Recombinant bacterium and application thereof in production of corydaline

Abstract

The invention discloses a recombinant bacterium and application thereof in production of benzylisoquinoline alkaloid. The recombinant bacterium contains berberbridge enzyme of which the activity is remarkably improved, de novo synthesis of the corydalis aureoflavum alkali and the benzylisoquinoline alkaloid is realized through multiple strategies, and the yield of the corydalis aureoflavum alkali and the benzylisoquinoline alkaloid is remarkably improved. The recombinant strain provided by the invention can be used for efficiently producing the corydaline and other benzylisoquinoline alkaloids, and has important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a recombinant bacterium and application thereof in producing chrysanthemine. Background Art

[0002] Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites with important research and medicinal value, including antitussive and anticancer noscapine, analgesics morphine and codeine, etc. At present, BIA compounds are mainly extracted from plants or chemically synthesized, but due to the low content of plant secondary metabolism and the existence of multiple stereo conformations of compounds, extraction, purification and chemical synthesis are very limited. Biosynthesis in microbial heterologous hosts has great potential to overcome these production barriers. The production of BIA compounds using Saccharomyces cerevisiae as a host has attracted widespread attention. However, it is still very challenging to reconstruct long heterologous pathways in yeast, and multiple optimizations are required, including identifying missing key enzymes, repositioning through enzyme engineering, or improving the efficiency of rate-limiting enzymes through cofactor engineering to increase production.

[0003] Scoulerine ((S)-Scoulerine) is an important intermediate that can further generate various types of compounds such as tetrahydropalmatine, sanguinarine, and noscapine. Therefore, constructing an engineered strain that produces high levels of scoulerine has important application value. Summary of the invention

[0004] The object of the present invention is to provide a recombinant bacterium for increasing the yield of benzylisoquinoline alkaloids (such as chrysocorydaline) or producing benzylisoquinoline alkaloids (such as chrysocorydaline).

[0005] The present invention firstly protects a recombinant bacterium, which may contain berberidase BBE with enhanced activity compared with that of the parent microorganism.

[0006] In the above recombinant bacteria, the recombinant bacteria may also contain or express a fusion protein of BBE and the endoplasmic reticulum C-terminal retention signal peptide HDEL, a fusion protein of BBE and the N-terminal Golgi target sequence, or BBE, preferably a fusion protein of BBE and the endoplasmic reticulum C-terminal retention signal HDEL.

[0007] In the above recombinant bacteria, the BBE may be PsBBE (AAC61839.1) derived from poppy (P. somniferum), CjBBE (BAM44344.1) derived from Coptis japonica (Coptis japonica) or CyBBE derived from Corydalis yanhusuo (Corydalis yanhusuo), preferably CyBBE.

[0008] The examples of the present invention found that CyBBE was transported from the Golgi apparatus to the vacuole, GOTS_CyBBE was retained in the Golgi apparatus, and CyBBE_ERTS was retrograded from the Golgi apparatus to the endoplasmic reticulum, thereby increasing the production of chrysocortin by 3 times.

[0009] In the above recombinant bacteria, the fusion protein of BBE and the endoplasmic reticulum C-terminal retention signal HDEL may be contained or expressed in vivo by introducing the encoding nucleic acid of the fusion protein into the starting bacteria.

[0010] In the above recombinant bacteria, the fusion protein of BBE and N-terminal Golgi target sequence may be contained or expressed in vivo by introducing the encoding nucleic acid of the fusion protein into the starting bacteria.

[0011] In the above-mentioned recombinant bacteria, the BBE may be contained or expressed in vivo by introducing the encoding nucleic acid of the BBE into the starting bacteria.

[0012] In the above recombinant bacteria, the body may contain or express tyrosine hydroxylase and dopa decarboxylase.

[0013] In the above recombinant bacteria, the tyrosine hydroxylase and dopa decarboxylase are contained or expressed in the body by introducing the tyrosine hydroxylase encoding nucleic acid and the dopa decarboxylase encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria.

[0014] In the above-mentioned recombinant bacteria, the introduction of the tyrosine hydroxylase encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria may be the introduction of an expression cassette containing the tyrosine hydroxylase encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria. The expression cassette containing the tyrosine hydroxylase encoding nucleic acid may include a promoter, a tyrosine hydroxylase encoding nucleic acid and a terminator.

[0015] In the above-mentioned recombinant bacteria, the introduction of the coding nucleic acid of DOPA decarboxylase into the starting bacteria or any of the above-mentioned recombinant bacteria may be the introduction of an expression cassette containing the coding nucleic acid of DOPA decarboxylase into the starting bacteria or any of the above-mentioned recombinant bacteria. The expression cassette containing the coding nucleic acid of DOPA decarboxylase may include a promoter, the coding nucleic acid of DOPA decarboxylase and a terminator.

[0016] In the above recombinant bacteria, the promoter may be TDH3p, CCW12p or TEF1p. The terminator may be IDP1t, TPS1t or ENO2t.

[0017] 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 a plasmid and / or integrated into the chromosome.

[0018] Specifically, the recombinant bacteria can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding tyrosine hydroxylase and an expression cassette containing a nucleic acid encoding dopa decarboxylase into the XI-5 site of the starting bacteria or any of the above-mentioned recombinant bacteria using a genome editing method.

[0019] 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.

[0020] In the above recombinant bacteria, the DOPA decarboxylase may specifically be DODC (genbank number: DAA64376.1).

[0021] Any of the above-mentioned recombinant bacteria may also contain or express higenamine synthase NCS or N-terminally truncated NCS enzyme.

[0022] In the above recombinant bacteria, the NCS enzyme may be CjNCS from Coptis japonica (genbank number: BAF45338.2).

[0023] In the above-mentioned recombinant bacteria, the N-terminally truncated NCS enzyme may 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.

[0024] In the above recombinant bacteria, the said body further contains or expresses higenamine synthase NCS or N-terminally truncated NCS enzyme, which can be obtained by introducing the encoding nucleic acid of NCS enzyme or the encoding nucleic acid of N-terminally truncated NCS enzyme into the starting bacteria or any of the above recombinant bacteria.

[0025] In the above-mentioned recombinant bacteria, the introduction of the coding nucleic acid of the NCS enzyme or the coding nucleic acid of the NCS enzyme with N-terminal truncations into the starting bacteria or any of the above-mentioned recombinant bacteria may be the introduction of an expression cassette containing the coding nucleic acid of the NCS enzyme or the coding nucleic acid of the NCS enzyme with N-terminal truncations into the starting bacteria or any of the above-mentioned recombinant bacteria. The expression cassette containing the coding nucleic acid of the NCS enzyme or the coding nucleic acid of the NCS enzyme with N-terminal truncations may include a promoter, the coding nucleic acid of the NCS enzyme or the coding nucleic acid of the NCS enzyme with N-terminal truncations, and a terminator.

[0026] In the above recombinant bacteria, the promoter may be TEF1p and the terminator may be TDH2t.

[0027] 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 a plasmid and / or integrated into the chromosome.

[0028] In the above recombinant bacteria, the NCS enzyme or N-terminally truncated NCS enzyme contained or expressed in the body may be increased copy number of the nucleic acid encoding the NCS enzyme or N-terminally truncated NCS enzyme in the starting bacteria or any of the above recombinant bacteria.

[0029] In the above recombinant bacteria, the number of copies of the nucleic acid encoding the N-terminally truncated NCS enzyme may be 3, 2 or 1, preferably 3.

[0030] Any of the above-mentioned recombinant bacteria may also contain or express tyrosine metabolic flux-related proteins.

[0031] In the above recombinant bacteria, the tyrosine metabolic flux-related protein can be EcAROL (genbank number NP_414922.1) from Escherichia coli, ARO4* (mutant ARO4 of yeast endogenous gene ARO4) K229L ), ARO7* (mutant of yeast endogenous gene ARO7 ARO7 G141S ), MtPDH1 from alfalfa (genbank number KM507076.1), ARO1 (endogenous yeast gene), ARO2 (endogenous yeast gene) and / or ARO3 (endogenous yeast gene).

[0032] In the above-mentioned recombinant bacteria, the tyrosine metabolic flux-related protein may be contained or expressed in vivo by introducing the encoding nucleic acid of the tyrosine metabolic flux-related protein into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0033] In the above-mentioned recombinant bacteria, the introduction of the nucleic acid encoding the protein related to tyrosine metabolic flux into the starting bacteria or any of the above-mentioned recombinant bacteria may be the introduction of the expression cassette of the nucleic acid encoding the protein related to tyrosine metabolic flux into the starting bacteria or any of the above-mentioned recombinant bacteria. The expression cassette may include a promoter, the nucleic acid encoding the protein related to tyrosine metabolic flux and a terminator.

[0034] In the above recombinant bacteria, the promoter may be TPI1p, PGK1p, TEF1p, TDH3p or tHXT7p. The terminator may be pYX212t, ADH1t, FBA1t, CYC1t or TDH2t.

[0035] 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 a plasmid and / or integrated into the chromosome.

[0036] In any of the above-mentioned recombinant bacteria, proteins related to the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid can also be knocked out or down-regulated.

[0037] In the above recombinant bacteria, the protein related to the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, YDR541C, AAD3, GRE2 and / or HFD1.

[0038] In the above recombinant bacteria, the proteins related to the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, GRE2 and HFD1.

[0039] Any of the above-mentioned recombinant bacteria may also contain or express aromatic amino acid decarboxylase.

[0040] In the above-mentioned recombinant bacteria, the aromatic amino acid decarboxylase may be PcAAS derived from Petroselinum crispum, RrAAS derived from Rhodiola Salidroside, or a mutant PsAAAD* derived from P. somniferum, preferably PsAAAD*.

[0041] In the above-mentioned recombinant bacteria, the aromatic amino acid decarboxylase may be contained or expressed in the body by introducing the encoding nucleic acid of the aromatic amino acid decarboxylase into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0042] In the above-mentioned recombinant bacteria, the introduction of the encoding nucleic acid of the aromatic amino acid decarboxylase into the starting bacteria or any of the above-mentioned recombinant bacteria can be the introduction of an expression cassette containing the encoding nucleic acid of the aromatic amino acid decarboxylase into the starting bacteria or any of the above-mentioned recombinant bacteria. The expression cassette may include a promoter, the encoding nucleic acid of the aromatic amino acid decarboxylase and a terminator.

[0043] The promoter may be FBA1p. The terminator may be TPS1t-ADH1t.

[0044] 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 a plasmid and / or integrated into the chromosome.

[0045] Any of the above-mentioned recombinant bacteria 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.

[0046] In the above-mentioned recombinant bacteria, the further containing or expressing of PAT and ADH in vivo may be achieved by introducing the nucleic acid encoding PAT and the nucleic acid encoding ADH into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0047] In the above recombinant bacteria, the introduction of the PAT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria may be the introduction of an expression cassette containing the PAT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria. The expression cassette may include a promoter, a PAT encoding nucleic acid and a terminator.

[0048] In the above-mentioned recombinant bacteria, the introduction of the ADH encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria may be the introduction of an expression cassette containing the ADH encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria. The expression cassette may include a promoter, an ADH encoding nucleic acid and a terminator. The promoter may be TDH3p, TEF2p or FBA1p. The terminator may be TDH2t or pYX212t.

[0049] 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 a plasmid and / or integrated into the chromosome.

[0050] Preferably, any of the above-mentioned recombinant bacteria may also contain or express AtPAT and MtncADH.

[0051] Any of the above-mentioned recombinant bacteria may also contain or express methyltransferase 6OMT and clavenine methyltransferase CNMT.

[0052] In the above recombinant bacteria, the 6OMT may be Ps6OMT (AAQ01669.1) from P. somniferum or Cy6OMT from C. yanhusuo. The CNMT may be PsCNMT (AAP45316.1) or CyCNMT (nucleotide sequence as shown in SEQ ID NO: 18) from P. somniferum.

[0053] In the above-mentioned recombinant bacteria, the methyltransferase 6OMT and the methyltransferase CNMT are also contained or expressed in the body, which can be obtained by introducing the encoding nucleic acid of 6OMT and the encoding nucleic acid of CNMT into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0054] In the above recombinant bacteria, the introduction of the 6OMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria may be the introduction of an expression cassette containing the 6OMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria. The expression cassette may include a promoter, the 6OMT encoding nucleic acid and a terminator.

[0055] In the above recombinant bacteria, the introduction of the CNMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria may be the introduction of an expression cassette containing the CNMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria. The expression cassette may include a promoter, a CNMT encoding nucleic acid and a terminator.

[0056] Any of the above recombinant bacteria may also contain or express NMCH in vivo. The NMCH may be EcNMCH (AAC39452.1) or CyNMCH derived from Eschscholzia californica.

[0057] In the above-mentioned recombinant bacteria, the in vivo further containing or expressing NMCH may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria by introducing the encoding nucleic acid of NMCH. The introduction of the encoding nucleic acid of NMCH into the starting bacteria or any of the above-mentioned recombinant bacteria may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria by introducing an expression cassette containing the encoding nucleic acid of NMCH into the starting bacteria or any of the above-mentioned recombinant bacteria. The expression cassette may include a promoter, the encoding nucleic acid of NMCH and a terminator.

[0058] The promoter may be TPI1p, PGK1p, TEF1p, tHXT7p, TDH3p, GPM1p or TEF2p.

[0059] The terminator may be pYX212t, ADH1t, CYC1t, FBA1t, TDH2t, PGI1t or ENO2t.

[0060] 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 a plasmid and / or integrated into the chromosome.

[0061] Preferably, any of the above-mentioned recombinant bacteria may also contain or express Ps6OMT, PsCNMT and CyNMCH.

[0062] In the above-mentioned recombinant bacteria, the NMCH further contained or expressed in vivo may be increased copy number of the nucleic acid encoding NMCH in the starting bacteria or any of the above-mentioned recombinant bacteria.

[0063] In the above recombinant bacteria, the copy number of the nucleic acid encoding NMCH may be 1-5, preferably 5.

[0064] Any of the above-mentioned recombinant bacteria may also contain or express methyltransferase 4'OMT.

[0065] In the above recombinant bacteria, the 4'OMT may be Ps4'OMT.

[0066] In the above recombinant bacteria, the further containing or expressing of 4'OMT in vivo may be achieved by introducing a 4'OMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria.

[0067] In the above recombinant bacteria, the introduction of the 4'OMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria may be the introduction of an expression cassette containing the 4'OMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria. The expression cassette may include a promoter, the 4'OMT encoding nucleic acid and a terminator.

[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 a plasmid and / or integrated into the chromosome.

[0069] In the above recombinant bacteria, the further containing or expressing of 4'OMT in vivo may be to increase the copy number of the nucleic acid encoding 4'OMT in the starting bacteria or any of the above recombinant bacteria.

[0070] In the above recombinant bacteria, the copy number of the nucleic acid encoding 4'OMT may be 2 or 1, preferably 2.

[0071] Any of the above-mentioned recombinant bacteria may also contain or express cytochrome P450 reductase ATR1 (nucleotide sequence as shown in SEQ ID NO: 55), ATR2 (nucleotide sequence as shown in SEQ ID NO: 56) or CPR, preferably ATR1.

[0072] In the above recombinant bacteria, the CPR may be PsCPR.

[0073] In the above-mentioned recombinant bacteria, the further containing or expressing ATR1 in the body may be the introduction of ATR1 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0074] In the above-mentioned recombinant bacteria, the further containing or expressing ATR2 in the body may be the introduction of ATR2 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0075] In the above-mentioned recombinant bacteria, the in vivo presence or expression of CPR may be achieved by introducing the encoding nucleic acid of CPR into the starting bacteria or any of the above-mentioned recombinant bacteria. The introduction of the encoding nucleic acid of CPR into the starting bacteria or any of the above-mentioned recombinant bacteria may be achieved by introducing the expression cassette containing the encoding nucleic acid of CPR into the starting bacteria or any of the above-mentioned recombinant bacteria. The introduction of ATR1 into the starting bacteria or any of the above-mentioned recombinant bacteria may be achieved by introducing the expression cassette containing ATR1 into the starting bacteria or any of the above-mentioned recombinant bacteria. The introduction of ATR2 into the starting bacteria or any of the above-mentioned recombinant bacteria may be achieved by introducing the expression cassette containing ATR2 into the starting bacteria or any of the above-mentioned recombinant bacteria. 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 a plasmid and / or integrated into the chromosome.

[0076] In the above recombinant bacteria, the introduction of the encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria may be the introduction of an expression cassette of the encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria. The expression cassette may include a promoter, an encoding nucleic acid and a terminator.

[0077] In the above recombinant bacteria, the promoter may be CCW12p and the terminator may be PGI1t.

[0078] 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 a plasmid and / or integrated into the chromosome.

[0079] Any of the above-mentioned recombinant bacteria may also contain or express catalase.

[0080] In the above recombinant bacteria, the catalase may be PRDX4.

[0081] In the above-mentioned recombinant bacteria, the inclusion or expression of catalase in the body may be achieved by introducing a nucleic acid encoding catalase into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0082] In the above-mentioned recombinant bacteria, the introduction of the encoding nucleic acid of catalase into the starting bacteria can be the introduction of the expression cassette of the encoding nucleic acid of catalase peroxiredoxinIV (α-mPRDX4_ERTS) with yeast α mating factor signal peptide fused to the N-terminus and ERTS signal peptide fused to the C-terminus into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0083] Any of the above-mentioned recombinant bacteria may also contain or express transcription factors that activate phospholipid biosynthesis or knock out transcription factors that inhibit phospholipid biosynthesis.

[0084] In the above recombinant bacteria, the knocked-out transcription factor that inhibits phospholipid biosynthesis may be the OPI1 gene.

[0085] In the above recombinant bacteria, the transcription factor that activates phospholipid biosynthesis may be the INO2 gene or the INO2 gene mutant INO2* (INO2_L119A).

[0086] The starting bacteria may be Saccharomyces cerevisiae. The Saccharomyces cerevisiae may specifically be the IMX581 yeast strain.

[0087] Any of the above-mentioned recombinant bacteria is a species of the genus Saccharomyces cerevisiae.

[0088] Any of the above-mentioned recombinant bacteria can be Saccharomyces cerevisiae.

[0089] The use of any of the above-mentioned recombinant bacteria in the production of chrysocolline, chondrine, norcoclaurine, chondrine, tetrahydroberberine, bamipine, berberine and / or papaverine also falls within the scope of protection of the present invention.

[0090] The present invention also protects a method for producing benzylisoquinoline alkaloids, which comprises fermenting and culturing any of the above-mentioned recombinant bacteria, collecting the fermentation product, and selectively purifying and collecting to obtain chrysoberyl, chondrine, norcoclaurine, chondrine, tetrahydroberberine, bamipine, berberine and / or papaverine.

[0091] In the above method, the benzylisoquinoline alkaloid is chrysocotyleine, cyperine, norcoclaurine, coclaurine, tetrahydroberberine, palmatine, berberine and / or papaverine.

[0092] The inventor of the present invention has constructed a recombinant bacterium for producing scoulerine after a large number of experiments. The synthetic pathway of scoulerine is divided into three modules for research, and the de novo synthesis of scoulerine is achieved by a variety of strategies, wherein the starting strain is the IMX581 yeast strain. Specifically, the present invention uses the NCS of norcoclaurine synthase to catalyze the formation of CC bonds between dopamine and 4-HPAA molecules and remove a molecule of water to produce the intermediate norcoclaurine ((S)-Norcoclaurine), and then generates scoulerine ((S)-Reticuline) through 3 methyltransferases (6OMT, CNMT and 4'OMT, respectively) and a P450 enzyme (NMCH), and finally catalyzes scoulerine to form scoulerine ((S)-Scoulerine) through BBE. Experiments have shown that the engineering strain constructed by the present invention can efficiently produce scoulerine. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 The biosynthetic pathway of golden corydaline.

[0094] Figure 2 The pathway and modification strategy for the de novo production of higenamine (module I) in Saccharomyces cerevisiae and the detection results of (v)-(viii) in step one.

[0095] Figure 3 These are the test results of (ii) and (iii) in step 1 of the embodiment.

[0096] Figure 4 This is the test result of step (VII) in step 1 of the embodiment.

[0097] Figure 5 This is the test result of step (eight) in the embodiment.

[0098] Figure 6 The optimization pathway and modification strategy for producing the taurocholine module (module II) and the detection results of (iv) and (v) in step 2.

[0099] Figure 7 This is the test result of step 2 (ii) in the embodiment.

[0100] Figure 8 The pathways and transformation strategies for BBE screening and optimization (module III) and the test results of steps (ii)-(iv) in step three.

[0101] Fig. 9 This is the test result of step 3 (a) in the embodiment. DETAILED DESCRIPTION

[0102] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0103] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0104] The names of the primers and their nucleotide sequences involved in the following examples are shown in Table 1.

[0105] Table 1

[0106]

[0107]

[0108]

[0109]

[0110] The strain names and genotypes involved in the following examples are shown in Table 2.

[0111] Table 2

[0112]

[0113]

[0114]

[0115] 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.

[0116] LC-MS / MS was performed on a Shimadzu Nexera UHPLC system, the mass spectrometer was a Sciex QTRAP 6500+, and the column was a Luna Omega 1.6 μm polar C18 Column (Phenomenex). Mobile phase A was 0.1% formic acid. Mobile phase B was acetonitrile, and the flow rate was set to 0.4 mL / min. The mass spectrometry conditions were set as follows: Curtain Gas (50); Collision Gas (Medium); IonSpray Voltage: -4500 V (4-HP acid and Tyrosol), 4500 V (Dopamine, S-NOR and S-RET); Temperature (450°C). The mobile phase gradient was: 0-4 min, 2% B-10% B, 4-6 min, 10% B-85% B; 6-7 min, 85% B; 7-7.1 min 85% B-2% B; 7.1-9 min, 2% B. Qualitative detection was performed in MRM mode, and quantitative detection was performed in Q3 MI mode or MS2 mode.

[0117] In the following examples, plasmid pMEL10 is a product of EUROSCARF collection, acc.no.P30779. Plasmid pMEL10 carries SNR52 promoter and gRNA backbone.

[0118] Example

[0119] The inventors of the present invention have constructed a recombinant bacterium for producing golden corydaline after a large number of experiments. The synthetic pathway of golden corydaline is divided into three modules for research, and the de novo synthesis of golden corydaline is achieved through a variety of strategies; wherein the starting strain is the IMX581 yeast strain, which is a cerevisiae strain with the Cas9 gene integrated under the TEF1 promoter, and the genotype is MATa ura3-52can1Δ::cas9-natNT2 TRP1 LEU2 HIS3, CEN.PK113-5D. In the following, the IMX581 yeast strain is referred to as IMX581.

[0120] The biosynthetic pathway of golden corydaline is shown in Figure 1 .

[0121] 1. De novo production of higenamine in Saccharomyces cerevisiae (Module I)

[0122] The approaches and transformation strategies for module 1 are shown in Figure 2 .

[0123] (I) Construction of XJ001 strain producing dopamine

[0124] IMX581 was coated on a YPD solid plate and inverted at 30°C for 48-72 hours to obtain a newly activated IMX581 yeast colony. Then, the gene fragment BvCYP76AD1* (genbank number AET43289.1) and the gene fragment DODC (genbank number DAA64376.1; the gene fragment DODC encodes dopa decarboxylase) from beet (Betavulgaris) were integrated into the XI-5 site of IMX581 using the CRISPR-Cas9 genome editing method.

[0125] 1. Construction of plasmid pMEL10-XI-5

[0126] The gRNA plasmid was constructed using the Gibson assembly method. The specific steps are as follows:

[0127] (1) Using plasmid pMEL10 as a template, PCR amplification was performed using a primer pair consisting of Vectorbackbone-F and Vectorbackbone-R to obtain linearized plasmid pMEL10.

[0128] (2) Artificially synthesizing XI-5-gRNA-F and XI-5-gRNA-R, then mixing them in equal amounts, treating them at 95° C. for 10 min, and cooling them to room temperature to obtain an annealed short nucleic acid sequence, which includes a 20 bp sequence homologous to the SNR52 promoter, a 20 bp gRNA sequence, and a 20 bp sequence homologous to the gRNA backbone.

[0129] (3) The annealed short nucleic acid sequence and the linearized plasmid pMEL10 were assembled according to standard Gibson method to obtain plasmid pMEL10-XI-5.

[0130] 2. Preparation of repair fragment (DonorDNA)

[0131] A. The repair fragment XI-5us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p is composed of upstream homology arm XI-5us (nucleotide sequence as shown in SEQ ID NO:33), terminator IDP1t (nucleotide sequence as shown in SEQ ID NO:3), gene fragment BvCYP76AD1*, promoter TDH3p (nucleotide sequence as shown in SEQ ID NO:1) and promoter CCW12p (nucleotide sequence as shown in SEQ ID NO:2) from 5' to 3'. The specific operation of preparing the repair fragment XI-5us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p by overlap method is as follows:

[0132] (1) The yeast genome or the synthetic gene was used as a template to amplify the upstream homology arm XI-5us, the terminator IDP1t, the gene fragment BvCYP76AD1*, the promoter TDH3p and the promoter CCW12p.

[0133] (2) The upstream homology arm XI-5us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p and promoter CCW12p were connected using the overlap system to obtain a template.

[0134] (2-1) Preparation of overlap system. The overlap system was 25 μL, consisting of 5 μL 5* PrimeSTAR buffer, 2 μL dNTP (2.5 mM each), upstream homology arm XI-5us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p, promoter CCW12p, Polymerase 0.5 μL and ddH 2 In the overlap system, the concentration of the upstream homology arm XI-5us was 0.03 pmol, and the molar ratio of the upstream homology arm XI-5us, the terminator IDP1t, the gene fragment BvCYP76AD1*, the promoter TDH3p and the promoter CCW12p was 1:3:5:3:1.

[0135] (2-2) Take the overlap system, react and obtain a product.

[0136] The reaction conditions were as follows: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 2 min, 15 cycles; and finally extension at 70°C for 10 min.

[0137] (2-3) Take 2 μl of the product obtained in step (2-2) and use it as a template to perform PCR amplification using a primer pair consisting of XI-5us-F and CCW12p-DODC-R1 to obtain the repair fragment XI-5us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p.

[0138] The reaction conditions were as follows: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 4 min, 35 cycles; and finally extension at 70°C for 10 min.

[0139] B. The repair fragment CCW12p-DODC-TPS1t-XI-5ds is composed of the promoter CCW12p, the gene fragment DODC, the terminator TPS1t (nucleotide sequence as shown in SEQ ID NO: 4) and the downstream homology arm XI-5ds (nucleotide sequence as shown in SEQ ID NO: 34) from 5' to 3'. The specific operation of preparing the repair fragment CCW12p-DODC-TPS1t-XI-5ds by the overlap method is as follows:

[0140] (1) The promoter CCW12p, gene fragment DODC, terminator TPS1t and downstream homology arm XI-5ds were amplified using yeast genome or synthetic genes as templates.

[0141] (2) The promoter CCW12p, gene fragment DODC, terminator TPS1t and downstream homology arm XI-5ds were connected using the overlap system to obtain a template.

[0142] (2-1) Prepare the overlap system. The overlap system is 25 μL, consisting of 5 μL 5*Prime STAR buffer, 2 μL dNTP (2.5 mM each), promoter CCW12p, gene fragment DODC, terminator TPS1t, downstream homology arm XI-5ds, Polymerase 0.5 μL and ddH 2 In the overlap system, the concentration of the promoter CCW12p was 0.03 pmol, and the molar ratio of the promoter CCW12p, gene fragment DODC, terminator TPS1t and downstream homology arm XI-5ds was 1:3:3:1.

[0143] (2-2) Take the overlap system, react and obtain a product.

[0144] The reaction conditions were as follows: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 2 min, 15 cycles; and finally extension at 70°C for 10 min.

[0145] (2-3) Take 2 μl of the product obtained in step (2-2) and use it as a template to perform PCR amplification using a primer pair consisting of CCW12p1-F and XI-5ds-R to obtain the repair fragment CCW12p-DODC-TPS1t-XI-5ds.

[0146] The reaction conditions were as follows: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 4 min, 35 cycles; and finally extension at 70°C for 10 min.

[0147] 3. Obtaining positive clones

[0148] (1) The plasmid pMEL10-XI-5 obtained in step 1, the repair fragment XI-5us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p and the repair fragment CCW12p-DODC-TPS1t-XI-5ds obtained in step 2 were added to the IMX581 yeast competent cells in sequence according to Table 3, mixed, and treated at 42°C for 30-60 min; centrifuged at room temperature and 10000g for 1 min, the cells were collected and resuspended with 100 μL of sterile water to obtain a resuspension.

[0149] Table 3

[0150] Components Addition amount PEG 3350 (50% (w / v) 260μL LiAc (concentration 1.0M) 36μL Salmon sperm single-stranded DNA (concentration 2.0 mg / ml) 50μL Plasmid pMEL10-XI-5 2μL (about 50ng) Repair fragment XI-5us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p 500-1000ng Repair fragment CCW12p-DODC-TPS1t-XI-5ds 500-1000ng water Refill system to 360 μL

[0151] Note: The repaired fragments must be denatured in a boiling water bath for 5 minutes and then placed on ice for 5 minutes.

[0152] (2) Place a drop of the resuspended liquid in the center of the SD-Ura plate and spread it evenly with a spreader until all the bacterial liquid is completely absorbed. Place the plate in an incubator at 30°C and culture it upside down for 2-3 days.

[0153] SD-Ura plate: add distilled water to 8 g of Ura minus medium and 15 g of agar powder, then dilute to 1 L with distilled water and sterilize at 121°C for 15 min; finally, add glucose sterilized by 0.22 μm filter membrane to a final concentration of 2%, and pour into a sterile plate to cool.

[0154] (2) Single clones were picked from the SD-Ura plate and colony PCR was performed. Specifically, the picked single clones were placed in 20mmol / L NaOH solution, lysed at 99℃ for 15min, then centrifuged for 2min, 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.

[0155] The reaction conditions were as follows: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 1 min, 30 cycles; and finally extension at 70°C for 10 min.

[0156] (3) The PCR amplification products were subjected to agarose gel electrophoresis respectively, and then the following judgment was made: if the PCR amplification product obtained by primer pair a for a single clone contained a DNA fragment of 1056 bp, the PCR amplification product obtained by primer pair b contained a DNA fragment of 919 bp, and the PCR amplification product obtained by primer pair c contained a DNA fragment of 1384 bp, then the single clone was a positive clone.

[0157] 4. Randomly select 3 positive clones and streak them on 5-FOA plates. Culture them in a 30°C incubator for 2-3 days. The plaques that grow are the strains that have lost the gRNA plasmid. The resulting strain is named XJ001 yeast strain (hereinafter referred to as XJ001). The genotype of the XJ001 yeast strain is IMX581 (XI-5::IDP1t-BvCYP76A1*-TDH3p+CCW12p-DODC-TPS1t).

[0158] 5-FOA plate: add distilled water to 8g yeast SC medium and 20g agar powder, then dilute to 1L with distilled water, sterilize at 121°C for 20min; finally, when the temperature of the medium drops to about 40°C, add 5-FOA mother solution (obtained by dissolving 5-fluoroorotic acid (5-FOA) powder with dimethyl sulfoxide (DMSO)) in a sterile clean bench and make the final concentration of 5-FOA 0.1%, pour into a sterile plate and cool.

[0159] (II) Screening of NCS enzymes

[0160] The effects of three N-terminally truncated CjNCS from Coptis japonica (genbank number BAF45338.2) (CjNCSN_Δ24, CjNCSN_Δ29 and CjNCSN_Δ35, i.e., the N-terminus of the amino acid sequence of CjNCS was truncated by 24, 29 and 35 amino acids, respectively) in XJ001 were compared. The efficiency comparison experiment of NCS enzyme was carried out in XJ001.

[0161] The expression cassette XI-3us-TEF1p-CjNCS_Δ24-TDH2t-XI-3ds consists of the upstream homology arm XI-3us (nucleotide sequence as shown in SEQ ID NO:35), the promoter TEF1p (nucleotide sequence as shown in SEQ ID NO:5), the coding gene of CjNCS_Δ24, the terminator TDH2t (nucleotide sequence as shown in SEQ ID NO:6) and the downstream homology arm XI-3ds (nucleotide sequence as shown in SEQ ID NO:36) from 5' to 3'.

[0162] 1. Construction of plasmid pMEL10-XI-3

[0163] According to the method in step (a), XI-5-gRNA-F was replaced with XI-3-gRNA-F, XI-5-gRNA-R was replaced with XI-3-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-XI-3.

[0164] 2. The plasmid pMEL10-XI-3 and expression cassette XI-3us-TEF1p-CjNCS_Δ24-TDH2t-XI-3ds were transferred into XJ001 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the expression cassette XI-3us-TEF1p-CjNCS_Δ24-TDH2t-XI-3ds was expressed.

[0165] us-TEF1p-CjNCS_Δ24-TDH2t-XI-3ds was integrated into the XI-3 site of XJ001, and the resulting strain was named XJ003 yeast strain (hereinafter referred to as XJ003). The genotype of the XJ003 yeast strain was XJ001 (XI-3::

[0166] TEF1p-CjNCS_Δ24-TDH2t).

[0167] 3. Replace the expression cassette XI-3us-TEF1p-CjNCS_Δ24-TDH2t-XI-3ds in step 2 with the expression cassette XI-3us-TEF1p-CjNCS_Δ29-TDH2t-XI-3ds, and keep the other steps unchanged, that is, integrate the expression cassette XI-3us-TEF1p-CjNCS_Δ29-TDH2t-XI-3ds into the XI-3 site of XJ001. The resulting strain is named XJ006 yeast strain (hereinafter referred to as XJ006). The genotype of the XJ006 yeast strain is XJ001 (XI-3::

[0168] TEF1p-CjNCS_Δ29-TDH2t).

[0169] The expression cassette XI-3us-TEF1p-CjNCS_Δ29-TDH2t-XI-3ds consists of the upstream homology arm XI-3us, the promoter TEF1p, the coding gene of CjNCS_Δ29, the terminator TDH2t and the downstream homology arm XI-3ds from 5' to 3'.

[0170] 4. Replace the expression cassette XI-3us-TEF1p-CjNCS_Δ24-TDH2t-XI-3ds in step 2 with the expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3ds, and keep the other steps unchanged, that is, integrate the expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3ds into the XI-3 site of XJ001. The resulting strain is named XJ007 yeast strain (hereinafter referred to as XJ007). The genotype of XJ007 yeast strain is XJ001 (XI-3::TEF1p-CjNCS_Δ35-TDH2t).

[0171] The expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3ds consists of the upstream homology arm XI-3us, the promoter TEF1p, the coding gene of CjNCS_Δ35, the terminator TDH2t and the downstream homology arm XI-3ds from 5' to 3'.

[0172] 5. Pick three single clones from XJ003, XJ006 and XJ007 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0173] 7. Detection of strain metabolic components

[0174] Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0175] Test results see Figure 3 Middle left picture. The results showed that XJ007, an engineered bacterium with the coding gene of CjNCS_Δ35 inserted, produced the highest content of higenamine.

[0176] (III) Tyrosine hydroxylase gene screening

[0177] CYP76AD1* and CYP76AD5 from Beta vulgaris, C3H from Arabidopsis thaliana and Zea mays, and HpaB and HpaC from Pseudomonas aeruginosa and Salmonella enterica were selected for efficiency comparison. The efficiency comparison of tyrosine hydroxylase was carried out in IMX581.

[0178] 1. Construction of plasmid pMEL10-XI-5

[0179] Same as step 1.

[0180] 2. Strain construction

[0181] The overlap 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 promoter is promoter TDH3p, promoter CCW12p or promoter TEF1p; the terminator is terminator TPS1t, terminator IDP1t or terminator ENO2t (nucleotide sequence is shown in SEQ ID NO: 7). The details are as follows:

[0182] (1) The upstream homology arm XI-5us, terminator IDP1t, CYP76AD1*, promoter TDH3p and promoter CCW12p were connected by the overlap method to obtain the gene expression cassette fragment XI-5us-IDP1t-CYP76AD1*-TDH3p-CCW12p (named as fragment 1). The promoter TDH3p, promoter CCW12p, DODC, terminator TPS1t and downstream homology arm XI-5ds were connected by the overlap method to obtain the gene expression cassette fragment TDH3p-CCW12p-DODC-TPS1t-XI-5ds (named as fragment 2). The plasmid pMEL10-XI-5, fragment 1 and fragment 2 obtained in step 1 were transferred into IMX581 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates, and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the genes CYP76AD1* and DODC were integrated into the XI-5 site of IMX581 to obtain strain XJ001.

[0183] (2) According to the method of the above step (1), CYP76AD1* was replaced by CYP76AD5, and the other steps remained unchanged, thereby obtaining the XJ013 strain integrated with CYP76AD5, referred to as XJ013.

[0184] (3) According to the method of the above step (1), CYP76AD1* was replaced by AtC3H, and the other steps remained unchanged, thereby obtaining the XJ011 strain integrated with AtC3H, referred to as XJ011.

[0185] (4) According to the method of the above step (1), CYP76AD1* was replaced by ZmC3H, and the other steps remained unchanged, thereby obtaining the XJ012 strain integrated with ZmC3H, referred to as XJ012.

[0186] (5) According to the method of the above step (1), CYP76AD1* was replaced with PaHpaB-PaHpaC (i.e., the fusion gene of PaHpaB and PaHpaC), and the other steps remained unchanged, thereby obtaining the XJ014 strain integrated with PaHpaB-PaHpaC, referred to as XJ014.

[0187] (6) According to the method of the above step (1), CYP76AD1* was replaced with PaHpaB-SeHpaC (i.e., the fusion gene of PaHpaB and SeHpaC), and the other steps remained unchanged, thereby obtaining the XJ015 strain integrated with PaHpaB-SeHpaC, referred to as XJ015.

[0188] (7) According to the method of the above step (1), CYP76AD1* was replaced with PaHpaC-PaHpaB (i.e., the fusion gene of PaHpaC and PaHpaB), and the other steps remained unchanged, thereby obtaining the XJ016 strain integrated with PaHpaC-PaHpaB, referred to as XJ016.

[0189] (8) According to the method of the above step (1), CYP76AD1* was replaced with SeHpaC-PaHpaB (i.e., the fusion gene of SeHpaC and PaHpaB), and the other steps remained unchanged, thereby obtaining the XJ017 strain integrated with SeHpaC-PaHpaB, referred to as XJ017.

[0190] (9) The upstream homology arm XI-5us, promoter TEF1p, PaHpaC, terminator ENO2t and terminator IDP1t were connected by the overlap method to obtain XI-5us-TEF1p-PaHpaC-ENO2t-IDP1t (named as fragment 3). The terminator ENO2t, terminator IDP1t, PaHpaB, promoter TDH3p and promoter CCW12p were connected by the overlap method to obtain ENO2t-IDP1t-PaHpaB-TDH3p-CCW12p (named as fragment 4); the promoter TDH3p, promoter CCW12p, DODC, terminator TPS1t and downstream homology arm XI-5ds were connected by the overlap method to obtain TDH3p-CCW12p-DODC-TPS1t-XI-5ds (named as fragment 5). The plasmid pMEL10-XI-5, fragments 3, 4 and 5 obtained in step 1 were transferred into IMX581 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates, and cultured in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the XI-5 site of IMX581, and the XJ018 strain integrated with PaHpaC and PaHpaB was obtained, referred to as XJ018.

[0191] (10) According to the method of the above step (9), PaHpaC was replaced by SeHpaC, and the other steps remained unchanged, thereby obtaining the XJ019 strain integrating PaHpaB and SeHpaC, referred to as XJ019.

[0192] 3. Pick three single clones from the strain obtained in step 2 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0193] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0194] Test results see Figure 3 Middle right picture (the strains from left to right are strains XJ001, XJ013, XJ011, XJ012, XJ014, XJ015, XJ016, XJ017, XJ018, and XJ019). The results show that the dopamine content of strain XJ013 is the highest. Therefore, the CYP76AD5 contained in this strain is the tyrosine hydroxylase with higher catalytic efficiency screened in this experiment.

[0195] (IV) Construction of XJ053 strain producing S-Nor

[0196] 1. Construction of XJ023 strain

[0197] Using IMX581 as the starting strain, strain XJ023 was constructed by increasing tyrosine metabolic flux, that is, integrating the target gene related to tyrosine metabolic flux into the chromosome of IMX581. The gene expression cassette consists of a promoter, a nucleic acid sequence of the target gene related to tyrosine metabolic flux, and a terminator. The target genes related to tyrosine metabolic flux are EcAROL from Escherichia coli (genbank number NP_414922.1), ARO4* (a mutant of the yeast endogenous gene ARO4), and ARO4* (a mutant of the yeast endogenous gene ARO4). K229L ), ARO7* (mutant of yeast endogenous gene ARO7 ARO7 G141S ), MtPDH1 (genbank number KM507076.1) from alfalfa, ARO1 (endogenous yeast gene), ARO2 (endogenous yeast gene) and ARO3 (endogenous yeast gene); promoters are promoter TPI1p (nucleotide sequence as shown in SEQ ID NO: 8), promoter PGK1p (nucleotide sequence as shown in SEQ ID NO: 9), promoter TEF1p, promoter TDH3p, promoter tHXT7p (nucleotide sequence as shown in SEQ ID NO: 20); terminators are terminator pYX212t (nucleotide sequence as shown in SEQ ID NO: 10), terminator ADH1t (nucleotide sequence as shown in SEQ ID NO: 11), terminator FBA1t (nucleotide sequence as shown in SEQ ID NO: 12), terminator CYC1t (nucleotide sequence as shown in SEQ ID NO: 13), terminator TDH2t. The specific steps are as follows:

[0198] (1) Construction of plasmid pMEL10-X-2

[0199] According to the method in step (a), XI-5-gRNA-F was replaced with X-2-gRNA-F, and XI-5-gRNA-R was replaced with X-2-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-X-2.

[0200] (2) Construction of plasmid pMEL10-X-4

[0201] According to the method in step (a), XI-5-gRNA-F was replaced with X-4-gRNA-F, and XI-5-gRNA-R was replaced with X-4-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-X-4.

[0202] (3) Plasmid pMEL10-X-2, expression cassette X-2us-TPI1p-EcaroL-pYX212t-ADH1t (composed of upstream homology arm X-2us, promoter TPI1p, nucleic acid sequence of Ecarol, terminator pYX212t and terminator ADH1t), expression cassette pYX212t-ADH1t-ARO7*-PGK1p-TEF1p (composed of terminator pYX212t, terminator ADH1t, nucleic acid sequence of ARO7*, promoter PGK1p and promoter TEF1p), expression cassette

[0203] PGK1p-TEF1p-ARO4*-CYC1t-FBA1t (composed of promoter PGK1p, promoter TEF1p, nucleic acid sequence of ARO4*, terminator CYC1t and terminator FBA1t) and expression cassette CYC1t-FBA1t-MtPDH1-tHXT7p-X-2ds (composed of terminator CYC1t, terminator FBA1t, nucleic acid sequence of MtPDH1, promoter tHXT7p and downstream homology arm X-2ds) was transferred into IMX581 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the X-2 site of IMX581 to obtain intermediate yeast; plasmid pMEL10-X-4, expression cassette X-4us-CYC1t-ARO1-TPI1p-TDH3p (consisting of upstream homology arm X-4us, terminator CYC1t, nucleic acid sequence of ARO1, promoter TPI1p and promoter TDH3p), expression cassette TPI1p-TDH3p-ARO2-ADH1t-TDH2t (consisting of promoter TPI1p, promoter TDH3p, nucleic acid sequence of ARO2, terminator ADH1t and terminator TDH2t) and expression cassette ADH1t-TDH2t-ARO3-TEF1p-X-4ds (consisting of terminator ADH1t, terminator TDH2t, nucleic acid sequence of ARO3, promoter TEF1p and downstream homology arm X-4ds) were transferred into intermediate yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30°C for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the X-4 site of the intermediate yeast, and the XJ023 strain was obtained.

[0204] 2. Construction of XJ053 strain

[0205] Integrate CYP76AD5, DODC and CjNCSN_Δ35 into the XJ023 strain to obtain the XJ053 strain. That is, the plasmid pMEL10-XI-5, the expression cassette XI-5us-IDP1t-BvCYP76AD5-TDH3p-CCW12p (consisting of the upstream homology arm XI-5us, the terminator IDP1t, the coding gene of BvCYP76AD5, the promoter TDH3p and the promoter CCW12p) and the expression cassette CCW12p-DODC-TPS1t-XI-5ds (consisting of the promoter CCW12p, DODC, the terminator TPS1t and the downstream homology arm XI-5ds) were transferred into the XJ023 yeast competent cells to obtain positive clones; randomly select positive clones and streak them on 5-FOA plates, culture them in a 30℃ incubator for 2-3 days, and the clones that grow are lost. The strain that lost the gRNA plasmid was integrated into the XI-5 site of the XJ023 strain to obtain the intermediate yeast; then the plasmid pMEL10-XI-3 and the expression cassette XI-3us-TEF1p-CjNCS_Δ35-TDH2t-XI-3ds (composed of the upstream homologous sequence XI-3us, the promoter TEF1p, the coding gene of CjNCS_Δ35, the terminator TDH2t and the downstream homologous arm XI-3ds) were transferred into the intermediate yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30°C for 2-3 days. The clones that grew were the strains that had lost the gRNA plasmid, that is, integrated into the XI-3 site, and the XJ053 strain was obtained.

[0206] 3. Select three single clones of XJ053 strain for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0207] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0208] The test results showed that the yield of S-NOR produced by strain XJ053 was 23.4 mg / L.

[0209] (V) Transformation of XJ053 strain to increase S-NOR production

[0210] By knocking out the byproduct metabolic flow genes in the XJ053 strain (knocking out ARI1, ADH6, YPR1, YDR541C, AAD3, GRE2 and HFD1), the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid was downregulated, and the S-NOR production was increased, that is, the competitive metabolic flow of aldehyde reductase or dehydrogenase for the substrate 4-HPAA was removed. The above strains were fermented and the S-NOR content was detected. The specific operation steps are as follows:

[0211] 1. Construction of plasmid

[0212] (1) Using the gRAN-SNR52P gene fragment (nucleotide sequence as shown in SEQ ID NO: 32) as a template, PCR amplification was performed using primer pair 1 (consisting of BsaI-ari1-gRNA-F and SNR52p-adh6-gRNA-R), primer pair 2 (consisting of SNR52p-adh6-gRNA-F and SNR52p-ypr1-gRNA-R), primer pair 3 (consisting of BsaI-ydc541c-gRNA-F and SNR52p-aad3-gRNA-R) and primer pair 4 (consisting 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, respectively.

[0213] (2) Gene fragment 1 and gene fragment 2 were Gibson-ligated with linearized plasmid pMEL10-BsaI (obtained by single digestion of plasmid pMEL10 with restriction endonuclease BsaI) to obtain recombinant plasmid pMEL10-ARI1+ADH6+YPR1. Gene fragment 3 was Gibson-ligated with linearized plasmid pMEL10-ARI1 (obtained by single digestion of plasmid pMEL10 with restriction endonuclease ARI1) to obtain recombinant plasmid pMEL10-YDR541C+AAD3. Gene fragment 4 was Gibson-ligated with linearized plasmid pMEL10-ARI1 to obtain recombinant plasmid pMEL10-GRE2+HFD1.

[0214] 2. Strain construction

[0215] The recombinant plasmid pMEL10-ARI1+ADH6+YPR1 and the DNA repair fragment (with a 60 bp overlapping homology arm sequence with the promoter and terminator of the target gene) were transferred into the competent cells of the XJ053 yeast strain to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, namely the XJ054 strain.

[0216] The recombinant plasmid pMEL10-YDR541C+AAD3 and the DNA repair fragment were transferred into the competent cells of the XJ053 yeast strain to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, namely the XJ055 strain.

[0217] The recombinant plasmid pMEL10-GRE2+HFD1 and the DNA repair fragment were transferred into the competent cells of the XJ053 yeast strain to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, namely the XJ056 strain.

[0218] The recombinant plasmid pMEL10-YDR541C+AAD3 and the DNA repair fragment were transferred into the competent cells of the XJ054 yeast strain to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, namely the XJ057 strain.

[0219] The recombinant plasmid pMEL10-GRE2+HFD1 and the DNA repair fragment were transferred into the competent cells of the XJ054 yeast strain to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, namely the XJ058 strain.

[0220] The recombinant plasmid pMEL10-YDR541C+AAD3 and the DNA repair fragment were transferred into the competent cells of the XJ058 yeast strain to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, namely the XJ059 strain.

[0221] 3. Pick three single clones from each strain obtained in step 2 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4, 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0222] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0223] Test results see Figure 2 Middle b. The results showed that the XJ058 strain had a normal growth OD value and the highest S-NOR production; that is, knocking out ARI1, ADH6, YPR1, GRE2, and HFD1 could remove the competitive metabolic flow of aldehyde reductase or dehydrogenase for the substrate 4-HPAA and increase the S-Nor production.

[0224] (VI) CjNCS_Δ35 copy number selection

[0225] One CjNCS_Δ35 gene fragment from Coptis japonica was integrated into the XJ058 strain (already containing one CjNCS_Δ35) to obtain the XJ062 strain, and two CjNC_SΔ35 gene fragments were integrated into the XJ058 strain to obtain the XJ063 strain (i.e., the XJ063 strain contains a total of three CjNC_SΔ35 gene fragments).

[0226] 1. Construction of strains XJ062 and XJ063

[0227] (1) Construction of plasmid pMEL10-XI-1

[0228] According to the method in step (a), XI-5-gRNA-F was replaced with XI-1-gRNA-F, XI-5-gRNA-R was replaced with XI-1-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-XI-1.

[0229] (2) Construction of plasmid pMEL10-LP1.T8

[0230] According to the method in step (a), XI-5-gRNA-F was replaced with LP1.T8-gRNA-F, and XI-5-gRNA-R was replaced with LP1.T8-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-LP1.T8.

[0231] (3) The plasmid pMEL10-XI-1 and the expression cassette XI-1-us-TDH3p-CjNCS_Δ35-CYC1t-XI-1-ds (consisting of the upstream homology arm XI-1-us (nucleotide sequence as shown in SEQ ID NO:51), the promoter TDH3p, the coding gene of CjNCS_Δ35, the terminator CYC1t and the downstream homology arm XI-1-ds (nucleotide sequence as shown in SEQ ID NO:52)) were transformed into the competent cells of the XJ058 strain to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the expression cassette XI-1-us-TDH3p-CjNCS_Δ35-CYC1t-XI-1-ds was integrated into the XI-1 site to obtain the XJ062 strain.

[0232] (4) The plasmid pMEL10-LP1.T8 and the expression cassette LP1.T8-us-TDH3p-CjNCS_Δ35-CYC1t-LP1.T8-ds (composed of the upstream homology arm LP1.T8-us (nucleotide sequence as shown in SEQ ID NO: 53), the promoter TDH3p, the coding gene of CjNCS_Δ35, the terminator CYC1t and the downstream homology arm LP1.T8-ds (nucleotide sequence as shown in SEQ ID NO: 54)) were transformed into the competent cells of the XJ062 strain to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the expression cassette LP1.T8-us-TDH3p-CjNCS_Δ35-CYC1t-LP1.T8-ds was integrated into the LP1.T8 site to obtain the strain XJ063.

[0233] 2. Pick 3 single clones from each strain obtained in step 1 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0234] 3. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0235] Test results see Figure 2 The results showed that the S-NOR content of strain XJ063 was the highest, reaching 214.8 mg / L, that is, the strain with a total of 3 CjNCS_Δ35 integrated had the highest product.

[0236] (VII) Use of aromatic amino acid decarboxylase (AAAD) to direct more product flow to 4-HPAA

[0237] The effects of endogenous Aro10, PcAAS from Petroselinum crispum, RrAAS from Rhodiola Salidroside, and mutant PsAAAD*(Y350F) from P. somniferum were compared in the XJ063 strain. The expression cassette consists of an upstream homology arm, a promoter, a target gene nucleic acid sequence, a terminator, and a downstream homology arm. The promoter is the promoter FBA1p (nucleotide sequence as shown in SEQ ID NO:14), the terminator is the terminator TPS1t-ADH1t (nucleotide sequence as shown in SEQ ID NO:15), and the target genes are the yeast endogenous genes Aro10, PcAAS (AAA33860.1), RrAAS (AUI41111.1), and PsAAAD* (AAC61842.1). The specific operations are as follows:

[0238] 1. Construction of strains

[0239] (1) Construction of plasmid pMEL10-X-3

[0240] According to the method in step (a), XI-5-gRNA-F was replaced with X-3-gRNA-F, and XI-5-gRNA-R was replaced with X-3-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-X-3.

[0241] (2) The plasmid pMEL10-X-3 and the expression cassette X-3us-FBA1p-ARO10-TPS1t-ADH1t-X-3ds (consisting of the upstream homology arm X-3us (nucleotide sequence as shown in SEQ ID NO:41), the promoter FBA1p, the yeast endogenous gene Aro10, the terminator sequence TPS1t-ADH1t and the downstream homology arm X-3ds (nucleotide sequence as shown in SEQ ID NO:42)) were transformed into XJ063 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates, and cultured in a constant temperature incubator at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the expression cassette X-3us-FBA1p-ARO10-TPS1t-ADH1t-X-3ds was integrated into the X-3 site to obtain the XJ0631 strain.

[0242] (3) The plasmid pMEL10-X-3 and the expression cassette X-3us-FBA1p-PcAAS-TPS1t-ADH1t-X-3ds (consisting of the upstream homology arm X-3us, the promoter FBA1p, PcAAS, the terminator sequence TPS1t-ADH1t and the downstream homology arm X-3ds) were transformed into XJ063 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the expression cassette X-3us-FBA1p-PcAAS-TPS1t-ADH1t-X-3ds was integrated into the X-3 site to obtain the XJ0632 strain.

[0243] (4) The plasmid pMEL10-X-3 and the expression cassette X-3us-FBA1p-PsAAAD*-TPS1t-ADH1t-X-3ds (consisting of the upstream homology arm X-3us, the promoter FBA1p, PsAAAD*, the terminator sequence TPS1t-ADH1t and the downstream homology arm X-3ds) were transformed into XJ063 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the expression cassette X-3us-FBA1p-PsAAAD*-TPS1t-ADH1t-X-3ds was integrated into the X-3 site to obtain the XJ0633 strain.

[0244] (5) The plasmid pMEL10-X-3 and the expression cassette X-3us-FBA1p-RrAAS-TPS1t-ADH1t-X-3ds (consisting of the upstream homology arm X-3us, the promoter FBA1p, RrAAS, the terminator sequence TPS1t-ADH1t and the downstream homology arm X-3ds) were transformed into XJ063 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the expression cassette X-3us-FBA1p-RrAAS-TPS1t-ADH1t-X-3ds was integrated into the X-3 site to obtain the XJ0634 strain.

[0245] 2. Pick 3 single clones from each strain obtained in step 1 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0246] 3. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0247] Test results see Figure 2 Medium D and Figure 4 The results showed that the XJ0633 strain produced the highest content of S-NOR, reaching 295.5 mg / L, that is, the strain with integrated PsAAAD* had the highest product among the strains, which was superior to other candidate aromatic amino acid decarboxylases (AAAD).

[0248] (VIII) Increasing the tyrosine supply in Saccharomyces cerevisiae by introducing plant tyrosine pathway genes prephenate transaminase (PAT) and arogenate dehydratase (ADH)

[0249] The expression cassette consists of an upstream homology arm, a promoter, a candidate gene nucleic acid sequence, a terminator and a downstream homology arm. The candidate PAT genes are AtPAT (NP_565529.1) from Arabidopsis thaliana, BvPAT1 (KMS97510.1) and BvPAT2 (KMT14107.1) from Beta vulgaris. The candidate ADH genes are BvADHα (AST12932.1) from Beta vulgari and MtncADH (ARV76496.1) from Medicago truncatula. The promoters are promoter TDH3p, promoter TEF2p (nucleotide sequence as shown in SEQ ID NO: 16) and promoter FBA1p; the terminators are terminator TDH2t and terminator pYX212t. The effects of different PAT and ADH combinations on the content of product S-NOR were tested in the XJ0633 strain. The specific operations are as follows:

[0250] 1. According to the method in step (a), replace XI-5-gRNA-F with 106a-gRNA-F, replace XI-5-gRNA-R with 106a-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-106a.

[0251] 2. Preparation of gene expression cassette fragments

[0252] (1) The upstream homology arm 106aus (nucleotide sequence as shown in SEQ ID NO: 43), promoter FBA1p, AtPAT, terminator TDH2t and terminator pYX212t were connected by the overlap method to obtain the gene expression cassette fragment 106aus-FBA1p-AtPAT-TDH2t-pYX212t (named as fragment 6). The downstream homology arm 106ads (nucleotide sequence as shown in SEQ ID NO: 44), promoter TEF2p, BvADHα and terminator pYX212t were connected by the overlap method to obtain the gene expression cassette fragment pYX212t-BvADHα-TEF2p-106ads (named as fragment 7).

[0253] (2) The downstream homology arm 106ads, promoter, TEF2p, MtncADH and terminator pYX212t were connected using the overlap method to obtain the gene expression cassette fragment pYX212t-MtncADH-TEF2p-106ads (named as fragment 8).

[0254] (3) The upstream homology arm 106aus, promoter FBA1p, BvPAT1, terminator TDH2t and terminator pYX212t were connected using the overlap method to obtain the gene expression cassette fragment 106aus-FBA1p-BvPAT1-TDH2t-pYX212t (named as fragment 9).

[0255] (4) The upstream homology arm 106aus, promoter FBA1p, BvPAT2, terminator TDH2t and terminator pYX212t were connected using the overlap method to obtain the gene expression cassette fragment 106aus-FBA1p-BvPAT2-TDH2t-pYX212t (named as fragment 10).

[0256] 3. Strain construction

[0257] The 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 in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of the XJ0633 strain, obtaining the XJ0635 strain.

[0258] The 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 in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of the XJ0633 strain, obtaining the XJ0636 strain.

[0259] The 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 in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of the XJ0633 strain, obtaining the XJ0637 strain.

[0260] The 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 in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of the XJ0633 strain, obtaining the XJ0638 strain.

[0261] The 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 in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the 106a site of the XJ0633 strain, obtaining the XJ0639 strain.

[0262] 4. Pick 3 single clones from each strain obtained in step 3 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0263] 5. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0264] Test results see Figure 2 Zhongehe Figure 5 The results showed that the S-NOR concentration of strain XJ0636 was the highest, reaching 315.9 mg / L; that is, the strain integrating AtPAT and MtncADH had the highest product, which was better than other candidate PAT and ADH combinations.

[0265] 2. Optimization of the oxadiazine production module (Module II)

[0266] Module II is the construction of the S-NOR to S-RET pathway. First, the 6OMT, CNMT and NMCH genes in the pathway are optimized. The expression cassette consists of a promoter, a nucleic acid sequence of a candidate gene and a terminator. The candidate genes are Ps6OMT (AAQ01669.1) from P. somniferum, PsCNMT (AAP45316.1) from P. somniferum, EcNMCH (AAC39452.1) from Eschscholzia californica, Cy6OMT (nucleotide sequence as shown in SEQ ID NO: 17) from C. yanhusuo, CyCNMT (nucleotide sequence as shown in SEQ ID NO: 18) and CyNMCH (nucleotide sequence as shown in SEQ ID NO: 19). The promoters are promoter TPI1p, promoter PGK1p, promoter TEF1p, promoter tHXT7p, promoter TDH3p, promoter GPM1p (nucleotide sequence as shown in SEQ ID NO: 21), and promoter TEF2p; the terminators are terminator pYX212t, terminator ADH1t, terminator CYC1t, terminator FBA1t, terminator TDH2t, terminator PGI1t (nucleotide sequence as shown in SEQ ID NO: 22), and terminator ENO2t. The candidate genes of different combinations were compared in the chassis strain XJ040. Finally, the candidate genes with the best combination were integrated into the strain XJ0636. The pathways and transformation strategies of module II are shown in Figure 6 .

[0267] (I) Construction of chassis strain XJ040

[0268] 1. Construction of plasmid

[0269] According to the method in step (a), XI-5-gRNA-F was replaced with X-2-gRNA-F, and XI-5-gRNA-R was replaced with X-2-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-X-2.

[0270] According to the method in step (a), XI-5-gRNA-F was replaced with X-4-gRNA-F, and XI-5-gRNA-R was replaced with X-4-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-X-4.

[0271] According to the method in step (a), XI-5-gRNA-F was replaced with XII-5-gRNA-F, and XI-5-gRNA-R was replaced with XII-5-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-XII-5.

[0272] 2. Construction of gene expression cassette

[0273] (1) The upstream homology arm X-2us (nucleotide sequence as shown in SEQ ID NO: 37), promoter TPI1p, the coding gene of EcAROL, terminator pYX212t and terminator ADH1t were connected by the overlap method to obtain the gene expression cassette fragment X-2us-TPI1p-EcaroL-pYX212t-ADH1t. The terminator pYX212t, terminator ADH1t, the coding gene of the endogenous gene mutant ARO7*, promoter PGK1p and promoter TEF1p were connected by the overlap method to obtain the gene expression cassette fragment pYX212t-ADH1t-ARO7*-PGK1p-TEF1p. The promoter PGK1p, the promoter TEF1p, the coding gene of the endogenous gene mutant ARO4*, the terminator CYC1t and the terminator FBA1t were connected by the overlap method to obtain the gene expression cassette fragment PGK1p-TEF1p-ARO4*-CYC1t-FBA1t. The terminator CYC1t, the terminator FBA1t, the coding gene of MtPDH1, the promoter tHXT7p and the downstream homology arm X-2ds (nucleotide sequence as shown in SEQ ID NO:38) were connected by the overlap method to obtain the gene expression cassette fragment

[0274] CYC1t-FBA1t-MtPDH1-tHXT7p-X-2ds.

[0275] (2) The upstream homology arm X-4us (nucleotide sequence as shown in SEQ ID NO: 39), terminator CYC1t, ARO1 coding gene, promoter TPI1p and promoter TDH3p were connected by overlap method to obtain gene expression cassette fragment X-4us-CYC1t-ARO1-TPI1p-TDH3p. The promoter TPI1p, promoter TDH3p, ARO2 coding gene, terminator ADH1t and terminator TDH2t were connected by overlap method to obtain gene expression cassette fragment TPI1p-TDH3p-ARO2-ADH1t-TDH2t. The terminator ADH1t, the terminator TDH2t, the coding gene of ARO3, the promoter TEF1p and the downstream homology arm X-4ds (nucleotide sequence as shown in SEQ ID NO: 40) were connected by the overlap method to obtain the gene expression cassette fragment ADH1t-TDH2t-ARO3-TEF1p-X-4ds.

[0276] (3) The upstream homology arm XII-5us (nucleotide sequence as shown in SEQ ID NO: 47), terminator PGI1t, Ps4'OMT coding gene (AAP45314.1), promoter TDH3p and promoter tHXT7p were connected by overlap method to obtain gene expression cassette fragment XII-5us-PGI1t-Ps4'OMT-TDH3p-tHXT7p. The promoter TDH3p, promoter tHXT7p, PsCPR coding gene, terminator CYC1t and downstream repair homology arm XII-5ds (nucleotide sequence as shown in SEQ ID NO: 48) were connected by overlap method to obtain gene expression cassette fragment TDH3p-tHXT7p-PsCPR-CYC1t-XII-5ds.

[0277] 3. Construction of XJ040 strain

[0278] Plasmid pMEL10-X-2, gene expression cassette fragment X-2us-TPI1p-EcaroL-pYX212t-ADH1t, gene expression cassette fragment pYX212t-ADH1t-ARO7*-PGK1p-TEF1p, gene expression cassette fragment

[0279] PGK1p-TEF1p-ARO4*-CYC1t-FBA1t and gene expression cassette fragment CYC1t-FBA1t-MtPDH1-tHXT7p-X-2ds were transformed into XJ003 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, they were integrated into the X-2 site of XJ003 to obtain intermediate yeast 1; plasmid pMEL10-X-4, gene expression cassette fragment X-4us-CYC1t-ARO1-TPI1p-TDH3p, gene expression cassette fragment TPI1p-TDH3p-ARO2-ADH1t-TDH2t and gene expression cassette fragment

[0280] ADH1t-TDH2t-ARO3-TEF1p-X-4ds was used to transform the competent cells of intermediate yeast 1 to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the X-4 site, and intermediate yeast 2 was obtained; the plasmid pMEL10-XII-5, the gene expression cassette fragment XII-5us-PGI1t-Ps4'OMT-TDH3p-tHXT7p and the gene expression cassette fragment TDH3p-tHXT7p-PsCPR-CYC1t-XII-5ds were used to transform the competent cells of intermediate yeast 2 to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the XII-5 site, and finally the chassis bacteria XJ040 was obtained.

[0281] (ii) Screening of different combinations of candidate genes in the chassis fungus XJ040.

[0282] 1. According to the method in step (a), replace XI-5-gRNA-F with XII-2-gRNA-F, and replace XI-5-gRNA-R with XII-2-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-XII-2.

[0283] 2. Construction of gene expression cassette

[0284] (1) Combination of Cy6OMT, CyCNMT and EcNMCH

[0285] The upstream homology arm XII-2-us (nucleotide sequence as shown in SEQ ID NO: 45), promoter GPM1p, Cy6OMT coding gene, terminator FBA1t and terminator ENO2t were connected by overlap method to obtain gene expression cassette XII-2-us-GPM1p-Cy6OMT-FBA1t-ENO2t. The terminator FBA1t, terminator ENO2t, CyCNMT coding gene, promoter TEF2p and promoter tHXT7p were connected by overlap method to obtain gene expression cassette FBA1t-ENO2t-CyCNMT-TEF2p-tHXT7p. The promoter TEF2p, promoter tHXT7p, the coding gene of EcNMCH, terminator ADH1t and downstream homology arm XII-2-ds (nucleotide sequence as shown in SEQ ID NO: 46) were connected using the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-EcNMCH-ADH1t-XII-2-ds.

[0286] (2) Combination of Cy6OMT, CyCNMT, and CyNMCH

[0287] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Cy6OMT, terminator FBA1t and terminator ENO2t were connected by the overlap method to obtain the gene expression cassette XII-2-us-GPM1p-Cy6OMT-FBA1t-ENO2t. The terminator FBA1t, terminator ENO2t, the coding gene of CyCNMT, promoter TEF2p and promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-CyCNMT-TEF2p-tHXT7p. The promoter TEF2p, promoter tHXT7p, the coding gene of CyNMCH, terminator ADH1t and the downstream homology arm XII-2-ds were connected by the overlap method to obtain the gene expression cassette TEF2p+tHXT7p-CyNMCH-ADH1t-XII-2-ds.

[0288] (3) Combination of Cy6OMT, PsCNMT and EcNMCH

[0289] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Cy6OMT, terminator FBA1t and terminator ENO2t were connected by the overlap method to obtain the gene expression cassette XII-2-us-GPM1p-Cy6OMT-FBA1t-ENO2t. The terminator FBA1t, terminator ENO2t, the coding gene of PsCNMT, promoter TEF2p and promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-PsCNMT-TEF2p-tHXT7p. The promoter TEF2p, promoter tHXT7p, the coding gene of EcNMCH, terminator ADH1t and the downstream homology arm XII-2-ds were connected by the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-EcNMCH-ADH1t-XII-2-ds.

[0290] (4) Combination of Cy6OMT, PsCNMT, and CyNMCH

[0291] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Cy6OMT, terminator FBA1t and terminator ENO2t were connected by the overlap method to obtain the gene expression cassette XII-2-us-GPM1p-Cy6OMT-FBA1t-ENO2t. The terminator FBA1t, terminator ENO2t, the coding gene of PsCNMT, promoter TEF2p and promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-PsCNMT-TEF2p-tHXT7p. The promoter TEF2p, promoter tHXT7p, the coding gene of CyNMCH, terminator ADH1t and the downstream homology arm XII-2-ds were connected by the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-CyNMCH-ADH1t-XII-2-ds.

[0292] (5) Combination of Ps6OMT, CyCNMT and EcNMCH

[0293] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Ps6OMT, terminator FBA1t and terminator ENO2t were connected by the overlap method to obtain the gene expression cassette XII-2-us-GPM1p-Ps6OMT-FBA1t-ENO2t. The terminator FBA1t, terminator ENO2t, the coding gene of CyCNMT, promoter TEF2p and promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-CyCNMT-TEF2p-tHXT7p. The promoter TEF2p, promoter tHXT7p, the coding gene of EcNMCH, terminator ADH1t and the downstream homology arm XII-2-ds were connected by the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-EcNMCH-ADH1t-XII-2-ds.

[0294] (6) Combination of Ps6OMT, CyCNMT and CyNMCH

[0295] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Ps6OMT, terminator FBA1t and terminator ENO2t were connected by the overlap method to obtain the gene expression cassette XII-2-us-GPM1p-Ps6OMT-FBA1t-ENO2t. The terminator FBA1t, terminator ENO2t, the coding gene of CyCNMT, promoter TEF2p and promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-CyCNMT-TEF2p-tHXT7p. The promoter TEF2p, promoter tHXT7p, the coding gene of CyNMCH, terminator ADH1t and the downstream homology arm XII-2-ds were connected by the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-CyNMCH-ADH1t-XII-2-ds.

[0296] (7) Combination of Ps6OMT, PsCNMT and EcNMCH

[0297] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Ps6OMT, terminator FBA1t and terminator ENO2t were connected by the overlap method to obtain the gene expression cassette XII-2-us-GPM1p-Ps6OMT-FBA1t-ENO2t. The terminator FBA1t, terminator ENO2t, the coding gene of PsCNMT, promoter TEF2p and promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-PsCNMT-TEF2p-tHXT7p. The promoter TEF2p, promoter tHXT7p, the coding gene of EcNMCH, terminator ADH1t and the downstream homology arm XII-2-ds were connected by the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-EcNMCH-ADH1t-XII-2-ds.

[0298] (8) Combination of Ps6OMT, PsCNMT and CyNMCH

[0299] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Ps6OMT, terminator FBA1t and terminator ENO2t were connected by the overlap method to obtain the gene expression cassette XII-2-us-GPM1p-Ps6OMT-FBA1t+ENO2t. The terminator FBA1t, terminator ENO2t, the coding gene of PsCNMT, promoter TEF2p and promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-PsCNMT-TEF2p-tHXT7p. The promoter TEF2p, promoter tHXT7p, the coding gene of CyNMCH, terminator ADH1t and the downstream homology arm XII-2-ds were connected by the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-CyNMCH-ADH1t-XII-2-ds.

[0300] 3. Transform XJ040 yeast competent cells with plasmid pMEL10-XII-2 and the gene expression cassette shown in step 2 (1) to obtain positive clones; randomly select positive clones and streak them on 5-FOA plates, and culture them in a 30°C incubator 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 the XJ040 strain, to obtain the XJ041 strain.

[0301] According to the above steps, the gene expression cassette shown in step 2 (1) is replaced with the gene expression cassette shown in step 2 (2) - the gene expression cassette shown in step 2 (8), and the other steps remain unchanged, thereby obtaining the XJ042 strain - the XJ048 strain in sequence.

[0302] 4. Pick 3 single clones from each strain obtained in step 3 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

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

[0304] Test results see Figure 7 The results showed that the XJ048 strain (a combination of Ps6OMT, PsCNMT, and CyNMCH) had the highest S-RET concentration. Therefore, the optimal combination screened in this experiment was Ps6OMT, PsCNMT, and CyNMCH.

[0305] (III) Preparation of strain XJ0662

[0306] The preferred Ps6OMT, PsCNMT and CyNMCH as well as Ps4'OMT and PsCPR were inserted into the XJ0636 strain to obtain the XJ0662 strain. The specific steps are as follows:

[0307] 1. According to the method in step (a), replace XI-5-gRNA-F with XII-5-gRNA-F, replace XI-5-gRNA-R with XII-5-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-XII-5.

[0308] 2. According to the method in step (a), replace XI-5-gRNA-F with XII-2-gRNA-F, and replace XI-5-gRNA-R with XII-2-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-XII-2.

[0309] 3. Construction of gene expression cassette

[0310] (1) The upstream homology arm XII-5-us, the terminator PGI1t, the coding gene of Ps4'OMT, the promoter TDH3p and the promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette XII-5-us-PGI1t-Ps4'OMT-TDH3p-tHXT7p (named as fragment 11). The promoter TDH3p, the promoter tHXT7p, the coding gene of PsCPR, the terminator CYC1t and the downstream homology arm XII-5-ds were connected by the overlap method to obtain the gene expression cassette TDH3p-tHXT7p-PsCPR-CYC1t-XII-5-ds (named as fragment 12).

[0311] (2) Using the overlap method, the upstream homology arm XII-2-us, the promoter GPM1p, the coding gene of Ps6OMT, the terminator FBA1t and the terminator ENO2t were connected to obtain a gene expression cassette

[0312] XII-2-us-GPM1p-Ps6OMT-FBA1t-ENO2t (named as fragment 13). The terminator FBA1t, the terminator ENO2t, the coding gene of PsCNMT, the promoter TEF2p and the promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-PsCNMT-TEF2p-tHXT7p (named as fragment 14). The promoter TEF2p, the promoter tHXT7p, the coding gene of CyNMCH, the terminator ADH1t and the downstream homology arm XII-2-ds were connected by the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-CyNMCH-ADH1t-XII-2-ds (named as fragment 15).

[0313] 4. Construction of strains XJ066 and XJ0662

[0314] The plasmid pMEL10-XII-5, fragment 11 and fragment 12 were transformed into XJ0636 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the XII-5 site of XJ0636, obtaining the XJ066 strain.

[0315] The plasmid pMEL10-XII-2, fragment 13, fragment 14 and fragment 15 were transformed into XJ066 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The grown clones were strains that had lost the gRNA plasmid, that is, integrated into the XII-2 site of XJ066, obtaining the XJ0662 strain.

[0316] (IV) CPR screening

[0317] 1. According to the method in step (a), replace XI-5-gRNA-F with PsCPR-gRNA-F, replace XI-5-gRNA-R with PsCPR-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-PsCPR.

[0318] 2. Transform the plasmid pMEL10-PsCPR and ATR1 fragment (nucleotide sequence as shown in SEQ ID NO: 55) into XJ0662 yeast competent cells to obtain positive clones; randomly select positive clones and streak them on 5-FOA plates, culture them in a 30°C incubator for 2-3 days, and the clones that grow out are strains that have lost the gRNA plasmid, that is, integrated into the PsCPR site of XJ0662, and obtain strain XJ0675. Transform the plasmid pMEL10-PsCPR and ATR2 fragment (nucleotide sequence as shown in SEQ ID NO: 56) into XJ0662 yeast competent cells to obtain positive clones; randomly select positive clones and streak them on 5-FOA plates, culture them in a 30°C incubator for 2-3 days, and the clones that grow out are strains that have lost the gRNA plasmid, that is, integrated into the PsCPR site of XJ0662, and obtain strain XJ0676.

[0319] 3. Pick three single clones from each strain obtained in step 2 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / LKH 2 PO 4 、0.5g / LMgSO 4 7H 2O, 20g / Lglucose, 2ml / Ltracemetalsolutions, 1ml / Lvitaminsolutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0320] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0321] Test results see Figure 6 The results showed that the S-RET content of strain XJ0675 was the highest compared with strains XJ0662 and XJ0676, which means that ATR1 was more efficient than ATR2 and PsCPR in this experiment.

[0322] 5. Impact of the number of candidate genes

[0323] The effects of Ps4'OMT and CyNMCH gene copy numbers on the content of product S-RET were compared in strain XJ0675. The specific steps are as follows:

[0324] 1. Construction of plasmid

[0325] (1) According to the method in step (a), XI-5-gRNA-F was replaced with HFD1-gRNA-F, and XI-5-gRNA-R was replaced with HFD1-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-HFD1.

[0326] (2) According to the method in step (a), XI-5-gRNA-F was replaced with 416d-gRNA-F, and XI-5-gRNA-R was replaced with 416d-gRNA-R, and the other steps remained unchanged to obtain plasmid pMEL10-416d.

[0327] (3) According to the method in step (a), XI-5-gRNA-F was replaced with ARI1-gRNA-F, and XI-5-gRNA-R was replaced with ARI1-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-ARI1.

[0328] (4) According to the method in step (a), XI-5-gRNA-F was replaced with ADH6-gRNA-F, and XI-5-gRNA-R was replaced with ADH6-gRNA-R, and the other steps remained unchanged to obtain plasmid pMEL10-ADH6.

[0329] (5) According to the method in step (a), XI-5-gRNA-F was replaced with YPR1-gRNA-F, and XI-5-gRNA-R was replaced with YPR1-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-YPR1.

[0330] 2. Construction of gene expression cassette

[0331] (1) The upstream homology arm HFD1-us (nucleotide sequence as shown in SEQ ID NO: 57), the terminator PGI1t, the coding gene of Ps4'OMT, the promoter TDH3p and the downstream homology arm HFD1-ds (nucleotide sequence as shown in SEQ ID NO: 58) were connected by the overlap method to obtain the gene expression cassette HFD1-us-PGI1t-Ps4'OMT-TDH3p-HFD1-ds (named as fragment 16).

[0332] (2) The upstream homology arm HFD1-us, terminator PGI1t, the coding gene of Ps4'OMT, promoter TDH3p and promoter tHXT7p were connected by overlap method to obtain the gene expression cassette HFD1-us-PGI1t-Ps4'OMT-TDH3p-tHXT7p (named as fragment 17). The promoter TDH3p, promoter tHXT7p, the coding gene of CyNMCH, terminator ADH1t and the downstream homology arm HFD1-ds were connected by overlap method to obtain the gene expression cassette TDH3p-tHXT7p-CyNMCH-ADH1t-HFD1-ds (named as fragment 18).

[0333] (3) The upstream homology arm 416d-us (nucleotide sequence as shown in SEQ ID NO: 59), the promoter tHXT7p, the coding gene of CyNMCH, the terminator ADH1t and the downstream homology arm 416d-ds (nucleotide sequence as shown in SEQ ID NO: 60) were connected using the overlap method to obtain the gene expression cassette 416d-us-LP1.T8-tHXT7p-CyNMCH-ADH1t-416d-ds (named as fragment 19).

[0334] (4) The upstream homologous arm ARI1-us (nucleotide sequence as shown in SEQ ID NO:61), the promoter tHXT7p, the coding gene of CyNMCH, the terminator ADH1t-LP4.T9 and the downstream homologous arm ARI1-ds (nucleotide sequence as shown in SEQ ID NO:62) were connected using the overlap method to obtain the gene expression cassette ARI1-us-LP3.T7-tHXT7p-CyNMCH-ADH1t-LP4.T9-ARI1-ds (named as fragment 20). The upstream homology arm ADH6-us (nucleotide sequence as shown in SEQ ID NO: 63), the promoter tHXT7p, the coding gene of CyNMCH, the terminator ADH1t and the downstream homology arm ADH6-ds (nucleotide sequence as shown in SEQ ID NO: 64) were connected using the overlap method to obtain the gene expression cassette ADH6us-LP3.T7-tHXT7p-CyNMCH-ADH1t-LP4.T9-ADH6-ds (named as fragment 21). The upstream homology arm YPR1-us (nucleotide sequence as shown in SEQ ID NO: 65), the promoter tHXT7p, the coding gene of CyNMCH, the terminator ADH1t and the downstream homology arm YPR1-ds (nucleotide sequence as shown in SEQ ID NO: 66) were connected using the overlap method to obtain the gene expression cassette YPR1-us-LP3.T7-tHXT7p-CyNMCH-ADH1t-LP4.T9-YPR1-ds (named as fragment 22).

[0335] 3. Strain construction

[0336] (1) The plasmid pMEL10-HFD1 and fragment 16 were transferred into the competent cells of XJ0675 to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates, and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the HFD1 site of XJ0675, and the XJ0685 strain was obtained.

[0337] (2) The plasmid pMEL10-HFD1, fragment 17 and fragment 18 were transferred into the competent cells of XJ0685 to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the HFD1 site of XJ0685, and the XJ0686 strain was obtained.

[0338] (3) The plasmid pMEL10-416d and fragment 19 were transferred into the competent cells of XJ0686 to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the 416d site of XJ0686, and the XJ0689 strain was obtained.

[0339] (4) Plasmid pMEL10-ARI1, plasmid pMEL10-ADH6 and plasmid pMEL10-YPR1 were respectively transferred into competent cells of XJ0685 with fragment 20, fragment 21 and fragment 22 to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the ARI1 site, ADH6 site and YPR1 site of XJ0685, and the XJ0690 strain was obtained.

[0340] (5) Plasmid pMEL10-ARI1, plasmid pMEL10-ADH6 and plasmid pMEL10-YPR1 were respectively transferred into competent cells of XJ0686 with fragment 20, fragment 21 and fragment 22 to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, integrated into the ARI1 site, ADH6 site and YPR1 site of XJ0686, and the XJ0691 strain was obtained.

[0341] 4. Pick 3 single clones from each strain obtained in step 3 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0342] 5. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0343] Test results see Figure 6 The results showed that the XJ0691 strain, which integrated two Ps4'OMT fragments and five CyNMCH fragments, had the highest S-RET content, reaching 425.2 mg / L.

[0344] 3. Screening and optimization of BBE (Module III)

[0345] The present invention adopts an endoplasmic reticulum engineering strategy to improve the activity of BBE in Saccharomyces cerevisiae. First, a BBE enzyme CyBBE (from Corydalis yanhusuo) with high catalytic efficiency is obtained through comparative screening; then the yeast endoplasmic reticulum retention signal is fused to the C-terminus of CyBBE (CyBBE_ERTS), so that it can be retrograded from the Golgi apparatus to the endoplasmic reticulum, thereby increasing the yield of golden corydaline by 3 times. In addition, by introducing mammalian source catalase peroxiredoxin IV (PRDX4) to mediate H 2 O 2 The degradation of CyBBE relieved the inhibitory effect of CyBBE and the endoplasmic reticulum expansion mediated by knocking out phospholipase OPI1 increased the potency of golden corydaline by 30% and 36%, respectively. The pathway and transformation strategy of module III are shown in Figure 8 .

[0346] (I) Screening for the best BBE gene

[0347] The candidate genes are PsBBE (AAC61839.1) from poppy (P.somniferum), CjBBE (BAM44344.1) from Coptis japonica, CyBBE (nucleotide sequence as shown in SEQ ID NO:23) from Corydalis yanhusuo, and StBBE (nucleotide sequence as shown in SEQ ID NO:24) from Stephania tetrandra. The candidate genes were respectively integrated into the low-yielding cyperine strain XJ048, and it was found that except StBBE, other expression candidate genes were able to produce golden corydaline, among which CyBBE had the highest activity. The expression cassette consists of an upstream homologous arm, a promoter, a nucleic acid sequence of the candidate gene, a terminator, and a downstream homologous arm. The promoter is the promoter CCW12p, and the terminator is the terminator PGI1t. The specific steps are as follows:

[0348] 1. Construction of plasmid

[0349] According to the method in step (a), XI-5-gRNA-F was replaced with XII-4-gRNA-F, and XI-5-gRNA-R was replaced with XII-4-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-XII-4.

[0350] 2. Strain construction

[0351] (1) The upstream homology arm XII-4-us (nucleotide sequence as shown in SEQ ID NO:49), the promoter CCW12p, the coding gene of PsBBE, the terminator PGI1t and the downstream homology arm XII-4-ds (nucleotide sequence as shown in SEQ ID NO:50) were connected by the overlap method to obtain the PsBBE gene expression cassette. The plasmid pMEL10-XII-4 and the PsBBE gene expression cassette were transferred into the competent cells of XJ048 yeast to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the PsBBE gene expression cassette was integrated into the XII-4 site of XJ048. The resulting strain was named XJ081 yeast strain.

[0352] (2) According to the above steps, the coding gene of PsBBE was replaced by the coding gene of CjBBE, and the other steps remained unchanged, that is, the CjBBE gene expression cassette was integrated into the XII-4 site of XJ048, and the resulting strain was named XJ082 yeast strain.

[0353] (3) According to the above steps, the coding gene of PsBBE was replaced with the coding gene of CyBBE, that is, the CyBBE gene expression cassette was integrated into the XII-4 site of XJ048, and the resulting strain was named XJ083 yeast strain.

[0354] (4) According to the above steps, the coding gene of PsBBE was replaced with the coding gene of StBBE, that is, the StBBE gene expression cassette was integrated into the XII-4 site of XJ048, and the resulting strain was named XJ084 yeast strain.

[0355] 3. Pick three single clones from each strain obtained in step 2 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0356] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0357] Test results see Fig. 9 The results showed that XJ081-XJ083 were all able to produce chrysocortin S-SCO, and the strain XJ083 produced the highest content of chrysocortin S-SCO, that is, CyBBE was the most efficient gene; XJ084 could not produce chrysocortin S-SCO.

[0358] (II) Optimization of CyBBE yeast in vivo expression localization

[0359] CyBBE was fused with the endoplasmic reticulum C-terminal retention signal HDEL (CyBBE_ERTS (nucleotide sequence as shown in SEQ ID NO: 25)) and the N-terminal Golgi target sequence (GOTS_CyBBE (nucleotide sequence as shown in SEQ ID NO: 26)) and tested in strain XJ0691. It was found that compared with the wild-type CyBBE, XJ0694 where GOTS_CyBBE was located and XJ0695 where CyBBE_ERTS was located increased the production of golden corydaline by 32.8% and 206%, respectively. Colocalization microscopy was then used to determine that the wild-type CyBBE was transported from the Golgi to the vacuole, GOTS_CyBBE was retained in the Golgi, and CyBBE_ERTS was retrograde from the Golgi to the endoplasmic reticulum. The specific steps are as follows:

[0360] 1. Strain construction

[0361] (1) The upstream homology arm XII-4-us, promoter CCW12p, nucleotide sequence of GOTS-CyBBE, terminator PGI1 and downstream homology arm XII-4-ds were connected by overlap method to obtain gene expression cassette XII-4-us-CCW12p-GOTS_CyBBE-PGI1t-XII-4-ds. The plasmid pMEL10-XII-4 and the gene expression cassette were transferred into XJ0691 yeast competent cells to obtain positive clones; 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, that is, the gene expression cassette was integrated into the XII-4 site of XJ0691. The resulting strain was named XJ0694 strain.

[0362] (2) According to the above steps, the nucleotide sequence of GOTS-CyBBE was replaced with the nucleotide sequence of CyBBE_ERTS, and the other steps remained unchanged. The resulting strain was named XJ0695 strain.

[0363] (3) According to the above steps, the nucleotide sequence of GOTS-CyBBE was replaced with the nucleotide sequence of CyBBE, and the other steps remained unchanged. The resulting strain was named XJ0696 strain.

[0364] 2. Pick 3 single clones from each strain obtained in step 1 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2 PO 4 、0.5g / L MgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0365] 3. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0366] 4. In order to explore the subcellular localization of CyBBE, GOTS-CyBBE with Golgi target sequence GOTS fused to the N-terminus, and CyBBE_ERTS with endoplasmic reticulum C-terminal retention signal HDEL fused to the C-terminus of CyBBE in Saccharomyces cerevisiae, the target genes (CyBBE, GOTS-CyBBE, CyBBE_ERTS) were fused with GFP tags, and ELO3-mRuBy2, VPH1-mRuBy, and CHS5-mRuBy2 were used as fluorescent tags for endoplasmic reticulum, vacuole, and Golgi, respectively, to detect the subcellular localization of the target genes. The specific steps are as follows:

[0367] ① Using CyBBE with a nucleotide sequence as shown in SEQ ID NO: 23 as a template, GPD-BBE1-F and BBE-1-GFP-R were used for PCR amplification to obtain CyBBE (named as fragment 23); using plasmid P416_GPD (purchased from the ATCC strain library, the website is https: / / www.atcc.org / search#q=P416&sort=relevancy&numberOfResults=24) as a template, BBE-1-GFP-F and GFP-GS-IDP1t-R were used for PCR amplification to obtain GFP_ERTS (named as fragment 24); using DNA fragment IDP1t-CCW12p (nucleotide sequence as shown in SEQ ID NO:72) as a template, IDP1t-rvF and CCW12p-R were used for PCR amplification to obtain IDP1t-CCW12p (named as fragment 25); the genomic DNA of the IMX581 yeast strain was used as a template, CCW12p-Elo3-F and Elo3-GS-mRuby2-R were used for PCR amplification to obtain Elo3 (named as fragment 26); the DNA fragment mRuBy2 (nucleotide sequence as shown in SEQ ID NO:73) was used as a template, GS-mRuBy2-F and mRuby2-CYC1t-R were used for PCR amplification to obtain mRuby2 (named as fragment 27); plasmid P416_GPD was used as a template, CYC1t-p416-F and GPD-p416-R were used for PCR amplification to obtain a linearized plasmid (named as fragment 28); the above adjacent fragments all have 25bp homology arms. Fragment 23-fragment 28 were used to construct plasmid according to the instructions of NEB's Gibson Assembly Kit to obtain the recombinant plasmid P416-GPD-CyBBE_GFP_ERTS+ELO3-mRuBy2.

[0368] ② Using plasmid P416_GPD as a template, BBE-1-GFP-F and GFP-IDP1t-R were used for PCR amplification to obtain fragment 29; using genomic DNA of IMX581 yeast strain as a template, CCW12p-Vph1-F and Vph1-GS-mRuby2-R were used for PCR amplification to obtain fragment 30. Fragments 29 and 30 were used to construct plasmids with fragments 23, 25, 27 and 28 in step ① according to the instructions of the Gibson Assembly kit of NEB Company to obtain the recombinant plasmid P416-GPD-CyBBE-GFP+VPH1-mRuBy2.

[0369] ③ Using plasmid P416_GPD as a template, GPD-Golgi-BBE1-F and BBE-1-GFP-R were used for PCR amplification to obtain Golgi-BBE1 (named as fragment 31); using the genomic DNA of the IMX581 yeast strain as a template, CCW12p-Chs5-F and Chs5-GS-mRuby2-R were used for PCR amplification to obtain CHS5 (named as fragment 32); fragment 31, fragment 32 and fragments 24, fragment 25, fragment 27 and fragment 28 in step ① were constructed according to the instructions of the Gibson Assembly kit of NEB Company to obtain the recombinant plasmid P416-GPD-GOTS_CyBBE-GFP+CHS5-mRuBy2.

[0370] ④Introduce the plasmids obtained in ①, ②, and ③ into XJ0691 to obtain recombinant yeast. Use co-localization microscopy to determine the localization of the target fragment in yeast.

[0371] Test results see Figure 8 Middle b. The results showed that the protein expressed by CyBBE was localized in the vacuole, GOTS_CyBBE was localized in the Golgi apparatus, and CyBBE_ERTS was localized in the endoplasmic reticulum, which means that CyBBE_ERTS retrograded from the Golgi apparatus to the endoplasmic reticulum. Compared with CyBBE, XJ0694 where GOTS_CyBBE was located and XJ0695 where CyBBE_ERTS was located increased the production of golden corydaline S-SCO by 32.8% and 206%, respectively. Combining the two results, it was shown that CyBBE_ERTS, which was fused with the endoplasmic reticulum C-terminal retention signal HDEL at the C-terminus, realized the function of retrograde from the Golgi apparatus to the endoplasmic reticulum, which increased the production of golden corydaline S-SCO (XJ0695, 76.5 mg / L).

[0372] (III) Using catalase PRDX4 to alleviate H 2 O 2 The inhibitory effect

[0373] There are research reports that H 2 O 2 Therefore, in this study, catalase PRDX4 (with yeast α-mating factor signal peptide fused to the N-terminus and ERTS signal peptide fused to the C-terminus, i.e., α-mPRDX4_ERTS) was introduced into Saccharomyces cerevisiae XJ0695 to decompose H 2 O 2 , improve the production of engineered bacteria S-RET (see Figure 8 The specific experimental steps are as follows:

[0374] 1. Construction of recombinant plasmid

[0375] (1) An artificially synthesized α-mPRDX4_ERTS fragment (nucleotide sequence as shown in SEQ ID NO: 27) was subjected to Gibson ligation with a linearized plasmid P416_GPD (plasmid P416_GPD was used as a template and a primer pair consisting of CYC1t-p416-F and GPD-p416-R was used for PCR amplification to obtain a linearized plasmid P416_GPD) to obtain a recombinant plasmid P416_GPD-α-mPRDX4_ERTS.

[0376] (2) Artificially synthesize α-mPRDX4(C127S)_ERTS (nucleotide sequence as shown in SEQ ID NO:28) and perform Gibson ligation with the linearized plasmid P416_GPD to obtain the recombinant plasmid P416_GPD-α-mPRDX4(C127S)_ERTS.

[0377] (3) Artificially synthesize α-mPRDX4(C248S)_ERTS (nucleotide sequence as shown in SEQ ID NO:29) and perform Gibson ligation with the linearized plasmid P416_GPD to obtain the recombinant plasmid P416_GPD-α-mPRDX4(C248S)_ERTS.

[0378] 2. Introduce the recombinant plasmid P416_GPD-α-mPRDX4_ERTS, recombinant plasmid P416_GPD-α-mPRDX4(C127S)_ERTS and recombinant plasmid P416_GPD-α-mPRDX4(C248S)_ERTS into XJ0695 respectively to obtain recombinant bacteria.

[0379] 3. Select three single clones from each strain obtained in step 2 and XJ0695 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / L KH 2PO 4 、0.5g / LMgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions, the solvent is water), then inoculate a single clone, and culture at 30℃, 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, and culture at 30℃, 220rpm for 72h to obtain a fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0380] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0381] Test results see Figure 8 The results showed that compared with XJ0695, the strain into which the catalase peroxiredoxinIV (α-mPRDX4_ERTS) was introduced with the yeast α mating factor signal peptide fused to its N-terminus and the ERTS signal peptide fused to its C-terminus produced the highest S-SCO production, which increased by 30% (up to 98.4 mg / L) compared with XJ0695.

[0382] (IV) Optimizing CyBBE expression through endoplasmic reticulum engineering

[0383] Since BBE is a FAD-dependent oxidase, it needs to undergo post-translational modification through a transport pathway including the endoplasmic reticulum, and optimizing the endoplasmic reticulum will affect the expression of BBE. This experiment studied the endoplasmic reticulum amplification project from four aspects, namely, knocking out the OPI1 gene encoding a negative regulator of phospholipid biosynthesis, knocking out the PAH1 gene encoding a Mg2+-dependent phosphatidic acid (PA) phosphatase, overexpressing the INO2 gene encoding a transcriptional activator of phospholipid biosynthesis, and the INO2 gene mutant INO2* (INO2_L119A).

[0384] 1. Construction of plasmid

[0385] (1) According to the method in step (a), XI-5-gRNA-F was replaced with FgF20-gRNA-F, and XI-5-gRNA-R was replaced with FgF20-gRNA-R, and the other steps remained unchanged to obtain plasmid pMEL10-FgF20.

[0386] (2) According to the method in step (a), XI-5-gRNA-F was replaced with PAH1-gRNA-F, and XI-5-gRNA-R was replaced with PAH1-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-PAH1.

[0387] (3) According to the method in step (a), replace XI-5-gRNA-F with OPI1-gRNA-F, and replace XI-5-gRNA-R with OPI1-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-OPI1.

[0388] 2. Construction of strains

[0389] (1) The plasmid pMEL10-FgF20 and the fragment FgF20-us-PDC1p-INO2-ENO2t-FgF20-ds (composed of the upstream homology arm FgF20-us (nucleotide sequence as shown in SEQ ID NO: 67), the promoter PDC1p (nucleotide sequence as shown in SEQ ID NO: 69), the INO2 gene (nucleotide sequence as shown in SEQ ID NO: 71), the terminator ENO2t (nucleotide sequence as shown in SEQ ID NO: 70) and the downstream homology arm FgF20-ds (nucleotide sequence as shown in SEQ ID NO: 68)) were transformed into the competent cells of XJ0695 yeast to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates, and cultured in a constant temperature incubator at 30° C. for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the fragment was integrated into the FgF20 site of XJ0695, and the XJ0786 strain (abbreviated as XJ0786) was obtained.

[0390] (2) According to the method of the above step (1), the INO2 gene is replaced with the INO2* gene (i.e., the mutant gene of INO2, INO2_L119A), and the other steps remain unchanged to obtain the XJ0787 strain (abbreviated as XJ0787).

[0391] (3) The plasmid pMEL10-PAH1 and the repair fragment a (the nucleotide sequence is shown in SEQ ID NO: 30, which has a 60 bp overlapping homology arm sequence with the promoter and terminator of the target gene) were transferred into the XJ0695 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the repair fragment a was integrated into the PAH1 gene site of XJ0695, thereby knocking out the PAH1 gene, and obtaining the XJ0791 strain (abbreviated as XJ0791).

[0392] (4) The plasmid pMEL10-OPI1 and the repair fragment b (the nucleotide sequence is shown in SEQ ID NO: 31, which has a 60 bp overlapping homology arm sequence with the promoter and terminator of the target gene) were transferred into the XJ0695 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates, and cultured in a 30°C incubator for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, the repair fragment b was integrated into the OPI1 gene site of XJ0695, thereby knocking out the OPI1 gene, and obtaining the XJ0796 strain (abbreviated as XJ0796).

[0393] 3. Pick three single clones from each strain obtained in step 2 for parallel experiments. Add 1 ml of Deft medium (solute and its concentration are 7.5 g / L (NH 4 ) 2 SO 4 , 14.4g / LKH 2 PO 4 、0.5g / LMgSO 4 7H 2 O, 20g / Lglucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, the solvent is water), then inoculate a single clone, culture at 30℃, 220rpm for 18-24h to obtain bacterial liquid. Inoculate the bacterial liquid into a shake flask (specification: 125mL) filled with 20mL Deft medium for expansion culture, culture at 30℃, 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0394] 4. Take 500 μL of fermentation broth, add 500 μL of 30% acetonitrile, vortex thoroughly, centrifuge at 13000 g for 5 min, and collect the supernatant for LC-MS / MS detection.

[0395] Test results see Figure 8 The results showed that the endoplasmic reticulum expansion mediated by overexpression of INO2, INO2* and knockout of OPI1 increased S-RET production by 17.1%, 17.6% and 35.7%, respectively, while the knockout of PAH1 decreased S-RET production. In other words, knockout of OPI1-mediated endoplasmic reticulum expansion had the best effect on increasing S-RET content. By knocking out OPI1, a transcription factor encoding a negative regulator of phospholipid biosynthesis, or knocking out PAH1, a phosphatase encoding a phospholipid biosynthesis, or overexpressing INO2 and INO2*, transcription factors encoding a positive regulator of phospholipid biosynthesis, *The above results indicate that overexpression of INO2, INO2* and knockout of OPI1-mediated ER expansion increase the production of taurocholine, that is, knockout of OPI1-mediated ER expansion has the best effect on increasing S-RET content.

[0396] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A recombinant bacterium for increasing the yield of benzylisoquinoline alkaloids, characterized in that: The recombinant bacteria contain berberisin BBE with enhanced activity compared with the parent microorganism.

2. The recombinant bacterium according to claim 1, characterized in that: The recombinant bacteria contain or express a fusion protein of BBE and the endoplasmic reticulum C-terminal retention signal peptide HDEL, a fusion protein of BBE and the N-terminal Golgi target sequence, or BBE.

3. The recombinant bacterium according to claim 1 or 2, characterized in that: The recombinant bacteria contain or express tyrosine hydroxylase and dopa decarboxylase.

4. The recombinant bacterium according to any one of claims 1 to 3, characterized in that: The recombinant bacteria also contain or express higenamine synthase NCS or NCS enzyme with N-terminal truncations.

5. The recombinant bacterium according to any one of claims 1 to 4, characterized in that: The recombinant bacteria also contain or express tyrosine metabolic flux-related proteins.

6. The recombinant bacterium according to any one of claims 1 to 5, characterized in that: The recombinant bacteria also knock out or down-regulate proteins related to the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid.

7. The recombinant bacterium according to any one of claims 1 to 6, characterized in that: The recombinant bacteria also contain or express aromatic amino acid decarboxylase.

8. The recombinant bacterium according to any one of claims 1 to 7, characterized in that: The recombinant bacteria also contain or express prephenate aminotransferase PAT and alloate dehydratase ADH.

9. The recombinant bacterium according to any one of claims 1 to 8, characterized in that: The recombinant bacteria also contain or express methyltransferase 6OMT and clavenine methyltransferase CNMT.

10. The recombinant bacterium according to any one of claims 1 to 9, characterized in that: The recombinant bacteria also contain or express one or more methyltransferases 4'OMT and N-methylcoclaurine 3'-hydroxylase NMCH.

11. The recombinant bacterium according to any one of claims 1 to 10, characterized in that: The recombinant bacteria also contain or express cytochrome P450 reductase ATR1, ATR2 or CPR.

12. The recombinant bacterium according to claim 1 or 2, characterized in that: The recombinant bacteria also contain or express catalase.

13. The recombinant bacterium according to claim 1 or 2, characterized in that: The recombinant bacteria also contain or express transcription factors that activate phospholipid biosynthesis or knock out transcription factors that inhibit phospholipid biosynthesis.

14. The recombinant bacterium according to any one of claims 1 to 13, characterized in that: The recombinant bacteria is a species of the genus Saccharomyces cerevisiae.

15. The recombinant bacterium according to any one of claims 1 to 14, characterized in that: The recombinant bacteria is Saccharomyces cerevisiae.

16. A method for producing benzylisoquinoline alkaloids, comprising the following steps: fermenting and culturing the recombinant bacteria according to any one of claims 1 to 15, collecting the fermentation product, and selectively purifying and collecting to obtain chrysoberyl, chondrine, norcoclaurine, chondrine, tetrahydroberberine, noscapine, bamipine, berberine and / or papaverine.

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