Recombinant strain and use thereof in production of benzophenanthridine alkaloids

Abstract

Disclosed are a recombinant strain and a use thereof in the production of benzophenanthridine alkaloids. The synthesis pathways of benzophenanthridine alkaloids were divided into multiple modules for study, and multiple strategies were used to achieve de novo synthesis of benzophenanthridine alkaloids.

Description

[0001] RECOMBINANT STRAIN AND USE THEREOF IN PRODUCTION OF BENZOPHENANTHRIDINE ALKALOIDS

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of biotechnology, in particular to a recombinant yeast strain and a use thereof in the production of benzophenanthridine alkaloids, such as sanguinarine or chelerythrine.

[0004] BACKGROUND

[0005] Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites that have important research and pharmaceutical values, including the antitussive and anti-cancer noscapine, and the analgesic agents morphine and codeine, etc. At present, BIA compounds are mainly obtained from plant extraction or chemical synthesis. However, due to the low content of plant secondary metabolism and the presence of multiple steric conformations of the compounds, both extraction purification and chemical synthesis have great limitations. Biosynthesis in a microbial heterologous host has great potential to overcome these production obstacles. The production of BIA compounds using the yeasts Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, or Rhodosporidium toruloides as a host has attracted much attention. However, reorganizing a relatively long heterologous pathway in the yeast remains a significant challenge, and entails multiple optimizations, including identifying a missing key enzyme, increasing the efficiency of a rate-limiting enzyme by means of enzyme engineered relocation or cofactor engineering, etc., so as to increase the yield.

[0006] Benzophenanthridine alkaloids (BZDAs) are an important class of BIA compounds, and are formed from spontaneous ring opening and rearrangement of protoberberine alkaloids. The benzophenanthridine alkaloids chelerythrine (CHE) and sanguinarine (SAN) have significant pharmacological activities, including antimicrobial, anti-cancer, anti-inflammatory, adrenolytic, and cyto-inhibitory effects, and have been used as natural growth promoters in animal husbandry. Berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the biosynthesis pathways of BZDAs, and can catalyze S-reticuline (S-RET) to produce S-scoulerine (S-SCO). Previous studies have shown that truncating PsBBE from Papaver somniferum and using a high-copy plasmid to increase the expression level of PsBBE did not increase its conversion efficiency. Thus, the enhancement of BBE activity in Saccharomyces cerevisiae remains a major challenge. Researchers have synthesized SAN in Saccharomyces cerevisiae, but not de novo. In addition, due to the lack of efficient conversion of DBOX, only trace amounts of SAN are converted from dihydrosanguinarine either spontaneously or by the yeast's own non-characteristic functional oxidase or upstream BBE. Therefore, the activities of BBE and DBOX are critical for the heterologous synthesis of sanguinarine and chelerythrine-related compounds.

[0007] SUMMARY

[0008] It is an objective of the present invention to provide a method for improving the yield of a benzophenanthridine alkaloid, such as sanguinarine, or chelerythrine, or chelirubine, in a yeast strain.

[0009] It is a particular objective of the present invention to provide a recombinant yeast strain for the production of a benzophenanthridine alkaloid, such as sanguinarine or chelerythrine.

[0010] The present invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0011] An aspect of the invention relates to a recombinant yeast strain for production of a benzylisoquinoline alkaloid, preferably scoulerine. The recombinant yeast strain comprises a heterologous berberine bridge enzyme (BBE) and a heterologousbenzophenanthridine oxidase (DBOX).

[0012] Another aspect of the invention relates to a method for producing a benzophenanthridine alkaloid. The method comprises fermentatively culturing a recombinant yeast strain according to above collecting a fermentation product from the fermentatively cultured recombinant yeast strain. The method also comprises obtaining the benzophenanthridine alkaloid therefrom.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows the biosynthesis pathways of sanguinarine and chelerythrine.

[0015] FIG. 2 shows the pathway and engineering strategy for the de novo production of norcoclaurine (module I) in Saccharomyces cerevisiae and the detection results for (V) to (VIII) in step I.

[0016] FIG. 3 shows the detection results for (II) and (III) in step I of the Example.

[0017] FIG. 4 shows the detection results for (VII) in step I of the Example. FIG. 5 shows the detection results for (VIII) in step I of the Example.

[0018] FIG. 6 shows the pathway and engineering strategy for the optimization of the reticuline- producing module (module II) and the detection results for (II), (IV) and (V) in step II.

[0019] FIG. 7 shows the detection results for (II) in step II of the Example.

[0020] FIG. 8 shows the pathway and engineering strategy for the screening and optimization of BBE (module III) and the detection results for (II) to (IV) in step III.

[0021] FIG. 9 shows the detection results for (I) in step III of the Example.

[0022] FIG. 10 shows the detection results for (I) and (II) in step IV of the Example.

[0023] FIG. 11 shows the detection results for (II) in step IV of the Example.

[0024] FIG. 12 shows the detection results for (I) in step V of the Example.

[0025] FIG. 13 shows the detection results for (II) in step V of the Example.

[0026] FIG. 14 shows the detection results for step VI of the Example.

[0027] FIG. 15 shows the detection results for (I) and (III) in step VII of the Example.

[0028] FIG. 16 shows the detection results for (II) in step VII of the Example.

[0029] FIG. 17 shows the detection results for (I) in step VIII of the Example.

[0030] FIG. 18 shows the detection results for (II) in step VIII of the Example.

[0031] FIG. 19 shows the detection results for (III) in step VIII of the Example.

[0032] DETAILED DESCRIPTION

[0033] As used herein, the terms “exogenous” or “heterologous” when used with respect to a nucleic acid (RNA or DNA), protein or gene refer to a nucleic acid, protein or gene, which occurs non-naturally as part of the cell, organism, genome, RNA or DNA sequence, into which it is introduced. Such an exogenous or heterologous gene or protein could be a gene or protein from another species or strain, a modified, mutated or evolved version of a gene or protein naturally occurring in the host cell or a chimeric version of a gene naturally occurring in the host cell or a fusion gene or protein. In these former cases, the modification, mutation or evolution causes a change in the nucleotide sequence of the gene or in the amino acid sequence of the protein to thereby obtain a modified, mutated or evolved gene with another nucleotide sequence as compared to the gene naturally occurring in the host cell or a modified, mutated or evovled protein with another amino acid seuqence as compared to the protein naturally occuring in the host cell. Evolved gene or protein refers to a genes or protein obtained by genetic modification, such as mutation or exposure to an evolutionary pressure, to derive a new gene with a different nucleotide sequence as compared to the wild type or native gene or a new protein with a different amino acid sequence as compared to the wild type or native protein. A chimeric gene is formed through the combination of portions of one or more coding sequences to produce a new gene. These modifications are distinct from a fusion gene, which merges whole gene sequences into a single reading frame and often retain their original functions. A fusion protein is a protein created through the joining of two or more genes that originally coded for separate proteins or polypeptides.

[0034] An "endogenous", "native" or "wild type" nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, a "wild type protein" is a protein that is naturally occurring in or endogenous to the organism or host cell.

[0035] The term "overexpress", or "over expression" as used herein refers to higher levels of activity of a gene, e.g., transcription of the gene; higher levels of translation of mRNA into protein; and / or higher levels of production of a gene product, e.g., polypeptide, than would be in the cell in its native or control, e.g., not transformed with the particular heterologous or recombinant polypeptides being overexpressed, state. A typical example of an overexpressed gene is a gene under transcription control of another promoter as compared to the native promoter of the gene. Also, or alternatively, other changes in the control elements of a gene, such as enhancers, could be used to overexpress the particular gene. Furthermore, modifications that affect, i.e., increase, the translation of the mRNA transcribed from the gene could, alternatively or in addition, be used to achieve an overexpressed gene as used herein. These terms can also refer to an increase in the number of copies of a gene and / or an increase in the amount of mRNA and / or gene product in the cell. It is further possible to achieve overexpression by including genes from different species encoding the same or homologous gene product, such as enzyme. Overexpression can result in levels that are 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 750%, 1000%, 1500%, 2000% or higher in the cell, or any range therein, as compared to control levels.

[0036] As an example overexpression is achieved by replacing a native promoter of a gene with another promoter having higher transcriptional activity in the host cell as compared to the native promoter. Another example of overexpression is to include multiple copies of gene in a host cells. These multiple copies of the gene could then be under transcriptional control of a common promoter or separate promoters. These two examples of overexpression can be combined. A "disrupted gene" as defined herein involves any mutation or modification to a gene resulting in a partial or fully non-functional gene and gene product. Such a mutation or modification includes, but is not limited to, a missense mutation, a nonsense mutation, a deletion, a substitution, an insertion, addition of a targeting sequence and the like. Furthermore, a disruption of a gene can be achieved also, or alternatively, by mutation or modification of control elements controlling the transcription of the gene, such as mutation or modification in a promoter, terminator and / or enhancement elements. In such a case, such a mutation or modification results in partially or fully loss of transcription of the gene, i.e., a lower or reduced transcription as compared to native and non- modified control elements. As a result a reduced, if any, amount of the gene product will be available following transcription and translation. Furthermore, disruption of a gene could also entail adding or removing a localization signal from the gene, resulting in decreased presence of the gene product in its native subcellular compartment. The objective of gene disruption is to reduce the available amount of the gene product, including fully preventing any production of the gene product, or to express a gene product that lacks or having lower enzymatic activity as compared to the native or wild type gene product.

[0037] As used herein the term "deletion" or "knock-out" refers to a gene that is inoperative or knocked out.

[0038] The term "attenuated activity" when related to an enzyme refers to a decrease in the activity of the enzyme in its native compartment compared to a control or wildtype state. Manipulations that result in attenuated activity of an enzyme include, but are not limited to, a missense mutation, a nonsense mutation, a deletion, a substitution, an insertion, addition of a targeting sequence, removal of a targeting sequence, or the like. A cell that contains modifications that result in attenuated enzyme activity will have a lower activity of the enzyme compared to a cell that does not contain such modifications. Attenuated activity of an enzyme may be achieved by encoding a nonfunctional gene product, e.g., a polypeptide having essentially no activity, e.g., less than about 10% or even 5% as compared to the activity of the wild type polypeptide.

[0039] The term "enhanced activity" when related to an enzyme refers to an increase in the activity of the enzyme in its optimal compartment compared to a control or wildtype state. Manipulations that result in enhanced activity of an enzyme include, but are not limited to, a mutation, addition of a targeting sequence, removal of a targeting sequence, or the like. A cell that contains modifications that result in enhanced enzyme activity will have a higher activity of the enzyme compared to a cell that does not contain such modifications.

[0040] As used herein, the term "genetically modified", when it is used with respect to an organism, refers to a host organism that has been genetically modified, as compared with an otherwise identical host organism that has not been so genetically modified. In principle, such "genetical modification" in accordance with the present disclosure may comprise any genetic modification that appropriately alters production of a gene product in a host organism as compared with an otherwise identical organism which is not subject to the modification. In certain embodiments, as described herein, the genetic modification comprises introducing genes into a host cell. Genetic modifications which boost the activity of a polypeptide include, but are not limited to: introducing one or more copies of a gene encoding the polypeptide (which may distinguish from any gene already present in the host cell encoding a polypeptide having the same activity); altering a gene present in the cell to increase transcription or translation of the gene (e.g., altering, adding additional sequence to, replacement of one or more nucleotides, deleting sequence from, or swapping for example, regulatory, a promoter or other sequence, in particular replacing the native or wild-type promoter of the gene with another promoter having higher transcriptional activity in the cell as compared to the native or wild-type promoter); and altering the sequence (e.g., noncoding or coding) of a gene encoding the polypeptide to boost activity (e.g., by increasing enzyme activity, decrease feedback inhibition, targeting a specific subcellular location, boost mRNA stability, boost protein stability). Genetic modifications that reduce activity of a polypeptide include, but are not limited to: deleting a portion or all of a gene encoding the polypeptide; inserting a nucleic acid sequence which disrupts a gene encoding the polypeptide; changing a gene present in the cell to reduce transcription or translation of the gene or stability of the mRNA or polypeptide encoded by the gene (for example, by adding additional sequence to, altering, deleting sequence from, replacement of one or more nucleotides, or swapping for example, replacement of one or more nucleotides, a promoter, regulatory or other sequence).

[0041] An aspect of the invention relates to a recombinant yeast strain for production of a benzylisoquinoline alkaloid, preferably scoulerine. The recombinant yeast strain comprises a heterologous berberine bridge enzyme (BBE) and a heterologousbenzophenanthridine oxidase (DBOX).

[0042] Various embodiments of the recombinant yeast strain, also referred to as recombinant strain A herein, are disclosed here below. The present invention firstly sets forth recombinant strain A used for the production of a benzophenanthridine alkaloid, the strain being obtained by engineering a yeast used for the production of scoulerine, recombinant strain A comprising or expressing in vivo berberine bridge enzyme (BBE) and benzophenanthridine oxidase (DBOX) having enhanced activity compared with a parental microorganism (i.e., a starting strain).

[0043] In the foregoing recombinant strain A, the recombinant strain A comprising or expressing in vivo berberine bridge enzyme (BBE) having enhanced activity compared with a parental microorganism may be recombinant strain A comprising or expressing in vivo a fusion protein of BBE with endoplasmic reticulum C-terminal retention signal peptide HDEL, a fusion protein of BBE with an N-terminal Golgi target sequence, or BBE. Preferred is the fusion protein of BBE with endoplasmic reticulum C-terminal retention signal HDEL.

[0044] In the foregoing recombinant strain A, the recombinant strain A comprising or expressing in vivo benzophenanthridine oxidase (DBOX) may be recombinant strain A comprising or expressing in vivo an N-terminal truncation of benzophenanthridine oxidase (DBOX).

[0045] In the foregoing recombinant strain A, the BBE may be PsBBE (GenBank, accession no. AAC61839, version AAC61839.1) derived from Papaver somniferum, CjBBE (GenBank, accession no. BAM44344, version BAM44344.1) derived from Coptis japonica, or CyBBE (amino acid sequence as shown in SEQ ID NO: 93) derived from Corydalis yanhusuo, and is preferably CyBBE.

[0046] It was found in the Example of the present invention that CyBBE was transported from the Golgi to the vacuole, and GOTS CyBBE (amino acid sequence as shown in SEQ ID NO: 94) was retained in the Golgi, whereas CyBBE ERTS (amino acid sequence as shown in SEQ ID NO: 95) was transported backward from the Golgi into the endoplasmic reticulum, thereby increasing the yield of scoulerine by 3 times.

[0047] In the foregoing recombinant strain A, the comprising or expressing in vivo the fusion protein of BBE with endoplasmic reticulum C-terminal retention signal HDEL may be by introducing a nucleic acid encoding the fusion protein into the starting strain.

[0048] In the foregoing recombinant strain A, the comprising or expressing in vivo the fusion protein of BBE with the N-terminal Golgi target sequence may be by introducing a nucleic acid encoding the fusion protein into the starting strain. In the foregoing recombinant strain A, the comprising or expressing in vivo BBE may be by introducing a nucleic acid encoding BBE into the starting strain.

[0049] In the foregoing recombinant strain A, the recombinant strain A comprising or expressing in vivo benzophenanthridine oxidase (DBOX) may be by introducing a nucleic acid encoding benzophenanthridine oxidase (DBOX) into the starting strain or any foregoing recombinant strain A.

[0050] In the foregoing recombinant strain A, the recombinant strain A comprising or expressing in vivo an N-terminal truncation of benzophenanthridine oxidase (DBOX) may be by introducing a nucleic acid encoding the N-terminal truncation of benzophenanthridine oxidase (DBOX) into the starting strain or any foregoing recombinant strain A.

[0051] In the foregoing recombinant strain A, the introducing a nucleic acid encoding benzophenanthridine oxidase (DBOX) into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding benzophenanthridine oxidase (DBOX) into the starting strain or any foregoing recombinant strain A.

[0052] In the foregoing recombinant strain A, the introducing a nucleic acid encoding the N- terminal truncation of benzophenanthridine oxidase (DBOX) into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding the N-terminal truncation of benzophenanthridine oxidase DBOX into the starting strain or any foregoing recombinant strain A.

[0053] The expression cassette comprising the nucleic acid encoding benzophenanthridine oxidase (DBOX) may comprise a promoter, the nucleic acid encoding benzophenanthridine oxidase (DBOX), and a terminator. The expression cassette comprising the nucleic acid encoding the N-terminal truncation of benzophenanthridine oxidase (DBOX) may comprise a promoter, the nucleic acid encoding the N-terminal truncation of benzophenanthridine oxidase (DBOX), and a terminator. The promoter may be the promoter PDClp or the promoter CCW12p. The terminator may be DITlt or TDH2t.

[0054] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome. In particular, recombinant strain A may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding benzophenanthridine oxidase (DBOX) into the XII- 1 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0055] In particular, recombinant strain A may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding the N-terminal truncation of benzophenanthridine oxidase (DBOX) into the FgF20 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0056] In the foregoing recombinant strain A, the benzophenanthridine oxidase (DBOX) may be McDB0X2 (GenBank, accession no. OVA00268, version OVA00268.1) derived from Macleaya cordata.

[0057] In the foregoing recombinant strain A, the N-terminal truncation of benzophenanthridine oxidase (DBOX) may be truncation of 29 amino acids from the N-terminus of McDB0X2 (i.e., McDBOX2_A29).

[0058] In the foregoing recombinant strain A, the expression cassette comprising the nucleic acid encoding benzophenanthridine oxidase (DBOX) may be the gene expression cassette XII- 1 us- DITlt-His-McDBOX2-PDClp-CCW12p. The expression cassette XII-1 us-DITlt-His- McDBOX2-PDClp-CCW12p may be obtained by ligating an upstream homology arm XII-1 us, the terminator DITlt, a His-tag sequence (CATCATCACCATCACCAT, SEQ ID NO: 214), the nucleic acid sequence of McDB0X2, the promoter PDClp, and the promoter CCW12p by using an overlap method.

[0059] In the foregoing recombinant strain A, the expression cassette comprising the nucleic acid encoding the N-terminal truncation of benzophenanthridine oxidase (DBOX) may be an McDBOX2_A29 expression cassette consisting of an upstream homology arm FgF20 us, the promoter PDClp, the nucleic acid sequence of McDBOX2_A29, the terminator DITlt, and a downstream homology arm FgF20 ds.

[0060] Any foregoing recombinant strain A may be any yeast. Preferably, the yeast to be modified can be selected from any known genus and species of yeast. In one embodiment, the yeast genus can be Saccharomyces, Kluyveromyces, Zygosaccharomyces, Candida, Hansenula, Torulopsis, Kloeckera, Pichia, Schizosaccharomyces, Trigonopsis, Brettanomyces, Debaromyces, Nadsonia, Upomyces, Cryptococcus, Aureobasidium, Trichosporon, Upomyces, Rhodotorula, Yarrowia, Phaffia, or Schwanniomyces, among others. In a further embodiment, the yeast can be Saccharomyces, Yarrowia, Zygosaccharomyces, Kluyveromyces or Pichia spp. In yet a further embodiment, the yeast can be Saccharomyces cerevisiae, Saccharomyces boulardii, Zygosaccharomyces bailii, Kluyveromyces lactis, and Yarrowia lipolytica.

[0061] Any foregoing recombinant strain A may be Saccharomyces cerevisiae.

[0062] Any foregoing starting strain may be a species of the Saccharomyces genus.

[0063] Any foregoing starting strain may be Saccharomyces cerevisiae.

[0064] The Saccharomyces cerevisiae may in particular be the IMX581 yeast strain.

[0065] A use of any foregoing recombinant strain A in the production of benzophenanthridine alkaloids is also set forth in the present invention.

[0066] The present invention further sets forth a method for producing a benzophenanthridine alkaloid, which may comprise the following steps: fermentatively culturing any foregoing recombinant strain A, collecting a fermentation product, and obtaining the benzophenanthridine alkaloid therefrom.

[0067] Any foregoing benzophenanthridine alkaloid may be sanguinarine or chelerythrine.

[0068] In the foregoing recombinant strain A, recombinant strain A comprises or expresses in vivo S-cheilanthifoline synthase (CFS) and S-stylopine synthase (SPS).

[0069] In the foregoing recombinant strain A, the comprising or expressing in vivo S- cheilanthifoline synthase (CFS) and S-stylopine synthase (SPS) may be by introducing a nucleic acid encoding S-cheilanthifoline synthase (CFS) and a nucleic acid encoding S-stylopine synthase (SPS) into the starting strain or any foregoing recombinant strain A.

[0070] In the foregoing recombinant strain A, the comprising or expressing in vivo S- cheilanthifoline synthase (CFS) and S-stylopine synthase (SPS) may be by introducing an expression cassette comprising the nucleic acid encoding S-cheilanthifoline synthase (CFS) and an expression cassette comprising the nucleic acid encoding S-stylopine synthase (SPS) into the starting strain or any foregoing recombinant strain A.

[0071] The expression cassette comprising the nucleic acid encoding S-cheilanthifoline synthase (CFS) may comprise a promoter, the nucleic acid encoding S-cheilanthifoline synthase (CFS), and a terminator. The expression cassette comprising the nucleic acid encoding S-stylopine synthase (SPS) may comprise a promoter, the nucleic acid encoding S-stylopine synthase (SPS), and a terminator. The promoter may be the promoter PDClp, the promoter GPMlp, the promoter TPIlp, or the promoter PGKlp. The terminator may be the terminator ENO2t, the terminator ADHlt, or the terminator FBAlt.

[0072] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0073] In particular, recombinant strain A may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding S-cheilanthifoline synthase (CFS) and the expression cassette comprising the nucleic acid encoding S-stylopine synthase (SPS) into the XI- 2 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0074] In the foregoing recombinant strain A, the S-cheilanthifoline synthase (CFS) may be EcCFS derived from Eschscholzia califomica, CyCFS (amino acid sequence as shown in SEQ ID NO: 100) derived from C. yanhusuo, or PsTNMT derived from P. somniferum, preferably EcCFS.

[0075] In the foregoing recombinant strain A, the S-stylopine synthase (SPS) may be EcSPS derived from E. califomica, CySPS (amino acid sequence as shown in SEQ ID NO: 101) derived from C. yanhusuo, or PsMSH derived from P. somniferunr, preferably EcSPS.

[0076] In the foregoing recombinant strain A, recombinant strain A comprises or expresses in vivo the N-methyltransferase TNMT.

[0077] In the foregoing recombinant strain A, the comprising or expressing in vivo the N- methyltransferase TNMT may be by introducing a nucleic acid encoding the N-methyltransferase TNMT into the starting strain or any foregoing recombinant strain A.

[0078] In the foregoing recombinant strain A, the comprising or expressing in vivo the N- methyltransferase TNMT may be by introducing an expression cassette comprising the nucleic acid encoding the N-methyltransferase TNMT into the starting strain or any foregoing recombinant strain A.

[0079] The expression cassette comprising the nucleic acid encoding the N-methyltransferase TNMT may comprise a promoter, the nucleic acid encoding the N-methyltransferase TNMT, and a terminator. The promoter may be the promoter PDClp, the promoter GPMlp, the promoter TPIlp, or the promoter PGKlp. The terminator may be the terminator ENO2t, the terminator ADHlt, or the terminator FBAlt. In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0080] In particular, recombinant strain A may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding the N-methyltransferase TNMT into the A7-2 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0081] In the foregoing recombinant strain A, the N-methyltransferase TNMT may be PsTNMT derived from P. somniferum.

[0082] In the foregoing recombinant strain A, recombinant strain A comprises or expresses in vivo A-c / .s-A-methylstylopine 14-hydroxylase (MSH).

[0083] In the foregoing recombinant strain A, the comprising or expressing in vivo S-cis-N- methylstylopine 14-hydroxylase (MSH) may be by introducing a nucleic acid encoding S-cis-N- methylstylopine 14-hydroxylase (MSH) into the starting strain or any foregoing recombinant strain A.

[0084] In the foregoing recombinant strain A, the comprising or expressing in vivo S-cis-N- methylstylopine 14-hydroxylase (MSH) may be by introducing an expression cassette comprising the nucleic acid encoding A-c / .s-A-methylstylopine 14-hydroxylase (MSH) into the starting strain or any foregoing recombinant strain A. The expression cassette comprising the nucleic acid encoding A-c / .s-A-methylstylopine 14-hydroxylase (MSH) may comprise a promoter, the nucleic acid encoding A-c / .s-A-methylstylopine 14-hydroxylase (MSH), and a terminator. The promoter may be the promoter PDClp, the promoter GPMlp, the promoter TPIlp, or the promoter PGKlp. The terminator may be the terminator ENO2t, the terminator ADHlt, or the terminator FBAlt.

[0085] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0086] In particular, recombinant strain A may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding A-c / .s-A-methylstylopine 14-hydroxylase (MSH) into the XI-2 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0087] In the foregoing recombinant strain A, the A-c / .s-A-methy Istylopine 14-hydroxylase (MSH) may be PsMSH derived from P. somniferum. In the foregoing recombinant strain A, recombinant strain A comprises or expresses in vivo protopine-6-hydroxylase (P6H).

[0088] In the foregoing recombinant strain A, the recombinant strain A comprising or expressing in vivo protopine-6-hydroxylase (P6H) may be by introducing a nucleic acid encoding protopine- 6-hydroxylase (P6H) into the starting strain or any foregoing recombinant strain A.

[0089] In the foregoing recombinant strain A, the introducing a nucleic acid encoding protopine- 6-hydroxylase (P6H) into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding protopine-6-hydroxylase (P6H) into the starting strain or any foregoing recombinant strain A.

[0090] The expression cassette comprising the nucleic acid encoding protopine-6-hydroxylase (P6H) may comprise a promoter, the nucleic acid encoding protopine-6-hydroxylase (P6H), and a terminator. The promoter may be the promoter PDClp or the promoter CCW12p. The terminator may be the terminator DITlt and the terminator TDH2t.

[0091] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0092] In particular, recombinant strain A may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding protopine-6-hydroxylase (P6H) into the XII- 1 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0093] In the foregoing recombinant strain A, the protopine-6-hydroxylase (P6H) may be EcP6H (GenBank, accession no. BAK20464, version BAK20464.1) derived from A. califomica.

[0094] In the foregoing recombinant strain A, recombinant strain A comprises or expresses in vivo a plasma membrane riboflavin transporter and / or FAD synthase.

[0095] In the foregoing recombinant strain A, the comprising or expressing in vivo a plasma membrane riboflavin transporter may be by introducing a nucleic acid encoding the plasma membrane riboflavin transporter into the starting strain or any foregoing recombinant strain A.

[0096] In the foregoing recombinant strain A, the comprising or expressing in vivo a plasma membrane riboflavin transporter may be by introducing an expression cassette comprising the nucleic acid encoding the plasma membrane riboflavin transporter into the starting strain or any foregoing recombinant strain A. The expression cassete comprising the nucleic acid encoding the plasma membrane riboflavin transporter may comprise a promoter, the nucleic acid encoding the plasma membrane riboflavin transporter, and a terminator. The promoter is the promoter GPD or the promoter CCW12p. The terminator is IDPlt or TDH2t.

[0097] In the foregoing recombinant strain A, the expression cassete is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0098] In particular, recombinant strain A may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding the plasma membrane riboflavin transporter into the 416d site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0099] In the foregoing recombinant strain A, the plasma membrane riboflavin transporter may be the plasma membrane riboflavin transporter MCH5 (nucleotide sequence as shown in SEQ ID NO: 78) derived from Saccharomyces cerevisiae.

[0100] In the foregoing recombinant strain A, the comprising or expressing in vivo FAD synthase may be by introducing a nucleic acid encoding FAD synthase into the starting strain or any foregoing recombinant strain A.

[0101] In the foregoing recombinant strain A, the comprising or expressing in vivo FAD synthase may be by introducing an expression cassette comprising the nucleic acid encoding FAD synthase into the starting strain or any foregoing recombinant strain A.

[0102] The expression cassete comprising the nucleic acid encoding FAD synthase may comprise a promoter, the nucleic acid encoding FAD synthase, and a terminator. The promoter is the promoter GPD or the promoter CCW12p. The terminator is IDPlt or TDH2t.

[0103] In the foregoing recombinant strain A, the expression cassete is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0104] In particular, recombinant strain A may be a strain obtained by integrating the expression cassete comprising the nucleic acid encoding FAD synthase into the 416d site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0105] In the foregoing recombinant strain A, the FAD synthase may be the FAD synthase BsRibc (nucleotide sequence as shown in SEQ ID NO: 79) derived from Bacillus subtilis. In the foregoing recombinant strain A, the may comprise or express in vivo tyrosine hydroxylase and DOPA decarboxylase.

[0106] In the foregoing recombinant strain A, the comprising or expressing in vivo tyrosine hydroxylase and DOPA decarboxylase may be by introducing a nucleic acid encoding tyrosine hydroxylase and a nucleic acid encoding DOPA decarboxylase into the starting strain or any foregoing recombinant strain A.

[0107] In the foregoing recombinant strain A, the introducing a nucleic acid encoding tyrosine hydroxylase into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding tyrosine hydroxylase into the starting strain or any foregoing recombinant strain A. The expression cassette comprising the nucleic acid encoding tyrosine hydroxylase may comprise a promoter, the nucleic acid encoding tyrosine hydroxylase, and a terminator.

[0108] In the foregoing recombinant strain A, the introducing a nucleic acid encoding DOPA decarboxylase into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding DOPA decarboxylase into the starting strain or any foregoing recombinant strain A. The expression cassette comprising the nucleic acid encoding DOPA decarboxylase may comprise a promoter, the nucleic acid encoding DOPA decarboxylase, and a terminator.

[0109] In the foregoing recombinant strain A, the promoter may be TDH3p, CCW12p, or TEFlp. The terminator may be IDPlt, TPS It, or ENO2t.

[0110] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0111] In particular, recombinant strain A may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding tyrosine hydroxylase and the expression cassette comprising the nucleic acid encoding DOPA decarboxylase into the XI-5 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0112] In the foregoing recombinant strain A, the tyrosine hydroxylase may be BvCYP76AD5 (GenBank, accession no. AJD87473, version AJD87473.1) derived from Beta vulgaris, BvCYP76ADl* that is a mutated version of BvCYP76AD (GenBank, accession no. AET43289, version AET43289.1) derived from Beta vulgaris with the W13L and F309L mutations, AtC3H (GenBank, accession no. NP_001030991, version NP_001030991.2) derived from Arabidopsis thaliana, ZmC3H (GenBank, accession no. ACG46606, version ACG46606.1) derived from Zea mays, PaHpaB (GenBank, accession no. WP 003104533, version WP 003104533.1) derived from Pseudomonas aeruginosa, PaHpaC (GenBank, accession no. WP 058166060, version WP 058166060.1) derived from Pseudomonas aeruginosa, or SeHpaC (GenBank, accession no. WP 001195556, version WP 001195556.1) derived from Salmonella, preferably BvCYP76AD5.

[0113] In the foregoing recombinant strain A, the DOPA decarboxylase may specifically be DODC (GenBank, accession no. DAA64376, version DAA64376.1) derived from Pseudomonas putida.

[0114] Any foregoing recombinant strain A may further comprise or express in vivo norcoclaurine synthase (NCS) or an N-terminally truncated NCS enzyme.

[0115] In the foregoing recombinant strain A, the NCS enzyme may be CjNCS (GenBank, accession no. BAF45338, version BAF45338.2) derived from Coptis japonica.

[0116] In the foregoing recombinant strain A, the N-terminally truncated NCS enzyme may specifically be CjNCSN_A35, CjNCSN_A24, or CjNCSN_A29, that is, 35, 24, or 29 amino acids, respectively, are truncated from the N-terminus of the amino acid sequence of CjNCS.

[0117] In the foregoing recombinant strain A, the further comprising or expressing in vivo norcoclaurine synthase (NCS) or an N-terminally truncated NCS enzyme may be by introducing a nucleic acid encoding the NCS enzyme or a nucleic acid encoding the N-terminally truncated NCS enzyme into the starting strain or any foregoing recombinant strain A.

[0118] In the foregoing recombinant strain A, the introducing a nucleic acid encoding the NCS enzyme or a nucleic acid encoding the N-terminally truncated NCS enzyme may be by introducing an expression cassette comprising the nucleic acid encoding the NCS enzyme or the nucleic acid encoding the N-terminally truncated NCS enzyme into the starting strain or any foregoing recombinant strain A. The expression cassette comprising the nucleic acid encoding the NCS enzyme or the nucleic acid encoding the N-terminally truncated NCS enzyme may comprise a promoter, the nucleic acid encoding the NCS enzyme or the nucleic acid encoding the N-terminally truncated NCS enzyme, and a terminator.

[0119] In the foregoing recombinant strain A, the promoter may be TEFlp. The terminator may be TDH2t. In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0120] In the foregoing recombinant strain A, the further comprising or expressing in vivo the NCS enzyme or an N-terminally truncated NCS enzyme may be by increasing the copy number of the nucleic acid encoding the NCS enzyme or the N-terminally truncated NCS enzyme in the starting strain or any foregoing recombinant strain A.

[0121] In the foregoing recombinant strain A, the copy number of the nucleic acid encoding the N-terminally truncated NCS enzyme may be 3, 2, or 1, preferably 3.

[0122] Any foregoing recombinant strain A may further comprise or express in vivo a tyrosine metabolic flow-related protein.

[0123] In the foregoing recombinant strain A, the tyrosine metabolic flow-related protein may be EcAROL (GenBank, accession no. NP_414922, version NP_414922.1) derived from Escherchia coli, AR04* (mutant ARO4K229Lof yeast endogenous gene AR04), AR07* (mutant ARO7G141Sof yeast endogenous gene AR07), MtPDHl (GenBank, accession no. KM507076, version KM507076.1) derived from barrel medic (Medicago truncatula), AR01 (yeast endogenous gene), AR02 (yeast endogenous gene), and / or AR03 (yeast endogenous gene).

[0124] In the foregoing recombinant strain A, the recombinant strain can be genetically modified for overexpression of the endogenous AR01, AR02 and / or AR03.

[0125] In the foregoing recombinant strain A, the further comprising or expressing in vivo a tyrosine metabolic flow-related protein may be by introducing a nucleic acid encoding the tyrosine metabolic flow-related protein into the starting strain or any foregoing recombinant strain A.

[0126] In the foregoing recombinant strain A, the introducing a nucleic acid encoding the tyrosine metabolic flow-related protein into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding the tyrosine metabolic flow-related protein into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding the tyrosine metabolic flow-related protein, and a terminator.

[0127] In the foregoing recombinant strain A, the promoter may be TPIlp, PGKlp, TEFlp, TDH3p, or tHXT7p. The terminator may be pYX212t, ADHlt, FBAlt, CYClt, or TDH2t. In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0128] Any foregoing recombinant strain A may further have proteins related to the synthesis activities of 4-hydroxyphenylethyl alcohol and 4-hydroxyphenylacetic acid knocked out or down regulated in vivo.

[0129] In the foregoing recombinant strain A, the proteins related to the synthesis activities of 4- hydroxyphenylethyl alcohol and 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, YDR541C, AAD3, GRE2, and / or HFD1.

[0130] In the foregoing recombinant strain A, the proteins related to the synthesis activities of 4- hydroxyphenylethyl alcohol and 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, GRE2, and HFDl.

[0131] Any foregoing recombinant strain A may further comprise or express in vivo an aromatic amino acid decarboxylase.

[0132] In the foregoing recombinant strain A, the aromatic amino acid decarboxylase may be PcAAS derived from Petroselinum crispum, RrAAS derived from Rhodiola Salidroside, or mutant PsAAAD* (mutant PSAAADY350F) derived from P. somniferum, preferably PsAAAD*.

[0133] In the foregoing recombinant strain A, the further comprising or expressing in vivo an aromatic amino acid decarboxylase may be by introducing a nucleic acid encoding the aromatic amino acid decarboxylase into the starting strain or any foregoing recombinant strain A.

[0134] In the foregoing recombinant strain A, the introducing a nucleic acid encoding the aromatic amino acid decarboxylase into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding the aromatic amino acid decarboxylase into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding the aromatic amino acid decarboxylase, and a terminator.

[0135] The promoter may be FBAlp. The terminator may be TPSlt-ADHlt.

[0136] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome. Any foregoing recombinant strain A may further comprise or express in vivo prephenate transaminase (PAT) and arogenate dehydratase (ADH). The PAT may be AtPAT (GenBank, accession no. NP_565529, version NP_565529.1) derived from Arabidopsis thaliana, BvPATl (GenBank, accession no. KMS97510, version KMS97510.1) derived from Beta vulgaris, or BvPAT2 (GenBank, accession no. KMT14107, version KMT14107.1) derived from B. vulgaris. The ADH may be BvADHa derived from Beta vulgaris or MtncADH derived from Medicago truncatula.

[0137] In the foregoing recombinant strain A, the further comprising or expressing in vivo PAT and ADH may be by introducing a nucleic acid encoding PAT and a nucleic acid encoding ADH into the starting strain or any foregoing recombinant strain A.

[0138] In the foregoing recombinant strain A, the introducing a nucleic acid encoding PAT into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding PAT into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding PAT, and a terminator.

[0139] In the foregoing recombinant strain A, the introducing a nucleic acid encoding ADH into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding ADH into the starting strain or any foregoing recombinant strain A. The expression cassette may include a promoter, the nucleic acid encoding ADH, and a terminator. The promoter may be TDH3p, TEF2p, or FBAlp. The terminator may be TDH2t or pYX212t.

[0140] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0141] Preferably, any foregoing recombinant strain A may further comprise or express in vivo AtPAT and MtncADH.

[0142] Any foregoing recombinant strain A may further comprise or express in vivo the methyltransferase (60MT) and coclaurine methyltransferase (CNMT).

[0143] In the foregoing recombinant strain A, the 60MT may be Ps6OMT (AAQO 1669.1) derived from 7*. somniferum or Cy60MT (amino acid sequence as shown in SEQ ID NO: 96) derived from C. yanhusuo. The CNMT may be PsCNMT (GenBank, accession no. AAP45316, version AAP45316.1) derived from P. somniferum or CyCNMT (nucleotide sequence as shown in SEQ ID NO: 18 and amino acid sequence as shown in SEQ ID NO: 97) derived from C. yanhusuo.

[0144] In the foregoing recombinant strain A, the further comprising or expressing in vivo the methyltransferase 6OMT and the methyltransferase CNMT may be by introducing a nucleic acid encoding 6OMT and a nucleic acid encoding CNMT into the starting strain or any foregoing recombinant strain A.

[0145] In the foregoing recombinant strain A, the introducing a nucleic acid encoding 60MT into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding 60MT into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding 60MT, and a terminator.

[0146] In the foregoing recombinant strain A, the introducing a nucleic acid encoding CNMT into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding CNMT into the starting strain or any foregoing recombinant strain A. The expression cassette may include a promoter, the nucleic acid encoding CNMT, and a terminator.

[0147] Any foregoing recombinant strain A may further comprise or express in vivo NMCH. The NMCH may be EcNMCH (GenBank, accession no. AAC39452, version AAC39452.1) derived from Eschscholzia califomica or CyNMCH (amino acid sequence as shown in SEQ ID NO: 98) derived from Corydalis yanhusuo.

[0148] In the foregoing recombinant strain A, the further comprising or expressing in vivo NMCH may be by introducing a nucleic acid encoding NMCH into the starting strain or any foregoing recombinant strain A. The introducing a nucleic acid encoding NMCH into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding NMCH into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding NMCH, and a terminator.

[0149] The promoter may be TPIlp, PGKlp, TEFlp, tHXT7p, TDH3p, GPMlp, or TEF2p.

[0150] The terminator may be pYX212t, ADHlt, CYClt, FBAlt, TDH2t, PGIlt, or ENO2t.

[0151] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome. Preferably, any foregoing recombinant strain A may further comprise or express in vivo Ps60MT, PsCNMT, and CyNMCH.

[0152] In the foregoing recombinant strain A, the further comprising or expressing in vivo NMCH may be by increasing the copy number of the nucleic acid encoding NMCH in the starting strain or any foregoing recombinant strain A.

[0153] In the foregoing recombinant strain A, the copy number of the nucleic acid encoding NMCH may be 1 to 5, preferably 5.

[0154] Any foregoing recombinant strain A may further comprise or express in vivo the methyltransferase 4’0MT.

[0155] In the foregoing recombinant strain A, the 4’ OMT may be Ps4’ OMT derived from Papaver somniferum.

[0156] In the foregoing recombinant strain A, the further comprising or expressing in vivo 4’ OMT may be by introducing a nucleic acid encoding 4’ OMT into the starting strain or any foregoing recombinant strain A.

[0157] In the foregoing recombinant strain A, the introducing a nucleic acid encoding 4’ OMT into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding 4’ OMT into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding 4’ OMT, and a terminator.

[0158] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0159] In the foregoing recombinant strain A, the further comprising or expressing in vivo 4’ OMT may be by increasing the copy number of the nucleic acid encoding 4 ’OMT in the starting strain or any foregoing recombinant strain A.

[0160] In the foregoing recombinant strain A, the copy number of the nucleic acid encoding 4’ OMT may be 2 or 1, preferably 2.

[0161] Any foregoing recombinant strain A may further comprise or express in vivo a cytochrome P450 reductase, such as ATR1 (nucleotide sequence as shown in SEQ ID NO: 55) derived from Arabidopsis thaliana, ATR2 (nucleotide sequence as shown in SEQ ID NO: 56) derived from Arabidopsis thaliana, or PsCPR derived from Papaver somniferum, preferably ATR1. In the foregoing recombinant strain A, the further comprising or expressing in vivo ATR1 may be by introducing ATR1 into the starting strain or any foregoing recombinant strain A.

[0162] In the foregoing recombinant strain A, the further comprising or expressing in vivo ATR2 may be by introducing ATR2 into the starting strain or any foregoing recombinant strain A.

[0163] In the foregoing recombinant strain A, the further comprising or expressing in vivo CPR may be by introducing a nucleic acid encoding CPR into the starting strain or any foregoing recombinant strain A. The introducing a nucleic acid encoding CPR into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding CPR into the starting strain or any foregoing recombinant strain A. The introducing ATR1 into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising ATR1 into the starting strain or any foregoing recombinant strain A. The introducing ATR2 into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising ATR2 into the starting strain or any foregoing recombinant strain A. In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0164] In the foregoing recombinant strain A, the introducing an encoding nucleic acid into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the encoding nucleic acid into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the encoding nucleic acid, and a terminator.

[0165] In the foregoing recombinant strain A, the promoter may be CCW12p. The terminator may be PGIlt.

[0166] In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0167] Any foregoing recombinant strain A may further comprise or express in vivo catalase.

[0168] In the foregoing recombinant strain A, the catalase may be PRDX4.

[0169] In the foregoing recombinant strain A, the comprising or expressing in vivo catalase may be by introducing a nucleic acid encoding catalase into the starting strain or any foregoing recombinant strain A. 1 In the foregoing recombinant strain A, the introducing a nucleic acid encoding catalase into the starting strain may be by introducing, into the starting strain or any foregoing recombinant strain A, an expression cassette comprising the nucleic acid encoding catalase peroxiredoxin IV (a-mPRDX4_ERTS) (amino acid sequence as shown in SEQ ID NO: 99) which is N-terminally fused with yeast a-mating factor signal peptide and C-terminally fused with ERTS signal peptide.

[0170] Any foregoing recombinant strain A may further comprise or express in vivo a transcription factor activating phospholipid biosynthesis or have a transcription factor that suppresses phospholipid biosynthesis knocked out in vivo.

[0171] In the foregoing recombinant strain A, the knocked-out transcription factor that suppresses phospholipid biosynthesis may be the OPI1 gene.

[0172] In the foregoing recombinant strain A, the transcription factor activating phospholipid biosynthesis may be the INO2 gene or the INO2 gene mutant INO2* (INO2L119A).

[0173] A use of any foregoing recombinant strain A in the production of sanguinarine, dihydrosanguinarine, protopine, A-c / .s-A-methylstylopine, and / or S-stylopine is also set forth in the present invention.

[0174] The present invention further sets forth a method for producing sanguinarine, dihydrosanguinarine, protopine, A-c / .s-A-methylstylopine, and / or S-stylopine, which comprises the following steps: fermentatively culturing any foregoing recombinant strain A, collecting a fermentation product, and obtaining sanguinarine, dihydrosanguinarine, protopine, S-cis-N- methylstylopine and / or S-stylopine therefrom.

[0175] Another aspect of the invention relates to a recombinant yeast strain for the production of dihydrosanguinarine. The recombinant yeast strain comprises the dihydrosanguinarine 10- hydroxylase CYP82P2 derived from Eschscholzia califomica.

[0176] Various embodiments of the recombinant yeast strain, also referred to as recombinant strain B herein, are disclosed here below.

[0177] The present invention further sets forth recombinant strain B, which is obtained by engineering a yeast used for the production of dihydrosanguinarine. Recombinant strain B comprises or expresses in vivo CYP82P2 (GenBank, accession no. BBD34756, version BBD34756.1) derived from Eschscholzia califomica. In the foregoing recombinant strain B, the comprising or expressing in vivo CYP82P2 may be by introducing the CYP82P2 gene (GenBank, accession no. BBD34756, version: BBD34756.1) into the yeast used for the production of dihydrosanguinarine.

[0178] In the foregoing recombinant strain B, the introducing the CYP82P2 gene (GenBank, accession no. BBD34756, version BBD34756.1) into the yeast used for the production of dihydrosanguinarine may be by introducing an expression cassette comprising the CYP82P2 gene into the yeast used for the production of dihydrosanguinarine. The expression cassette may comprise a promoter, the CYP82P2 gene, and a terminator. The promoter may be the promoter GPD. The terminator may be the terminator IDPlt.

[0179] Any foregoing recombinant strain B further comprises or expresses in vivo 10- hydroxymethyltransferase OMT.

[0180] In the foregoing recombinant strain B, the comprising or expressing in vivo 10- hydroxymethyltransferase OMT may be by introducing a gene encoding 10- hydroxymethyltransferase OMT into the yeast used for the production of dihydrosanguinarine.

[0181] In the foregoing recombinant strain B, the introducing a gene encoding 10- hydroxymethyltransferase OMT into the yeast used for the production of dihydrosanguinarine may be by introducing an expression cassette comprising the gene encoding 10- hydroxymethyltransferase OMT into the yeast used for the production of dihydrosanguinarine. The expression cassette may comprise a promoter, the gene encoding 10- hydroxymethyltransferase OMT, and a terminator. The promoter may be the promoter CCW12p. The terminator may be the terminator CYC It.

[0182] The 10-hydroxymethyltransferase OMT may be Ecl 1 OMT (GenBank, accession no. BB A20642, version BBA20642.1 ) derived from Eschscholzia californica or Ec2OMT (GenBank, accession no. BBA20644, version BBA20644.1) derived from E. californica, preferably Ecl 1 OMT.

[0183] The expression cassette comprising the gene encoding 10-hydroxymethyltransferase OMT may be an Ecl lOMT expression cassette (comprising the promoter CCW12p, Ecl lOMT (GenBank, accession no. BBA20642, version BBA20642.1), and the terminator CYClt) or an Ec2OMT expression cassette (comprising the promoter CCW12p, Ec2OMT (GenBank, accession no. BBA20642, version BBA20642.1), and the terminator CYClt. In the foregoing recombinant strain B, the yeast used for the production of dihydrosanguinarine may be a yeast used for the production of sanguinarine.

[0184] In the foregoing recombinant strain B, the yeast used for the production of dihydrosanguinarine may be any foregoing recombinant strain A.

[0185] A use of any foregoing recombinant strain B in the production of chelirubine and / or 10- hydroxydihydrosanguinarine is also set forth in the present invention.

[0186] The present invention further sets forth a method for producing chelirubine and / or 10- hydroxydihydrosanguinarine, which comprises the following steps: fermentatively culturing any foregoing recombinant strain B, collecting a fermentation product, and obtaining the chelirubine and / or 10-hydroxydihydrosanguinarine therefrom.

[0187] Yet another aspect of the invention relates to a recombinant yeast strain for production of chelerythrine, allocryptopine, and / or S -tetrahydroberberine. The recombinant yeast strain comprises a heterologous berberine bridge enzyme (BBE), a heterologousbenzophenanthridine oxidase (DBOX) and a heterologous O-methyltransferase (OMT).

[0188] Various embodiments of the recombinant yeast strain, also referred to as recombinant strain C herein, are disclosed here below. The present invention further sets forth a recombinant strain, which is any foregoing recombinant strain A (referring to any foregoing recombinant strain A comprising or expressing in vivo berberine bridge enzyme (BBE) and benzophenanthridine oxidase (DBOX) having enhanced activity compared with a parental microorganism (i.e., a starting strain) comprising or expressing in vivo an O-methyltransferase. For the purpose of differentiation, this recombinant strain is designated as recombinant strain C.

[0189] In the foregoing recombinant strain C, the comprising or expressing in vivo an O- methyltransferase may be by introducing a nucleic acid encoding the O-methyltransferase into the starting strain or any foregoing recombinant strain A.

[0190] In the foregoing recombinant strain C, the comprising or expressing in vivo an O- methyltransferase may be by introducing an expression cassette comprising the nucleic acid encoding the O-methyltransferase into the starting strain or any foregoing recombinant strain A.

[0191] The expression cassette comprising the nucleic acid encoding the O-methyltransferase may comprise a promoter, the nucleic acid encoding the O-methyltransferase, and a terminator. The promoter may be the promoter PDClp, the promoter GPMlp, the promoter TPIlp, or the promoter PGKlp. The terminator may be the terminator ENO2t, the terminator IDPlt, the terminator ADHlt, or the terminator FBAlt.

[0192] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0193] In particular, recombinant strain C may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding an O-methyltransferase into the XI-2 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0194] In the foregoing recombinant strain C, the O-methyltransferase may be TfS9OMT (GenBank, accession no. AAU20770, version AAU20770.1) derived from Thalictrum flavum.

[0195] In the foregoing recombinant strain C, recombinant strain C comprises or expresses in vivo S-tetrahydroberberine synthase (CAS).

[0196] In the foregoing recombinant strain C, the comprising or expressing in vivo S- tetrahydroberberine synthase (CAS) may be by introducing a nucleic acid encoding S- tetrahydroberberine synthase (CAS) into the starting strain or any foregoing recombinant strain A.

[0197] In the foregoing recombinant strain C, the comprising or expressing in vivo S- tetrahydroberberine synthase (CAS) may be by introducing an expression cassette comprising the nucleic acid encoding S-tetrahydroberberine synthase (CAS) into the starting strain or any foregoing recombinant strain A.

[0198] The expression cassette comprising the nucleic acid encoding S-tetrahydroberberine synthase (CAS) may comprise a promoter, the nucleic acid encoding S-tetrahydroberberine synthase (CAS), and a terminator. The promoter may be the promoter PDClp, the promoter GPMlp, the promoter TPIlp, or the promoter PGKlp. The terminator may be the terminator ENO2t, the terminator IDPlt, the terminator ADHlt, or the terminator FBAlt.

[0199] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0200] In particular, recombinant strain C may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding S-tetrahydroberberine synthase (CAS) into the XI- 2 site of the starting strain or any foregoing recombinant strain A by using a genome editing method. In the foregoing recombinant strain C, the S -tetrahydroberberine synthase (CAS) may be CjCAS (GenBank, accession no. BAB68769, version BAB68769.1) derived from Coptis japonica or CyCAS (amino acid sequence as shown in SEQ ID NO: 102) derived from Corydalis yanhusuo.

[0201] In the foregoing recombinant strain C, recombinant strain C comprises or expresses in vivo the N-methyltransferase TNMT.

[0202] In the foregoing recombinant strain C, the comprising or expressing in vivo the N- methyltransferase TNMT may be by introducing a nucleic acid encoding the N-methyltransferase TNMT into the starting strain or any foregoing recombinant strain A.

[0203] In the foregoing recombinant strain C, the comprising or expressing in vivo the N- methyltransferase TNMT may be by introducing an expression cassette comprising the nucleic acid encoding the N-methyltransferase TNMT into the starting strain or any foregoing recombinant strain A.

[0204] The expression cassette comprising the nucleic acid encoding the N-methyltransferase TNMT may comprise a promoter, the nucleic acid encoding the N-methyltransferase TNMT, and a terminator. The promoter may be the promoter PDClp, the promoter GPMlp, the promoter TPIlp, or the promoter PGKlp. The terminator may be the terminator ENO2t, the terminator ADHlt, or the terminator FBAlt.

[0205] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0206] In particular, recombinant strain C may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding the N-methyltransferase TNMT into the A7-2 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0207] In the foregoing recombinant strain C, the N-methyltransferase TNMT may be PsTNMT derived from P. somniferum.

[0208] In the foregoing recombinant strain C, recombinant strain A comprises or expresses in vivo A-c / .s-A-methylstylopine 14-hydroxylase (MSH).

[0209] In the foregoing recombinant strain C, the comprising or expressing in vivo S-cis-N- methylstylopine 14-hydroxylase (MSH) may be by introducing a nucleic acid encoding S-cis-N- methylstylopine 14-hydroxylase (MSH) into the starting strain or any foregoing recombinant strain A. In the foregoing recombinant strain C, the comprising or expressing in vivo S-cis-N- methylstylopine 14-hydroxylase (MSH) may be by introducing an expression cassette comprising the nucleic acid encoding A-cv.s-A-methylstylopine 14-hydroxylase (MSH) into the starting strain or any foregoing recombinant strain A. The expression cassette comprising the nucleic acid encoding A-c / .s-A-methylstylopine 14-hydroxylase (MSH) may comprise a promoter, the nucleic acid encoding A-c / .s-A-methylstylopine 14-hydroxylase (MSH), and a terminator. The promoter may be the promoter PDClp, the promoter GPMlp, the promoter TPIlp, or the promoter PGKlp. The terminator may be the terminator ENO2t, the terminator ADHlt, or the terminator FBAlt.

[0210] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0211] Specifically, recombinant strain C may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding A-c / .s-A-methylstylopine 14-hydroxylase (MSH) into the XI-2 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0212] In the foregoing recombinant strain C, the A-c / .s-A-methy Istylopine 14-hydroxylase (MSH) may be PsMSH derived from P. somniferum.

[0213] In the foregoing recombinant strain C, recombinant strain A comprises or expresses in vivo protopine-6-hydroxylase (P6H).

[0214] In the foregoing recombinant strain C, the recombinant strain A comprising or expressing in vivo protopine-6-hydroxylase (P6H) may be by introducing a nucleic acid encoding protopine- 6-hydroxylase (P6H) into the starting strain or any foregoing recombinant strain A.

[0215] In the foregoing recombinant strain C, the introducing a nucleic acid encoding protopine- 6-hydroxylase (P6H) into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding protopine-6-hydroxylase (P6H) into the starting strain or any foregoing recombinant strain A.

[0216] The expression cassette comprising the nucleic acid encoding protopine-6-hydroxylase (P6H) may comprise a promoter, the nucleic acid encoding protopine-6-hydroxylase (P6H), and a terminator. The promoter may be the promoter PDClp or the promoter CCW12p. The terminator may be the terminator DITlt and the terminator TDH2t. In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0217] In particular, recombinant strain C may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding protopine-6-hydroxylase (P6H) into the XII- 1 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0218] In the foregoing recombinant strain C, the protopine-6-hydroxylase (P6H) may be EcP6H (GenBank, accession no. BAK20464, version BAK20464.1) derived from A. califomica.

[0219] In the foregoing recombinant strain C, recombinant strain C comprises or expresses in vivo a plasma membrane riboflavin transporter and / or FAD synthase.

[0220] In the foregoing recombinant strain C, the comprising or expressing in vivo a plasma membrane riboflavin transporter may be by introducing a nucleic acid encoding the plasma membrane riboflavin transporter into the starting strain or any foregoing recombinant strain A.

[0221] In the foregoing recombinant strain C, the comprising or expressing in vivo a plasma membrane riboflavin transporter may be by introducing an expression cassette comprising the nucleic acid encoding the plasma membrane riboflavin transporter into the starting strain or any foregoing recombinant strain A.

[0222] The expression cassette comprising the nucleic acid encoding the plasma membrane riboflavin transporter may comprise a promoter, the nucleic acid encoding the plasma membrane riboflavin transporter, and a terminator. The promoter is the promoter GPD or the promoter CCW12p. The terminator is IDPlt or TDH2t.

[0223] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0224] In particular, recombinant strain C may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding the plasma membrane riboflavin transporter into the 416d site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0225] In the foregoing recombinant strain C, the plasma membrane riboflavin transporter may be the plasma membrane riboflavin transporter MCH5. In the foregoing recombinant strain C, the comprising or expressing in vivo FAD synthase may be by introducing a nucleic acid encoding FAD synthase into the starting strain or any foregoing recombinant strain A.

[0226] In the foregoing recombinant strain C, the comprising or expressing in vivo FAD synthase may be by introducing an expression cassette comprising the nucleic acid encoding FAD synthase into the starting strain or any foregoing recombinant strain A.

[0227] The expression cassette comprising the nucleic acid encoding FAD synthase may comprise a promoter, the nucleic acid encoding FAD synthase, and a terminator. The promoter is the promoter GPD or promoter the CCW12p. The terminator is IDPlt or TDH2t.

[0228] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0229] In particular, recombinant strain C may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding FAD synthase into the 416d site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0230] In the foregoing recombinant strain C, the FAD synthase may be the FAD synthase BsRibc (GenBank, accession no. WP_003245551, version WP_003245551.1) derived from Bacillus subtilis.

[0231] In the foregoing recombinant strain C, recombinant strain C may comprise or express tyrosine hydroxylase and DOPA decarboxylase.

[0232] In the foregoing recombinant strain C, the comprising or expressing in vivo tyrosine hydroxylase and DOPA decarboxylase may be by introducing a nucleic acid encoding tyrosine hydroxylase and a nucleic acid encoding DOPA decarboxylase into the starting strain or any foregoing recombinant strain A.

[0233] In the foregoing recombinant strain C, the introducing a nucleic acid encoding tyrosine hydroxylase into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding tyrosine hydroxylase into the starting strain or any foregoing recombinant strain A. The expression cassette comprising the nucleic acid encoding tyrosine hydroxylase may comprise a promoter, the nucleic acid encoding tyrosine hydroxylase, and a terminator. In the foregoing recombinant strain C, the introducing a nucleic acid encoding DOPA decarboxylase into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding DOPA decarboxylase into the starting strain or any foregoing recombinant strain A. The expression cassette comprising the nucleic acid encoding DOPA decarboxylase may comprise a promoter, the nucleic acid encoding DOPA decarboxylase, and a terminator.

[0234] In the foregoing recombinant strain C, the promoter may be TDH3p, CCW12p, or TEFlp. The terminator may be IDPlt, TPS It, or ENO2t.

[0235] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0236] In particular, recombinant strain C may be a strain obtained by integrating the expression cassette comprising the nucleic acid encoding tyrosine hydroxylase and the expression cassette comprising the nucleic acid encoding DOPA decarboxylase into the XI-5 site of the starting strain or any foregoing recombinant strain A by using a genome editing method.

[0237] In the foregoing recombinant strain C, the tyrosine hydroxylase may be BvCYP76AD5 (GenBank, accession no. AJD87473, version AJD87473.1) derived from Beta vulgaris, BvCYP76ADl* that is a mutated version of BvCYP76ADl (GenBank, accession no. AET43289, version AET43289.1) derived from Beta vulgaris with the W13L and F309L mutations, AtC3H (GenBank, accession no. NP_001030991, version NP_001030991.2) derived from Arabidopsis thaliana, ZmC3H (GenBank, accession no. ACG46606, version ACG46606.1) derived from Zea mays, PaHpaB (GenBank, accession no. WP 003104533, version WP 003104533.1) derived from Pseudomonas aeruginosa, PaHpaC (GenBank, accession no. WP 058166060, version WP 058166060.1) derived from Pseudomonas aeruginosa, or SeHpaC (GenBank, accession no. WP 001195556, version WP 001195556.1) derived from Salmonella, preferably BvCYP76AD5.

[0238] In the foregoing recombinant strain C, the DOPA decarboxylase may specifically be DODC (GenBank, accession no. DAA64376, version DAA64376.1) derived from Pseudomonas putida.

[0239] Any foregoing recombinant strain C may further comprise or express in vivo norcoclaurine synthase (NCS) or an N-terminally truncated NCS enzyme. In the foregoing recombinant strain C, the NCS enzyme may be CjNCS (GenBank, accession no. BAF45338, version BAF45338.2) derived from Coptis japonica.

[0240] In the foregoing recombinant strain C, the N-terminally truncated NCS enzyme may specifically be CjNCSN_A35, CjNCSN_A24, or CjNCSN_A29, that is, 35, 24, or 29 amino acids are truncated, respectively, from the N-terminus of the amino acid sequence of CjNCS.

[0241] In the foregoing recombinant strain C, the further comprising or expressing in vivo norcoclaurine synthase (NCS) or an N-terminally truncated NCS enzyme may be by introducing a nucleic acid encoding the NCS enzyme or a nucleic acid encoding the N-terminally truncated NCS enzyme into the starting strain or any foregoing recombinant strain A.

[0242] In the foregoing recombinant strain C, the introducing the nucleic acid encoding the NCS enzyme or the nucleic acid encoding the N-terminally truncated NCS enzyme may be by introducing an expression cassette comprising the nucleic acid encoding the NCS enzyme or the nucleic acid encoding the N-terminally truncated NCS enzyme into the starting strain or any foregoing recombinant strain A. The expression cassette comprising the nucleic acid encoding the NCS enzyme or the nucleic acid encoding the N-terminally truncated NCS enzyme may comprise a promoter, the nucleic acid encoding the NCS enzyme or the nucleic acid encoding the N- terminally truncated NCS enzyme, and a terminator.

[0243] In the foregoing recombinant strain C, the promoter may be TEFlp. The terminator may be TDH2t.

[0244] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0245] In the foregoing recombinant strain C, the further comprising or expressing in vivo the NCS enzyme or an N-terminally truncated NCS enzyme may be by increasing the copy number of the nucleic acid encoding the NCS enzyme or the N-terminally truncated NCS enzyme in the starting strain or any foregoing recombinant strain A.

[0246] In the foregoing recombinant strain C, the copy number of the nucleic acid encoding the N-terminally truncated NCS enzyme may be 3, 2, or 1, preferably 3.

[0247] Any foregoing recombinant strain C may further comprise or express in vivo a tyrosine metabolic flow-related protein. In the foregoing recombinant strain C, the tyrosine metabolic flow-related protein may be EcAROL (GenBank No. NP_414922.1) derived from E. coli, AR04* (mutant ARO4K229Lof yeast endogenous gene AR04), AR07* (mutant ARO7G141Sof yeast endogenous gene AR07), MtPDHl derived from barrel medic (Medicago truncatula) (GenBank, accession no. KM507076, version KM507076.1), AR01 (yeast endogenous gene), AR02 (yeast endogenous gene), and / or AR03 (yeast endogenous gene).

[0248] In the foregoing recombinant strain C, the recombinant strain can be genetically modified for overexpression of the endogenous AR01, AR02 and / or AR03.

[0249] In the foregoing recombinant strain C, the further comprising or expressing in vivo a tyrosine metabolic flow-related protein may be by introducing a nucleic acid encoding the tyrosine metabolic flow-related protein into the starting strain or any foregoing recombinant strain A.

[0250] In the foregoing recombinant strain C, the introducing the nucleic acid encoding the tyrosine metabolic flow-related protein into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding the tyrosine metabolic flow-related protein into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding the tyrosine metabolic flow-related protein, and a terminator.

[0251] In the foregoing recombinant strain C, the promoter may be TPIlp, PGKlp, TEFlp, TDH3p, or tHXT7p. The terminator may be pYX212t, ADHlt, FBAlt, CYClt, or TDH2t.

[0252] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0253] Any foregoing recombinant strain C may further have proteins related to the synthesis activities of 4-hydroxyphenylethyl alcohol and 4-hydroxyphenylacetic acid knocked out or down regulated in vivo.

[0254] In the foregoing recombinant strain C, the proteins related to the synthesis activities of 4- hydroxyphenylethyl alcohol and 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, YDR541C, AAD3, GRE2, and / or HFD1.

[0255] In the foregoing recombinant strain C, the proteins related to the synthesis activities of 4- hydroxyphenylethyl alcohol and 4-hydroxyphenylacetic acid may be ARI1, ADH6, YPR1, GRE2, and HFDl. Any foregoing recombinant strain C may further comprise or express in vivo an aromatic amino acid decarboxylase.

[0256] In the foregoing recombinant strain C, the aromatic amino acid decarboxylase may be PcAAS derived from Petroselinum crispum, RrAAS derived from Rhodiola Salidroside, or mutant PsAAAD* derived from P. somniferum, preferably PsAAAD*.

[0257] In the foregoing recombinant strain C, the further comprising or expressing in vivo an aromatic amino acid decarboxylase may be by introducing a nucleic acid encoding the aromatic amino acid decarboxylase into the starting strain or any foregoing recombinant strain A.

[0258] In the foregoing recombinant strain C, the introducing a nucleic acid encoding the aromatic amino acid decarboxylase into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding the aromatic amino acid decarboxylase into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding the aromatic amino acid decarboxylase, and a terminator.

[0259] The promoter may be FBAlp. The terminator may be TPSlt-ADHlt.

[0260] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0261] Any foregoing recombinant strain C may further comprise or express in vivo prephenate transaminase (PAT) and arogenate dehydratase (ADH). The PAT may be AtPAT (GenBank, accession no. NP_565529, version NP_565529.1) derived from Arabidopsis thaliana, BvPATl (GenBank, accession no. KMS97510, version KMS97510.1) derived from Beta vulgaris, or BvPAT2 (GenBank, accession no. KMT14107, version KMT14107.1) derived from B. vulgaris. The ADH may be BvADHa derived from B. vulgaris or MtncADH derived from Medicago truncatula.

[0262] In the foregoing recombinant strain C, the further comprising or expressing in vivo PAT and ADH may be by introducing a nucleic acid encoding PAT and a nucleic acid encoding ADH into the starting strain or any foregoing recombinant strain A.

[0263] In the foregoing recombinant strain C, the introducing a nucleic acid encoding PAT into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding PAT into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding PAT, and a terminator.

[0264] In the foregoing recombinant strain C, the introducing a nucleic acid encoding ADH into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding ADH into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding ADH, and a terminator. The promoter may be TDH3p, TEF2p, or FBAlp. The terminator may be TDH2t or pYX212t.

[0265] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0266] Preferably, any foregoing recombinant strain C may further comprise or express in vivo AtPAT and MtncADH.

[0267] Any foregoing recombinant strain C may further comprise or express in vivo the methyltransferase 60MT and coclaurine methyltransferase (CNMT).

[0268] In the foregoing recombinant strain C, the 60MT may be Ps6OMT (GenBank, accession no. AAQ01669, version AAQ01669.1) derived from P. somniferum or Cy60MT derived from C. yanhusuo. The CNMT may be PsCNMT (GenBank, accession no. AAP45316, version AAP45316.1) derived from P. somniferum or CyCNMT (nucleotide sequence as shown in SEQ ID NO: 18 and amino acid sequence as shown in SEQ ID NO: 97) derived from C. yanhusuo.

[0269] In the foregoing recombinant strain C, the further comprising or expressing in vivo the methyltransferase 60MT and the methyltransferase CNMT may be by introducing a nucleic acid encoding 60MT and a nucleic acid encoding CNMT into the starting strain or any foregoing recombinant strain A.

[0270] In the foregoing recombinant strain C, the introducing a nucleic acid encoding 60MT into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding 60MT into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding 60MT, and a terminator.

[0271] In the foregoing recombinant strain C, the introducing a nucleic acid encoding CNMT into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassete comprising the nucleic acid encoding CNMT into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding CNMT, and a terminator.

[0272] Any foregoing recombinant strain C may further comprise or express in vivo NMCH. The NMCH may be EcNMCH (GenBank, accession no. AAC39452, version AAC39452.1) derived from Eschscholzia califomica or CyNMCH (amino acid sequence as shown in SEQ ID NO: 98) derived from Corydalis yanhusuo.

[0273] In the foregoing recombinant strain C, the further comprising or expressing in vivo NMCH may be by introducing a nucleic acid encoding NMCH into the starting strain or any foregoing recombinant strain A. The introducing a nucleic acid encoding NMCH into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassete comprising the nucleic acid encoding NMCH into the starting strain or any foregoing recombinant strain A. The expression cassete may comprise a promoter, the nucleic acid encoding NMCH, and a terminator.

[0274] The promoter may be TPIlp, PGKlp, TEFlp, tHXT7p, TDH3p, GPMlp, or TEF2p.

[0275] The terminator may be pYX212t, ADHlt, CYClt, FBAlt, TDH2t, PGIlt or ENO2t.

[0276] In the foregoing recombinant strain C, the expression cassete is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0277] Preferably, any foregoing recombinant strain C may further comprise or express in vivo Ps6OMT, PsCNMT, and CyNMCH.

[0278] In the foregoing recombinant strain C, the further comprising or expressing in vivo NMCH may be by increasing the copy number of the nucleic acid encoding NMCH in the starting strain or any foregoing recombinant strain A.

[0279] In the foregoing recombinant strain C, the copy number of the nucleic acid encoding NMCH may be 1 to 5, preferably 5.

[0280] Any foregoing recombinant strain C may further comprise or express in vivo the methyltransferase 4’0MT.

[0281] In the foregoing recombinant strain C, the 4’ OMT may be Ps4’ OMT derived from Papaver somniferum. In the foregoing recombinant strain C, the further comprising or expressing in vivo 4’ OMT may be by introducing a nucleic acid encoding 4’ OMT into the starting strain or any foregoing recombinant strain A.

[0282] In the foregoing recombinant strain C, the introducing a nucleic acid encoding 4 ’OMT into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding 4’ OMT into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the nucleic acid encoding 4’ OMT, and a terminator.

[0283] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0284] In the foregoing recombinant strain C, the further comprising or expressing in vivo 4’ OMT may be by increasing the copy number of the nucleic acid encoding 4 ’OMT in the starting strain or any foregoing recombinant strain A.

[0285] In the foregoing recombinant strain C, the copy number of the nucleic acid encoding 4’ OMT may be 2 or 1, preferably 2.

[0286] Any foregoing recombinant strain C may further comprise or express in vivo the cytochrome P450 reductase ATR1, ATR2, or CPR, preferably ATR1.

[0287] In the foregoing recombinant strain C, the CPR may be PsCPR.

[0288] In the foregoing recombinant strain C, the further comprising or expressing in vivo ATR1 may be by introducing ATR1 into the starting strain or any foregoing recombinant strain A.

[0289] In the foregoing recombinant strain C, the further comprising or expressing in vivo ATR2 may be by introducing ATR2 into the starting strain or any foregoing recombinant strain A.

[0290] In the foregoing recombinant strain C, the further comprising or expressing in vivo CPR may be by introducing a nucleic acid encoding CPR into the starting strain or any foregoing recombinant strain A. The introducing a nucleic acid encoding CPR into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the nucleic acid encoding CPR into the starting strain or any foregoing recombinant strain A. The introducing ATR1 into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising ATR1 into the starting strain or any foregoing recombinant strain A. The introducing ATR2 into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising ATR2 into the starting strain or any foregoing recombinant strain A. In the foregoing recombinant strain A, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0291] In the foregoing recombinant strain C, the introducing an encoding nucleic acid into the starting strain or any foregoing recombinant strain A may be by introducing an expression cassette comprising the encoding nucleic acid into the starting strain or any foregoing recombinant strain A. The expression cassette may comprise a promoter, the encoding nucleic acid, and a terminator.

[0292] In the foregoing recombinant strain C, the promoter may be CCW12p. The terminator may be PGIlt.

[0293] In the foregoing recombinant strain C, the expression cassette is introduced into the starting strain or any foregoing recombinant strain A in the form of a plasmid and / or in the form of being integrated into a chromosome.

[0294] Any foregoing recombinant strain C may further comprise or express in vivo catalase.

[0295] In the foregoing recombinant strain C, the catalase may be PRDX4.

[0296] In the foregoing recombinant strain C, the comprising or expressing in vivo catalase may be by introducing a nucleic acid encoding catalase into the starting strain or any foregoing recombinant strain A.

[0297] In the foregoing recombinant strain C, the introducing a nucleic acid encoding catalase into the starting strain may be by introducing, into the starting strain or any foregoing recombinant strain A, an expression cassette comprising the nucleic acid encoding catalase peroxiredoxin IV (a-mPRDX4_ERTS) which is N-terminally fused with yeast a-mating factor signal peptide and C- terminally fused with ERTS signal peptide.

[0298] Any foregoing recombinant strain C may further comprise or express in vivo a transcription factor that activates phospholipid biosynthesis or have a transcription factor that suppresses phospholipid biosynthesis knocked out in vivo.

[0299] In the foregoing recombinant strain C, the knocked-out transcription factor that suppresses phospholipid biosynthesis may be the OPI1 gene.

[0300] In the foregoing recombinant strain C, the transcription factor that activates phospholipid biosynthesis may be the INO2 gene or the INO2 gene mutant INO2* (INO2L119A). The recombinant strain C may be any yeast. Preferably, recombinant strain C can be selected from any known genus and species of yeast. In one embodiment, the yeast genus can be Saccharomyces, Kluyveromyces, Zygosaccharomyces, Candida, Hansemda, Torulopsis, Kloeckera, Pichia, Schizosaccharomyces, Trigonopsis, Brettanomyces, Debaromyces, Nadsonia, Upomyces, Cryptococcus, Aureobasidium, Trichosporon, Upomyces, Rhodotorula, Yarrowia, Phaffia, or Schwanniomyces, among others. In a further embodiment, the yeast can be Saccharomyces, Yarrowia, Zygosaccharomyces, Kluyveromyces or Pichia spp. In yet a further embodiment, the yeast can be Saccharomyces cerevisiae, Saccharomyces boulardii, Zygosaccharomyces bailii, Kluyveromyces lactis, and Yarrowia lipolytica.

[0301] Any foregoing recombinant strain C may be a species of the Saccharomyces genus.

[0302] Any foregoing recombinant strain C may be Saccharomyces cerevisiae.

[0303] Any foregoing starting strain C may be a species of the Saccharomyces genus.

[0304] Any foregoing starting strain C may be Saccharomyces cerevisiae.

[0305] The Saccharomyces cerevisiae may specifically be the IMX581 yeast strain.

[0306] A use of any foregoing recombinant strain C in the production of chelerythrine, allocryptopine, and / or S-tetrahydroberberine is also set forth in the present invention.

[0307] The present invention further provides a method for producing chelerythrine, allocryptopine, and / or S-tetrahydroberberine, which comprises the following steps: fermentatively culturing any foregoing recombinant strain C, collecting a fermentation product, and obtaining the chelerythrine, allocryptopine, and / or S-tetrahydroberberine therefrom.

[0308] The present inventors, after conducting extensive experiments, constructed recombinant strains for producing benzophenanthridine alkaloids (such as sanguinarine or chelerythrine). The present inventors conducted research by dividing the synthesis pathways of sanguinarine and chelerythrine into multiple modules, and achieved de novo synthesis of sanguinarine and chelerythrine through multiple strategies. For example, in order to increase BBE activity, a novel endoplasmic reticulum engineering strategy was developed to increase the yield of the key intermediate S-Scoulerine by 3 times. McDB0X2 was identified as the only DBOX gene at present reported to act in Saccharomyces cerevisiae, and the reconstruction of the entire biosynthesis pathways of benzophenanthridine alkaloids was completed. By means of performing an N- terminal truncation operation on DBOX to increase cytoplasmic expression, performing endoplasmic reticulum expansion, and increasing the supply of the cofactor FAD, the yield of sanguinarine and chelerythrine was increased, finally obtaining a yield of sanguinarine of 3.8 mg / L and a yield of chelerythrine of 38.1 mg / L. The starting strain may be the IMX581 yeast strain. Experiments demonstrated that the engineered strains constructed in the present invention can produce sanguinarine and chelerythrine at high efficiency. The present invention has important value in applications.

[0309] The present invention will be described in further detail below with reference to a specific Example, which is provided solely for purpose of illustrating the present invention rather than limiting the scope of the present invention. The Example provided below can serve as a guide for those of ordinary skill in the art to make further improvements and is not intended to limit the present invention in any manner.

[0310] The experimental methods in the following Example are conventional methods, unless otherwise specified, and are performed according to the techniques or conditions described in documents in the art or according to product manuals. The materials, reagents, etc., used in the following Example are commercially available, unless otherwise specified.

[0311] The names of primers and the nucleotide sequences thereof involved in the following Example are shown in Table 1.

[0312] Table 1

[0313]

[0314]

[0315] The names of strains and the genotypes thereof involved in the following Example are shown in Table 2.

[0316] Table 2

[0317]

[0318] IMX581 yeast strain (described in the following document: Mans R et al., CRISPR / Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res 2015, 15, fov004.) can be purchased from the euroscarf website, specifically http: / / www.euroscarf.de / plasmid_details. php?accno=Y40593.

[0319] The LC-MS / MS is a Shimadzu Nexera UHPLC system, the mass spectrometry detector is a Sciex QTRAP 6500+, and the chromatographic column is a Luna Omega 1.6 pm polar Cl 8 100 A column (Phenomenex). Mobile phase A is 0.1% formic acid. Mobile phase B is acetonitrile, flow rate setting 0.4 mL / min. Mass spectrometry conditions settings: Curtain Gas (50); Collision Gas (Medium); lonSpray Voltage: -4500 V (4-HP acid and Tyrosol), 4500 V (Dopamine, S-NOR and S-RET); temperature (450°C), mobile phase gradient: 0 to 4 min, 2% B to 10% B, 4 to 6 min, 10% B to 85% B; 6 to 7 min, 85% B; 7 to 7.1 min 85% B to 2% B; 7.1 to 9 min 2% B. MRM mode is used for qualitative detection, and Q3 MI mode or MS2 mode is used for quantitative detection.

[0320] In the Example below, plasmid pMELlO is a product of EUROSCARF collection, acc. no. P30779. Plasmid pMELlO harbors SNR52 promoter and a gRNA backbone.

[0321] EXAMPLE

[0322] The present inventors, after conducting extensive experiments, constructed recombinant strains for producing the benzophenanthridine alkaloid sanguinarine SAN. The present inventors conducted research by dividing the synthesis pathway of sanguinarine into five modules, and achieved de novo synthesis of sanguinarine through multiple strategies. For example, in order to increase BBE activity, a novel endoplasmic reticulum engineering strategy was developed to increase the yield of the key intermediate S-Scoulerine by 206%; McDB0X2 was identified as the only DBOX gene reported to act in Saccharomyces cerevisiae, and the reconstruction of the entire biosynthesis pathways of benzophenanthridine alkaloids was completed; by performing N- terminal truncation operation on DBOX to increase cytoplasmic expression, performing endoplasmic reticulum expansion, and increasing the supply of the cofactor FAD, the yield of sanguinarine was increased, finally obtaining a yield of sanguinarine of 3.8 mg / L. The starting strain was IMX581 yeast strain, which was a Saccharomyces cerevisiae strain integrating a Cas9 gene under TEF1 promoter, the genotype being MATa ura3-52 canid:: cas9-natNT2 TRP1 LEU2 HIS3, CEN.PK113-5D. Hereinafter, IMX581 yeast strain is referred to as IMX581 for short.

[0323] The biosynthesis pathway of sanguinarine is shown in FIG. 1.

[0324] I. De novo production of norcoclaurine in Saccharomyces cerevisiae (Module I)

[0325] The pathway and engineering strategy of module I are shown in FIG. 2.

[0326] (I) Construction of dopamine-producing strain XJ001

[0327] IMX581 was spread onto a YPD solid plate, and the plate was incubated upside down at 30°C for 48 h to 72 h to obtain freshly activated IMX581 yeast colonies. Then, gene fragment BvCYP76ADl* (BVCYP76AD1W13L’F309L) (BvCYP76ADl, GenBank, accession no. AET43289, version AET43289.1) derived from Beta vulgaris and gene fragment DODC (GenBank, accession no. DAA64376, version DAA64376.1; gene fragment DODC encoding DOPA decarboxylase) were integrated into the A7-5 site of IMX581 by using CRISPR-Cas9 genome editing method.

[0328] 1. Construction of plasmid pMEL10-XI-5

[0329] Gibson assembly method was used to perform gRNA plasmid construction. The specific steps were as follows:

[0330] (1) Plasmid pMELlO was used as a template, and a primer pair consisting of Vector backbone-F and Vector backbone-R was used to perform PCR amplification to obtain linearized plasmid pMELlO.

[0331] (2) XI-5-gRNA-F and XI-5-gRNA-R were artificially synthesized, then mixed in equal amounts, treated at 95°C for 10 min, and cooled to room temperature to obtain an annealed short nucleic acid sequence, which comprised a 20 bp sequence homologous to SNR52 promoter, a 20 bp gRNA sequence, and a 20 bp sequence homologous to the gRNA backbone.

[0332] (3) The annealed short nucleic acid sequence and linearized plasmid pMELlO were assembled according to standard Gibson to obtain plasmid pMEL10-XI-5.

[0333] 2. Preparation of repair fragments (Donor DNA)

[0334] A. Repair fragment XI-5 us-IDPlt-BvCYP76ADl*-TDH3p-CCW12p, comprised, sequentially from 5' to 3', upstream homology arm XI-5 us (nucleotide sequence as shown in SEQ ID NO: 33), terminator IDPlt (nucleotide sequence as shown in SEQ ID NO: 3), gene fragment BvCYP76ADl*, promoter TDH3p (nucleotide sequence as shown in SEQ ID NO: 1), and promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The specific operation for preparing repair fragment XI-5 us-IDPlt-BvCYP76ADl*-TDH3p-CCW12p by using overlap method is as follows:

[0335] (1) Yeast genome or synthetic genes were used as templates respectively to perform amplification to obtain upstream homology arm XI-5 us, terminator IDPlt, gene fragment BvCYP76ADl*, promoter TDH3p, and promoter CCWlp.

[0336] (2) The upstream homology arm XI-5 us, terminator IDPlt, gene fragment BvCYP76ADl*, promoter TDH3p, and promoter CCW12p were ligated by using an overlap system to obtain a template.

[0337] (2-1) Preparation of the overlap system. The overlap system was 25 pL consisting of 5 pL of 5*Prime STAR buffer, 2 pL of dNTP (2.5 mM each), upstream homology arm XI-5 us, terminator IDPlt, gene fragment BvCYP76ADl*, promoter TDH3p, promoter CCW12p, 0.5 pL of Polymerase, and ddfhO. In the overlap system, the concentration of upstream homology arm XI-5 us was 0.03 pmol, and the molar ratio of upstream homology arm XI-5 us, terminator IDPlt, gene fragment BvCYP76ADl*, promoter TDH3p, and promoter CCW12p was 1 :3:5:3: 1.

[0338] (2-2) The overlap system was allowed to react to obtain a product.

[0339] The reaction conditions were: 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; final extension at 70°C for 10 min.

[0340] (2-3) 2 pl of the product obtained in step (2-2) was used as a template, and a primer pair consisting of XI-5us-F and CCW12p-DODC-Rl was used to perform PCR amplification to obtain repair fragment XI-5 us-IDPlt-BvCYP76ADl*-TDH3p-CCW12p.

[0341] The reaction conditions were: 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; final extension at 70°C for 10 min.

[0342] B. Repair fragment CCW12p-DODC-TPSlt-XI-5 ds comprised, sequentially from 5' to 3', promoter CCW12p, gene fragment DODC, terminator TPS It (nucleotide sequence as shown in SEQ ID NO: 4), and the downstream homology arm XI-5 ds (nucleotide sequence as shown in SEQ ID NO: 34). The specific operation for preparing repair fragment CCW12p-DODC-TPSlt- XI-5 ds by using overlap method is as follows:

[0343] (1) Yeast genome or synthetic genes were used as templates, respectively to perform amplification to obtain promoter CCW12p, gene fragment DODC, terminator TPS It, and downstream homology arm XI-5 ds.

[0344] (2) Promoter CCW12p, gene fragment DODC, terminator TPS It, and downstream homology arm XI-5 ds were amplified by using an overlap system to obtain a template.

[0345] (2-1) Preparation of the overlap system. The overlap system was 25 pL consisting of 5 pL of 5*Prime STAR buffer, 2 pL of dNTP (2.5 mM each), promoter CCW12p, gene fragment DODC, terminator TPS It, downstream homology arm XI-5 ds, 0.5 pL of Polymerase, and ddFEO. In the overlap system, the concentration of promoter CCW12p was 0.03 pmol, and the molar ratio of promoter CCW12p, gene fragment DODC, terminator TPS It and downstream homology arm XI-5 ds was 1 :3:3: 1.

[0346] (2-2) The overlap system was allowed to react to obtain a product. The reaction conditions were: 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; final extension at 70°C for 10 min.

[0347] (2-3) 2 .1 of the product obtained in step (2-2) was used as a template, and a primer pair consisting of CCW12pl-F and XI-5 ds-R was used to perform PCR amplification to obtain repair fragment CCW12p-DODC-TPSlt-XI-5 ds.

[0348] The reaction conditions were: 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; final extension at 70°C for 10 min.

[0349] 3. Obtainment of positive clones

[0350] (1) Plasmid pMEL10-XI-5 obtained in step 1 and repair fragment XI-5 us-IDPlt- BvCYP76ADl*-TDH3p-CCW12p and repair fragment CCW12p-DODC-TPSlt-XI-5 ds) obtained in step 2 were sequentially added to IMX581 yeast competent cells according to Table 3. After mixing homogeneously, the yeast cells were treated at 42°C for 30 min to 60 min, and centrifuged at 10,000 g for 1 min at room temperature. The yeast cells were collected, and resuspended in 100 pL of sterile water to obtain a resuspension.

[0351] Table 3

[0352] Note: The repair fragments were required to be denatured in a boiling water bath for 5 min prior to being placed on ice for 5 min. (2) The resuspension was dropped onto the center of a SD-Ura plate, and was spread uniformly with a spreader until the entire spread yeast solution was absorbed completely. The plate was placed in an incubator at 30°C and cultured in an inverted state for 2 to 3 d.

[0353] The SD-Ura plate: Distilled water was added to 8 g of Ura minus culture medium and 15 g of agar powder. Then the volume was made up to 1 L with distilled water, and the solution was sterilized at 121°C for 15 min. Finally, glucose having been sterilized by using a 0.22 pm filter was added to a final concentration of 2%, and the resulting solution was poured into a sterile plate and allowed to cool.

[0354] (2) Monoclones were individually picked from the SD-Ura plate, and colony PCR was performed. In particular, each of the picked monoclones was placed in a 20 mmol / L NaOH solution, lysed at 99°C for 15 min, then centrifuged for 2 min, and the supernatant was collected. The supernatant was used as a template, and primer pair a (consisting of XI-5-v-F and IDPlt-r-v- R), primer pair b (consisting of DODC-TPSlt-F and XI-5-v-R), and primer pair c (consisting of AtC3H-TDH3p-r-F and CCW12p-DODC-Rl) were used respectively to perform PCR amplification to obtain PCR amplification products.

[0355] The reaction conditions were: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, and extension at 72°C for 1 min, 30 cycles; final extension at 70°C for 10 mm.

[0356] (3) The PCR amplification products were respectively subjected to agarose gel electrophoresis, and then judgment was made as follows: for a particular monoclone, if the PCR amplification product obtained using primer pair a contained a 1056 bp DNA fragment, the PCR amplification product obtained using primer pair b contained a 919 bp DNA fragment, and the PCR amplification product obtained using primer pair c contained a 1384 bp DNA fragment, then the monoclone is a positive clone.

[0357] 4. Three positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2-3 d. The grown plaque was the strain that lost the gRNA plasmid, and the obtained strain was named XJ001 yeast strain (hereinafter referred to XJ001 for short). The genotype of XJ001 yeast strain was IMX581 (XI-5:: IDP lt-BvCYP76Al*-TDH3p + CCW12p-DODC-TPSlt).

[0358] The 5-FOA plate: To 8 g of yeast SC culture medium and 20 g of agar powder were added distilled water. Then, the volume was made up to 1 L with distilled water, and the solution was sterilized at 121 °C for 20 min. Finally, after the temperature of the culture medium decreased to about 40°C, 5-FOA mother solution (obtained by dissolving 5-fluoroorotic acid (5-FOA) powder with dimethyl sulfoxide (DMSO)) was added in a sterile ultraclean bench to a final 5-FOA concentration of 0.1%. The resulting solution was poured into a sterile plate and allowed to cool.

[0359] (II) Screening of NCS enzyme

[0360] Three N-terminally truncated CjNCS derived from Coptis japonica (GenBank, accession no. BAF45338, version BAF45338.2) (CjNCSN_A24, CjNCSN_A29 and CjNCSN_A35, respectively, that is, with 24, 29, and 35 amino acids, respectively, being truncated from the N- terminus of the amino acid sequence of CjNCS) were compared for their effects in XJ001. Comparative experiments on NCS enzyme efficiency were performed in XJ001.

[0361] Expression cassette XI-3 us-TEFlp-CjNCS_A24-TDH2t-XI-3 ds comprised, sequentially from 5' to 3', upstream homology arm XI-3 us (nucleotide sequence as shown in SEQ ID NO: 35), promoter TEFlp (nucleotide sequence as shown in SEQ ID NO: 5), the encoding gene of CjNCS_A24, terminator TDH2t (nucleotide sequence as shown in SEQ ID NO: 6), and downstream homology arm XI-3 ds (nucleotide sequence as shown in SEQ ID NO: 36).

[0362] 1. Construction of plasmid pMEL10-XI-3

[0363] Plasmid pMEL10-XI-3 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with XI-3-gRNA-F, and XI-5-gRNA-R was replaced with XI-3-gRNA- R, whereas the other parts of the method remained unchanged.

[0364] 2. Plasmid pMEL10-XI-3 and expression cassette XI-3 us-TEFlp-CjNCS_A24-TDH2t- XI-3 ds were transformed into XJ001 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, expression cassette XI-3 us-TEFlp-CjNCS_A24-TDH2t-XI-3 ds was integrated into the XI-3 site of XJ001. The resulting strain was named XJ003 yeast strain (hereinafter referred to as XJ003 for short), and the genotype of XJ003 yeast strain was X 1001 (XI-3:: TEF Ip-CjNCS 24-TDH2i).

[0365] 3. Expression cassette XI-3 us-TEFlp-CjNCS_A24-TDH2t-XI-3 ds in step 2 was replaced with expression cassette XI-3 us-TEFlp-CjNCS_A29-TDH2t-XI-3 ds, and the other parts of the method remained unchanged. That is, expression cassette XI-3 us-TEFlp-CjNCS_A29-TDH2t- XI-3 ds was integrated into the XI-3 site of XJ001. The resulting strain was named XJ006 yeast strain (hereinafter referred to as XJ006 for short), and the genotype of XJ006 yeast strain was XJ001 (XI-3:: TEFlp-CjNCS 29-TDH2f).

[0366] Expression cassette XI-3 us-TEFlp-CjNCS_A29-TDH2t-XI-3 ds comprised, sequentially from 5' to 3', upstream homology arm XI-3 us, promoter TEFlp, the encoding gene of CjNCS_A29, terminator TDH2t, and downstream homology arm XI-3 ds.

[0367] 4. Expression cassette XI-3 us-TEFlp-CjNCS_A24-TDH2t-XI-3 ds in step 2 was replaced with expression cassette XI-3 us-TEFlp-CjNCS_A35-TDH2t-XI-3 ds, and the other parts of the method remained unchanged. That is, expression cassette XI-3 us-TEFlp-CjNCS_A35-TDH2t- XI-3 ds was integrated into the XI-3 site of XI001. The resulting strain was named XI007 yeast strain (hereinafter referred to as XI007 for short), and the genotype of XI007 yeast strain was XJ001 (XI-3:: TEFlp-CjNCS 35-TDH2f).

[0368] Expression cassette XI-3 us-TEFlp-CjNCS_A35-TDH2t-XI-3 ds comprised, sequentially from 5' to 3', upstream homology arm XI-3 us, promoter TEFlp, the coding gene of CjNCS_A35, terminator TDH2t, and downstream homology arm XI-3 ds.

[0369] 5. Three monoclones of each of XI003, XI006 and XI007 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^FEO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0370] 6. Detection of metabolic components of the strains

[0371] 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0372] The detection results are shown in the left panel of FIG. 3. The results indicated that XI007, i.e., the engineered strain into which the encoding gene of CjNCS_A35 was inserted, produced the highest amount of norcoclaurine.

[0373] (Ill) Screening of tyrosine hydroxylase genes CYP76AD1* and CYP76AD5 derived from Beta vulgaris, C3H derived iromArabidopsis thaliana and Zea mays, and HpaB and HpaC derived from Pseudomonas aeruginosa and Salmonella enterica were selected for efficiency comparison. The efficiency comparison of tyrosine hydroxylase was performed in IMX581.

[0374] 1. Construction of plasmid pMEL10-XI-5

[0375] The same as in step (I) 1.

[0376] 2. Construction of strains

[0377] Fragments were ligated by using overlap method to form a complete gene expression cassette. The gene expression cassette consisted of a promoter, the nucleic acid sequence of a candidate gene, and a terminator. The candidate gene was: CYP76AD1* (GenBank, accession no. AET43289, version AET43289.1), DODC (GenBank, accession no. DAA64376, version DAA64376.1), CYP76AD5 (GenBank, accession no. AJD87473, version AJD87473.1), AtC3H (GenBank, accession no. NP 001030991, version NP 001030991.2), ZmC3H (GenBank, accession no. ACG46606, version ACG46606.1), PaHpaB (GenBank, accession no. WP_003104533, version, WP_003104533.1), PaHpaC (GenBank, accession no. WP_058166060, version WP 058166060.1), or SeHpaC (GenBank, accession no. WP 001195556, version WP 001195556.1); the promoter was the promoter TDH3p, the promoter CCW12p, or the promoter TEFlp; the terminator was the terminator TPS It, the terminator IDPlt, or the terminator ENO2t (nucleotide sequence as shown in SEQ ID NO: 7). The specific procedures were as follows:

[0378] (1) Upstream homology arm XI-5 us, terminator IDPlt, CYP76AD1*, promoter TDH3p and promoter CCW12p were ligated by using overlap method to obtain gene expression cassette fragment XI-5 z / .s-IDP lt-CYP76AD l *-TDH3p-CCW12p (named Fragment 1). Promoter TDH3p, promoter CCW12p, DODC, terminator TPSlt and downstream homology arm XI-5 ds were ligated by using overlap method to obtain gene expression cassette fragment TDH3p-CCW12p- DODC-TPSlt-XI-5 ds. (named Fragment 2). Plasmid pMEL10-XI-5 obtained in step 1, Fragment 1 and Fragment 2 were transformed into IMX581 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, genes CYP76AD1* and DODC were integrated into the XI-5 site in IMX581, affording XJ001 strain. (2) XJ013 strain integrated with CYP76AD5, named XJ013 for short, was obtained according to the method of the above step (1), except that CYP76AD1* was replaced with CYP76AD5, whereas the other parts of the method remained unchanged.

[0379] (3) XJ011 strain integrated with AtC3H, named XJ011 for short, was obtained according to the method of the above step (1), except that CYP76AD1* was replaced with AtC3H, whereas the other parts of the method remained unchanged.

[0380] (4) XJ012 strain integrated with ZmC3H, named XJ012 for short, was obtained according to the method of the above step (1), except that CYP76AD1* was replaced with ZmC3H, whereas the other parts of the method remained unchanged.

[0381] (5) XJ014 strain integrated with PaHpaB-PaHpaC, named XJ014 for short, was obtained according to the method of the above step (1 ), except that CYP76AD1 * was replaced with PaHpaB- PaHpaC (that is, a fusion gene of PaHpaB and PaHpaC), whereas the other parts of the method remained unchanged.

[0382] (6) XJ015 strain integrated with PaHpaB-SeHpaC, named XJ015 for short, was obtained according to the method of the above step (1 ), except that CYP76AD1 * was replaced with PaHpaB- SeHpaC (that is, a fusion gene of PaHpaB and SeHpaC), whereas the other parts of the method remained unchanged.

[0383] (7) XJ016 strain integrated with PaHpaC-PaHpaB, named XJ016 for short, was obtained according to the method of the above step (1), except that CYP76AD1* was replaced with PaHpaC-PaHpaB (that is, a fusion gene of PaHpaC and PaHpaB), whereas the other parts of the method remained unchanged.

[0384] (8) XJ017 strain integrated with SeHpaC-PaHpaB, named XJ017 for short, was obtained according to the method of the above step (1 ), except that CYP76AD1 * was replaced with SeHpaC- PaHpaB (that is, a fusion gene of SeHpaC and PaHpaB), whereas the other parts of the method remained unchanged.

[0385] (9) Upstream homology arm XI-5 us, promoter TEFlp PaHpaC, terminator ENO2t and terminator IDPlt were ligated by using overlap method to obtain XI-5 us-TEFlp-PaHpaC-ENO2t- IDPlt (named Fragment 3). Terminator ENO2t, terminator IDPlt, PaHpaB, promoter TDH3p and promoter CCW12p were ligated by using overlap method to obtain ENO2t-IDPlt-PaHpaB- TDH3p-CCW12p (named Fragment 4); Promoter TDH3p, promoter CCW12p, DODC, terminator TPS It and downstream homology arm XI-5 ds were ligated using overlap method to obtain TDH3p-CCW12p-DODC-TPSlt-l -5 ds (named Fragment 5). Plasmid pMEL10-XI-5 obtained in step 1, Fragment 3 and Fragment 4 were transformed intoIMX581 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XI-5 site of IMX581 afforded XJ0018 strain into which PaHpaC and PaHpaB were integrated, named XJ018 for short.

[0386] (10) XJ019 strain integrated with PaHpaB and SeHpaC, named XJ019 for short, was obtained according to the method of the above step (9), except that PaHpaC was replaced with SeHpaC, whereas the other parts of the method remained unchanged.

[0387] 3. Three monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4’7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0388] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0389] The detection results are shown in the right panel of FIG. 3 (the strains were, respectively, from left to right, XJ001, XJ013, XJ011, XJ012, XJ014, XJ015, XJ016, XJ017, XJ018, and XJ019). Results indicated that XJ013 strain had the highest amount of dopamine. Therefore, CYP76AD5 comprised in this strain was the tyrosine hydroxylase having a high catalytic efficiency, obtained by screening in this experiment.

[0390] (IV) Construction of S -Nor-producing strain XJ053

[0391] 1. Construction of XJ023 strain

[0392] IMX581 was used as a starting strain to construct strain XJ023 by increasing the tyrosine metabolic flux. That is, a gene of interest related to the tyrosine metabolic flux was integrated into the chromosome of IMX581. The gene expression cassette consisted of a promoter, the gene of interest related to the tyrosine metabolic flux, and a terminator. The genes of interest related to the tyrosine metabolic flux was EcAROL derived from E. coli (GenBank, accession no. NP_414922, version NP_414922.1), ARCH* (mutant ARO4K229Lof yeast endogeneous gene AR04), AR07* (mutant ARO7G141Sof yeast endogeneous gene AR07), MtPDHl derived from barrel medic (GenBank, accession no. KM507076, version KM507076.1), AR01 (yeast endogenous gene), AR02 (yeast endogenous gene), or AR03 (yeast endogenous gene); the promoter is promoter TPIlp (nucleotide sequence as shown in SEQ ID NO: 8), promoter PGKlp (nucleotide sequence as shown in SEQ ID NO: 9), promoter TEFlp, promoter TDH3p, or promoter tHXT7p (nucleotide sequence as shown in SEQ ID NO: 20); the terminator was terminator pYX212t (nucleotide sequence as shown in SEQ ID NO: 10), terminator ADHlt (nucleotide sequence as shown in SEQ ID NO: 11), terminator FBAlt (nucleotide sequence as shown in SEQ ID NO: 12), terminator CYClt (nucleotide sequence as shown in SEQ ID NO: 13), or terminator TDH2t. The specific steps were as follows:

[0393] (1) Construction of plasmid pMEL10-X-2

[0394] Plasmid pMEL10-X-2 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with X-2-gRNA-F, and XI-5-gRNA-R was replaced with X-2-gRNA-R, whereas the other parts of the method remained unchanged.

[0395] (2) Construction of plasmid pMEL10-X-4

[0396] Plasmid pMEL10-XI-4 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with X-4-gRNA-F, and XI-5-gRNA-R was replaced with X-4-gRNA-R, whereas the other parts of the method remained unchanged.

[0397] (3) Plasmid pMEL10-X-2, expression cassette X-2 us-TPIlp-EcaroL-pYX212t-ADHlt (consisting of the upstream homology arm X-2 us, promoter TPIlp, the nucleic acid sequence of EcAROL, terminator pYX212t and terminator ADHlt), expression cassette pYX212t-ADHlt- ARO7*-PGKlp-TEFlp (consisting of terminator pYX212t, terminator ADHlt, the nucleic acid sequence of ARO7*, promoter PGKlp and promoter TEFlp), expression cassette PGKlp-TEFlp- ARO4*-CYClt-FBAlt (consisting of promoter PGKlp, promoter TEFlp, the nucleic acid sequence of ARO4*, terminator CYTlt and terminator FBAlt) and expression cassette CYClt- FBAlt-MtPDHl-tHXT7p-X-2 ds (consisting of terminator CYClt, terminator FBAlt, the nucleic acid sequence of MtPDHl, promoter tHXT7p and downstream homology arm X-2 ds) were transformed into IMX581 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the

[0398] X-2 site of IMX581 afforded an intermediate yeast. Plasmid pMEL10-X-4, expression cassette X- 4 us-CYClt-AROl-TPIlp-TDH3p (consisting of upstream homology arm X-4 us, terminator CYClt, the nucleic acid sequence of AR01, promoter TPIlp and promoter TDH3p), expression cassette TPIlp-TDH3p-ARO2-ADHlt-TDH2t (consisting of promoter TPIlp, promoter TDH3p, the nucleic acid sequence of AR02, terminator ADHlt and terminator TDH2t) and expression cassette ADHlt-TDH2t-ARO3-TEFlp-X-4 ds (consisting of terminator ADHlt, terminator TDH2t, the nucleic acid sequence of AR03, promoter TEFlp and downstream homology arm X- 4 ds) were transformed into the intermediate yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the X-4 site of the intermediate yeast afforded XJ023 strain.

[0399] 2. Construction of XJ053 strain

[0400] CYP76AD5, DODC and CjNCSN_A35 were integrated into XJ023 strain to obtain XJ053 strain. That is, plasmid pMEL10-XI-5, expression cassette XI-5 us-IDPlt-BvCYP76AD5-TDH3p- CCW12p (consisting of upstream homology arm XI-5 us, terminator IDPlt, the encoding gene of BvCYP76AD5, promoter TDH3p and promoter CCW12p) and expression cassette CCW12p- DODC-TPSlt-XI-5 ds (consisting of promoter CCW12p, DODC, terminator TPS It and downstream homology arm XI-5 ds) were transformed into XJ023 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XI-5 site of XJ023 strain afforded an intermediate yeast. Then, plasmid pMEL10-XI-3 and expression cassette XI-3 us-TEFlp-CjNCS_A35-TDH2t-

[0401] XI-3 ds (consisting of upstream homologous sequence XI-3 us, promoter TEFlp, the encoding gene of CjNCS_A35, terminator TDH2t and downstream homology arm XI-3 ds) were transformed into the intermediate yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XI-3 site afforded XJ053 strain. 3. Three monoclones of XJ053 strain were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NFL^SC , 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4-7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0402] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0403] The detection results indicated that XJ053 strain produced S-NOR at a yield of 23.4 mg / L.

[0404] (V) Engineering of XJ053 strain to increase the yield of S-NOR

[0405] The yield of S-NOR was increased by knocking out the genes of by-product metabolic fluxes in XJ053 strain (knocking out ARI1, ADH6, YPR1, YDR541C, AAD3, GRE2 and HFDI) and down regulating the activities in 4-hydroxyphenethyl alcohol and 4-hydroxyphenylacetic acid synthesis. That is, the competitive metabolic flux of aldehyde reductase or dehydrogenase for the substrate 4-HPAA was removed. The resulting strain was fermentatively cultured, and the amount of S-NOR was measured. The specific operation steps were as follows:

[0406] 1. Construction of plasmids

[0407] (1) gRAN-SNR52P gene fragment (nucleotide sequence as shown in SEQ ID NO: 32) was used as a template, and primer pair 1 (consisting of BsaLaril-gRNA-F and SNR52p-adh6-gRNA- R), primer pair 2 (consisting of SNR52p-adh6-gRNA-F and SNR52p-yprl-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-hfdl-gRNA-R) were used respectively to perform PCR amplification to obtain gene fragment 1, gene fragment 2, gene fragment 3, and gene fragment 4, respectively.

[0408] (2) Gene fragment 1, gene fragment 2 and linearized plasmid pMELlO-Bsal (obtained by single digestion of plasmid pMELl 0 with restriction endonuclease Bsal) were subjected to Gibson ligation to obtain recombinant plasmid pMEL10-ARIl+ADH6+YPRl.Gene fragment 3 and linearized plasmid pMELlO-ARIl (obtained by single digestion of plasmid pMELlO with restriction endonuclease ARI 1) were subjected to Gibson ligation to obtain recombinant plasmid pMEL10-YDR541C + AAD3. Gene fragment 4 and linearized plasmid pMELlO-ARIl were subjected to Gibson ligation to obtain recombinant plasmid pMEL10-GRE2 + HFD1.

[0409] 2. Construction of strains

[0410] Recombinant plasmid pMEL10-ARIl+ADH6+YPRl and a DNA repair fragment (which had a 60 bp homology arm sequence overlapped with the promoter and terminator of the target gene) were transformed into XJ053 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid, thereby affording XJ054 strain.

[0411] Recombinant plasmid pMEL10-YDR541C+AAD3 and the DNA repair fragment were transformed into XJ053 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid, thereby affording XJ055 strain.

[0412] Recombinant plasmid pMEL10-GRE2+HFDl and the DNA repair fragment were transformed into XJ053 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid, thereby affording XJ056 strain.

[0413] Recombinant plasmid pMEL10-YDR541C+ AAD3 and the DNA repair fragment were transformed into XJ054 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid, thereby affording XJ057 strain.

[0414] Recombinant plasmid pMEL10-GRE2+HFDl and the DNA repair fragment were transformed into XJ054 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid, thereby affording XJ058 strain. Recombinant plasmid pMEL10-YDR541C+AAD3 and the DNA repair fragment were transformed into XJ058 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid, thereby affording XJ059 strain.

[0415] 3. Three monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL VFLO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0416] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0417] The detection results are shown in FIG. 2b. The results indicated that the growth OD value of XJ058 strain was normal and the yield of S-NOR was the highest. That is, knockout of ARI1, ADH6, YPR1, GRE2 and HFD1 could remove the competitive metabolic flux of aldehyde reductase or dehydrogenase for the substrate 4-HPAA and increase the yield of S-Nor.

[0418] (VI) Selection of the copy number of CjNCS_A35

[0419] One more CjNCS_A35 gene fragment derived from Coptis japonica was integrated into XJ058 strain (already containing one CjNCS_A35) to obtain XJ062 strain, and two more CjNCS_A35 gene fragments were integrated into XJ058 strain to obtain XJ063 strain (that is, XJ063 strain contained three CjNCS_A35 gene fragments in total).

[0420] 1. Construction of XJ062 strain and XJ063 strain

[0421] (1) Construction of plasmid pMELlO-XI-1

[0422] Plasmid pMEL10-XI-l was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with XI-l-gRNA-F, and XI-5-gRNA-R was replaced with XI-l-gRNA- R, whereas the other parts of the method remained unchanged. (2) Construction of plasmid pMEL10-LPl.T8

[0423] Plasmid pMEL10-LPl.T8 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with LPl.T8-gRNA-F, and XI-5-gRNA-R was replaced with LP1.T8- gRNA-R, whereas the other parts of the method remained unchanged.

[0424] (3) Plasmid pMEL10-XI-l and expression cassette XI-l-us-TDH3p-CjNCS_A35-CYClt- XI-1-ds (consisting of upstream homology arm Xi-l-us (nucleotide sequence as shown in SEQ ID NO: 51), promoter TDH3p, the encoding gene of CjNCS_A35, terminator CYClt and downstream homology arm XI-1 -ds (nucleotide sequence as shown in SEQ ID NO: 52)) were transformed into XJ058 strain competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, expression cassette XI-l-us-TDH3p- CjNCS_A35-CYClt-XI-l-ds was integrated into the XI-1 site, affording XJ062 strain.

[0425] (4) Plasmid pMEL10-LPl.T8 and expression cassette LPl.T8-us-TDH3p-CjNCS_A35- CYClt-LPl.T8-ds (consisting of upstream homology arm LPl.T8-us (nucleotide sequence as shown in SEQ ID NO: 53), promoter TDH3p, the encoding gene of CjNCS_A35, terminator CYClt and downstream homology arm LPl.T8-ds (nucleotide sequence as shown in SEQ ID NO: 54)) were transformed into XJ062 strain competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, expression cassette LPl.T8-us-TDH3p-CjNCS_A35-CYClt-LPl.T8-ds was integrated into the LP1.T8 site, affording XJ063 strain.

[0426] 2. Three monoclones of each of the strains obtained in step 1 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4’7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured. 3. 500 jj.L of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0427] The detection results are shown in FIG. 2c. The results indicated that XJ063 strain had the highest amount of S-NOR, reaching 214.8 mg / L. That is, the strain in which three CjNCS_A35’s were integrated in total had the highest product amount.

[0428] (VII) Use of an aromatic amino acid decarboxylase (AAAD) to direct more product flow to 4-HPAAA

[0429] The effects of endogenous ArolO, PcAAS derived from Petroselinum crispum, RrAAS derived from Rhodiola Salidroside and mutant PsAAAD* (Y350F) derived from P. somniferum were compared in XJ063 strain. The expression cassette consisted of an upstream homology arm, a promoter, the nucleic acid sequence of a gene of interest, a terminator, and a downstream homology arm. The promoter was promoter FBAlp (nucleotide sequence as shown in SEQ ID NO:

[0430] 14), the terminator was terminator TPSlt-ADHlt (nucleotide sequence as shown in SEQ ID NO:

[0431] 15), and the gene of interest was yeast endogenous gene ArolO, PcAAS (GenBank, accession no. AAA33860, version AAA33860.1), RrAAS (GenBank, accession no. AUI41111, version AUI41111.1) or PsAAAD * (GenBank, accession no. AAC61842, version AAC61842.1). The specific operation was as follows:

[0432] 1. Construction of strains

[0433] (1) Construction of plasmid pMEL10-X-3

[0434] Plasmid pMEL10-X-3 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with X-3-gRNA-F, and XI-5-gRNA-R was replaced with X-3-gRNA-R, whereas the other parts of the method remained unchanged.

[0435] (2) Plasmid pMEL10-X-3 and expression cassette X-3 us-FBAlp-ARO 10-TPSlt-ADH It- X-3 ds (consisting of upstream homology arm X-3 us (nucleotide sequence as shown in SEQ ID NO: 41), promoter FBAlp, yeast endogeneous gene ArolO, terminator sequence TPSlt-ADH It and downstream homology arm X-3 ds (nucleotide sequence as shown in SEQ ID NO: 42)) were transformed into XJ063 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, expression cassette X-3 us-FBAlp-ARO 10-TPS It- ADH lt-X-3 ds was integrated into the XI-3 site, affording XJ0631 strain.

[0436] (3) Plasmid pMEL10-X-3 and expression cassette X-3 us-FBAlp-PcAAS-TPSlt-ADHlt-X- 3 ds (consisting of upstream homology arm X-3 us, promoter FBAlp, PcAAS, terminator sequence TPSlt-ADHlt and downstream homology arm X-3 ds) were transformed into XJ063 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, expression cassette X-3 us-FBAlp-PcAAS-TPSlt- ADH lt-X-3 ds was integrated into the X-3 site, affording XJ0632 strain.

[0437] (4) Plasmid pMEL10-X-3 and expression cassette X-3 us-FBAlp-ARO 10-TPSlt-ADH lt- X-3 ds (consisting of upstream homology arm X-3 us, promoter FBAlp, PsAAAD*, terminator sequence TPSlt-ADHlt and downstream homology arm X-3 ds) were transformed into XJ063 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, expression cassette X-3 us-FBAlp- PsAAAD*-TPSlt-ADH lt-X-3 ds was integrated into the X-3 site, affording XJ0633 strain.

[0438] (5) Plasmid pMEL10-X-3 and expression cassette X-3 us-FBAlp-RrAAS-TPSlt-ADH It-X- 3 ds (consisting of upstream homology arm X-3 us, promoter FBAlp, RrAAS, terminator sequence TPSlt-ADHlt and downstream homology arm X-3 ds) were transformed into XJ063 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, expression cassette X-3 us-FBAlp-RrAAS-TPSlt- ADH lt-X-3 ds was integrated into the X-3 site, affording XJ0634 strain.

[0439] 2. Three monoclones of each of the strains obtained in step 1 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4’7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0440] 3. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0441] The detection results are shown in FIG. 2d and FIG. 4. The results indicated that XJ063 strain had the highest amount of S-NOR, reaching 295.5 mg / L. That is, the strain in which PsAAAD* was integrated had the highest product amount, which was superior to other candidate aromatic amino acid decarboxylases (AAAD).

[0442] (VIII) Increase of supply of Saccharomyces cerevisiae tyrosine by introducing plant tyrosine pathway genes for prephenate transaminase (PAT) and arogenate dehydratase (ADH).

[0443] The expression cassette consisted of an upstream homology arm, a promoter, the nucleic acid sequence of a candidate gene, a terminator, and a downstream homology arm. The candidate PAT gene was AtPAT (GenBank, accession no. NP_565529, version NP_565529.1) derived from A. thaliana, BvPATl (GenBank, accession no. KMS97510, version KMS97510.1) derived from Beta vulgaris or BvPAT2 (GenBank, accession no. KMT1410, version KMT14107.1). The candidate ADH gene was BvADHa (GenBank, accession no. AST12932, version AST12932.1) derived from Beta vulgari and MtncADH (GenBank, accession no. ARV76496, version ARV76496.1) derived from Medicago truncatula. The promoter was promoter TDH3p, promoter TEF2p (nucleotide sequence as shown in SEQ ID NO: 16) or promoter FBAlp. The terminator was terminator TDH2t or terminator pYX212t.The effect of different combinations of PAT and ADH on the amount of the product S-NOR was determined in XJ0633 strain. The specific operation was as follows:

[0444] 1. Plasmid pMEL10-106a was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with 106a-gRNA-F, and XI-5-gRNA-R was replaced with 106a- gRNA-R, whereas the other parts of the method remained unchanged.

[0445] 2. Preparation of gene expression cassette fragments

[0446] (1) Upstream homology arm 106a us (nucleotide sequence as shown in SEQ ID NO: 43), promoter FBAlp, AtPAT, terminator TDH2t and terminator pYX212t were ligated by using overlap method to obtain gene expression cassette fragment 106a us-FBAlp-AtPAT-TDH2t- pYX212t (named Fragment 6). Downstream homology arm 106a ds (nucleotide sequence as shown in SEQ ID NO: 44), promoter TEF2p, BvADHa, and terminator pYX212t were ligated using overlap method to obtain gene expression cassette fragment pYX212t-BvADHa-TEF2p- 106a ds (named Fragment 7).

[0447] (2) Downstream homology arm 106a ds, promoter TEF2p, MtncADH, and terminator pYX212t were ligated using overlap method to obtain gene expression cassette fragment pYX212t-MtncADH-TEF2p-106a ds (named Fragment 8).

[0448] (3) Upstream homology arm 106a us, promoter FBAlp, BvPATl, terminator TDH2t and terminator pYX212t were ligated by using overlap method to obtain gene expression cassette fragment 106a us-FBAlp-BvPATl-TDH2t-pYX212t (named Fragment 9).

[0449] (4) Upstream homology arm 106a us, promoter FBAlp, BvPAT2, terminator TTDH2tand terminator pYX212t were ligated by using overlap method to obtain gene expression cassette fragment 106a us-FBAlp-BvPAT2-TDH2t-pYX212t (named Fragment 10).

[0450] 3. Construction of strains

[0451] Plasmid pMEL10-106a, Fragment 6 and Fragment 7 were transformed into XJ0633 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the 106a site of XJ0633 strain afforded XJ0635 strain.

[0452] Plasmid pMEL10-106a, Fragment 6 and Fragment 8 were transformed into XJ053 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the 106a site of XJ0633 strain afforded XJ0636 strain.

[0453] Plasmid pMEL10-106a, Fragment 9 and Fragment 7 were transformed into XJ0633 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the 106a site of XJ0633 strain afforded XJ0637 strain.

[0454] Plasmid pMEL10-106a, Fragment 9 and Fragment 8 were transformed into XJ0633 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the 106a site of XJ0633 strain afforded XJ0638 strain.

[0455] Plasmid pMEL10-106a, Fragment 10 and Fragment 8 were transformed into XJ053 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the 106a site of XJ0633 strain afforded XJ0639 strain.

[0456] 4. Three monoclones of each of the strains obtained in step 3 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^FLO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODeoonm of the fermentation broth was measured.

[0457] 5. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0458] The detection results are shown in FIG. 2e and FIG. 5. The results indicated that XJ0636 strain had the highest concentration of S-NOR, reaching 315.9 mg / L. That is, the strain in which AtPAT and MtncADH were integrated had the highest product amount, better than other combinations of PAT and ADH.

[0459] II. Optimization of reticuline-producing module (Module II)

[0460] Module II is for the construction of the S-NOR to S-RET pathway. Firstly, optimization was made on 60MT, CNMT, and NMCH genes in this pathway. The expression cassette consisted of a promoter, the nucleic acid sequence of a candidate gene, and a terminator. The candidate gene was Ps6OMT (GenBank, accession no. AAQ01669, version AAQ01669.1) derived from P. somniferum, PsCNMT (GenBank, accession no. AAP45316, version AAP45316.1) derived from P. somniferum, EcNMCH (GenBank, accession no. AAC39452, version AAC39452.1) derived from Eschscholzia califomica, Cy6OMT derived from C. yanhusuo (nucleotide sequence as shown in SEQ ID NO: 17), CyCNMT (nucleotide sequence as shown in SEQ ID NO: 18), or CyNMCH (nucleotide sequence as shown in SEQ ID NO: 19). The promoter was promoter TPIlp, promoter PGKlp, promoter TEFlp, promoter tHXT7p, promoter TDH3p, promoter GPMlp (nucleotide sequence as shown in SEQ ID NO: 21), or promoter TEF2p. The terminator was terminator pYX212t, terminator ADHlt, terminator CYClt, terminator FBAlt, terminator TDH2t, terminator PGIlt (nucleotide sequence as shown in SEQ ID NO: 22), or terminator ENO2t. Different combinations of the candidate genes were compared in chassis strain XJ040. Finally, the candidate genes determined to be the optimal combination was integrated into XJ0636 strain. The pathway and engineering strategy of module II are shown in FIG. 6.

[0461] (I) Construction of chassis strain XJ040

[0462] 1. Construction of plasmids

[0463] Plasmid pMEL10-X-2 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with X-2-gRNA-F, and XI-5-gRNA-R was replaced with X-2-gRNA-R, whereas the other parts of the method remained unchanged.

[0464] Plasmid pMEL10-XI-4 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with X-4-gRNA-F, and XI-5-gRNA-R was replaced with X-4-gRNA-R, whereas the other parts of the method remained unchanged.

[0465] Plasmid pMEL10-XII-5 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with XII-5-gRNA-F, and XI-5-gRNA-R was replaced with XII-5-gRNA- R, whereas the other parts of the method remained unchanged.

[0466] 2. Construction of gene expression cassettes

[0467] (1) Upstream homology arm X-2 us (nucleotide sequence as shown in SEQ ID NO: 37), promoter TPIlp, the encoding gene of EcAROL, terminator pYX212t and terminator ADHlt were ligated by using overlap method to obtain gene expression cassette fragment X-2 us-TPIlp- EcaroL-pYX212t- ADHlt. Terminator pYX212t, terminator ADHlt, the encoding gene of the endogenous gene mutant AR07*, promoter PGKlp, and promoter TEFlp were ligated by using overlap method to obtain gene expression cassette fragment pYX212t- ADHlt- AR07*-PGKlp- TEFlp. Promoter PGKlp, promoter TEFlp, the encoding gene of the endogenous gene mutant AR04*, terminator CYClt, and terminator FBAlt were ligated using overlap method to obtain gene expression cassette fragment PGKlp-TEFlp-ARO4*-CYClt-FBAlt. Terminator CYClt, terminator FBAlt, the encoding gene of MtPDHl, promoter tHXT7p, and downstream homology

[0468] 1 arm X-2 ds (nucleotide sequence as shown in SEQ ID NO: 38) were ligated using overlap method to obtain gene expression cassette fragment CYClt-FBAlt-MtPDHl-tHXT7p-X-2 ds.

[0469] (2) Upstream homology arm X-4 us (nucleotide sequence as shown in SEQ ID NO: 39), terminator CYC It, the encoding gene of AR01, promoter TPIlp, and promoter TDH3p were ligated using overlap method to obtain gene expression cassette fragment X-4 us-CYClt-AROl- TPIlp-TDH3p. Promoter TPIlp, promoter TDH3p, the encoding gene of AR02, terminator ADHlt, and terminator TDH2t were ligated by overlap method to obtain gene expression cassette fragment TPIlp-TDH3p-ARO2-ADHlt-TDH2t. Terminator ADHlt, terminator TDH2t, the encoding gene of AR03, promoter TEFlp, and downstream homology arm X-4 ds (nucleotide sequence as shown in SEQ ID NO: 40) were ligated using overlap method to obtain gene expression cassette fragment ADHlt- TDH2t-ARO3-TEFlp-X-4 ds.

[0470] (3) Upstream homologous XII-5 us (nucleotide sequence as shown in SEQ ID NO: 47), terminator PGHt, the encoding gene of Ps4’OMT (GenBank, accession no. AAP45314, version AAP45314.1), promoter TDH3p, and promoter tHXT7p were ligated using overlap method to obtain gene expression cassette XII-5 us-PGIlt-Ps4’OMT-TDH3p-tHXT7p. Promoter TDH3p, promoter tHXT7p, the encoding gene of PsCPR, terminator CYC It, and downstream homology arm XII-5 ds (nucleotide sequence as shown in SEQ ID NO: 48) were ligated by using overlap method to obtain gene expression cassette fragment TDH3p-tHXT7p-PsCPR-CYClt-XII-5 ds.

[0471] 3. Construction of XJ040 strain

[0472] Plasmid pMEL10-X-2, gene expression cassette fragment X-2 us-TPIlp-EcaroL- pYX212t- ADHlt, gene expression cassette fragment pYX212t-ADHlt-ARO7*-PGKlp-TEFlp, gene expression cassette fragment PGKlp-TEFlp-ARO4*-CYClt-FBAlt, and gene expression cassette fragment CYClt-FBAlt-MtPDHl-tHXT7p-X-2 ds were transformed into XJ003 yeast competent cells to obtain a positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the X-2 site of XJ003 strain afforded an intermediate yeast 1. Plasmid pMEL10-X-4, gene expression cassette fragment X-4 us-CYClt-AROl-TPIlp-TDH3p, gene expression cassette fragment TPHp-TDH3p-ARO2- ADHlt-TDH2t, and gene expression cassette fragment ADHlt-TDH2t-ARO3-TEFlp-X-4 ds were transformed into the intermediate yeast 1 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the X-4 site afforded an intermediate yeast 2. Plasmid pMEL10-XII-5, gene expression cassette fragment XII-5 us-PGIlt-Ps4’OMT-TDH3p-tHXT7p, and gene expression cassette fragment TDH3p-tHXT7p-PsCPR-CYClt-XII-5 ds were transformed into the intermediate yeast 2 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XII-5 site finally afforded chassis strain XJ040.

[0473] (II) Screening of different combinations of candidate genes in chassis strain XJ040.

[0474] 1. Plasmid pMELlO-XII-2 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with XII-2-gRNA-F, and XI-5-gRNA-R was replaced with XII -2- gRNA-R, whereas the other parts of the method remained unchanged.

[0475] 2. Construction of gene expression cassettes

[0476] (1) Combination of Cy60MT, CyCNMT, and EcNMCH

[0477] Upstream homology arm XII-2-us (nucleotide sequence as shown in SEQ ID NO: 45), promoter GPMlp, the encoding gene of Cy60MT, terminator FBAlt and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XII-2-us-GPMlp-Cy6OMT- FBAlt-ENO2t. Terminator FBAlt, terminator ENO2t, the encoding gene of CyCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t-CyCNMT-TEF2p-tHXT7p. Promoter TEF2p, promoter tHXT7p, the encoding gene of EcNMCH, terminator ADHlt, and downstream homology arm XII-2-ds (nucleotide sequence as shown in SEQ ID NO: 46) were ligated by using overlap method to obtain gene expression cassette TEF2p-tHXT7p-EcNMCH-ADHlt-XII-2-ds.

[0478] (2) Combination of Cy60MT, CyCNMT, and CyNMCH

[0479] Upstream homology armXII-2-us, promoter GPMlp, the encoding gene of Cy60MT, terminator FBAlt and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XII-2-us-GPMlp-Cy6OMT-FBAlt-ENO2t. Terminator FBAlt, terminator ENO2t, the encoding gene of CyCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t-CyCNMT-TEF2p- tHXT7p. Promoter TEF2p, promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm XII-2-ds were ligated by using overlap method to obtain gene expression cassette TEF2p+tHXT7p-CyNMCH-ADHlt-XII-2-ds.

[0480] (3) Combination of Cy60MT, PsCNMT, and EcNMCH

[0481] Upstream homology arm XII -2 -us, promoter GPMlp, the encoding gene of Cy60MT, terminator FBAlt and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XII-2-us-GPMlp-Cy6OMT-FBAlt-ENO2t. Terminator FBAlt, terminator ENO2t, the encoding gene of PsCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t-PsCNMT-TEF2p- tHXT7p. Promoter TEF2p, promoter tHXT7p, the encoding gene of EcNMCH, terminator ADHlt, and downstream homology arm XII-2-ds were ligated by using overlap method to obtain gene expression cassette TEF2p-tHXT7p-EcNMCH-ADHlt-XII-2-ds.

[0482] (4) Combination of Cy60MT, PsCNMT, CyNMCH

[0483] Upstream homology arm XII -2 -us, promoter GPMlp, the encoding gene of Cy60MT, terminator FBAlt and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XII-2-us-GPMlp-Cy6OMT-FBAlt-ENO2t. Terminator FBAlt, terminator ENO2t, the encoding gene of PsCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t-PsCNMT-TEF2p- tHXT7p. Promoter TEF2p, promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm XII-2-ds were ligated by using overlap method to obtain gene expression cassette TEF2p-tHXT7p-CyNMCH-ADHlt-XII-2-ds.

[0484] (5) Combination of Ps6OMT, CyCNMT, and EcNMCH

[0485] Upstream homology arm XII -2 -us, promoter GPMlp, the encoding gene of Ps6OMT, terminator FBAlt, and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XII-2-us-GPMlp-Ps6OMT-FBAlt-ENO2t. Terminator FBAlt, terminator ENO2t, the encoding gene of CyCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t-CyCNMT-TEF2p- tHXT7p. Promoter TEF2p, promoter tHXT7p, the encoding gene of EcNMCH, terminator ADHlt, and downstream homology arm XII-2-ds were ligated by using overlap method to obtain gene expression cassette TEF2p-tHXT7p-EcNMCH-ADHlt-XII-2-ds.

[0486] (6) Combination of Ps6OMT, CyCNMT, and CyNMCH Upstream homology arm XII -2 -us, promoter GPMlp, the encoding gene of Ps6OMT, terminator FBAlt, and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XII-2-us-GPMlp-Ps6OMT-FBAlt-ENO2t. Terminator FBAlt, terminator ENO2t, the encoding gene of CyCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t-CyCNMT-TEF2p- tHXT7p. Promoter TEF2p, promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm XII-2-ds were ligated by using overlap method to obtain gene expression cassette TEF2p-tHXT7p-CyNMCH-ADHlt-XII-2-ds.

[0487] (7) Combination of Ps6OMT, PsCNMT, and EcNMCH

[0488] Upstream homology arm XII -2 -us, promoter GPMlp, the encoding gene of Ps6OMT, terminator FBAlt, and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XII-2-us-GPMlp-Ps6OMT-FBAlt-ENO2t. Terminator FBAlt, terminator ENO2t, the encoding gene of PsCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t-PsCNMT-TEF2p- tHXT7p. Promoter TEF2p, promoter tHXT7p, the encoding gene of EcNMCH, terminator ADHlt, and downstream homology arm XII-2-ds were ligated by using overlap method to obtain gene expression cassette TEF2p-tHXT7p-EcNMCH-ADHlt-XII-2-ds.

[0489] (8) Combination of Ps6OMT, PsCNMT, and CyNMCH

[0490] Upstream homology arm XII -2 -us, promoter GPMlp, Ps6OMT, the encoding gene of terminator FBAlt, and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XH-2-us-GPMlp-Ps6OMT-FBAlt+ENO2t. Terminator FBAlt, terminator ENO2t, the encoding gene of PsCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t-PsCNMT-TEF2p- tHXT7p. Promoter TEF2p, promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm XII-2-ds were ligated by using overlap method to obtain gene expression cassette TEF2p-tHXT7p-CyNMCH-ADHlt-XII-2-ds.

[0491] 3. Plasmid pMELlO-X II-2 and the gene expression cassette shown in step 2 (1) were transformed into XJ040 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XII-2 site of XJ040 strain afforded XJ041 strain. XJ042 strain to XJ048 strain were sequentially obtained according to the above procedure, except that the gene expression cassette shown in step 2 (1) was replaced with the gene expression cassettes shown in step 2 (2) to the gene expression cassette shown in step 2 (8), respectively, whereas the other parts of the above procedure remained unchanged.

[0492] 4. Three monoclones of each of the strains obtained in step 3 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^LLO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0493] 5. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0494] The detection results are shown in FIG. 7. The results indicated that XJ048 strain (combination of Ps6OMT, PsCNMT, and CyNMCH) had the highest S-RET concentration. Therefore, the optimal combination obtained by screening in this experiment was Ps6OMT, PsCNMT and CyNMCH.

[0495] (Ill) Preparation of XJ0662 Strain

[0496] The optimized Ps6OMT, PsCNMT, and CyNMCH, together with Ps4' OMT and PsCPR, were inserted into the XJ0636 strain to obtain the XJ0662 strain. The specific steps were as follows:

[0497] 1. Plasmid pMELlO-XII-5 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with XII-5-gRNA-F, and XI-5-gRNA-R was replaced with XII-5- gRNA-R, whereas the other parts of the method remained unchanged.

[0498] 2. Plasmid pMEL10-XII-2 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with XII-2-gRNA-F, and XI-5-gRNA-R was replaced with XII -2- gRNA-R, whereas the other parts of the method remained unchanged.

[0499] 3. Construction of gene expression cassettes (1) Upstream homology arm XII-5-us, terminator PGIlt, the encoding gene of Ps4’OMT, promoter TDH3p, and promoter tHXT7p were ligated using overlap method to obtain gene expression cassette XII-5-us-PGIlt-Ps4’OMT-TDH3p - tHXT7p (named Fragment 11). Promoter TDH3p, promoter tHXT7p, the encoding gene of PsCPR, terminator CYClt, and downstream homology arm XII-5-ds were ligated by using overlap method to obtain gene expression cassette TDH3p-tHXT7p-PsCPR-CYClt-XII-5-ds (named Fragment 12).

[0500] (2) Upstream homology arm XII-2-us, promoter GPMlp, the encoding gene of Ps6OMT, terminator FBAlt, and terminator ENO2t were ligated by using overlap method to obtain gene expression cassette XII-2-us-GPMlp-Ps6OMT-FBAlt-ENO2t (named Fragment 13). Terminator FBAlt, terminator ENO2t, the encoding gene of PsCNMT, promoter TEF2p, and promoter tHXT7p were ligated by using overlap method to obtain gene expression cassette FBAlt-ENO2t- PsCNMT-TEF2p-tHXT7p (named Fragment 14). Promoter TEF2p, promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm XII-2-ds were ligated by using overlap method to obtain gene expression cassette TEF2p-tHXT7p-CyNMCH- ADHlt-XII-2-ds (named Fragment 15).

[0501] 4. Construction of XJ066 and XJ0662 strains

[0502] Plasmid pMEL10-XII-5, Fragment 11 and Fragment 12 were transformed into XJ0636 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XII-5 site of XJ0636 strain afforded XJ066 strain.

[0503] Plasmid pMEL10-XII-2, Fragment 13, Fragment 14, and Fragment 15 were transformed into XJ066 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XII-2 site of XJ066 strain afforded XJ0662 strain.

[0504] (IV) Screening of CPR

[0505] 1. Plasmid pMELlO-PsCPR was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with PsCPR-gRNA-F, and XI-5-gRNA-R was replaced with PsCPR- gRNA-R, whereas the other parts of the method remained unchanged. 2. Plasmid pMELlO-PsCPR and ATR1 fragment (nucleotide sequence as shown in SEQ ID NO: 55) were transformed into XJ0662 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the PsCPR site of XJ0662 afforded XJ0675 strain. Plasmid pMELlO-PsCPR and ATR2 fragment (nucleotide sequence as shown in SEQ ID NO: 56) were transformed into XJ0662 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the PsCPR site of XJ0662 afforded XJ0676 strain.

[0506] 3. Three monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4’7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0507] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0508] The detection results are shown in FIG. 6b. The results indicated that XJ0675 strain had the highest amount of S-RET compared with XJ0662 strain and XJ0676 strain. That is, ATR1 was more efficient than ATR2 and PsCPR in this experiment.

[0509] (V) Effect of the number of candidate genes

[0510] The effects of the copy number of Ps4’OMT gene and that of CyNMCH gene on the amount of the product S-RET were compared in strain XJ0675. The specific steps were as follows:

[0511] 1. Plasmid construction (1) Plasmid pMELlO-HFDl was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with HFDl-gRNA-F, and XI-5-gRNA-R was replaced with HFD1- gRNA-R, whereas the other parts of the method remained unchanged.

[0512] (2) Plasmid pMEL10-416d was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with 416d-gRNA-F, and XI-5-gRNA-R was replaced with 416d- gRNA-R, whereas the other parts of the method remained unchanged.

[0513] (3) Plasmid pMELlO-ARIl was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with ARIl-gRNA-F, and XI-5-gRNA-R was replaced with ARI1- gRNA-R, whereas the other parts of the method remained unchanged.

[0514] (4) Plasmid pMEL10-ADH6 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with ADH6-gRNA-F, and XI-5-gRNA-R was replaced with ADH6-gRNA-R, whereas the other parts of the method remained unchanged.

[0515] (5) Plasmid pMELlO-YPRl was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with YPRl-gRNA-F, and XI-5-gRNA-R was replaced with YPR1- gRNA-R, whereas the other parts of the method remained unchanged.

[0516] 2. Construction of gene expression cassettes

[0517] (1) Upstream homology arm HFDl-us (nucleotide sequence as shown in SEQ ID NO: 57), terminator PGIlt, the encoding gene of Ps4’0MT, promoter TDH3p, and downstream homology arm HFDl-ds (nucleotide sequence as shown in SEQ ID NO: 58) were ligated using overlap method to obtain gene expression cassette HFDl-us-PGIlt-Ps4’OMT-TDH3p-HFDl-ds (named Fragment 16).

[0518] (2) Upstream homology arm HFDl-us, terminator PGIlt, the encoding gene of Ps4’0MT, promoter TDH3p, and promoter tHXT7p were ligated using overlap method to obtain gene expression cassette HFDl-us-PGIlt-Ps4’OMT-TDH3p-tHXT7p (named Fragment 17). Promoter TDH3p, promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm HFDl-ds were ligated by using overlap method to obtain gene expression cassette TDH3p-tHXT7p-CyNMCH-ADHlt-HFDl-ds (named Fragment 18).

[0519] (3) Upstream homology arm 416d-us (nucleotide sequence as shown in SEQ ID NO: 59), promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm 416d-ds (nucleotide sequence as shown in SEQ ID NO: 60) were ligated by using overlap method to obtain gene expression cassette 416d-us-LPl.T8-tHXT7p-CyNMCH-ADHlt- 416d-ds (named Fragment 19).

[0520] (4) Upstream homology arm ARIl-us (nucleotide sequence as shown in SEQ ID NO: 61), promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt-LP4.T9, and downstream homology arm ARIl-ds (nucleotide sequence as shown in SEQ ID NO: 62) were ligated by using overlap method to obtain gene expression cassette ARIl-us-LP3.T7-tHXT7p-CyNMCH-ADHlt- LP4.T9-ARIl-ds (named Fragment 20). Upstream homology arm ADH6-us (nucleotide sequence as shown in SEQ ID NO: 63), promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm ADH6-ds (nucleotide sequence as shown in SEQ ID NO: 64) were ligated by using overlap method to obtain gene expression cassette ADH6us-LP3.T7- tHXT7p-CyNMCH-ADHlt-LP4.T9-ADH6-ds (named Fragment 21). Upstream homology arm YPRl-us (nucleotide sequence as shown in SEQ ID NO: 65), promoter tHXT7p, the encoding gene of CyNMCH, terminator ADHlt, and downstream homology arm YPRl-ds (nucleotide sequence as shown in SEQ ID NO: 66) were ligated by using overlap method to obtain gene expression cassette YPRl-us-LP3.T7-tHXT7p-CyNMCH-ADHlt-LP4.T9-YPRl-ds (named Fragment 22).

[0521] 3. Construction of strains

[0522] (1) Plasmid pMELlO-HFDl and Fragment 16 were transformed into XJ0675 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5- FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the HFD1 site of XJ0675 afforded XJ0685 strain.

[0523] (2) Plasmid pMELlO-HFDl, Fragment 17 and Fragment 18 were transformed into XJ0685 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the HFD1 site of XJ0685 strain afforded XJ0686 strain.

[0524] (3) Plasmid pMEL10-416d and Fragment 19 were transformed into XJ0686 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5- FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the 416d site of XJ0686 afforded XJ0689 strain.

[0525] (4) Plasmid pMELlO-ARIl, plasmid pMEL10-ADH6 and plasmid pMELlO-YPRl, together with Fragment 20, Fragment 21, and Fragment 22 respectively, were transformed into XJ0685 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the ARI1 site, ADH6 site and YPR1 site of XJ0685 afforded XJ0690 strain.

[0526] (5) Plasmid pMELlO-ARIl, plasmid pMEL10-ADH6 and plasmid pMELlO-YPRl, together with Fragment 20, Fragment 21, and Fragment 22 respectively, were transformed into XJ0686 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the ARI1 site, ADH6 site and YPR1 site of XJ0686 afforded XJ0691 strain.

[0527] 4. Three monoclones of each of the strains obtained in step 3 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4’7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODeoonm of the fermentation broth was measured.

[0528] 5. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0529] The detection results are shown in FIG. 6c. The results indicated that XJ0691 strain in which two Ps4’OMT fragments and five CyNMCH fragments were integrated had the highest amount of S-RET, reaching 425.2 mg / L.

[0530] III. Screening and optimization of BBE (Module III) The present invention adopted an endoplasmic reticulum engineering strategy to increase the activity of BBE in Saccharomyces cerevisiae. Firstly, the BBE enzyme CyBBE (derived from Corydalis yanhusuo) having higher catalytic activity was obtained by performing comparison and screening. Then, a yeast endoplasmic reticulum retention signal was fused to the C terminus of CyBBE (CyBBE ERTS), such that CyBBE was transported backward from the Golgi into the endoplasmic reticulum, thereby increasing the yield of scoulerine by 3 times. In addition, mitigation of the inhibitory effect of CyBBE by introducing mammalian-derived catalase peroxiredoxin IV (PRDX4) to mediate H2O2 decomposition, and endoplasmic reticulum expansion mediated by knocking out phospholipase OPI1 increased the titer of scoulerine by 30% and 36%, respectively. The pathway and engineering strategy of module III are shown in FIG. 8.

[0531] (I) Screening of optimal BBE genes

[0532] The candidate genes were PsBBE (GenBank, accession no. AAC61839, version AAC61839.1) derived from somniferum, CjBBE (GenBank, accession no. BAM44344, version BAM44344.1) derived from Coptis japonica, CyBBE (nucleotide sequence as shown in SEQ ID No: 23) derived from Corydalis yanhusuo, and StBBE (nucleotide sequence as shown in SEQ ID No: 24) derived from Stephania tetrandra. The candidate genes were respectively integrated into reticuline low-producing strain XJ048, and it was found that the expressed candidate genes except StBBE were all able to produce reticuline, among which CyBBE had the highest activity. The expression cassette consisted of an upstream homology arm, a promoter, the nucleic acid sequence of a candidate gene, a terminator, and a downstream homology arm. The promoter is promoter CCW12p, and the terminator is terminator PGIlt. The specific steps were as follows:

[0533] 1. Plasmid construction

[0534] Plasmid pMEL10-XII-4 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with XII-4-gRNA-F, and XI-5-gRNA-R was replaced with XII-4-gRNA- R, whereas the other parts of the method remained unchanged.

[0535] 2. Construction of strains

[0536] (1) Upstream homology arm XII-4-us (nucleotide sequence as shown in SEQ ID NO: 49), promoter CCW12p, the encoding gene of PsBBE, terminator PGIlt, and downstream homology arm XII-4-ds (nucleotide sequence as shown in SEQ ID NO: 50) were ligated using overlap method to obtain PsBBE gene expression cassette. Plasmid pMELlO-XII-4 and PsBBE gene expression cassette were transformed into XJ048 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, PsBBE gene expression cassette was integration into the XII-4 site of XJ048, affording a yeast strain, which was named XJ081 strain.

[0537] (2) A yeast strain was obtained according to the above procedure, except that the encoding gene of PsBBE was replaced with the encoding gene of CjBBE, whereas the other parts of the above procedure remained unchanged. That is, CjBBE gene expression cassette was integrated into the XII-4 site of XI048, and the yeast strain obtained was named XI082 strain.

[0538] (3) A yeast strain was obtained according to the above procedure, except that the encoding gene of PsBBE was replaced with the encoding gene of CyBBE. That is, CyBBE gene expression cassette was integrated into the XII-4 site of XI048, and the yeast strain obtained was named XI083 strain.

[0539] (4) A yeast strain was obtained according to the above procedure, except that the encoding gene of PsBBE was replaced with the encoding gene of StBBE. That is, StBBE gene expression cassette was integrated into the XII-4 site of XI048, and the yeast strain obtained was named XI084 strain.

[0540] 3. Three monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL TFEO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0541] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0542] The detection results are shown in FIG. 9. The results indicated that XI081 to XI083 were all able to produce scoulerine S-SCO. XI083 strain produced the highest amount of scoulerine S- SCO, that is, CyBBE was the gene having the highest efficiency. XJ084 was not able to produce scoulerine S-SCO.

[0543] (b) Optimization of localization of expression of CyBBE in yeast

[0544] CyBBE was fused to endoplasmic reticulum C-terminal retention signal HDEL (CyBBE ERTS (nucleotide sequence as shown in SEQ ID NO: 25)) and to N-terminal Golgi target sequence (GOTS CyBBE (nucleotide sequence as shown in SEQ ID NO: 26)), respectively, and test was performed in XJ0691 strain. It was found that XJ0694 comprising GOTS CyBBE and XJ0695 comprising CyBBE ERTS increased the yield of scoulerine by 32.8% and 206%, respectively, compared with wild-type CyBBE. Afterwards, it was determined by using a colocalization microscope that wild-type CyBBE was transported from the Golgi to the vacuole, GOTS CyBBE retained in the Golgi, whereas CyBBE ERTS was transported backward from the Golgi into the endoplasmic reticulum. The specific steps were as follows:

[0545] 1. Construction of strains

[0546] (1) Upstream homology arm XII-4-us, promoter CCW12p, the nucleotide sequence of GOTS-CyBBE, terminator PGI1, and the downstream homology arm XII-4-ds were ligated by overlap method to obtain gene expression cassette XII-4-us-CCW12p-GOTS_CyBBE-PGIlt-XII- 4-ds. Plasmid pMEL10-XII-4 and the gene expression cassette were transformed into XJ0691 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the gene expression cassette was integrated into the XII-4 site of XJ0691, affording a strain which was named XJ0694 strain.

[0547] (2) A strain was obtained according to the above step, except that the nucleotide sequence of GOTS-CyBBE was replaced with the nucleotide sequence of CyBBE ERTS, whereas the other parts of the step remained unchanged. The strain obtained was named XJ0695 strain.

[0548] (3) A strain was obtained according to the above step, except that the nucleotide sequence of GOTS-CyBBE was replaced with the nucleotide sequence of CyBBE, whereas the other parts of the step remained unchanged. The strain obtained was named XJ0696 strain.

[0549] 2. Three monoclones of each of the strains obtained in step 1 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4’7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0550] 3. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0551] 4. In order to investigate the subcellular localization of CyBBE, GOTS-CyBBE which was N-terminally fused with Golgi target sequence GOTS, and CyBBE ERTS which was C-terminally fused with endoplasmic reticulum C-terminal retention signal HDEL in Saccharomyces cerevisiae, a GFP tag was used to fuse the genes of interest (CyBBE, GOTS-CyBBE, and CyBBE ERTS), and ELO3-mRuBy2, VPHl-mRuBy, and CHS5-mRuBy2 were used as fluorescent tags for the endoplasmic reticulum, vacuole, and Golgi, respectively to determine the subcellular localization of the genes of interest. The specific steps were as follows:

[0552] (1) CyBBE having the nucleotide sequence as shown in SEQ ID NO: 23 was used as a template, and GPD-BBE1 -F and BBE-1 -GFP-R were used to perform PCR amplification to obtain CyBBE (named Fragment 23). Plasmid P416 GPD (purchased from ATCC strain library, URL: https: / / www.atcc.org / search#q=P416&sort=relevancy&numberOfResults=24) was used as a template, and BBE-1 -GFP-F and GFP-GS-IDPlt-R were used to perform PCR amplification to obtain GFP ERTS (named Fragment 24). DNA fragment DPlt-CCW12p (nucleotide sequence as shown in SEQ ID NO: 91) was used as a template, and IDPlt-r-v-F and CCW12p-R were used to perform PCR amplification to obtain IDPlt-CCW12p (named Fragment 25). The genomic DNA of IMX581 yeast strain was used as a template, and CCW12p-Elo3-F and Elo3-GS-mRuby2-R were used to perform PCR amplification to obtain Elo3 (named Fragment 26). DNA fragment mRuBy2 (nucleotide sequence as shown in SEQ ID NO: 92) was used as a template, and GS- mRuBy2-F and mRuby2-CYClt-R were used to perform PCR amplification to obtain mRuby2 (named Fragment 27). Plasmid P416 GPD was used as a template, and CYClt-p416-F and GPD- p416-R were used to perform PCR amplification to obtain a linearized plasmid (named Fragment 28). The above adjacent fragments all had a 25 bp homology arm. Fragment 23 to Fragment 28 were used to perform plasmid construction according to the instruction of the Gibson Assembly Kit of NEB Corporation to obtain recombinant plasmid P416-GPD-CyBBE_GFP_ERTS+ELO3- mRuBy2.

[0553] (2) Plasmid P416 GPD was used as a template, and BBE-l-GFP-F and GFP-IDPlt-R were used to perform PCR amplification to obtain Fragment 29. The genomic DNA of IMX581 yeast strain was used as a template, and CCW12p-Vphl-F and Vphl-GS-mRuby2-R were used to perform PCR amplification to obtain Fragment 30. Fragment 29 and Fragment 30 were used together with Fragment 23, Fragment 25, Fragment 27, and Fragment 28 obtained in step (1) to perform plasmid construction according to the instruction of the Gibson Assembly Kit of NEB Corporation to obtain recombinant plasmid P416-GPD-CyBBE-GFP+VPHl-mRuBy2.

[0554] (3) Plasmid P416 GPD was used as a template, and GPD-Golgi-BBEl-F and BBE-l-GFP- R were used to perform PCR amplification to obtain Golgi-BBEl (named Fragment 31). The genomic DNA of IMX581 yeast strain was used as a template, and CCW12p-Chs5-F and Chs5- GS-mRuby2-R were used to perform PCR amplification to obtain CHS5 (named Fragment 32). Fragment 31 and Fragment 32 were used together with Fragment 24, Fragment 25, Fragment 27, and Fragment 28 obtained in step (1) to perform plasmid construction according to the instruction of the Gibson Assembly Kit of NEB Corporation to obtain recombinant plasmid P416-GPD- GOTS_CyBBE-GFP+CHS5-mRuBy2.

[0555] (4) The plasmids obtained in (1), (2) and (3) were introduced into XJ0691 to obtain a recombinant yeast. A colocalization microscope was used to determine the localization of the fragment of interest in the yeast.

[0556] The detection results are shown in FIG. 8b. The results indicated that the CyBBE-expressed protein was localized in the vacuole, GOTS CyBBE was localized in the Golgi, and CyBBE ERTS was localized in the endoplasmic reticulum, suggesting that CyBBE ERTS was transported backward from the Golgi into the endoplasmic reticulum. XJ0694 comprising GOTS CyBBE and XJ0695 comprising CyBBE ERTS increased the yield of scoulerine S-SCO by 32.8% and 206%, respectively, compared with CyBBE. The above two kinds of results suggested that CyBBE ERTS which was C-terminally fused with endoplasmic reticulum C- terminal retention signal HDEL fulfilled the function of transporting backward from the Golgi into the endoplasmic reticulum, such that the yield of scoulerine S-SCO was increased (XJ0695, 76.5mg / L).

[0557] (Ill) Mitigation of the inhibitory effect of H2O2 by using catalase PRDX4 It has been reported that H2O2 may inhibit the function of BBE. Hence, in this study, catalase PRDX4 (N-terminally fused with yeast a-mating factor signal peptide and C-terminally fused with ERTS signal peptide, i.e., a-mPRDX4_ERTS) was introduced into Saccharomyces cerevisiae XJ0695 to decompose H2O2 so as to increase the yield of S-RET of the engineered strain (see FIG. 8C). The specific experimental steps were as follows:

[0558] 1. Construction of recombinant plasmids

[0559] (1) Artificially synthesized a-mPRDX4 ERTS fragment (nucleotide sequence as shown in SEQ ID NO: 27) and linearized plasmid P416 GPD (plasmid P416 GPD was used as a template, and a primer pair consisting of CYClt-p416-F and GPD-p416-R were used to perform PCR amplification to obtain the linearized plasmid P416 GPD) were subjected to Gibson ligation to obtain recombinant plasmid P416 GPD-a-mPRDX4 ERTS.

[0560] (2) Artificially synthesized a-mPRI)X4(Q \ 27 )_ERTS (nucleotide sequence as shown in SEQ ID NO: 28) and linearized plasmid P416 GPD were subjected to Gibson ligation to obtain recombinant plasmid P416 GPD-a-mPRDX4(C127S) ERTS.

[0561] (3) Artificially synthesized a-mPRI)X4(Q24iXP)_ERTS (nucleotide sequence as shown in SEQ ID NO: 29) and linearized plasmid P416 GPD were subjected to Gibson ligation to obtain recombinant plasmid P416 GPD-a-mPRDX4(C248 ERTS.

[0562] 2. Recombinant plasmid P416 3PD-a-mPRDX4 ERTS, recombinant plasmid P416 GPD-a-mPRDX4(C127S) ERTS and recombinant plasmid P416 GPD-a- mPRDX4(C248' ERTS were respectively introduced into XJ0695 to obtain recombinant strains.

[0563] 3. Three monoclones of each of the strains obtained in step 2 and XJ0695 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO44-7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, and 1 ml / L of vitamin solutions, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0564] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection. The detection results are shown in FIG. 8c. The results indicated that the strain into which catalase peroxiredoxin IV (a-mPRDX4_ERTS) N-terminally fused with yeast a-mating factor signal peptide and C-terminally fused with ERTS signal peptide was introduced produced the highest yield of S-SCO compared with XJ0695. The yield of S-SCO increased by 30% (reaching 98.4 mg / L) compared with XJ0695.

[0565] (IV) Optimization of CyBBE expression by means of endoplasmic reticulum engineering Since BBE is an FAD-dependent oxidase, post-translational modification through a transport pathway including the endoplasmic reticulum is required, and optimization of endoplasmic reticulum results will affect BBE expression. In this experiment, endoplasmic reticulum expansion engineering was investigated in four aspects: knockout of OPI1 gene encoding negative regulation of phospholipid biosynthesis, knockout of PAH1 gene encoding Mg2+-dependent phosphatidic acid (PA) phosphatase, and overexpression of INO2 gene and INO2 gene mutant INO2* (INO2 L119A) encoding transcription activators of phospholipid biosynthesis.

[0566] 1. Construction of plasmids

[0567] (1) Plasmid pMEL10-FgF20 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with FgF20-gRNA-F, and XI-5-gRNA-R was replaced with FgF20-gRNA-R, whereas the other parts of the method remained unchanged.

[0568] (2) Plasmid pMELlO-PAHl was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with PAHl-gRNA-F, and XI-5-gRNA-R was replaced with PAH1- gRNA-R, whereas the other parts of the method remained unchanged.

[0569] (3) Plasmid pMELlO-OPIl was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with OPIl-gRNA-F, and XI-5-gRNA-R was replaced with OPI1- gRNA-R, whereas the other parts of the method remained unchanged.

[0570] 2. Construction of strains

[0571] (1) Plasmid pMEL10-FgF20 and fragment FgF20-us-PDClp-IN02-EN02t-FgF20-ds (consisting of upstream homology arm FgF20-us (nucleotide sequence as shown in SEQ ID NO: 67), promoter PDClp (nucleotide sequence as shown in SEQ ID NO: 69), INO2 gene (nucleotide sequence as shown in SEQ ID NO: 70), terminator ENO2t (nucleotide sequence as shown in SEQ ID NO: 7) and downstream homology arm FgF20-ds (nucleotide sequence as shown in SEQ ID NO: 68)) were transformed into XJ0695 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragment was integrated into the FgF20 site of XJ0695, affording XJ0786 strain (referred to as XJ0786 for short).

[0572] (2) XJ0787 strain (referred to as XJ0787 for short) was obtained according to the method of step (I) 1, except that INO2 gene was replaced with INO2* gene (that is, INO2 mutant gene, IN02 L119A), whereas the other parts of the method remained unchanged.

[0573] (3) Plasmid pMELlO-PAHl and repair fragment a (nucleotide sequence as shown in SEQ ID NO: 30, which had a 60 bp homology arm sequence overlapped with the promoter and terminator of the target gene) were transformed into XJ0695 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, repair fragment a was integrated into the PAH1 gene site of XJ0695 to knock out PAH1 gene, affording XJ0791 strain (referred to as XJ0791 for short).

[0574] (4) Plasmid pMELlO-OPIl and repair fragment b (nucleotide sequence as shown in SEQ ID NO: 31, which had a 60 bp homology arm sequence overlapped with the promoter and terminator of the target gene) were transformed into XJ0695 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, repair fragment b was integrated into the OPI1 gene site of XJ0695 to knock out OPI1 gene, affording XJ0796 strain (referred to as XJ0796 for short).

[0575] 3. Three monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^FEO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured. 4. 500 jj.L of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0576] The detection results are shown in FIG. 8d. The results indicate that the endoplasmic reticulum expansion process mediated by overexpression of INO2 and INO2* and knockout of OPI1 increased the yield of S-RET by 17.1%, 17.6% and 35.7%, while knockout of PAH1 decreased the yield of S-RET. That is, the endoplasmic reticulum expansion mediated by the knockout OPI1 had the best effect in increasing the amount of S-RET. Endoplasmic reticulum expansion was mediated by knockout of transcription factor OPI1 encoding negative regulation of phospholipid biosynthesis or knockout of phosphatase PAH1 encoding phospholipid biosynthesis, or by overexpression of transcription activators INO2 and INO2* encoding positive regulation of phospholipid biosynthesis. The results indicated that the endoplasmic reticulum expansion process mediated by overexpression of INO2 and INO2* and knockout of OPI1 increased the yield of reticuline, and the endoplasmic reticulum expansion mediated by knockout of OPI1 had the best effect in increasing the amount of S-RET.

[0577] IV. Production of sanguinarine from scoulerine (Module IV)

[0578] The biosynthesis pathway from scoulerine to sanguinarine requires seven steps to complete: scoulerine is catalyzed by two CYP450’s (S-cheilanthifoline synthase, CFS; S-stylopine synthase, SPS) to generate S-stylopine, which is catalyzed by an V-methyltransferase (TNMT) to generate V-methylstylopine, which is hydroxylated by CYP450 (Vc / .s-A-methylstylopine 14- hydroxylase, MSH) to generate protopine (PRO), which is catalytically rearranged by CYP450 (protopine 6-hydroxylase, P6H) to generate dihydrosanguinarine, which is finally oxidized by benzophenanthridine oxidase (DBOX) to generate sanguinarine (FIG. 1). In this experiment, protopine-producing strains were first constructed, and the combination of EcCFS and EcSPS was screened and shown to exhibit a higher enzyme activity. Then, from the protopine-producing strains, benzophenanthridine oxidase McDB0X2 was screened, which was the only gene, among all candidate FDA-dependent oxidases, which played a key role in the conversion of dihydrosanguinarine to sanguinarine.

[0579] (I) Construction of protopine-producing strains

[0580] EcCFS and EcSPS derived from E. californica, CyCFS and CySPS derived from C. yanhusuo, and PsTNMT and PsMSH derived from P. somniferum were respectively selected and integrated into scoulerine-producing XJ0695 strain. Fermentative culture was performed, and the amounts of protopine in the strain were determined and compared. The combination of EcCFS and EcSPS was screened and shown to exhibit a higher enzyme activity. The specific steps were as follows:

[0581] 1. Construction of plasmid pMEL10-XI-2

[0582] Plasmid pMEL10-XI-2 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with XI-2-gRNA-F, and XI-5-gRNA-R was replaced with XI-2-gRNA- R, whereas the other parts of the method remained unchanged.

[0583] 2. Preparation of gene expression cassettes

[0584] An encoding nucleic acid expression cassette comprised an upstream homology arm, a promoter, the nucleic acid sequence of a gene of interest, a terminator, and a downstream homology arm. The gene of interest was EcCFS (GenBank, accession no. BAG75113, version BAG75113.1), EcSPS (GenBank, accession no. BAD98250, version BAD98250.1), CyCFS (nucleotide sequence as shown in SEQ ID NO: 71), CySPS (nucleotide sequence as shown in SEQ ID NO: 72), PsTNMT (GenBank, accession no. ACO90237, version ACO90237.1), or PsMSH (GenBank, accession no. AGC92398, version AGC92398.1). The promoter was promoter PDClp (nucleotide sequence as shown in SEQ ID NO: 69), promoter GPMlp (nucleotide sequence as shown in SEQ ID NO: 21), promoter TPIlp (nucleotide sequence as shown in SEQ ID NO: 8), or promoter PGKlp (nucleotide sequence as shown in SEQ ID NO: 9). The terminator was terminator ENO2t (nucleotide sequence as shown in SEQ ID NO: 7), terminator ADHlt (nucleotide sequence as shown in SEQ ID NO: 11), or terminator FBAlt (nucleotide sequence as shown in SEQ ID NO: 12).

[0585] (1) Upstream homology arm XI-2 us (nucleotide sequence as shown in SEQ ID NO: 73), promoter PDClp, the nucleic acid sequence of EcCFS, terminator ENO2t and terminator IDPlt (nucleotide sequence as shown in SEQ ID NO: 3) were ligated by using overlap method to obtain gene expression cassette XI-2 us-PDClp-EcCFS-ENO2t-IDPl (named Fragment 33). Terminator ENO2t, terminator IDPlt, the nucleic acid sequence of EcSPS, promoter GPMlp, and promoter TPIlp were ligated by using overlap method to obtain gene expression cassette ENO2t-IDPlt- EcSPS-GPMlp-TPIlp (named Fragment 34). Promoter GPMlp, promoter TPIlp, the nucleic acid sequence of PsTNMT, terminator ADHlt, and terminator FBAlt were ligated by using overlap method to obtain gene expression cassette GPMlp-TPIlp-PsTNMT-ADHlt-FBAlt (named Fragment 35). Terminator ADHlt, terminator FBAlt, the nucleic acid sequence of PsMSH, promoter PGKlp, and downstream homology arm XI-2 ds (nucleotide sequence as shown in SEQ ID NO: 74) were ligated by using overlap method to obtain gene expression cassette ADHlt- FBAlt-PsMSH-PGKlp-XI-2 ds (named Fragment 36).

[0586] (2) Gene expression cassette ENO2t-IDPlt-CySPS-GPMlp-TPIlp (named Fragment 37) was obtained according to the method of step (I) 1, except that EcSPS was replaced with CySPS, whereas the other parts of the method remained unchanged.

[0587] (3) Gene expression cassette X-2 us-PDClp-CyCFS-ENO2t-IDPl (named Fragment 38) was obtained according to the method of step (I) 1, except that EcCFS was replaced with CyCFS, whereas the other parts of the method remained unchanged.

[0588] 3. Construction of strains

[0589] (1) Plasmid pMEL10-XI-2, together with Fragment 33, Fragment 34, Fragment 35, and Fragment 36, was transformed into XJ0695 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the XI-2 site of XJ0695 strain, affording XJ0743 strain.

[0590] (2) Plasmid pMEL10-XI-2, together with gene expression cassette Fragment 33, Fragment 37, Fragment 35, and Fragment 36, was transformed into XJ0695 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the XI-2 site of XJ0695 strain, affording XJ0744 strain.

[0591] (3) Plasmid pMEL10-XI-2, together with gene expression cassette Fragment 38, Fragment 34, Fragment 35, and Fragment 36, were transformed into XJ0695 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the XI-2 site of XJ0695 strain, affording XJ0745 strain.

[0592] (4) Plasmid pMEL10-XI-2, together with gene expression cassette Fragment 38, Fragment 37, Fragment 35, and Fragment 36, was transformed into XJ0695 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the XI-2 site of XJ0695 strain, affording XJ0746 strain.

[0593] 4. Three positive monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^LLO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0594] 5. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0595] The detection results are shown in FIG. lOa-d. Results indicated that XJ0743 strain, XJ0744 strain, XJ0745 strain, and XJ0746 strain all produced protopine, with XJ0743 strain producing the highest amount of protopine. That is, in this experiment, the combination of EcCFS and EcSPS exhibited the optimum activity in the engineered strain.

[0596] (II) Screening of benzophenanthridine oxidase McDB0X2

[0597] Codon-optimized genes for AmSTOX from Argemone Mexicana, BwSTOX from Berberiis wilsoniae, CjTHBO from C. japonica, PsFADOX from P. somniferum, and McDBOXl and McDB0X2 genes from Macleaya cordata were synthesized in vitro, and were respectively transformed, together with EcP6H from E. califomica, into protopine-producing XJ0743 strain. Then, Western blotting was used to determine protein expression amount, and it was found that protein expression was good for BwSTOX and McDB0X2. However, it was detected that even XJ07437 strain (as a control) expressing only EcP6H could produce the target product sanguinarine, whereas dihydrosanguinarine could not be detected in XJ0750 strain into which McDB0X2 was introduced, confirming that McDB0X2 played a key role in the conversion of dihydrosanguinarine into sanguinarine. The specific steps were as follows: 1. Plasmid pMEL10-XII-l was obtained according to the method of step (I) 1, except that

[0598] XI-5-gRNA-F was replaced with XII-l-gRNA-F, and XI-5-gRNA-R was replaced with XII-1 - gRNA-R, whereas the other parts of the method remained unchanged.

[0599] 2. Preparation of gene expression cassettes and construction of strains

[0600] An encoding nucleic acid expression cassette comprised an upstream homology arm, a promoter, the nucleic acid sequence of a gene of interest, a terminator sequence, and a downstream homology arm. The gene of interest was AmSTOX (GenBank, accession no. ADY15027, version ADY15027.1), BwSTOX (GenBank, accession no. ADY15026, version ADY15026.1), CjSTOX (GenBank, accession no. BAJ40864, version BAJ40864.1), PsFADOX5 (GenBank, accession no. AGL44334, version AGL44334.1), StBBE (nucleotide sequence as shown in SEQ ID NO: 24), McDBOXl (GenBank, accession no. OVA00267, version OVA00267.1), McDB0X2 (GenBank, accession no. OVA00268, version OVA00268.1), or EcP6H (GenBank, accession no. BAK20464, version BAK20464.1).The promoter was promoter PDClp (nucleotide sequence as shown in SEQ ID NO: 69) or promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminator was terminator DIT It (nucleotide sequence as shown in SEQ ID NO: 75), or terminator TDH2t (nucleotide sequence as shown in SEQ ID NO: 6). His-tag sequence was CATCATCACCATCACCAT (SEQ ID NO: 214).

[0601] (1) Upstream homology arm XII-1 us (nucleotide sequence as shown in SEQ ID NO: 76), terminator DITlt, His-tag sequence, the nucleic acid sequence of AmSTOX, promoter PDClp, and promoter CCW12p were ligated by using overlap method to obtain gene expression cassette

[0602] XII-1 us-DITlt-His-AmSTOX-PDClp-CCW12p (named Fragment 39). Promoter PDClp, promoter CCW12p, the nucleic acid sequence of EcP6H, terminator TDH2t, and downstream homology arm XII-1 ds (nucleotide sequence as shown in SEQ ID NO: 77) were ligated by using overlap method to obtain gene expression cassettePDClp-CCW12p-EcP6H-TDH2t-XII-lds (named Fragment 40). Plasmid pMELlO-XII-1, together with Fragment 39 and Fragment 40, was transformed into XJ0743 strain competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the XII-1 site of XJ0743 strain, affording XJ07431 strain.

[0603] (2) XJ07432 strain, XJ07433 strain, XJ07434 strain, XJ07435 strain, XJ07436 strain and XJ0750 strain were respectively obtained according to the method of step (I) 1, except that the AmSTOX sequence was replaced with the BwSTOX sequence, CjSTOX sequence, PsFADOX5 sequence, StBBE sequence, McDBOXl sequence and McDB0X2 sequence, respectively, whereas the other parts of the method remained unchanged.

[0604] (3) Plasmid pMELlO-XII-1 and gene expression cassette fragment XII-1 us-CCW12p- EcP6H-TDH2t-XII-l ds (consisting of upstream homology arm promoter XI 1-1 us, promoter CCW12p, the nucleic acid sequence of EcP6H, terminator TDH2t, and downstream homology arm XI 1-1 ds) were transformed into XJ0743 strain competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the gene expression cassette was integrated into the XII-1 site of XI0743 strain, affording XI07437 strain into which only EcP6H gene was inserted, as a control strain.

[0605] 3. Three positive monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^FLO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0606] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0607] 5. Determination of protein expression by Western blotting

[0608] (1) Each of the strains obtained in step 2 was inoculated in 1 mL of delft culture medium, and was cultured at 30°C and 220 rpm overnight to obtain a preculture.

[0609] (2) The preculture was transferred to 5 mL of fresh delft culture medium at a ratio of 1:50 and was cultured at 30°C and 220 rpm to obtain a cultured strain solution having an ODgoonm of about 1. (3) The cells in the cultured strain solution were collected, first washed twice with PBS buffer, then resuspended by adding 200 pL of acid-washed glass beads and 300 pL of PBS buffer, and then disrupted using a FastPrep rapid sample preparation apparatus to obtain a sample.

[0610] (4) The sample was added with 4 x NuPAGE™ LDS sample buffer (ThermoFisher), and boiled at 95°C for 10 minutes. Protein electrophoresis was run at 150 V for 1 hour.

[0611] (5) The protein was transferred to a Trans-Blot® Turbo™ PVDF transfer membrane (BioRad) and then blocked with PBST (PBS + 0.1% Tween 20) containing 5% milk at room temperature for 2 hours. The membrane was washed 3 times with PBST, and was incubated with 6xHis-Tag monoclonal antibody (His. H8) (ThermoFisher) or anti-GAPDH antibody (G-9) (Santa Cruz Biotechnology) at room temperature for 1 hour. Then, the membrane was washed 3 times with PBST, and was incubated with HRP-conjugated anti-mouse IgG (h+L) secondary antibody (Invitrogen) at room temperature for 1 hour. Afterwards, the membrane was washed 3 times with PBST, and was incubated with West Pico plus HRP substrate (ThermoFisher) for 5 minutes. Finally, analysis was conducted using a ChemiDoc XRS image analyzer (Bio-Rad).

[0612] The detection results are shown in FIG. 10 e-g and FIG. 11. The results indicated that the size of BwSTOX and McDB0X2 proteins were correct, suggesting good expression, the band of PsFADOX5 was diffuse, and none of AmSTOX, CjTHBO, and McDBOXl was expressed. Determination of the metabolic components of the strains indicated that all of the strains produced sanguinarine, including the control strain. However, no dihydrosanguinarine was determined in XJ0750 strain, indicating that McDBOX2 gene fulfilled the function of converting dihydrosanguinarine into sanguinarine in the yeast. That is, among the benzophenanthridine oxidase genes obtained by screening in this experiment, McDBOX2 gene was the only benzophenanthridine oxidase gene that fulfilled the function in the yeast.

[0613] V. Optimization of the pathway from scoulerine to sanguinarine

[0614] Research has found that McDBOX2 would decrease the ODeoonm of a strain and increase the accumulation amount of the intermediate product reticuline, suggesting that McDBOX2 had some inhibitory effect on the function of BBE. Therefore, the present invention adopted the following strategy for pathway optimization: (1) truncation of 29 amino acids from the N-terminus of McDBOX2 to increase its expression in the cytosol, with the finding that compared with XJ0750 strain (XJ07437 + McDBOX2), XJ07502 strain (XJ07437+McDBOX2A29) was able to grow normally and accumulated less of the intermediate product reticuline, and was able to convert all dihydrosanguinarine to sanguinarine; (2) overexpression of plasma membrane riboflavin transporter MCH5 and FAD synthase BsRibc to increase FAD supply; endoplasmic reticulum expansion mediated by knockout of phospholipase OPI1. Finally, XJ07504 strain having a yield of sanguinarine reaching 3.8 mg / L was obtained.

[0615] (I) Truncation of 29 amino acids from the N-terminus of McDB0X2 (GenBank: OVA00268.1) contributed to increasing the yield of sanguinarine.

[0616] (1) Plasmid pMEL10-FgF20 was used to transform the gene expression cassette of McDBOX2_A29 (consisting of upstream homology arm FgF20 us (nucleotide sequence as shown in SEQ ID NO: 67), promoter PDClp, the nucleic acid sequence of McDBOX2_A29, terminator DITlt and downstream homology arm FgF20 ds (nucleotide sequence as shown in SEQ ID NO: 68) into XJ07437 strain competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragment was integrated into the FgF20 site, affording XJ07502 strain.

[0617] 2. Three positive monoclones of XJ0750 strain and of XJ07502 strain were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^FEO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0618] 3. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0619] The detection results are shown in FIG. 12. The experiment showed that the growth ODgoonm value of the strain containing McDB0X2 gene (XJ0750 strain) was significantly reduced as compared with the strain not containing McDB0X2 gene (XJ07437 strain), suggesting a significant inhibitory effect of McDB0X2 gene on the growth of the yeast strain; whereas the growth ODgoonm value of the strain containing McDBOX2_A29 (XJ0752 strain) showed very little reduction in the strain growth ODgoonm value, suggesting that truncation of 29 amino acids from the N-terminus of McDBOX2 contributed to mitigating the inhibitory effect of the gene on the growth of the strain (FIG. 12a). The metabolic components of the strains showed that, compared with XJ07437 strain and XJ0750 strain, XJ0752 strain accumulated the least of the intermediate S-RET (a substrate for the BBE enzyme in the synthesis pathway), whereas the final product SAN was the highest (FIG. 12b). Further, compared with the control strain XJ07437 strain, both XJ0750 strain and XJ0752 strain were able to completely convert the intermediate dihydrosanguinarine to sanguinarine (FIG. 12c). Thus, it can be seen that truncation of 29 amino acids from the N-terminus of McDBOX2 could effectively mitigate the inhibitory effect of McDBOX2 on the BBE step and increase the yield of SAN.

[0620] (II) Overexpression of plasma membrane riboflavin transporter MCH5 and FAD synthase BsRibc to increase FAD supply, and endoplasmic reticulum expansion mediated by knockout of phospholipase OPI1, contributed to the increase in the yield of sanguinarine

[0621] Since both BBE and McDB0X2 are FAD-dependent oxidases, there are competition for FAD supply and competition for endoplasmic reticulum. Therefore, in this experiment, the yield of sanguinarine was increased by the following two approaches: overexpression of 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. The specific steps were as follows:

[0622] 1. Construction of XJ053 strain (overexpression of MCH5 and BsRibc)

[0623] An encoding nucleic acid expression cassette comprised an upstream homology arm, a promoter, the nucleic acid sequence of a gene of interest, a terminator, and a downstream homology arm. The gene of interest was plasma membrane riboflavin transporter MCH5, FAD synthase BsRibc, or phospholipase OPI1. The promoter was promoter GPDp (nucleotide sequence as shown in SEQ ID NO: 81) or promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminator was DITlt (nucleotide sequence as shown in SEQ ID NO: 3), or TDH2t (nucleotide sequence as shown in SEQ ID NO: 6).

[0624] Upstream homology arm 416d us (nucleotide sequence as shown in SEQ ID NO: 59), promoter GPD, the nucleic acid sequence of MCH5, terminator IDPlt, and promoter CCW12p were ligated by using overlap method to obtain gene expression cassette 416d-GPD-MCH5-IDPlt- CCW12p (named Fragment 41). Terminator IDPlt, promoter CCW12p, the nucleic acid sequence of BsRibc, terminator CYC It, and downstream homology arm 416d ds (nucleotide sequence as shown in SEQ ID NO: 60) were ligated by using overlap method to obtain gene expression cassette IDPlt-CCW12p-BsRibc-CYClt-416d ds (named Fragment 42). Fragment 41 and Fragment 42, together with plasmid pMEL10-416d, were transformed into XJ07502 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the 416d site, affording XJ07503 strain.

[0625] 2. Construction of XJ07504 strain (knocking out phospholipase OPI1)

[0626] Plasmid pMELlO-OPIl and DNA repair fragment c (nucleotide sequence as shown in SEQ ID NO: 82, which had a 60 bp homology arm sequence overlapped with the promoter and terminator of the target gene) were integrated into the site of OPI1 gene, thereby knocking out OPI1 gene, affording XJ07504 strain.

[0627] 3. Three positive monoclones of XJ07502 strain, of XJ07503 strain, and of XJ07504 strain were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4-7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0628] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0629] The detection results are shown in FIG. 13. Results showed that, compared with XJ07502 strain and XJ07503 strain, XJ07504 strain had the highest amount of sanguinarine, with the yield of sanguinarine reaching 3.8 mg / L. That is, overexpression of MCH5, overexpression of BsRibc, and knockout of phospholipase OPI1 contributed to increasing the yield of sanguinarine.

[0630] VI. Construction of the pathway of chelirubine (Module V) The downstream derivative chelirubine of sanguinarine SAN has aroused people’s interest for exhibiting an anti-proliferative effect on several cancer cell lines and being useful as a DNA fluorescent probe. Two additional reaction steps are required for converting dihydrosanguinarine to chelirubine, namely hydroxylation and methylation reactions, as shown in Module V in FIG. 1. Although it has been reported that three CYP82P hydroxylases and two O-methyltransferases may be involved in this pathway, the chelirubine synthesis pathway has not been fully characterized. The inventors of the present invention have screened pathway candidate genes in engineered sanguinarine-producing yeast strains, including three P450s derived from E. californica (CYP82P2, CYP82P3, and CYP82P4) and two OMTs (Ec2OMT and Ecl 10MT). First, CYP82P2, CYP82P3, and CYP82P4 were respectively expressed in sanguinarine-producing XJ0822 strain containing McDBOX2. It was found that 10-hydroxysanguinarine having a molecular weight m / z of 348.0 was detected only in the strain expressing CYP82P2, and it was presumed that CYP82P2 catalyzed dihydrosanguinarine to produce 10-hydroxydihydrosanguinarine, which was then oxidized by McDBOX2 to produce 10-hydroxysanguinarine having the molecular weight. Later, two O-methyltransferases Ec2OMT and Ecl lOMT were identified in the sanguinarine-producing strains. It has been verified that the combination of CYP82P2 and Ecl 10MT could catalyze the last two steps of chelirubine biosynthesis. The specific steps were as follows:

[0631] (I) Functional screening of p450 genes

[0632] 1. Gibson assembly method was used to perform recombinant plasmid construction. The specific steps were as follows:

[0633] (1) Plasmid P416 GPD (purchased from ATCC strain library, URL https: / / www.atcc.org / search#q=P416&sort=relevancy&numberOfResults=24) was used as a template, and a primer pair consisting of CYClt-p416-F and GPD-p416-R were used to perform PCR amplification to obtain linearized plasmid P416 GPD, i.e., linearized initial plasmid.

[0634] (2) CYP82P2 fragment (GenBank, accession no. BBD34756, version BBD34756.1), CYP82P3 fragment (GenBank, accession no. BBD34757, version BBD34757.1), and CYP82P4 fragment (GenBank, accession no. BBD34758, version BBD34758.1) were artificially synthesized.

[0635] (3) CYP82P2 fragment, CYP82P3 fragment, CYP82P4 fragment, and the linearized initial plasmid were assembled according to the standard Gibson to obtain recombinant plasmid P416 GPD-CYP82P2, recombinant plasmid P416 GPD-CYP82P3, and recombinant plasmid P416 GPD-CYP82P4, respectively.

[0636] 2. Construction of strains

[0637] Recombinant plasmid P416 GPD-CYP82P2, recombinant plasmid P416 GPD-CYP82P3, recombinant plasmid P416 GPD-CYP82P4, and plasmid P416 GPD were respectively introduced into sanguinarine-producing XJ0822 strain to obtain XJ0851 strain, XJ0852 strain, XJ0853 strain, and XJ0854 strain, respectively.

[0638] The preparation method for XJ0822 strain was as follows: (1) artificially synthesized DNA fragment LP1.T8 (nucleotide sequence as shown in SEQ ID NO: 90) was used as a template, and a primer pair consisting of FgF20-LPl.T8-F and LPl.T8-CCW12p-R were used to perform PCR amplification to obtain Fragment FgF20 us-LPl.T8; (2) the genomic DNA of strain XJ0750 was used as a template, and primer pair xl consisting of CCW12pl-F and TDH2t-FBAlt-R, primer pair x2 consisting of FBAlt400-r-R and PGKlp-PDClp-R, primer pair x3 consisting of XI-2 us- PDClp-F and IDPlt-r-v-R, and primer pair consisting of IDPlt-r-F and GPMlp-FgF20-R were respectively used to perform PCR amplification to obtain Fragment CCW12p-EcP6H-TDH2t- FBAlt, Fragment FBAlt-PsMSH-PGKlp-PDClp, Fragment PDClp-EcCFS-ENO2t-IDPlt and Fragment IDPlt-EcSPS-GPMlp-FgF20 ds, respectively; (3) plasmid pMEL10-FgF20 together with the five fragments was transformed into XJ0750 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the FgF20 site of XJ0750 strain, affording XJ0778 strain; (4) then, plasmid pMELlO-OPIl and DNA repair fragment c (nucleotide sequence as shown in SEQ ID NO: 82, which had a 60 bp homology arm sequence overlapped with the promoter and terminator of the target gene) were integrated into the site of OPI1 gene of XJ0778 strain, thereby knocking out OPI1 gene, and finally affording XJ0822 strain.

[0639] 3. XJ0851 strain, XJ0852 strain, XJ0853 strain, and XJ0854 strain were activated on plates, and three monoclones of each strain were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NEL^SCL, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO44-7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, and 1 ml / L of vitamin solutions, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0640] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0641] The detection results are shown in FIG. 14a-c. The results indicated that CYP82P2, CYP82P3, and CYP82P4 were respectively expressed in the sanguinarine-producing XJ0822 strain, and 10-hydroxysanguinarine having a molecular weight m / z of 348.0 was detected only in CYP82P2-expressing XJ0851 strain. That is, among the above candidate P450 genes, CYP82P2 was the only gene that could catalyze the hydroxylation reaction of dihydrosanguinarine. Presumably, the reaction process was: CYP82P2 catalyzed dihydrosanguinarine to produce 10- hydroxydihydrosanguinarine, which was then oxidized by McDB0X2 to produce 10- hydroxysanguinarine having the molecular weight m / z of 348.0.

[0642] (II) Screening of the function of 10-hydroxymethyltransferase OMT

[0643] 1. Construction of plasmids

[0644] (1) Artificially synthesized CYP82P2 expression cassette (comprising promoter GPD, CYP82P2 fragment, and terminator IDPlt), Ecl 1 OMT expression cassette (comprising promoter CCW12p, Ecl lOMT (GenBank, accession no. BBA20642, version BBA20642.1), and terminator CYClt) were used to perform plasmid construction (adjacent fragments all had a 25 bp homology arm) according to the instruction of the Gibson Assembly Kit of NEB Corporation so as to construct same into linearized plasmid P416 GPD, thereby obtaining recombinant plasmid P416_GPD-CYP82P2-IDPlt-CCW12p-Ecl lOMT-CYClt. s(2) Recombinant plasmid P416_GPD-CYP82P2-IDPlt-CCW12p-Ec2OMT-CYClt was obtained according to the method of step (I) 1, except that Ecl 1 OMT was replaced with Ec2OMT (GenBank: BBA20644.1), whereas the other parts of the method remained unchanged.

[0645] 2. Construction of strains

[0646] (1) Recombinant plasmid P416_GPD-CYP82P2-IDPlt-CCW12p-Ecl lOMT-CYClt was introduced into sanguinarine-producing XJ0832 strain to obtain a XJ0855 strain.

[0647] The preparation method for XJ0832 strain was as follows: (A) upstream homology arm FgF7 us (nucleotide sequence as shown in SEQ ID NO: 83), promoter PDClp, ATR1 fragment, terminator CYC It, and terminator DITlt were ligated by using overlap method to obtain gene expression cassette FgF7 us-PDClp-ATRl-CYClt-DITlt. (B) terminator CYClt, terminator DITlt, mPRDX4 (nucleotide sequence as shown in SEQ ID NO: 27), promoter TEFlp, and downstream homology arm FgF7 ds (nucleotide sequence as shown in SEQ ID NO: 84) were ligated by using overlap method to obtain gene expression cassette CYClt-DITlt-mPRDX4- TEFlp-FgF7 ds. (C) plasmid pMEL10-FgF7 together with the above two gene expression cassettes was transformed into XJ0822 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the FgF7 site of XJ0822 strain, affording XJ0829 strain. (D) Upstream homology arm 416d us, promoter GPD, the nucleic acid sequence of MCH5, terminator IDPlt, and promoter CCW12p were ligated by using overlap method to obtain gene expression cassette 416d-GPD- MCH5-IDPlt-CCW12p. (E) promoter CCW12p, the nucleotide sequence of BsRibc, terminator CYClt, and downstream homology arm 416d ds were ligated by using overlap method to obtain gene expression cassette CCW12p-BsRibc-CYClt-416d ds. (F) plasmid pMEL10-416d together with the above two gene expression cassettes was transformed into XJ0829 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the 416d site of XJ0829 strain, affording XJ0832 strain.

[0648] (2) Construction of plasmid pMEL10-FgF7

[0649] Plasmid pMEL10-FgF7 was obtained according to the method of step (I) 1, except that XI- 5-gRNA-F was replaced with FgF7-gRNA-F, and XI-5-gRNA-R was replaced with FgF7-gRNA- R, whereas the other parts of the method remained unchanged.

[0650] (3) Recombinant plasmid P416_GPD-CYP82P2-IDPlt-CCW12p-Ec2OMT-CYClt was introduced into sanguinarine-producing XJ0832 strain to obtain a XJ0856 strain.

[0651] (4) Recombinant plasmid P416 GPD-CYP82P2 was introduced into sanguinarine- producing XJ0832 strain to obtain XJ0857 strain.

[0652] (5) Plasmid P416 GPD was introduced into sanguinarine-producing XJ0832 strain to obtain XJ0858 strain. 3. Three monoclones of XJ0855 strain, of XJ0856 strain, of XJ0857, and of XJ0858 strain were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO44-7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, and 1 ml / L of vitamin solutions, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0653] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0654] The detection results are shown in FIG. 14d-f. Results indicated that O-methyltransferases Ec2OMT and Ecl lOMT were identified in XJ0832 strain, the hydroxylated product 10- hydroxydihydrosanguinarine was found in both the strain expressing CYP82P2 alone and the strain co-expressing CYP82P2 and Ec2OMT, and a peak having a molecular weight m / z of 362.0, which was identified as chelirubine, was found in the strain expressing CYP82P2 and Ecl 10MT. The results indicated that Ecl 10MT fulfilled the function of oxymethylation in the last step of the chelirubine biosynthesis pathway.

[0655] VII. Production of chelerythrine from scoulerine (Module VI)

[0656] The biosynthesis pathway from scoulerine to chelerythrine requires a total of six catalyzed reactions: scoulerine is first catalyzed by an O-methyltransferase (S9OMT) and CYP450 (CAS) to produce S -tetrahydroberberine, which then underwent TNMT, MSH, P6H, and DBOX to produce chelerythrine. In the present application, a strain producing the intermediate allocryptopine was first constructed: mutant gene TfS9OMT* derived from Thalictrum flavum, CjCAS derived from C. japonica (or CyCAS derived from C. yanhusuo), and PsTNMT and PsMSH genes derived from P. somniferum were introduced into scoulerine-producing XJ0695 strain. It was found that XJ0741 strain into which CjCAS derived from C. japonica was introduced could produce a higher amount of allocryptopine. Then, the functional gene flavoprotein oxidase McDB0X2 was screened from existing XJ0743 strain producing the same type of compound, i.e., protopine. Afterwards, three P6H genes were respectively co-expressed with McDB0X2, and the yields were compared, confirming that the chelerythrine XJ0747 strain expressing EcP6H had the highest activity. The specific operation steps were as follows:

[0657] (I) Construction of strains producing the intermediate allocryptopine

[0658] 1. Plasmid pMEL10-XI-2 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with XI-2-gRNA-F, and XI-5-gRNA-R was replaced with XI -2- gRNA-R, whereas the other parts of the method remained unchanged.

[0659] 2. Preparation of gene expression cassettes

[0660] A nucleic acid-encoding expression cassette comprised a promoter, the nucleic acid sequence of a gene of interest, and a terminator. The gene of interest was TfS9OMT* (GenBank, accession no. AAU20770, version AAU20770.1), CjCAS (GenBank, accession no. BAB68769, version BAB68769.1), PsTNMT (GenBank, accession no. ACO90237, version ACO90237.1), PsMSH (GenBank, accession no. AGC92398, version AGC92398.1), or CyCAS (nucleotide sequence as shown in SEQ ID NO: 85). The promoter was promoter PDClp (nucleotide sequence as shown in SEQ ID NO: 69), promoter GPMlp (nucleotide sequence as shown in SEQ ID NO: 21), promoter TPIlp (nucleotide sequence as shown in SEQ ID NO: 8), or promoter PGKlp (nucleotide sequence as shown in SEQ ID NO: 9). The terminator was terminator ENO2t (nucleotide sequence as shown in SEQ ID NO: 7), terminator IDPlt ((nucleotide sequence as shown in SEQ ID NO: 3), terminator ADHlt (nucleotide sequence as shown in SEQ ID NO: 11), or terminator FBAlt (nucleotide sequence as shown in SEQ ID NO: 12).

[0661] Upstream homology arm X-2 us (nucleotide sequence as shown in SEQ ID NO: 37), promoter PDClp, the nucleic acid sequence of TfS9OMT*, terminator ENO2t, and terminator IDPlt were ligated by using overlap method to obtain gene expression cassette X-2 us-PDClp- TfS9OMT*-ENO2t-IDPl (named Fragment 33). Terminator ENO2t, terminator IDPlt, the nucleic acid sequence of CjCAS, promoter GPMlp, and promoter TPIlp were ligated by using overlap method to obtain gene expression cassette ENO2t-IDPlt-CjCAS-GPMlp-TPIlp (named Fragment 34). Promoter GPMlp, promoter TPIlp, the nucleic acid sequence of PsTNMT, terminator ADHlt, and terminator FBAlt were ligated by using overlap method to obtain gene expression cassette GPMlp-TPIlp-PsTNMT-ADHlt-FBAlt (named Fragment 35). Terminator ADHlt, terminator FBAlt, the nucleic acid sequence of PsMSH, promoter PGKlp, and downstream homology arm X-2 ds (nucleotide sequence as shown in SEQ ID NO: 38) were ligated by using overlap method to obtain gene expression cassette ADHlt-FBAlt-PsMSH-PGKlp-X-2 ds (named Fragment 36). Terminator ENO2t, terminator IDPlt, the nucleic acid sequence of CyCAS, promoter GPMlp, and promoter TPIlp were ligated by using overlap method to obtain gene expression cassette ENO2t-IDPlt-CyCAS-GPMlp-TPIlp (named Fragment 37).

[0662] 3. Construction of strains

[0663] Plasmid pMEL10-XI-2 together with Fragment 33, Fragment 34, Fragment 35, and Fragment 36 was transformed into XJ0695 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XI-2 site of XJ0695 strain afforded XJ0741 strain (referred to as XJ0741 for short).

[0664] Plasmid pMEL10-XI-2 together with Fragment 33, Fragment 37, Fragment 35, and Fragment 36 was transformed into XJ0695 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XI-2 site of XJ0695 strain afforded XJ0742 strain (referred to as XJ0742 for short).

[0665] 4. Three positive monoclones of each of the strains obtained in step 3 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^FLO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0666] 5. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0667] The detection results are shown in FIG. 15a, b, c, and d. The results indicated that compared with allocryptopine, both XJ0741 strain and XJ0742 strain were able to produce allocryptopine, and XJ0741 strain into which CjCAS from C. japonica was introduced was able to produce allocryptopine at a higher amount. It can thus be seen that CjCAS was the gene more favorable for increasing the amount of the final product in an engineered stain, and hence CjCAS was selected as an element for pathway construction.

[0668] (II) Screening of flavoprotein oxidase McDB0X2

[0669] Codon optimized AmSTOX from Argemone Mexicana, BwSTOX from Berberis wilsoniae, CjTHBO from C. japonica, PsFADOX from P. somniferum, and McDBOXl and McDBOX2 genes from Macleaya cordata were synthesized in vitro respectively, and were respectively transformed to protopine-producing XJ0743 strain together with EcP6H from E. californica (the preparation method of XJ0743 strain was as follows: (1) upstream homology arm XI -2 us (nucleotide sequence as shown in SEQ ID NO: 73), promoter PDClp (nucleotide sequence as shown in SEQ ID NO: 69), the nucleotide sequence of EcCFS (GenBank, accession no. BAG75113, version BAG75113.1), terminator ENO2t (nucleotide sequence as shown in SEQ ID NO: 7), and terminator IDPlt (nucleotide sequence as shown in SEQ ID NO: 3) were ligated by using overlap method to obtain gene expression cassette XI -2 us-PDClp-EcCFS-ENO2t-IDPl. (2) Terminator ENO2t, terminator IDPlt, the nucleic acid sequence of EcSPS (GenBank, accession no. BAD98250, version BAD98250.1), promoter GPMlp (nucleotide sequence as shown in SEQ ID NO: 21), and promoter TPIlp (nucleotide sequence as shown in SEQ ID NO: 8) were ligated by using overlap method to obtain gene expression cassette ENO2t-IDPlt-EcSPS-GPMlp-TPIlp. (3) Promoter GPMlp, promoter TPIlp, the nucleic acid sequence of PsTNMT (ACO90237.1), terminator ADHlt (nucleotide sequence as shown in SEQ ID NO: 11), and terminator FBAlt (nucleotide sequence as shown in SEQ ID NO: 12) were ligated by using overlap method to obtain gene expression cassette GPMlp-TPIlp-PsTNMT-ADHlt-FBAlt. (4) Terminator ADHlt, terminator FBAlt, the nucleic acid sequence of PsMSH (GenBank, accession no. AGC92398, version AGC92398.1), promoter PGKlp, and downstream homology arm XI-2 ds (nucleotide sequence as shown in SEQ ID NO: 74) were ligated by using overlap method to obtain gene expression cassette ADHlt-FBAlt-PsMSH-PGKlp-XI-2 ds. (5) Plasmid pMEL10-XI-2 together with the four gene expression cassettes was transformed into XJ0695 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the XI-2 site of XJ0695 strain, affording XJ0743 strain). Western blotting was used to determine protein expression amount, and it was found that BwSTOX and McDBOX2 proteins were well expressed. However, it was detected that even the control XJ07437 strain expressing only EcP6H could produce the target product sanguinarine, whereas dihydrosanguinarine could not be detected in XJ0750 strain into which McDBOX2 was introduced. This result confirmed that McDBOX2 played a key role in the conversion of dihydrosanguinarine into sanguinarine. The specific steps were as follows:

[0670] 1. Plasmid pMELlO-XII-1 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with XII-l-gRNA-F, and XI-5-gRNA-R was replaced with XII-1 - gRNA-R, whereas the other parts of the method remained unchanged.

[0671] 2. Construction of strains

[0672] A nucleic acid-encoding expression cassette comprised a promoter, the nucleic acid sequence of a gene of interest, and a terminator. The gene of interest was AmSTOX (GenBank, accession no. ADY15027, version ADY15027.1), EcP6H (GenBank, accession no. BAK20464, version BAK20464.1), BwSTOX (GenBank, accession no. ADY15026, version ADY15026.1), CjSTOX (GenBank, accession no. BAJ40864, version BAJ40864.1), PsFADOX5 (GenBank, accession no. AGL44334, version AGL44334.1), StBBE (nucleotide sequence as shown in SEQ ID NO: 24, McDBOXl (GenBank, accession no. OVA00267, version OVA00267.1), or McDBOX2 (GenBank, accession no. OVA00268, version OVA00268.1). The promoter was promoter PDClp (nucleotide sequence as shown in SEQ ID NO: 69) or promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminator was terminator DITlt (nucleotide sequence as shown in SEQ ID NO: 75) or terminator TDH2t (nucleotide sequence as shown in SEQ ID NO:6). His-tag sequence was CATCATCACCATCACCAT (SEQ ID NO: 214).

[0673] (1) Upstream homology arm XII-1 us (nucleotide sequence as shown in SEQ ID NO: 76), terminator DITlt, His-tag sequence (CATCATCACCATCACCAT, SEQ ID NO: 214), the nucleic acid sequence of AmSTOX, promoter PDClp, and promoter CCW12p were ligated by using overlap method to obtain gene expression cassette XII-1 us-DITlt-His-AmSTOX-PDClp- CCW12p (named Fragment 38). Promoter PDClp, promoter CCW12p, the nucleic acid sequence of EcP6H, terminator TDH2t, and downstream homology arm XII-1 ds (nucleotide sequence as shown in SEQ ID NO: 77) were ligated by using overlap method to obtain gene expression cassettePDClp-CCW12p-EcP6H-TDH2t-XII-l ds (named Fragment 39). Plasmid pMELlO-XII- 1 together with Fragment 38 and Fragment 39 was transformed into XJ0743 strain competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XII- 1 site of XJ0743 strain afforded XJ07431 strain.

[0674] (2) XJ07432 strain, XJ07433 strain, XJ07434 strain, XJ07435 strain, XJ07436 strain, and XJ0750 strain were respectively obtained according to the method of step (I) 1, except that 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 PsFAD0X5, the nucleic acid sequence of StBBE, the nucleic acid sequence of McDBOXl, and the nucleic acid sequence of McDB0X2, respectively, whereas the other parts of the method remained unchanged.

[0675] (3) Plasmid pMELlO-XII-1 together with gene expression cassette fragment XI 1-1 us- CCW12p-EcP6H-TDH2t-XII-l ds (consisting of upstream homology arm promoter XI 1-1 us, promoter CCW12p, the nucleic acid sequence of EcP6H, terminator TDH2t, and downstream homology arm XI 1-1 ds) was transformed into XJ0743 strain competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XII-1 site of XJ0743 strain afforded XJ07437 strain into which only EcP6H gene was inserted, as a control strain.

[0676] 3. Three positive monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NFLi^SCE, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL ^FLO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODeoonm of the fermentation broth was measured.

[0677] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0678] 5. Determination of protein expression by Western blotting

[0679] Ill (1) Each of the strains obtained in step 2 was inoculated in 1 mL of delft culture medium, and was cultured at 30°C and 220 rpm overnight to obtain a preculture.

[0680] (2) The preculture was transferred to 5 mL of fresh delft culture medium at a ratio of 1:50 and was cultured at 30°C and 220 rpm to obtain a cultured strain solution having an ODgoonm of about 1.

[0681] (3) The cells in the cultured strain solution were collected, first washed twice with PBS buffer, then resuspended by adding 200 pL of acid-washed glass beads and 300 pL of PBS buffer, and then disrupted using a FastPrep rapid sample preparation apparatus to obtain a sample.

[0682] (4) The sample was added with 4 x NuPAGE™ LDS sample buffer (ThermoFisher), and boiled at 95°C for 10 minutes. Protein electrophoresis was run at 150 V for 1 hour.

[0683] (5) The protein was transferred to a Trans-Blot® Turbo™ PVDF transfer membrane (BioRad) and then blocked with PBST (PBS + 0.1% Tween 20) containing 5% milk at room temperature for 2 hours. The membrane was washed 3 times with PBST, and was incubated with 6xHis-Tag monoclonal antibody (His. H8) (ThermoFisher) or anti-GAPDH antibody (G-9) (Santa Cruz Biotechnology) at room temperature for 1 hour. Then, the membrane was washed 3 times with PBST, and was incubated with HRP-conjugated anti-mouse IgG (h+L) secondary antibody (Invitrogen) at room temperature for 1 hour. Then, the membrane was washed 3 times with PBST, and was incubated with West Pico plus HRP substrate (ThermoFisher) for 5 minutes. Finally, analysis was conducted using a ChemiDoc XRS image analyzer (Bio-Rad).

[0684] The detection results are shown in FIG. 16. The results of Western blotting indicated that the size of BwSTOX and McDB0X2 proteins were correct, suggesting good expression, the band of PsFADOX5 was diffuse, and none of AmSTOX, CjTHBO, and McDBOXl was expressed (see FIG. 16c and d). Determination of the metabolic components of the strains indicated that (see FIG. 16a and b) all of the strains produced sanguinarine, including the control strain. However, no dihydrosanguinarine was determined in XJ0750 strain, indicating that McDB0X2 gene fulfilled the function of converting dihydrosanguinarine into sanguinarine in the yeast. That is, among the flavoprotein oxidase genes obtained by screening in this experiment, McDB0X2 gene was the only flavoprotein oxidase gene that fulfilled the function of catalyzing the oxidation of dihydrosanguinarine to produce sanguinarine in the yeast.

[0685] (c) Screening of P6H genes P6H genes from three different sources were respectively verified in XJ0741 strain together with McDB0X2 screened in step (II). Under the same fermentation conditions, XJ0747 strain expressing EcP6H had the highest amount of the final product chelerythrine. This experiment identified EcP6H as an element for constructing this pathway. The specific steps were as follows:

[0686] 1. Plasmid pMEL10-XII-l was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with XII-l-gRNA-F, and XI-5-gRNA-R was replaced with XII-1 - gRNA-R, whereas the other parts of the method remained unchanged.

[0687] 2. Construction of strains

[0688] A nucleic acid-encoding expression cassette comprised a promoter, the nucleic acid sequence of a gene of interest, and a terminator. The gene of interest was EcP6H (GenBank, accession no. BAK20464, version BAK20464.1), McP6H (GenBank, accession no. OVA18597, version 0VA18597.1), CyP6H (nucleotide sequence as shown in SEQ ID NO: 86), or McDB0X2 (GenBank, accession no. OVA00268, version OVA00268.1).The promoter was promoter PDClp (nucleotide sequence as shown in SEQ ID NO: 69) or promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminator was terminator DITlt (nucleotide sequence as shown in SEQ ID NO: 75), or terminator TDH2t (nucleotide sequence as shown in SEQ ID NO: 6). His-tag sequence was CATCATCACCATCACCAT (SEQ ID NO: 214).

[0689] (1) Upstream homology arm XII-1 us, terminator DITlt, His-tag sequence (CATCATCACCATCACCAT, SEQ ID NO: 214), the nucleic acid sequence of McDB0X2, promoter PDClp, and promoter CCW12p were ligated by using overlap method to obtain gene expression cassette XII-1 us-DITlt-His-McDBOX2-PDClp-CCW12p. Promoter PDClp, promoter CCW12p, the nucleic acid sequence of EcP6H, terminator TDH2t, and downstream homology arm XII-1 ds were ligated by using overlap method to obtain gene expression cassette PDClp-CCW12p-EcP6H-TDH2t-XII-l ds. Plasmid pMELlO-XII-1 was used to transform the two gene expression cassette into XJ0741 yeast competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XII-1 site of XJ0741 strain afforded XJ0747 strain.

[0690] (2) XJ0748 strain and XJ0749 strain were obtained according to the method of step (I) 1, except that the nucleic acid sequence of EcP6H was replaced with the nucleic acid sequence of McP6H and the nucleic acid sequence of CyP6H respectively, whereas the other parts of the method remained unchanged.

[0691] 3. Three positive monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NEL^SCL, 14.4 g / L of KH2PO4, 0.5 g / L of MgSCL VIfcO, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0692] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0693] The detection results are shown in FIG. 15e, f, and g. The results showed that XJ0747 strain, XJ0748 strain, and XJ0749 strain were all able to produce the final product chelirubine, and XJ0747 strain expressing EcP6H had the highest amount of the final product chelirubine. Therefore, EcP6H was the most suitable gene element among the three candidate P6H genes for the construction of an engineered strain for chelirubine.

[0694] VIIL Optimization of the McDBOX step in the chelirubine pathway

[0695] Research has found that McDB0X2 would decrease the ODgoonm of a strain and increase the accumulation amount of the intermediate product reticuline, suggesting that McDB0X2 had some inhibitory effect on the function of BBE. The present invention adopted the following strategies for perform pathway optimization: (1) overexpression of plasma membrane riboflavin transporter MCH5 and FAD synthase BsRibc to increase FAD supply, with the finding that the yield of chelirubine was increased by 24.4% and 39.2%, presumably due to increased McDB0X2 activity resulting from expression of MCH5 and BsRibc; (2) truncation of 29 amino acids from the N-terminus of McDB0X2 to increase the expression thereof in the cytosol, with the finding that XJ07472 strain after McDB0X2 truncation (XJ07411+McDBOX2A29) could convert all dihydrochelirubine to chelirubine; and (3) knockout of phospholipase OPIlto mediate endoplasmic reticulum expansion. Finally, XJ07474 strain having a yield of chelirubine reaching 38.1 mg / L was obtained.

[0696] (I) Increasing FAD supply

[0697] Genes for increasing FAD supply was introduced into XJ0774 strain by means of plasmid transformation (XJ0774 strain was prepared as follows: (1) Artificially synthesized DNA fragment LP1.T8 (nucleotide sequence as shown in SEQ ID NO: 90) was used as a template, and a primer pair consisting of FgF20-LPl.T8-F and LPl.T8-CCW12p-R were used to perform PCR amplification to obtain Fragment FgF20 us-LPl.T8. (2) The genomic DNA of XJ0747 strain was used as a template, and primer pair Y1 consisting of CCW12pl-F and TDH2t-FBAlt-R, primer pair Y2 consisting of FBAlt400-r-RandPGKlp-IDPlt-R, and primer pair Y3 consisting of IDPlt- r-F and GPM1 p-FgF20-R were used respectively to perform PCR amplification to obtain Fragment CCW12p-EcP6H-TDH2t-FBAlt, Fragment FBAlt-PsMSH-PGKlp, and Fragment IDPlt-EcSPS- GPMlp-FgF20 ds, respectively. (3) Plasmid pMEL10-FgF20 together with the above four fragments was transformed into XJ0747 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, the fragments were integrated into the FgF20 site of XJ0747 strain, affording XJ0774 strain.) The genes for increasing FAD supply include carrying FAD ER localization transport protein FLCI (yeast endogenous gene), FLC2 (yeast endogenous gene), FLC3 (yeast endogenous gene), and YOR365C (yeast endogenous gene), mitochondrial FAD transport protein FLX1 (yeast endogenous gene), plasma membrane riboflavin transporter MCH5 (nucleotide sequence as shown in SEQ ID NO: 78), FAD biosynthesis related gene FMN1 (yeast endogenous gene) and FADI (yeast endogenous gene), and Bacillus subtilis-derived FAD synthase BsRiba (nucleotide sequence as shown in SEQ ID NO: 87) and BsRibc (nucleotide sequence as shown in SEQ ID NO: 79). The effect of the above candidate genes was evaluated by comparing the yield of CHE in the strains under the same fermentation conditions. The specific steps were as follows:

[0698] 1. Construction of recombinant plasmids

[0699] (l)Yeast genomic DNA was used as a template, and primer pair 11 (consisting of GPD- FLC1-F and FLCI -CYC It-R), primer pair 12 (consisting of GPD-FLC2-F and FLC2-CYClt-R), primer pair 13 (consisting of GPD-YOR3-F and YOR365C-CYClt-R), primer pair 14 (consisting of GPD-FLC3-F and FLC3-CYClt-R), primer pair 15 (consisting of GPD-MCH5-F and MCH5- CYClt-R), and primer pair 16 (consisting of GPD-FLX1-F and FLXl-CYClt-R) were respectively used to perform PCR amplification (the enzyme for the PCR amplification was PrimeStar HS DNA polymerase (Takara Co., R010B), to obtain Fragment FLCI, Fragment FLC2, Fragment YOR365C, Fragment FLC3, Fragment MCH5, and Fragment FLX1, respectively.

[0700] The reaction procedure was: 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; extension at 70°C for 10 min.

[0701] (2) Bacillus subtilis genomic DNA was used as template, and primer pair 17 (consisting of GPD-BsRibc-F and BsRibc-CYClt-R) and primer pair 18 (consisting of GPD-BsRiba-F and BsRiba-CYClt-R) were respectively used to perform PCR amplification (the enzyme for the PCR amplification was PrimeStar HS DNA polymerase) to obtain Fragment BsRibc and Fragment BsRiba, respectively.

[0702] The reaction procedure was: 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; extension at 70°C for 10 min.

[0703] (3) Fragment FLCI, Fragment FLC2, Fragment YOR365C, Fragment FLC3, Fragment MCH5, Fragment FLX1, Fragment BsRibc, Fragment BsRiba, and artificially synthesized fusion fragment of FMN1 and FADI (nucleotide sequence as shown in SEQ ID NO: 88) were respectively subjected to Gibson ligation with linearized plasmid P416 GPD to obtain 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, and recombinant plasmid P416 GPD- FMN1+FAD1, respectively.

[0704] Plasmid P416 GPD (purchased from ATCC strain library, URL https: / / www.atcc.org / search#q=P416&sort=relevancy&numberOfResults=24) was used as a template, and a primer pair consisting of CYClt-p416-F and GPD-p416-R were used to perform PCR amplification to obtain linearized plasmid P416 GPD.

[0705] (2) Plasmids (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) were introduced into XJ0774 strain to obtain recombinant strains.

[0706] 3. Three positive monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO447H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, and 1 ml / L of vitamin solutions, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0707] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0708] The detection results are shown in FIG. 17. The results indicated that, compared with the recombinant strain into which plasmid P416 GPD was introduced (i.e., control strain), the recombinant strains into which recombinant plasmid P416 GPD-MCH5 and recombinant plasmid P416_GPD-BsRibc were introduced increased the yield of CHE by 24.4% and 39.2%, respectively, whereas the recombinant strains into which other recombinant plasmids were introduced had no significant effect on CHE production. However, the amount of S-RET was not decreased in the strain expressing MCH5 or BsRibc, suggesting that the expression of MCH5 or BsRibc might have increased McDB0X2 activity at this stage, resulting in the increased yield of CHE, rather than mitigating the limitation of McDBOX on the catalytic effect of CyBBE (see FIG. 17b). The results indicated that overexpression of MCH5 fragment or BsRibc fragment could increase FAD supply and increase the yield of CHE in an engineered strain.

[0709] (II) Truncation of 29 amino acids from the N-terminus of McDB0X2 to increase the expression thereof in cytosol

[0710] 29 amino acids were truncated from the N-terminus of McDB0X2 (GenBank, accession no. OVA00268, version OVA00268.1), and the effect of the N-terminal truncation on the activity was compared. The control group was XJ07411 strain containing no McDB0X2 andXJ0747 strain containing McDB0X2, and the experimental group was XJ07472 strain containing McDBOX2_A29. The growth curve, CHE amount, intermediate conversion, etc., of the three strains under the same fermentation conditions were determined to evaluate whether truncation of 29 amino acids from the N-terminus of McDB0X2 could facilitate the construction of target engineered strains. The specific steps were as follows:

[0711] 1. Plasmid construction

[0712] (1) Plasmid pMEL10-XII-l was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with XII-l-gRNA-F, and XI-5-gRNA-R was replaced with XII-1- gRNA-R, whereas the other parts of the method remained unchanged.

[0713] (2) Plasmid pMEL10-FgF20 was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with FgF20-gRNA-F, and XI-5-gRNA-R was replaced with FgF20-gRNA-R, whereas the other parts of the method remained unchanged.

[0714] 2. Construction of strains

[0715] An encoding nucleic acid expression cassette comprised an upstream homology arm, a promoter, the nucleic acid sequence of a gene of interest, a terminator, and a downstream homology arm. The promoter was promoter PDClp (nucleotide sequence as shown in SEQ ID NO: 69) or promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminator was terminator DITlt (nucleotide sequence as shown in SEQ ID NO: 75), or terminator TDH2t (nucleotide sequence as shown in SEQ ID NO: 6).

[0716] (1) Plasmid pMEL10-XII-l and the gene expression cassette fragment of EcP6H (consisting of upstream homology arm XII- 1 us, promoter CCW12p, the nucleic acid sequence of EcP6H, terminator TDH2t, and downstream homology arm XII- Ids) were transformed into XJ0741 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the XII-1 site of XJ0741 strain afforded XJ07411 strain (comprising no McDB0X2 gene).

[0717] (2) Plasmid pMELl 0-FgF20 and the gene expression cassette fragment of McDBOX2_A29 (consisting of upstream homology arm FgF20 us (nucleotide sequence as shown in SEQ ID NO: 67), promoter PDClp, the nucleic acid sequence of McDBOX2_A29, terminator DITlt, and downstream homology arm FgF20 ds (nucleotide sequence as shown in SEQ ID NO: 68) were transformed into XJ07411 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the FgF20 site of XJ07411 strain afforded XJ07472 strain (i.e., XJ07411+McDBOX2_A29).

[0718] 3. Three positive monoclones of each of the strains obtained in step 2 were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4’7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0719] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0720] 5. Determination of protein expression by Western blotting

[0721] (1) Each of the strains obtained in step 2 was inoculated in 1 mL of delft culture medium, and was cultured at 30°C and 220 rpm overnight to obtain a preculture.

[0722] (2) The preculture was transferred to 5 mL of fresh delft culture medium at a ratio of 1:50 and was cultured at 30°C and 220 rpm to obtain a cultured strain solution having an ODgoonm of about 1.

[0723] (3) The cells in the cultured strain solution were collected, first washed twice with PBS buffer, then resuspended by adding 200 pL of acid-washed glass beads and 300 pL of PBS buffer, and then disrupted using a FastPrep rapid sample preparation apparatus to obtain a sample.

[0724] (4) The sample was added with 4 x NuPAGE™ LDS sample buffer (ThermoFisher), and boiled at 95°C for 10 minutes. Protein electrophoresis was run at 150 V for 1 hour.

[0725] (5) The protein was transferred to a Trans-Blot® Turbo™ PVDF transfer membrane (BioRad) and then blocked with PBST (PBS + 0.1% Tween 20) containing 5% milk at room temperature for 2 hours. The membrane was washed 3 times with PBST, and was incubated with 6xHis-Tag monoclonal antibody (His. H8) (ThermoFisher) or anti-GAPDH antibody (G-9) (Santa Cruz Biotechnology) at room temperature for 1 hour. Then, the membrane was washed 3 times with PBST, and was incubated with HRP-conjugated anti-mouse IgG (h+L) secondary antibody (Invitrogen) at room temperature for 1 hour. Afterwards, the membrane was washed 3 times with PBST, and was incubated with West Pico plus HRP substrate (ThermoFisher) for 5 minutes. Finally, analysis was conducted using a ChemiDoc XRS image analyzer (Bio-Rad).

[0726] The results of Western blotting analysis are shown in FIG. 18. The results showed that the strain expressing McDBOX2_A29 exhibited a protein band having a size close to the theoretical 56.8 kDa of McDBOX2A29, whereas the strain expressing McDB0X2 exhibited a blot of multiple bands (see FIG. 18a); the strain expressing McDBOX2_A29 exhibited no significant difference in growth ODsoonm value compared with the strain expressing McDBOX2 (FIG. 18b); the amount of the intermediate RET was not increased (FIG. 18c). The above results indicated that truncation of 29 amino acids from the N-terminus of McDBOX2 to increase the expression thereof in cytosol could effectively resolve the inhibition of McDBOX2 gene on the growth of cells and on the conversion of the intermediate RET.

[0727] (Ill) Integration of FAD enhancement strategies

[0728] Chelerythrine engineered strains were constructed using the McDBOX2 N-terminal truncation (McDBOX2_A29) strategy and the endoplasmic reticulum expansion strategy. On the basis of XJ07472 strain (comprising the complete CHE pathway of McDBOX2_A29), plasma membrane riboflavin transporter MCH5 (nucleotide sequence as shown in SEQ ID NO: 78) and FAD synthase BsRibc (nucleotide sequence as SEQ ID NO: 79) were overexpressed to increase FAD supply, and phospholipase OPI1 was knocked out to mediate endoplasmic reticulum expansion, finally affording XJ07474 strain which had a yield of chelerythrine reaching 38.1 mg / L. The specific steps were as follows:

[0729] 1. Overexpression ofMCH5 and BsRibc (construction of XJ07473 strain)

[0730] A nucleic acid-encoding expression cassette consisted of an upstream homology arm, a promoter, the nucleic acid sequence of a gene of interest, a terminator, and a downstream homology arm. The promoter was promoter GPD (nucleotide sequence as shown in SEQ ID NO: 81) or promoter CCW12p (nucleotide sequence as shown in SEQ ID NO: 2). The terminator was terminator IDPlt (nucleotide sequence as shown in SEQ ID NO: 3), or terminator CYClt (nucleotide sequence as shown in SEQ ID NO: 13).

[0731] (1) Upstream homology arm 416d us (nucleotide sequence as shown in SEQ ID NO: 59), promoter GPD, the nucleic acid sequence of MCH5, terminator IDPlt, and promoter CCW12p were ligated by using overlap method to obtain gene expression cassette 416d us-GPD-MCH5- IDPlt-CCW12p (named Fragment 40). Terminator IDPlt, promoter CCW12p, the nucleic acid sequence of BsRibc, terminator CYC It, and downstream homology arm 416d ds (nucleotide sequence as shown in SEQ ID NO: 60) were ligated by using overlap method to obtain gene expression cassette IDPlt-CCW12p-BsRibc-CYClt-416d ds (named Fragment 41).

[0732] (2) Plasmid pMEL10-416d together with Fragment 40 and Fragment 41 together was transformed into XJ07472 competent cells to obtain positive clones. The positive clones were randomly selected and streaked onto a 5-FOA plate, and were cultured in an incubator at 30°C for 2 to 3 d. The grown clone was the strain that lost the gRNA plasmid. That is, integration into the 416d site of XJ07472 strain afforded XJ07473 strain.

[0733] 2 Knockout of phospholipase OPI1 (construction of XJ07474 strain)

[0734] (1) Plasmid pMELlO-OPIl was obtained according to the method of step (I) 1, except that XI-5-gRNA-F was replaced with OPIl-gRNA-F, and XI-5-gRNA-R was replaced with OPI1- gRNA-R, whereas the other parts of the method remained unchanged.

[0735] (2) Plasmid pMELlO-OPIl was used to integrate repair fragment LP3.T7 (nucleotide sequence as shown in SEQ ID NO: 89) into the site of OPI1 gene of XJ07473 strain to knock out OPI1 gene, affording XJ07474 strain.

[0736] 3. Three monoclones of XJ07472 strain, of XJ07473 strain, and of XJ07474 strain were picked for parallel experiments. 1 ml of Deft culture medium (solutes and concentrations thereof: 7.5 g / L of (NH4)2SO4, 14.4 g / L of KH2PO4, 0.5 g / L of MgSO4-7H2O, 20 g / L of glucose, 2 ml / L of trace metal solutions, 1 ml / L of vitamin solutions and 60 mg / L of uracil, the solvent being water) was added to a 14 mL shake tube. Then, the monoclone was inoculated, and cultured at 30°C and 220 rpm for 18 to 24 h to obtain a strain solution. The strain solution was inoculated into a shake flask (spec. 125 mL) containing 20 mL of Deft culture medium for scale-up culture, and was cultured at 30°C and 220 rpm for 72 h to obtain a fermentation broth. The ODgoonm of the fermentation broth was measured.

[0737] 4. 500 pL of the fermentation broth was added with 500 pL of 30% acetonitrile. The mixture was vortexed well, and was centrifuged at 13000 g for 5 min. The supernatant was collected for LC-MS / MS detection.

[0738] The detection results are shown in FIG. 19. The results indicated that by integrating FAD enhancement strategy (overexpression of MCH5 and BsRibc), McDB0X2 N-terminal truncation (McDBOX2_A29) strategy, and endoplasmic reticulum expansion strategy (knockout of phospholipase OPI1), XJ07474 strain having a yield of chelerythrine reaching 38.1 mg / L was finally obtained.

[0739] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be carried out in a wider scope under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the present invention has been shown and described in particular embodiments, it will be understood that further modifications may be made to the present invention. In conclusion, in light of the principle of the present invention, the present application is intended to encompass any variations, uses, or modifications to the present invention, including alterations that depart from the disclosed scope of the present application but are made by using conventional techniques known in the art.

Claims

CLAIMS1. A recombinant yeast strain for production of a benzylisoquinoline alkaloid, preferably scoulerine, characterized in that the recombinant yeast strain comprises: a heterologous berberine bridge enzyme (BBE); and a heterologousbenzophenanthridine oxidase (DBOX).

2. The recombinant yeast strain according to claim 1, wherein the heterologous BBE is selected from the group consisting of PsBBE derived from Papaver somniferum, CjBBE derived from Coptis japonica and CyBBE derived from Corydalis yanhusuo, preferably CyBBE derived from C. yanhusuo.

3. The recombinant yeast strain according to claim 1 or 2, wherein the recombinant yeast strain comprises the heterologous BBE in the form of a fusion protein of the heterologous BBE with an endoplasmic reticulum C-terminal retention signal peptide (HDEL), and / or a fusion protein of the heterologous BBE with an N-terminal Golgi target sequence.

4. The recombinant yeast strain according to any one of claims 1 to 3, wherein the heterologous DBOX is McDB0X2 derived from Macleaya cordata.

5. The recombinant yeast strain according to any one of claims 1 to 4, wherein the recombinant yeast strain comprises the heterologous DBOX in the form of an N-terminal truncation of the heterologous DBOX, preferably McDB0X2 derived from Macleaya cordata truncated by 29 amino acids from the N-terminus of McDB0X2 (McDBOX2_A29).

6. The recombinant yeast strain according to any one of claims 1 to 5, further comprising: a heterologous S-cheilanthifoline synthase (CFS), preferably selected from the group consisting of EcCFS derived from Eschscholzia califomica, CyCFS derived from Corydalis yanhusuo, and PsTNMT derived imm Papaver somniferum, more preferably EcCFS; and a heterologous S-stylopine synthase (SPS), preferably selected from the group consisting of EcSPS derived from E. califomica, CySPS derived from C. yanhusuo, or PsMSH derived from P. somniferum, more preferably EcSPS.

7. The recombinant yeast strain according to any one of claims 1 to 5, further comprising a heterologous O-methyltransferase, preferably TfS9OMT derived from Thalictrum flavum.

8. The recombinant yeast strain according to any one of claims 1 to 5, 7, further comprising a heterologous S -tetrahydroberberine synthase (CAS), preferably selected from the group consisting of CjCAS derived from Coptis japonica and CyCAS derived from Corydalis yanhusuo .

9. The recombinant yeast strain according to any one of claims 1 to 8, further comprising a heterologous A-methyltransferase (TNMT), preferably PsTNMT derived from Papaver somniferum.

10. The recombinant yeast strain according to any one of claims 1 to 9, further comprising a heterologous S-cis A-methylstylopine 14-hydroxylase (MSH), preferably PsMSH derived from Papaver somniferum.

11. The recombinant yeast strain according to any one of claims 1 to 10, further comprising a heterologous protopine-6-hydroxylase (P6H), preferably EcP6H derived from Eschscholzia califomica.

12. The recombinant yeast strain according to any one of claims 1 to 11, further comprising the dihydrosanguinarine 10-hydroxylase CYP82P2 derived from Eschscholzia califomica.

13. The recombinant yeast strain according to any one of claims 1 to 12, further comprising a heterologous 10-hydroxymethyltransferase (OMT), preferably selected from the group consisting of Ecl lOMT derived from E. califomica and Ec2OMT derived from E. califomica, preferably Ecl 1 OMT.

14. The recombinant yeast strain according to any one of claims 1 to 13, further comprising: a plasma membrane riboflavin transporter, preferably the plasma membrane riboflavin transporter MCH5 and / ora heterologous FAD synthase, preferably BsRibc derived from Bacillus subtilis.

15. The recombinant yeast strain according to any one of claims 1 to 14, further comprising: a heterologous tyrosine hydroxylase, preferably selected from the group consisting of BvCYP76AD5 derived from Beta vulgaris, BvCYP76ADl* that is a mutated version of BvCYP76ADl derived from Beta vulgari with the W13L and F309L mutations, AtC3H derived from Arabidopsis thaliana, ZmC3H derived from Zea mays, PaHpaB derived from Pseudomonas aeruginosa, PaHpaC derived from Pseudomonas aeruginosa and SeHpaC derived from Salmonella, more preferably BvCYP76AD5, and a heterologous DOPA decarboxylase preferably DOPA decarboxylase (DODC) derived from Pseudomonas putida, and / or a heterologous norcoclaurine synthase (NCS), preferably CjNCS derived from Coptis japonica, or an N-terminally truncated NCS, preferably selected from the group consisting CjNCSN_A35 corresponding to CjNCS derived from C. japonica with 35 amino acids truncated from the N-terminus of CjNCS, CjNCSN_A24 corresponding to CjNCS derived from C. japonica with 24 amino acids truncated from the N-terminus of CjNCS, and CjNCSN_A29 corresponding to CjNCS derived from C. japonica with 29 amino acids truncated from the N-terminus of CjNCS; and / or a tyrosine metabolic flow-related protein, preferably a heterologous tyrosine metabolic flow- related protein selected from the group consisting of EcAROL derived from Escherichia coli, ARCH* that is a mutated version of endogenous AR04 with the K229L mutation, AR07* that is a mutated version of endogenous AR07 with the G141S mutation, and MtPDHl derived from Medicago truncatukr, and / or at least one endogenous gene encoding for a respective protein involved in the synthesis activities of 4-hydroxyphenylethyl alcohol and 4-hydroxyphenylacetic acid is knocked out or down regulated in the recombinant yeast strain, optionally wherein the respective protein is selected from the group consisting of ARI1, ADH6, YPR1, YDR541C, AAD3, GRE2, and HFD1; and / or a heterologous aromatic amino acid decarboxylase, preferably selected from the group consisting of PcAAS derived from Petroselinum crispum, RrAAS derived from Rhodiola Salidroside, and PsAAAD* that is a mutated version of PsAAAD derived from Papaver somniferum with the Y350F mutation; and / ora heterologous prephenate transaminase (PAT), preferably selected from the group consisting of AtPAT derived from Arabidopsis thaliana, BvPATl derived from Beta vulgaris and BvPAT2 derived from B. vulgaris, more preferably AtPAT, and a heterologous arogenate dehydratase (ADH), preferably selected from the group consisting of BvADHa derived from B. vulgaris and MtncADH derived from Medicago truncatula, more preferably MtncADH; and / or a heterologous methyltransferase (60MT), preferably selected from the group consisting of Ps6OMT derived from Papaver somniferum and Cy60MT derived from Corydalis yanhusuo, more preferably Ps6OMT, and a heterologous coclaurine methyltransferase (CNMT), preferably selected from the group consisting of PsCNMT derived from P. somniferum and CyCNMT derived from C. yanhusuo, more preferably PsCNMT; and / or a heterologous methyltransferase (4’0MT), preferably Ps4’OMT derived from Papaver somniferum, and a heterologous N-methylcoclaurine 3'-hydroxylase (NMCH), preferably selected from the group consisting of EcNMCH derived from Eschscholzia califomica and CyNMCH derived from C. yanhusuo, more preferably CyNMCH; and / or a cytochrome P450 reductase, preferably selected from the group consisting of ATR1 derived from Arabidopsis thaliana, ATR2 derived from A. thaliana, and PsCPR derived from Papaver somniferum, more preferably ATR1; and / or a catalase, preferably a catalase N-terminally fused with a yeast oc -mating factor signal peptide and C-terminally fused with an endoplasmic reticulum retention signal peptide; and / or a transcription factor activating phospholipid biosynthesis preferably wherein the transcription factor is selected from the group consisting of endogenous INO2 and INO2* that is mutated version of endogenous INO2 with the LI 19A mutation or has an endogenous transcription factor, preferably IPI1, suppressing phospholipid biosynthesis knocked.

16. The recombinant yeast strain according to any one of claims 1 to 15, wherein the recombinant yeast strain A is from a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Candida, Hansenula, Torulopsis, Kloeckera, Pichia, Schizosaccharomyces, Trigonopsis, Brettanomyces, Debaromyces, Nadsonia, Upomyces, Cryptococcus, Aureobasidium, Trichosporon, Upomyces, Rhodotorula, Yarrowia, Phaffia, or Schwanniomyces, preferably selected from the group consisting of Saccharomyces, Yarrowia, Zygosaccharomyces, Kluyveromyces and Pichia.

17. The recombinant yeast strain according to claim 16, wherein the recombinant yeast strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Zygosaccharomyces bailii, Kluyveromyces lactis, and Yarrow ia lipolytica, preferably Saccharomyces cerevisiae.

18. A method for producing a benzophenanthridine alkaloid, comprising the following steps: fermentatively culturing a recombinant yeast strain according to any one of claims 1 to 17; collecting a fermentation product from the fermentatively cultured recombinant yeast strain; and obtaining the benzophenanthridine alkaloid therefrom.

19. The method according to claim 18, wherein fermentatively culturing comprises fermentatively culturing a recombinant yeast strain according to any one of claims 1 to 6, or 9-17 when dependent on any one of claims 1-6; and the benzophenanthridine alkaloid is selected from the group consisting of sanguinarine, dihydrosanguinarine, protopine, -cA-A-methylstylopine, S-stylopine, chelirubine, 10- hydroxydihydrosanguinarine20. The method according to claim 18, wherein fermentatively culturing comprises fermentatively culturing a recombinant yeast strain according to any one of claims 1 to 5, 7, 8, or 9-11, or 14-17 when dependent on any one of claims 1-5, 7, 8; and the benzophenanthridine alkaloid is selected from the group consisting of chelerythrine, allocryptopine, and / or S -tetrahydroberberine.

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