Recombinant bacterium and application thereof in production of benzophenanthridine alkaloid

Abstract

The invention discloses a recombinant bacterium and application of the recombinant bacterium in production of benzophenanthridine alkaloid. The synthetic route of the benzophenanthridine alkaloid is divided into a plurality of modules for research, and de novo synthesis of the benzophenanthridine alkaloid is realized through a plurality of strategies, for example, in order to improve the activity of BBE, a new endoplasmic reticulum engineering strategy is developed, so that the yield of a key intermediate S-Soulerine is increased by 3 times; the McDBOX2 is identified as the only reported DBOX gene which plays a role in saccharomyces cerevisiae at present, and the reconstruction of the whole biosynthetic pathway of the benzophenanthridine alkaloid is completed; by carrying out N-terminal truncation operation on DBOX, cytoplasmic expression and endoplasmic reticulum amplification are improved, and supply of a cofactor FAD is increased, so that the yield of benzophenanthridine alkaloid is increased. Experiments prove that the recombinant strain constructed by the invention can be used for efficiently producing benzophenanthridine alkaloid. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant bacterium and an application thereof in producing benzophenanthridine alkaloids (such as sanguinarine or chelerythrine). 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] Benzophenanthridine alkaloids (BZDAs) are an important type of BIAs compounds, which are formed by spontaneous ring opening and rearrangement of protoberberine alkaloids. The benzophenanthridine alkaloids chelerythrine (CHE) and sanguinarine (SAN) have significant pharmacological activities, including antibacterial, anticancer, anti-inflammatory, adrenal lytic and cytostatic effects, and have been used as natural growth promoters in animal feeding. Berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the biosynthetic pathway of BZDAs, which catalyzes S-retein (S-RET) to S-scorine (S-SCO). Previous studies have shown that intercepting PsBBE from opium poppy and increasing the expression level of PsBBE by adding high-copy plasmids does not improve its conversion efficiency. Therefore, it is still a great challenge to improve the activity of BBE in Saccharomyces cerevisiae. Some researchers have synthesized SAN in Saccharomyces cerevisiae, but not from scratch. In addition, due to the lack of efficient conversion by DBOX, only trace amounts of SAN are converted from dihydrosanguinarine either spontaneously or by uncharacterized functional oxidases of yeast itself or by upstream BBE. Therefore, the activities of BBE and DBOX are key to the heterologous synthesis of sanguinarine and chelerythrine-related compounds. Summary of the invention

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

[0005] The present invention firstly protects a recombinant fungus A for producing benzophenanthridine alkaloids, which is obtained by transforming a yeast for producing chrysanthemine; the recombinant fungus A contains or expresses berberidase BBE and benzophenanthridine oxidase DBOX with enhanced activity compared with the parent microorganism (i.e., the starting bacteria).

[0006] In the above-mentioned recombinant bacteria A, the berberidase BBE contained or expressed in the body of the recombinant bacteria A with enhanced activity than that of the parent microorganism can be a fusion protein of BBE contained or expressed in the body of the recombinant bacteria A 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, it is a fusion protein of BBE and the endoplasmic reticulum C-terminal retention signal HDEL.

[0007] In the above-mentioned recombinant bacterial A, the recombinant bacterial A containing or expressing the benzophenanthridine oxidase DBOX in the body may be an N-terminal truncation of the benzophenanthridine oxidase DBOX contained or expressed in the body of the recombinant bacterial A.

[0008] In the above recombinant bacteria A, the BBE can 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.

[0009] 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.

[0010] In the above recombinant bacteria A, the fusion protein containing or expressing BBE and the endoplasmic reticulum C-terminal retention signal HDEL in vivo can be obtained by introducing the encoding nucleic acid of the fusion protein into the starting bacteria.

[0011] In the above recombinant bacteria A, the fusion protein containing or expressing BBE and N-terminal Golgi target sequence in vivo can be obtained by introducing the encoding nucleic acid of the fusion protein into the starting bacteria.

[0012] In the above-mentioned recombinant bacteria A, the containing or expressing of BBE in vivo may be the introduction of the encoding nucleic acid of the BBE into the starting bacteria.

[0013] In the above-mentioned recombinant bacterial A, the recombinant bacterial A contains or expresses the benzophenanthridine oxidase DBOX in the body, which can be achieved by introducing the encoding nucleic acid of the benzophenanthridine oxidase DBOX into the starting bacteria or any of the above-mentioned recombinant bacterial A.

[0014] In the above-mentioned recombinant bacterial strain A, the recombinant bacterial strain A contains or expresses an N-terminal truncation of benzophenanthridine oxidase DBOX in vivo, which can be achieved by introducing the encoding nucleic acid of the N-terminal truncation of benzophenanthridine oxidase DBOX into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0015] In the above-mentioned recombinant Bacterium A, the introduction of the nucleic acid encoding the benferidine oxidase DBOX into the starting bacteria or any of the above-mentioned recombinant Bacterium A may be the introduction of an expression cassette containing the nucleic acid encoding the benferidine oxidase DBOX into the starting bacteria or any of the above-mentioned recombinant Bacterium A.

[0016] In the above-mentioned recombinant bacteria A, the introduction of the nucleic acid encoding the N-terminal truncation of benzophenanthridine oxidase DBOX into the starting bacteria or any of the above-mentioned recombinant bacteria A can be the introduction of an expression cassette containing the nucleic acid encoding the N-terminal truncation of benzophenanthridine oxidase DBOX into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0017] The expression cassette containing the nucleic acid encoding the benferidine oxidase DBOX may include a promoter, a nucleic acid encoding the benferidine oxidase DBOX and a terminator. The expression cassette containing the nucleic acid encoding the N-terminal truncation of the benferidine oxidase DBOX may include a promoter, a nucleic acid encoding the N-terminal truncation of the benferidine oxidase DBOX and a terminator. The promoter may be promoter PDC1p or promoter CCW12p. The terminator may be DIT1t or TDH2t.

[0018] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0019] Specifically, the recombinant Bacterium A can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding benferidine oxidase DBOX into the XII-1 site of the starting bacterium or any of the above-mentioned recombinant Bacterium A using a genome editing method.

[0020] Specifically, the recombinant Bacterium A can be a strain obtained by integrating an expression cassette of a nucleic acid encoding an N-terminal truncation of benzophenone oxidase DBOX into the FgF20 site of a starting bacterium or any of the above-mentioned recombinant Bacterium A using a genome editing method.

[0021] In the above-mentioned recombinant bacteria A, the benferidine oxidase DBOX may be McDBOX2 (genbank number: OVA00268.1).

[0022] In the above recombinant bacteria A, the N-terminal truncation of the benferidine oxidase DBOX can be the N-terminal truncation of McDBOX2 with 29 amino acids (ie, McDBOX2_Δ29).

[0023] In the above-mentioned recombinant bacteria A, the expression cassette containing the nucleic acid encoding the benferidine oxidase DBOX can be a gene expression cassette XII-1us-DIT1t-His-McDBOX2-PDC1p-CCW12p. Expression cassette XII-1

[0024] us-DIT1t-His-McDBOX2-PDC1p-CCW12p can be obtained by connecting the upstream homology arm XII-1us, terminator DIT1t, His tag sequence (CATCATCACCATCACCAT), the nucleic acid sequence of McDBOX2, promoter PDC1p and promoter CCW12p using the overlap method.

[0025] In the above recombinant bacteria A, the expression cassette containing the nucleic acid encoding the N-terminal truncation of benferidine oxidase DBOX can be a McDBOX2_Δ29 gene expression cassette consisting of an upstream homology arm FgF20 us, a promoter PDC1p, a nucleic acid sequence of McDBOX2_Δ29, a terminator DIT1t and a downstream homology arm FgF20 ds.

[0026] Any of the above-mentioned recombinant bacteria A can be a species of the genus Saccharomyces cerevisiae.

[0027] Any of the above-mentioned recombinant bacteria A can be Saccharomyces cerevisiae.

[0028] Any of the above starting bacteria may be a species of the genus Saccharomyces cerevisiae.

[0029] Any of the above starting bacteria can be Saccharomyces cerevisiae.

[0030] The cerevisiae yeast may specifically be the IMX581 yeast strain.

[0031] The use of any of the above-mentioned recombinant bacteria in the production of benzophenanthridine alkaloids also falls within the protection of the present invention.

[0032] The present invention also protects a method for producing benzophenanthridine alkaloids, which may include the following steps: fermenting and culturing any of the above-mentioned recombinant bacteria, collecting the fermentation products, and obtaining benzophenanthridine alkaloids therefrom.

[0033] Any of the above-mentioned benzophenanthridine alkaloids may be sanguinarine or chelerythrine.

[0034] In the above-mentioned recombinant fungus A, the recombinant fungus A contains or expresses papaverine synthase CFS and papaverine synthase SPS.

[0035] In the above-mentioned recombinant fungus A, the inclusion or expression of cholerae synthase CFS and papaverine synthase SPS in the body can be achieved by introducing the encoding nucleic acid of cholerae synthase CFS and the encoding nucleic acid of papaverine synthase SPS into the starting bacteria or any of the above-mentioned recombinant fungus A.

[0036] In the above-mentioned recombinant fungus A, the presence or expression of papaverine synthase CFS and papaverine synthase SPS in the body can be achieved by introducing an expression cassette containing the nucleic acid encoding papaverine synthase CFS and an expression cassette containing the nucleic acid encoding papaverine synthase SPS into the starting bacteria or any of the above-mentioned recombinant fungus A.

[0037] The expression cassette containing the encoding nucleic acid of spatholina synthase CFS may include a promoter, the encoding nucleic acid of spatholina synthase CFS and a terminator. The expression cassette containing the encoding nucleic acid of papaverine synthase SPS may include a promoter, the encoding nucleic acid of papaverine synthase SPS and a terminator. The promoter may be promoter PDC1p, promoter GPM1p, promoter TPI1p or promoter PGK1p. The terminator may be terminator ENO2t, terminator ADH1t or terminator FBA1t.

[0038] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0039] Specifically, the recombinant strain A can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding styraxine synthase CFS and an expression cassette containing a nucleic acid encoding papaverine synthase SPS into the XI-2 site of the starting bacteria or any of the above-mentioned recombinant strains A using a genome editing method.

[0040] In the above-mentioned recombinant bacteria A, the choline synthase CFS can be EcCFS derived from E. californica, CyCFS derived from C. yanhusuo or PsTNMT derived from Papaver somiferum; preferably EcCFS.

[0041] In the above-mentioned recombinant bacteria A, the papaverine synthase SPS can be EcSPS derived from E. californica, CySPS derived from C. yanhusuo or PsMSH derived from Papaver somiferum; preferably EcSPS.

[0042] In the above-mentioned recombinant fungus A, the recombinant fungus A contains or expresses N-methyltransferase TNMT in the body.

[0043] In the above-mentioned recombinant bacterial strain A, the presence or expression of N-methyltransferase TNMT in the body may be achieved by introducing the encoding nucleic acid of N-methyltransferase TNMT into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0044] In the above-mentioned recombinant Bacterium A, the containing or expressing of N-methyltransferase TNMT in vivo may be achieved by introducing an expression cassette of a nucleic acid encoding N-methyltransferase TNMT into the starting bacteria or any of the above-mentioned recombinant Bacterium A.

[0045] The expression cassette containing the encoding nucleic acid of N-methyltransferase TNMT may include a promoter, the encoding nucleic acid of N-methyltransferase TNMT and a terminator. The promoter may be promoter PDC1p, promoter GPM1p, promoter TPI1p or promoter PGK1p. The terminator may be terminator ENO2t, terminator ADH1t or terminator FBA1t.

[0046] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0047] Specifically, the recombinant strain A can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding N-methyltransferase TNMT into the XI-2 site of the starting bacteria or any of the above-mentioned recombinant strain A using a genome editing method.

[0048] In the above recombinant strain A, the N-methyltransferase TNMT may be PsTNMT derived from poppy (P. somiferum).

[0049] In the above-mentioned recombinant fungus A, the recombinant fungus A contains or expresses N-methyl papaverine 14-hydroxylase MSH in its body.

[0050] In the above-mentioned recombinant Bacterium A, the containing or expressing of N-methyl papaverine 14-hydroxylase MSH in the body may be achieved by introducing the encoding nucleic acid of N-methyl papaverine 14-hydroxylase MSH into the starting bacteria or any of the above-mentioned recombinant Bacterium A.

[0051] In the above-mentioned recombinant bacteria A, the in vivo containing or expressing N-methyl papaverine 14-hydroxylase MSH can be obtained by introducing an expression cassette containing a nucleic acid encoding N-methyl papaverine 14-hydroxylase MSH into the starting bacteria or any of the above-mentioned recombinant bacteria A. The expression cassette containing a nucleic acid encoding N-methyl papaverine 14-hydroxylase MSH may include a promoter, a nucleic acid encoding N-methyl papaverine 14-hydroxylase MSH and a terminator. The promoter may be promoter PDC1p, promoter GPM1p, promoter TPI1p or promoter PGK1p. The terminator may be terminator ENO2t, terminator ADH1t or terminator FBA1t.

[0052] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0053] Specifically, the recombinant strain A can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding N-methyl papaverine 14-hydroxylase MSH into the XI-2 site of the starting strain or any of the above-mentioned recombinant strain A using a genome editing method.

[0054] In the above recombinant strain A, the N-methyl papaverine 14-hydroxylase MSH can be PsMSH derived from poppy (P. somiferum).

[0055] In the above-mentioned recombinant bacterial A, the recombinant bacterial A contains or expresses proopiopine-6-hydroxylase P6H in the body.

[0056] In the above-mentioned recombinant fungus A, the recombinant fungus A contains or expresses protopine-6-hydroxylase P6H in vivo, which can be achieved by introducing the encoding nucleic acid of protopine-6-hydroxylase P6H into the starting bacteria or any of the above-mentioned recombinant fungus A.

[0057] In the above-mentioned recombinant Bacterium A, the introduction of the nucleic acid encoding protopine-6-hydroxylase P6H into the starting bacteria or any of the above-mentioned recombinant Bacterium A may be the introduction of an expression cassette containing the nucleic acid encoding protopine-6-hydroxylase P6H into the starting bacteria or any of the above-mentioned recombinant Bacterium A.

[0058] The expression cassette containing the nucleic acid encoding protopine-6-hydroxylase P6H may include a promoter, the nucleic acid encoding protopine-6-hydroxylase P6H and a terminator. The promoter may be promoter PDC1p or promoter CCW12p. The terminator may be terminator DIT1t and terminator TDH2t.

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

[0060] Specifically, the recombinant strain A can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding protoopine-6-hydroxylase P6H into the XII-1 site of the starting bacteria or any of the above-mentioned recombinant strain A using a genome editing method.

[0061] In the above-mentioned recombinant bacteria A, the protopine-6-hydroxylase P6H can be EcP6H (BAK20464) derived from California poppy.

[0062] In the above-mentioned recombinant fungus A, the recombinant fungus A contains or expresses a plasma membrane riboflavin transporter and / or FAD synthase.

[0063] In the above-mentioned recombinant bacterial strain A, the plasma membrane riboflavin transporter contained or expressed in the body can be obtained by introducing the encoding nucleic acid of the plasma membrane riboflavin transporter into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0064] In the above-mentioned recombinant bacteria A, the plasma membrane riboflavin transporter contained or expressed in the body can be obtained by introducing an expression cassette of a nucleic acid encoding the plasma membrane riboflavin transporter into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0065] The expression cassette containing the nucleic acid encoding the plasma membrane riboflavin transporter may include a promoter, a nucleic acid encoding the plasma membrane riboflavin transporter and a terminator. The promoter is promoter GPD or promoter CCW12p. The terminator is IDP1t or TDH2t.

[0066] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0067] Specifically, the recombinant Bacterium A can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding a plasma membrane riboflavin transporter into the 416d site of a starting bacterium or any of the above-mentioned recombinant Bacterium A using a genome editing method.

[0068] In the above-mentioned recombinant bacteria A, the plasma membrane riboflavin transporter can be the plasma membrane riboflavin transporter MCH5 (nucleotide sequence as shown in SEQ ID NO: 78).

[0069] In the above-mentioned recombinant bacterial strain A, the in vivo presence or expression of FAD synthase may be the introduction of a nucleic acid encoding FAD synthase into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0070] In the above-mentioned recombinant bacterial strain A, the in vivo presence or expression of FAD synthase may be achieved by introducing an expression cassette containing a nucleic acid encoding FAD synthase into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0071] The expression cassette containing the nucleic acid encoding FAD synthase may include a promoter, the nucleic acid encoding FAD synthase and a terminator. The promoter is promoter GPD or promoter CCW12p. The terminator is IDP1t or TDH2t.

[0072] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0073] Specifically, the recombinant Bacterium A can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding FAD synthase into the 416d site of the starting bacterium or any of the above-mentioned recombinant Bacterium A using a genome editing method.

[0074] In the above-mentioned recombinant bacteria A, the FAD synthase may be FAD synthase BsRibc (nucleotide sequence as shown in SEQ ID NO: 79).

[0075] In the above recombinant bacteria A, the body may contain or express tyrosine hydroxylase and dopa decarboxylase.

[0076] In the above-mentioned recombinant bacterial strain A, the tyrosine hydroxylase and dopa decarboxylase contained or expressed in the body can be obtained by introducing the tyrosine hydroxylase encoding nucleic acid and the dopa decarboxylase encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0077] In the above-mentioned recombinant bacteria A, the introduction of the tyrosine hydroxylase encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria A 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 A. The expression cassette containing the tyrosine hydroxylase encoding nucleic acid may include a promoter, a tyrosine hydroxylase encoding nucleic acid and a terminator.

[0078] In the above-mentioned recombinant bacteria A, the introduction of the coding nucleic acid of DOPA decarboxylase into the starting bacteria or any of the above-mentioned recombinant bacteria A 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 A. 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.

[0079] In the above recombinant bacteria A, the promoter may be TDH3p, CCW12p or TEF1p. The terminator may be IDP1t, TPS1t or ENO2t.

[0080] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0081] Specifically, the recombinant strain A 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 strain A using a genome editing method.

[0082] In the above-mentioned recombinant bacteria A, 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.

[0083] In the above recombinant bacteria A, the DOPA decarboxylase may specifically be DODC (genbank number DAA64376.1).

[0084] Any of the above-mentioned recombinant bacteria A may also contain or express higenamine synthase NCS or N-terminally truncated NCS enzyme.

[0085] In the above-mentioned recombinant bacteria A, the NCS enzyme may be CjNCS from Coptis japonica (genbank number BAF45338.2).

[0086] In the above-mentioned recombinant bacteria A, 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.

[0087] In the above-mentioned recombinant fungus A, the body also contains or expresses higenamine synthase NCS or N-terminally truncated NCS enzyme, which can be obtained by introducing the encoding nucleic acid of the NCS enzyme or the encoding nucleic acid of the N-terminally truncated NCS enzyme into the starting bacteria or any of the above-mentioned recombinant fungus A.

[0088] In the above-mentioned recombinant bacteria A, 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 A can 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 A. 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.

[0089] In the above recombinant bacteria A, the promoter may be TEF1p and the terminator may be TDH2t.

[0090] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0091] In the above-mentioned recombinant bacteria A, the NCS enzyme or the N-terminally truncated NCS enzyme contained or expressed in the body can be increased copy number of the nucleic acid encoding the NCS enzyme or the N-terminally truncated NCS enzyme in the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0092] In the above recombinant bacteria A, the number of copies of the nucleic acid encoding the N-terminally truncated NCS enzyme may be 3, 2 or 1, preferably 3.

[0093] Any of the above-mentioned recombinant bacteria A may also contain or express tyrosine metabolic flux-related proteins.

[0094] In the above recombinant strain A, 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).

[0095] In the above-mentioned recombinant bacterial A, the tyrosine metabolic flux-related protein may be contained or expressed in the body by introducing the encoding nucleic acid of the tyrosine metabolic flux-related protein into the starting bacteria or any of the above-mentioned recombinant bacterial A.

[0096] In the above-mentioned recombinant bacteria A, the introduction of the nucleic acid encoding the tyrosine metabolic flux-related protein into the starting bacteria or any of the above-mentioned recombinant bacteria A may be the introduction of the expression cassette of the nucleic acid encoding the tyrosine metabolic flux-related protein into the starting bacteria or any of the above-mentioned recombinant bacteria A. The expression cassette may include a promoter, the nucleic acid encoding the tyrosine metabolic flux-related protein and a terminator.

[0097] In the above recombinant bacteria A, the promoter may be TPI1p, PGK1p, TEF1p, TDH3p or tHXT7p. The terminator may be pYX212t, ADH1t, FBA1t, CYC1t or TDH2t.

[0098] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0099] In any of the above-mentioned recombinant bacteria A, proteins related to the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid can also be knocked out or down-regulated.

[0100] In the above-mentioned recombinant bacteria A, 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.

[0101] In the above recombinant bacteria A, the proteins related to the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, GRE2 and HFD1.

[0102] Any of the above-mentioned recombinant bacteria may also contain or express aromatic amino acid decarboxylase.

[0103] In the above-mentioned recombinant bacteria A, the aromatic amino acid decarboxylase can be PcAAS derived from Petroselinum crispum, RrAAS derived from Rhodiola Salidroside, or a mutant PsAAAD* derived from P. somniferum, preferably PsAAAD*.

[0104] In the above-mentioned recombinant bacterial strain A, the aromatic amino acid decarboxylase is contained or expressed in the body, which can be obtained by introducing the encoding nucleic acid of the aromatic amino acid decarboxylase into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0105] In the above-mentioned recombinant bacteria A, 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 A 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 A. The expression cassette may include a promoter, the encoding nucleic acid of the aromatic amino acid decarboxylase and a terminator.

[0106] The promoter may be FBA1p. The terminator may be TPS1t-ADH1t.

[0107] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0108] 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.

[0109] In the above-mentioned recombinant bacterial strain A, the PAT and ADH are also contained or expressed in the body by introducing the PAT encoding nucleic acid and the ADH encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0110] In the above recombinant bacteria A, the introduction of the PAT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria A 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 A. The expression cassette may include a promoter, a PAT encoding nucleic acid and a terminator.

[0111] In the above-mentioned recombinant bacteria A, the introduction of the ADH encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria A can 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 A. 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.

[0112] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0113] Preferably, any of the above-mentioned recombinant bacteria A may also contain or express AtPAT and MtncADH.

[0114] Any of the above-mentioned recombinant bacteria may also contain or express methyltransferase 6OMT and clavenine methyltransferase CNMT.

[0115] In the above recombinant bacteria A, 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.

[0116] In the above-mentioned recombinant bacterial strain A, 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 bacterial strain A.

[0117] In the above recombinant Bacterium A, the introduction of the 6OMT encoding nucleic acid into the starting bacteria or any of the above recombinant Bacterium A can be the introduction of an expression cassette containing the 6OMT encoding nucleic acid into the starting bacteria or any of the above recombinant Bacterium A. The expression cassette may include a promoter, a 6OMT encoding nucleic acid and a terminator.

[0118] In the above recombinant Bacteria A, the introduction of the CNMT encoding nucleic acid into the starting bacteria or any of the above recombinant Bacteria A can be the introduction of an expression cassette containing the CNMT encoding nucleic acid into the starting bacteria or any of the above recombinant Bacteria A. The expression cassette may include a promoter, a CNMT encoding nucleic acid and a terminator.

[0119] Any of the above-mentioned recombinant bacteria may also contain or express NMCH in vivo. The NMCH may be EcNMCH (AAC39452.1) or CyNMCH derived from Eschscholzia californica.

[0120] In the above-mentioned recombinant bacteria A, the in vivo further contains or expresses NMCH, which may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A by introducing the encoding nucleic acid of NMCH into the starting bacteria or any of the above-mentioned recombinant bacteria A. The introduction of the encoding nucleic acid of NMCH into the starting bacteria or any of the above-mentioned recombinant bacteria A may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A by introducing an expression cassette containing the encoding nucleic acid of NMCH. The expression cassette may include a promoter, the encoding nucleic acid of NMCH and a terminator.

[0121] The promoter may be TPI1p, PGK1p, TEF1p, tHXT7p, TDH3p, GPM1p or TEF2p.

[0122] The terminator may be pYX212t, ADH1t, CYC1t, FBA1t, TDH2t, PGI1t or ENO2t.

[0123] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0124] Preferably, any of the above-mentioned recombinant bacteria may also contain or express Ps6OMT, PsCNMT and CyNMCH.

[0125] In the above-mentioned recombinant bacterial A, the NMCH contained or expressed in the body can be increased copy number of the nucleic acid encoding NMCH in the starting bacteria or any of the above-mentioned recombinant bacterial A.

[0126] In the above-mentioned recombinant bacteria A, the copy number of the nucleic acid encoding NMCH can be 1-5, preferably 5.

[0127] Any of the above-mentioned recombinant bacteria A may also contain or express methyltransferase 4'OMT.

[0128] In the above recombinant bacteria A, the 4'OMT may be Ps4'OMT.

[0129] In the above-mentioned recombinant bacterial strain A, the 4'OMT may be contained or expressed in the body by introducing the encoding nucleic acid of 4'OMT into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0130] In the above recombinant Bacteria A, the introduction of the 4'OMT encoding nucleic acid into the starting bacteria or any of the above recombinant Bacteria A can 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 A. The expression cassette may include a promoter, a 4'OMT encoding nucleic acid and a terminator.

[0131] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0132] In the above-mentioned recombinant bacteria A, the 4'OMT contained or expressed in the body can be increased copy number of the nucleic acid encoding 4'OMT in the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0133] In the above recombinant bacteria A, the copy number of the nucleic acid encoding 4'OMT can be 2 or 1, preferably 2.

[0134] Any of the above-mentioned recombinant bacteria A 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.

[0135] In the above-mentioned recombinant bacteria A, the CPR may be PsCPR.

[0136] In the above-mentioned recombinant bacterial strain A, the ATR1 contained or expressed in the body may be introduced into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0137] In the above-mentioned recombinant bacterial strain A, the ATR2 contained or expressed in the body may be introduced into the starting bacteria or any of the above-mentioned recombinant bacterial strain A.

[0138] In the above-mentioned recombinant fungus A, 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 fungus A. The introduction of the encoding nucleic acid of CPR into the starting bacteria or any of the above-mentioned recombinant fungus A 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 fungus A. The introduction of ATR1 into the starting bacteria or any of the above-mentioned recombinant fungus A may be achieved by introducing the expression cassette containing ATR1 into the starting bacteria or any of the above-mentioned recombinant fungus A. The introduction of ATR2 into the starting bacteria or any of the above-mentioned recombinant fungus A may be achieved by introducing the expression cassette containing ATR2 into the starting bacteria or any of the above-mentioned recombinant fungus A. In the above-mentioned recombinant fungus A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant fungus A in the form of a plasmid and / or integrated into the chromosome.

[0139] In the above recombinant Bacteria A, the introduction of the encoding nucleic acid into the starting bacteria or any of the above recombinant Bacteria A may be the introduction of an expression cassette of the encoding nucleic acid into the starting bacteria or any of the above recombinant Bacteria A. The expression cassette may include a promoter, an encoding nucleic acid and a terminator.

[0140] In the above recombinant bacteria A, the promoter may be CCW12p and the terminator may be PGI1t.

[0141] In the above-mentioned recombinant Bacterium A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant Bacterium A in the form of a plasmid and / or integrated into the chromosome.

[0142] Any of the above-mentioned recombinant bacteria A may also contain or express catalase.

[0143] In the above-mentioned recombinant bacteria A, the catalase may be PRDX4.

[0144] In the above-mentioned recombinant bacterial strain A, 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 bacterial strain A.

[0145] In the above-mentioned recombinant bacteria A, 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 peroxiredoxin IV (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 A.

[0146] Any of the above-mentioned recombinant bacteria A may also contain or express transcription factors that activate phospholipid biosynthesis or knock out transcription factors that inhibit phospholipid biosynthesis.

[0147] In the above-mentioned recombinant bacteria A, the knocked-out transcription factor that inhibits phospholipid biosynthesis may be the OPI1 gene.

[0148] In the above-mentioned recombinant bacteria A, the transcription factor that activates phospholipid biosynthesis may be the INO2 gene or the INO2 gene mutant INO2* (INO2_L119A).

[0149] The use of any of the above-mentioned recombinant bacteria A in the production of sanguinarine, dihydrosanguinarine, protoopine, N-methyl papaverine and / or papaverine also falls within the scope of protection of the present invention.

[0150] The present invention also protects a method for producing sanguinarine, dihydrosanguinarine, protopine, N-methyl papaverine and / or papaverine, comprising the following steps: fermenting and culturing any of the above-mentioned recombinant bacteria A, collecting the fermentation product, and obtaining sanguinarine, dihydrosanguinarine, protopine, N-methyl papaverine and / or papaverine therefrom.

[0151] The present invention also protects a recombinant bacteria B, which is obtained by transforming a yeast used for producing dihydrosanguinarine; the recombinant bacteria B contains or expresses CYP82P2 (genbank: BBD34756.1).

[0152] In the above recombinant bacteria B, the yeast containing or expressing CYP82P2 in vivo may be a yeast into which the CYP82P2 gene (genbank: BBD34756.1) is introduced for producing sanguinarine.

[0153] In the above-mentioned recombinant bacteria B, the yeast for producing dihydrosanguinarine by introducing the CYP82P2 gene (genbank: BBD34756.1) can be a yeast for producing dihydrosanguinarine by introducing an expression cassette containing the CYP82P2 gene. The expression cassette may include a promoter, a CYP82P2 gene and a terminator. The promoter may be promoter GPD. The terminator may be terminator IDP1t.

[0154] Any of the above-mentioned recombinant bacteria B also contains or expresses 10-hydroxymethyltransferase OMT.

[0155] In the above recombinant bacteria B, the 10-hydroxymethyltransferase OMT contained or expressed in the body may be a yeast for producing sanguinarine, wherein the gene encoding the 10-hydroxymethyltransferase OMT is introduced into the yeast.

[0156] In the above-mentioned recombinant bacteria B, the introduction of the coding gene of 10-hydroxymethyltransferase OMT into the yeast for producing dihydrosanguinarine can be the introduction of an expression cassette containing the coding gene of 10-hydroxymethyltransferase OMT into the yeast for producing dihydrosanguinarine. The expression cassette may include a promoter, a coding gene of 10-hydroxymethyltransferase OMT and a terminator. The promoter may be promoter CCW12p. The terminator may be terminator CYC1t.

[0157] The 10-hydroxymethyltransferase OMT may be Ec11OMT (genbank: BBA20642.1) or Ec2OMT (genbank: BBA20644.1), preferably Ec11OMT.

[0158] The expression cassette containing the gene encoding 10-hydroxymethyltransferase OMT can be an Ec11OMT expression cassette (including promoter CCW12p, Ec11OMT (genbank: BBA20642.1) and terminator CYC1t) or an Ec2OMT expression cassette (including promoter CCW12p, Ec2OMT (genbank: BBA20642.1) and terminator CYC1t).

[0159] In any of the above-mentioned recombinant bacteria B, the yeast used to produce dihydrosanguinarine can be a yeast used to produce sanguinarine.

[0160] In any of the above-mentioned recombinant bacteria B, the yeast used to produce dihydrosanguinarine can be any of the above-mentioned recombinant bacteria A.

[0161] The use of any of the above-mentioned recombinant bacteria B in the production of chelerythrine and / or 10-hydroxydihydrosanguinis also falls within the protection scope of the present invention.

[0162] The present invention also protects a method for producing chelerythrine and / or 10-hydroxydihydrosanguiner, comprising the following steps: fermenting and culturing any of the above-mentioned recombinant bacteria B, collecting the fermentation product, and obtaining chelerythrine and / or 10-hydroxydihydrosanguiner therefrom.

[0163] The present invention also protects a recombinant bacterium, which is any of the above-mentioned recombinant bacteria A (referring to any of the above-mentioned recombinant bacteria A containing or expressing berberidase BBE and benzophenone oxidase DBOX with enhanced activity compared with the parent microorganism (i.e., the starting bacteria), and the recombinant bacteria in the subsequent invention content are the same as herein) containing or expressing O-methyltransferase. For distinction, the recombinant bacteria is named recombinant bacteria C.

[0164] In the above-mentioned recombinant bacteria C, the O-methyltransferase contained or expressed in the body may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A by a nucleic acid encoding the O-methyltransferase.

[0165] In the above-mentioned recombinant bacteria C, the O-methyltransferase contained or expressed in vivo may be obtained by introducing an expression cassette containing a nucleic acid encoding the O-methyltransferase into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0166] The expression cassette containing the nucleic acid encoding the O-methyltransferase may include a promoter, the nucleic acid encoding the O-methyltransferase and a terminator. The promoter may be promoter PDC1p, promoter GPM1p, promoter TPI1p or promoter PGK1p. The terminator may be terminator ENO2t, terminator IDP1t, terminator ADH1t or terminator FBA1t.

[0167] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0168] Specifically, the recombinant strain C can be a strain obtained by integrating an expression cassette containing an O-methyltransferase encoding nucleic acid into the XI-2 site of the starting strain or any of the above-mentioned recombinant strains A using a genome editing method.

[0169] In the above recombinant bacteria C, the O-methyltransferase may be TfS9OMT (AAU20770.1) derived from Thalictrum flavum.

[0170] In the above-mentioned recombinant bacteria C, the recombinant bacteria C contains or expresses tetrahydroberberine synthase CAS in the body.

[0171] In the above-mentioned recombinant bacteria C, the tetrahydroberberine synthase CAS contained or expressed in the body can be obtained by introducing the encoding nucleic acid of the tetrahydroberberine synthase CAS into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0172] In the above-mentioned recombinant bacteria C, the tetrahydroberberine synthase CAS may be contained or expressed in vivo by introducing an expression cassette containing a nucleic acid encoding the tetrahydroberberine synthase CAS into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0173] The expression cassette containing the nucleic acid encoding tetrahydroberberine synthase CAS may include a promoter, the nucleic acid encoding tetrahydroberberine synthase CAS and a terminator. The promoter may be promoter PDC1p, promoter GPM1p, promoter TPI1p or promoter PGK1p. The terminator may be terminator ENO2t, terminator IDP1t, terminator ADH1t or terminator FBA1t.

[0174] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0175] Specifically, the recombinant strain C can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding tetrahydroberberine synthase CAS into the XI-2 site of the starting strain or any of the above-mentioned recombinant strains A using a genome editing method.

[0176] In the above recombinant bacteria C, the tetrahydroberberine synthase CAS can be CjCAS (BAB68769.1) derived from Coptis chinensis or CyCAS derived from Corydalis yanhusuo.

[0177] In the above-mentioned recombinant bacteria C, the recombinant bacteria C contains or expresses N-methyltransferase TNMT.

[0178] In the above-mentioned recombinant bacteria C, the presence or expression of N-methyltransferase TNMT in the body may be achieved by introducing the encoding nucleic acid of N-methyltransferase TNMT into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0179] In the above-mentioned recombinant bacteria C, the containing or expressing of N-methyltransferase TNMT in vivo may be achieved by introducing an expression cassette containing a nucleic acid encoding N-methyltransferase TNMT into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0180] The expression cassette containing the encoding nucleic acid of N-methyltransferase TNMT may include a promoter, the encoding nucleic acid of N-methyltransferase TNMT and a terminator. The promoter may be promoter PDC1p, promoter GPM1p, promoter TPI1p or promoter PGK1p. The terminator may be terminator ENO2t, terminator ADH1t or terminator FBA1t.

[0181] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0182] Specifically, the recombinant strain C can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding N-methyltransferase TNMT into the XI-2 site of the starting strain or any of the above-mentioned recombinant strains A using a genome editing method.

[0183] In the above recombinant strain C, the N-methyltransferase TNMT may be PsTNMT derived from poppy (P. somiferum).

[0184] In the above recombinant bacteria C, the recombinant bacteria A contains or expresses N-methyl papaverine 14-hydroxylase MSH.

[0185] In the above recombinant bacteria C, the presence or expression of N-methyl papaverine 14-hydroxylase MSH in the body may be obtained by introducing the encoding nucleic acid of N-methyl papaverine 14-hydroxylase MSH into the starting bacteria or any of the above recombinant bacteria A.

[0186] In the above-mentioned recombinant bacteria C, the presence or expression of N-methyl papaverine 14-hydroxylase MSH in the body can be achieved by introducing an expression cassette containing a nucleic acid encoding N-methyl papaverine 14-hydroxylase MSH into the starting bacteria or any of the above-mentioned recombinant bacteria A. The expression cassette containing a nucleic acid encoding N-methyl papaverine 14-hydroxylase MSH may include a promoter, a nucleic acid encoding N-methyl papaverine 14-hydroxylase MSH, and a terminator. The promoter may be promoter PDC1p, promoter GPM1p, promoter TPI1p, or promoter PGK1p. The terminator may be terminator ENO2t, terminator ADH1t, or terminator FBA1t.

[0187] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0188] Specifically, the recombinant strain C can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding N-methyl papaverine 14-hydroxylase MSH into the XI-2 site of the starting strain or any of the above-mentioned recombinant strains A using a genome editing method.

[0189] In the above recombinant bacteria C, the N-methyl papaverine 14-hydroxylase MSH can be PsMSH derived from poppy (P. somiferum).

[0190] In the above-mentioned recombinant bacteria C, the recombinant bacteria A contains or expresses proopioline-6-hydroxylase P6H.

[0191] In the above-mentioned recombinant bacteria C, the recombinant bacteria A contains or expresses protopine-6-hydroxylase P6H in vivo, which can be achieved by introducing the encoding nucleic acid of protopine-6-hydroxylase P6H into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0192] In the above-mentioned recombinant bacteria C, the introduction of the nucleic acid encoding protopine-6-hydroxylase P6H into the starting bacteria or any of the above-mentioned recombinant bacteria A may be the introduction of an expression cassette containing the nucleic acid encoding protopine-6-hydroxylase P6H into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0193] The expression cassette containing the nucleic acid encoding protopine-6-hydroxylase P6H may include a promoter, the nucleic acid encoding protopine-6-hydroxylase P6H and a terminator. The promoter may be promoter PDC1p or promoter CCW12p. The terminator may be terminator DIT1t and terminator TDH2t.

[0194] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0195] Specifically, the recombinant strain C can be a strain obtained by integrating an expression cassette containing the nucleic acid encoding protoopine-6-hydroxylase P6H into the XII-1 site of the starting strain or any of the above-mentioned recombinant strains A using a genome editing method.

[0196] In the above recombinant bacteria C, the protopine-6-hydroxylase P6H may be EcP6H (BAK20464) derived from California poppy.

[0197] In the above-mentioned recombinant bacteria C, the recombinant bacteria C contains or expresses a plasma membrane riboflavin transporter and / or FAD synthase.

[0198] In the above-mentioned recombinant bacteria C, the presence or expression of the plasma membrane riboflavin transporter in the body may be achieved by introducing the encoding nucleic acid of the plasma membrane riboflavin transporter into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0199] In the above-mentioned recombinant bacteria C, the presence or expression of the plasma membrane riboflavin transporter in the body can be achieved by introducing an expression cassette of a nucleic acid encoding the plasma membrane riboflavin transporter into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0200] The expression cassette containing the nucleic acid encoding the plasma membrane riboflavin transporter may include a promoter, a nucleic acid encoding the plasma membrane riboflavin transporter and a terminator. The promoter is promoter GPD or promoter CCW12p. The terminator is IDP1t or TDH2t.

[0201] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0202] Specifically, the recombinant bacteria C can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding a plasma membrane riboflavin transporter into the 416d site of the starting bacteria or any of the above-mentioned recombinant bacteria A using a genome editing method.

[0203] In the above-mentioned recombinant bacteria C, the plasma membrane riboflavin transporter may be the plasma membrane riboflavin transporter MCH5.

[0204] In the above-mentioned recombinant bacteria C, the FAD synthase contained or expressed in the body may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A by introducing the encoding nucleic acid of the FAD synthase.

[0205] In the above-mentioned recombinant bacteria C, the in vivo presence or expression of FAD synthase may be achieved by introducing an expression cassette containing a nucleic acid encoding FAD synthase into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0206] The expression cassette containing the nucleic acid encoding FAD synthase may include a promoter, the nucleic acid encoding FAD synthase and a terminator. The promoter is promoter GPD or promoter CCW12p. The terminator is IDP1t or TDH2t.

[0207] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0208] Specifically, the recombinant strain C can be a strain obtained by integrating an expression cassette containing a nucleic acid encoding FAD synthase into the 416d site of the starting bacteria or any of the above-mentioned recombinant strains A using a genome editing method.

[0209] In the above-mentioned recombinant bacteria C, the FAD synthase may be FAD synthase BsRibc.

[0210] The above-mentioned recombinant bacteria C may contain or express tyrosine hydroxylase and dopa decarboxylase in the body.

[0211] The above-mentioned recombinant bacteria C, wherein the tyrosine hydroxylase and dopa decarboxylase are contained or expressed in the body, can be obtained by introducing the tyrosine hydroxylase encoding nucleic acid and the dopa decarboxylase encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0212] In the above-mentioned recombinant bacteria C, the introduction of the tyrosine hydroxylase encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria A 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 A. The expression cassette containing the tyrosine hydroxylase encoding nucleic acid may include a promoter, a tyrosine hydroxylase encoding nucleic acid and a terminator.

[0213] In the above recombinant bacteria C, the introduction of the coding nucleic acid of DOPA decarboxylase into the starting bacteria or any of the above recombinant bacteria A 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 recombinant bacteria A. 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.

[0214] In the above recombinant strain C, the promoter may be TDH3p, CCW12p or TEF1p. The terminator may be IDP1t, TPS1t or ENO2t.

[0215] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0216] Specifically, the recombinant strain C 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 strain or any of the above-mentioned recombinant strains A using a genome editing method.

[0217] In the above-mentioned recombinant bacteria C, 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.

[0218] In the above recombinant bacteria C, the DOPA decarboxylase may specifically be DODC (genbank number DAA64376.1).

[0219] Any of the above-mentioned recombinant bacteria C may also contain or express higenamine synthase NCS or N-terminally truncated NCS enzyme.

[0220] In the above recombinant bacteria C, the NCS enzyme may be CjNCS from Coptis japonica (genbank number BAF45338.2).

[0221] In the above recombinant bacteria C, 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.

[0222] In the above-mentioned recombinant bacteria C, the body also contains or expresses higenamine synthase NCS or N-terminally truncated NCS enzyme, which can be obtained by introducing the encoding nucleic acid of the NCS enzyme or the encoding nucleic acid of the N-terminally truncated NCS enzyme into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0223] In the above-mentioned recombinant bacteria C, 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 A 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 A. 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.

[0224] In the above recombinant strain C, the promoter may be TEF1p and the terminator may be TDH2t.

[0225] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0226] In the above-mentioned recombinant bacteria C, the NCS enzyme or the N-terminally truncated NCS enzyme contained or expressed in the body can be increased copy number of the nucleic acid encoding the NCS enzyme or the N-terminally truncated NCS enzyme in the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0227] In the above recombinant strain C, the number of copies of the nucleic acid encoding the N-terminally truncated NCS enzyme may be 3, 2 or 1, preferably 3.

[0228] Any of the above-mentioned recombinant bacteria C may also contain or express tyrosine metabolic flux-related proteins.

[0229] In the above recombinant strain C, 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).

[0230] In the above-mentioned recombinant bacteria C, the tyrosine metabolic flux-related protein may be contained or expressed in the body 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 A.

[0231] In the above recombinant bacteria C, the introduction of the nucleic acid encoding the tyrosine metabolic flux-related protein into the starting bacteria or any of the above recombinant bacteria A may be the introduction of an expression cassette of the nucleic acid encoding the tyrosine metabolic flux-related protein into the starting bacteria or any of the above recombinant bacteria A. The expression cassette may include a promoter, a nucleic acid encoding the tyrosine metabolic flux-related protein, and a terminator.

[0232] In the above recombinant strain C, the promoter may be TPI1p, PGK1p, TEF1p, TDH3p or tHXT7p. The terminator may be pYX212t, ADH1t, FBA1t, CYC1t or TDH2t.

[0233] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0234] In any of the above-mentioned recombinant bacteria C, proteins related to the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid can also be knocked out or down-regulated.

[0235] In the above recombinant strain C, 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.

[0236] In the above recombinant strain C, the proteins related to the synthesis activity of 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, GRE2 and HFD1.

[0237] Any of the above-mentioned recombinant bacteria C may also contain or express aromatic amino acid decarboxylase.

[0238] In the above-mentioned recombinant bacteria C, 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*.

[0239] In the above-mentioned recombinant bacteria C, 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 A.

[0240] In the above recombinant bacteria C, the introduction of the nucleic acid encoding the aromatic amino acid decarboxylase into the starting bacteria or any of the above recombinant bacteria A may be the introduction of an expression cassette containing the nucleic acid encoding the aromatic amino acid decarboxylase into the starting bacteria or any of the above recombinant bacteria A. The expression cassette may include a promoter, the nucleic acid encoding the aromatic amino acid decarboxylase, and a terminator.

[0241] The promoter may be FBA1p. The terminator may be TPS1t-ADH1t.

[0242] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0243] Any of the above-mentioned recombinant bacteria C 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.

[0244] In the above-mentioned recombinant bacteria C, the PAT and ADH may be contained or expressed in the body by introducing the PAT encoding nucleic acid and the ADH encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0245] In the above recombinant bacteria C, the introduction of the PAT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria A 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 A. The expression cassette may include a promoter, a PAT encoding nucleic acid and a terminator.

[0246] In the above-mentioned recombinant bacteria C, the introduction of the ADH encoding nucleic acid into the starting bacteria or any of the above-mentioned recombinant bacteria A can 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 A. 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.

[0247] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0248] Preferably, any of the above-mentioned recombinant bacteria C may also contain or express AtPAT and MtncADH.

[0249] Any of the above-mentioned recombinant bacteria C may also contain or express methyltransferase 6OMT and clavenine methyltransferase CNMT.

[0250] In the above recombinant bacteria C, the 6OMT may be Ps6OMT (AAQ01669.1) from P. somniferum or Cy6OMT from C. yanhusuo. The CNMT may be PsCNMT (AAP45316.1) or CyCNMT from P. somniferum.

[0251] In the above-mentioned recombinant bacteria C, 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 A.

[0252] In the above recombinant bacteria C, the introduction of the 6OMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria A can be the introduction of an expression cassette containing the 6OMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria A. The expression cassette may include a promoter, a 6OMT encoding nucleic acid and a terminator.

[0253] In the above recombinant bacteria C, the introduction of the CNMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria A 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 A. The expression cassette may include a promoter, a CNMT encoding nucleic acid and a terminator.

[0254] Any of the above-mentioned recombinant bacteria C may also contain or express NMCH in vivo. The NMCH may be EcNMCH (AAC39452.1) or CyNMCH derived from Eschscholzia californica.

[0255] In the above-mentioned recombinant bacteria C, the in vivo further contains or expresses NMCH, which may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A by introducing the encoding nucleic acid of NMCH into the starting bacteria or any of the above-mentioned recombinant bacteria A. The introduction of the encoding nucleic acid of NMCH into the starting bacteria or any of the above-mentioned recombinant bacteria A may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A by introducing an expression cassette containing the encoding nucleic acid of NMCH. The expression cassette may include a promoter, the encoding nucleic acid of NMCH and a terminator.

[0256] The promoter may be TPI1p, PGK1p, TEF1p, tHXT7p, TDH3p, GPM1p or TEF2p.

[0257] The terminator may be pYX212t, ADH1t, CYC1t, FBA1t, TDH2t, PGI1t or ENO2t.

[0258] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0259] Preferably, any of the above-mentioned recombinant bacteria C may also contain or express Ps6OMT, PsCNMT and CyNMCH.

[0260] In the above-mentioned recombinant bacteria C, the NMCH contained or expressed in the body can be increased copy number of the nucleic acid encoding NMCH in the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0261] In the above recombinant bacteria C, the copy number of the nucleic acid encoding NMCH can be 1-5, preferably 5.

[0262] Any of the above-mentioned recombinant bacteria C may also contain or express methyltransferase 4'OMT.

[0263] In the above recombinant strain C, the 4'OMT may be Ps4'OMT.

[0264] In the above recombinant bacteria C, the further containing or expressing 4'OMT in the body may be achieved by introducing the encoding nucleic acid of 4'OMT into the starting bacteria or any of the above recombinant bacteria A.

[0265] In the above recombinant bacteria C, the introduction of the 4'OMT encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria A can 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 A. The expression cassette may include a promoter, a 4'OMT encoding nucleic acid and a terminator.

[0266] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0267] In the above recombinant bacteria C, the 4'OMT contained or expressed in the body can be increased copy number of the nucleic acid encoding 4'OMT in the starting bacteria or any of the above recombinant bacteria A.

[0268] In the above recombinant strain C, the copy number of the nucleic acid encoding 4'OMT can be 2 or 1, preferably 2.

[0269] Any of the above-mentioned recombinant bacteria C may also contain or express cytochrome P450 reductase ATR1, ATR2 or CPR, preferably ATR1.

[0270] In the above-mentioned recombinant bacteria C, the CPR may be PsCPR.

[0271] In the above-mentioned recombinant bacteria C, the ATR1 is also contained or expressed in the body, which may be obtained by introducing ATR1 into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0272] In the above-mentioned recombinant bacteria C, the ATR2 contained or expressed in the body may be introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0273] In the above-mentioned recombinant bacteria C, 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 A. The introduction of the encoding nucleic acid of CPR into the starting bacteria or any of the above-mentioned recombinant bacteria A may be achieved by introducing an expression cassette containing the encoding nucleic acid of CPR into the starting bacteria or any of the above-mentioned recombinant bacteria A. The introduction of ATR1 into the starting bacteria or any of the above-mentioned recombinant bacteria A may be achieved by introducing an expression cassette containing ATR1 into the starting bacteria or any of the above-mentioned recombinant bacteria A. The introduction of ATR2 into the starting bacteria or any of the above-mentioned recombinant bacteria A may be achieved by introducing an expression cassette containing ATR2 into the starting bacteria or any of the above-mentioned recombinant bacteria A. In the above-mentioned recombinant bacteria A, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0274] In the above recombinant bacteria C, the introduction of the encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria A may be the introduction of an expression cassette of the encoding nucleic acid into the starting bacteria or any of the above recombinant bacteria A. The expression cassette may include a promoter, an encoding nucleic acid and a terminator.

[0275] In the above recombinant strain C, the promoter may be CCW12p and the terminator may be PGI1t.

[0276] In the above-mentioned recombinant bacteria C, the expression cassette is introduced into the starting bacteria or any of the above-mentioned recombinant bacteria A in the form of a plasmid and / or integrated into the chromosome.

[0277] Any of the above-mentioned recombinant bacteria C may also contain or express catalase.

[0278] In the above-mentioned recombinant bacteria C, the catalase may be PRDX4.

[0279] In the above-mentioned recombinant bacteria C, the presence or expression of catalase in the body may be achieved by introducing the encoding nucleic acid of catalase into the starting bacteria or any of the above-mentioned recombinant bacteria A.

[0280] In the above-mentioned recombinant bacteria C, 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 peroxiredoxin IV (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 A.

[0281] Any of the above-mentioned recombinant bacteria C may also contain or express transcription factors that activate phospholipid biosynthesis or knock out transcription factors that inhibit phospholipid biosynthesis.

[0282] In the above-mentioned recombinant bacteria C, the knocked-out transcription factor that inhibits phospholipid biosynthesis may be the OPI1 gene.

[0283] In the above recombinant strain C, the transcription factor that activates phospholipid biosynthesis may be the INO2 gene or the INO2 gene mutant INO2* (INO2_L119A).

[0284] Any of the above-mentioned recombinant bacteria C can be a species of the genus Saccharomyces cerevisiae.

[0285] Any of the above-mentioned recombinant bacteria C can be Saccharomyces cerevisiae.

[0286] Any of the above starting bacteria C is a species of Saccharomyces cerevisiae.

[0287] Any of the above starting bacteria C is Saccharomyces cerevisiae.

[0288] The cerevisiae yeast may specifically be the IMX581 yeast strain.

[0289] The use of any of the above-mentioned recombinant bacteria C in the production of chelerythrine, allocryptopine and / or tetrahydroberberine also falls within the protection scope of the present invention.

[0290] The present invention also provides a method for producing chelerythrine, allocryptopine and / or tetrahydroberberine, comprising the following steps: fermenting and culturing any of the above-mentioned recombinant bacteria C, collecting the fermentation product, and obtaining chelerythrine, allocryptopine and / or tetrahydroberberine therefrom.

[0291] The inventors of the present invention have constructed a recombinant bacterium for producing benzophenanthridine alkaloids (such as sanguinarine or chelerythrine) through a large number of experiments. The synthetic pathways of sanguinarine and chelerythrine are divided into multiple modules for research, and the de novo synthesis of sanguinarine and chelerythrine is achieved through multiple strategies. For example, in order to improve the activity of BBE, a new endoplasmic reticulum engineering strategy is developed, thereby increasing the yield of the key intermediate S-Scoulerine by 3 times; McDBOX2 is identified as the only DBOX gene reported to function in Saccharomyces cerevisiae, and the reconstruction of the entire biosynthetic pathway of benzophenanthridine alkaloids is completed; the yield of sanguinarine and chelerythrine is increased by increasing the N-terminal truncation of DBOX to improve cytoplasmic expression, endoplasmic reticulum amplification and increasing the supply of cofactor FAD, and finally the yield of sanguinarine is 3.8 mg / L and the yield of chelerythrine is 38.1 mg / L; wherein the starting strain can be the IMX581 yeast strain. Experiments have shown that the engineered strain constructed by the present invention can efficiently produce sanguinarine and chelerythrine. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0292] Figure 1 It is the biosynthetic pathway of sanguinarine and chelerythrine.

[0293] 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.

[0294] Figure 3 These are the test results of (ii) and (iii) in step 1 of the embodiment.

[0295] Figure 4 This is the test result of step (VII) in step 1 of the embodiment.

[0296] Figure 5 This is the test result of step (eight) in the embodiment.

[0297] Figure 6 The optimization pathway and modification strategy for producing the choline module (module II) and the detection results of (ii), (iv) and (v) in step 2.

[0298] Figure 7 This is the test result of step 2 (ii) in the embodiment.

[0299] 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.

[0300] Fig. 9 This is the test result of step 3 (a) in the embodiment.

[0301] Fig.10 The test results of (a) and (b) in step 4 of the embodiment are shown in FIG.

[0302] Fig.11 This is the test result of step 4 (ii) in the embodiment.

[0303] Fig.12 This is the test result of step 5 (a) in the embodiment.

[0304] Fig.13 This is the test result of step 5 (ii) in the embodiment.

[0305] Fig.14 This is the detection result of step six of the embodiment.

[0306] Fig.15 These are the test results of (a) and (c) in step seven of Example 1.

[0307] Fig.16 This is the test result of step 7 (ii) in the embodiment.

[0308] Fig.17 This is the test result of step eight (a) in the embodiment.

[0309] Fig.18 This is the test result of step eight (ii) in the embodiment.

[0310] Fig.19 This is the test result of step eight (three) in the embodiment. DETAILED DESCRIPTION

[0311] 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.

[0312] 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.

[0313] The names of the primers and their nucleotide sequences involved in the following examples are shown in Table 1.

[0314] Table 1

[0315]

[0316]

[0317]

[0318]

[0319] The strain names and genotypes involved in the following examples are shown in Table 2.

[0320] Table 2

[0321]

[0322]

[0323]

[0324]

[0325] IMX581 yeast strain (described in the following literature: Mans R et al. CRISPR / Cas9: amolecular 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.

[0326] 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); Ion Spray 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 used MRM mode, and quantitative detection used Q3 MI mode or MS2 mode.

[0327] In the following examples, plasmid pMEL10 is a product of EUROSCARF collection, acc.no.P30779. Plasmid pMEL10 carries SNR52 promoter and gRNA backbone.

[0328] Example

[0329] The inventors of the present invention have constructed a recombinant bacterium for producing benzophenanthridine alkaloid sanguinarine SAN through a large number of experiments. The synthesis pathway of sanguinarine is divided into 5 modules for research, and de novo synthesis of sanguinarine is achieved through a variety of strategies. For example, in order to improve the activity of BBE, a new endoplasmic reticulum engineering strategy is developed, thereby increasing the yield of the key intermediate S-Scoulerine by 206%; McDBOX2 is identified as the only DBOX gene reported to function in Saccharomyces cerevisiae, and the reconstruction of the entire biosynthetic pathway of benzophenanthridine alkaloids is completed; the yield of sanguinarine is increased by increasing the cytoplasmic expression, endoplasmic reticulum amplification and increasing the supply of cofactor FAD by performing N-terminal truncation operation on DBOX, and finally obtaining a recombinant bacterium with a sanguinarine yield of 3.8 mg / L. The starting strain is the IMX581 yeast strain, which is a Saccharomyces cerevisiae strain in which the Cas9 gene is integrated under the TEF1 promoter, and the genotype is MATa ura3-52 can1Δ::cas9-natNT2 TRP1LEU2HIS3, CEN.PK113-5D. In the following, the IMX581 yeast strain is referred to as IMX581.

[0330] The biosynthetic pathway of sanguinarine is shown in Figure 1 .

[0331] 1. De novo production of higenamine in Saccharomyces cerevisiae (Module I)

[0332] The approaches and transformation strategies for module 1 are shown in Figure 2 .

[0333] (I) Construction of XJ001 strain producing dopamine

[0334] 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 (Beta vulgaris) were integrated into the XI-5 site of IMX581 using the CRISPR-Cas9 genome editing method.

[0335] 1. Construction of plasmid pMEL10-XI-5

[0336] The gRNA plasmid was constructed using the Gibson assembly method. The specific steps are as follows:

[0337] (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.

[0338] (2) Artificially synthesizing XI-5-gRNA-F and XI-5-gRNA-R, then mixing equal amounts, treating at 95° C. for 10 min, and cooling 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.

[0339] (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.

[0340] 2. Preparation of repair fragment (Donor DNA)

[0341] A. Repair fragment XI-5us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p. From 5' to 3', the upstream homology arm XI-5us (nucleotide sequence as shown in SEQ ID NO:33), the terminator IDP1t (nucleotide sequence as shown in SEQ ID NO:3), the gene fragment BvCYP76AD1*, the promoter TDH3p (nucleotide sequence as shown in SEQ ID NO:1) and the promoter CCW12p (nucleotide sequence as shown in SEQ ID NO:2) are sequentially included. The repair fragment XI-5us was prepared by the overlap method.

[0342] The specific operation of us-IDP1t-BvCYP76AD1*-TDH3p-CCW12p is as follows:

[0343] (1) Using yeast genome or synthetic genes as templates, upstream homology arm XI-5us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p and promoter CCW12p were amplified.

[0344] (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.

[0345] (2-1) Preparation of overlap system. The overlap system is 25 μL, consisting of 5*Prime STARbuffer 5 μL, dNTP (2.5 mM each) 2 μL, upstream homology arm XI-5us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p, promoter CCW12p, Polymerase 0.5 μL and ddH2O. In the overlap system, the concentration of upstream homology arm XI-5us is 0.03 pmol, and the molar ratio of upstream homology arm XI-5us, terminator IDP1t, gene fragment BvCYP76AD1*, promoter TDH3p and promoter CCW12p is 1:3:5:3:1.

[0346] (2-2) Take the overlap system, react and obtain a product.

[0347] 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.

[0348] (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.

[0349] 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.

[0350] 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:

[0351] (1) The promoter CCW12p, gene fragment DODC, terminator TPS1t and downstream homology arm XI-5ds were amplified using yeast genome or synthetic gene as template.

[0352] (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.

[0353] (2-1) Preparation of overlap system. The overlap system is 25 μL, consisting of 5*Prime STARbuffer 5 μL, dNTP (2.5 mM each) 2 μL, promoter CCW12p, gene fragment DODC, terminator TPS1t, downstream homology arm XI-5ds, Polymerase 0.5 μL and ddH2O. In the overlap system, the concentration of promoter CCW12p is 0.03 pmol, and the molar ratio of promoter CCW12p, gene fragment DODC, terminator TPS1t and downstream homology arm XI-5ds is 1:3:3:1.

[0354] (2-2) Take the overlap system, react and obtain a product.

[0355] 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.

[0356] (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.

[0357] 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.

[0358] 3. Obtaining positive clones

[0359] (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.

[0360] Table 3

[0361] Components Addition amount PEG3350 (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

[0362] Note: The repaired fragments must be denatured in a boiling water bath for 5 minutes and then placed on ice for 5 minutes.

[0363] (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.

[0364] 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.

[0365] (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.

[0366] 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.

[0367] (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.

[0368] 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).

[0369] 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.

[0370] (II) Screening of NCS enzymes

[0371] 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 enzymes was carried out in XJ001.

[0372] 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'.

[0373] 1. Construction of plasmid pMEL10-XI-3

[0374] 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.

[0375] 2. The plasmid pMEL10-XI-3 and the expression cassette XI-3us-TEF1p-CjNCS_Δ24-TDH2t-XI-3ds were transferred into the 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 grown clones were strains that had lost the gRNA plasmid, that is, the expression cassette XI-3us-TEF1p-CjNCS_Δ24-TDH2t-XI-3ds was integrated into the XI-3 site of XJ001. The resulting strain was named XJ003 yeast strain (hereinafter referred to as XJ003), and the genotype of the XJ003 yeast strain was XJ001 (XI-3::TEF1p-CjNCS_Δ24-TDH2t).

[0376] 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), and the genotype of XJ006 yeast strain is XJ001 (XI-3::TEF1p-CjNCS_Δ29-TDH2t).

[0377] 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'.

[0378] 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).

[0379] 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'.

[0380] 5. Pick 3 single clones from XJ003, XJ006 and XJ007 respectively for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / LMgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain fermentation liquid. Determine the OD of the fermentation liquid 600nm .

[0381] 7. Detection of strain metabolic components

[0382] 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.

[0383] 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.

[0384] (III) Tyrosine hydroxylase gene screening

[0385] 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.

[0386] 1. Construction of plasmid pMEL10-XI-5

[0387] Same as step 1.

[0388] 2. Strain construction

[0389] 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:

[0390] (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.

[0391] (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.

[0392] (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.

[0393] (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.

[0394] (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.

[0395] (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.

[0396] (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.

[0397] (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.

[0398] (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.

[0399] (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.

[0400] 3. Pick 3 single clones from the strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain fermentation broth. Determine the OD of the fermentation broth 600nm .

[0401] 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.

[0402] 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.

[0403] (IV) Construction of XJ053 strain producing S-Nor

[0404] 1. Construction of XJ023 strain

[0405] 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:

[0406] (1) Construction of plasmid pMEL10-X-2

[0407] 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.

[0408] (2) Construction of plasmid pMEL10-X-4

[0409] 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.

[0410] (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 PGK1p-T EF1p-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) were transformed into IMX581 yeast competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates for 3 The cells were cultured in a 0°C incubator for 2-3 days. The clones that grew out were strains that had lost the gRNA plasmid, that is, they were integrated into the X-2 site of IMX581 to obtain intermediate yeast. The plasmid pMEL10-X-4, the expression cassette X-4us-CYC1t-ARO1-TPI1p-TDH3p (composed of the upstream homology arm X-4us, the terminator CYC1t, the nucleic acid sequence of ARO1, the promoter TPI1p and the promoter TDH3p), and the expression cassette TPI1p-TDH3p-ARO2-ADH1t-TDH2t (composed of the promoter TPI1p, the promoter TDH3p, the ARO 2 nucleic acid sequence, 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 box 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.

[0411] 2. Construction of XJ053 strain

[0412] 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.

[0413] 3. Select three single clones of XJ053 strain for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, and the solvent is water) to a 14mL shake tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0414] 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.

[0415] The test results showed that the yield of S-NOR produced by strain XJ053 was 23.4 mg / L.

[0416] (V) Transformation of XJ053 strain to increase S-NOR production

[0417] 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:

[0418] 1. Construction of plasmid

[0419] (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.

[0420] (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.

[0421] 2. Strain construction

[0422] 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.

[0423] 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] 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.

[0428] 3. Pick 3 single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0429] 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.

[0430] 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.

[0431] (VI) CjNCS_Δ35 copy number selection

[0432] 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).

[0433] 1. Construction of strains XJ062 and XJ063

[0434] (1) Construction of plasmid pMEL10-XI-1

[0435] 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.

[0436] (2) Construction of plasmid pMEL10-LP1.T8

[0437] 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.

[0438] (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.

[0439] (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.

[0440] 2. Pick 3 single clones from each strain obtained in step 1 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shake tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0441] 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.

[0442] 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.

[0443] (VII) Use of aromatic amino acid decarboxylase (AAAD) to direct more product flow to 4-HPAA

[0444] 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:

[0445] 1. Construction of strains

[0446] (1) Construction of plasmid pMEL10-X-3

[0447] 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.

[0448] (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.

[0449] (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.

[0450] (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.

[0451] (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.

[0452] 2. Pick 3 single clones from each strain obtained in step 1 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shake tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0453] 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.

[0454] 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).

[0455] (VIII) Increasing the tyrosine supply in Saccharomyces cerevisiae by introducing plant tyrosine pathway genes prephenate transaminase (PAT) and arogenate dehydratase (ADH)

[0456] 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:

[0457] 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.

[0458] 2. Preparation of gene expression cassette fragments

[0459] (1) The upstream homology arm 106a us (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).

[0460] (2) The downstream homology arm 106a ds, promoter, TEF2p, MtncADH and terminator pYX212t were connected using the overlap method to obtain the gene expression cassette fragment pYX212t-MtncADH-TEF2p-106a ds (named as fragment 8).

[0461] (3) The upstream homology arm 106a us, promoter FBA1p, BvPAT1, terminator TDH2t and terminator pYX212t were connected by the overlap method to obtain the gene expression cassette fragment 106a us-FBA1p-BvPAT1-TDH2t-pYX212t (named as fragment 9).

[0462] (4) The upstream homology arm 106a us, promoter FBA1p, BvPAT2, terminator TDH2t and terminator pYX212t were connected using the overlap method to obtain the gene expression cassette fragment 106a us-FBA1p-BvPAT2-TDH2t-pYX212t (named as fragment 10).

[0463] 3. Strain construction

[0464] 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.

[0465] 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.

[0466] 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.

[0467] 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.

[0468] 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.

[0469] 4. Pick 3 single clones from each strain obtained in step 3 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shake tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0470] 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.

[0471] 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 that integrated AtPAT and MtncADH had the highest product, which was better than other candidate PAT and ADH combinations.

[0472] 2. Optimization of the oxadiazine production module (Module II)

[0473] 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 .

[0474] (I) Construction of chassis strain XJ040

[0475] 1. Construction of plasmid

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 2. Construction of gene expression cassette

[0480] (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 CYC1t-FBA1t-MtPDH1-tHXT7p-X-2ds.

[0481] (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.

[0482] (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.

[0483] 3. Construction of XJ040 strain

[0484] 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

[0485] 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

[0486] 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.

[0487] (ii) Screening of different combinations of candidate genes in the chassis fungus XJ040.

[0488] 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.

[0489] 2. Construction of gene expression cassette

[0490] (1) Combination of Cy6OMT, CyCNMT and EcNMCH

[0491] 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.

[0492] (2) Combination of Cy6OMT, CyCNMT, and CyNMCH

[0493] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Cy6OMT, terminator FBA1t and terminator ENO2t were connected by overlap method to obtain a gene expression cassette.

[0494] XII-2-us-GPM1p-Cy6OMT-FBA1t-ENO2t. The terminator FBA1t, the terminator ENO2t, the coding gene of CyCNMT, the promoter TEF2p and the promoter tHXT7p were connected by the overlap method to obtain the gene expression cassette FBA1t-ENO2t-CyCNMT-TEF2p-tHXT7p. 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.

[0495] (3) Combination of Cy6OMT, PsCNMT, and EcNMCH

[0496] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Cy6OMT, terminator FBA1t and terminator ENO2t were connected by overlap method to obtain a gene expression cassette.

[0497] XII-2-us-GPM1p-Cy6OMT-FBA1t-ENO2t. 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. The promoter TEF2p, the promoter tHXT7p, the coding gene of EcNMCH, 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-EcNMCH-ADH1t-XII-2-ds.

[0498] (4) Combination of Cy6OMT, PsCNMT, and CyNMCH

[0499] The upstream homology arm XII-2-us, promoter GPM1p, the coding gene of Cy6OMT, terminator FBA1t and terminator ENO2t were connected by overlap method to obtain a gene expression cassette.

[0500] XII-2-us-GPM1p-Cy6OMT-FBA1t-ENO2t. 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. 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.

[0501] (5) Combination of Ps6OMT, CyCNMT and EcNMCH

[0502] 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.

[0503] (6) Combination of Ps6OMT, CyCNMT and CyNMCH

[0504] 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.

[0505] (7) Combination of Ps6OMT, PsCNMT and EcNMCH

[0506] 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.

[0507] (8) Combination of Ps6OMT, PsCNMT and CyNMCH

[0508] 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.

[0509] 3. Transform XJ040 yeast competent cells with plasmid pMEL10-X II-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.

[0510] 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.

[0511] 4. Pick 3 single clones from each strain obtained in step 3 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0512] 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.

[0513] 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.

[0514] (III) Preparation of strain XJ0662

[0515] 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:

[0516] 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.

[0517] 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.

[0518] 3. Construction of gene expression cassette

[0519] (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).

[0520] (2) 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 (named as fragment 13). 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 (named as fragment 14). The promoter TEF2p, promoter tHXT7p, the coding gene of CyNMCH, terminator ADH1t and downstream homology arm XII-2-ds were connected using the overlap method to obtain the gene expression cassette TEF2p-tHXT7p-CyNMCH-ADH1t-XII-2-ds (named as fragment 15).

[0521] 4. Construction of strains XJ066 and XJ0662

[0522] 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.

[0523] 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.

[0524] (IV) CPR screening

[0525] 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.

[0526] 2. Transform XJ0662 yeast competent cells with plasmid pMEL10-PsCPR and ATR1 fragment (nucleotide sequence as shown in SEQ ID NO:55) 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 are strains that have lost the gRNA plasmid, that is, integrated into the PsCPR site of XJ0662, to obtain strain XJ0675. Transform XJ0662 yeast competent cells with plasmid pMEL10-PsCPR and ATR2 fragment (nucleotide sequence as shown in SEQ ID NO:56) 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 are strains that have lost the gRNA plasmid, that is, integrated into the PsCPR site of XJ0662, to obtain strain XJ0676.

[0527] 3. Pick 3 single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0528] 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.

[0529] 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.

[0530] 5. Impact of the number of candidate genes

[0531] 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:

[0532] 1. Construction of plasmid

[0533] (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.

[0534] (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.

[0535] (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.

[0536] (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.

[0537] (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.

[0538] 2. Construction of gene expression cassette

[0539] (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).

[0540] (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

[0541] TDH3p-tHXT7p-CyNMCH-ADH1t-HFD1-ds (designated as fragment 18).

[0542] (3) Using the overlap method, 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 to obtain a gene expression cassette

[0543] 416d-us-LP1.T8-tHXT7p-CyNMCH-ADH1t-416d-ds (designated as fragment 19).

[0544] (4) Using the overlap method, the upstream homology 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 homology arm ARI1-ds (nucleotide sequence as shown in SEQ ID NO: 62) were connected to obtain a gene expression cassette

[0545] 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 by 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 by the overlap method to obtain a gene expression cassette.

[0546] YPR1-us-LP3.T7-tHXT7p-CyNMCH-ADH1t-LP4.T9-YPR1-ds (designated as fragment 22).

[0547] 3. Strain construction

[0548] (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.

[0549] (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.

[0550] (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.

[0551] (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.

[0552] (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.

[0553] 4. Pick 3 single clones from each strain obtained in step 3 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0554] 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.

[0555] 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.

[0556] 3. Screening and optimization of BBE (Module III)

[0557] The present invention adopts an endoplasmic reticulum engineering strategy to improve the activity of BBE in Saccharomyces cerevisiae. First, the BBE enzyme CyBBE (derived 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 production of chrysanthemum corydaline by 3 times. In addition, the inhibitory effect of CyBBE is alleviated by introducing mammalian source catalase peroxiredoxin IV (PRDX4) to mediate H2O2 decomposition and endoplasmic reticulum expansion mediated by knocking out phospholipase OPI1, which increases the potency of chrysanthemum corydaline by 30% and 36%, respectively. The pathway and transformation strategy of module III are shown in Figure 8 .

[0558] (I) Screening for the best BBE gene

[0559] 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:

[0560] 1. Construction of plasmid

[0561] 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.

[0562] 2. Strain construction

[0563] (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.

[0564] (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.

[0565] (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.

[0566] (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.

[0567] 3. Pick 3 single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0568] 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.

[0569] 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.

[0570] (II) Optimization of CyBBE yeast in vivo expression localization

[0571] 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:

[0572] 1. Strain construction

[0573] (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.

[0574] (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.

[0575] (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.

[0576] 2. Pick 3 single clones from each strain obtained in step 1 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shake tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0577] 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.

[0578] 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:

[0579] 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:91) 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:92) 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.

[0580] 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.

[0581] 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.

[0582] 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.

[0583] 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).

[0584] (III) Using catalase PRDX4 to alleviate the inhibitory effect of H2O2

[0585] Previous studies have reported that H2O2 inhibits the function of BBE. 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 H2O2 and increase the production of engineered bacteria S-RET (see Figure 8 The specific experimental steps are as follows:

[0586] 1. Construction of recombinant plasmid

[0587] (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.

[0588] (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.

[0589] (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.

[0590] 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.

[0591] 3. Pick 3 single clones from each strain obtained in step 2 and XJ0695 for parallel experiments. Add 1 ml of Deft medium (solutes and their concentrations are 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / LMgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and the solvent is water) to a 14 mL shake tube, then inoculate a single clone, and culture at 30°C and 220 rpm for 18-24 hours to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125 mL) containing 20 mL of Deft medium for expansion culture, and culture at 30°C and 220 rpm for 72 hours to obtain a fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0592] 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.

[0593] Test results see Figure 8 The results showed that compared with XJ0695, the strain into which the catalase peroxiredoxin IV (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.

[0594] (IV) Optimizing CyBBE expression through endoplasmic reticulum engineering

[0595] 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).

[0596] 1. Construction of plasmid

[0597] (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.

[0598] (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.

[0599] (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.

[0600] 2. Construction of strains

[0601] (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: 70), the terminator ENO2t (nucleotide sequence as shown in SEQ ID NO: 7) 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.

[0602] (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).

[0603] (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).

[0604] (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).

[0605] 3. Pick 3 single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / Luracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0606] 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.

[0607] 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.

[0608] 4. Production of sanguinarine from golden corydaline (Module IV)

[0609] The biosynthetic pathway from cheilanthifoline to sanguinarine requires seven steps: cheilanthifoline is catalyzed by two CYP450s (S-cheilanthifoline synthase, CFS; S-stylopine synthase, SPS) to generate papaverine, which is then catalyzed by N-methyltransferase (TNMT) to generate N-methyl papaverine, and then hydroxylated by CYP450 (S-cis-N-methylstylopine14-hydroxylase, MSH) to generate protopine (PRO), which is then rearranged by CYP450 (protopine6-hydroxylase, P6H) to generate dihydrosanguinarine, and finally oxidized by benferidine oxidase (DBOX) to generate sanguinarine ( Figure 1 ). In this experiment, we first constructed a strain that produces protopine, screened out the combination of EcCFS and EcSPS, and found that it showed higher enzyme activity. Then, we screened out the benzophenone oxidase McDBOX2 in the strain that produces protopine. It is the only gene among all candidate FAD-dependent oxidases that can play a key role in the conversion of dihydrosanguinarine to sanguinarine.

[0610] (I) Construction of Protopine-producing Strains

[0611] EcCFS and EcSPS from E.californica, CyCFS and CySPS from C.yanhusuo, and PsTNMT and PsMSH from P.somiferum were selected and integrated into the XJ0695 strain producing golden corydaline. The content of protopine in the strains was compared through fermentation culture and content detection, and the combination of EcCFS and EcSPS was screened to show higher enzyme activity. The specific steps are as follows:

[0612] 1. Construction of plasmid pMEL10-XI-2

[0613] According to the method of step 1 (a), XI-5-gRNA-F was replaced with XI-2-gRNA-F, and XI-5-gRNA-R was replaced with XI-2-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-XI-2.

[0614] 2. Preparation of gene expression cassette

[0615] The encoding nucleic acid expression cassette includes an upstream homology arm, a promoter, a nucleic acid sequence of a target gene, a terminator and a downstream homology arm. The target genes are EcCFS (BAG75113), EcSPS (BAD98250), CyCFS (nucleotide sequence as shown in SEQ ID NO:71), CySPS (nucleotide sequence as shown in SEQ ID NO:72), PsTNMT (ACO90237.1) and PsMSH (AGC92398.1). The promoters are promoter PDC1p (nucleotide sequence as shown in SEQ ID NO:69), promoter GPM1p (nucleotide sequence as shown in SEQ ID NO:21), promoter TPI1p (nucleotide sequence as shown in SEQ ID NO:8) and promoter PGK1p (nucleotide sequence as shown in SEQ ID NO:9). The terminators include terminator ENO2t (nucleotide sequence shown in SEQ ID NO: 7), terminator ADH1t (nucleotide sequence shown in SEQ ID NO: 11) and terminator FBA1t (nucleotide sequence shown in SEQ ID NO: 12).

[0616] (1) The upstream homology arm XI-2us (nucleotide sequence shown in SEQ ID NO: 73), promoter PDC1p, nucleic acid sequence of EcCFS, terminator ENO2t and terminator IDP1t (nucleotide sequence shown in SEQ ID NO: 3) were connected by overlap method to obtain gene expression cassette XI-2us-PDC1p-EcCFS-ENO2t-IDP1 (named as fragment 33). The terminator ENO2t, terminator IDP1t, nucleic acid sequence of EcSPS, promoter GPM1p and promoter TPI1p were connected by overlap method to obtain gene expression cassette ENO2t-IDP1t-EcSPS-GPM1p-TPI1p (named as fragment 34). The promoter GPM1p, promoter TPI1p, nucleic acid sequence of PsTNMT, terminator ADH1t and terminator FBA1t were connected by overlap method to obtain gene expression cassette GPM1p-TPI1p-PsTNMT-ADH1t-FBA1t (named as fragment 35). The terminator ADH1t, terminator FBA1t, nucleic acid sequence of PsMSH, promoter PGK1p and downstream homology arm XI-2ds (nucleotide sequence as shown in SEQ ID NO: 74) were connected by overlap method to obtain gene expression cassette ADH1t-FBA1t-PsMSH-PGK1p-XI-2ds (named as fragment 36).

[0617] (2) Following the method of step (1), EcSPS was replaced by CySPS, and the other steps remained unchanged to obtain the gene expression cassette ENO2t-IDP1t-CySPS-GPM1p-TPI1p (named as fragment 37).

[0618] (3) Following the method of step (1), replace EcCFS with CyCFS, and keep other steps unchanged to obtain the gene expression cassette X-2us-PDC1p-CyCFS-ENO2t-IDP1 (named as fragment 38).

[0619] 3. Construction of strains

[0620] (1) The plasmid pMEL10-XI-2 was transformed into XJ0695 competent cells together with fragments 33, 34, 35 and 36 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, the fragment was integrated into the XI-2 site of the XJ0695 strain to obtain the XJ0743 strain.

[0621] (2) The plasmid pMEL10-XI-2 and the gene expression cassette fragments 33, 37, 35 and 36 were transformed into XJ0695 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 fragment was integrated into the XI-2 site of the XJ0695 strain, and the XJ0744 strain was obtained in turn.

[0622] (3) The plasmid pMEL10-XI-2 and the gene expression cassette fragments fragment 38, fragment 34, fragment 35 and fragment 36 were transformed into XJ0695 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 fragment was integrated into the XI-2 site of the XJ0695 strain, and the XJ0745 strain was obtained in turn.

[0623] (4) The plasmid pMEL10-XI-2 and the gene expression cassette fragments fragment 38, fragment 37, fragment 35 and fragment 36 were transformed into XJ0695 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 fragment was integrated into the XI-2 site of the XJ0695 strain, and the XJ0746 strain was obtained in turn.

[0624] 4. Pick 3 positive single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0625] 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.

[0626] Test results see Fig.10 The results showed that strains XJ0743, XJ0744, XJ0745 and XJ0746 can all produce protopine, among which strain XJ0743 produced the highest content of protopine. That is, in this experiment, the combination of EcCFS and EcSPS showed the best activity in the engineered bacteria.

[0627] (II) Screening of benzophenone oxidase McDBOX2

[0628] Codon-optimized Argemone Mexicana AmSTOX, Berberis wilsoniae BwSTOX, C. japonica CjTHBO, Papaver PsFADOX, Macleaya cordata McDBOX1 and McDBOX2 genes were synthesized in vitro and transferred into the original opium alkaloid-producing strain XJ0743 together with EcP6H from Echinops californica. The protein expression was then detected by Western blot, and it was found that BwSTOX and McDBOX2 proteins were well expressed. However, the detection found that even in the XJ07437 strain that only expressed EcP6H (as a control), the target product sanguinarine could be produced, while dihydrosanguinarine could not be detected in the XJ0750 strain into which McDBOX2 was introduced, confirming that McDBOX2 plays a key role in the conversion of dihydrosanguinarine to sanguinarine. The specific steps are as follows:

[0629] 1. According to the method in step 1 (a), replace XI-5-gRNA-F with XII-1-gRNA-F, and replace XI-5-gRNA-R with XII-1-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-XII-1.

[0630] 2. Preparation of gene expression cassette and strain construction

[0631] The encoding nucleic acid expression cassette includes an upstream homology arm, a promoter, a nucleic acid sequence of a target gene, a terminator sequence, and a downstream homology arm. The target genes are AmSTOX (ADY15027.1), BwSTOX (ADY15026.1), CjSTOX (BAJ40864.1), PsFADOX5 (AGL44334.1), StBBE (nucleotide sequence as shown in SEQ ID NO: 24), McDBOX1 (OVA00267.1), McDBOX2 (OVA00268.1), and EcP6H (BAK20464). The promoters are promoter PDC1p (nucleotide sequence as shown in SEQ ID NO: 69) and promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminators are terminator DIT1t (nucleotide sequence as shown in SEQ ID NO: 75) and terminator TDH2t (nucleotide sequence as shown in SEQ ID NO: 6). The His tag sequence is CATCATCACCATCACCAT.

[0632] (1) The upstream homology arm XII-1us (nucleotide sequence as shown in SEQ ID NO: 76), terminator DIT1t, His tag sequence, AmSTOX nucleic acid sequence, promoter PDC1p and promoter CCW12p were connected by the overlap method to obtain the gene expression cassette XII-1us-DIT1t-His-AmSTOX-PDC1p-CCW12p (named as fragment 39). The promoter PDC1p, promoter CCW12p, EcP6H nucleic acid sequence, terminator TDH2t and downstream homology arm XII-1ds (nucleotide sequence as shown in SEQ ID NO: 77) were connected by the overlap method to obtain the gene expression cassette PDC1p-CCW12p-EcP6H-TDH2t-XII-1ds (named as fragment 40). The plasmid pMEL10-XII-1 was transformed into the competent cells of the XJ0743 strain together with fragments 39 and 40 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 XII-1 site of the XJ0743 strain to obtain the XJ07431 strain.

[0633] (2) According to the above step (1), the AmSTOX sequence was replaced with the BwSTOX sequence, CjSTOX sequence, PsFADOX5 sequence, StBBE sequence, McDBOX1 sequence and McDBOX2 sequence respectively, and the other steps remained unchanged, thereby obtaining the XJ07432 strain, the XJ07433 strain, the XJ07434 strain, the XJ07435 strain, the XJ07436 strain and the XJ0750 strain in turn.

[0634] (3) The plasmid pMEL10-XII-1 and the gene expression cassette fragment XII-1us-CCW12p-EcP6H-TDH2t-XII-1ds (consisting of the upstream homology arm promoter X11-1us, promoter CCW12p, the nucleic acid sequence of EcP6H, terminator TDH2t and downstream homology arm X11-1ds) were transformed into the competent cells of the XJ0743 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 gene expression cassette was integrated into the XII-1 site of the XJ0743 strain, and the control strain XJ07437 strain with only the EcP6H gene inserted was obtained.

[0635] 3. Pick 3 positive single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0636] 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.

[0637] 5. Western blot detection of protein expression

[0638] (1) Each strain obtained in step 2 was inoculated into 1 mL of delft medium and cultured overnight at 30°C and 220 rpm to obtain a preculture.

[0639] (2) The pre-culture was transferred to 5 mL of fresh Delft medium at a ratio of 1:50 and cultured at 30°C and 220 rpm to obtain the OD 600nm About 1% of the culture solution.

[0640] (3) Collect cells from the culture solution, wash twice with PBS buffer, add 200 μL of acid-washed glass beads and 300 μL of PBS buffer to resuspend, and then use FastPrep rapid sample preparation instrument to disrupt the cells to obtain a sample.

[0641] (4) Add 4×NuPAGE to the sample TM LDS sample buffer (ThermoFisher) was boiled at 95°C for 10 minutes, and protein electrophoresis was performed at 150V for 1 hour.

[0642] (5) Transfer protein to Trans- Turbo TM After PVDF transfer membrane (Bio-Rad), block with PBST (PBS + 0.1% Tween 20) containing 5% milk at room temperature for 2 hours. Wash the membrane 3 times with PBST and incubate with 6×His-Tag monoclonal antibody (His.H8) (ThermoFisher) or anti-GAPDH antibody (G-9) (Santa Cruz Biotechnology) at room temperature for 1 hour. Then wash the membrane 3 times with PBST and incubate with HRP-conjugated anti-mouse IgG (h + L) secondary antibody (Invitrogen) at room temperature for 1 hour. Then wash the membrane 3 times with PBST and incubate with West Pico plus HRP substrate (ThermoFisher) for 5 minutes, and finally analyze with ChemiDoc XRS image analyzer (Bio-Rad).

[0643] Test results see Fig.10 Zhongeg and Fig.11. The results showed that BwSTOX and McDBOX2 proteins were well expressed with the correct protein size, PsFADOX5 bands were diffuse, while AmSTOX, CjTHBO and McDBOX1 were not expressed. The detection of strain metabolic components showed that all strains, including the control strain, produced sanguinarine. However, dihydrosanguinarine was not detected in the XJ0750 strain, which indicated that the McDBOX2 gene played a role in converting dihydrosanguinarine to produce sanguinarine in yeast, that is, the McDBOX2 gene was the only benzophenone oxidase gene screened in this experiment that functioned in yeast.

[0644] 5. Optimizing the pathway from golden corydaline to sanguinarine

[0645] The study found that the expression of McDBOX2 would reduce the OD of the strain 600nm And the accumulation of the intermediate product sanguinarine was increased, indicating that McDBOX2 had a certain inhibitory effect on the function of BBE. Therefore, the present invention adopts the following strategies for pathway optimization: (1) 29 amino acids were truncated at the N-terminus of McDBOX2 to increase its expression in the cytoplasm. It was found that compared with the XJ0750 strain (XJ07437+McDBOX2), the XJ07502 strain (XJ07437+McDBOX2Δ29) could grow normally and accumulate less intermediate product sanguinarine, and could convert all dihydrosanguinarine into sanguinarine; (2) Overexpression of plasma membrane riboflavin transporter MCH5 and FAD synthase BsRibc to increase FAD supply; knockout phospholipase OPI1-mediated endoplasmic reticulum expansion. Finally, the XJ07504 strain with a sanguinarine production of 3.8 mg / L was obtained.

[0646] (I) McDBOX2 (genbank: OVA00268.1) N-terminal truncation of 29 amino acids helps to increase sanguinarine production

[0647] 1. The gene expression cassette of McDBOX2_Δ29 (consisting of the upstream homology arm FgF20 us (nucleotide sequence as shown in SEQ ID NO: 67), the promoter PDC1p, the nucleic acid sequence of McDBOX2_Δ29, the terminator DIT1t and the downstream homology arm FgF20 ds (nucleotide sequence as shown in SEQ ID NO: 68) was transformed into the competent cells of the XJ07437 strain using the plasmid pMEL10-FgF20 to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature box 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 to obtain the XJ07502 strain.

[0648] 2. Select 3 positive single clones from strains XJ0750 and XJ07502 for parallel experiments. Add 1 ml of Deft medium (solutes and their concentrations are 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / LMgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions and 60 mg / Luracil, and the solvent is water) to a 14 mL shake tube, then inoculate the single clone, and culture at 30°C and 220 rpm for 18-24 hours to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125 mL) containing 20 mL of Deft medium for expansion culture, and culture at 30°C and 220 rpm for 72 hours to obtain a fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0649] 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.

[0650] Test results see Fig.12 The experiment showed that the strain containing McDBOX2 gene (XJ0750 strain) had a higher growth OD than the strain without McDBOX2 gene (XJ07437 strain). 600nm The value decreased significantly, indicating that the McDBOX2 gene had a significant inhibitory effect on the growth of yeast strains; while the strain containing McDBOX2_Δ29 (XJ0752 strain) had a growth OD 600nm The value decreased very little, indicating that the 29 amino acids truncated at the N-terminus of McDBOX2 helped to alleviate the growth inhibitory effect of this gene on the strain ( Fig.12 The metabolic composition of the strains showed that compared with strains XJ07437 and XJ0750, strain XJ0752 accumulated the least amount of intermediate S-RET (substrate of the BBE enzyme in the synthesis pathway) and the highest amount of final product SAN ( Fig.12 Compared with the control strain XJ07437, strains XJ0750 and XJ0752 can completely convert the intermediate dihydrosanguinarine to sanguinarine ( Fig.12 (c) Thus, 29 amino acids truncated at the N-terminus of McDBOX2 can effectively alleviate the inhibitory effect of McDBOX2 on the BBE step and increase the SAN yield.

[0651] (II) Overexpression of plasma membrane riboflavin transporter MCH5 and FAD synthase BsRibc to increase FAD supply; knockout of phospholipase OPI1 to mediate endoplasmic reticulum expansion helps to increase sanguinarine production

[0652] Since both BBE and McDBOX2 are FAD-dependent oxidases, there is competition for FAD supply and competition for the endoplasmic reticulum membrane. Therefore, in this experiment, the production of sanguinarine was increased from two angles: overexpression of the plasma membrane riboflavin transporter MCH5 (nucleotide sequence as shown in SEQ ID NO: 78) and FAD synthase BsRibc (nucleotide sequence as shown in SEQ ID NO: 79) to increase FAD supply and knockout of phospholipase OPI1 (nucleotide sequence as shown in SEQ ID NO: 80) to mediate endoplasmic reticulum expansion.

[0653] The specific steps are as follows:

[0654] 1. Construction of XJ07503 strain (overexpressing MCH5 and BsRibc)

[0655] The encoding nucleic acid expression cassette includes an upstream homology arm, a promoter, a nucleic acid sequence of a target gene, a terminator, and a downstream homology arm. The target genes are plasma membrane riboflavin transporter MCH5, FAD synthase BsRibc, and phospholipase OPI1. The promoters are promoter GPD (nucleotide sequence as shown in SEQ ID NO:81) and promoter CCW12p (nucleotide sequence as shown in SEQ ID NO:2). The terminators are IDP1t (nucleotide sequence as shown in SEQ ID NO:3) and TDH2t (nucleotide sequence as shown in SEQ ID NO:6).

[0656] The upstream homology arm 416d us (nucleotide sequence shown in SEQ ID NO: 59), promoter GPD, nucleic acid sequence of MCH5, terminator IDP1t and promoter CCW12p were connected by overlap method to obtain gene expression cassette 416d-GPD-MCH5-IDP1t-CCW12p (named as fragment 41). The terminator IDP1t, promoter CCW12p, nucleic acid sequence of BsRibc, terminator CYC1t and downstream homology arm 416d ds (nucleotide sequence shown in SEQ ID NO: 60) were connected by overlap method to obtain gene expression cassette IDP1t-CCW12p-BsRibc-CYC1t-416d ds (named as fragment 42). Fragment 41 and fragment 42 and plasmid pMEL10-416d were transformed into XJ07502 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 fragment was integrated into the 416d site to obtain the XJ07503 strain.

[0657] 2. Construction of strain XJ07504 (knockout of phospholipase OPI1)

[0658] The plasmid pMEL10-OPI1 and the DNA repair fragment c (nucleotide sequence as shown in SEQ ID NO: 82, which has a 60 bp overlapping homology arm sequence with the promoter and terminator of the target gene) were integrated into the OPI1 gene site to knock out the OPI1 gene and obtain the XJ07504 strain.

[0659] 3. Select three positive single clones from strains XJ07502, XJ07503 and XJ07504 for parallel experiments. Add 1 ml of Deft medium (solutes and their concentrations are 7.5 g / L (NH4) 2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions and 60 mg / L uracil, and the solvent is water) to a 14 mL shake tube, then inoculate the single clone, and culture at 30°C and 220 rpm for 18-24 hours to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125 mL) containing 20 mL of Deft medium for expansion culture, and culture at 30°C and 220 rpm for 72 hours to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0660] 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.

[0661] Test results see Fig.13 The results showed that compared with strains XJ07502 and XJ07503, strain XJ07504 had the highest sanguinarine content, with a sanguinarine yield of 3.8 mg / L; that is, overexpression of MCH5, overexpression of BsRibc and knockout of phospholipase OPI1 were beneficial to increasing sanguinarine yield.

[0662] 6. Pathway to construct Chelirubine (Module V)

[0663] Chelirubine, a downstream derivative of sanguinarine SAN, has shown antiproliferative effects on several cancer cell lines and can be used as a DNA fluorescent probe, which has attracted people's interest. Two additional reaction steps are required to convert dihydrosanguinarine to chelidonine, namely hydroxylation and methylation reactions, such as Figure 1 As shown in module V. Although three CYP82P hydroxylases and two O-methyltransferases have been reported to be possibly involved in the pathway, the pathway for the synthesis of chelidonine has not been fully characterized. The inventors of the present invention screened candidate genes for the pathway in the engineered yeast strain producing chelidonine: including three P450s (CYP82P2, CYP82P3 and CYP82P4) from California poppy and two OMTs (Ec2OMT and Ec11OMT). First, CYP82P2, CYP82P3 and CYP82P4 were expressed in the XJ0822 strain containing McDBOX2 producing chelidonine, and it was found that 10-hydroxysanguinarine with a molecular weight of m / z 348.0 was detected only in the strain expressing CYP82P2, and it was speculated that CYP82P2 catalyzed dihydrosanguinarine to produce 10-hydroxydihydrosanguinarine; and then oxidized by McDBOX2 to produce 10-hydroxysanguinarine. Later, two O-methyltransferases, Ec2OMT and Ec11OMT, were identified in the hematoxylin-producing strain. It was verified that the combination of CYP82P2 and Ec11OMT can catalyze the last two steps of Chelirubine biosynthesis.

[0664] The specific steps are as follows:

[0665] (I) Screening of p450 gene functions

[0666] 1. Use Gibson assembly method to construct recombinant plasmid. The specific steps are as follows:

[0667] (1) Plasmid P416_GPD (purchased from ATCC strain library, website:

[0668] https: / / www.atcc.org / search#q=P416&sort=relevancy&numberOfResults=24) 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, i.e., a linearized initial plasmid.

[0669] (2) Artificially synthesized CYP82P2 fragment (genbank: BBD34756.1), CYP82P3 fragment (genbank: BBD34757.1) and CYP82P4 fragment (genbank: BBD34758.1).

[0670] (3) The CYP82P2 fragment, CYP82P3 fragment, CYP82P4 fragment and the linearized initial plasmid were assembled according to the standard Gibson assembly to obtain the recombinant plasmid P416_GPD-CYP82P2, recombinant plasmid P416_GPD-CYP82P3 and recombinant plasmid P416_GPD-CYP82P4 respectively.

[0671] 2. Strain construction

[0672] The recombinant plasmid P416_GPD-CYP82P2, recombinant plasmid P416_GPD-CYP82P3, recombinant plasmid P416_GPD-CYP82P4 and plasmid P416_GPD were introduced into the hematocrit-producing XJ0822 strain, respectively, to obtain the XJ0851 strain, the XJ0852 strain, the XJ0853 strain and the XJ0854 strain in turn.

[0673] The preparation method of XJ0822 strain is as follows: (1) using the artificially synthesized DNA fragment LP1.T8 (nucleotide sequence as shown in SEQ ID NO: 90) as a template, using the primer pair consisting of FgF20-LP1.T8-F and LP1.T8-CCW12p-R to perform PCR amplification to obtain the fragment FgF20 us-LP1.T8; (2) Using the genomic DNA of strain XJ0750 as a template, PCR amplification was performed using primer pair x1 consisting of CCW12p1-F and TDH2t-FBA1t-R, primer pair x2 consisting of FBA1t400-rR and PGK1p-PDC1p-R, primer pair x3 consisting of XI-2us-PDC1p-F and IDP1t-rvR, and primer pair x4 consisting of IDP1t-rF and GPM1p-FgF20-R, respectively, to obtain fragments CCW12p-EcP6H-TDH2t-FBA1t, fragment FBA1t-PsMSH-PGK1p-PDC1p, fragment PDC1p-EcCFS-ENO2t-IDP1t, and fragment IDP1t-EcSPS-GPM1p-FgF20-R. ds; (3) transforming the plasmid pMEL10-FgF20 and the five fragments into XJ0750 competent cells to obtain positive clones; randomly selecting positive clones and streaking them on 5-FOA plates, culturing them in a constant temperature incubator at 30°C for 2-3 days, and the clones that grew were strains that had lost the gRNA plasmid, that is, integrating the fragment into the FgF20 site of the XJ0750 strain to obtain the XJ0778 strain; (4) then integrating the plasmid pMEL10-OPI1 and the DNA repair fragment c (the nucleotide sequence is shown in SEQ ID NO: 82, which has a 60 bp overlapping homology arm sequence with the promoter and terminator of the target gene) into the OPI1 gene site of the XJ0778 strain, thereby knocking out the OPI1 gene, and finally obtaining the XJ0822 strain.

[0674] 3. After activating strains XJ0851, XJ0852, XJ0853 and XJ0854 on the plate, pick three single clones for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metalsolutions, 1ml / L vitamin solutions, and the solvent is water) to a 14mL shake tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth600nm .

[0675] 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.

[0676] Test results see Fig.14 ac. The results showed that CYP82P2, CYP82P3 and CYP82P4 were expressed in the sanguinarine-producing strain XJ0822, and 10-Hydroxysanguinarine with a molecular weight of m / z 348.0 was detected only in the strain XJ0851 expressing CYP82P2, that is, the CYP82P2 gene was the only gene among the candidate P450 genes that could catalyze the hydroxylation reaction of dihydrosanguinarine. The reaction process was speculated to be that CYP82P2 catalyzed dihydrosanguinarine to produce 10-hydroxydihydrosanguinarine, which was then oxidized by McDBOX2 to produce 10-Hydroxysanguinarine with a molecular weight of m / z 348.0.

[0677] (II) Functional screening of 10-hydroxymethyltransferase (OMT)

[0678] 1. Plasmid construction

[0679] (1) The artificially synthesized CYP82P2 expression cassette (including promoter GPD, CYP82P2 fragment and terminator IDP1t) and the Ec11OMT expression cassette (including promoter CCW12p, Ec11OMT (genbank: BBA20642.1) and terminator CYC1t) were constructed according to the instructions of the Gibson Assembly kit of NEB (adjacent fragments had 25 bp homology arms) and constructed into the linearized plasmid P416_GPD to obtain the recombinant plasmid P416_GPD-CYP82P2-IDP1t-CCW12p-Ec11OMT-CYC1t.

[0680] (2) According to the above step (1), Ec11OMT was replaced with Ec2OMT (genbank: BBA20644.1), and other steps remained unchanged to obtain the recombinant plasmid P416_GPD-CYP82P2-IDP1t-CCW12p-Ec2OMT-CYC1t.

[0681] 2. Strain construction

[0682] (1) The recombinant plasmid P416_GPD-CYP82P2-IDP1t-CCW12p-Ec11OMT-CYC1t was introduced into the hematoxylin-producing strain XJ0832 to obtain the strain XJ0855.

[0683] The preparation method of XJ0832 strain is as follows: ① Using the overlap method to connect the upstream homology arm FgF7us (nucleotide sequence as shown in SEQ ID NO:83), promoter PDC1p, ATR1 fragment, terminator CYC1t and terminator DIT1t to obtain the gene expression cassette FgF7us-PDC1p-ATR1-CYC1t-DIT1t; ② Using the overlap method to connect the terminator CYC1t, terminator DIT1t, mPRDX4 (nucleotide sequence as shown in SEQ ID NO:27), promoter TEF1p and downstream homology arm FgF7ds (nucleotide sequence as shown in SEQ ID NO:84) to obtain the gene expression cassette CYC1t-DIT1t-mPRDX4-TEF1p-FgF7 ds; ③ The plasmid pMEL10-FgF7 and the above two gene expression cassettes were transformed into XJ0822 competent cells to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates, and cultured in a constant temperature box 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 FgF7 site of the XJ0822 strain to obtain the XJ0829 strain; ④ The upstream homology arm 416d us, promoter GPD, the nucleic acid sequence of MCH5, the terminator IDP1t and the promoter CCW12p were connected by the overlap method to obtain the gene expression cassette 416d-GPD-MCH5-IDP1t-CCW12p; ⑤ The promoter CCW12p, the nucleic acid sequence of BsRibc, the terminator CYC1t and the downstream homology arm 416d ds were connected by the overlap method to obtain the gene expression cassette CCW12p-BsRibc-CYC1t-416d ds; ⑥ Transform the plasmid pMEL10-416d together with the above two gene expression cassettes into XJ0829 competent cells to obtain positive clones; randomly select positive clones and streak them on 5-FOA plates, and culture them in a constant temperature box at 30°C for 2-3 days. The clones that grow are strains that have lost the gRNA plasmid, that is, the fragment is integrated into the 416d site of the XJ0829 strain to obtain the XJ0832 strain.

[0684] (2) Construction of plasmid pMEL10-FgF7

[0685] According to the method in step (a), XI-5-gRNA-F was replaced with FgF7-gRNA-F, XI-5-gRNA-R was replaced with FgF7-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-FgF7.

[0686] (3) The recombinant plasmid P416_GPD-CYP82P2-IDP1t-CCW12p-Ec2OMT-CYC1t was introduced into the hematoxylin-producing strain XJ0832 to obtain the strain XJ0856.

[0687] (4) The recombinant plasmid P416_GPD-CYP82P2 was introduced into the hematoxylin-producing strain XJ0832 to obtain the strain XJ0857.

[0688] (5) The plasmid P416_GPD was introduced into the hematocrit-producing strain XJ0832 to obtain the strain XJ0858.

[0689] 3. Select three single clones from strains XJ0855, XJ0856, XJ0857 and XJ0858 for parallel experiments. Add 1 ml of Deft medium (solutes and their concentrations are 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitamin solutions, and the solvent is water) to a 14 mL shake tube, then inoculate a single clone, and culture at 30°C and 220 rpm for 18-24 hours to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125 mL) containing 20 mL of Deft medium for expansion culture, and culture at 30°C and 220 rpm for 72 hours to obtain a fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0690] 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.

[0691] Test results see Fig.14 The results showed that O-methyltransferases Ec2OMT and Ec11OMT were identified in the XJ0832 strain, and the hydroxylated product 10-hydroxydihydrosanguinarine was found in the strains expressing CYP82P2 alone and co-expressing CYP82P2 and Ec2OMT, while a peak with a molecular weight of m / z 362.0 was found in the strain expressing CYP82P2 and Ec11OMT, which was identified as chelirubine. The results showed that Ec11OMT played the role of the last step of oxygen methylation in the chelirubine biosynthesis pathway.

[0692] VII. Production of chelerythrine from chrysanthemine (Module VI)

[0693] The biosynthetic pathway from chrysotoxine to chelerythrine requires a total of 6 catalytic reactions: first, chrysotoxine is catalyzed by O-methyltransferase (S9OMT) and CYP450 (CAS) to produce tetrahydroberberine, and then chelerythrine is produced through (TNMT, MSH, P6H, DBOX). The present application first constructs a strain producing the intermediate Allocryptopine: in the chrysotoxine-producing strain XJ0695, the mutant gene TfS9OMT* from Thalictrum flavum, CjCAS from Japanese Coptis chinensis (or CyCAS from Corydalis yanhusuo), and PsTNMT and PsMSH genes from poppy are introduced, and it is found that the strain XJ0741 introduced with Japanese Coptis chinensis CjCAS can produce a higher content of allocryptopine. Then, the functional gene flavoprotein oxidase McDBOX2 was screened out from the existing strain XJ0743 that produces the same type of compound protopine; then the three P6H genes were co-expressed with McDBOX2, and the yields were compared to determine that the chelerythrine XJ0747 strain expressing EcP6H had the highest activity. The specific steps are as follows:

[0694] (I) Construction of a strain producing the intermediate Allocryptopine

[0695] 1. According to the method in step 1 (a), XI-5-gRNA-F was replaced with XI-2-gRNA-F, XI-5-gRNA-R was replaced with XI-2-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-XI-2.

[0696] 2. Preparation of gene expression cassette

[0697] The encoding nucleic acid expression cassette includes a promoter, a nucleic acid sequence of a target gene, and a terminator. The target genes are TfS9OMT* (AAU20770.1), CjCAS (BAB68769.1), PsTNMT (ACO90237.1), PsMSH (AGC92398.1), and CyCAS (nucleotide sequence as shown in SEQ ID NO: 85). The promoters are promoter PDC1p (nucleotide sequence as shown in SEQ ID NO: 69), promoter GPM1p (nucleotide sequence as shown in SEQ ID NO: 21), promoter TPI1p (nucleotide sequence as shown in SEQ ID NO: 8), and promoter PGK1p (nucleotide sequence as shown in SEQ ID NO: 9). The terminators include terminator ENO2t (nucleotide sequence as shown in SEQ ID NO: 7), terminator IDP1t (nucleotide sequence as shown in SEQ ID NO: 3), terminator ADH1t (nucleotide sequence as shown in SEQ ID NO: 11) and terminator FBA1t (nucleotide sequence as shown in SEQ ID NO: 12).

[0698] The upstream homology arm X-2us (nucleotide sequence as shown in SEQ ID NO: 37), promoter PDC1p, nucleic acid sequence of TfS9OMT*, terminator ENO2t and terminator IDP1t were connected by overlap method to obtain gene expression cassette X-2us-PDC1p-TfS9OMT*-ENO2t-IDP1 (named as fragment 33). The terminator ENO2t, terminator IDP1t, nucleic acid sequence of CjCAS, promoter GPM1p and promoter TPI1p were connected by overlap method to obtain gene expression cassette ENO2t-IDP1t-CjCAS-GPM1p-TPI1p (named as fragment 34). The promoter GPM1p, promoter TPI1p, nucleic acid sequence of PsTNMT, terminator ADH1t and terminator FBA1t were connected by overlap method to obtain gene expression cassette GPM1p-TPI1p-PsTNMT-ADH1t-FBA1t (named as fragment 35). The terminator ADH1t, the terminator FBA1t, the nucleic acid sequence of PsMSH, the promoter PGK1p 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 ADH1t-FBA1t-PsMSH-PGK1p-X-2ds (named as fragment 36). The terminator ENO2t, the terminator IDP1t, the nucleic acid sequence of CyCAS, the promoter GPM1p and the promoter TPI1p were connected by the overlap method to obtain the gene expression cassette ENO2t-IDP1t-CyCAS-GPM1p-TPI1p (named as fragment 37).

[0699] 3. Strain construction

[0700] The plasmid pMEL10-XI-2 and fragments 33, 34, 35 and 36 were transferred into 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 grown clones were strains that had lost the gRNA plasmid, that is, integrated into the XI-2 site of the XJ0695 strain, and the XJ0741 strain (abbreviated as XJ0741) was obtained.

[0701] The plasmid pMEL10-XI-2 and fragments 33, 37, 35 and 36 were transferred into 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 grown clones were strains that had lost the gRNA plasmid, that is, integrated into the XI-2 site of the XJ0695 strain, and the XJ0742 strain (abbreviated as XJ0742) was obtained.

[0702] 4. Pick 3 positive single clones from each strain obtained in step 3 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0703] 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.

[0704] Test results see Fig.15a, b, c and d. The results showed that compared with Allocryptopine, both XJ0741 and XJ0742 strains can produce Allocryptopine, and XJ0741 strain introduced with Japanese Coptis chinensis CjCAS can produce a higher content of Allocryptopine. It can be seen that CjCAS is a gene that is more conducive to increasing the content of the final product in the engineered bacteria, and CjCAS was selected as the element for pathway construction.

[0705] (II) Screening of flavoprotein oxidase McDBOX2

[0706] Codon-optimized Argemone Mexicana AmSTOX, Berberis wilsoniae BwSTOX, C. japonica CjTHBO, Papaver PsFADOX, Macleaya cordata McDBOX1 and McDBOX2 genes were synthesized in vitro and transferred into the proopioline-producing strain XJ0743 together with EcP6H from Ectopus californicus (the preparation method of the XJ0743 strain is as follows: (1) using the overlap method to connect the upstream homology arm XI-2us (nucleotide sequence shown in SEQ ID NO: 73), the promoter PDC1p (nucleotide sequence shown in SEQ ID NO: 69), the nucleic acid sequence of EcCFS (BAG75113), the terminator ENO2t (nucleotide sequence shown in SEQ ID NO: 7) and the terminator IDP1t (nucleotide sequence shown in SEQ ID NO: 10); and the codon-optimized Argemone Mexicana AmSTOX, Berberis wilsoniae BwSTOX, C. japonica CjTHBO, Papaver PsFADOX, Macleaya cordata McDBOX1 and McDBOX2 genes were synthesized in vitro and transferred into the proopioline-producing strain XJ0743 together with EcP6H from Ectopioides californicus. NO:3), to obtain the gene expression cassette XI-2us-PDC1p-EcCFS-ENO2t-IDP1; (2) using the overlap method to connect the terminator ENO2t, the terminator IDP1t, the nucleic acid sequence of EcSPS (BAD98250), the promoter GPM1p (nucleotide sequence as shown in SEQ ID NO:21) and the promoter TPI1p (nucleotide sequence as shown in SEQ ID NO:8), to obtain the gene expression cassette ENO2t-IDP1t-EcSPS-GPM1p-TPI1p; (3) using the overlap method to connect the promoter GPM1p, the promoter TPI1p, the nucleic acid sequence of PsTNMT (ACO90237.1), the terminator ADH1t (nucleotide sequence as shown in SEQ ID NO:11) and the terminator FBA1t (nucleotide sequence as shown in SEQ ID NO:12). NO:12), and obtained the gene expression cassette GPM1p-TPI1p-PsTNMT-ADH1t-FBA1t; (4) using the overlap method to connect the terminator ADH1t, terminator FBA1t, the nucleic acid sequence of PsMSH (AGC92398.1), the promoter PGK1p and the downstream homology arm XI-2ds (the nucleotide sequence is shown in SEQ ID NO:74), and obtained the gene expression cassette ADH1t-FBA1t-PsMSH-PGK1p-XI-2ds; (5) the plasmid pMEL10-XI-2 and the four gene expression cassettes were transformed into XJ0695 competent cells to obtain positive clones; the positive clones were randomly selected and streaked on 5-FOA plates, and cultured in a constant temperature incubator at 30°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 XI-2 site of the XJ0695 strain to obtain the XJ0743 strain).The protein expression was detected by Western blot technology, and it was found that BwSTOX and McDBOX2 proteins were well expressed. However, it was found that even in the control strain XJ07437 that only expressed EcP6H, the target product sanguinarine could be produced, while dihydrosanguinarine could not be detected in the XJ0750 strain into which McDBOX2 was introduced. This result confirmed that McDBOX2 plays a key role in the conversion of dihydrosanguinarine to sanguinarine. The specific steps are as follows:.

[0707] 1. According to the method in step 1 (a), replace XI-5-gRNA-F with XII-1-gRNA-F, and replace XI-5-gRNA-R with XII-1-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-XII-1.

[0708] 2. Strain construction

[0709] The encoding nucleic acid expression cassette includes a promoter, a nucleic acid sequence of a target gene, and a terminator. The target genes are AmSTOX (ADY15027.1), EcP6H (BAK20464), BwSTOX (ADY15026.1), CjSTOX (BAJ40864.1), PsFADOX5 (AGL44334.1), StBBE (nucleotide sequence as shown in SEQ ID NO: 24), McDBOX1 (OVA00267.1) and McDBOX2 (OVA00268.1). The promoters are promoter PDC1p (nucleotide sequence as shown in SEQ ID NO: 69) and promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminators are terminator DIT1t (nucleotide sequence as shown in SEQ ID NO: 75) and terminator TDH2t (nucleotide sequence as shown in SEQ ID NO: 6). The His tag sequence is CATCATCACCATCACCAT.

[0710] (1) The upstream homology arm XII-1us (nucleotide sequence as shown in SEQ ID NO: 76), terminator DIT1t, His tag sequence (CATCATCACCATCACCAT), AmSTOX nucleic acid sequence, promoter PDC1p and promoter CCW12p were connected by the overlap method to obtain the gene expression cassette XII-1us-DIT1t-His-AmSTOX-PDC1p-CCW12p (named as fragment 38). The promoter PDC1p, promoter CCW12p, EcP6H nucleic acid sequence, terminator TDH2t and downstream homology arm XII-1ds (nucleotide sequence as shown in SEQ ID NO: 77) were connected by the overlap method to obtain the gene expression cassette PDC1p-CCW12p-EcP6H-TDH2t-XII-1ds (named as fragment 39). The plasmid pMEL10-XII-1 and fragments 38 and 39 were transferred into the competent cells of the XJ0743 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 grown clones were strains that had lost the gRNA plasmid, that is, integrated into the XII-1 site of the XJ0743 strain, obtaining the XJ07431 strain.

[0711] (2) According to the method of step (1), the nucleic acid sequence of AmSTOX was replaced with the nucleic acid sequence of BwSTOX, the nucleic acid sequence of CjSTOX, the nucleic acid sequence of PsFADOX5, the nucleic acid sequence of StBBE, the nucleic acid sequence of McDBOX1 and the nucleic acid sequence of McDBOX2, respectively, while the other steps remained unchanged, and the XJ07432 strain, XJ07433 strain, XJ07434 strain, XJ07435 strain, XJ07436 strain and XJ0750 strain were obtained in turn.

[0712] (3) The plasmid pMEL10-XII-1 and the gene expression cassette X11-1 us-CCW12p-EcP6H-TDH2t-XII-1ds (composed of the upstream homology arm promoter X11-1 us, promoter CCW12p, the nucleic acid sequence of EcP6H, terminator TDH2t and downstream homology arm X11-1 ds) were transferred into the competent cells of the XJ0743 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, that is, integrated into the XII-1 site of the XJ0743 strain, obtaining the control strain XJ07437 strain with only the EcP6H gene inserted.

[0713] 3. Pick 3 positive single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0714] 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.

[0715] 5. Western blot detection of protein expression

[0716] (1) Each strain obtained in step 2 was inoculated into 1 mL of delft medium and cultured overnight at 30°C and 220 rpm to obtain a preculture.

[0717] (2) The pre-culture was transferred to 5 mL of fresh Delft medium at a ratio of 1:50 and cultured at 30°C and 220 rpm to obtain the OD 600nm About 1% of the culture solution.

[0718] (3) Collect cells from the culture solution, wash twice with PBS buffer, add 200 μL of acid-washed glass beads and 300 μL of PBS buffer to resuspend, and then use FastPrep rapid sample preparation instrument to disrupt the cells to obtain a sample.

[0719] (4) Add 4×NuPAGE to the sample TM LDS sample buffer (ThermoFisher) was boiled at 95°C for 10 minutes, and protein electrophoresis was performed at 150V for 1 hour.

[0720] (5) Transfer protein to Trans- Turbo TMAfter PVDF transfer membrane (Bio-Rad), block with PBST (PBS + 0.1% Tween 20) containing 5% milk at room temperature for 2 hours. Wash the membrane 3 times with PBST and incubate with 6×His-Tag monoclonal antibody (His.H8) (ThermoFisher) or anti-GAPDH antibody (G-9) (Santa Cruz Biotechnology) at room temperature for 1 hour. Then wash the membrane 3 times with PBST and incubate with HRP-conjugated anti-mouse IgG (h + L) secondary antibody (Invitrogen) at room temperature for 1 hour. Finally, wash the membrane 3 times with PBST and incubate with West Pico plus HRP substrate (ThermoFisher) for 5 minutes, and finally analyze with ChemiDoc XRS image analyzer (Bio-Rad).

[0721] The experimental results are shown in Fig.16 Western blot results showed that BwSTOX and McDBOX2 proteins were well expressed with the correct protein size, PsFADOX5 bands were diffuse, while AmSTOX, CjTHBO and McDBOX1 were not expressed (see Fig.16 c and d). The strain metabolic component detection showed (see Fig.16 In a and b), all strains including the control strain produced sanguinarine. However, no dihydrosanguinarine was detected in the XJ0750 strain, which indicates that the McDBOX2 gene plays a role in converting dihydrosanguinarine to produce sanguinarine in yeast, that is, the McDBOX2 gene is the only flavoprotein oxidase gene screened in this experiment that plays a role in catalyzing the oxidation of dihydrosanguinarine to produce sanguinarine in yeast.

[0722] (III) Screening of P6H gene

[0723] The P6H genes from three different sources and McDBOX2 selected in step (ii) were respectively verified in the XJ0741 strain. Under the same fermentation conditions, the XJ0747 strain expressing EcP6H had the highest content of the final product chelerythrine. This experiment determined that EcP6H was the construction element of this pathway. The specific steps are as follows:

[0724] 1. According to the method in step 1 (a), replace XI-5-gRNA-F with XII-1-gRNA-F, and replace XI-5-gRNA-R with XII-1-gRNA-R, and keep other steps unchanged to obtain plasmid pMEL10-XII-1.

[0725] 2. Strain construction

[0726] The encoding nucleic acid expression cassette includes a promoter, a nucleic acid sequence of a target gene, and a terminator. The target genes are EcP6H (BAK20464), McP6H (OVA18597), CyP6H (nucleotide sequence as shown in SEQ ID NO: 86), and McDBOX2 (OVA00268.1). The promoters are promoter PDC1p (nucleotide sequence as shown in SEQ ID NO: 69) and promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminators are terminator DIT1t (nucleotide sequence as shown in SEQ ID NO: 75) and terminator TDH2t (nucleotide sequence as shown in SEQ ID NO: 6). The His tag sequence is CATCATCACCATCACCAT.

[0727] (1) The upstream homology arm XII-1us, terminator DIT1t, His tag sequence (CATCATCACCATCACCAT), McDBOX2 nucleic acid sequence, promoter PDC1p and promoter CCW12p were connected by the overlap method to obtain the gene expression cassette XII-1us-DIT1t-His-McDBOX2-PDC1p-CCW12p. The promoter PDC1p, promoter CCW12p, EcP6H nucleic acid sequence, terminator TDH2t and downstream homology arm XII-1ds were connected by the overlap method to obtain the gene expression cassette PDC1p-CCW12p-EcP6H-TDH2t-XII-1ds. Plasmid pMEL10-XII-1 was used to transform the two gene expression cassettes into XJ0741 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-1 site of the XJ0741 strain, obtaining the XJ0747 strain.

[0728] (2) According to the method of step (1), the nucleic acid sequence of EcP6H was replaced by the nucleic acid sequence of McP6H and the nucleic acid sequence of CyP6H respectively, and the other steps remained unchanged, thereby obtaining the XJ0748 strain and the XJ0749 strain respectively.

[0729] 3. Pick 3 positive single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth. 600nm .

[0730] 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.

[0731] Test results see Fig.15 e, f and g. The results showed that strains XJ0747, XJ0748 and XJ0749 could all produce the final product chelerythrine, and the final product chelerythrine content of strain XJ0747 expressing EcP6H was the highest. Therefore, EcP6H is the most suitable gene element for the construction of chelerythrine engineering bacteria among the three candidate P6H genes.

[0732] 8. Optimization of the McDBOX step in the chelerythrine pathway

[0733] The study found that the expression of McDBOX2 would reduce the OD of the strain 600nm And the accumulation of the intermediate product chondrine was increased, indicating that McDBOX2 had a certain inhibitory effect on the function of BBE. The present invention adopts the following strategies for pathway optimization: (1) Overexpression of plasma membrane riboflavin transporter MCH5 and FAD synthase BsRibc to increase FAD supply, and it was found that the production of chelerythrine could be increased by 24.4% and 39.2%, which is speculated to be due to the increase in McDBOX2 activity caused by the expression of MCH5 and BsRibc; (2) Truncation of the N-terminal 29 amino acids of McDBOX2 to increase its expression in the cytoplasm, and it was found that the XJ07472 strain (XJ07411+McDBOX2Δ29) after truncating McDBOX2 could convert all dihydrochelerythrine into chelerythrine; (3) Knockout of phospholipase OPI1 to mediate endoplasmic reticulum expansion. Finally, the XJ07474 strain with a chelerythrine production of 38.1 mg / L was obtained.

[0734] 1. Increasing the supply of FAD

[0735] By plasmid transformation, the XJ0774 strain (the preparation method of the XJ0774 strain is as follows: (1) using the artificially synthesized DNA fragment LP1.T8 (nucleotide sequence as shown in SEQ ID NO: 90) as a template, using the primer pair consisting of FgF20-LP1.T8-F and LP1.T8-CCW12p-R to perform PCR amplification to obtain the fragment FgF20 us-LP1.T8; (2) Using the genomic DNA of strain XJ0747 as a template, PCR amplification was performed using primer pair Y1 consisting of CCW12p1-F and TDH2t-FBA1t-R, primer pair Y2 consisting of FBA1t400-rR and PGK1p-IDP1t-R, and primer pair Y3 consisting of IDP1t-rF and GPM1p-FgF20-R, respectively, to obtain fragments CCW12p-EcP6H-TDH2t-FBA1t, fragment FBA1t-PsMSH-PGK1p, and fragment IDP1t-EcSPS-GPM1p-FgF20-R. ds; (3) transforming the plasmid pMEL10-FgF20 and the above four fragments into XJ0747 competent cells to obtain positive clones; randomly selecting positive clones and streaking them on 5-FOA plates, culturing them in a constant temperature box at 30°C for 2-3 days, and the clones that grew out were strains that had lost the gRNA plasmid, that is, integrating the fragment into the FgF20 site of the XJ0747 strain to obtain the XJ0774 strain) into which genes for enhancing FAD supply were introduced, including carrying FAD ER localization transport proteins FLC1 (endogenous yeast gene), FLC2 (endogenous yeast gene), FLC3 (endogenous yeast gene) and YOR365C (endogenous yeast gene), mitochondrial FAD transport protein FLX1 (endogenous yeast gene), plasma membrane riboflavin transporter MCH5 (nucleotide sequence as shown in SEQ ID NO:78), FAD biosynthesis-related genes FMN1 (yeast endogenous gene) and FAD1 (yeast endogenous gene), FAD synthase BsRiba (nucleotide sequence as shown in SEQ ID NO:87) and BsRibc (nucleotide sequence as shown in SEQ ID NO:79) from Bacillus subtilis. The effects of the above candidate genes were evaluated by comparing the CHE production of the strains under the same fermentation conditions. The specific steps are as follows:

[0736] 1. Construction of recombinant plasmid

[0737] (1) Using yeast genomic DNA as a template, PCR amplification was performed using primer pair 11 (consisting of GPD-FLC1-F and FLC1-CYC1t-R), primer pair 12 (consisting of GPD-FLC2-F and FLC2-CYC1t-R), primer pair 13 (consisting of GPD-YOR365C-F and YOR365C-CYC1t-R), primer pair 14 (consisting of GPD-FLC3-F and FLC3-CYC1t-R), primer pair 15 (consisting of GPD-MCH5-F and MCH5-CYC1t-R), and primer pair 16 (consisting of GPD-FLX1-F and FLX1-CYC1t-R), respectively (the enzyme for PCR amplification was PrimeStar HS DNA polymerase (Takara, R010B) was used to obtain fragments FLC1, FLC2, YOR365C, FLC3, MCH5 and FLX1 in sequence.

[0738] The reaction program was as follows: denaturation at 95°C for 5 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 2 min, 30 cycles; and extension at 70°C for 10 min.

[0739] (2) Using Bacillus subtilis genomic DNA as a template for amplification, PCR amplification was performed using primer pair 17 (consisting of GPD-BsRibc-F and BsRibc-CYC1t-R) and primer pair 18 (consisting of GPD-BsRiba-F and BsRiba-CYC1t-R), respectively (the enzyme used for PCR amplification was PrimeStar HSDNA polymerase), to obtain fragments BsRibc and BsRiba, respectively.

[0740] The reaction program was as follows: denaturation at 95°C for 5 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 2 min, 30 cycles; and extension at 70°C for 10 min.

[0741] (3) Fragment FLC1, fragment FLC2, fragment YOR365C, fragment FLC3, fragment MCH5, fragment FLX1, fragment BsRibc, fragment BsRiba and the artificially synthesized fusion fragment of FMN1 and FAD1 (nucleotide sequence as shown in SEQ ID NO:88) were Gibson ligated with the linearized plasmid P416_GPD to obtain recombinant plasmids P416_GPD-FLC1, P416_GPD-FLC2, recombinant plasmid P416_GPD-YOR365C, recombinant plasmid P416_GPD-FLC3, recombinant plasmid P416_GPD-MCH5, recombinant plasmid P416_GPD-FLX1, recombinant plasmid P416_GPD-BsRiba, recombinant plasmid P416_GPD-BsRibc and recombinant plasmid P416_GPD-FMN1+FAD1, respectively.

[0742] Plasmid P416_GPD (purchased from the ATCC strain library, website: https: / / www.atcc.org / search#q=P416&sort=relevancy&numberOfResults=24) 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 linearized plasmid P416_GPD.

[0743] 2. Introduce the plasmid (plasmid P416_GPD, recombinant plasmid P416_GPD-FLC1, P416_GPD-FLC2, recombinant plasmid P416_GPD-YOR365C, recombinant plasmid P416_GPD-FLC3, recombinant plasmid P416_GPD-MCH5, recombinant plasmid P416_GPD-FLX1, recombinant plasmid P416_GPD-BsRiba, recombinant plasmid P416_GPD-BsRibc or recombinant plasmid P416_GPD-FMN1+FAD1) into the XJ0774 strain to obtain recombinant bacteria.

[0744] 3. Pick 3 positive single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0745] 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.

[0746] Test results see Fig.17 . The results showed that compared with the recombinant bacteria introduced with plasmid P416_GPD (i.e., the control strain), the CHE production of the recombinant bacteria introduced with recombinant plasmid P416_GPD-MCH5 and recombinant plasmid P416_GPD-BsRibc increased by 24.4% and 39.2%, respectively, while the recombinant bacteria introduced with other recombinant plasmids had no significant effect on CHE production. However, the S-RET content of the strains expressing MCH5 and BsRibc did not decrease, indicating that the expression of MCH5 or BsRibc may increase the activity of McDBOX2 at this stage, thereby leading to an increase in CHE production, rather than alleviating the restriction of McDBOX on the catalytic effect of CyBBE (see Fig.17 (b) The results showed that overexpression of MCH5 fragment or BsRibc fragment could enhance FAD supply and increase CHE production of engineered bacteria.

[0747] (ii) Truncation of the N-terminus of McDBOX2 by 29 amino acids to increase its expression in the cytoplasm

[0748] By truncating 29 amino acids at the N-terminus of McDBOX2 (genbank: OVA00268.1), the effect of N-terminal truncation on activity was compared. The control group was the XJ07411 strain without McDBOX2 and the XJ0747 strain containing McDBOX2, and the experimental group was the XJ07472 strain containing McDBOX2_Δ29. By detecting the growth curves, CHE content, and conversion of intermediates of the three strains under the same fermentation conditions, it was evaluated whether truncating the N-terminal 29 amino acids of McDBOX2 was beneficial to the construction of the target engineering bacteria. The specific steps are as follows:

[0749] 1. Construction of plasmid

[0750] (1) According to the method in step 1 (a), XI-5-gRNA-F was replaced with XII-1-gRNA-F, and XI-5-gRNA-R was replaced with XII-1-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-XII-1.

[0751] (2) 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.

[0752] 2. Strain construction

[0753] The encoding nucleic acid expression cassette includes an upstream homology arm, a promoter, a nucleic acid sequence of a target gene, a terminator, and a downstream homology arm. The promoters are promoter PDC1p (nucleotide sequence as shown in SEQ ID NO:69) and promoter CCW12p (nucleotide sequence as shown in SEQ ID NO:2). The terminators are terminator DIT1t (nucleotide sequence as shown in SEQ ID NO:75) and terminator TDH2t (nucleotide sequence as shown in SEQ ID NO:6).

[0754] (1) The plasmid pMEL10-XII-1 and the gene expression cassette fragment of EcP6H (consisting of the upstream homology arm XII-1us, the promoter CCW12p, the nucleic acid sequence of EcP6H, the terminator TDH2t and the downstream homology arm XII-1ds) were transformed into XJ0741 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 XII-1 site of the XJ0741 strain, and the XJ07411 strain (without the McDBOX2 gene) was obtained.

[0755] (2) The plasmid pMEL10-FgF20 and the gene expression cassette fragment of McDBOX2_Δ29 (consisting of the upstream homology arm FgF20us (nucleotide sequence as shown in SEQ ID NO: 67), the promoter PDC1p, the nucleic acid sequence of McDBOX2_Δ29, the terminator DIT1t and the downstream homology arm FgF20 ds (nucleotide sequence as shown in SEQ ID NO: 68) were transformed into XJ07411 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, integrated into the FgF20 site of the XJ07411 strain, and the XJ07472 strain (i.e., XJ07411+McDBOX2_Δ29) was obtained.

[0756] 3. Pick 3 positive single clones from each strain obtained in step 2 for parallel experiments. Add 1ml of Deft medium (solutes and their concentrations are 7.5g / L (NH4)2SO4, 14.4g / L KH2PO4, 0.5g / LMgSO4·7H2O, 20g / L glucose, 2ml / L trace metal solutions, 1ml / L vitamin solutions and 60mg / L uracil, and the solvent is water) to a 14mL shaking tube, then inoculate the single clone, and culture at 30℃ and 220rpm for 18-24h to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125mL) containing 20mL of Deft medium for expansion culture, and culture at 30℃ and 220rpm for 72h to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0757] 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.

[0758] 5. Western blot detection of protein expression

[0759] (1) Each strain obtained in step 2 was inoculated into 1 mL of delft medium and cultured overnight at 30°C and 220 rpm to obtain a preculture.

[0760] (2) The pre-culture was transferred to 5 mL of fresh Delft medium at a ratio of 1:50 and cultured at 30°C and 220 rpm to obtain the OD 600nm About 1% of the culture solution.

[0761] (3) Collect cells from the culture solution, wash twice with PBS buffer, add 200 μL of acid-washed glass beads and 300 μL of PBS buffer to resuspend, and then use FastPrep rapid sample preparation instrument to disrupt the cells to obtain a sample.

[0762] (4) Add 4×NuPAGE to the sample TM LDS sample buffer (ThermoFisher) was boiled at 95°C for 10 minutes, and protein electrophoresis was performed at 150V for 1 hour.

[0763] (5) Transfer protein to Turbo TM After PVDF transfer membrane (Bio-Rad), block with PBST (PBS + 0.1% Tween 20) containing 5% milk at room temperature for 2 hours. Wash the membrane 3 times with PBST and incubate with 6×His-Tag monoclonal antibody (His.H8) (ThermoFisher) or anti-GAPDH antibody (G-9) (Santa Cruz Biotechnology) at room temperature for 1 hour. Then wash the membrane 3 times with PBST and incubate with HRP-conjugated anti-mouse IgG (h + L) secondary antibody (Invitrogen) at room temperature for 1 hour. Then wash the membrane 3 times with PBST and incubate with West Pico plus HRP substrate (ThermoFisher) for 5 minutes, and finally analyze with ChemiDoc XRS image analyzer (Bio-Rad).

[0764] Western blot analysis results are shown in Fig.18 The results showed that a protein band appeared in the strain expressing McDBOX2_Δ29, and its size was close to the theoretical 56.8 kDa of McDBOX2Δ29, while the strain expressing McDBOX2 had multiple bands (see Fig.18 (a); Growth OD of the strain expressing McDBOX2_Δ29 compared to the strain expressing McDBOX2 600nm There is no significant difference in the values ​​( Fig.18 b); the intermediate RET content did not increase ( Fig.18 (c) The above results indicate that truncating the N-terminus of McDBOX2 by 29 amino acids to increase its expression in the cytoplasm can effectively resolve the inhibition of cell growth by the McDBOX2 gene and the inhibition of the conversion of the intermediate RET.

[0765] 3. Integration of FAD enhancement strategy

[0766] The McDBOX2 N-terminal truncation (McDBOX2_Δ29) strategy and the endoplasmic reticulum expansion strategy were used to construct the chelerythrine engineering bacteria. Based on the XJ07472 strain (containing the complete CHE pathway of McDBOX2_Δ29), the plasma membrane riboflavin transporter MCH5 (nucleotide sequence as shown in SEQ ID NO:78) and FAD synthase BsRibc (nucleotide sequence as shown in SEQ ID NO:79) were overexpressed to increase the FAD supply, and the phospholipase OPI1 was knocked out to mediate the endoplasmic reticulum expansion, and finally the XJ07474 strain with a chelerythrine production of 38.1 mg / L was obtained. The specific steps are as follows:

[0767] 1. Overexpression of MCH5 and BsRibc (construction of XJ07473 strain)

[0768] The encoding nucleic acid expression cassette is composed of an upstream homology arm, a promoter, a nucleic acid sequence of a target gene, a terminator, and a downstream homology arm. The promoter is a promoter GPD (nucleotide sequence as shown in SEQ ID NO: 81) and a promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminator is a terminator IDP1t (nucleotide sequence as shown in SEQ ID NO: 3) and a terminator CYC1t (nucleotide sequence as shown in SEQ ID NO: 13).

[0769] (1) The upstream homology arm 416d us (nucleotide sequence shown in SEQ ID NO: 59), promoter GPD, nucleic acid sequence of MCH5, terminator IDP1t and promoter CCW12p were connected by overlap method to obtain gene expression cassette 416dus-GPD-MCH5-IDP1t-CCW12p (named as fragment 40). The terminator IDP1t, promoter CCW12p, nucleic acid sequence of BsRibc, terminator CYC1t and downstream homology arm 416d ds (nucleotide sequence shown in SEQ ID NO: 60) were connected by overlap method to obtain gene expression cassette IDP1t-CCW12p-BsRibc-CYC1t-416d ds (named as fragment 41).

[0770] (2) The plasmid pMEL10-416d was transformed into XJ07472 competent cells together with fragment 40 and fragment 41 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 the XJ07472 strain, and the XJ07473 strain was obtained.

[0771] 2. Knockout of phospholipase OPI1 (construction of strain XJ07474)

[0772] (1) According to the method in step 1 (a), XI-5-gRNA-F was replaced with OPI1-gRNA-F, and XI-5-gRNA-R was replaced with OPI1-gRNA-R, and other steps remained unchanged to obtain plasmid pMEL10-OPI1.

[0773] (2) The repair fragment LP3.T7 (nucleotide sequence as shown in SEQ ID NO: 89) was integrated into the OPI1 gene site of the XJ07473 strain using plasmid pMEL10-OPI1, thereby knocking out the OPI1 gene to obtain the XJ07474 strain.

[0774] 3. Pick 3 single clones from each of the XJ07472, XJ07473 and XJ07474 strains for parallel experiments. Add 1 ml of Deft medium (solutes and their concentrations are 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, 1 ml / L vitaminsolutions and 60 mg / L uracil, and the solvent is water) to a 14 mL shake tube, then inoculate the single clone, and culture at 30°C and 220 rpm for 18-24 hours to obtain a bacterial solution. Inoculate the bacterial solution into a shake flask (specification: 125 mL) containing 20 mL of Deft medium for expansion culture, and culture at 30°C and 220 rpm for 72 hours to obtain a fermentation broth. Determine the OD of the fermentation broth 600nm .

[0775] 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.

[0776] Test results see Fig.19 The results showed that by integrating the FAD enhancement strategy (overexpression of MCH5 and BsRibc), the N-terminal truncation of McDBOX2 (McDBOX2_Δ29) strategy and the endoplasmic reticulum expansion strategy (knockout of phospholipase OPI1), the XJ07474 strain with a chelerythrine production of 38.1 mg / L was finally obtained.

[0777] 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 fungus A obtained by transforming a yeast used to produce golden corydaline; characterized in that: The recombinant bacteria A contains or expresses berberidase BBE and benzophenanthridine oxidase DBOX with enhanced activities compared with the parent microorganism.

2. The recombinant bacteria A according to claim 1, characterized in that: The recombinant bacteria A contains or expresses a berberidase BBE with enhanced activity compared with the parent microorganism. The recombinant bacteria A contains or expresses 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 bacteria A according to claim 1, characterized in that: The recombinant bacteria A contains or expresses the benzophenanthridine oxidase DBOX, which is an N-terminal truncation of the benzophenanthridine oxidase DBOX contained or expressed in the recombinant bacteria A.

4. The recombinant bacteria A according to any one of claims 1 to 3, characterized in that: The recombinant bacteria A contains or expresses papaverine synthase CFS and papaverine synthase SPS.

5. The recombinant bacteria A according to any one of claims 1 to 4, characterized in that: The recombinant bacteria A also contains or expresses N-methyltransferase TNMT.

6. The recombinant bacteria A according to any one of claims 1 to 5, characterized in that: The recombinant bacteria A also contains or expresses N-methyl papaverine 14-hydroxylase MSH.

7. The recombinant bacteria A according to any one of claims 1 to 6, characterized in that: The recombinant bacteria A also contains or expresses proopiopine-6-hydroxylase P6H.

8. The recombinant bacteria A according to any one of claims 1 to 7, characterized in that: The recombinant bacteria A also contains or expresses a plasma membrane riboflavin transporter and / or FAD synthase.

9. The recombinant bacteria A according to any one of claims 1 to 3, characterized in that: The recombinant bacteria A contains or expresses O-methyltransferase in vivo.

10. The recombinant bacteria A according to claim 1, 2, 3 or 9, characterized in that: The recombinant bacteria A also contains or expresses tetrahydroberberine synthase CAS.

11. The recombinant bacteria A according to claim 1, 2, 3, 9 or 10, characterized in that: The recombinant bacteria A also contains or expresses tetrahydroberberine N-methyltransferase TNMT.

12. The recombinant bacteria A according to claim 1, 2, 3, 9, 10 or 11, characterized in that: The recombinant bacteria A also contains or expresses N-methyl papaverine 14-hydroxylase MSH.

13. The recombinant bacteria A according to claim 1, 2, 3, 9, 10, 11 or 12, characterized in that: The recombinant bacteria A also contains or expresses proopiopine-6-hydroxylase P6H.

14. The recombinant bacteria A according to claim 1, 2, 3, 9, 10, 11, 12 or 13, characterized in that: The recombinant bacteria A also contains or expresses a plasma membrane riboflavin transporter and / or FAD synthase.

15. The recombinant bacteria A according to any one of claims 1 to 14, characterized in that: The yeast used for producing golden corydaline contains or expresses tyrosine hydroxylase and dopa decarboxylase; and / or the body also contains or expresses higenamine synthase NCS or N-terminally truncated NCS enzyme; and / or the body also contains or expresses tyrosine metabolic flux-related proteins; and / or knocking out or down-regulating proteins related to 4-hydroxyphenylethanol and 4-hydroxyphenylacetic acid synthesis activities in vivo; and / or also contain or express aromatic amino acid decarboxylase in the body; and / or the body also contains or expresses prephenate aminotransferase PAT and alloate dehydratase ADH; and / or the body also contains or expresses methyltransferase 6OMT and clavaline methyltransferase CNMT; and / or the body also contains or expresses more than one methyltransferase 4'OMT and N-methylcoclaurine 3'-hydroxylase NMCH; and / or also contain or express cytochrome P450 reductase ATR1, ATR2 or CPR in the body; and / or also contain or express catalase in the body; And / or the body also contains or expresses transcription factors that activate phospholipid biosynthesis or knocks out transcription factors that inhibit phospholipid biosynthesis.

16. The recombinant bacteria A according to any one of claims 1 to 15, characterized in that: The recombinant bacteria A is a species of the genus Saccharomyces cerevisiae.

17. The recombinant bacteria A according to any one of claims 1 to 16, characterized in that: The recombinant bacteria A is Saccharomyces cerevisiae.

18. Use of the recombinant bacteria A according to any one of claims 1 to 3 in the production of benzophenanthridine alkaloids.

19. A method for producing benzophenanthridine alkaloids, comprising the following steps: fermenting and culturing the recombinant bacteria A according to any one of claims 1 to 3, collecting the fermentation product, and obtaining the benzophenanthridine alkaloids therefrom.

20. Use of the recombinant bacteria A according to claim 1, 2, 3, 4, 5, 6, 7, 8, 15, 16 or 17 in the production of sanguinarine, dihydrosanguinarine, protopine, N-methyl papaverine and / or papaverine.

21. A method for producing sanguinarine, dihydrosanguinarine, protopine, N-methyl papaverine and / or papaverine, comprising the steps of: fermenting and culturing the recombinant bacteria A according to claim 1, 2, 3, 4, 5, 6, 7, 8, 15, 16 or 17, collecting the fermentation product, and obtaining sanguinarine, dihydrosanguinarine, protopine, N-methyl papaverine and / or papaverine therefrom.

22. Use of the recombinant bacteria A of claim 1, 2, 3, 9, 10, 11, 12, 13, 14, 15, 16 or 17 in the production of chelerythrine, allocryptopine and / or tetrahydroberberine.

23. A method for producing chelerythrine, allocryptopine and / or tetrahydroberberine, comprising the following steps: fermenting and culturing the recombinant bacteria A according to claim 1, 2, 3, 9, 10, 11, 12, 13, 14, 15, 16 or 17, collecting the fermentation product, and obtaining chelerythrine, allocryptopine and / or tetrahydroberberine therefrom.

24. A recombinant bacterium B obtained by transforming a yeast used for producing dihydrosanguinarine; characterized in that: The recombinant bacteria B contains or expresses CYP82P2 in vivo.

25. The recombinant bacterium B according to claim 24, characterized in that: The recombinant bacteria B contains or expresses 10-hydroxymethyltransferase OMT.

26. The recombinant bacterium B according to claim 24 or 25, characterized in that: The yeast used to produce dihydrosanguinarine is the recombinant yeast A described in claim 1, 2, 3, 4, 5, 6, 7, 8, 15, 16 or 17.

27. Use of the recombinant bacterium B according to any one of claims 24 to 26 in the production of chelerythrine and / or 10-hydroxydihydrosanguina.

28. A method for producing chelerythrine and / or 10-hydroxydihydrosanguina, comprising the following steps: fermenting and culturing the recombinant bacterium B according to any one of claims 24 to 26, collecting the fermentation product, and obtaining chelerythrine and / or 10-hydroxydihydrosanguina therefrom.

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