CN113621530A - Genetic engineering yeast for producing wogonin compounds, and construction method and application thereof - Google Patents

Abstract

The invention relates to a genetic engineering yeast for producing wogonin compounds, a construction method and application thereof. Exogenous genes are introduced into the yeast engineering bacteria through a gene recombination technology to obtain the yeast engineering bacteria capable of producing wogonin, and the wogonin compounds are produced by taking chrysin as a precursor. The yeast engineering bacteria have the characteristics of low metabolic background, strong heterologous expression capacity, capability of synthesizing end products in whole cells, easiness in separating the end products and the like, and provide a new idea for industrial production of flavonoid drugs.

Description

Genetic engineering yeast for producing wogonin compounds, and construction method and application thereof

The application is a divisional application of Chinese patent application with the application number of 202010263807.X and the application date of 2020, 4 and 7.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a genetic engineering yeast for producing wogonin compounds, a construction method and application thereof.

Background

The flavonoid compound is widely present in higher plants and ferns, belongs to a secondary metabolite of plants, and is an important active ingredient of many traditional Chinese herbal medicine plants. Flavonoids have diverse biological functions due to the particularity and diversity of their structures: treating cardiovascular and cerebrovascular system diseases, reducing blood lipid, reducing cholesterol, inhibiting thrombosis, and dilating coronary artery; improving the sensitivity of thyroid to estrogen, strengthening the function of thyroid C cells secreting calcitonin, and finally inhibiting bone resorption to treat osteoporosis; removing excessive free radicals in the metabolic process, and protecting liver cells from being damaged by electrophilic free radical compounds and poisons; inhibiting the generation of free radicals, reducing lipid peroxidation, and stimulating the action of antioxidase.

Scutellaria baicalensis Georgi (Scutellaria baicalensis Georgi) is a perennial herb of Scutellaria of Labiatae, and the root of Scutellaria baicalensis has been used as a medicine by Chinese for more than two thousand years, wherein the baicalein flavonoid molecule with the most remarkable pharmacological activity is specific to the root. The root-specific flavones are structurally different from other flavones and mainly show the deletion of 4' -OH on the B ring. Besides the traditional anti-inflammatory, antiviral and antioxidant effects, the baicalein flavonoid molecules can also play an active role in the treatment of serious diseases such as cancer, Parkinson's disease, Alzheimer's disease and the like. For example, baicalein can induce pancreatic cancer cell apoptosis by down-regulating anti-apoptotic Mcl-1 protein to achieve an anti-cancer effect, and can also inhibit the fiber formation of alpha-nucleoprotein to resist Parkinson's disease; baicalin can protect neuronal cells from oxidative stress induced by amyloid beta protein to help prevent Alzheimer's disease, and selectively block proliferation of human lung cancer cells by regulating signal molecules; wogonin can regulate the redox states of malignant tumor and normal lymphocyte to achieve the effect of distinguishing tumor cells, etc. At present, various marketed drugs contain baicalein and baicalin components, such as baicalein aluminum capsules, baicalin capsules, bear gall and scutellaria eye drops and the like.

The most common preparation method of flavonoids is directly extracting from plants by physical or chemical means. The plant extraction method is relatively direct and simple to operate, but the large consumption of plant raw materials and organic reagents can cause the method to be deeply limited by climatic and regional factors and damage human bodies and the environment. The chemical synthesis method involves violent reaction, has potential safety hazard and has limited application prospect. At present, the biological method for de novo synthesis of baicalein and other scutellaria baicalensis root specific flavones has few cases, is obviously limited by low activity of heterologous expression plant enzyme, has low flavone yield and limited promotion space, and cannot realize further industrialized application.

Prokaryotic escherichia coli has been applied to heterologous synthesis of some compounds, but the research of the inventor aiming at baicalein compounds finds that the adaptability of plant enzymes and escherichia coli is low, the expression of some plant enzymes in the escherichia coli is not ideal, and the production of the baicalein compounds in the escherichia coli is difficult by using some synthetases from scutellaria as a species source. Some of the enzymes required for the reaction are often inactive or less active when expressed in E.coli, requiring extensive expression optimisation such as the addition of fusion tags to promote correct folding, but even then often have less than optimal results. For example, production of baicalein compounds introduces enzymes of cytochrome P450 family, which are difficult to express in e.coli or must be modified in a complex way to exert partial activity. In the process of synthesizing baicalein by using the escherichia coli engineering strain, L-phenylalanine precursor is required to be manually added for synthesis, and the synthesis efficiency is low. In addition, endotoxin from E.coli potentially leads to difficulties and higher requirements for the pharmaceutical purification process.

In the prior art, although some researches are available in the aspect of expressing compounds by yeast cells, the yeast cells have not been successfully used for synthesizing baicalein compounds in the field, especially pichia pastoris cells. This may lie in that more genes need to be introduced for expressing the compounds, the catalytic activity of the protein encoded by partial genes is not high, high expression is needed to meet the requirement of synthesis capacity, and the excessive dosage of exogenous genes causes a series of physiological problems of yeast cells. For example, there is a problem of instability due to excessive foreign genes; protein folding stress adversely affects physiology; overexpression of proteins consumes carbon and energy sources of host cells and imposes a certain metabolic burden on the host. Although pichia pastoris has been applied to the expression of a large number of foreign genes, there is still a bottleneck in the art to use it for stable co-expression of a plurality of foreign genes and to achieve efficient production.

Therefore, there is a need in the art to further research and develop a novel chassis host and a production process of baicalein and other baicalein root-specific flavonoids so as to obtain high-efficiency expression, simplify the operation in the process and reduce the cost.

Disclosure of Invention

The invention aims to provide a genetic engineering yeast for producing baicalein compounds, a construction method and application thereof. The technical scheme of the invention is the only technical scheme that the whole cell can synthesize the baicalin de novo from the basic carbon source at present.

In a first aspect of the present invention, there is provided a method for producing baicalein, the method comprising: (1) providing a yeast engineering bacterium, wherein an exogenous expression cassette of the following genes is transformed in the yeast engineering bacterium: PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1, F6H; and (2) culturing the yeast engineering bacteria in the step (1) to produce the baicalein.

In a second aspect of the present invention, there is provided a method for producing baicalin, the method comprising: (1) providing a yeast engineering bacterium, wherein an exogenous expression cassette of the following genes is transformed in the yeast engineering bacterium: expression cassettes for PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1, F6H, UBGT; and (2) culturing the engineered yeast strain of (1) to produce baicalin.

In another aspect of the present invention, there is provided a method for producing wogonin, the method comprising: (1) providing a yeast engineering bacterium, wherein an exogenous expression cassette of the following genes is transformed in the yeast engineering bacterium: F8H, PFOMT 5; and (2) culturing the engineered yeast strain of (1), and adding chrysin as a precursor to produce wogonin.

In another preferred embodiment, the 4CL, CHS-2, CHI, FNSII-2, F6H, UBGT, F8H and PFO MT5 are derived from Scutellaria baicalensis Georgi (Sb).

In another preferred embodiment, the PAL is from Rhodotorula toruloides (Rt).

In another preferred embodiment, the CPR1 is from Arabidopsis thaliana (At).

In another preferred embodiment, the yeast engineering bacteria is pichia pastoris; preferably, when PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1 and F6H are transformed in the engineered yeast, a gene fragment of PAL +4CL, a gene fragment of CHS-2+ CHI + FNSII-2 and a gene fragment of F6H + CPR1 are prepared and introduced into the engineered yeast.

In another preferred example, when the UBGT is introduced into the engineered yeast, a gene segment of UBGT + ENO1 is set; the ENO1 is a gene fragment that helps provide an integration site for UBGT. Preferably, the ENO1 can be obtained by primer amplification of the sequences of SEQ ID NO. 31 and SEQ ID NO. 32.

In another preferred embodiment, the function of KU70 gene is down-regulated or deleted in pichia pastoris.

In another preferred example, in the production of baicalein or baicalin, the culture in step (2) is carried out using a yeast culture medium, preferably supplemented with a carbon source during the culture.

In another preferred example, when baicalein or baicalin is produced, the carbon source is glucose; more preferably, glucose is added 1 time every 12 to 36 hours to a final concentration of 1 to 4% (v/v).

In another preferred embodiment, the precursor L-phenylalanine and/or malonyl-CoA is not added when baicalein or baicalin is produced.

In another preferred example, when baicalein or baicalin is produced, the culture time in the step (2) is 100-200 hours.

In another preferred embodiment, in the production of wogonin, the culture in step (2) is carried out using a yeast medium, preferably supplemented with a carbon source during the culture.

In another preferred example, when wogonin is produced, the carbon source is glucose; more preferably, glucose is added 1 time every 12 to 36 hours to a final concentration of 1 to 4% (v/v).

In another preferred example, when wogonin is produced, chrysin is added into a yeast culture medium during initial culture, and the final concentration of the fermentation liquid is 50-800 μ M; preferably 100 to 500 μ M; more preferably 150-300 μ M (e.g., 200 μ M, 250 μ M, etc.).

In another preferred embodiment, the cultivation time in step (2) is 55-95 hours when producing wogonin.

In another preferred embodiment, the production of wogonin is carried out by adding glucose 1 time every 18 to 30 hours (more preferably 22 to 26 hours) to a final concentration of 1.5 to 3% (v/v).

In another preferred embodiment, the yeast medium is a YPD medium.

In another preferred embodiment, the cultivation temperature is 30. + -. 2 ℃ and more preferably 30. + -. 1 ℃.

In another preferred embodiment, the rotation speed during the cultivation is 200. + -.50 r/min, more preferably 200. + -.20 r/min.

In another preferred embodiment, the cultivation time in step (2) is 120-180 hours, preferably 130-160 hours, such as 140, 144, 150, 155 hours, when producing baicalein or baicalin.

In another preferred embodiment, the cultivation time in step (2) is 60-90 hours, preferably 65-80 hours, such as 70, 72, 75, 80 hours, when producing wogonin.

In another preferred embodiment, the PAL gene has the nucleotide sequence shown in SEQ ID NO. 1, or a degenerate sequence thereof, or a nucleotide sequence encoding an homologous functional protein which is identical to the nucleotide sequence shown in SEQ ID NO. 1 by more than 70% (preferably more than 80%, more preferably more than 90%, more preferably more than 93%, more preferably more than 95%, more preferably more than 97%).

In another preferred embodiment, the 4CL gene has the nucleotide sequence shown in SEQ ID NO. 2, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, and more preferably 97% or more) identical to the sequence shown in SEQ ID NO. 2.

In another preferred embodiment, the CHS-2 gene has the nucleotide sequence shown in SEQ ID NO. 3, or a degenerate sequence thereof, or a nucleotide sequence encoding a functional homologue having a homology of 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more) to the sequence shown in SEQ ID NO. 3.

In another preferred embodiment, the CHI gene has the nucleotide sequence shown in SEQ ID NO. 4, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is identical to the nucleotide sequence shown in SEQ ID NO. 4 by more than 70% (preferably more than 80%, more preferably more than 90%, more preferably more than 93%, more preferably more than 95%, more preferably more than 97%).

In another preferred embodiment, the FNSII-2 gene has the nucleotide sequence shown in SEQ ID NO:5, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more; more preferably 90% or more; more preferably 93% or more; more preferably 95% or more; more preferably 97% or more) identical to the sequence shown in SEQ ID NO: 5.

In another preferred embodiment, the F6H gene has the nucleotide sequence shown in SEQ ID NO. 6, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more; more preferably 90% or more; more preferably 93% or more; more preferably 95% or more; more preferably 97% or more) identical to the sequence shown in SEQ ID NO. 6.

In another preferred embodiment, the CPR1 gene has the nucleotide sequence shown in SEQ ID NO. 7, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is identical to the nucleotide sequence shown in SEQ ID NO. 7 by more than 70% (preferably more than 80%, more preferably more than 90%, more preferably more than 93%, more preferably more than 95%, more preferably more than 97%).

In another preferred embodiment, the UBGT gene has the nucleotide sequence shown in SEQ ID NO. 8, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more) identical to the sequence shown in SEQ ID NO. 8.

In another preferred embodiment, the F8H gene has the nucleotide sequence shown in SEQ ID NO. 9, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more; more preferably 90% or more; more preferably 93% or more; more preferably 95% or more; more preferably 97% or more) identical to the sequence shown in SEQ ID NO. 9.

In another preferred embodiment, the PFOMT5 gene has the nucleotide sequence shown in SEQ ID NO:10, or a degenerate sequence thereof, or a nucleotide sequence encoding a functional homologue having a homology of 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more) to the sequence shown in SEQ ID NO: 10.

In another preferred embodiment, the pichia pastoris is GS 115.

In another aspect of the present invention, there is provided a yeast engineering bacterium for producing baicalein, comprising an exogenous expression cassette of the following genes: PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1, F6H.

In another aspect of the present invention, there is provided a yeast engineering bacterium for producing baicalin, which comprises an exogenous expression cassette of the following genes: PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1, F6H. Optionally also an expression cassette for UBGT.

In another aspect of the present invention, there is provided a yeast engineering bacterium for producing wogonin, comprising an exogenous expression cassette for the following genes: F8H, PFOMT 5.

In another preferred embodiment, the yeast engineering bacteria is pichia pastoris.

In another preferred embodiment, the 4CL, CHS-2, CHI, FNSII-2, F6H, UBGT is from Scutellaria baicalensis.

In another preferred example, the F8H and PFO MT5 are from scutellaria baicalensis.

In another preferred embodiment, the PAL is from Rhodotorula toruloides.

In another preferred embodiment, the CPR1 is from arabidopsis thaliana.

In another aspect of the invention, there is provided the use of a combination of genes comprising the following groups of genes: PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1 and F6H, and optionally UBGT, F8H and/or PFO MT5, which are used for transforming into yeast engineering bacteria to prepare baicalein compounds.

In another preferred embodiment, the combination of genes is placed in one or more expression constructs. For example, the gene fragment of PAL +4CL, the gene fragment of CHS-2+ CHI + FNSII-2, the gene fragment of F6H + CPR1 are placed in separate constructs.

In another preferred embodiment, the genome combination is PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1 and F6H, and the baicalein compound is baicalein.

In another preferred embodiment, the combination of genes is PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1, F6H and UBGT, and the baicalein compound is baicalin.

In another preferred embodiment, the genome is F8H and PFO MT5, and the baicalein compound is wogonin.

In another preferred embodiment, the 4CL, CHS-2, CHI, FNSII-2, F6H, UBGT, F8H and/or PFOMT5 is from Scutellaria baicalensis, the PAL is from Rhodotorula toruloides, and/or the CPR1 is from Arabidopsis thaliana.

In another aspect of the present invention, there is provided a kit for producing baicalein, comprising: PAL, 4CL, CHS-2, CHI, FNSII-2, F6H and CPR1 genes or expression constructs (including expression plasmids) containing said genes or yeast cells; or wherein it comprises: PAL, 4CL, CHS-2, CHI, FNSII-2, F6H, CPR1 and UBGT genes or expression constructs (including expression plasmids) containing the same or yeast cells; or wherein it comprises: F8H and PFOMT5 genes or expression constructs (including expression plasmids) or yeast cells containing said genes; preferably, the 4CL, CHS-2, CHI, FNSII-2, F6H, UBGT, F8H and/or PFOMT5 are from Scutellaria baicalensis, the PAL is from Rhodotorula toruloides, and/or the CPR1 is from Arabidopsis thaliana.

In another aspect of the present invention, there is provided a recombinant expression vector or expression construct for the production of baicalein, said recombinant expression vector comprising expression cassettes for the following genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR 1.

In another aspect of the present invention, there is provided a recombinant expression vector or expression construct for producing baicalin, said recombinant expression vector comprising expression cassettes for the following genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR1, SbUBGT;

in another aspect of the present invention, there is provided a recombinant expression vector or expression construct for the production of wogonin, said recombinant expression vector comprising an expression cassette for: F8H and PFOMT 5.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 shows the synthesis of baicalein, baicalin and wogonin.

FIG. 2 shows the fermentation of strains producing baicalein, baicalin and wogonin in shake flasks.

A. The shaking flask fermentation process of recombinant strains for producing baicalein records the cell biomass and baicalein yield which change along with the fermentation time.

B. The shaking flask fermentation process of recombinant strains for producing baicalin records the cell biomass and baicalin yield which change along with the fermentation time.

C. The shake flask fermentation process of the recombinant strain producing wogonin records the cell biomass and wogonin yield as a function of fermentation time.

Detailed Description

The inventor conducts intensive research, introduces a series of exogenous genes into a yeast engineering strain (preferably pichia pastoris) through a gene recombination technology, and obtains the yeast engineering strain capable of producing baicalein, baicalin and wogonin. The yeast engineering bacteria have the characteristics of low metabolic background, strong heterologous expression capacity, no need of adding a precursor, whole-cell synthesis of a final product, easy separation of the final product and the like, and provide a new idea for industrial production of flavonoid drugs.

The invention provides a gene engineering yeast for producing baicalein compounds. By heterologous expression in engineered yeast strains of the following enzymes: phenylalanine ammonia lyase (Pal), P-coumaroyl-coa ligase (4CL), chalcone synthase (CHS-2), chalcone isomerase (CHI), flavone synthase (FNSII-2), flavone 6-hydroxylase (F6H), cytochrome P450 reductase (CPR1), which enables the de novo synthesis of baicalein in yeast cells. Furthermore, the integration of baicalein 7-O-glucosyltransferase (UBGT) on the basis of the strain can realize the de novo synthesis of baicalin in yeast cells. The intermediate product chrysin in the synthesis process of baicalein is used as a substrate. The present invention also realizes the synthesis of wogonin from chrysin in yeast cells by heterologous expression of flavone 8-hydroxylase (F8H), baicalein phenylpropanoids and flavonoid O-methyltransferase (PFOMT 5). The synthetic route constructed in the present invention is shown in FIG. 1.

The invention realizes that the microorganism utilizes the basic carbon source to synthesize the baicalein and the baicalin from the beginning for the first time, and the microorganism utilizes the chrysin to synthesize the wogonin for the first time. The recombinant yeast engineering bacteria constructed in the invention has good adaptability with plant enzymes, has unique advantages compared with prokaryotic escherichia coli in heterologous expression of plant-derived flavone synthetase, can obtain good expression and exert catalytic activity without modifying enzyme structure, and has development and application potentials.

Term(s) for

As used herein, the term "expression cassette" or "gene expression cassette" refers to a gene expression system that contains all the necessary elements required for expression of a polypeptide of interest, typically including the following elements: a promoter, a gene sequence encoding a polypeptide, a terminator; in addition, the protein also can selectively comprise a signal peptide coding sequence and the like; these elements are operatively connected.

As used herein, the term "operably linked" or "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the term "expression construct" or "expression construct" refers to a recombinant DNA molecule comprising a desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. The "construct" is typically contained in an expression vector.

As used herein, the term "exogenous" or "heterologous" refers to the relationship between two or more nucleic acids or protein sequences from different sources, or the relationship between a protein (or nucleic acid) from a different source and a host cell. For example, a nucleic acid is exogenous to a host cell if the combination of the nucleic acid and the host cell is not normally naturally occurring. A particular sequence is "foreign" to the cell or organism into which it is inserted.

As used herein, the term "baicalein compound" includes baicalein, baicalin, or wogonin. The "baicalein compound" can also be a variation on the compounds disclosed herein, for example, the parent nuclear structure of the compound remains unchanged, but substitution of groups (e.g., aliphatic hydrocarbon groups containing 1 to 4 carbon atoms, preferably 1 to 2 carbon atoms) occurs at individual (e.g., 1 to 3, 1 to 2) positions.

Gene and expression system thereof

In the invention, the high-efficiency production of baicalein or baicalin in the yeast engineering bacteria is realized by transforming seven gene combinations (PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1, F6H) or eight gene combinations (PAL, 4CL, CHS-2, CHI, FNSII-2, CPR1, F6H, UBGT) into the yeast engineering bacteria. In a preferred embodiment of the present invention, the seven-gene combination comprises the following genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR 1; the eight-gene combination comprises the following genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR1, SbUBGT. The present inventors succeeded in co-expressing these genes in yeast cells and producing compounds with high activity, since the genes of the species Scutellaria baicalensis except for RtPAL and AtCPR1 are derived from the above genes.

In the invention, the high-efficiency production of wogonin in the yeast engineering bacteria is realized by transforming two gene combinations (F8H, PFO MT5) into the yeast engineering bacteria. The two gene combinations comprise the following genes: SbF8H, SbPFOMT 5.

The gene of the present invention may be naturally occurring, for example, it may be isolated or purified from a plant or microorganism. In addition, the gene can also be artificially prepared, for example, the gene can be obtained according to the conventional genetic engineering recombination technology, or the gene can be obtained by an artificial synthesis method.

The nucleotide sequence of the gene can be the same as the sequence shown in SEQ ID NO. 1-10, or can be a degenerate variant thereof. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes a protein with identical function, but differs from a sequence selected from the group consisting of those shown in SEQ ID NOs: 1-10. The present invention includes the natural sequences of the above genes, and also includes sequences subjected to codon optimization. In the invention, the natural gene sequence is adopted for recombinant expression at present, and the yield can be expected to be further improved by optimizing by using a plurality of codon optimization methods known in the field and establishing optimized engineered yeast. These further optimization techniques based on the solution of the present invention should also be covered in the solution of the present invention.

The genes may include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The invention also relates to variants of said genes, which encode polypeptides which differ in amino acid sequence from their corresponding wild-type polypeptide, being fragments, analogues or derivatives of the wild-type polypeptide. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Also, the polynucleotides that hybridize to the polypeptide encoded by the polynucleotide have the same biological functions and activities as the corresponding wild-type polypeptide.

Each gene of the present invention is preferably obtained from Rhodotorula toruloides, Scutellaria baicalensis, and Arabidopsis thaliana. The present invention may also include other genes obtained from other microorganisms that are highly homologous (e.g., have greater than 70%, such as 80%, 90%, 95%, or even 98% sequence identity) to the corresponding genes in Rhodotorula toruloides, Scutellaria baicalensis, and Arabidopsis thaliana, and are also contemplated within the scope of the present invention. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

The full-length sequence of each gene of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombination method, or an artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the sequences can be amplified. When the sequence is longer, two or more PCR amplifications can be carried out, and then the amplified fragments are spliced together according to the correct sequence.

The invention also relates to a vector comprising said polynucleotide, and a host cell genetically engineered with said vector.

In the present invention, the sequence of each gene may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

The sequences of the genes can be respectively inserted into recombinant expression vectors, and a plurality of the recombinant expression vectors are co-transferred into host cells; the expression cassettes of multiple genes can also be inserted into the same recombinant expression vector in tandem and transferred into host cells. The recombinant expression vector may further comprise an expression control sequence operably linked to the sequence of the gene to facilitate expression of the protein. It is understood that recombinant expression vectors can be conveniently constructed by those skilled in the art having the benefit of the teachings of the present invention. The obtained recombinant expression vector is also included in the present invention.

In the expression regulation sequence or the expression cassette, an inducible or constitutive promoter can be applied according to different requirements, and the inducible promoter can realize more controllable protein expression and compound production, thereby being beneficial to industrial application.

As a preferred mode of the present invention, there is provided an expression vector (expression construct) comprising an expression cassette of the following genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR 1; there is also provided another expression vector (expression construct) comprising an expression cassette for the following genes: SbUBGT; and another expression vector (expression construct) comprising an expression cassette for the following genes: SbF8H, SbPFOMT 5.

The expression vector (expression construct) can be established using techniques familiar to those skilled in the art. Once the desired selected gene is known, one skilled in the art can proceed to create an expression construct. The gene sequences may be inserted into different expression constructs (e.g., expression vectors) or into the same expression construct, so long as the encoded polypeptide is efficiently expressed and active after transfer into a cell. In a preferred embodiment of the present invention, the expression vector is pGAP Z α.

Vectors containing the appropriate gene sequences and appropriate promoter or control sequences described above may be used to transform appropriate host cells to enable expression of the protein. In the invention, the host cell is preferably a yeast engineering bacterium, more preferably pichia pastoris, such as pichia pastoris GS 115.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a eukaryote, the following DNA transformation methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method, and the medium used in the culture can be a yeast medium well known in the art. The culture is performed under conditions suitable for growth of the yeast cells.

The present invention also provides a kit for biosynthesizing a baicalein compound, which comprises: PAL, 4CL, CHS-2, CHI, FNSII-2, F6H and CPR1 genes or expression constructs (including expression plasmids) containing said genes or yeast cells; or comprises the following steps: PAL, 4CL, CHS-2, CHI, FNSII-2, F6H, CPR1 and UBGT genes or expression constructs (including expression plasmids) containing the same or yeast cells; or comprises the following steps: F8H and PFOMT5 genes or expression constructs (including expression plasmids) or yeast cells containing the genes. More preferably, the kit further comprises instructions for performing the biosynthetic method.

The recombinant pichia pastoris strain obtained by the gene recombination technology has the characteristics of low metabolic background, strong heterologous expression capability, no need of adding a precursor additionally, capability of synthesizing a final product in a whole cell manner, easiness in separation of the final product and the like, solves the problems in the synthesis by the traditional biological and chemical methods to a great extent, and provides a new idea for industrially producing flavonoid drugs. Particularly, the invention realizes the successful expression of various genes from the scutellaria in the yeast engineering bacteria, which is closer to the natural processing mode of the baicalein in plants, and ensures the high-efficiency production of the baicalein compounds.

Method for synthesizing baicalein, baicalin and wogonin

The structural formula of baicalein is shown in the following formula (I), the structural formula of baicalin is shown in the following formula (II), and the structural formula of wogonin is shown in the following formula (III).

The invention discloses a method for producing baicalein, baicalin and wogonin by microbial heterologous synthesis. The method comprises the following steps: transforming seven genes (preferably RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR1) into engineering yeast bacteria to produce baicalein; alternatively, eight genes (preferably RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR1, SbUBGT) are transformed into the yeast engineering bacteria to produce baicalin; alternatively, wogonin is produced by transforming a yeast engineering bacterium with two genes (preferably SbF8H, SbPFOMT 5).

The invention also provides a fermentation culture method of the recombinant yeast engineering bacteria for producing the baicalein or the baicalin. The yeast engineering bacteria for producing baicalein or baicalin can be cultured by using a yeast culture medium, and a carbon source is preferably supplemented during the culture; preferably the carbon source is glucose; preferably, 1 time of glucose is added every 12 to 36 hours; more preferably to a final concentration of 1-4% (v/v). Particularly, when the recombinant yeast engineering bacteria are produced, the enzyme activity is ideal and the endogenous mechanism of yeast cells is mobilized, so the technical scheme of the invention can produce the baicalein compounds on the basis of not adding precursors of L-phenylalanine and/or malonyl coenzyme A; this is advantageous for large-scale production, and simplifies the process (e.g., the preparation of the culture medium is simpler and clearer, and the separation and purification of the product at the later stage are more convenient), and reduces the cost. In a preferred embodiment of the present invention, the culture time is optimized to be 100-200 hours, preferably 120-180 hours, and more preferably 130-160 hours according to the production characteristics of the recombinant yeast engineering bacteria for producing baicalein or baicalin.

In one embodiment of the present invention, a preferred method for producing baicalein or baicalin is provided, comprising: culturing the recombinant strain in liquid YPD culture at 30 deg.C and 200r/min to logarithmic phase, collecting thallus, washing with sterile water twice, transferring to YPD culture medium, and culturing at 30 deg.C and 200r/min for 144 h. 2% glucose was added to the broth every 24h during fermentation.

The invention also provides a fermentation culture method of the recombinant yeast engineering bacteria for producing wogonin. The yeast engineering bacteria for producing wogonin can be cultured by using a yeast culture medium, and a carbon source is preferably supplemented during the culture; preferably the carbon source is glucose; preferably, 1 time of glucose is added every 12 to 36 hours; more preferably to a final concentration of 1-4% (v/v). In a preferred embodiment of the present invention, the present inventors have optimized the culture time to be 55-95 hours, preferably 60-90 hours, more preferably 65-80 hours according to the production characteristics of the recombinant engineered yeast strain for producing wogonin.

In one embodiment of the present invention, a preferred method for producing wogonin is provided, comprising: culturing the recombinant strain in liquid YPD culture at 30 deg.C and 200r/min to logarithmic phase, collecting thallus, washing with sterile water twice, transferring to YPD culture medium, adding 200 μ M chrysin as precursor, and culturing at 30 deg.C and 200r/min for 72 h. 2% glucose was added to the broth every 24h during fermentation.

After obtaining the fermentation product, extracting baicalein, baicalin, or wogonin from the fermentation product may employ techniques known in the present invention. The product can be analytically identified using high performance liquid chromatography to confirm that the desired compound is obtained.

The invention has the main advantages that:

the invention uses recombinant pichia pastoris for heterologous production of baicalein, thereby solving the problems that a plant extraction method consumes a large amount of plant raw materials, is limited by seasonal regional factors, has low extraction efficiency and the like; adverse factors such as more byproducts, low activity of target products, large environmental pollution and the like in the chemical synthesis method are avoided; compared with the method of using escherichia coli as a chassis host, the method solves the problem of low heterologous expression activity of plant enzymes, is more suitable for efficient synthesis of flavonoids such as baicalein and the like, and opens up a new way for industrial production of flavonoids such as baicalein and the like. Therefore, the recombinant pichia pastoris strain has the potential of industrial production of baicalein.

The invention realizes the technical breakthrough of using the recombinant yeast to carry out whole-cell de novo biosynthesis of the baicalein and the baicalin, and the basic carbon source can meet the production requirement without adding an amino acid precursor, thereby providing a new way for producing the baicalein and other flavonoid compounds.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Culture medium

YPD liquid medium: 20.0g/L of glucose, 20.0g/L of peptone and 10.0g/L of yeast extract;

YPD solid Medium: 20.0g/L of glucose, 20.0g/L of peptone, 10.0g/L of yeast extract and 20.0g/L of agar;

YND solid Medium: 20.0g/L glucose, 6.7g/L nitrogen source (YNB) without amino yeast and 20.0g/L agar;

example 1 construction of expression plasmid

1、P_GAPConstruction of plasmids expressing respective genes

The genes RtPAL, AtCPR1 and SbUBGT are composed of threonineThe national jingzhi biotechnology limited was synthesized and constructed directly on the vector plasmid pGAP Z α (Invitrogen), plasmid name: pGAP _ RtPal, pGAP _ AtCPR1pGAP _ SbUBGT. The original plasmids for the other genes (Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, SbF8H, SbPFOMT5) were obtained from the Shanghai research center for plant science, Zhongkobai. Designing specific primers, and carrying out PCR amplification by using original plasmids of all genes as templates and using corresponding primers in table 1 to obtain related genes (Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, SbF8H and SbPFOA 5) derived from scutellaria baicalensis; using AsuII and KpnI to double-enzyme cut the plasmid pGAP Z alpha, adopting a seamless assembly kit to integrate each gene into the P in the plasmid pGAP Z alpha respectively_GAPDownstream, sequencing correctly to obtain a DNA fragment having P_GAPPlasmid expressing each gene, plasmid name: pGAP _ Sb4CL, pGAP _ SbCHS-2, pGAP _ SbCHI, pGAP _ SbFNSII-2, pGAP _ SbF6H, pGAP _ SbF8H, pGAP _ SbPFOMT 5.

Wherein the gene sequence of the RtPAL is shown as SEQ ID NO. 1;

the gene sequence of Sb4CL is shown as SEQ ID NO. 2;

the gene sequence of SbCHS-2 is shown as SEQ ID NO. 3;

the gene sequence of SbCHI is shown in SEQ ID NO. 4;

the gene sequence of SbFNSII-2 is shown as SEQ ID NO. 5;

the gene sequence of SbF6H is shown in SEQ ID NO. 6.

The gene sequence of AtCPR1 is shown in SEQ ID NO. 7.

The gene sequence of SbUBGT is shown as SEQ ID NO. 8;

the gene sequence of SbF8H is shown as SEQ ID NO. 9;

the gene sequence of SbPFOMT5 is shown in SEQ ID NO: 10.

The primers used for PCR amplification are shown in Table 1.

TABLE 1

2. Construction of Gene-Combined plasmid

The plasmids pGAP _ RtpAl, pGAP _ SbFNSII-2, pGAP _ SbF6H and pGAP _ SbF8H constructed in the above "1" were digested with BamH I, and the linearized plasmids were recovered, respectively.

The plasmids pGAP _ Sb4CL, pGAP _ SbCHI, pGAP _ AtCPR1 and pGAP _ SbPFMT 5 constructed in the above "1" were used as templates, and BamH I in Table 1 was used as primers to obtain complete expression cassettes of Sb4CL, SbCHI, AtCPR1 and SbPFMT 5 genes including promoters and terminators by PCR amplification and recovery. Linearized plasmids and gene expression cassettes can be ligated by means of a seamless assembly kit to give plasmids pGAP _ Rtpal + Sb4CL, pGAP _ SbCHI + SbFNSII-2, pGAP _ SbF6H + AtCPR1, pGAP _ SbF8H + SbPFOMT 5.

BtgZ I is used for enzyme digestion of the plasmid pGAP _ SbCHI + SbFNSII-2, and the linearized plasmid is recovered. The plasmid pGAP _ SbCHS-2 constructed in the above "1" was used as a template, BtgZ I in Table 1 was used as a primer, and the complete expression cassette of SbCHS-2 gene including a promoter and a terminator was recovered by PCR amplification. The linearized plasmid and the expression cassette of the gene can be connected by a seamless assembly kit to obtain the plasmid pGAP _ SbCHS-2+ SbCHI + SbFNSII-2.

3. Construction of Gene-Combined plasmid containing homology arm

Linearized plasmids were recovered using ApaI and XhoI double digestion of plasmids pDAG2, pDTg1 and pDFg1 (see Liu Q, Shi X, Song L, et al. CRISPR-Cas 9 mediated genomic polymorphism in Pichia pastoris [ J ]. Microbiological Cell Factories,2019,18: 144). The plasmids pGAP _ RtPal + Sb4CL, pGAP _ SbCHS-2+ SbCHI + SbFNSII-2, pGAP _ SbF6H + AtCPR1 and pGAP _ SbF8H + SbPFOMT5 constructed in the above "2" were used as templates, and ApaI to SpeI in Table 1 were used as primers to amplify by PCR to obtain fragments containing a plurality of gene expression cassettes, and the linearized plasmids and the expression cassette fragments of the genes were ligated by means of a seamless assembly kit to obtain plasmids pDAG2_ RtPal + Sb4CL, pDTg1_ SbCHS-2+ SbCHI + SbFNSII-2, pDFg1_ SbF6H + AtCPR1, and pDAG2_ SbF8H + SbPFO 5.

4. Construction of plasmid containing ENO1 integration site

The plasmid pGAP _ SbUBGT constructed in the above "1" was digested with Nsi I, and the linearized plasmid was recovered. The genome of Pichia pastoris GS115 is used as a template, ENO1 fragments are amplified by corresponding primers in the table 1, and linearized plasmids and ENO1 fragments can be connected by a seamless assembly kit to obtain plasmids pGAP _ SbUBGT + ENO 1.

Example 2 preparation of target recombinant Yeast

1. PCR validation of recombinant strains

Electrically transforming each DNA fragment and the corresponding gRNA plasmid into pichia pastoris GS 115-delta ku70 (see 201910403132.1), recovering, coating on an YND solid culture medium, culturing for 5 days, selecting a fresh bacterial colony to culture in a YPD liquid culture medium, extracting the genome of each transformant by using a yeast genome extraction kit, and identifying the condition that each gene synthesized by baicalein is integrated into the pichia pastoris genome through cloning PCR reaction.

Cloning PCR reaction conditions:

(1) initial denaturation at 95 ℃ for 5 min;

(2) denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 1min/kb, and circulating reaction for 30 times;

(3) finally, the extension is carried out for 7min at 72 ℃.

2. Obtaining of recombinant bacteria containing 7 exogenous genes

Specific primers were designed, and the plasmids pDAG2_ RtPal + Sb4CL, pDTG1_ SbCHS-2+ SbCHI + SbFNSII-2 and pDFg1_ SbF6H + AtCPR1 were used as templates, and the corresponding primers in Table 2 were used to obtain each gene fragment containing the homologous arm by PCR amplification, and were electrically transformed into Pichia pastoris competence with the circular plasmid 3.5k-PFg1-PAg2+ pTg1 (see Liu Q, Shi X, Song L, et al. CRISPR-Cas 9 mediated genomic polymorphism in Pichia pastoris [ J ]. Microbiological Cell Factories,2019,18:144), and the recombinant was screened by YND solid culture to obtain Pichia pastoris engineered yeast transformed with 7 foreign genes (SbRtPAL, Sb4CL, CHS-2, CHI, SbFNI-4832, SbF6, SbCPR) 1. The strain was streaked onto YPD solid medium and PCR validation of the recombinant strain was performed again to ensure loss of the circular plasmid.

TABLE 2

3. Obtaining of recombinant bacteria containing 8 exogenous genes

The recombinant yeast containing 7 exogenous genes obtained in the above 2 was made competent, and the linearized plasmid pGAP _ SbUBGT + ENO1 was electrically transformed into the competent state, and recombinants were selected using a solid YPD medium having bleomycin resistance, to obtain Pichia pastoris genetically engineered bacteria into which 8 exogenous genes (RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR1, SbUBGT) were transferred.

4. Obtaining of recombinant bacteria containing 2 exogenous genes

Specific primers are designed, plasmid pDAG2_ SbF8H + SbPFOMT5 is used as a template, corresponding primers in table 2 are used for PCR amplification to obtain each gene fragment containing a homology arm, the gene fragment and a circular plasmid 3.5k-PAg2 (see Liu Q, Shi X, Song L, et al. CRISPR-Cas 9 mediated genomic integration in Pichia pastoris [ J ]. Microbiological Cell Factories,2019,18:144) are simultaneously electrically transformed into a Pichia pastoris competence, YND solid culture is used for screening recombinants, and Pichia pastoris genetically engineered bacteria transformed into 2 exogenous genes (SbF8H, SbPFOMT5) are obtained. The strain was streaked onto YPD solid medium and PCR validation of the recombinant strain was performed again to ensure loss of the circular plasmid.

Example 3 fermentation Process of 250ml Erlenmeyer flask of recombinant engineering bacteria

The bacterial strain for producing the baicalein: it contains 7 kinds of exogenous genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR 1.

The strains for producing baicalin: it contains 8 kinds of exogenous genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR1, SbUBGT.

Strain for producing wogonin: it contains 2 kinds of exogenous genes: SbF8H, SbPFOMT 5.

The recombinant strain was cultured in a liquid YPD medium at 30 ℃ and 200r/min until logarithmic phase, the collected strain was washed twice with sterile water and transferred to a 250mL Erlenmeyer flask containing 50mL of YPD liquid medium (no precursor was added to the strain producing baicalein or baicalin, 200. mu.M chrysin was added to the strain producing wogonin, and the strain producing baicalein or baicalin was cultured at 30 ℃ and 200r/min (144 hours for the strain producing baicalein or baicalin and 72 hours for the strain producing wogonin). 2% (v/v) glucose was added to the liquid medium every 24h during the fermentation.

Example 4 extraction and identification of fermentation products of recombinant Strain

Culturing the recombinant strain in YPD liquid culture medium to logarithmic phase, and taking 50OD (OD value is Yeast concentration unit, 1OD is about 5x 10)⁷And (4) yeast cells. OD value was measured by an ultraviolet spectrophotometer at a wavelength of 600 nm), washed twice with sterile water, and transferred to 50mL of YPD liquid medium. The strain for producing baicalein or baicalin does not need to be added with precursor, and the strain for producing wogonin also needs to be added with 200 mu M chrysin. Carrying out fermentation culture at the culture temperature of 30 ℃ and the rotation speed of 200 r/min; culturing the strain for producing baicalein or baicalin for 144 hours, and culturing the strain for producing wogonin for 72 hours; wherein 2% glucose was added every 24 h. 10mL of fermentation liquid after fermentation culture is extracted with equal volume of ethyl acetate for three times. The upper ethyl acetate organic phase was collected, distilled and dissolved in 1mL of methanol for High Performance Liquid Chromatography (HPLC) analysis.

The high performance liquid chromatography analysis is completed by reverse high performance liquid chromatography Agilent 1100, a C18 column is used as a chromatographic column, the column temperature is 30 ℃, the ultraviolet detection wavelength is 275nm, and the elution conditions are shown in Table 3.

TABLE 3 HPLC mobile phase conditions for the target product

Example 5 cellular Biomass and product yield during fermentation

Fermentation was carried out according to the 250ml Erlenmeyer flask fermentation culture method of example 3. Sampling is carried out at intervals of 24h during the fermentation period, and the cell biomass and the baicalein, baicalin or wogonin product amount are measured.

The fermentation and production conditions of the baicalein-producing strain are shown in FIG. 2A, and the cell biomass of the baicalein-producing strain gradually increases with the increase of the fermentation time, and the yield of the baicalein also gradually increases, reaches the highest value at about 144 hours of fermentation, and is about 35 mg/L. The method is the highest yield of the baicalein synthesized by whole cells from the beginning at present, and the production requirement can be met only by a basic carbon source glucose without additionally adding phenylalanine as a precursor during fermentation.

The fermentation and production conditions of the baicalin-producing strain are shown in FIG. 2B, and the cell biomass of the baicalin-producing strain gradually increases along with the increase of the fermentation time, and the yield of the baicalin also gradually increases, reaches the highest value at about 144 hours of fermentation, and is about 13 mg/L. The method is the highest yield of the baicalin synthesized by whole cells from the beginning at present, and the production requirement can be met only by a basic carbon source glucose without additionally adding phenylalanine as a precursor during fermentation.

The fermentation and production of the strain producing wogonin are shown in FIG. 2C, and the cell biomass of the strain producing wogonin gradually increased with the increase of fermentation time, and the production of wogonin also gradually increased, reaching the maximum value at about 72 hours of fermentation, and the yield was about 0.55 mg/L.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the above teachings of the present invention, and such equivalents may also fall within the scope of the appended claims of the present application.

Sequence listing

<110> university of eastern China's science and technology, Shanghai Chenshan vegetable garden

<120> genetic engineering yeast for producing wogonin compounds, construction method and application thereof

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gttccgtctc tcgccgttaa ctggaagggc aagaccgccg aggagttgac ggagtcagac 240

gactttttca gggaaatcgt gtctggtcct ttcgagaaat tcaccaaggt gacgatgatt 300

ctgccattga cggggaagca atactcggag aaagttgcag agaactgcgt tgcgtactgg 360

aaagccgtag ggaaatacac ggatgctgaa tcggaagcaa tcgacaagtt tctccaagtg 420

ttcaaggacg aaacgttcgc gcctggagct tccatcctct tcacccaatc gccggccggc 480

tccttgacga ttagcttctc gaaagacgga tcgattccag agcaagggaa ggcagtaata 540

gagaacaaac agctgtcgga ggcagtgctg gagtcgatca tcggaaagca tggtgtgtcg 600

ccgtcggcga agcagagttt ggcggcaaga ctatcggagt tgttttaa 648

<210> 5

<211> 1509

<212> DNA

<213> Scutellaria baicalensis (Scutellaria baicalensis Georgi)

<400> 5

atggacttag tagaagtcac actctacgcc gccctcttcc tcctctccgc cgccttcctc 60

ctcctaatct tcgccggaga ccgctcctcc ccaccgggcc cgttccctct tccgatcatc 120

gggcacctcc acctcctcgg cccgaaactc caccaaagct tccacgggct gtcccaacgg 180

cacggcccgc tgatgcaaat ccggctgggc tccatcaact gcgtggtggc gtcgacgccg 240

gagctggcta aagagttcct caaaaccaac gagctggtgt tctcctcgcg caaacactcg 300

accgccattg atatcgtcac ctacaattcc tccttcgcct tctccccata cgggccctac 360

tggaagtaca tcaagaagct ctgcacctac gagctgctcg gagcgagaaa cctccaccat 420

ttccagccca tcaggacctt cgaggtccac acttttctcc ggcttctcat ggagaagagc 480

gaatccgggg agagctttaa tgtcaccgag gagctcatta agctcaccag caacgtcatg 540

tccaatatga tgctcggcac caggtgctcc gccacagacg gcgaggcgga ggcggctagg 600

acggtgatcc gggaggtgac ggagatcttc ggggagttcg atgctgctga tatcatctgg 660

ttctgtaaga acttcgattt gcagggaata aggaagaggt cggaggatat tcagagaagg 720

tatgatgctt tgctggaaaa gattatcacc gacagagaga agctccggcg gagccaccgc 780

ggcggcgagg ccaaggattt tcttgatatc tttctggata taatggacag tggcaactct 840

gaggtgaaat tcagcagaga acatctcaaa gctttgattt tggatttctt caccgctggt 900

acagacacaa cggcgatcag cacagagtgg gcaatagcag aactgatgaa caacccaaag 960

gtactaaaga aagcacaaga agagatccag aaagtggtgg gatcttgtag attgatggat 1020

gaatcagacg cccctaatct cccatacctt gaggccatca tcaaggagac cttcaggctc 1080

caccctccga tcccgatgct tgcccggaaa tccgtctccg attgcgtcat cgacggctac 1140

aacatcccgg cgagcactct cctcttcgtc aacatttggt ccattggtcg gaaccccgag 1200

tgttgggaca gccccttctc cttccggccc gaacgcttct tcgagaagga caacgcttcg 1260

atcgatatca aggggcagca cttccagctg ctgccttttg gaacgggcag gaggggctgc 1320

ccgggaatgc ttttggccat acaggagctg ctacttatca taggcactat gattcagtgc 1380

tttgattggg aattgcctga gggttcgggc cctgtcgaca tgaccgaacg ggccgggttg 1440

actgctccac gggctgaaga tttgatttgt cgggtctcct gccgggttga cccgaaaatt 1500

gttttctag 1509

<210> 6

<211> 1554

<212> DNA

<213> Scutellaria baicalensis (Scutellaria baicalensis Georgi)

<400> 6

atggagttga gctctgtcat ctatggcgcc atcgccttgc tctctctttt ctactgttac 60

ctacacttct ccaagccaaa aaagagttcc ctgaacgcgc cgccggaggc tggcggcgcg 120

cgcttcatca ccggccacct ccacctgatg gacggccgct ccgcctccga caaacttcct 180

cacataaacc tgggtttgct tgcagaccag cacggcccaa tcttcacgat caggctgggc 240

gtgcaccgag ccgtggtggt gagcagctgg gagctagcca aggagatctt caccacccac 300

gacacggcgg tcatggctcg gccccggctc atagccgacg actacctgag ctacgacgga 360

gcctcgcttg ggttctcacc ctacggacca tactggcgcg aaatccgcaa gctggtcacc 420

accgagctgc tctcggctcg ccggatcgag ctgcagcgag ccacgcgtgt gcgtgagatc 480

acgcagttca ccggcgagct ctacaagctg tgggaggaga agaaagacgg gtctgggagg 540

gttttggtgg acatgaagca gtggctgggg aacttgagcc tcaatctggt gtcgaggatg 600

gtggtgggca agaggttcta cggcggcgac gactcggaga cgaccaagag gtggcggggc 660

gtcatgaggg agttcttcca gctcatcgga cagttcatcc ccggcgacgg gcttccgttt 720

ctccggtggc tcgacttggg tgggttcgag aagaggacga gggacactgc ctatgagctg 780

gataagatca tcgcaatgtg gctggcggag tatcggaaaa gggaatattc cggcgacgac 840

aaggagcagt gcttcatggc cctcatgctc tcactcgtgc aagctaatcc cactctgcaa 900

ctccactacg atgctgatac catcatcaaa gctacttgcc aggttttgat atcggcggca 960

agtgacacga cgacggtgat cctaatctgg gtaatttccc tcctactaaa caatgctgat 1020

gtcctaaaaa aggttcaaga agaactggac gaacaagtgg ggagagaaag acgagtggaa 1080

gaatcggaca taagcaattt accctacctc caagcggtgg tgaaggagac aatgaggcta 1140

taccctccgg cccccttcgc cggagtacga gccttcagcg aagactgcac cgtgggaggc 1200

taccacatcc agaaaggcac gtttttgata gtcaatctgt ggaagctgca tcgagaccct 1260

cgtgtgtggt ccgatgatgc cttggagttc aaaccacaac ggttttttga caaaaaggtg 1320

gaggttaagg gtcaggactt cgagttgatg ccatttggtg gtggtcgaag aatgtgtccc 1380

ggctcgaacc tgggcatgca catggtgcac tttgtgctgg ctaacatatt gcaggccttt 1440

gacataacca ctgggtccac tgtggatatg accgagagtg ttgggttgac caacatgaaa 1500

gccacaccac ttgatgctat tctcactcca aggttgtcac ctactctata ttaa 1554

<210> 7

<211> 2079

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 7

atgacctctg ccttgtacgc ctctgatctt tttaagcaat tgaagtctat catgggaact 60

gactctctta gtgacgatgt cgtccttgtc atcgccacta cctctcttgc tcttgttgcc 120

ggtttcgtcg tcttgctttg gaaaaagacc actgccgaca gatctggaga acttaagcct 180

ttgatgatcc ctaagtcttt gatggccaag gatgaggatg acgaccttga ccttggttct 240

ggaaagacta gagtttctat cttcttcgga actcaaactg gaactgctga aggattcgcc 300

aaggcccttt ctgaagagat caaggctaga tacgagaagg ctgccgtcaa ggtcatcgac 360

cttgacgatt acgccgctga tgatgaccag tatgaagaga agcttaagaa ggagactctt 420

gccttttttt gcgtcgctac ctacggtgac ggtgaaccaa ccgacaacgc tgctagattt 480

tctaagtggt ttaccgagga aaatgaaaga gacatcaaac ttcagcagtt ggcctacgga 540

gtctttgcct tgggtaatag acagtacgag cattttaaca agattggtat tgtccttgat 600

gaagaacttt gtaagaaagg tgccaagagg cttattgagg ttggattggg tgacgacgac 660

cagagtatcg aagacgactt taacgcttgg aaggagtctc tttggtctga gcttgacaag 720

cttcttaagg acgaggacga caagtctgtc gccaccccat ataccgctgt cattccagaa 780

tatagagtcg tcacccacga tccaaggttc actactcaga aatctatgga atctaatgtt 840

gctaatggaa acaccaccat tgacatccac catccatgca gagtcgatgt cgccgtccaa 900

aaagagttgc acacccatga gagtgataga tcttgcattc acttggagtt cgacatctct 960

aggaccggta tcacctatga gaccggtgat cacgtcggag tttacgccga gaaccacgtc 1020

gaaatcgtcg aggaagccgg aaagttgttg ggacatagtc ttgatttggt tttttctatt 1080

cacgctgata aggaagacgg atctccattg gagagtgctg tccctccacc tttccccgga 1140

ccatgtacct tgggaaccgg tcttgccaga tacgccgacc ttttgaaccc accaaggaag 1200

tctgctttgg tcgctttggc tgcttacgct accgagccaa gtgaagccga gaagcttaaa 1260

cacttgacca gtccagatgg taaggacgaa tactctcagt ggattgtcgc ctctcagagg 1320

agtttgcttg aagtcatggc cgcctttcct agtgctaagc ctcctcttgg tgtctttttc 1380

gccgctatcg ccccaagatt gcagccaagg tactatagta tcagttcttg ccaagattgg 1440

gccccatcta gggttcatgt caccagtgcc cttgtctatg gaccaactcc aactggtagg 1500

attcacaagg gtgtctgcag tacttggatg aagaacgctg ttccagccga aaagtctcac 1560

gagtgttctg gtgcccctat cttcatcaga gctagtaatt ttaaattgcc ttctaaccca 1620

tctaccccta tcgtcatggt tggtcccgga actggtcttg ccccttttag aggttttttg 1680

caagaaagga tggcccttaa agaggacggt gaggagcttg gttcttctct tcttttcttt 1740

ggttgcagaa atagacaaat ggattttatt tatgaagatg aattgaacaa cttcgtcgac 1800

caaggagtca tctctgagct tattatggct ttctctaggg agggagccca gaaagaatac 1860

gttcagcata agatgatgga gaaagccgcc caagtctggg atcttatcaa agaggaggga 1920

tacttgtatg tctgtggaga cgctaaggga atggctaggg acgtccatag aacccttcac 1980

actatcgtcc aagaacaaga gggagttagt agttctgagg ccgaagccat cgtcaagaag 2040

ttgcaaaccg agggtaggta tcttagggac gtttggtga 2079

<210> 8

<211> 1431

<212> DNA

<213> Scutellaria baicalensis (Scutellaria baicalensis Georgi)

<400> 8

atgggtcagc ttcacattgt ccttgtccct atgatcgccc acggtcacat gatccctatg 60

ttggacatgg ctaaactttt tagttctagg ggtgttaaga ccaccatcat cgctactcca 120

gccttcgctg aaccaattag aaaagctagg gagagtggtc acgacatcgg attgaccacc 180

accaagttcc cacctaaggg atctagtctt ccagataata ttagaagttt ggaccaagtt 240

accgacgact tgttgcctca cttctttaga gctcttgagt tgttgcaaga accagtcgag 300

gagattatgg aggacttgaa gccagattgc ttggtcagtg acatgttcct tccatggacc 360

accgacagtg ccgctaagtt cggtatccca agattgcttt tccacggaac cagtcttttc 420

gctaggtgct ttgccgagca gatgagtatc cagaagcctt ataagaatgt cagttctgac 480

tctgagccat tcgtccttag aggtttgcca cacgaggtct ctttcgtcag aacccagatt 540

ccagactacg aattgcaaga gggaggagac gacgccttct ctaagatggc caaacagatg 600

agggacgccg acaagaagag ttatggtgac gttatcaact cttttgaaga attggagtct 660

gaatacgctg actataataa aaatgtcttc ggaaagaaag cttggcacat cggtcctctt 720

aagttgttca acaatagagc tgagcagaag tcttctcaga ggggtaagga gagtgccatc 780

gatgaccacg agtgccttgc ttggcttaac tctaagaagc caaattctgt cgtttacatg 840

tgcttcggaa gtatggccac cttcactcca gctcaacttc acgagaccgc tgtcggactt 900

gagagtagtg gtcaagattt catctgggtc gtcagaaacg gtggtgagaa tgaggactgg 960

ttgccacaag gattcgagga gagaatcaaa ggtaagggac ttatgattag gggttgggct 1020

ccacaagtta tgatcttgga ccacccaagt accggtgctt tcgtcaccca ctgcggatgg 1080

aactctacct tggagggtat ctgcgccgga cttccaatgg tcacttggcc agtctttgct 1140

gagcagttct ataatgagaa gcttgtcacc gaagtcctta agaccggagt ctctgtcggt 1200

aacaagaagt ggcaaagggt cggagaagga gtcggaagtg aagccgttaa ggaggccgtt 1260

gagagagtca tggttggaga tggagccgcc gaaatgagaa gtagggcctt gtactacaag 1320

gaaatggcta gaaaagccgt cgaggaggga ggaagttctt acaataacct taacgctttg 1380

atcgaagagt tgagtgccta cgtccctcct atgaaacaag gattgaattg a 1431

<210> 9

<211> 1581

<212> DNA

<213> Scutellaria baicalensis (Scutellaria baicalensis Georgi)

<400> 9

atgctatatg acaacagcca atacttagcc atggaattca gttctgccat ttatggagcc 60

attgccttct ttcttttctt atactattgc ctgctctata caacatcttc caagccaaat 120

accagagctt acaaagcacc gccggaggct ggcggggcgc gcctcttttc cggccatctc 180

catctcatgg ctggtggcac caccggtgaa cttccccaca tcaacttggc taacttagca 240

gataaacacg gcccagtttt cacaatccgg ctgggcgtaa agcgagcctt ggtggtgagc 300

agttgggaat cagccaaaga actgttcact acctgtgacg tggcggtgtc gtcccgtcca 360

cgtatgaagg cggcgaagct cttaggctat gatttcgcca tgtttgggtt cgccccatac 420

ggtgcatact ggcgtgagct gcgaaaactg atctcagttg agcttctctc aactcgtaga 480

atggagttgc agaaagaggt tcgagattca gaaactaggg agtccattaa agagctttat 540

aagctttggg aagagagaag tgagggttcg gacagtgtgt tggtggacat gaagcagtgg 600

tttggggact tgaacttgaa tgttgtcctg agaatggtgg cggggaagag atggatcggc 660

ccggagacgg ggcggtggag ggaggtgctg agggatttct tctacttggc ggggatgttt 720

gttccggccg atgcctttcc gtttcttggg tggttggatt taggcggaca tgagaagaga 780

atgaggcaaa ctgctaaaga attggacgga attgttggag gatgggtggc ggagcatcgg 840

gaaaaggaat attccggcga agataagcca aaggatttcg tcgatgttat gttgtctgtt 900

gtacaaggtt caagtcttcg agctgactat gatgctgata ccatcatcaa agcaacatgc 960

gaggctttga ttgtgggtgg aagtgacact acaacagtga tgctaatatg gacactttct 1020

ttcctattga ataatcggca tgttctgaga aaggctcaag aagagctgga caagcatgta 1080

ggaagggaaa gacgtgtaaa ccaatcagac atcaacaact tagtctatct tcaggcaata 1140

gttaaagaga ctttgagatt ataccctgct ggccccattg gtgggatacg agagttcacc 1200

caagattgcc aagtcggagg ctaccacgtt cctaaaggca cgtggctgat cgtgaacttg 1260

tggaagttgc atcgagatcc caaggtttgg tcagaagatt gtttggaatt taggccagaa 1320

cgatttctaa ataaaaacat agatgtcagg ggtcaagatt tcgagttgat cccatttggt 1380

ggtggtcgga ggatttgccc cggggctaat tttgggctgc atatgctgca tttggtgttg 1440

gctaacttgc tgcaagcttt cgagctcacc actgtgtccg atcaagtgat tgatatgacg 1500

gagagtgctg gaatgacaaa tatgaaagcc acaccactta atgttcttgt tgccccaagg 1560

ttgtctccca ctctttactg a 1581

<210> 10

<211> 705

<212> DNA

<213> Scutellaria baicalensis (Scutellaria baicalensis Georgi)

<400> 10

atgatcgaga agtacaaccg caccattctc cagagcgaca ctctcctcaa gtacattttg 60

gaaacgagtg cctttcccag agaacatgaa cagcttaagg agctgagaga agctactgtt 120

gaaaagtaca aagcttggag tttgatgaat gtgcctgctg atgaggggca gtttatttcg 180

atgcttttga aaataatgaa tgcaaagaag acaattgaaa ttggggtttt cactggatac 240

tcacttctct ctacagctct tgctcttcct catgatggca aaataatagc aattgatcca 300

gacaaagaag catatgagac tggtctgccc agcattcaga aggccaacat ggctcacaaa 360

attcacttct tcgattctcc tgccacacaa atcttggatg atctcatcgc caagggagaa 420

gaaggcacgt tcgattttgc atttgtggat gcagacaaag aaaactacat gaattatcac 480

gagcaattat tgaaactggt taagattggg ggagtgatcg ggtacgacaa caccctatgg 540

ttcggtacgg tggcatcgcc tgagacggag gagatgttgg agtttgtgaa gagtagtcga 600

gcccatatgg tggagttgaa ctcttttctt gcaaccgatt ctcgtatcga gttagcccat 660

ctttctatcg gagatggact tgctttgtgc aagcgtctca aatga 705

<210> 11

<211> 40

<212> DNA

<213> primers (Primer)

<400> 11

tcaattgaac aactatttcg atggagactg tagaaaacca 40

<210> 12

<211> 40

<212> DNA

<213> primers (Primer)

<400> 12

gccgccgcgg ctcgaggtac atttgaaaga gtagctgctt 40

<210> 13

<211> 40

<212> DNA

<213> primers (Primer)

<400> 13

tcaattgaac aactatttcg atggtgacag ttgaagaatt 40

<210> 14

<211> 40

<212> DNA

<213> primers (Primer)

<400> 14

gccgccgcgg ctcgaggtac attgagaggc acactatgca 40

<210> 15

<211> 39

<212> DNA

<213> primers (Primer)

<400> 15

tcaattgaac aactatttcg atgtctgctt cgccatccg 39

<210> 16

<211> 40

<212> DNA

<213> primers (Primer)

<400> 16

gccgccgcgg ctcgaggtac aaacaactcc gatagtcttg 40

<210> 17

<211> 40

<212> DNA

<213> primers (Primer)

<400> 17

tcaattgaac aactatttcg atggacttag tagaagtcac 40

<210> 18

<211> 40

<212> DNA

<213> primers (Primer)

<400> 18

gccgccgcgg ctcgaggtac gaaaacaatt ttcgggtcaa 40

<210> 19

<211> 40

<212> DNA

<213> primers (Primer)

<400> 19

tcaattgaac aactatttcg atggagttga gctctgtcat 40

<210> 20

<211> 40

<212> DNA

<213> primers (Primer)

<400> 20

gccgccgcgg ctcgaggtac atatagagta ggtgacaacc 40

<210> 21

<211> 40

<212> DNA

<213> primers (Primer)

<400> 21

tcaattgaac aactatttcg atgctatatg acaacagcca 40

<210> 22

<211> 40

<212> DNA

<213> primers (Primer)

<400> 22

gccgccgcgg ctcgaggtac gtaaagagtg ggagacaacc 40

<210> 23

<211> 40

<212> DNA

<213> primers (Primer)

<400> 23

tcaattgaac aactatttcg atgatcgaga agtacaaccg 40

<210> 24

<211> 40

<212> DNA

<213> primers (Primer)

<400> 24

gccgccgcgg ctcgaggtac tttgagacgc ttgcacaaag 40

<210> 25

<211> 42

<212> DNA

<213> primers (Primer)

<400> 25

gaccttcgtt tgtgcggatc agatcttttt tgtagaaatg tc 42

<210> 26

<211> 40

<212> DNA

<213> primers (Primer)

<400> 26

gctatggtgt gtgggggatc tctcacttaa tcttctgtac 40

<210> 27

<211> 40

<212> DNA

<213> primers (Primer)

<400> 27

ttactcttcc agattttctc agatcttttt tgtagaaatg 40

<210> 28

<211> 40

<212> DNA

<213> primers (Primer)

<400> 28

gcgatgcgcg gagtccgaga tctcacttaa tcttctgtac 40

<210> 29

<211> 40

<212> DNA

<213> primers (Primer)

<400> 29

ggcagtaatt gatatactag attttggtca tgcatgagat 40

<210> 30

<211> 40

<212> DNA

<213> primers (Primer)

<400> 30

gacctaccct acgacgggcc ttgaagctat ggtgtgtggg 40

<210> 31

<211> 40

<212> DNA

<213> primers (Primer)

<400> 31

taagggattt tggtcatgca aaagagtgag aggaaagtac 40

<210> 32

<211> 40

<212> DNA

<213> primers (Primer)

<400> 32

aaaagatctg atctcatgca caaaagcctg ctagatgtgc 40

<210> 33

<211> 23

<212> DNA

<213> primers (Primer)

<400> 33

ggggtgagcc tcaaggtata tag 23

<210> 34

<211> 21

<212> DNA

<213> primers (Primer)

<400> 34

gggatagcca tcgtttcgaa t 21

<210> 35

<211> 27

<212> DNA

<213> primers (Primer)

<400> 35

ctatgaccat gattacgaat tcgagct 27

<210> 36

<211> 20

<212> DNA

<213> primers (Primer)

<400> 36

tgcctgcagg tcgactctag 20

<210> 37

<211> 20

<212> DNA

<213> primers (Primer)

<400> 37

catgattacg aattcgagct 20

<210> 38

<211> 20

<212> DNA

<213> primers (Primer)

<400> 38

gtcgactcta gacggactca 20

Claims (10)

1. A method of producing wogonin, comprising:

(1) providing a yeast engineering bacterium, wherein an exogenous expression cassette of the following genes is transformed in the yeast engineering bacterium: F8H, PFOMT 5; and

(2) culturing the yeast engineering bacteria in the step (1), and adding chrysin as a precursor to produce wogonin.

2. The method of claim 1, wherein the F8H and/or PFOMT5 is from Scutellaria baicalensis (Scutellaria baicalensis Georgi, Sb).

3. The method of claim 1, wherein the engineered yeast is pichia pastoris.

4. The method of claim 3, wherein KU70 gene function is down-regulated or deleted in Pichia pastoris.

5. The method of claim 1, wherein the culturing in step (2) is carried out using a yeast medium, and the carbon source is supplemented during the culturing.

6. The method of claim 5, wherein the carbon source is glucose; more preferably, the glucose is added 1 time every 12 to 36 hours until the final concentration is 1 to 4% (v/v); preferably, during initial culture, chrysin is added into a yeast culture medium, and the final concentration in a fermentation liquid is 50-800 mu M; preferably, the cultivation time in step (2) is 55-95 hours.

7. The yeast engineering bacteria for producing wogonin is characterized by comprising an exogenous expression cassette of the following genes: F8H, PFOMT 5.

8. The engineered yeast strain of claim 7, wherein the engineered yeast strain is Pichia pastoris; preferably, the F8H and/or PFO MT5 is from Scutellaria baicalensis.

9. Use of a combination of genes comprising the following group of genes: F8H and/or PFO MT5, the genome combination is used for transforming into yeast engineering bacteria to prepare wogonin compounds; preferably, the F8H and/or PFO MT5 is from Scutellaria baicalensis.

10. A kit for producing wogonin, comprising: F8H and PFOMT5 genes or expression constructs or yeast cells containing the genes, and chrysin or a culture medium containing the chrysin; preferably, the F8H and/or PFO MT5 is from Scutellaria baicalensis.

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