQ ID NO:20)) were then transformed into IMX581 yeast competent cells to obtain positive clones. (as shown in NO:21), the expression cassette TPI1p-TDH3p-ARO2-ADH1t-TDH2t (composed of the nucleic acid sequences of the promoters TPI1p, TDH3p, and ARO2, and the terminator ADH1t and TDH2t), and the expression cassette ADH1t-TDH2t-ARO3-TEF1p-X-4ds (composed of the nucleic acid sequences of the terminator ADH1t, TDH2t, and ARO3, the promoter TEF1p, and the downstream homologous arm X-4ds (nucleotide sequence as shown in SEQ ID NO:21) (composed of the terminator CYC1t, the promoter TDH3p, the nucleic acid sequences of ARO2, and the terminator ADH1t and TDH2t), and the expression cassette ADH1t-TDH2t-ARO3-TEF1p-X-4ds). The composition (shown in NO:22) was transferred into competent intermediate yeast cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the X-4 site of intermediate yeast, resulting in strain XJ023.

[0194] 2. Construction of strain XJ053

[0195] Integrating CYP76AD5, DODC, and CjNCSN_Δ35 into strain XJ023 yielded strain XJ053. Specifically, plasmid pMEL10-XI-5, expression cassette XI-5 us-IDP1t-BvCYP76AD5-TDH3p-CCW12p (composed of the upstream homologous arm XI-5 us, terminator IDP1t, the coding gene for BvCYP76AD5, promoter TDH3p, and promoter CCW12p) and expression cassette CCW12p-DODC-TPS1t-XI-5 ds (composed of promoter CCW12p, DODC, terminator TPS1t, and downstream homologous arm XI-5) were integrated. The pMEL10-XI-3 plasmid and expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3 ds (composed of the upstream homologous sequence XI-3us, promoter TEF1p, the coding gene of CjNCS_Δ35, terminator TDH2t, and downstream homologous arm XI-3 ds) were then transformed into competent intermediate yeast cells to obtain positive clones. The pMEL10-XI-3 plasmid and expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3 ds (composed of the upstream homologous sequence XI-3us, promoter TEF1p, the coding gene of CjNCS_Δ35, terminator TDH2t, and downstream homologous arm XI-3 ds) were then transformed into competent intermediate yeast cells to obtain positive clones. The pMEL10-XI-3 plasmid and expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3 ds were then transformed into competent intermediate yeast cells to obtain positive clones. The pMEL10-XI-3 plasmid and expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3 ds (composed of the upstream homologous sequence XI-3us, promoter TEF1p, terminator TDH2t, and downstream homologous arm XI-3 ds) were then transformed into positive clones.

[0196] 3. Three single clones of strain XJ053 were selected for parallel experiments. 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) was added to a 14 mL shaker tube. A single clone was then inoculated and cultured at 30°C and 220 rpm for 18-24 h to obtain the bacterial culture. The bacterial culture was then transferred to a 125 mL shaker flask containing 20 mL of Delft medium for further culture and incubated at 30°C and 220 rpm for 72 h to obtain the fermentation broth. The OD of the fermentation broth was measured. 600nm .

[0197] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and dilute to an appropriate concentration for LC-MS / MS detection.

[0198] The test results showed that strain XJ053 produced 23.4 mg / L of (S)-NOR.

[0199] (v) Modify strain XJ053 to increase (S)-NOR yield.

[0200] By knocking out byproduct metabolic flux genes in strain XJ053 (knockout of ARI1, ADH6, YPR1, YDR541C, AAD3, GRE2, and HFD1), the synthesis capacity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid was downregulated, thereby increasing (S)-NOR production. This effectively removed the competitive metabolic flux against the substrate 4-HPAA by aldehyde reductases or dehydrogenases. The strain was then fermented, and the (S)-NOR content was measured. The specific operational steps are as follows:

[0201] 1. Construction of plasmids

[0202] (1) Using the gRAN-SNR52P gene fragment (nucleotide sequence as shown in SEQ ID NO:23) as a template, PCR amplification was performed using primer pair 1 (composed of BsaI-ari1-gRNA-F and SNR52p-adh6-gRNA-R), primer pair 2 (composed of SNR52p-adh6-gRNA-F and SNR52p-ypr1-gRNA-R), primer pair 3 (composed of BsaI-ydc541c-gRNA-F and SNR52p-aad3-gRNA-R) and primer pair 4 (composed of BsaI-gre2-gRNA-F and SNR52p-hfd1-gRNA-R) to obtain gene fragment 1, gene fragment 2, gene fragment 3 and gene fragment 4 in sequence.

[0203] (2) Gene fragment 1 and gene fragment 2 were ligated with the linearized plasmid pMEL10-BsaI (obtained by digesting plasmid pMEL10 with restriction endonuclease BsaI) via Golden Gate to obtain the recombinant plasmid pMEL10-ARI1+ADH6+YPR1. Gene fragment 3 was ligated with the linearized plasmid pMEL10-BsaI (obtained by digesting plasmid pMEL10 with restriction endonuclease BsaI) via Golden Gate to obtain the recombinant plasmid pMEL10-YDR541C+AAD3. Gene fragment 4 was ligated with the linearized plasmid pMEL10-BsaI via Golden Gate to obtain the recombinant plasmid pMEL10-GRE2+HFD1.

[0204] 2. Construction of DNA Repair Fragments

[0205] The fragments were joined using the overlap method to form a complete DNA repair fragment. The DNA repair fragment consists of the upstream sequence of the knockout gene, the gene sequence carrying the exogenous gRNA cleavage site, and the downstream sequence of the knockout gene. The gene sequences carrying the exogenous gRNA cleavage site are: LP1.T8, LP2.T10, LP3.T7, and LP4.T9.

[0206] The upstream homologous arms ari1 us (nucleotide sequence as shown in SEQ ID NO:24), LP3.T7 (nucleotide sequence as shown in SEQ ID NO:25), and the downstream homologous arm ari1 ds (nucleotide sequence as shown in SEQ ID NO:26) were ligated using the overlap method to obtain the DNA repair fragment ari1 us-LP3.T7-ari1 ds.

[0207] The upstream homologous arm adh6 us (nucleotide sequence as shown in SEQ ID NO:27), LP3.T7, and the downstream homologous arm adh6 ds (nucleotide sequence as shown in SEQ ID NO:28) were ligated using the overlap method to obtain the DNA repair fragment adh6 us-LP3.T7-adh6 ds.

[0208] The upstream homologous arm ypr1 us (nucleotide sequence as shown in SEQ ID NO:29), LP3.T7, and the downstream homologous arm ypr1 ds (nucleotide sequence as shown in SEQ ID NO:30) were ligated using the overlap method to obtain the DNA repair fragment ypr1 us-LP3.T7-ypr1 ds.

[0209] The upstream homologous arms ydr541c us (nucleotide sequence as shown in SEQ ID NO:31), LP2.T10 (nucleotide sequence as shown in SEQ ID NO:32), and the downstream homologous arm ydr541c ds (nucleotide sequence as shown in SEQ ID NO:33) were ligated using the overlap method to obtain the DNA repair fragment ydr541c us-LP2.T10-ydr541c ds.

[0210] The upstream homologous arm aad3 us (nucleotide sequence as shown in SEQ ID NO:34), LP2.T10, and the downstream homologous arm aad3 ds (nucleotide sequence as shown in SEQ ID NO:35) were ligated using the overlap method to obtain the DNA repair fragment aad3 us-LP2.T10-aad3 ds.

[0211] The upstream homologous arms gre2 us (nucleotide sequence as shown in SEQ ID NO:36), LP1.T8 (nucleotide sequence as shown in SEQ ID NO:37), and the downstream homologous arm gre2 ds (nucleotide sequence as shown in SEQ ID NO:38) were ligated using the overlap method to obtain the DNA repair fragment gre2 us-LP1.T8-gre2 ds.

[0212] The upstream homologous arms hfd1us (nucleotide sequence as shown in SEQ ID NO:39), LP4.T9 (nucleotide sequence as shown in SEQ ID NO:40), and the downstream homologous arm hfd1ds (nucleotide sequence as shown in SEQ ID NO:41) were ligated using the overlap method to obtain the DNA repair fragment hfd1us-LP4.T9-hfd1ds.

[0213] 3. Strain construction

[0214] The recombinant plasmid pMEL10-ARI1+ADH6+YPR1, along with the DNA repair fragments ari1 us-LP3.T7-ari1 ds, adh6 us-LP3.T7-adh6 ds, and ypr1 us-LP3.T7-ypr1 ds, were transformed into competent cells of yeast strain XJ053 to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30°C for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid, thus yielding strain XJ054.

[0215] The recombinant plasmid pMEL10-YDR541C+AAD3, the DNA repair fragment ydr541c us-LP2.T10-ydr541cds, and the DNA repair fragment aad3 us-LP2.T10-aad3 ds were transformed into competent cells of yeast strain XJ053 to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, which were the strains XJ055.

[0216] The recombinant plasmid pMEL10-GRE2+HFD1, the DNA repair fragment gre2 us-LP1.T8-gre2 ds, and the DNA repair fragment hfd1 us-LP4.T9-hfd1 ds were transformed into competent cells of yeast strain XJ053 to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, which were the strains XJ056.

[0217] The recombinant plasmid pMEL10-YDR541C+AAD3, the DNA repair fragment ydr541c us-LP2.T10-ydr541cds, and the DNA repair fragment aad3 us-LP2.T10-aad3 ds were transformed into competent cells of yeast strain XJ054 to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, which were the strains XJ057.

[0218] The recombinant plasmid pMEL10-GRE2+HFD1, the DNA repair fragment gre2 us-LP1.T8-gre2 ds, and the DNA repair fragment hfd1 us-LP4.T9-hfd1 ds were transformed into competent cells of yeast strain XJ054 to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, which were the strains XJ058.

[0219] The recombinant plasmid pMEL10-YDR541C+AAD3, the DNA repair fragment ydr541c us-LP2.T10-ydr541cds, and the DNA repair fragment aad3 us-LP2.T10-aad3 ds were transformed into competent cells of yeast strain XJ058 to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, which were the strains XJ059.

[0220] 4. Select three single clones from each strain obtained in step 3 for parallel experiments. Add 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) to a 14 mL shaker tube, then inoculate with a single clone and incubate at 30°C and 220 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a shaker flask (125 mL) containing 20 mL of Delft medium for expansion culture, and incubate at 30°C and 220 rpm for 72 h to obtain the fermentation broth. Measure the OD of the fermentation broth. 600nm .

[0221] 5. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and dilute to an appropriate concentration for LC-MS / MS detection.

[0222] Test results are shown Figure 2 b. The results showed that strain XJ058 had a normal growth OD value and the highest (S)-NOR yield; that is, knocking out ARI1, ADH6, YPR1, GRE2 and HFD1 can remove the competitive metabolic flux for aldehyde reductase or dehydrogenase of substrate 4-HPAA and increase (S)-NOR yield.

[0223] (vi) Selection of CjNCS_Δ35 copy number

[0224] One CjNCS_Δ35 gene fragment from Coptis japonica was integrated into strain XJ058 (which already contained one CjNCS_Δ35) to obtain strain XJ062. Two more CjNC_SΔ35 gene fragments were integrated into strain XJ058 to obtain strain XJ063 (i.e., strain XJ063 contains a total of three CjNC_SΔ35 gene fragments).

[0225] 1. Construction of strains XJ062 and XJ063

[0226] (1) Construction of plasmid pMEL10-XI-1

[0227] Following the method in step (I), replace XI-5-gRNA-F with XI-1-gRNA-F and XI-5-gRNA-R with XI-1-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-XI-1.

[0228] (2) Construction of plasmid pMEL10-LP1.T8

[0229] Following the method in step (I), replace XI-5-gRNA-F with LP1.T8-gRNA-F and XI-5-gRNA-R with LP1.T8-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-LP1.T8.

[0230] (3) The plasmid pMEL10-XI-1 and the expression cassette XI-1 us-TDH3p-CjNCS_Δ35-CYC1t-XI-1 ds (composed of the upstream homologous arm XI-1 us (nucleotide sequence as shown in SEQ ID NO:42), the promoter TDH3p, the coding gene of CjNCS_Δ35, the terminator CYC1t, and the downstream homologous arm XI-1 ds (nucleotide sequence as shown in SEQ ID NO:43)) were transformed into competent cells of strain XJ058 to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The expression cassette XI-1 us-TDH3p-CjNCS_Δ35-CYC1t-XI-1 ds was integrated into the XI-1 site to obtain strain XJ062.

[0231] (4) The plasmid pMEL10-LP1.T8 and the expression cassette LP1.T8-us-TDH3p-CjNCS_Δ35-CYC1t-LP1.T8-ds (composed of the upstream homologous arm LP1.T8 us (nucleotide sequence as shown in SEQ ID NO:44), the promoter TDH3p, the coding gene of CjNCS_Δ35, the terminator CYC1t, and the downstream homologous arm LP1.T8 ds (nucleotide sequence as shown in SEQ ID NO:45)) were transformed into competent cells of strain XJ062 to obtain positive clones. The positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The expression cassette LP1.T8-us-TDH3p-CjNCS_Δ35-CYC1t-LP1.T8-ds was integrated into the LP1.T8 site to obtain strain XJ063.

[0232] 2. Select three single clones from each strain obtained in step 1 for parallel experiments. Add 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) to a 14 mL shaker tube, then inoculate with a single clone and incubate at 30°C and 220 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a shaker flask (125 mL) containing 20 mL of Delft medium for expansion culture, and incubate at 30°C and 220 rpm for 72 h to obtain the fermentation broth. Measure the OD of the fermentation broth. 600nm .

[0233] 3. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and dilute to an appropriate concentration for LC-MS / MS detection.

[0234] Test results are shown Figure 2 The results showed that strain XJ063 had the highest (S)-NOR content, reaching 214.8 mg / L, indicating that the strain that integrated a total of 3 CjNCS_Δ35 molecules produced the highest product.

[0235] (vii) Use aromatic amino acid decarboxylase (AAAD) to direct more metabolic flux toward 4-HPAA

[0236] The effects of endogenous Aro10, PcAAS derived from *Petroselinum crispum*, RrAAS derived from *Rhodiola saridroside*, and the mutant PsAAAD* (Y350F) derived from *P. somniferum* were compared in strain XJ063. The expression cassette consists of an upstream homologous arm, a promoter, the target gene nucleic acid sequence, a terminator, and a downstream homologous arm. The promoter is FBA1p, the terminator is TPS1t-ADH1t, and the target genes are the yeast endogenous genes Aro10, PcAAS (AAA33860.1), RrAAS (AUI41111.1), and PsAAAD* (AAC61842.1). Specific procedures are as follows:

[0237] 1. Construction of strains

[0238] (1) Construction of plasmid pMEL10-X-3

[0239] Following the method in step (I), replace XI-5-gRNA-F with X-3-gRNA-F and XI-5-gRNA-R with X-3-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-X-3.

[0240] (2) The plasmid pMEL10-X-3 and the expression cassette X-3 us-FBA1p-ARO10-TPS1t-ADH1t-X-3 ds (composed of the upstream homologous arm X-3 us (nucleotide sequence as shown in SEQ ID NO:46), the promoter FBA1p (nucleotide sequence as shown in SEQ ID NO:47), the yeast endogenous gene Aro10, the terminator sequence TPS1t-ADH1t (nucleotide sequence as shown in SEQ ID NO:48), and the downstream homologous arm X-3 ds (nucleotide sequence as shown in SEQ ID NO:49)) were transformed into XJ063 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30℃ for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid, i.e., the expression cassette X-3 us-FBA1p-ARO10-TPS1t-ADH1t-X-3 ds was integrated into the X-3 site to obtain strain XJ0631.

[0241] (3) The plasmid pMEL10-X-3 and the expression cassette X-3 us-FBA1p-PcAAS-TPS1t-ADH1t-X-3 ds (composed of the upstream homologous arm X-3 us, promoter FBA1p, PcAAS, terminator sequence TPS1t-ADH1t and downstream homologous arm X-3 ds) were transformed into XJ063 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30℃ incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The expression cassette X-3 us-FBA1p-PcAAS-TPS1t-ADH1t-X-3 ds was integrated into the X-3 site to obtain strain XJ0632.

[0242] (4) The plasmid pMEL10-X-3 and the expression cassette X-3 us-FBA1p-PsAAAD*-TPS1t-ADH1t-X-3 ds (composed of the upstream homologous arm X-3 us, promoter FBA1p, PsAAAD*, terminator sequence TPS1t-ADH1t and downstream homologous arm X-3 ds) were transformed into XJ063 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30℃ incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The expression cassette X-3 us-FBA1p-PsAAAD*-TPS1t-ADH1t-X-3 ds was integrated into the X-3 site to obtain strain XJ0633.

[0243] (5) The plasmid pMEL10-X-3 and the expression cassette X-3 us-FBA1p-RrAAS-TPS1t-ADH1t-X-3 ds (composed of the upstream homologous arm X-3 us, promoter FBA1p, RrAAS, terminator sequence TPS1t-ADH1t and downstream homologous arm X-3 ds) were transformed into XJ063 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30℃ incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The expression cassette X-3 us-FBA1p-RrAAS-TPS1t-ADH1t-X-3 ds was integrated into the X-3 site to obtain strain XJ0634.

[0244] 2. Select three single clones from each strain obtained in step 1 for parallel experiments. Add 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) to a 14 mL shaker tube, then inoculate with a single clone and incubate at 30°C and 220 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a shaker flask (125 mL) containing 20 mL of Delft medium for expansion culture, and incubate at 30°C and 220 rpm for 72 h to obtain the fermentation broth. Measure the OD of the fermentation broth. 600nm .

[0245] 3. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and dilute to an appropriate concentration for LC-MS / MS detection.

[0246] Test results are shown Figure 2 d and Figure 4 The results showed that strain XJ0633 produced the highest content of (S)-NOR, reaching 295.5 mg / L, indicating that the strain integrating PsAAAD* produced the highest product, which was superior to other candidate aromatic amino acid decarboxylases (AAAD).

[0247] (viii) Increasing tyrosine supply in Saccharomyces cerevisiae by introducing the plant tyrosine pathway genes prephenatetransaminase (PAT) and aromate dehydratase (ADH).

[0248] The expression cassette consists of an upstream homologous arm, a promoter, candidate gene nucleotide sequences, a terminator, and a downstream homologous arm. The candidate PAT genes are AtPAT (NP_565529.1) from *Arabidopsis thaliana*, BvPAT1 (KMS97510.1) and BvPAT2 (KMT14107.1) from *Betavulgaris*. The candidate ADH genes are BvADHα (AST12932.1) from *Betavulgaris* and MtncADH (ARV76496.1) from *Medicago truncatula*. The promoters are TDH3p, TEF2p (nucleotide sequences shown in SEQ ID NO:51), and FBA1p; the terminators are TDH2t and pYX212t. The effects of different PAT and ADH combinations on the (S)-NOR content of the product were tested in strain XJ0633. The specific steps are as follows:

[0249] 1. Following the method in step (I), replace XI-5-gRNA-F with 106a-gRNA-F and XI-5-gRNA-R with 106a-gRNA-R, keeping all other steps unchanged, to obtain plasmid pMEL10-106a.

[0250] 2. Preparation of gene expression cassette fragments

[0251] (1) The upstream homologous arm 106a us (nucleotide sequence as shown in SEQ ID NO:50), promoter FBA1p, AtPAT, terminator TDH2t, and terminator pYX212t were ligated using the overlap method to obtain the gene expression cassette fragment 106aus-FBA1p-AtPAT-TDH2t-pYX212t (named fragment 6). The terminator pYX212t, BvADHα, promoter TEF2p (nucleotide sequence as shown in SEQ ID NO:51), and downstream homologous arm 106a ds (nucleotide sequence as shown in SEQ ID NO:52) were ligated using the overlap method to obtain the gene expression cassette fragment pYX212t-BvADHα-TEF2p-106a ds (named fragment 7).

[0252] (2) The downstream homologous arm 106a ds, promoter, TEF2p, MtncADH and terminator pYX212t were connected by the overlap method to obtain the gene expression cassette fragment pYX212t-MtncADH-TEF2p-106a ds (named fragment 8).

[0253] (3) The upstream homologous arm 106a us, promoters FBA1p and BvPAT1, terminator TDH2t and terminator pYX212t were connected by the overlap method to obtain the gene expression cassette fragment 106a us-FBA1p-BvPAT1-TDH2t-pYX212t (named fragment 9).

[0254] (4) The upstream homologous arm 106a us, promoter FBA1p, BvPAT2, terminator TDH2t and terminator pYX212t were connected by the overlap method to obtain the gene expression cassette fragment 106a us-FBA1p-BvPAT2-TDH2t-pYX212t (named fragment 10).

[0255] 3. Strain construction

[0256] Plasmid pMEL10-106a, fragment 6, and fragment 7 were transformed into XJ0633 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of XJ0633 strain, to obtain strain XJ0635.

[0257] Plasmid pMEL10-106a, fragment 6, and fragment 8 were transformed into XJ0633 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of XJ0633 strain, to obtain strain XJ0636.

[0258] Plasmid pMEL10-106a, fragment 9, and fragment 7 were transformed into XJ0633 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of XJ0633 strain, resulting in strain XJ0637.

[0259] Plasmid pMEL10-106a, fragment 9, and fragment 8 were transformed into XJ0633 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of XJ0633 strain, resulting in strain XJ0638.

[0260] Plasmid pMEL10-106a, fragment 10, and fragment 8 were transformed into XJ0633 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of XJ0633 strain, resulting in strain XJ0639.

[0261] 4. Select three single clones from each strain obtained in step 3 for parallel experiments. Add 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) to a 14 mL shaker tube, then inoculate with a single clone and incubate at 30°C and 220 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a shaker flask (125 mL) containing 20 mL of Delft medium for expansion culture, and incubate at 30°C and 220 rpm for 72 h to obtain the fermentation broth. Measure the OD of the fermentation broth. 600nm .

[0262] 5. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and dilute to an appropriate concentration for LC-MS / MS detection.

[0263] Test results are shown Figure 2 China and Figure 5 The results showed that strain XJ0636 had the highest (S)-NOR concentration, reaching 315.9 mg / L; that is, the strain that integrated AtPAT and MtncADH had the highest product, which was superior to other candidate PAT and ADH combinations.

[0264] II. Production of (R,S)-lindoldhamine from (S)-NOR (Module II)

[0265] Module II describes the pathway for the production of the alkaloid (R,S)-lindoldhamine from (S)-NOR. First, methyltransferase 6OMT is introduced into the platform bacteria used for (S)-NOR production to generate the precursor (S)-coclaurine ((S)-COC), which is the precursor for the synthesis of the alkaloid. Then, isomerases DCS and DCR are sequentially introduced, along with the P450 reduction chaperone NCP1, to synthesize (R)-coclaurine ((R)-COC). Finally, CYP80Q4 is introduced to catalyze the production of (R,S)-lindoldhamine. The expression cassette consists of a promoter, the nucleic acid sequences of candidate genes, and a terminator. The candidate genes are Ps6OMT (AAQ01669.1) from *P. somniferum*, PrDCS from *Papaver rhoeas*, PbDCR from *Papaverbracteatum*, and CYP80Q4 from *Stephania tetrandra*.

[0266] (I) Construction of engineered strain XJ06360 for the production of (S)-COC

[0267] 1. Construction of plasmids

[0268] Following the method in step 1(a), replace XI-5-gRNA-F with XII-2-gRNA-F and XI-5-gRNA-R with XII-2-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-XII-2.

[0269] 2. Gene expression cassette construction

[0270] The upstream homologous arm XII-2 us (nucleotide sequence as shown in SEQ ID NO:53), the promoter GPM1p (nucleotide sequence as shown in SEQ ID NO:54), the gene encoding Ps6OMT, the terminator FBA1t, and the downstream homologous arm XII-2 ds (nucleotide sequence as shown in SEQ ID NO:55) were ligated using the overlap method to obtain the gene expression cassette XII-2us-GPM1p-Ps6OMT-FBA1t-XII-2 ds.

[0271] 3. Transform XJ0636 yeast competent cells with plasmid pMEL10-X II-2 and gene expression cassette XII-2 us-GPM1p-Ps6OMT-FBA1t-XII-2 ds to obtain positive clones; randomly select positive clones and streak them on 5-FOA plates, and incubate them at 30℃ for 2-3 days. The clones that grow are strains that have lost the gRNA plasmid, that is, integrated into the XII-2 site of strain XJ0636, to obtain strain XJ06360.

[0272] (II) Construction of engineered strain XJ06388 for the production of dibenzylisoquinoline alkaloids (R,S)-lindoldhamine ((R,S)-lindoldhamine)

[0273] The isomerases DCS, DCR, P450 reduction chaperone NCP1, and dimerase CYP80Q4 derived from Stephania tetrandra were introduced into the XJ06360 strain that produces (S)-COC. The recombinant strain was then tested to determine whether it could synthesize the ring-opening bisbenzylisoquinoline alkaloid (R,S)-black shell alkaloid.

[0274] 1. Construction of plasmids

[0275] Following the method in step 1(a), replace XI-5-gRNA-F with LP4.T9-gRNA-F and XI-5-gRNA-R with LP4.T9-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-LP4.T9.

[0276] 2. Gene expression cassette construction

[0277] (1) The upstream homologous arm hfd1us (nucleotide sequence as shown in SEQ ID NO:56), promoter GPD1p (nucleotide sequence as shown in SEQ ID NO:57), the gene encoding PrDCS (nucleotide sequence as shown in SEQ ID NO:58), terminator IDP1t and promoter CCW12p were linked using the overlap method to obtain the gene expression cassette hfd1us-GPD1p-PrDCS-IDP1t-CCW12p.

[0278] (2) The terminator IDP1t, promoter CCW12p, the coding gene of NCP1 (yeast endogenous gene), terminator CYC1t and downstream homologous arm hfd1 ds (nucleotide sequence as shown in SEQ ID NO:59) were linked using the overlap method to obtain the gene expression cassette IDP1t-CCW12p-NCP1-CYC1t-hfd1 ds.

[0279] 3. Strain construction

[0280] Plasmid pMEL10-LP4.T9, gene expression cassette hfd1us-GPD1p-PrDCS-IDP1t-CCW12p, and gene expression cassette IDP1t-CCW12p-NCP1-CYC1t-hfd1ds were transformed into XJ06360 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the hfd1 site of XJ06360 strain, resulting in strain XJ06385.

[0281] 4. Plasmid construction

[0282] Following the method in step 1(a), replace XI-5-gRNA-F with 416d-gRNA-F and XI-5-gRNA-R with 416d-gRNA-R, keeping all other steps unchanged, to obtain plasmid pMEL10-416d.

[0283] 5. Gene expression cassette construction

[0284] (1) The upstream homologous arm 416dus (nucleotide sequence as shown in SEQ ID NO:60), promoter GPD1p, the coding gene of CYP80Q4 (nucleotide sequence as shown in SEQ ID NO:61), terminator IDP1t and promoter CCW12p were linked by the overlap method to obtain the gene expression cassette 416dus-GPD1p-CYP80Q4-IDP1t-CCW12p.

[0285] (2) The terminator IDP1t, the promoter CCW12p, the gene encoding PbDCR (nucleotide sequence as shown in SEQ ID NO:62), the terminator CYC1t, and the downstream homologous arm 416d ds (nucleotide sequence as shown in SEQ ID NO:63) were linked using the overlap method to obtain the gene expression cassette IDP1t-CCW12p-PbDCR-CYC1t-416d ds.

[0286] 6. Transform XJ06385 yeast competent cells with plasmid pMEL10-416d, gene expression cassette 416d us-GPD1p-CYP80Q4-IDP1t-CCW12p and gene expression cassette IDP1t-CCW12p-PbDCR-CYC1t-416d ds to obtain positive clones; randomly select positive clones and streak them on 5-FOA plates and incubate them at 30℃ for 2-3 days. The clones that grow are strains that have lost the gRNA plasmid, that is, integrated into the 416d site of strain XJ06385, to obtain strain XJ06388.

[0287] 7. Select three single clones of strains XJ06360, XJ06385, and XJ06388 constructed in step (I) for parallel experiments. Add 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) to a 14 mL shaker tube, then inoculate with a single clone and incubate at 30℃ and 220 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a 125 mL shaker flask containing 20 mL of Delft medium for further culture and incubate at 30℃ and 220 rpm for 72 h to obtain the fermentation broth. Measure the OD of the fermentation broth. 600nm .

[0288] 8. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and dilute to an appropriate concentration for LC-MS / MS detection.

[0289] Test results are shown Figure 6 (XJ06360 is strain XJ06360, XJ06360+(PrDCS+NCP1) is strain XJ06385, XJ06360+(PrDCS+NCP1)+(CYP80Q4+PbDCR) is strain XJ06388) and Figure 7 (XJ06388 is strain XJ06388). The results showed that strain XJ06360 could synthesize (S)-COC, strain XJ06385 could synthesize the intermediate 1,2-dehydroCOC; and strain XJ06388 could synthesize the bisbenzylisoquinoline alkaloid (R,S)-lindoldhamine.

[0290] III. Production of cyclized bisnoraromoline and / or bisnorobamegine (Module III)

[0291] Four P450 cyclases derived from *Stephania tetrandra* (CYP82BC1, CYP82BC2, CYP82BC3, and CYP82BC4) were introduced into strain XJ06388 to test whether the engineered strain could synthesize the cyclized alkaloids bisnoraromoline and bisnorobamegine. The structural formulas of bisnoraromoline and / or bisnorobamegine are shown below. Figure 1 The expression cassette consists of an upstream homologous arm, a promoter, the target gene nucleic acid sequence, a terminator, and a downstream homologous arm. The promoter is CCW12p, the terminator is PGI1t, and the target genes are the synthetic CYP82BC1, CYP82BC2, CYP82BC3, and CYP82BC4.

[0292] 1. Construction of plasmid pMEL10-XII-4

[0293] Following the method in step 1(a), replace XI-5-gRNA-F with XII-4-gRNA-F and XI-5-gRNA-R with XII-4-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-XII-4.

[0294] 2. Gene expression cassette construction

[0295] The upstream homologous arm XII-4 us (nucleotide sequence as shown in SEQ ID NO:64), the gene encoding promoter CCW12p and CYP82BC1 (nucleotide sequence as shown in SEQ ID NO:65), the terminator PGI1t (nucleotide sequence as shown in SEQ ID NO:66), and the downstream homologous arm XII-4 ds (nucleotide sequence as shown in SEQ ID NO:67) were ligated using the overlap method to obtain the gene expression cassette XII-4 us-CCW12p-CYP82BC1-PGI1t-XII-4 ds.

[0296] Following the steps described above, replace CYP82BC1 with CYP82BC2 (nucleotide sequence as shown in SEQ ID NO:68), and keep all other steps unchanged to obtain the gene expression cassette XII-4 us-CCW12p-CYP82BC2-PGI1t-XII-4 ds.

[0297] Following the steps described above, replace CYP82BC1 with CYP82BC3 (nucleotide sequence as shown in SEQ ID NO:69), and keep all other steps unchanged to obtain the gene expression cassette XII-4 us-CCW12p-CYP82BC3-PGI1t-XII-4 ds.

[0298] Following the steps described above, replace CYP82BC1 with CYP82BC4 (nucleotide sequence as shown in SEQ ID NO:70), and keep all other steps unchanged to obtain the gene expression cassette XII-4 us-CCW12p-CYP82BC4-PGI1t-XII-4 ds.

[0299] 3. Strain construction

[0300] The plasmid pMEL10-XII-4 and the gene expression cassette XII-4 us-CCW12p-CYP82BC1-PGI1t-XII-4 ds were transformed into XJ06388 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The expression cassette XII-4-us-CCW12p-CYP82BC1-PGI1t-XII-4-ds was integrated into the XII-4 site to obtain strain XJ06390.

[0301] Following the steps described above, the gene expression cassettes XII-4 us-CCW12p-CYP82BC1-PGI1t-XII-4 ds were replaced with gene expression cassettes XII-4 us-CCW12p-CYP82BC2-PGI1t-XII-4 ds, XII-4 us-CCW12p-CYP82BC3-PGI1t-XII-4 ds, and XII-4 us-CCW12p-CYP82BC4-PGI1t-XII-4 ds, respectively, to obtain strains XJ06391, XJ06389, and XJ06392.

[0302] 4. Three single clones of strains XJ06389, XJ06390, XJ06391, and XJ06392 were selected for parallel experiments. 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) was added to a 14 mL shaker tube. A single clone was then inoculated and cultured at 30°C and 220 rpm for 18–24 h to obtain the bacterial culture. The bacterial culture was then transferred to a 125 mL shaker flask containing 20 mL of Delft medium for further culture and incubated at 30°C and 220 rpm for 72 h to obtain the fermentation broth. The OD of the fermentation broth was measured. 600nm .

[0303] 5. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and dilute to an appropriate concentration for LC-MS / MS detection.

[0304] Test results are shown Figure 7 (XJ06388+CYP82BC1 is strain XJ06390, XJ06388+CYP82BC2 is strain XJ06391, XJ06388+CYP82BC3 is strain XJ06389, and XJ06388+CYP82BC4 is strain XJ06392). The results showed that strains XJ06389, XJ06390, XJ06391, and XJ06392 could all produce bisnorobamegine, m / z567[M+H]. + (Retention time 2.6 min), and strains XJ06390 and XJ06392 can also synthesize additional bisnoraromoline, m / z 567 [M+H]. + (Retention time 2.4 min). It can be seen that strains XJ06389, XJ06390, XJ06391 and XJ06392 can all synthesize the cyclized bisnorobamegine alkaloid.

[0305] IV. Synthesis of the dibenzylisoquinoline alkaloids isotetracycline and obaberine (Module IV)

[0306] The inventors of this application discovered that the rate-limiting steps for the synthesis of the dibenzylisoquinoline alkaloid isotetracin are: (1) (S)-COC is catalyzed to (R)-COC by isomerases DCS and DCR; (2) CYP80Q4 catalysis. To improve the metabolic flux for isotetracin synthesis, on the one hand, the copy number of the enzymes catalyzing the rate-limiting step is increased to improve the precursor level; on the other hand, four methylating enzymes are introduced to synthesize the compounds isotetracin and obaberine. The structural formula of obaberine is shown below. Figure 1 The specific steps are as follows:

[0307] (a) Increase the copy number of PrDCS, PbDCR, and CYP80Q4

[0308] 1. Construction of plasmid pMEL10-LP3.T7

[0309] Following the method in step 1(a), replace XI-5-gRNA-F with LP3.T7-gRNA-F and XI-5-gRNA-R with LP3.T7-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-LP3.T7.

[0310] 2. Gene expression cassette construction

[0311] (1) The upstream homologous arm ari1us, promoter GPD1p, the coding gene of PrDCS, terminator IDP1t and promoter CCW12p were connected by the overlap method to obtain the gene expression cassette ari1-us-GPD1p-PrDCS-IDP1t-CCW12p.

[0312] (2) The terminator IDP1t, the promoter CCW12p, the coding genes of PbDCR, the terminator CYC1t, and the downstream homologous arms ari1ds were connected by the overlap method to obtain the gene expression cassette IDP1t-CCW12p-PbDCR-CYC1t-ari1ds.

[0313] (3) The gene expression cassettes ari1us-GPD1p-PrDCS-IDP1t-CCW12p and IDP1t-CCW12p-PbDCR-CYC1t-ari1 ds were linked using the overlap method to obtain the complete gene expression cassette ari1us-GPD1p-PrDCS-IDP1t-CCW12p-PbDCR-CYC1t-ari1 ds.

[0314] Following the steps above, replace ari1 us with adh6 us and ari1 ds with adh6 ds, keeping all other steps unchanged, to obtain the gene expression cassette adh6 us-GPD1p-PrDCS-IDP1t-CCW12p-PbDCR-CYC1t-adh6 ds.

[0315] Following the steps above, replace adh6 us with ypr1 us and adh6 ds with ypr1 ds, keeping all other steps unchanged, to obtain the gene expression cassette ypr1 us-GPD1p-PrDCS-IDP1t-CCW12p-PbDCR-CYC1t-ypr1 ds.

[0316] 3. Strain construction

[0317] The plasmid pMEL10-LP3.T7, the gene expression cassette ari1-us-GPD1p-PrDCS-IDP1t-CCW12p-PbDCR-CYC1t-ari1-ds, the gene expression cassette adh6-us-GPD1p-PrDCS-IDP1t-CCW12p-PbDCR-CYC1t-adh6-ds, and the gene expression cassette ypr1-us-GPD1p-PrDCS-IDP1t-CCW12p-PbDCR-CYC1t-ypr1-ds were transformed Positive clones were obtained by inoculating XJ06388 yeast competent cells. Randomly selected positive clones were streaked onto 5-FOA plates and cultured at 30°C for 2-3 days. The resulting clones were strains that had lost their gRNA plasmids. Three copies of PrDCS and PbDCR were then integrated into the ari1, adh6, and ypr1 sites, respectively (the knockout of these three sites simultaneously introduces the exogenous gRNA cleavage site LP3.T7. Therefore, the required plasmid for introducing three copies of the isomerase is pMEL10-LP3.T7), resulting in strain XJ06398.

[0318] 4. Construction of plasmid pMEL10-LP2.T10

[0319] Following the method in step 1(a), replace XI-5-gRNA-F with LP2.T10-gRNA-F and XI-5-gRNA-R with LP2.T10-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-LP2.T10.

[0320] 5. Gene expression cassette construction

[0321] (1) The upstream homologous arm ydr541cus, the promoter GPD1p, the coding gene of CYP80Q4, the terminator IDP1t and the promoter CCW12p were connected by the overlap method to obtain the gene expression cassette ydr541cus-GPD1p-CYP80Q4-IDP1t-CCW12p.

[0322] (2) The terminator IDP1t, the promoter CCW12p, the coding gene of PbDCR, the terminator CYC1t and the downstream homologous arm ydr541c ds were connected by the overlap method to obtain the gene expression cassette IDP1t-CCW12p-PbDCR-CYC1t-ydr541c ds.

[0323] (3) The gene expression cassettes ydr541cus-GPD1p-CYP80Q4-IDP1t-CCW12p and IDP1t-CCW12p-PbDCR-CYC1t-ydr541c ds were linked using the overlap method to obtain the complete gene expression cassette ydr541cus-GPD1p-CYP80Q4-IDP1t-CCW12p-PbDCR-CYC1t-ydr541c ds.

[0324] 6. Strain construction

[0325] The plasmid pMEL10-LP2.T10 and the gene expression cassette ydr541cus-GPD1p-CYP80Q4-IDP1t-CCW12p-PbDCR-CYC1t-ydr541c ds were transformed into XJ06398 yeast competent cells to obtain positive clones. Randomly selected positive clones were streaked onto 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were from strains that had lost the gRNA plasmid, i.e., the gene expression cassette ydr541cus-GPD1p-CYP80Q4-IDP1t-CCW12p-PbDCR-CYC1t-ydr541c ds. The ds were integrated into the ydr541c site (when the ydr541c site is knocked out, the exogenous gRNA cleavage site LP2.T10 is introduced simultaneously. Therefore, when introducing the gene expression cassette ydr541cus-GPD1p-CYP80Q4-IDP1t-CCW12p-PbDCR-CYC1t-ydr541c ds, the required plasmid is pMEL10-LP2.T10), resulting in strain XJ06399.

[0326] (ii) Add four methyltransferases (CNMT6, T5, T7 and OMT10)

[0327] 1. Construction of plasmid pMEL10-XI-2

[0328] Following the method in step 1(a), replace XI-5-gRNA-F with XI-2-gRNA-F and XI-5-gRNA-R with XI-2-gRNA-R, while keeping all other steps unchanged, to obtain plasmid pMEL10-XI-2.

[0329] 2. Gene expression cassette construction

[0330] (1) The coding gene of upstream homologous arm XI-2 us (nucleotide sequence as shown in SEQ ID NO:71), promoter PDC1p (nucleotide sequence as shown in SEQ ID NO:72), CNMT6 (nucleotide sequence as shown in SEQ ID NO:73), terminator ENO2t and terminator IDP1t were connected by the overlap method to obtain the gene expression cassette XI-2 us-PDC1p-CNMT6-ENO2t-IDP1t.

[0331] (2) The terminator ENO2t, terminator IDP1t, the coding gene of T5 (nucleotide sequence as shown in SEQ ID NO:74), promoter GPM1p and promoter TPI1p were linked by the overlap method to obtain the gene expression cassette ENO2t-IDP1t-T5-GPM1p-TPI1p.

[0332] (3) The promoters GPM1p, TPI1p, T7 (nucleotide sequence as shown in SEQ ID NO:75), terminator ADH1t and terminator FBA1t were linked by the overlap method to obtain the gene expression cassette GPM1p-TPI1p-T7-ADH1t-FBA1t.

[0333] (4) The terminator ADH1t, terminator FBA1t, the coding gene of OMT10 (nucleotide sequence as shown in SEQ ID NO:76), promoter PGK1p and downstream homologous arm XI-2 ds (nucleotide sequence as shown in SEQ ID NO:77) were linked using the overlap method to obtain the gene expression cassette ADH1t-FBA1t-OMT10-PGK1p-XI-2 ds.

[0334] 3. Strain construction

[0335] Plasmids pMEL10-XI-2, gene expression cassettes XI-2 us-PDC1p-CNMT6-ENO2t-IDP1t, ENO2t-IDP1t-T5-GPM1p-TPI1p, GPM1p-TPI1p-T7-ADH1t-FBA1t, and ADH1t-FBA1t-OMT10-PGK1p-XI-2 ds were transformed into XJ06399 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmids. The four methyltransferases CNMT6, T5, T7, and OMT10 were integrated into the XI-2 site to obtain strain XJ06320.

[0336] (III) De novo synthesis of isotetracycline and obaberine in yeast

[0337] 1. Strain construction

[0338] The plasmid pMEL10-XII-4 and gene expression cassette XII-4us-CCW12p-CYP82BC1-PGI1t-XII-4 ds constructed in step 3 were transformed into XJ06320 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The gene expression cassette XII-4us-CCW12p-CYP82BC1-PGI1t-XII-4 ds was integrated into the XII-4 site to obtain strain XJ06326.

[0339] Following the steps described above, the gene expression cassettes XII-4 us-CCW12p-CYP82BC1-PGI1t-XII-4 ds were replaced with the gene expression cassettes XII-4 us-CCW12p-CYP82BC2-PGI1t-XII-4 ds, XII-4 us-CCW12p-CYP82BC3-PGI1t-XII-4 ds, and XII-4 us-CCW12p-CYP82BC4-PGI1t-XII-4 ds constructed in step three, respectively, to obtain strains XJ06327, XJ06325, and XJ06328.

[0340] 2. Three single clones of strains XJ06325, XJ06326, XJ06327, and XJ06328 were selected for parallel experiments. 1 ml of Delft medium (solutes and concentrations of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and 60 mg / L uracil, solvent: water) was added to a 14 mL shaker tube. A single clone was then inoculated and cultured at 30°C and 220 rpm for 18–24 h to obtain the bacterial culture. The bacterial culture was then transferred to a 125 mL shaker flask containing 20 mL of Delft medium for further culture and incubated at 30°C and 220 rpm for 72 h to obtain the fermentation broth. The OD of the fermentation broth was measured. 600nm .

[0341] 3. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000g for 5 min, collect the supernatant and dilute to an appropriate concentration for LC-MS / MS detection.

[0342] Test results are shown Figure 8 and Figure 9 The results showed that strains XJ06325, XJ06326, XJ06327, and XJ06328 could all produce isotetracycline, with strain XJ06325 producing the highest yield of isotetracycline, reaching 3.5 mg / L. Furthermore, strains XJ06326 and XJ06328 were found to synthesize another bisbenzylisoquinoline alkaloid, obaberine, with the same m / z as isotetracycline.

[0343] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A recombinant bacterium, characterized in that: The recombinant bacteria contain or express tyrosine hydroxylase and / or dopa decarboxylase in vivo.

2. The recombinant bacteria according to claim 1, characterized in that: The recombinant bacteria also contain or express norcodonol alkaloid synthase or N-terminally truncated norcodonol alkaloid synthase.

3. The recombinant bacteria according to claim 1 or 2, characterized in that: The recombinant bacteria also contain or express tyrosine metabolic flux-related proteins.

4. The recombinant bacteria according to any one of claims 1 to 3, characterized in that: The recombinant bacteria also have proteins associated with the synthesis of 4-hydroxyphenylethanol and / or 4-hydroxyphenylacetic acid knocked out or downregulated in vivo.

5. The recombinant bacteria according to any one of claims 1 to 4, characterized in that: The recombinant bacteria also contain or express aromatic amino acid decarboxylases.

6. The recombinant bacteria according to any one of claims 1 to 5, characterized in that: The recombinant bacteria also contain or express prephenylacetic acid aminotransferase and alloacetic acid dehydratase.

7. The recombinant bacteria according to any one of claims 1 to 6, characterized in that: The recombinant bacteria also contain or express methyltransferase 6OMT, isomerase, P450 reduction chaperone, and / or dimerase.

8. The recombinant bacteria according to any one of claims 1 to 7, characterized in that: The recombinant bacteria also contain or express P450 cyclase.

9. The recombinant bacteria according to any one of claims 1 to 8, characterized in that: The recombinant bacteria also contain or express methyltransferases CNMT6, T5, T7 and OMT10.

10. The recombinant bacteria according to any one of claims 1 to 9, characterized in that: The recombinant strain is a species of Saccharomyces cerevisiae.

11. The recombinant bacteria according to any one of claims 1 to 10, characterized in that: The recombinant strain is Saccharomyces cerevisiae.

12. The use of the recombinant bacteria according to any one of claims 1 to 11 in the production of dibenzylisoquinoline alkaloids or in increasing the yield of dibenzylisoquinoline alkaloids.

13. A method for producing dibenzylisoquinoline alkaloids, comprising the following steps: fermenting and culturing the recombinant bacteria according to any one of claims 1 to 11, collecting the fermentation product, and obtaining dibenzylisoquinoline alkaloids therefrom.

14. The application according to claim 12 or the method according to claim 13, characterized in that: The bisbenzylisoquinoline alkaloids are (R,S)-bisnoraromoline, bisnorobamegine, isotetracycline, and / or obaberine.

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