Saccharomyces cerevisiae genetically engineered bacterium for synthesizing verbascoside from beginning and application of saccharomyces cerevisiae genetically engineered bacterium

Abstract

The invention relates to the field of genetic engineering, in particular to a saccharomyces cerevisiae genetically engineered bacterium for producing verbascoside through de novo synthesis and application of the saccharomyces cerevisiae genetically engineered bacterium. The genetically engineered bacterium contains genes capable of coding a chorismate synthase mutant ARO4K229L, a chorismate mutase mutant ARO7G141S, shikimic acid kinase AROL, aromatic acetaldehyde synthase AAS, UDP glucosyltransferase UGT, tyrosine ammonia lyase TAL, 4-coumaric acid: coenzyme A ligase 4CL, acyltransferase BAHD-AT, rhamnose biosynthase RHM2, UDP rhamnosyltransferase URT, cytochrome P450 reductase CPR and cytochrome P450 enzyme CYP. The genetically engineered bacterium can be used for producing verbascoside from beginning by fermentation. In an experiment, the highest de novo synthesis yield of verbascoside of the genetically engineered bacterium can reach 5.7 mg / L, and the genetically engineered bacterium has a relatively high practical value.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to a genetically engineered yeast strain for synthesizing verbascoside from scratch and application of the genetically engineered strain in producing verbascoside. Background Art

[0002] Phenylpropanoid glycosides (PhGs) are a class of natural phenolic acid compounds widely distributed in Lamiaceae plants, with antioxidant, anti-cancer, learning and memory enhancement functions. In terms of chemical structure, this type of compound uses salidroside as the parent core structure, and undergoes acylation, glycosylation and other modifications on the core glucose group. Among them, PhGs in which the C3 hydroxyl group of the glucose group is replaced by a rhamnosyl group are the most representative, and "star molecules" such as verbascoside and echinacoside all belong to this type of compound.

[0003] Verbascoside is a plant phenolic acid compound and an important active substance in many medicinal plants. It has been used in human civilization for a long time. For example, verbascoside is one of the main active ingredients of traditional Chinese medicinal plants Rehmannia glutinosa and Cistanche deserticola. The Chinese Pharmacopoeia stipulates that the content of verbascoside is one of the reference standards for the quality of these medicinal materials. In addition, verbascoside is one of the important active substances in the extracts of commonly used medicinal plants such as Echinacea and Plantago in North America and Europe. In the past three decades, pharmacological studies on verbascoside have shown that verbascoside has antioxidant, anti-inflammatory, antibacterial, and anti-tumor effects, and plays an important role in the repair of nervous system damage, digestive system regulation, and immune system regulation. Therefore, it has great potential in the development of new drugs.

[0004] At present, the production of verbascoside mainly comes from plant extraction. This type of method has some inherent limitations, such as the restrictions of cultivated land area, weather environment, long production cycle, and separation and purification problems caused by low content and complex compound components. The chemical synthesis of verbascoside has been reported, but it has problems such as many synthesis steps, low yield, and serious environmental pollution.

[0005] With the advancement of synthetic biology technology, the production of plant natural products by environmentally friendly microbial fermentation has become a development trend. However, there is no report on the engineering strain of Saccharomyces cerevisiae that produces verbascoside and its application. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide a genetically engineered yeast of Saccharomyces cerevisiae capable of producing verbascoside, wherein the genetically engineered bacteria can produce verbascoside by microbial fermentation.

[0007] The inventors of the present invention have constructed a gene encoding a chorismate synthase mutant ARO4 through extensive research. K229L Gene, Chorismate mutase mutant ARO7 G141S The invention provides a genetically engineered yeast strain of the present invention, which contains the gene, shikimate kinase AROL gene, aromatic acetaldehyde synthase AAS gene, UDP glucose syltransferase UGT gene, tyrosine ammonia lyase TAL gene, 4-coumaric acid: coenzyme A ligase 4CL gene, acyltransferase BAHD-AT gene, rhamnose biosynthetic enzyme RHM2 gene, UDP rhamnosyltransferase URT gene, cytochrome P450 reductase CPR gene, and cytochrome P450 enzyme CYP gene. In the presence of glucose, the microbial de novo synthesis of verbascoside is achieved. And the yield of verbascoside obtained by the genetically engineered bacteria provided by the invention can reach the milligram level. Among them, the molecular formula of verbascoside C 29 H 36 O 15 , molecular weight 624.59, CAS number 61276-17-3, structural formula shown in Formula I, and its English name is Verbascoside.

[0008] O

[0009] Formula I

[0010] Based on the above, on the one hand, the present invention provides a genetically engineered strain of Saccharomyces cerevisiae for producing the above-mentioned verbascoside, wherein the genetically engineered strain contains a mutant ARO4 encoding a chorismate synthase. K229L The gene, chorismate mutase mutant ARO 7 G141S The genes of 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 biosynthetic enzyme RHM2, and UDP rhamnosyltransferase URT were identified.

[0011] Preferably, the genetically engineered bacteria also contain a gene for cytochrome P450 reductase CPR and a gene for cytochrome P450 enzyme CY P.

[0012] In a second aspect, the present invention provides the use of the genetically engineered yeast Saccharomyces cerevisiae as described above in the production of verbascoside.

[0013] In a third aspect, the present invention provides a method for synthesizing verbascoside from scratch, wherein the genetically engineered bacteria as described above are cultured in a medium containing glucose to constitutively and highly express exogenous genes to produce verbascoside.

[0014] The genetically engineered bacteria for producing verbascoside of the present invention can produce the compound verbascoside from scratch in the presence of glucose, with a maximum yield of 5.7 mg / L. Therefore, the present invention provides a genetically engineered yeast for producing verbascoside from scratch, which lays a foundation for the large-scale industrial production of verbascoside and has important scientific research value and social benefits.

[0015] Detailed description of the invention: The present invention is described in detail below.

[0016] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0017] In the first aspect, the present invention discloses a genetically engineered yeast for producing verbascoside from scratch, wherein the genetically engineered yeast contains a mutant ARO4 encoding a chorismate synthase. K229L Gene, Chorismate mutase mutant ARO7 G141S gene, shikimate kinase AROL gene, aromatic acetaldehyde synthase AAS gene, UDP glucosyltransferase UGT gene, tyrosine ammonia lyase TAL gene, 4-coumaric acid: coenzyme A ligase 4CL gene, acyltransferase BAHD-AT gene, rhamnose biosynthetic enzyme RHM 2 gene, UDP rhamnosyltransferase URT gene, cytochrome P450 reductase CPR gene, cytochrome P450 enzyme CYP gene.

[0018] According to the present invention, when the genetically engineered bacteria are in the presence of glucose, the compound verbascoside can be produced de novo.

[0019] According to a specific embodiment of the present invention, the exogenous gene of the genetically engineered bacteria is capable of encoding a chorismate synthase mutant ARO4 K229L Gene, Chorismate mutase mutant ARO7 G141S Gene, shikimate kinase AROL gene, aromatic acetaldehyde synthase AAS gene, UDP glucose transferase UGT gene, tyrosine ammonia lyase TAL gene, 4-coumaric acid: CoA ligase 4CL gene, acyltransferase BAHD-AT gene, rhamnose biosynthetic enzyme RHM2 gene, UDP rhamnosyltransferase URT gene, cytochrome P450 reductase CPR gene, cytochrome P450 enzyme CYP gene, which can synthesize the compound verbascoside shown in the above formula I from scratch in the presence of glucose.

[0020] According to the present invention, the term "chorismate synthase ARO4" is chorismate mutase, abbreviated as ARO7, which is an enzyme that catalyzes phosphoenolpyruvate and 4-phosphate erythrose to produce dihydroxyacetone phosphate, which can be from various sources, microbial sources, plant sources, and its corresponding amino acid sequence and its mutant sequence that releases feedback inhibition are well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not go into details.

[0021] According to the present invention, the term "chorismate mutase ARO7" is 3-deoxy-7-phosphoheptulonate synthase, abbreviated as ARO4, which is an enzyme that catalyzes chorismate to produce prephenate, and can be of various origins, including microbial origin and plant origin. Its corresponding amino acid sequence and its mutant sequence that releases feedback inhibition are well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not go into details.

[0022] According to the present invention, the term "shikimate kinase AROL" is aromatic aldehyde synthase, abbreviated as AROL, which is an enzyme that catalyzes shikimic acid to produce shikimate-3-phosphate. It can be from various sources, such as microbial sources and plant sources. Its corresponding amino acid sequence is well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not go into details.

[0023] According to the present invention, the term "aromatic acetaldehyde synthase AAS" is shikimate kinase, abbreviated as AAS, which is an enzyme that catalyzes tyrosine to produce 4-hydroxyphenylacetaldehyde, which can be from various sources, microbial sources, plant sources, and its corresponding amino acid sequence is well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not go into details.

[0024] According to the present invention, the term "UDP glucose transferase UGT" is UDP-glycosyltransferase, abbreviated as UGT, which catalyzes the activation of glucose groups in vivo to connect to different receptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids and small molecules, thereby giving the glycosylated products many biological functions. It can be from various sources, such as microbial sources and plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not repeat them.

[0025] According to the present invention, the term "tyrosine ammonia lyase TAL" is Tyrosine ammonilyase, abbreviated as TAL, which is an enzyme that catalyzes tyrosine to produce p-coumaric acid. It can be from various sources, such as microbial sources and plant sources. Its corresponding amino acid sequence is well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, which will not be elaborated in the present invention.

[0026] According to the present invention, the term "4-coumaric acid: CoA ligase 4CL" is hydroxycinnamate: CoAligase, abbreviated as 4CL, which is a key enzyme in the phenylpropanoid metabolic pathway in the biosynthesis of monolignols and flavonoids. It can be from various sources, for example, microbial sources, plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not repeat them.

[0027] According to the present invention, the term "acyltransferase BAHD-AT" is a coenzyme A-dependent acyltransferase, abbreviated as BAHD-AT, which catalyzes the activation of acyl groups in vivo to connect to different receptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids and small molecules, thereby giving the acylated products many biological functions. It can be from various sources, such as microbial sources and plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not repeat them.

[0028] According to the present invention, the term "rhamnose biosynthetic enzyme RHM2" is rhamnose biosynthetic enzyme 2, abbreviated as RHM2, which is an enzyme that catalyzes the production of UDP-rhamnose, which can be from various sources, such as microbial sources and plant sources, and its corresponding amino acid sequence is well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, which will not be repeated in the present invention.

[0029] According to the present invention, the term "UDP rhamnosyltransferase URT" is UDP-rhamnosyltransferase, abbreviated as URT, which catalyzes the activation of rhamnosyl groups in vivo to connect to different receptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids and small molecules, thereby giving the glycosylated products many biological functions. It can be from various sources, such as microbial sources and plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or by querying in relevant public literature, and the present invention will not repeat them.

[0030] According to the present invention, the term "cytochrome P450 enzyme CYP" is a cytochrome P450 enzyme, abbreviated as CYP, which catalyzes the hydroxylation reaction of different molecules in the body, such as proteins, nucleic acids, oligosaccharides, lipids and small molecules, thereby giving the hydroxylated products many biological functions. It can be from various sources, such as microbial sources and plant sources, and its corresponding amino acid sequence is well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not repeat them.

[0031] According to the present invention, the term "cytochrome P450 enzyme reductase CPR" is Cytochrome P450 reductase, abbreviated as CPR, which is responsible for transferring electrons from NADPH to CYP and other heme proteins in the body, thereby giving CYP and other hemoglobin the function of catalyzing oxidation reactions. It can be from various sources, such as microbial sources and plant sources, and its corresponding amino acid sequences are well known to those skilled in the art and can be obtained by conventional technical means in the art, for example, by searching in a database (such as NCBI) or querying in relevant public literature, and the present invention will not repeat them.

[0032] According to the present invention, the term "mutant" means that the amino acid site in the original sequence is mutated so that its function can relieve substrate feedback inhibition.

[0033] It is well known that among the 20 different amino acids constituting proteins, except for Met (ATG) or Trp (TGG) which are respectively encoded by a single codon, the other 18 amino acids are respectively encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are mostly more than one codons that determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the composition of the amino acid, so the nucleotide sequence of the gene encoding the same protein can be different. Those skilled in the art can completely deduce the nucleotide sequence of the gene that can encode them based on the known codon table and the amino acid sequence of the corresponding enzyme, and obtain the nucleotide sequence by biological methods (such as PCR method, mutation method) or chemical synthesis method.

[0034] According to a preferred embodiment of the present invention, the chorismate synthase mutant ARO4 encoding the enzyme that releases feedback inhibition K229L The gene is from Saccharomyces cerevisiae, and its sequence is shown in SEQ ID No: 1 in patent CN109988722B.

[0035] According to a preferred embodiment of the present invention, the mutant ARO7 encoding chorismate mutase G141S The gene is from Saccharomyces cerevisiae, and its sequence is shown in SEQ ID No: 2 in patent CN109988722B.

[0036] According to a preferred embodiment of the present invention, the gene of shikimate kinase AROL is from Escherichia coli MG1655, and its sequence is shown as SEQ ID No: 3 in reference patent CN109988722B.

[0037] According to a preferred embodiment of the present invention, the gene encoding aromatic acetaldehyde synthase AAS is the PcAAS gene from curly parsley; preferably, the aromatic acetaldehyde synthase AAS is the wild-type PcAAS with GenBank accession number AAA33860.1, and the sequence of the codon-optimized PcAAS gene refers to the nucleotide sequence shown in SEQ ID No: 4 in patent CN109988722B.

[0038] According to a preferred embodiment of the present invention, the gene encoding UDP glucose transferase UGT is UDP glucose transferase U8GT33; preferably, the gene capable of encoding UDP glucose transferase UGT is the RrU8GT33 gene from Rhodiola rosea, and its GenBank accession number is MF674558; more preferably, the gene capable of encoding UDP glucose transferase UGT is a codon-optimized RrU8GT33 gene, and the nucleotide sequence is shown in SEQ ID No.1.

[0039] According to a preferred embodiment of the present invention, the gene encoding tyrosine ammonia lyase TAL is the Fj TAL gene from Flavobacterium johnsonii, and its GenBank accession number is KR095307.1; preferably, the gene capable of encoding tyrosine ammonia lyase TAL is a codon-optimized FjTAL gene, and the nucleotide sequence is shown in SEQ ID No.2.

[0040] According to a preferred embodiment of the present invention, the gene encoding 4-coumaric acid:CoA ligase is the At4CL gene from Arabidopsis thaliana, and its GenBank accession number is AY099747.1.

[0041] According to a preferred embodiment of the present invention, the BAHD-AT encoding the acyltransferase is the acyltransferase SiAT; preferably, the gene capable of encoding the BAHD-AT is the SiAT gene from sesame, and its GenBank accession number is XM_011085581.2.

[0042] According to a preferred embodiment of the present invention, the gene encoding the rhamnose biosynthetic enzyme RHM2 is the AtRHM2 gene from Arabidopsis thaliana, and its GenBank accession number is AJ565874.1.

[0043] According to a preferred embodiment of the present invention, the gene encoding UDP rhamnosyltransferase URT is UDP rhamnosyltransferase UGT79G15; preferably, the gene capable of encoding UDP rhamnosyltransferase UGT is the SiUGT79G15 gene from sesame, and its GenBank accession number is XM_011084410.2.

[0044] According to a preferred embodiment of the present invention, the gene encoding the cytochrome P450 reductase CPR is from the AtCPR1 gene of Arabidopsis thaliana, and its GenBank accession number is NM_117326.4; preferably, the gene capable of encoding the cytochrome P450 reductase CPR is a truncated atCPR1, and the nucleotide sequence is shown in SEQ ID No.3.

[0045] According to a preferred embodiment of the present invention, the CYP98A20 gene encoding the cytochrome P450 enzyme has a GenBank accession number of NM_001304410.1.

[0046] In the present invention, the above gene is subjected to Saccharomyces cerevisiae codon optimization, so that the target protein can be expressed more effectively in the Saccharomyces cerevisiae expression system.

[0047] According to the present invention, the strain used to construct the genetically engineered bacteria can be various strains conventionally used in the art, for example, fungi, such as yeast, or bacteria, such as bacillus. According to a preferred embodiment of the present invention, the strain is Saccharomyces cerevisiae, and there is no special requirement for the type of Saccharomyces cerevisiae used to construct the Saccharomyces cerevisiae expression strain, and it can be various Saccharomyces cerevisiae commonly used in the art that can express the target gene. In order to better express the target gene, the Escherichia coli is preferably Saccharomyces cerevisiae BY4742, and more preferably Saccharomyces cerevisiae with high yield of salidroside and p-coumaric acid.

[0048] The high-yield salidroside-producing yeast ST1 is a chorismate synthase mutant ARO4 integrated at multiple sites in the genome. K229L Chorismate mutase mutant ARO7 G141S The saccharomyces cerevisiae with the gene of , the gene of shikimate kinase AROL, the gene of aromatic acetaldehyde synthase PcA AS, and the gene of UDP glucosyltransferase RrU8GT33 can be constructed by various methods, for example, including the λRed recombination system, the CRISPR-Cas9 recombination system, and RNAi. Preferably, the saccharomyces cerevisiae of the present invention is constructed using the λRed recombination system. The saccharomyces cerevisiae with high salidroside production constructed by the present invention is suitable for biosynthesis of verbascoside.

[0049] According to the present invention, the construction method of the genetically engineered bacteria can be completed by conventional technical means in the art, for example, by introducing a vector into the corresponding strain into which a corresponding gene is inserted. The present invention has no special requirements for the type of expression vector, and can be various expression vectors commonly used in the art that can express the target gene in the strain, such as plasmids, etc. It should be understood by those skilled in the art that the construction method of the expression vector can adopt various methods commonly used in the art, such as connecting the target gene to the vector after enzyme digestion, which will not be repeated here.

[0050] Among them, the genes used to construct the genetically engineered bacteria of the present invention can be cloned into one vector and introduced into Saccharomyces cerevisiae, or cloned into different vectors and introduced into Saccharomyces cerevisiae. Due to the carrying capacity of the vector and the carrying capacity of the number of exogenous vectors of Saccharomyces cerevisiae, it is preferred that the genes used to construct the genetically engineered bacteria of the present invention are cloned into 2-3 vectors and introduced into Saccharomyces cerevisiae.

[0051] In addition, the mutant ARO4 encoding chorismate synthase K229L Chorismate mutase mutant ARO7 G141S The genes of 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 biosynthetic enzyme RHM2, UDP rhamnosyltransferase URT, cytochrome P450 reductase CPR and cytochrome P450 enzyme CYP are integrated into the genome of the host bacteria through a transformation method, and more preferably, multiple copies are integrated into the rDNA site of the yeast genome.

[0052] There is no particular limitation on the order in which the genes used to construct the genetically engineered bacteria of the present invention are arranged in the vector, as long as they can be effectively expressed.

[0053] In a second aspect, the present invention provides the use of the genetically engineered bacteria as described above in the production of verbascoside.

[0054] In a third aspect, the present invention provides a method for producing verbascoside, wherein the genetically engineered bacteria as described above are cultured in a medium containing glucose, and the exogenous gene is constitutively highly expressed to produce verbascoside.

[0055] According to the present invention, the culture conditions are conventional culture conditions, such as using a culture medium containing an amino acid auxotrophic type as a selection marker, and culturing and fermenting at 30° C. During fermentation culture, the above gene can be translated into the corresponding protein, and the corresponding protein can play its role.

[0056] Among them, taking the genetically engineered bacteria as Saccharomyces cerevisiae as an example, the culture medium can be Sc culture medium (the 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, and those skilled in the art can select according to actual conditions.

[0057] According to the present invention, when the obtained genetically engineered bacteria are cultured to produce verbascoside, it is also preferred to activate the constructed genetic engineering to prepare seed solution, for example, culturing at 30°C for 12-20 hours in a culture medium containing an amino acid auxotrophic type as a selection marker.

[0058] According to a preferred embodiment of the present invention, the above Sc medium can be used for activation of genetically engineered bacteria, that is, to prepare seed solution, and the above Sc medium can be used for production of verbascoside. Wherein, the inoculation amount of the seed solution can also be a conventional choice in the art, for example, the inoculation amount is 1% by volume, that is, 1 mL of the seed solution is added to every 100 mL of Sc liquid medium.

[0059] According to the present invention, the amino acid auxotrophy is an antibiotic used as a screening marker on the expression vector. Taking the commonly used transformation vector of Saccharomyces cerevisiae as an example, the amino acid auxotrophy, for example, can be histidine, leucine, uracil; the concentration of the amino acid is a conventional choice 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.

[0060] According to the present invention, it can be understood that those skilled in the art can select corresponding genetically engineered bacteria according to the type of compound to be produced.

[0061] Specifically, when it is necessary to produce verbascoside, the exogenous gene can be selected to encode the chorismate synthase mutant AR O4 K229L Chorismate mutase mutant ARO7 G141S The genes of 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 biosynthetic enzyme RHM2, UDP rhamnosyltransferase URT, cytochrome P450 reductase CPR, and cytochrome P450 enzyme CYP can be cultured under conditions containing glucose to produce verbascoside ( Figure 1 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention, but they do not constitute a limitation of the present invention. In the accompanying drawings:

[0063] Figure 1 The schematic diagram of the biosynthesis pathway of salidroside and verbascoside in the Saccharomyces cerevisiae expression strain of the present invention is shown.

[0064] Figure 2 The present invention is a multi-copy integration of RrU8GT33-Leu-ARO7 into the rDNA site of the genomic yeast BY4742 G141S -ARO4K229L Schematic representation of the -AROL-PcAAS fragment.

[0065] Figure 3 It is the HPLC detection chart and MS spectrum of the fermentation liquid a of Saccharomyces cerevisiae ST1. Peak 1 is salidroside, and the detection wavelength is 224nm.

[0066] Figure 4 It is the HPLC detection chart and MS spectrum of the fermentation liquid b of Saccharomyces cerevisiae ST2. Peak 1 is verbascoside, and the verbascoside standard is used as the control. The detection wavelength is 312nm. DETAILED DESCRIPTION

[0067] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0068] In the following examples, Escherichia coli DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and Escherichia coli DH5α was used for the construction of all vectors and gene cloning in the present invention.

[0069] The Saccharomyces cerevisiae expression vectors pCf301, pCf302 and pCf303 were deposited in our laboratory.

[0070] Max Super-Fidelity DNA Polymerase was purchased from Vazyme.

[0071] ClonExpress Multis One Step Cloning Kit was purchased from Vazyme.

[0072] All primers and genes were synthesized by Suzhou Genewise Biotechnology Co., Ltd.

[0073] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual", or according to the conditions recommended by the manufacturers of the corresponding biological instruments.

[0074] Chorismate synthase mutant ARO4 K229L The gene is from Saccharomyces cerevisiae, and its sequence is shown in patent CN109988722B;

[0075] Chorismate mutase mutant ARO7 G141S The gene is from Saccharomyces cerevisiae, and its sequence is shown in patent CN109988722B;

[0076] The gene of shikimate kinase AROL comes from Escherichia coli MG1655, and its sequence is shown in patent CN109988722B;

[0077] The gene encoding aromatic acetaldehyde synthase AAS is PcAAS from curly parsley, and its sequence is shown in patent CN109988722B;

[0078] The encoded UDP glucose transferase UGT is UDP glucose transferase RrU8GT33, and its sequence is shown in SEQ ID No.1.

[0079] The gene encoding tyrosine ammonia lyase TAL is FjTAL from Flavobacterium johnsonii, and its sequence is shown in SEQ ID No.2.

[0080] The At4CL gene encoding 4-coumaric acid:CoA ligase is from Arabidopsis thaliana, and its GenBank accession number is AY099747.1.

[0081] The BAHD-AT encoding the acyltransferase is acyltransferase SiAT, and its GenBank accession number is XM_011085581.2.

[0082] The gene encoding the rhamnose biosynthetic enzyme RHM2 is AtRHM2 from Arabidopsis thaliana, and its GenBank accession number is AJ565874.1.

[0083] The gene encoding UDP rhamnosyltransferase URT is UDP rhamnosyltransferase SiUGT79G15, and its GenBank accession number is XM_011084410.2.

[0084] The gene encoding the cytochrome P450 reductase CPR comes from the AtCPR1 gene of Arabidopsis thaliana, and its truncated sequence is shown in SEQ ID No.3.

[0085] The gene encoding the cytochrome P450 enzyme is SiCYP98A20 from sesame, and its GenBank accession number is NM_001304410.1.

[0086] Example 1

[0087] This example is used to illustrate the construction of a high-yield salidroside yeast chassis cell: five key enzyme encoding genes for the biosynthesis of salidroside (exogenous genes include the gene of shikimate kinase AROL, the gene of aromatic acetaldehyde synthase PcAAS, the gene of chorismate synthase mutant ARO4 K229L Chorismate mutase mutant ARO7 G141SThe genes for tyrosol and salidroside were introduced into Saccharomyces cerevisiae to overexpress these genes; the metabolic flow from glucose to tyrosine was regulated to enhance the biosynthesis of tyrosol and salidroside, thereby achieving the construction of a pathway for Saccharomyces cerevisiae to synthesize salidroside from glucose from scratch ( Figure 1 ) to obtain a salidroside brewer's yeast chassis strain. In addition, based on the above-mentioned glucose synthesis of salidroside through the shikimic acid pathway, the gene of tyrosine ammonia lyase TAL, the gene of 4-coumaric acid: coenzyme A ligase 4CL, the gene of acyltransferase BAHD-AT, the gene of rhamnose biosynthetic enzyme RHM2, the gene of UDP rhamnosyltransferase URT, the gene of cytochrome P450 reductase CPR, and the gene of cytochrome P450 enzyme SiCYP98A20 are further expressed in the chassis strain, and the brewer's yeast can be cultured under the condition of containing glucose to produce verbascoside ( Figure 1 ).

[0088] Saccharomyces cerevisiae expression fragment RrU8GT33-Leu-ARO7 G141S -ARO4 K229L -AROL-PcAAS, the preparation method of the expression fragment is as follows:

[0089] Using primers RrU8GT33-F (SEQ ID No. 4) and RrU8GT33-R (SEQ ID No. 5) as guides and the synthetic gene RrU8GT33 (SEQ ID No. 1) as a template, PCR was performed to amplify the fragment of RrU8GT33.

[0090] Using primers Leu-F (SEQ ID No.6) and AROL-R (SEQ ID No.7) as guides, pCf302-ARO7 G141S -ARO4 K229L PCR was performed using AROL as a template to amplify Leu-ARO7 G141S -ARO4 K229L -ARO1 fragment. pCf302-ARO7 G141S -ARO4 K229L -AROL plasmid is deposited in our laboratory, and its sequence is shown in patent CN109988722B.

[0091] Using primers PcAAS-F (SEQ ID No. 8) and PcAAS-R (SEQ ID No. 9) as guides and the synthetic gene PcAAS (its sequence is referenced in patent CN109988722B) as a template, PCR was performed to amplify the PcAAS fragment.

[0092] Using primers rDNA-down-F (SEQ ID No. 10) and rDNA-down-R (SEQ ID No. 11) as guides and the Saccharomyces cerevisiae BY4742 genome as a template, the downstream fragment of the recombinant homology arm was amplified; using rDNA-up-F (SEQ ID No. 12) and rDNA-up-R (SEQ ID No. 13) as guides and the Saccharomyces cerevisiae BY4742 genome as a template, the upstream fragment of the recombinant homology arm was amplified;

[0093] The reaction system of PCR amplification reaction is: 2×Phanta Max Buffer 25μL, dNTP Mix (10mMeach) 1μL, 10μM forward primer 2μL, 10μM reverse primer 2μL, template 1μL, Phanta Max Super-Fidelity DNA Polymerase 1μL, water 18μL.

[0094] The reaction program of PCR amplification reaction was: denaturation at 98°C for 2 minutes; denaturation at 98°C for 20 seconds, annealing at 56°C for 20 seconds, extension at 72°C for 5 minutes, 30 cycles; extension at 72°C for 10 minutes.

[0095] Prepare the competent state of Saccharomyces cerevisiae BY4742, the preparation method being:

[0096] Take out BY4742 stored in a -80℃ refrigerator, inoculate it into YPD medium and culture it at 30℃ overnight. The next morning, transfer it to fresh YPD medium at a ratio of 1:100 and culture it at 30℃ for 6-7h. When OD600 is about 0.8, collect the bacteria at 5000rpm for 5min, wash them twice with an equal volume of ddH2O water, then wash them once with 1ml 0.1M AcLi, and finally resuspend them with 320ul 0.1M AcLi to obtain yeast competent cells.

[0097] The above fragments were transformed into Saccharomyces cerevisiae BY4742 using the lithium acetate transformation method, and the transformation method is specifically as follows:

[0098] Add the above fragments to the EP tube (total volume is 10ul) at a molar ratio of 1:1:1:1:1, mix well, add 2ul salmon sperm DNA, mix well; add 12ul prepared BY4742 yeast competent cells and 100ul transformation buffer (50% PEG3350, 800ul; 1M AcLi, 100ul; 10×TE, 100ul; DMSO, 50ul), mix well, stand at 30℃ for 30min; heat shock at 42℃ for 15min, stand on ice for 5min; remove the supernatant at 800g for 1min, add 1ml fresh YPD medium, and culture at 30℃ for 2h; remove the supernatant at 2000g for 1min, wash twice with ddH2O water, spread on the Sc solid plate containing Leu auxotrophy, and culture at 30℃ for 48h. Screening for simultaneous expression of RrU8GT33-Leu-ARO7 using Leu auxotrophy G141S -ARO4 K229L -AROL-PcAAS fragment transformed strain ST1, which can produce high salidroside, Saccharomyces cerevisiae BY4742 genome rDNA site multiple copies of RrU8GT33-Leu-ARO7 G141S -ARO4 K229L The schematic diagram of the -AROL-PcAAS fragment is shown in Figure 2 shown.

[0099] Example 2

[0100] This example is used to illustrate the reconstruction of the Saccharomyces cerevisiae expression vector Ver1 in the high-yield salidroside Saccharomyces cerevisiae, and the preparation method of the vector is as follows:

[0101] PCR was performed using primers SiAT-F (SEQ ID No. 14) / SiAT-R (SEQ ID No. 15) as guides and the synthetic SiAT gene (Genbank accession number: XM_011085581.2) as a template to amplify the SiAT gene, which was then seamlessly cloned into the plasmid pCf303 digested with SpeI to construct pCf303-SiAT.

[0102] Using primers at4CL-F (SEQ ID No. 16) / at4CL-R (SEQ ID No. 17) as guides and the synthetic at4CL gene (Genbank accession number: AY099747.1) as a template, PCR was performed to amplify the at4CL gene, which was then seamlessly cloned into the plasmid pCf303-SiAT after digestion with XmaI to construct pCf303-SiAT-at4CL.

[0103] Using primers FjTAL-F (SEQ ID No. 18) / FjTAL-R (SEQ ID No. 19) as guides and the synthetic FjTAL gene (sequence as SEQ ID No. 2) as a template, PCR was performed to amplify the FjTAL gene; using primers pCf303-F (SEQ ID No. 20) / pCf303-R (SEQ ID No. 21) as guides and the synthetic pCf303-SiAT-at4CL as a template, PCR was performed to amplify the backbone fragment. The two fragments were seamlessly cloned and connected to obtain the expression vector Ver1: pCf303-SiAT-at4CL-FjTAL.

[0104] The reaction system of PCR amplification reaction was: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP Mix (10 mM each), 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.

[0105] The reaction program of PCR amplification reaction was: denaturation at 98°C for 2 minutes; denaturation at 98°C for 20 seconds, annealing at 56°C for 20 seconds, extension at 72°C for 5 minutes, 30 cycles; extension at 72°C for 10 minutes.

[0106] The reaction system for seamless cloning is: 5×CE MultiS Buffer 4μL, ExnaseMultiS 2μL, linearized vector fragment pCf303 / pCf303-SiAT / pCf303-SiAT-at4CL 4μL, gene fragment SiAT / at4CL / FjTAL 10μL. React at 37℃ for 30min; cool to 4℃ or immediately put on ice. Transform 20μL of the reaction solution into E. coli competent cells DH5α to obtain positive clones, which are confirmed to be correct by sequencing, and the constructed vector Ver1: pCf303-FjTAL-at4CL-SiAT is obtained.

[0107] The Saccharomyces cerevisiae expression vector Ver2 is pCf301-atRHM2-SiUGT79G15-atCPR1-SiCYP98A20, and the preparation method of the vector is as follows:

[0108] Using primers atRHM2-F (SEQ ID No. 22) and atRHM2-R (SEQ ID No. 23) as guides and the synthetic gene atRHM2 (Genbank accession number: AJ565874.1) as a template, PCR was performed to amplify the atRHM2 fragment, which was then seamlessly cloned into the plasmid pCf301 after NheI digestion to construct pCf301-atRHM2.

[0109] Using primers SiUGT79G15-F (SEQ ID No. 24) and SiUGT79G15-R (SEQ ID No. 25) as guides and the synthetic gene SiUGT79G15 (Genbank accession number: XM_011084410.2) as a template, PCR was performed to amplify the SiUGT79G15 fragment, which was then seamlessly cloned into the plasmid pCf301-atRHM2 after SpeI digestion to construct pCf301-atRHM2-SiUGT79G15.

[0110] Using primers atCP1-F (SEQ ID No.26) and atCPR1-R (SEQ ID No.27) as guides and the synthetic truncated CRP1 gene (SEQ ID No.3) as a template, PCR was performed to amplify the atCPR1 fragment, which was seamlessly cloned into the plasmid pCf301 digested with BamHI and XhoI to construct pCf301-atCPR1.

[0111] Using primers SiCYP98A20-F (SEQ ID No. 28) and SiCYP98A20-R (SEQ ID No. 29) as guides and the synthetic gene SiCYP98A20 (Genbank accession number: NM_001304410.1) as a template, PCR was performed to amplify the SiCYP98A20 fragment, which was then seamlessly cloned into the plasmid pCf301-atCPR1 after SpeI digestion to construct pCf301-atCPR1-SiCYP98A20.

[0112] Using primers atCPR1-SiCYP98A20-F (SEQ ID No.30) and atCPR1-SiCYP98A20-R (SEQ ID No.31) as guides and plasmid pCf301-atCPR1-SiCYP98A20 as a template, PCR was performed to amplify the atCPR1-SiCYP98A20 fragment; using primers pCf301-F (SEQ ID No.32) and pCf301-R (SEQ ID No.33) as guides and plasmid pCf301-atRHM2-SiUGT79G15 as a template, PCR was performed to amplify the backbone fragment; the two fragments were connected by seamless cloning to construct the expression vector Ver2: pCf301-atCPR1-SiCYP98A20-atCPR1-SiCYP98A20.

[0113] The reaction system of PCR amplification reaction was: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP Mix (10 mM each), 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.

[0114] The reaction program of PCR amplification reaction was: denaturation at 98°C for 2 minutes; denaturation at 98°C for 20 seconds, annealing at 56°C for 20 seconds, extension at 72°C for 5 minutes, 30 cycles; extension at 72°C for 10 minutes.

[0115] The reaction system for seamless cloning was: 4 μL of 5×CE MultiS Buffer, 2 μL of Exnase MultiS, 4 μL of linearized vector fragment pCf301 / pCf301-atRHM2 / pCf301-atCPR1 / pCf301-atRHM2-SiUGT79G15, 10 μL of gene fragment atRHM2 / SiUGT79G15 / atCPR1 / SiCYP98A20 / atCPR1-SiCYP98A20. The reaction was carried out at 37°C for 30 min, and then the temperature was lowered to 4°C or immediately placed on ice for cooling. 20 μL of the reaction solution was transformed into E. coli competent cells DH5α to obtain positive clones, which were confirmed to be correct by sequencing, and the constructed expression vector Ver2: pCf301-atCPR1-SiCYP98A20-atCPR1-SiCYP98A20 was obtained.

[0116] The vectors Ver1 (pCf303-SiAT-at4CL-FjTAL) and Ver2 (pCf301-atCPR1-SiCYP98A20-atCPR1-SiCYP98A20) were co-transformed into the Saccharomyces cerevisiae ST1 strain according to the method of Example 1 to obtain the Saccharomyces cerevisiae ST2 strain that produces verbascoside.

[0117] Test Case

[0118] This test example is used to illustrate the fermentation culture and product identification of the recombinant strains of Saccharomyces cerevisiae ST1 and ST2.

[0119] 1. Fermentation culture

[0120] 1) The Saccharomyces cerevisiae expression strains ST1 and ST2 constructed in Examples 1 and 2 above were inoculated into 5 ml of Sc liquid medium (Yeast Nitrogen Base 6.7 g / L, Dropoutmix 2 g / L, glucose 20 g / L) containing amino acid auxotrophic types (histidine, leucine, uracil, final concentrations of 50 mg / L, 100 mg / L, 100 mg / L, respectively) as selection markers, and cultured at 30°C for 12 hours to obtain Saccharomyces cerevisiae seed liquid.

[0121] 2) The seed liquid of the cerevisiae strain ST1 obtained in step (1) was inoculated into 50 mL of Sc liquid culture medium containing the amino acid auxotrophic type (histidine, leucine, uracil, final concentrations of 50 mg / L, 100 mg / L, 100 mg / L, respectively) as the screening marker at an inoculum volume of 1%, and cultured at 30° C. for 24-72 hours to obtain a fermentation liquid a containing salidroside.

[0122] 3) The seed liquid of the cerevisiae strain ST2 obtained in step (1) was inoculated into 50 mL of Sc liquid culture medium containing the amino acid auxotrophic type (histidine, leucine, uracil, final concentrations of 50 mg / L, 100 mg / L, 100 mg / L, respectively) as the screening marker at an inoculum volume of 1%, and cultured at 30° C. for 24-72 hours to obtain a fermentation liquid b containing verbascoside.

[0123] 2. Product testing

[0124] 1) HPLC detection of products: 1 mL of fermentation broth a and b were centrifuged at 12000 rpm for 5 min, and the supernatant was taken for HPLC analysis. The analysis conditions were as follows: Instrument: Shimadzu liquid chromatograph, determination conditions included: C18 column (4.6×250 mm); detection wavelength: salidroside (224 nm) / verbascoside (312 nm); mobile phase A = water (containing 0.1% volume formic acid), 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 increased evenly 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 50 μL.

[0125] The results of fermentation broth a and b are shown in Figure 3 and Figure 4 .in, Figure 3 : Peak 1 appearing in fermentation broth a (strain ST1) is salidroside; Figure 4 : Peak 1 appearing in fermentation broth b (strain ST2) is verbascoside.

[0126] 2) LC-MS analysis of the product: The peaks detected in each fermentation broth in step (1) were subjected to LC-MS analysis, wherein the conditions for the LC-MS analysis included: C18 column (4.6×250 mm); detection wavelength: salidroside (224 nm) / verbascoside (312 nm); mobile phase A = water (containing 5 mM ammonium acetate), 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 increased evenly 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 50 μL. ESI negative ion source, molecular weight scanning range 50-1000.

[0127] The results are as follows Figure 3 and Figure 4 shown. Figure 3 : The MS spectrum of peak 1 in fermentation broth a has the MS characteristic peak 301.1331 of salidroside. Figure 4 : The MS spectrum of peak 2 in fermentation broth b has the MS characteristic peak 623.2054 of verbascoside.

[0128] It was determined that the yield of salidroside in the fermentation liquid a of strain ST1 could reach up to 1.5+0.03 g / L, and the yield of verbascoside in the fermentation liquid b of strain ST2 could reach up to 5.7+0.84 mg / L.

[0129] Therefore, the Saccharomyces cerevisiae producing verbascoside of the present invention can ferment and produce verbascoside with a maximum yield of 5.7 + 0.84 mg / L. Therefore, the present invention provides a new biosynthetic pathway for producing verbascoside, and these compounds, as important active ingredients of medicinal plants such as Rehmannia glutinosa, Cistanche deserticola, Echinacea purpurea, etc., have good application prospects, lay the foundation for large-scale industrial production, and have important economic value and social benefits.

[0130] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A genetically engineered strain of Saccharomyces cerevisiae for synthesizing verbascoside de novo, characterized in that: The genetically engineered bacteria contain a gene capable of expressing a mutant ARO4 encoding chorismate synthase. K229L Chorismate mutase mutant ARO7 G141S genes, genes for shikimate kinase AROL, genes for aromatic acetaldehyde synthase AAS, genes for UDP glucosyltransferase UGT, genes for tyrosine ammonia lyase TAL, genes for 4-coumaric acid: Coenzyme A ligase 4CL, genes for acyltransferase BAHD-AT, genes for rhamnose biosynthetic enzyme RHM2, genes for UDP rhamnosyltransferase URT, genes for cytochrome P450 reductase CPR, and genes for cytochrome P450 enzyme CYP.

2. The genetically engineered yeast of claim 1, characterized in that: The chorismate synthase mutant ARO4 K229L The gene is from Saccharomyces cerevisiae; the encoding chorismate mutase mutant ARO7 G141S The gene is from Saccharomyces cerevisiae; the gene encoding shikimate kinase AROL is from Saccharomyces cerevisiae.

3. The genetically engineered yeast of claim 1, characterized in that: The gene encoding aromatic acetaldehyde synthase AAS is the PcAAS gene from curly parsley; preferably, the aromatic acetaldehyde synthase AAS is a codon-optimized PcAAS gene; the UDP glucosyltransferase UGT is UDP glucosyltransferase U8GT33; preferably, the gene capable of encoding UDP glucosyltransferase UGT is the RrU8GT33 gene from Rhodiola rosea, and its GenBank accession number is MF674558; more preferably, the gene capable of encoding UDP glucosyltransferase UGT is a codon-optimized RrU8GT33 gene, and the nucleotide sequence is shown in SEQ ID No.

1.

4. The genetically engineered yeast of claim 1, characterized in that: The gene encoding tyrosine ammonia lyase TAL is the FjTAL gene from Flavobacterium johnsonii, preferably, its GenBank accession number is KR095307.1; more preferably, the gene capable of encoding tyrosine ammonia lyase TAL is a codon-optimized FjTAL gene, and the nucleotide sequence is shown in SEQID No.2; the gene encoding 4-coumaric acid: Coenzyme A ligase is the At4CL gene from Arabidopsis thaliana, preferably, its GenBank accession number is AY099747.1; the BAHD-AT encoding acyltransferase is the acyltransferase SiAT; preferably, the gene capable of encoding BAHD-AT is the SiAT gene from sesame, preferably, its GenBank accession number is XM_011085581.

2.

5. The genetically engineered yeast of claim 1, characterized in that: The gene encoding the rhamnose biosynthetic enzyme RHM2 is the AtRHM2 gene from Arabidopsis thaliana, and preferably its GenBank accession number is AJ565874.

1.

6. The genetically engineered yeast of claim 1, characterized in that: The encoded UDP rhamnosyltransferase URT is UDP rhamnosyltransferase UGT79G15; preferably, the gene capable of encoding UDP rhamnosyltransferase UGT is SiUGT79G15 gene from sesame, preferably, its GenBank accession number is XM_011084410.2; the gene encoding cytochrome P450 reductase CPR is AtCPR1 gene from Arabidopsis thaliana, preferably, its GenBank accession number is NM_117326.4; more preferably, the gene capable of encoding cytochrome P450 reductase CPR is truncated atCPR1, and the nucleotide sequence is shown in SEQ ID No.3; the CYP98A20 gene encoding cytochrome P450 enzyme, preferably, its GenBank accession number is NM_001304410.

1.

7. The genetically engineered bacterium according to any one of claims 1 to 6, characterized in that: It is Saccharomyces cerevisiae; preferably, the Saccharomyces cerevisiae is a Saccharomyces cerevisiae with high yield of salidroside and / or p-coumaric acid; more preferably, the Saccharomyces cerevisiae is Saccharomyces cerevisiae BY4742.

8. The genetically engineered bacterium according to any one of claims 1 to 6, characterized in that: ARO4 encoding chorismate synthase mutant K229L Chorismate mutase mutant ARO7 G141S The genes of 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 biosynthetic enzyme RHM2, UDP rhamnosyltransferase URT, cytochrome P450 reductase CPR and cytochrome P450 enzyme CYP are introduced into the host bacteria through a transformation method, specifically by cloning the genes in one or more vectors and introducing them into the host bacteria, preferably, cloning the genes in 2-3 vectors and introducing them into the host bacteria; further preferably, the introduced genes are integrated into the genome of the host bacteria, more preferably, multiple copies are integrated into the rDNA site of the yeast genome.

9. Use of the genetically engineered yeast Saccharomyces cerevisiae according to any one of claims 1 to 8 in producing verbascoside.

10. A method for synthesizing verbascoside from scratch, characterized in that: The genetically engineered yeast of any one of claims 1 to 8 is cultured in a Sc medium containing glucose to constitutively and highly express exogenous genes to produce verbascoside; optionally, the step of isolating verbascoside is also included.

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