Saccharomyces cerevisiae genetically engineered bacterium for de novo synthesis of verbascoside and echinacoside and application of saccharomyces cerevisiae genetically engineered bacterium

Abstract

The invention relates to the field of gene engineering, in particular to saccharomyces cerevisiae gene engineering bacteria for producing verbascoside and echinacoside through de novo synthesis and application of the saccharomyces cerevisiae gene engineering bacteria. The genetically engineered bacterium contains a coded chorismate synthase mutant, a chorismate mutase mutant, shikimic acid kinase, cytochrome b5, glucose-6-phosphate-dehydrogenase, aromatic acetaldehyde synthase, UDP (User Datagram Protocol) glucosyltransferase, tyrosine ammonia lyase, phenylalanine ammonia lyase, cinnamate hydroxylase, 4-coumaric acid: coenzyme A ligase and acyltransferase. The invention relates to a gene encoding a UDP glucosyltransferase mutant, and more preferably, the gene encoding the UDP glucosyltransferase mutant comprises genes of a UDP glucosyltransferase mutant, a UDP glucosyltransferase mutant, a UDP glucosyltransferase mutant and a UDP glucosyltransferase mutant. Under a shake flask condition, verbascoside produced by the strain reaches 231 mg / L, echinacoside produced by the strain reaches 184 mg / L, and the strain has industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a Saccharomyces cerevisiae genetically engineered strain for de novo synthesis of verbascoside and echinacoside, and the application of this genetically engineered strain in the production of verbascoside and echinacoside. Background Technology

[0002] Phenylpropanoid glycosides (PhGs) are a class of natural phenolic acids widely distributed in plants of the order Lamiaceae, possessing antioxidant, anticancer, and memory-enhancing functions. Structurally, they are water-soluble natural glycosides containing hydroxymethoxy-substituted phenethyl and hydroxymethoxy-substituted cinnamic groups, typically with a central glucose core, and also exhibiting ester and oxyglycosidic bonds. PhGs with the C3 hydroxyl group of glucose replaced by a rhamnosyl group are the most representative; verbascoside and echinacoside are examples of this class of compounds.

[0003] Verbascoside and echinacoside are phenolic acid compounds from plants. As important representatives of complex phenylethanoid glycosides, they have long been used as important active substances in various medicinal plants. For example, verbascoside and echinacoside are among the main active ingredients of Rehmannia glutinosa and Cistanche deserticola, traditional Chinese medicinal plants. The Chinese Pharmacopoeia stipulates that the content of verbascoside is one of the reference standards for the quality of these medicinal materials. Pharmacological studies over the past thirty years have shown that verbascoside and echinacoside have antioxidant, anti-inflammatory, antibacterial, antitumor, and free radical scavenging effects. They also play an important role in the repair of nervous system damage, the regulation of the digestive system, the regulation of the immune system, and the improvement of memory. Therefore, they have great potential in new drug development.

[0004] Currently, the production of verbascoside and echinacoside mainly relies on plant extraction. This method has inherent limitations, such as being constrained by factors like arable land area, weather conditions, and long production cycles, as well as separation and purification problems caused by low content and complex compound composition. The chemical synthesis of verbascoside has been reported, but due to its complex compound structure, it faces challenges such as multiple synthetic steps, low yield, and low recovery rate.

[0005] With the advancement of synthetic biology technology, the production of plant natural products through environmentally friendly microbial fermentation has become a development trend, offering advantages such as short growth cycles, small footprint, simple separation and purification, immunity to environmental influences, and stable production. However, there are currently no reports on engineered strains of *Saccharomyces cerevisiae* for producing verbascoside and echinacoside, nor on their applications. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects in the prior art and provide a genetically engineered Saccharomyces cerevisiae strain capable of producing verbascoside and echinacoside. This genetically engineered strain can produce verbascoside and echinacoside using glucose as a carbon source through microbial fermentation.

[0007] Through extensive research, the inventors of this invention constructed a mutant ARO4 that encodes branching acid synthase. K229L Gene, branching acid mutase mutant ARO7 G141S Genes including shikimate kinase AROL gene, aromatic acetaldehyde synthase AAS gene, UDP glucosyltransferase UGT gene, tyrosine ammonia-lyase TAL gene, phenylalanine ammonia-lyase PAL gene, cinnamate hydroxylase C4H gene, 4-coumaric acid:coenzyme A ligase 4CL gene, acyltransferase BAHD-AT gene, rhamnose biosynthetic enzyme RHM2 gene, UDP rhamnose syltransferase URT gene, cytochrome P450 reductase CPR gene, cytochrome P450 enzyme CYP gene, and UDP glucosyltransferase mutant FsGT61. F144H A genetically engineered *Saccharomyces cerevisiae* strain was developed. Using glucose as the sole carbon source, it achieved de novo microbial synthesis of verbascoside and echinacoside. Furthermore, the yield of verbascoside obtained through the genetically engineered strain provided by this invention can reach the gram level. The molecular formula of verbascoside is C1. 29 H 36 O 15 With a molecular weight of 624.59 and CAS number 61276-17-3, its structural formula is shown in Formula I, and its English name is Ver bascoside. The molecular formula of echinacoside is C1. 35 H 46 O 20 It has a molecular weight of 786.26, CAS number 82854-37-3, structural formula shown in Formula II, and its English name is Echinacoside.

[0008]

[0009] Based on the above, in one aspect, the present invention provides a genetically engineered *Saccharomyces cerevisiae* strain for producing verbascoside or echinacoside as described above, wherein the genetically engineered strain contains a mutant ARO4 capable of encoding cladistic acid synthase. K229L The gene, branch acid mutase mutant ARO7 G141SGenes of shikimate kinase AROL, aromatic acetaldehyde synthase AAS, UD P glucosyltransferase UGT, tyrosine ammonia-lyase TAL, phenylalanine ammonia-lyase PAL, cinnamate hydroxylase C4H, 4-coumaric acid:coenzyme A ligase 4CL, acyltransferase BAHD-AT, cytochrome P450, rhamnose biosynthetic enzyme RHM2, and UDP rhamnose syltransferase URT.

[0010] Among them, the branched acid synthase mutant ARO4 K229L The gene, branch acid mutase mutant ARO7 G141S The genes for shikimate kinase AROL and acetaldehyde kinase AAS and UDP-glucosyltransferase UGT can enhance the shikimate pathway and increase the synthesis of precursors phenylalanine and tyrosine; the genes for acetaldehyde synthase AAS and UDP-glucosyltransferase UGT can catalyze the synthesis of the intermediate rhodioloside; the genes for tyrosine ammonia-lyase TAL, phenylalanine ammonia-lyase PAL, and cinnamic acid hydroxylase C4H catalyze the synthesis of the intermediate p-coumaric acid; the genes for acyltransferase BAHD-AT and rhamnose biosyntheticase RHM2 catalyze the synthesis of the intermediate laurin B; cytochrome P450 can catalyze the conversion of laurin B to verbascoside; and finally, the gene for UDP-rhamnose kinase URT catalyzes the conversion of verbascoside to echinacoside.

[0011] Preferably, the genetically engineered bacteria also contain genes for cytochrome P450 reductase CPR and cytochrome P450 enzyme CYP, for the synthesis of verbascoside.

[0012] More preferably, the genetically engineered bacteria also contain the UDP-glucosyltransferase mutant FsGT61. F144H The gene is used for the synthesis of echinacoside.

[0013] Secondly, the present invention provides the application of the genetically engineered Saccharomyces cerevisiae as described above in the production of verbascoside and echinacoside.

[0014] Thirdly, the present invention provides a method for de novo synthesis of verbascoside and echinacoside, wherein the genetically engineered bacteria described above are cultured in a medium with glucose as the sole carbon source, constitutively overexpressing the exogenous gene to efficiently produce verbascoside and echinacoside.

[0015] The genetically engineered bacteria of this invention, which produce verbascoside or echinacoside, can de novo produce the compounds verbascoside and echinacoside in the presence of glucose, achieving maximum shake-flask yields of 230.64 mg / L for verbascoside and 184.19 mg / L for echinacoside. Therefore, based on the relevant metabolic pathways, this invention constructs them in *Saccharomyces cerevisiae*, including selecting a more suitable *Saccharomyces cerevisiae* chassis for plant cytochrome P450 expression, and optimizing metabolic pathways, including optimizing the supply of precursor rhodioloside and UDP-rhamnose, and increasing the transcription level of restriction genes, to achieve efficient de novo synthesis of verbascoside and echinacoside. While de novo synthesis of verbascoside and echinacoside by heterologous microorganisms has not been reported before, this invention is the first to achieve de novo synthesis of complex phenylethanoid glycosides (verascoside and echinacoside) in a heterologous host, using only glucose as a precursor and achieving high yields. Therefore, this invention provides a genetically engineered Saccharomyces cerevisiae strain for de novo production of verbascoside and echinacoside, laying the foundation for the large-scale industrial production of verbascoside and echinacoside, and possessing significant scientific research value and industrial application prospects. Detailed description of the invention:

[0017] The present invention will now be described in detail.

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] In a first aspect, the present invention discloses a Saccharomyces cerevisiae genetically engineered strain for de novo production of verbascoside, the genetically engineered strain containing a cladization acid synthase mutant ARO4. K229L Gene, branching acid mutase mutant ARO7 G141S Genes including shikimate kinase AROL gene, aromatic acetaldehyde synthase AAS gene, UDP glucosyltransferase UGT gene, tyrosine ammonia-lyase TAL gene, phenylalanine ammonia-lyase PAL gene, cinnamate hydroxylase gene C4H, 4-coumaric acid:coenzyme A ligase 4CL1 gene, acyltransferase BAHD-AT gene, rhamnose biosyntheticase RHM2 gene, UDP rhamnose syltransferase URT gene, cytochrome P450 reductase CPR gene, and cytochrome P450 enzyme CYP gene, further containing the ability to express the UDP glucosyltransferase mutant FsGT61. F144H The genes.

[0020] According to the present invention, the genetically engineered bacteria can de novo produce the compounds verbascoside and echinacoside in the presence of glucose.

[0021] According to a specific embodiment of the present invention, the exogenous gene of the genetically engineered bacteria is capable of encoding the branching acid synthase mutant ARO4. K229L Gene, branching acid mutase mutant ARO7 G141S The genes, including shikimate kinase AROL gene, aromatic acetaldehyde synthase AAS gene, UDP glucosyltransferase UGT gene, tyrosine ammonia-lyase TAL gene, phenylalanine ammonia-lyase PAL gene, cinnamate hydroxylase C4H gene, 4-coumaric acid:coenzyme A ligase 4CL1 gene, acyltransferase BAHD-AT gene, rhamnose biosyntheticase RHM2 gene, UDP rhamnose syltransferase URT gene, cytochrome P450 reductase CPR gene, and cytochrome P450 enzyme CYP gene, can de novo synthesize verbascoside, the compound shown in Formula I above, in the presence of glucose. Preferably, the UDP glucosyltransferase mutant FsGT61 is introduced. F144H It can synthesize echinacoside, a compound shown in Formula II above, de novo using glucose as the sole carbon source.

[0022] According to the present invention, the term "branched acid synthase ARO4" is a branched acid mutase, abbreviated as ARO4, which is an enzyme that catalyzes the formation of dihydroxypyruvate from phosphoenolpyruvate and erythrose 4-phosphate. It can be from various sources, including microbial and plant sources. Its corresponding amino acid sequence and its mutant sequence that relieves feedback inhibition are well known to those skilled in the art and can be obtained using conventional technical means in the art. For example, it can be obtained by searching in databases (such as NCBI) or by querying relevant published literature. The present invention will not elaborate further.

[0023] According to the present invention, the term "branching acid mutase ARO7" is 3-deoxy-7-phosphoheptulonate synthase, abbreviated as ARO4, which is an enzyme that catalyzes the production of prephenyl acid from branching acid. It can be from various sources, including microbial and plant sources. Its corresponding amino acid sequence and its mutant sequence that relieves feedback inhibition are well known to those skilled in the art and can be obtained using conventional techniques in the field. For example, it can be obtained by searching in databases (such as NCBI) or by querying relevant published literature. The present invention will not elaborate further.

[0024] According to the present invention, the term "shikimic acid kinase AROL" is aromatic aldehyde synthase, abbreviated AROL, which is an enzyme that catalyzes the production of shikimic acid from shikimic acid to shikimic acid-3-phosphate. It can be from various sources, including microbial and plant sources. Its corresponding amino acid sequence is well known to those skilled in the art and can be obtained using conventional technical means in the art. For example, it can be obtained by searching in databases (such as NCBI) or by querying relevant published literature. The present invention will not elaborate further.

[0025] According to the present invention, the term "aromatic acetaldehyde synthase AAS" is shikimate kinase, abbreviated as AAS, which is an enzyme that catalyzes the production of 4-hydroxyphenylacetaldehyde from tyrosine. It can be from various sources, including microbial and plant sources. Its corresponding amino acid sequence is well known to those skilled in the art and can be obtained using conventional technical means in the field. For example, it can be obtained by searching in databases (such as NCBI) or by querying relevant published literature. The present invention will not elaborate further.

[0026] According to the present invention, the term "UDP-glycosyltransferase UGT" is UDP-glycosyltransferase, abbreviated as UGT. In vivo, its catalytically activated glucose groups are linked to various receptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids, and small molecules, thereby endowing the glycosylated products with a variety of biological functions. It can be from various sources, such as microbial or plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained using conventional techniques, such as searching databases (e.g., NCBI) or relevant published literature. Further details are omitted here.

[0027] According to the present invention, the term "tyrosine ammonilyase TAL" is an abbreviation for Tyrosine ammonilyase, which is an enzyme that catalyzes the production of p-coumaric acid from tyrosine. It can be from various sources, including microbial and plant sources. Its corresponding amino acid sequence is well known to those skilled in the art and can be obtained using conventional technical means in the field. For example, it can be obtained by searching in databases (such as NCBI) or by querying relevant published literature. The present invention will not elaborate further.

[0028] According to the present invention, the term "phenylalanine ammonia-lyase PAL" is phenylalanine ammonia-lyase, abbreviated as PAL, which is an enzyme that catalyzes the production of cinnamic acid from phenylalanine. It can be from various sources, including microbial and plant sources. Its corresponding amino acid sequence is well known to those skilled in the art and can be obtained using conventional technical means in the art. For example, it can be obtained by searching in databases (such as NCBI) or by querying relevant published literature. The present invention will not elaborate further.

[0029] According to the present invention, the term "cinnamate-4-hydroxylase" is an enzyme that catalyzes the formation of p-coumaric acid from cinnamic acid. It can be from various sources, including microbial and plant sources. Its corresponding amino acid sequence is well known to those skilled in the art and can be obtained using conventional technical means in the field. For example, it can be obtained by searching in databases (such as NCBI) or by querying relevant published literature. The present invention will not elaborate further.

[0030] According to the present invention, the term "4-coumaric acid:coagulant ligase 4CL" is hydroxycinnamate:CoAligase, abbreviated as 4CL, which is a key enzyme in the phenylpropanoid metabolic pathway in the biosynthesis of monolignins and flavonoids. It can be from various sources, such as microbial or plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained using conventional techniques, such as searching databases (e.g., NCBI) or relevant published literature. Further details are omitted here.

[0031] According to the present invention, the term "acyltransferase BAHD-AT" refers to a coenzyme A-dependent acyltransferase, abbreviated as BAHD-AT. In vivo, its acyl group, upon catalytic activation, attaches to various receptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids, and small molecules, thereby endowing the acylated products with a variety of biological functions. It can be from various sources, such as microbial or plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained using conventional techniques, such as searching databases (e.g., NCBI) or relevant published literature. Further details are omitted here.

[0032] According to the present invention, the term "rhamnose biosynthetic enzyme RHM2" is rhamnose biosynthetic enzyme 2, abbreviated as RH M2, which is an enzyme that catalyzes the production of UDP-rhamnose. It can be from various sources, including microbial and plant sources. Its corresponding amino acid sequence is well known to those skilled in the art and can be obtained using conventional technical means in the field. For example, it can be obtained by searching in databases (such as NCBI) or by querying relevant published literature. The present invention will not elaborate further.

[0033] According to the present invention, the term "UDP-rhamnosyltransferase URT" is UDP-rhamnosyltransferase, abbreviated as URT. In vivo, its catalytically activated rhamnosyl group attaches to various receptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids, and small molecules, thereby endowing the glycosylated products with a variety of biological functions. It can be from various sources, such as microbial or plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained using conventional techniques, such as searching databases (e.g., NCBI) or relevant published literature. Further details are omitted here.

[0034] According to the present invention, the term "cytochrome P450 enzyme (CYP)" refers to cytochrome P450 enzyme, abbreviated as CYP. CYP catalyzes the hydroxylation of various molecules in vivo, such as proteins, nucleic acids, oligosaccharides, lipids, and small molecules, thereby conferring many biological functions upon the hydroxylated products. CYP can come from various sources, such as microbial or plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained using conventional techniques, such as searching databases (e.g., NCBI) or relevant published literature. Further details are omitted here.

[0035] According to the present invention, the term "cytochrome P450 reductase CPR" is Cytochrome P450 reductase, abbreviated as CPR. In vivo, it is responsible for transferring electrons from NADPH to CYP and other heme proteins, thereby composing CYP and other heme proteins with the function of catalyzing oxidation reactions. It can be from various sources, such as microbial or plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained using conventional techniques, such as searching in databases (e.g., NCBI) or relevant published literature. Further details are omitted here.

[0036] According to the present invention, the term "mutant" refers to a mutation at an amino acid site in the original sequence that enables the enzyme to relieve substrate feedback inhibition or to enable the glycosyltransferase to recognize new substrates and perform catalytic activity.

[0037] It is known that of the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG), which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D, page 950). That is, due to the degeneracy of the genetic codon, most amino acids are determined by more than one codon. The substitution of the third nucleotide in a triplet codon usually does not change the amino acid composition; therefore, genes encoding the same protein can have different nucleotide sequences. Those skilled in the art can deduce the nucleotide sequences of genes encoding these proteins based on known codon tables and the amino acid sequences of the corresponding enzymes, and obtain these nucleotide sequences through biological methods (such as PCR and mutagenesis) or chemical synthesis.

[0038] According to a preferred embodiment of the present invention, a branched acid synthase mutant ARO4 that relieves feedback inhibition is encoded. K229L The gene is derived from Saccharomyces cerevisiae, and its sequence is shown in SEQ ID No: 1 of patent CN109988722B.

[0039] According to a preferred embodiment of the present invention, the branched acid mutase mutant ARO7 is encoded. G141S The gene is derived from Saccharomyces cerevisiae, and its sequence is shown in SEQ ID No: 2 of patent CN109988722B.

[0040] According to a preferred embodiment of the present invention, the gene for shikimic acid kinase AROL is derived from Escherichia coli MG1655, and its sequence is shown in SEQ ID No: 3 of patent CN109988722B.

[0041] According to a preferred embodiment of the present invention, the aromatic acetaldehyde synthase AAS is a PcAAS gene from parsley; preferably, the aromatic acetaldehyde synthase AAS is wild-type PcAAS with GenBank accession number AAA33860.1, and the sequence of its codon-optimized PcAAS gene is referenced to the nucleotide sequence shown in SEQ ID No: 4 of patent CN109988722B.

[0042] According to a preferred embodiment of the present invention, the gene encoding UDP glucosyltransferase UGT is UDP glucosyltransferase UGT33; preferably, the gene encoding UDP glucosyltransferase UGT is the RrUG T33 gene from Rhodiola rosea, with GenBank accession number MF674558; more preferably, the gene encoding UDP glucosyltransferase UGT is the codon-optimized RrUGT33 gene.

[0043] According to a preferred embodiment of the present invention, the gene encoding tyrosine ammonia-lyase TAL is the FjTAL gene from Flavobacterium johnsonii, with GenBank accession number KR095307.1; preferably, the gene encoding tyrosine ammonia-lyase TAL is a codon-optimized FjTAL gene.

[0044] According to a preferred embodiment of the present invention, the gene encoding phenylalanine ammonia-lyase PAL is the AtPAL2 gene from Arabidopsis thaliana, with GenBank accession number AEE79055.1; preferably, the gene encoding tyrosine ammonia-lyase PAL is the codon-optimized AtPAL2 gene.

[0045] According to a preferred embodiment of the present invention, the gene encoding cinnamic acid hydroxylase C4H is the AtC4H gene from Arabidopsis thaliana, with GenBank accession number AEC08397.1; preferably, the gene capable of encoding cinnamic acid hydroxylase gene C4H is a codon-optimized AtC4H gene.

[0046] According to a preferred embodiment of the present invention, the gene encoding 4-coumaric acid:coenzyme A ligase is the At4CL1 gene from Arabidopsis thaliana, with GenBank accession number AY099747.1. Preferably, the gene encoding tyrosine ammonia-lyase 4CL is a codon-optimized At4CL1 gene.

[0047] According to a preferred embodiment of the present invention, the BAHD-AT encoding the acyltransferase is the acyltransferase SiAT; preferably, the gene encoding BAHD-AT is the SiAT gene from sesame, with GenBank accession number XM_011085581.2. Preferably, the gene encoding the acyltransferase BAHD-AT is the codon-optimized SiAT gene.

[0048] According to a preferred embodiment of the present invention, the gene encoding rhamnose biosynthetic enzyme RHM2 is the AtRH M2 gene from Arabidopsis thaliana, with GenBank accession number AJ565874.1. Preferably, the gene encoding rhamnose biosynthetic enzyme RHM2 is a codon-optimized AtRHM2 gene.

[0049] According to a preferred embodiment of the present invention, the gene encoding UDP-rhamnosyltransferase URT is UDP-rhamnosyltransferase UGT79G15; preferably, the gene encoding UDP-rhamnosyltransferase UGT is the SiUGT79G15 gene from sesame, with GenBank accession number XM_011084410.2. Preferably, the gene encoding UDP-rhamnosyltransferase URT is the codon-optimized SiUGT79G15 gene.

[0050] According to a preferred embodiment of the present invention, the gene encoding the cytochrome P450 reductase CPR is derived from the AtCPR1 gene of Arabidopsis thaliana, with GenBank accession number NM_117326.4; preferably, the gene encoding the cytochrome P450 reductase CPR is a codon-optimized AtCPR1 gene.

[0051] According to a preferred embodiment of the present invention, the CYP98A20 gene encoding cytochrome P450 enzyme has a GenBank accession number of NM_001304410.1. Preferably, the gene encoding cytochrome P450 enzyme is a codon-optimized CYP98A20 gene.

[0052] According to a preferred embodiment of the present invention, the UDP glucosyltransferase UGT is encoded by the UDP glucosyltransferase mutant FsGT61. F144H Preferably, the gene encoding UDP glucosyltransferase UGT is FsGT61 from Forsythia suspensa. F144H Gene; nucleotide sequence as shown in SEQ ID No. 1.

[0053] In this invention, the above-mentioned gene is codon optimized in Saccharomyces cerevisiae, which can enable the target protein to be expressed more effectively in the Saccharomyces cerevisiae expression system.

[0054] According to the present invention, the strain used to construct the genetically engineered bacteria can be any strain commonly used in the art, such as fungi, like yeast, or bacteria, like bacilli. In a preferred embodiment of the present invention, the strain is *Saccharomyces cerevisiae*, and there are no special requirements regarding the type of *Saccharomyces cerevisiae* used to construct the expression strain; it can be any commonly used *Saccharomyces cerevisiae* capable of expressing the target gene. To ensure better expression of the target gene, the strain is preferably *Saccharomyces cerevisiae* BY4742, and more preferably a *Saccharomyces cerevisiae* strain that produces high levels of coumaric acid.

[0055] According to the present invention, the method for constructing the genetically engineered bacteria can be accomplished using conventional techniques in the art, such as by introducing a vector containing the corresponding gene into a specific strain. The present invention does not have special requirements regarding the type of expression vector; it can be any commonly used expression vector capable of expressing the target gene in a strain, such as plasmids. Those skilled in the art should understand that the construction method of the expression vector can employ various methods commonly used in the art, such as ligating the target gene into a vector after enzyme digestion, which will not be elaborated further here.

[0056] The gene used to construct the genetically engineered bacteria of the present invention can be cloned into one vector and introduced into *Saccharomyces cerevisiae*, or it can be cloned into different vectors and introduced into *Saccharomyces cerevisiae*. Considering the capacity of the vectors and the number of exogenous vectors that *Saccharomyces cerevisiae* can handle, preferably, the gene used to construct the genetically engineered bacteria of the present invention is cloned into 2-3 vectors and introduced into *Saccharomyces cerevisiae*. More preferably, the gene used to construct the genetically engineered bacteria of the present invention is integrated into the *Saccharomyces cerevisiae* genome for stable expression.

[0057] In addition, the branched acid synthase mutant ARO4 K229L The gene, branch acid mutase mutant ARO7 G141S The genes for shikimate kinase AROL, aromatic acetaldehyde synthase AAS, UDP-glucosyltransferase UGT, tyrosine ammonia-lyase TAL, phenylalanine ammonia-lyase PAL, cinnamate hydroxylase C4H, 4-coumaric acid:coenzyme A ligase 4CL, acyltransferase BAHD-AT, rhamnose biosyntheticase RHM2, UDP-rhamnose syltransferase URT, cytochrome P450 reductase CPR, cytochrome P450 enzyme CYP, and UDP-glucosyltransferase mutant FsGT61 are included. F144H The gene is integrated into the host bacterium's genome through transformation methods, more preferably into multiple copies of the yeast genome rDNA site and other designated sites.

[0058] There are no particular restrictions on the order of the genes used to construct the genetically engineered bacteria of the present invention in the vector, as long as they can be effectively expressed.

[0059] Secondly, the present invention provides the application of the genetically engineered bacteria described above in the production of verbascoside and echinacoside.

[0060] Thirdly, the present invention provides a method for producing verbascoside and echinacoside, wherein the genetically engineered bacteria described above are cultured in a glucose-containing medium, and the exogenous gene is constitutively highly expressed to produce verbascoside and echinacoside.

[0061] According to the present invention, the culture conditions are conventional, such as using a culture medium containing auxotrophic amino acids as selection markers, and culturing and fermenting at 30°C. During fermentation, the aforementioned genes can be translated into corresponding proteins, enabling the corresponding proteins to perform their functions.

[0062] Taking Saccharomyces cerevisiae as an example, the culture medium can be Sc medium (solvent is water, and the solutes and their final concentrations are: Yeast Nitrogen Base 6.7g / L, Dropoutmix 2g / L, glucose 20g / L liquid culture medium, which can be selected by those skilled in the art according to the actual situation).

[0063] According to the present invention, when the genetically engineered bacteria produced verbascoside and echinacoside by cultivation, it is also preferable to activate the constructed genetic engineering to prepare seed liquid, for example, by culturing at 30°C and 220 rpm for 24 h in a culture medium containing amino acid auxotrophs as screening markers.

[0064] According to a preferred embodiment of the present invention, the above-mentioned Sc culture medium can be used for the activation of genetically engineered bacteria, i.e., the preparation of seed culture. The above-mentioned Sc culture medium can also be used for the production of verbascoside and echinacoside. The inoculation amount of the seed culture can also be a conventional choice in the art; for example, an inoculation amount of 1% by volume, i.e., 1 mL of the seed culture is added to every 100 mL of Sc liquid culture medium.

[0065] According to the present invention, the amino acid auxotroph is an antibiotic used as a screening marker on the expression vector. Taking a commonly used transformation vector of Saccharomyces cerevisiae as an example, the amino acid auxotroph may be, for example, histidine, leucine, uracil, and lysine. The concentration of the amino acid is conventionally selected in the art. For example, the amount of histidine added is such that its final concentration is 50 mg / L; the amount of leucine added is such that its final concentration is 100 mg / L; the amount of uracil added is such that its final concentration is 100 mg / L; and the amount of lysine added is such that its final concentration is 100 mg / L.

[0066] According to the present invention, it is understood that those skilled in the art can select appropriate genetically engineered bacteria based on the type of compound to be produced.

[0067] Specifically, when it is necessary to produce verbascoside, the exogenous gene can be selected as the ARO4 mutant that encodes clade acid synthase. K229L The gene, branch acid mutase mutant ARO7 G141SThe genes for shikimate kinase AROL, aromatic acetaldehyde synthase AAS, UDP-glucosyltransferase UGT, tyrosine ammonia-lyase TAL, 4-coumaric acid:coenzyme A ligase 4CL, acyltransferase BAHD-AT, rhamnose biosyntheticase RHM2, UDP-rhamnose syltransferase URT, cytochrome P450 reductase CPR, and cytochrome P450 enzyme CYP can be cultured under glucose-containing conditions to produce verbascoside. Figure 1 ).

[0068] Specifically, when it is necessary to produce echinacoside, the exogenous gene can be selected as the ARO4 mutant that encodes clade acid synthase. K229L The gene, branch acid mutase mutant ARO7 G141S The genes for echinacoside, shikimate kinase AROL, aromatic acetaldehyde synthase AAS, UDP-glucosyltransferase UGT, tyrosine ammonia-lyase TAL, 4-coumaric acid:coenzyme A ligase 4CL, acyltransferase BAHD-AT, rhamnose biosyntheticase RHM2, UDP-rhamnose syltransferase URT, cytochrome P450 reductase CPR, cytochrome P450 enzyme CYP, and mutant genes of UDP-glucosyltransferase UGT can be cultured under glucose-containing conditions. Figure 1 ). Attached Figure Description

[0069] The accompanying drawings are used to provide a further understanding of the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof.

[0070] Figure 1 This invention relates to the multiple copy integration of the *Saccharomyces cerevisiae* BY4742 genome rDNA site into FjTAL-AtPAL2-AtC4H-Leu-ARO7. G141S -ARO4 K229L A schematic diagram of the -AROL fragment, which can produce high levels of p-coumaric acid.

[0071] Figure 2 The image shows the HPLC chromatogram of the fermentation broth a of Saccharomyces cerevisiae. Peak 1 is verbascoside, and the detection wavelength is 328 nm.

[0072] Figure 3 The image shows the HPLC chromatogram of the fermentation broth b of Saccharomyces cerevisiae. Peak 1 represents echinacoside, and the detection wavelength is 328 nm. Detailed Implementation

[0073] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0074] In the following examples, Escherichia coli DH5α was purchased from Beijing TransGen Biotech Co., Ltd., and was used for the construction of all vectors and the cloning of genes in this invention.

[0075] The Saccharomyces cerevisiae expression vectors pCf301, pCf302, pCf303, pCf304, and pCf305 are preserved in our laboratory.

[0076] Max Super-Fidelity DNA Polymerase was purchased from Vazyme.

[0077] The ClonExpress Multis One Step Cloning Kit was purchased from Vazyme.

[0078] All primers and genes were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0079] Unless otherwise specified, the experimental methods described in the following examples were performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual, or as recommended by the manufacturer of the corresponding biological product.

[0080] ARO4, a branched acid synthase mutant K229L The gene comes from Saccharomyces cerevisiae, and its sequence is shown in patent CN109988722B;

[0081] ARO7, a branched acid mutase mutant G141S The gene comes from Saccharomyces cerevisiae, and its sequence is shown in patent CN109988722B;

[0082] The gene for shikimic acid kinase AROL is derived from Escherichia coli MG1655, and its sequence is shown in patent CN109988722B.

[0083] The gene encoding aromatic acetaldehyde synthase AAS is the PcAAS gene from parsley, and its sequence is shown in patent CN109988722B.

[0084] The UDP-glucosyltransferase UGT is encoded as UDP-glucosyltransferase RrUGT33, with GenBank accession number MF674558.

[0085] The gene encoding the tyrosine ammonia-lyase TAL is FjTAL from Flavobacterium johnsonii, with GenBank accession number KR095307.1.

[0086] The gene encoding 4-coumaric acid:coenzyme A ligase is the At4CL gene from Arabidopsis thaliana, with GenBank accession number AY099747.1.

[0087] The BAHD-AT encoding the acyltransferase is the acyltransferase SiAT, and its GenBank accession number is XM_011085581.2.

[0088] The gene encoding rhamnose biosynthesizer RHM2 is the AtRHM2 gene from Arabidopsis thaliana, with GenBank accession number AJ565874.1.

[0089] The gene encoding UDP rhamnosyltransferase URT is UDP rhamnosyltransferase SiUGT79G15, and its GenBank accession number is XM_011084410.2.

[0090] The gene encoding the cytochrome P450 reductase CPR is derived from the AtCPR1 gene in Arabidopsis thaliana, with GenBank accession number NM_117326.4.

[0091] The gene encoding cytochrome P450 enzyme is SiCYP98A20, derived from sesame, with GenBank accession number NM_001304410.1.

[0092] The UGT mutant encoding UDP glucosyltransferase is the UDP glucosyltransferase mutant FsGT61. F144H Its sequence is shown in SEQ ID No. 1.

[0093] Example 1

[0094] This example illustrates the construction of verbascoside-enzyme Saccharomyces cerevisiae cells: The six key enzyme encoding genes for the biosynthesis of p-coumaric acid (exogenous genes include the gene for shikimate kinase AROL and the branching acid synthase mutant ARO4) are used. K229L The gene, branch acid mutase mutant ARO7 G141SThe genes phenylalanine ammonia-lyase AtPAL2, cinnamic acid hydroxylase AtC4H, and tyrosine ammonia-lyase FjTAL were introduced into *Saccharomyces cerevisiae*, and these genes were overexpressed in the yeast. This regulated shikimic acid surging metabolic flux to enhance the biosynthesis of tyrosol and p-coumaric acid, thereby constructing a pathway for the de novo synthesis of p-coumaric acid from glucose in *Saccharomyces cerevisiae*, resulting in a p-coumaric acid-producing *Saccharomyces cerevisiae* chassis strain. Furthermore, based on the above-mentioned glucose-to-shikimic acid synthesis pathway for p-coumaric acid, the genes for UDP-glucosyltransferase RrUGT33, 4-coumaric acid:coenzyme A ligase 4CL, acyltransferase BAHD-AT, rhamnose biosyntheticase RHM2, UDP-rhamnoseyltransferase URT, cytochrome P450 reductase CPR, and cytochrome P450 enzyme SiCYP98A20 were further expressed in the chassis strain. This allows for the cultivation of *Saccharomyces cerevisiae* under glucose-containing conditions to produce verbascoside (…). Figure 1 Furthermore, based on the aforementioned synthesis of verbascoside from glucose via the shikimic acid pathway, a mutant FsGT61, which further expresses UDP-glucosyltransferase, was developed in the chassis bacteria. F144H The gene allows for the cultivation of Saccharomyces cerevisiae to produce echinacoside under glucose-containing conditions.

[0095] Saccharomyces cerevisiae expression fragment FjTAL-AtPAL2-AtC4H-Leu-ARO7 G141S -ARO4 K229L -AROL, the preparation method of this expression fragment is as follows:

[0096] Guided by primers p.rDNA.F (SEQ ID No. 2) and p1.r (SEQ ID No. 3), and using pESC-304-ARO4... K229L -EcAROL-ARO7 G141S PCR was performed using ARO7 as a template, and the amplification was obtained. G141S -ARO4 K229L -AROL fragment. Plasmid pcf304-ARO4 K229L -EcAROL-ARO7 G141S The plasmid is deposited in our laboratory, and its sequence is shown in patent CN109988722B.

[0097] Guided by primers p2.f (SEQ ID No. 4) and P.rDNA.R (SEQ ID No. 5), PCR was performed using pcf301-AtC4H-FjTAL-AtPAL2 as a template to amplify the Leu2-AtC4H-FjTAL-AtPAL2 fragment. The pcf301-AtC4H-FjTAL-AtPAL2 plasmid is deposited in our laboratory.

[0098] Guided by primers rDNA-down-F (SEQ ID No. 6) and rDNA-down-R (SEQ ID No. 7), and using the genome of *Saccharomyces cerevisiae* BY4742 as a template, the downstream fragment of the recombinant homologous arm was amplified; guided by primers rDNA-up-F (SEQ ID No. 8) and rDNA-up-R (SEQ ID No. 9), and using the genome of *Saccharomyces cerevisiae* BY4742 as a template, the upstream fragment of the recombinant homologous arm was amplified.

[0099] The PCR amplification reaction system was as follows: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP Mix (10 mMeach), 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of template, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 18 μL of water.

[0100] The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 20 s, 56℃ annealing for 20 s, 72℃ extension for 5 min, 30 cycles; 72℃ extension for 10 min.

[0101] The preparation method for competent cells of Saccharomyces cerevisiae BY4742 is as follows:

[0102] Remove the BY4742 cells stored at -80℃ and streak them on YPD plates for 72 hours to allow single colonies to grow. Then, pick a single colony and inoculate it into YPD liquid medium, incubating overnight at 30℃ and 220 rpm. The next morning, transfer it to fresh YPD medium to achieve an initial OD600 of 0.2. Incubate at 30℃ and 220 rpm for 6-7 hours. When the OD600 reaches approximately 0.8, indicating the yeast is in mid-log phase, collect the cells at 5000 rpm for 5 minutes. Wash twice with an equal volume of ddH2O water, then once with 1 ml of 0.1 M AcLi. Finally, resuspend the cells in 320 μL of 0.1 M AcLi to obtain competent yeast cells.

[0103] The above fragments were converted into Saccharomyces cerevisiae BY4742 using a lithium acetate conversion method. The specific conversion method is as follows:

[0104] Add the above fragments in an equimolar ratio of approximately 1 μg to a sterile EP tube (total volume 10 μL), mix well, add 2 μL of denatured salmon sperm DNA, and mix thoroughly; add 12 μL of prepared BY4742 yeast competent cells and 100 μL of transformation buffer (800 μL of 50% PEG3350; 100 μL of 1M AcLi; 100 μL of 10×TE; 50 μL of DMSO), mix thoroughly, and incubate at 30°C for 30 min; heat shock at 42°C for 15 min, and incubate on ice for 5 min; centrifuge at 800g for 1 min and discard the supernatant, add 1 mL of fresh YPD medium, and incubate at 30°C for 2 h; centrifuge at 2000g for 1 min and discard the supernatant, wash twice with sterile ddH2O water, spread on Sc solid plates containing Leu auxotrophs, and incubate at 30°C for 48-72 h. Using Leu auxotrophic screening to simultaneously express FjTAL-AtPAL2-AtC4H-Leu-ARO7 G141S -ARO4 K229L The ECH8 strain, transformed with the AROL fragment, is capable of high-yield production of coumaric acid and multiple copies of the FjTAL-AtPAL2-AtC4H-Leu-ARO7 rDNA site in the Saccharomyces cerevisiae BY4742 genome. G141S -ARO4 K229L -A schematic diagram of the AROL fragment is shown below. Figure 1 As shown.

[0105] Example 2

[0106] This example illustrates the reconstruction of the verbascoside synthesis pathway in high-yield p-coumaric acid-producing Saccharomyces cerevisiae. The Saccharomyces cerevisiae expression fragments are: L1: AtRHM2-URA3-RrUGT33-SIRT-AtC4L1, L2: SiAT, and L3: CYP98A20-AtCPR1-PcAAS.

[0107] Plasmid pESC304 is an expression plasmid with three sets of strong constitutive promoters (GPM1, ENO2, TIP1) selected by URA3. Plasmid pESC304 was linearized by double digestion with restriction endonucleases PacI and BcuI. Then, AtRHM2, with homologous fragments at both ends, was integrated into the linear vector using a one-step cloning method to obtain plasmid pESC304-AtRH M2 (specific information: pESC-304-URA3-P). GPM1 -AtRHM2-T ENO2Using primers ATHRM-F (SEQ ID No. 10) / ATTHRM-R (SEQ ID No. 11) as guides and pESC304-AtRHM2 as a template, PCR was performed to amplify the first part of fragment L1: the AtRHM2 expression cassette.

[0108] Plasmid pcf303 is an HIS3-labeled expression plasmid containing three sets of strong constitutive promoters (PGK1, TEF1, TDH3). Plasmid pcf303 was linearized using restriction endonucleases, and then RrUGT33 (with homologous fragments at both ends), At4CL1 (with homologous fragments at both ends), and SiRT (with homologous fragments at both ends) were integrated into a linear vector using a one-step cloning method to obtain plasmid pcf303-RrUGT33-At4CL1-SiRT (specific information: pcf303-URA3-P). PGK1 -SiRT-T ADH1 ,P TD H3 -RrUGT33-T TEF1 ,P TEF1 -At4CL1-T CYC1 Using primers HIS-F (SEQ ID No. 12) / HIS-R (SEQ ID No. 13) as guides and pcf303-RrUGT33-At4CL1-SiRT as a template, PCR was performed to amplify the latter part of fragment L2: the RrUGT33-At4CL1-SiRT expression cassette.

[0109] Plasmid pcf301 is an expression plasmid labeled with URA3 and containing three sets of strong constitutive promoters (PGK1, TEF1, TDH3). Plasmid pcf303 was linearized using the restriction endonuclease AatII, and then SiAT with homologous fragments at both ends was integrated into the linear vector using a one-step cloning method to obtain plasmid pcf301-SiAT (specific information: pcf301-URA3-P). TEF1 -SiAT-T CYC1 Using primers SIAT-F (SEQ ID No. 14) / SIAT-R (SEQ ID No. 15) as guides and pcf301-SiAT as a template, PCR was performed to amplify fragment L2:SiAT.

[0110] In addition, pcf301 was linearized by enzyme digestion. Then, using a one-step cloning method, AtCRP1 with homologous fragments at both ends, CYP98A20 with homologous fragments at both ends, and PcAAS with homologous fragments at both ends were integrated into a linear vector to obtain the plasmid pcf301-AtCRP1-CYP98A20-PcAAS (specific information: pcf301-URA3-P).PGK1 -CYP98A20-T ADH1 ,P T EF1 -AtCPR1-T CYC1 ;P TDH3 -PcAAS-T TEF1 Then, using primers CYP-F (SEQ ID No. 16) / CYP-R (SEQ ID No. 17) as guides and pcf301-AtCPR1-CYP98A20-PcAAS as template, PCR was performed to amplify fragment L3:AtCPR1-CYP98A20.

[0111] The PCR amplification reaction system was as follows: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP Mix (10 mMeach), 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of template, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 18 μL of water.

[0112] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 5-10 min, 30 cycles; 72℃ extension for 10 min.

[0113] The amplified fragments L1, L2, and L3 were sequentially transformed into the genome of *Saccharomyces cerevisiae* strain ECH8 according to the method in Example 1, and then plated on auxotrophic Sc ​​plates, resulting in a strain with successful gene integration after 72 hours of culture. After multiple yeast transformations, *Saccharomyces cerevisiae* strain ECH18, which produces verbascoside, was finally obtained.

[0114] Example 3

[0115] This example illustrates the reconstruction of the echinacoside synthesis pathway in *Saccharomyces cerevisiae*, a high-yield producer of verbascosides. F144H

[0116] pcf301 was linearized by BamHI digestion, and FsGT 61 with homologous fragments at both ends was cloned using a one-step cloning method. F144H Integrating into a linear vector, the plasmid pcf301-FsGT61 was obtained. F144H (Specific information: pcf301-URA3-P) TDH3 -Fs GT61 F144H -T TEF1Guided by primers FSGT-F (SEQ ID No. 18) / FSGT-R (SEQ ID No. 19), pcf301-FsGT61 F144H PCR was performed using the template to amplify the L4 fragment SiAT-FsGT61. F144H Expression box.

[0117] The PCR amplification reaction system was as follows: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP Mix (10 mMeach), 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of template, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 18 μL of water.

[0118] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 5-10 min, 30 cycles; 72℃ extension for 10 min.

[0119] The amplified fragment L4 was transformed into Saccharomyces cerevisiae strain ECH18 according to the method in Example 1. After being cultured upside down on a Sc-URA plate for 72 hours, a strain successfully integrated into the genome was obtained, resulting in Saccharomyces cerevisiae strain ECH21 that produces echinacoside.

[0120] Test case

[0121] This test case illustrates the fermentation culture and product analysis of recombinant Saccharomyces cerevisiae strains ECH18 and ECH21.

[0122] 1. Fermentation culture

[0123] 1) The Saccharomyces cerevisiae expression strains ECH18 and ECH21 constructed in Examples 2 and 3 above were inoculated into 3 mL of Sc liquid medium (Yeast Nitrogen Base 6.7 g / L, Dropout mix 2 g / L, glucose 20 g / L) containing amino acid auxotrophs (histidine, leucine, uracil, and lysine, with final concentrations of 50 mg / L, 100 mg / L, 100 mg / L, and 100 mg / L, respectively) as selection markers, and cultured at 30 °C and 220 rpm for 24 h to obtain Saccharomyces cerevisiae seed culture.

[0124] 2) The seed culture of Saccharomyces cerevisiae strain ECH18 obtained in step (1) was inoculated into 50 mL of S C liquid medium (non-amino acid auxotrophic type) at an inoculation rate of 1%, and cultured at 30℃ and 220 rpm for 72 h to obtain fermentation broth a containing verbascoside.

[0125] 3) The seed culture of the Saccharomyces cerevisiae strain ECH21 obtained in step (1) was inoculated into 50 mL of liquid medium containing Sc (non-amino acid auxotrophic type) at an inoculation rate of 1%, and cultured at 30℃ and 220 rpm for 72 h to obtain fermentation broth b containing echinacoside.

[0126] 2. Product Detection

[0127] 1) HPLC detection of the products: Take 0.5 mL each of fermentation broth a and b, add an equal volume of anhydrous ethanol, vortex for 10 min, centrifuge at 12000 rpm for 5 min, collect the supernatant, filter the supernatant through a 0.22 μm filter, and perform HPLC analysis. The analytical conditions are as follows: Instrument: Shimadzu HPLC system; Measurement conditions include: C18 column (4.6 × 250 mm); Detection wavelength: verbascoside and echinacoside (312 nm); Mobile phase A = water (containing 0.1% formic acid by volume), B = acetonitrile; Flow rate = 1 mL / min; Gradient elution conditions: 0-5 min, 5% volume B; 5-18 min, 5% volume B to 13.2% volume B (the concentration of B increases uniformly within 5-18 min); 18-43 min, 18% volume B to 21.6% volume B; 43-50 min, 100% volume B; 50-57 min, 5% volume B. Injection volume: 20 μL.

[0128] The results for fermentation broths a and b are shown below. Figure 2 and Figure 3 .in, Figure 2 Peak 1 in fermentation broth a (ECH18) is verbascoside; Figure 3 Peak 1 in fermentation broth b (strain ECH21) is echinacoside.

[0129] The highest yield of verbascoside in fermentation broth a of strain ECH18 was determined to be 230.64 mg / L. The highest yield of echinacoside in fermentation broth b of strain ECH21 was 184.19 mg / L.

[0130] Therefore, the de novo synthesis yield of verbascoside by genetically engineered bacteria under shake-flask conditions reached a maximum of 230.64 mg / L, and the shake-flask yield of echinacoside reached a maximum of 184.19 mg / L. Thus, this invention provides a novel biosynthetic pathway for the production of verbascoside and echinacoside. These compounds, as important active ingredients in medicinal plants such as Rehmannia glutinosa, Cistanche deserticola, and Echinacea purpurea, have excellent application prospects, laying the foundation for large-scale industrial production and possessing significant economic and social benefits.

[0131] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A genetically engineered Saccharomyces cerevisiae bacterium for synthesizing verbascoside de novo, characterized in that, The genetically engineered bacteria contain genes capable of expressing a mutant of shikimate synthase ARO4 K229L , a mutant of shikimate mutase ARO7 G141S , a gene of shikimate kinase AROL, a gene of cytochrome b5 CYB5, a gene of glucose-6-phosphate-dehydrogenase ZWF1, a gene of aromatic acetaldehyde synthase AAS, a gene of UDP-glucosyltransferase UGT, a gene of tyrosine ammonia-lyase TAL, a gene of 4-coumaric acid:coenzyme A ligase 4CL, a gene of acyltransferase BAHD-AT, a gene of phenylalanine ammonia-lyase PAL, a gene of cinnamic acid hydroxylase C4H, a gene of rhamnose biosynthesis enzyme RHM2, a gene of UDP-rhamnose glycosyltransferase URT, a gene of cytochrome P450 reductase CPR, and a gene of cytochrome P450 enzyme CYP, and further contain a gene capable of expressing a mutant of UDP-glucosyltransferase FsGT61 F144H .

2. The genetically engineered Saccharomyces cerevisiae of claim 1, wherein, The encoding branched acid synthase mutant ARO4 K229L The gene is derived from *Saccharomyces cerevisiae*; the gene encoding the branched acid mutase mutant ARO7 G141S The gene for shikimate kinase AROL is from Saccharomyces cerevisiae; the gene encoding cytochrome b5 CYB5 is from Saccharomyces cerevisiae; and the gene encoding glucose-6-phosphate dehydrogenase ZWF1 is from Saccharomyces cerevisiae.

3. The genetically engineered Saccharomyces cerevisiae of claim 1, wherein, The coding aromatic acetaldehyde synthase AAS is the PcAAS gene from Petroselinum crispum; preferably, the aromatic acetaldehyde synthase AAS is a codon-optimized PcAAS gene; the UDP-glucosyltransferase UGT is the UDP-glucosyltransferase RrUGT33; preferably, the gene capable of encoding the UDP-glucosyltransferase UGT is the RrUGT33 gene from Rhodiola rosea, with GenBank accession number MF674558; more preferably, the gene capable of encoding the UDP-glucosyltransferase UGT is a codon-optimized RrUGT33 gene.

4. The genetically engineered Saccharomyces cerevisiae of claim 1, wherein, The gene capable of encoding the tyrosine ammonia-lyase TAL is the FjTAL gene from Flavobacterium johnsoniae, preferably with GenBank accession number KR095307.1; more preferably, the gene capable of encoding the tyrosine ammonia-lyase TAL is a codon-optimized FjTAL gene; the gene capable of encoding the 4-coumaric acid: coenzyme A ligase is the At4CL1 gene from Arabidopsis thaliana, preferably with GenBank accession number AY099747.1; more preferably, the gene capable of encoding the 4-coumaric acid: coenzyme A ligase is a codon-optimized At4CL1 gene; The BAHD-AT capable of encoding the acyltransferase is the acyltransferase SiAT; preferably, the gene capable of encoding the BAHD-AT is the SiAT gene from Sesamum indicum, preferably with GenBank accession number XM_011085581.2; more preferably, the gene capable of encoding the BAHD-AT is a codon-optimized SiAT gene.

5. The Saccharomyces cerevisiae genetically engineered strain according to claim 1, characterized in that, The gene capable of encoding the rhamnose biosynthesis enzyme RHM2 is the AtRHM2 gene from Arabidopsis thaliana, preferably with GenBank accession number AJ565874.1; more preferably, the gene capable of encoding the rhamnose biosynthesis enzyme RHM2 is a codon-optimized AtRHM2 gene; The PAL capable of encoding the deaminase is the phenylalanine ammonia-lyase gene AtPAL2; preferably, the gene capable of encoding AtPAL2 is the AtPAL2 gene from Arabidopsis thaliana, preferably with GenBank accession number AEE79055.1; more preferably, the gene capable of encoding the PAL is a codon-optimized AtPAL2 gene; The gene C4H capable of encoding the cinnamic acid hydroxylase is the cinnamic acid hydroxylase gene AtC4H; preferably, the gene capable of encoding C4H is the AtC4H gene from Arabidopsis thaliana, preferably with GenBank accession number AEC08397.1; more preferably, the gene capable of encoding C4H is a codon-optimized AtC4H gene. 6.The Saccharomyces cerevisiae genetically engineered bacterium of claim 1, characterized in that, The encoded UDP rhamnose transferase URT is UDP rhamnose transferase UGT79G15; preferably, the gene capable of encoding UDP rhamnose transferase UGT is SiUGT79G15 gene from sesame, preferably, its GenBank accession number is XM_011084410.2; more preferably, the gene encoding the UDP rhamnose transferase URT is a codon-optimized SiUGT79G15 gene; The gene encoding cytochrome P450 reductase CPR is from AtCPR1 gene of Arabidopsis thaliana, preferably, its GenBank accession number is NM_117326.4; the CYP98A20 gene encoding cytochrome P450 enzyme, preferably, its GenBank accession number is NM_001304410.

1. More preferably, the CYP98A20 gene encoding cytochrome P450 enzyme is a codon-optimized CYP98A20 gene. The encoded UDP glucose transferase UGT is a mutant FsGT61 of UDP glucose transferase F144H ; the nucleotide sequence is shown in SEQ ID No.

1. 7.The genetically engineered bacterium according to any one of claims 1 to 6, characterized in that, The starting strain is Saccharomyces cerevisiae; preferably, the Saccharomyces cerevisiae is a high-p-coumaric acid-producing Saccharomyces cerevisiae.

8. The method for constructing genetically engineered bacteria according to any one of claims 1 to 7, wherein Gene encoding shikimate synthase mutant ARO4 K229L Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase mutant ARO7 G141S Gene encoding shikimate dehydrogenase Preferably, the gene is introduced into the host strain by cloning the gene in one or more vectors, preferably, the gene is cloned into 2-3 vectors and introduced into the host strain; Or the introduced gene expression frame is integrated into the genome of the host strain by CRISPR / CAS9 or homologous recombination method; more preferably, it is integrated into the rDNA site or designated site of the yeast genome in multiple copies.

9. The use of the genetically engineered Saccharomyces cerevisiae strain of any one of claims 1 to 7 in the production of verbascoside and / or echinacoside.

10. A method of synthesizing from scratch verbascoside and echinacoside, characterized in that, The genetically engineered Saccharomyces cerevisiae strain of any one of claims 1 to 7 is cultured in Sc medium containing glucose to express the exogenous gene and produce verbascoside or echinacoside; optionally, the step of isolating verbascoside or echinacoside is further included. The genetically engineered Saccharomyces cerevisiae strain of any one of claims 1 to 7 is cultured in Sc medium containing glucose to express the exogenous gene and produce verbascoside or echinacoside; optionally, the step of isolating verbascoside or echinacoside is further included.

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