CN114561312A

CN114561312A - Recombinant yeast for synthesizing ursolic acid and construction method thereof

Info

Publication number: CN114561312A
Application number: CN202210432868.3A
Authority: CN
Inventors: 王颖; 贾男; 余源; 李敬知; 姜尧竹; 李春
Original assignee: Tsinghua University; Beijing Institute of Technology BIT
Current assignee: Tsinghua University; Beijing Institute of Technology BIT
Priority date: 2022-04-24
Filing date: 2022-04-24
Publication date: 2022-05-31
Anticipated expiration: 2042-04-24
Also published as: CN114561312B

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a recombinant yeast for synthesizing ursolic acid, a construction method and application thereof.

Description

Recombinant yeast for synthesizing ursolic acid and construction method thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to saccharomyces cerevisiae for efficiently synthesizing ursolic acid, and a construction method and application thereof.

Background

Ursolic Acid (UA) is a natural pentacyclic triterpenoid carboxylic acid, and is present in various fruits and plants, such as apple, hawthorn, glossy privet fruit, and the like. Research shows that ursolic acid has many important pharmacological functions and physiological activities, for example, by mediating some pharmacological processes and regulating some signal pathways, ursolic acid can be used for preventing the development of some chronic diseases, and has the functions of resisting oxidation, resisting inflammation, resisting cancer and protecting liver, and has the capability of inducing apoptosis. However, the main industrial source of ursolic acid is still extracted from plants, which is bound by objective factors such as land, climate, etc., and the problems of farmland occupation, organic reagent consumption, etc. are caused, thus the large-scale popularization and application of ursolic acid are severely limited. With the development of the disciplines of synthetic biology, metabolic engineering and the like, the production of triterpenoids by using microbial cell factories is the current focus.

Saccharomyces cerevisiae (C.,)Saccharomyces cerevisiae) The strain is selected as a chassis host because of the advantages of clear genetic background, mature gene operation technology, safety, no toxicity and the like. For example: patent CN111205993A discloses a recombinant yeast for producing ursolic acid and oleanolic acid, a construction method and application thereof, and discloses that CYP716A12, CYP716AL1 and AtCPR1 are respectively used for improving the yield of the ursolic acid and the oleanolic acid.

Disclosure of Invention

The application further constructs a synthetic approach of the ursolic acid by key enzyme excavation and heterologous expression; the capability of yeast for synthesizing ursolic acid is enhanced by increasing the supply of precursors such as acetyl coenzyme A and the like; the cofactor supply of the ursolic acid synthesis path is further improved by analyzing the relationship between the synthesis path and the cofactor of the endogenous metabolic network, so that the synthesis efficiency of the ursolic acid is improved; and finally, exploring the most suitable pH and the most suitable nutrient components through fermentation optimization. The method aims at high yield of ursolic acid in saccharomyces cerevisiae, solves the problem of low synthesis efficiency of the ursolic acid in the saccharomyces cerevisiae from the perspective of supplying cofactors and precursors, and also provides a new idea and method for efficient synthesis of other triterpene compounds.

The invention aims to provide saccharomyces cerevisiae for efficiently synthesizing ursolic acid and a construction method thereof. The inventor creatively discovers that the yield of the synthesized ursolic acid can be improved by converting cytochrome P450 enzyme Ej8656 into the saccharomyces cerevisiae for synthesizing alpha-amyrin. In order to enhance the supply of precursor acetyl-CoA, acetaldehyde dehydrogenase ALD6 is overexpressed, and mitochondrial transporter MPC2 is knocked out to obtain Saccharomyces cerevisiae strain WN 29; the relation between the synthesis pathway and the endogenous cofactor metabolism network is analyzed, and WN85 is obtained by overexpression of glyceraldehyde-3-phosphate dehydrogenase GaPC from Kluyveromyces lactis and NADH-rHMGR from the silicate bacillus.

In a first aspect of the present invention, there is provided a recombinant yeast for synthesizing ursolic acid.

Preferably, the recombinant yeast expresses a CYP450 enzyme. The CYP450 enzyme is derived from loquat.

Preferably, the recombinant yeast further expresses CPR enzyme. The CPR enzyme includes but is not limited to CPR enzyme derived from alfalfa (preferably Medicago truncatula), Glycyrrhiza glabra and/or Lotus corniculatus.

In one embodiment of the invention, the recombinant yeast expresses CYP450 enzyme and/or CPR enzyme.

Preferably, the recombinant yeast overexpresses a gene in the acetyl-coa precursor synthesis pathway, overexpresses a cofactor supplying gene, and/or reduces expression of a gene in the acetyl-coa precursor consuming pathway.

The genes of the acetyl-CoA precursor synthesis pathway are selected from coding genes of acetyl-CoA synthase (preferably ACS1/ACS 2), alcohol dehydrogenase (preferably ADH 2) and/or acetaldehyde dehydrogenase (preferably ALD 6);

the cofactor supply gene is selected from isocitrate dehydrogenase (preferably IDP2 derived from Saccharomyces cerevisiae), malate dehydrogenase (preferably MAE1 derived from Saccharomyces cerevisiae), and acetaldehyde dehydrogenase (preferably rice-based rot fungus) ((Dickeyazeae) ADA, E.coli: (Escherichia coli) eutE) and glyceraldehyde-3-phosphate dehydrogenase (preferably Kluyveromyces lactis (K.lactis) ((R))Kluyveromyces lactis) GaPC of (a) and/or NADH-dependent hydroxymethylglutarate reductase (preferably from Bacillus silicate: (b))Silicibaterpomeroyi) rHMGR) of (a);

the genes of the acetyl-CoA precursor consumption pathway are selected from encoding genes of mitochondrial porin (preferably POR 2), mitochondrial transporter (preferably MPC 2) and/or citrate synthase (preferably CIT 2).

In a specific embodiment of the invention, said recombinant yeast expresses CYP450 enzyme Ej8656 from loquat and CPR enzyme MtCPR from medicago truncatula.

In a specific embodiment of the invention, the recombinant yeast expresses CYP450 enzyme Ej8656 derived from loquat, CPR enzyme MtCPR derived from medicago truncatula, Rhizoctonia solani: (Rhizobium solani)Dickeyazeae) Acetaldehyde dehydrogenase ADA and a knock-out mitochondrial transporter gene MPC 2.

In a specific embodiment of the present invention, the recombinant yeast expresses CYP450 enzyme Ej8656 derived from loquat, CPR enzyme MtCPR derived from medicago truncatula, and Rhizoctonia solani (Zygospora oryzae) ((R))Dickeyazeae) Acetaldehyde dehydrogenase ADA, acetaldehyde dehydrogenase ALD6 and a knock-out mitochondrial transporter gene MPC 2.

In one embodiment of the present invention, the recombinant yeast expresses CYP450 enzyme Ej8656 derived from loquat and CPR enzyme LjCPR derived from lotus japonicus.

In one embodiment of the present invention, the recombinant yeast expresses CYP450 enzyme derived from loquat, CPR enzyme LjCPR derived from Lotus japonicus, glyceraldehyde-3-phosphate dehydrogenase GaPC derived from Kluyveromyces lactis, and/or NADH-dependent hydroxymethylglutarate reductase rHMGR derived from Bacillus silicate.

Preferably, the recombinant yeast is a yeast capable of synthesizing alpha-amyrin.

Preferably, the recombinant yeast is selected from any one of saccharomyces cerevisiae, pichia pastoris, candida lipolytica, yarrowia lipolytica, hansenula anomala, schizosaccharomyces pombe, rhodotorula glutinis, candida tropicalis or candida utilis.

In one embodiment of the present invention, the recombinant yeast is Saccharomyces cerevisiae.

In a second aspect of the present invention, there is provided a plasmid for constructing the above recombinant yeast, said plasmid comprising genes encoding CYP450 enzyme and CPR enzyme, said CYP450 enzyme being derived from loquat.

Preferably, the CPR enzyme is derived from alfalfa (preferably medicago truncatula), licorice or crowtoe.

In a third aspect of the present invention, there is provided a method for constructing the recombinant yeast.

Preferably, the construction method comprises introducing a gene encoding a CYP450 enzyme into the yeast. The CYP450 enzyme is derived from loquat.

Preferably, the construction method comprises introducing genes encoding CYP450 enzyme and CPR enzyme into the yeast. The coupling of CYP450 enzyme and CPR enzyme can raise the yield of ursolic acid.

The construction method comprises introducing a gene of an acetyl-CoA precursor synthesis pathway, and/or introducing a cofactor supply gene, and/or knocking out a gene of an acetyl-CoA precursor consumption pathway into the recombinant yeast.

The genes of the acetyl-CoA precursor synthesis pathway are selected from coding genes of acetyl-CoA synthase (preferably ACS1/ACS 2), alcohol dehydrogenase (preferably ADH 2) and/or acetaldehyde dehydrogenase (preferably ALD 6).

The cofactor supply gene is selected from isocitrate dehydrogenase (preferably IDP2 derived from Saccharomyces cerevisiae), malate dehydrogenase (preferably MAE1 derived from Saccharomyces cerevisiae), and acetaldehyde dehydrogenaseEnzyme (preferably rice-based rot fungi) ((Dickeyazeae) ADA, E.coli: (Escherichia coli) eutE) and glyceraldehyde-3-phosphate dehydrogenase (preferably Kluyveromyces lactis (K.lactis) ((R))Kluyveromyces lactis) GaPC of (a) and/or NADH-dependent hydroxymethylglutarate reductase (preferably from Bacillus silicate: (b))Silicibaterpomeroyi) rHMGR) of (1).

In a specific embodiment of the present invention, the construction method comprises introducing into a recombinant yeast a gene encoding CYP450 enzyme Ej8656 derived from Eriobotrya japonica and CPR enzyme MtCPR derived from Medicago truncatula.

In a specific embodiment of the invention, the construction method comprises the expression of CYP450 enzyme Ej8656 from loquat, CPR enzyme MtCPR from medicago truncatula, Rhizoctonia solani (Rhizoctonia solani) (B.oryzae) from medicago truncatula into recombinant yeastDickeyazeae) The gene coding for acetaldehyde dehydrogenase ADA and the knock-out mitochondrial transporter gene MPC 2.

In a specific embodiment of the invention, the construction method comprises introducing CYP450 enzyme Ej8656 from loquat, CPR enzyme MtCPR from medicago truncatula, and Rhizoctonia solani (Zygospora oryzae) into the recombinant yeastDickeyazeae) The genes encoding acetaldehyde dehydrogenase ADA and ALD6, and the knock-out mitochondrial transporter gene MPC 2.

In a specific embodiment of the present invention, the construction method comprises introducing into a recombinant yeast a gene encoding CYP450 enzyme Ej8656 derived from Eriobotrya japonica and CPR enzyme LjCPR derived from Lotus corniculatus.

In one embodiment of the present invention, the construction method comprises introducing into a recombinant yeast a gene encoding CYP450 enzyme derived from Eriobotrya japonica, CPR enzyme LjCPR derived from Lotus corniculatus, glyceraldehyde-3-phosphate dehydrogenase GaPC derived from Kluyveromyces lactis, and/or NADH-dependent hydroxymethylglutarate reductase rHMGR derived from Bacillus silicate.

Preferably, a vector (preferably a plasmid) containing the gene to be introduced is constructed and then transformed into yeast.

Preferably, each of the introduced genes is in single or multiple copies.

Preferably, the construction method comprises overexpressing a cofactor supplying gene of the ursolic acid synthesis pathway and/or a gene of the acetyl-CoA precursor synthesis pathway by constructing a high-copy plasmid.

In a fourth aspect of the present invention, a method for synthesizing ursolic acid is provided.

Preferably, the method comprises the step of fermenting and culturing the recombinant yeast.

Preferably, the fermentation medium contains galactose. Further preferably, the medium of the fermentation also contains glucose or a disaccharide or polysaccharide or other type of carbon source that can be broken down into glucose.

In the fifth aspect of the invention, the invention provides an application of the recombinant yeast and/or the construction method of the recombinant yeast in the synthesis of ursolic acid.

The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that specify the steps of the recited process, as well as other steps that are not materially affected.

All combinations of items described herein as "and/or" including "connected by this term are to be considered as if each combination had been individually listed herein. For example, "A and/or B" includes "A", "A and B", and "B". Also for example, "A, B and/or C" encompasses "a," B, "" C, "" a and B, "" a and C, "" B and C, "and" a and B and C.

The application is abbreviated and fully called:

CYP 450: cytochromeP450, cytochrome P450.

CPR: cytochrome P450 oxidoreductase.

rHMGR: hydroxymethyl glutarate reductase.

NADH: nicotinamide adenine dinucleotide, reduced form of Nicotinamide adenine dinucleotide and reduced coenzyme I, wherein N refers to Nicotinamide, A refers to adenine and D refers to dinucleotide.

GaPC: glyceraldehyde-3-phosphate dehydrogenase in the cytoplasm.

ADH 2: alcohol dehydrogenase 2, alcohol dehydrogenase 2.

The saccharomyces cerevisiae engineering bacteria have the following advantages:

1. the invention utilizes saccharomyces cerevisiae (Saccharomyces cerevisiae) As a chassis host, the catalytic efficiency of cytochrome P450 enzyme is improved and the yield of ursolic acid is improved by strengthening the precursor supply and analyzing the relationship between the synthesis path and the endogenous cofactor metabolism network.

2. The expression of the ursolic acid synthase in the invention is induced by galactose, thereby realizing the production of the ursolic acid at a specific stage and reducing the content of the ursolic acid on the saccharomyces cerevisiae (C)Saccharomyces cerevisiae) Damage to cells and increase yield.

3. Saccharomyces cerevisiae (C. cerevisiae) of the present inventionSaccharomyces cerevisiae) The engineering bacteria directly synthesize a large amount of ursolic acid by using glucose and galactose, so that the high-efficiency synthesis of the plant secondary metabolite in the saccharomyces cerevisiae is realized, the problems of low yield of early chemical synthesis and separation extraction are solved, and the industrial production of the ursolic acid is promoted.

Drawings

FIG. 1: a plasmid construction flow chart of a saccharomyces cerevisiae cell factory for efficiently synthesizing ursolic acid.

FIG. 2: and (3) detecting the ursolic acid production of the constructed ursolic acid producing saccharomyces cerevisiae engineering strain WN 1.

FIG. 3: and (3) gas mass spectrometry detection results of the ursolic acid produced by the constructed ursolic acid producing saccharomyces cerevisiae engineering strain.

FIG. 4: the constructed saccharomyces cerevisiae engineering strains WN1, WN7, WN29 and WN85 for producing the yield result of the ursolic acid in a 100mL shake flask.

FIG. 5: and the constructed saccharomyces cerevisiae engineering strain WN85 for producing the ursolic acid is used for producing the yield result of the ursolic acid by batch feeding fermentation in a 5L fermentation tank.

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: construction of plasmid of cytochrome P450 enzyme Ej8656 and construction of saccharomyces cerevisiae chassis

The sequence of cytochrome P450 enzyme Ej8656 from loquat was analyzed by using an online codon analysis website (http:// www.jcat.de /) to synthesize a codon-optimized gene (the sequence of Ej8656 after codon optimization is shown in SEQ ID NO: 27) in Saccharomyces cerevisiae by codon preference, and the gene was ligated to plasmid pESC-Leu together with Medicago truncatula source MtCPR and Lotus japonicus source LjCPR. The PCR reaction system shown in Table 1 and the PCR primer pair P shown in Table 2 were used_GAL10-Ej8656-T_ADH1、P_GAL1-MtCPR-T_CYC1、P_GAL1-LjCPR-T_CYC1Three expression cassettes were amplified, wherein P_GAL10、P_GAL1Represents a promoter; t is_ADH1、T_CYC1Represents a terminator.

TABLE 1 PCR reaction System

TABLE 2 PCR primers

The amplification procedure is shown in Table 3.

TABLE 3 amplification procedure

Step 2 to step 4, repeat 30 cycles. After the completion of the PCR reaction, the target fragment was recovered by electrophoresis using 1% agarose gel.

The empty vectors pRS415 (single-copy plasmid) and pRS42K (multi-copy plasmid) were digested with restriction enzymes and recovered, and the digestion system is shown in Table 4.

TABLE 4 plasmid digestion System

The amplified expression cassette was assembled with a linear vector using the Gibson assembly method, see Table 5 for the Gibson system.

TABLE 5 Gibson Assembly System

Each fraction was divided into 6.5. mu.l, and added at an equimolar ratio of 2.5. mu.l of the total volume of the amplified fragment and the linear vector, and 1. mu.l of Taq DNA ligase was added to the reaction mixture, and the total reaction volume was 10. mu.l. The reaction system is placed in a PCR instrument, reacted for 1 hour at 50 ℃ and then quickly placed on ice after the reaction is finished. Mu.l of the reaction was transformed into 50. mu.l of E.coli competent cells. The heat shock transformation procedure of Escherichia coli was as follows:

(1) coli Top10 competent cells were removed from a-80 ℃ freezer and thawed on ice for 5 min.

(2) Add 10. mu.l of the reaction to the cell suspension, mix by gentle rotation, ice-wash for 30 min.

(3) The competent cells were placed in a 42 ℃ water bath for 90 seconds and then quickly transferred to an ice bath to cool for 2 minutes without shaking.

(4) To the competent cells, 500. mu.l of fresh LB medium without antibiotics was added, mixed well and placed at 37 ℃ and 200rpm in a shaker for 60 minutes.

(5) Under aseptic condition, taking a proper amount of bacterial liquid, and uniformly coating the bacterial liquid on a fresh LB solid culture medium plate containing corresponding antibiotics by using a coating rod. After the culture, plasmids were extracted.

Plasmids pRS415-Leu-Ej8656-MtCPR (the construction process is shown in figure 1), pRS42K-KanMX-Ej8656-MtCPR (the KanMX represents a geneticin screening marker for screening positive strains), and pRS415-Leu-Ej8656-LjCPR are respectively transformed into saccharomyces cerevisiae to construct a synthetic ursolic acid strain, wherein a strain transformed into pRS415-Leu-Ej8656-MtCPR is named WN1 (single copy), a strain transformed into pRS42K-KanMX-Ej8656-MtCPR is named WN5 (multiple copies), a strain transformed into pRS415-Leu-Ej8656-LjCPR is named WN7 (single copy), and the lithium acetate transformation steps are as follows:

(1) the saccharomyces cerevisiae cells to be transformed are inoculated into 2-5ml SD-His-Ura-Trp liquid culture medium and cultured for 16h overnight at the temperature of 30 ℃ and 200 rpm.

(2) The overnight cultured Saccharomyces cerevisiae cells to be transformed were inoculated in a volume ratio of 10% into fresh SD-His-Ura-Trp liquid medium at 200rpm and 30 ℃ for 4-5 hours.

(3) The cell culture broth was added to a 1.5ml centrifuge tube and centrifuged at 4000rpm for 3 minutes.

(4) The supernatant was discarded, resuspended in 1ml of sterile water, and centrifuged at 4000rpm for 3 minutes.

(5) The supernatant was discarded, resuspended in 1ml sterile water, and centrifuged at 4000rpm for 3 minutes.

(6) The supernatant was removed, resuspended in 1ml of 100mM lithium acetate and allowed to stand for 5 minutes.

(7) The above-mentioned heavy suspension was centrifuged at 4000rpm for 3 minutes, and the supernatant was removed by a pipette.

(8) Salmon sperm DNA was boiled for 5 minutes and rapidly cooled on ice.

(9) To the cells, the transformation mixture was added as in Table 6 below, where PEG3350 had to be added first to protect the cells from exposure to excessive concentrations of lithium acetate.

TABLE 6 Yeast transformation mixtures

(10) Whirling the centrifugal tube for 1 minute to fully and uniformly mix the conversion system;

(11) placing the centrifuge tube in an incubator at 30 ℃ for incubation for 30 minutes; then heat shock is carried out in water bath at 42 ℃ for 20-30 minutes;

(12) after centrifugation at 4000rpm for 3 minutes, the supernatant was removed by a pipette, and the cell pellet was collected.

(13) Resuspend and centrifuge with sterile water and wash twice.

(14) The supernatant was discarded and 100. mu.l of sterile water was slowly added to resuspend the cells.

(15) And (3) uniformly smearing the cell resuspension liquid on a solid culture medium plate with a corresponding screening pressure by using a coating rod, and culturing for 2-4 days in an incubator at 30 ℃.

WN1, WN5, WN7 were fermented at 30 ℃ for 5 days in a 100mL shake flask containing 10mLSD medium at pH 6.2. After fermentation, detection is carried out on WN1 and WN7 strains through GC-MS, and the results are shown in figure 4, wherein the yield of ursolic acid of the WN1 strain is 33.23mg/L, the yield of ursolic acid of the WN7 strain is 43.09mg/L, and the yield of ursolic acid of the WN5 strain is not generated. The gas chromatogram of WN1 strain for producing ursolic acid is shown in FIG. 2, and the gas mass spectrometry detection result is shown in FIG. 3.

Example 2 preparation of high expression plasmid for enhancing supply of precursor acetyl coenzyme A or cofactor supplying Gene and Strain synthesizing Ursolic acid

Using the primers in Table 7 and yeast genomic DNA as a template, acetaldehyde dehydrogenase gene ALD6 and promoter P were amplified by PCR_PGK1A terminator T_CYC1Construction of the expression cassette P by OE-PCR_PGK1-ALD6-T_CYC1The PCR reaction system and reaction conditions are shown in tables 1 and 3. The empty vector pRS42K was digested with restriction enzymes and recovered, and the digestion system is shown in Table 8. The amplified expression cassette was assembled with a linear vector using the Gibson assembly method, see Table 5 for the Gibson system. Mu.l of the reaction mixture was transformed into 50. mu.l of E.coli competent cells to construct plasmid pRS42K-KanMX-ALD6, and the E.coli heat shock transformation procedure was as described in example 1.

TABLE 7 PCR primers

TABLE 8 plasmid cleavage System

Knockout of MPC2 gene:

knockout of MPC2 gene can reduce mitochondrial depletion of pyruvate and acetyl coa. Assembling metabolic pathway key gene expression cassette P by the same method as constructing plasmid gene expression cassette_PYK1-ADA-T_ENO2Wherein P is_PYK1Represents a promoter, T_ENO2Represents a terminator, ADA is derived from Phycomyces oryzae (A)Dickeyazeae) The acetaldehyde dehydrogenase of (1). Meanwhile, pGAPZaA plasmid is taken as a template, and the BleoR screening gene expression cassette P is amplified by PCR_TEF1-BleoR-T_CYC1Wherein P is_TEF1Represents a promoter, T_CYC1Represents a terminator, and the BleoR represents a bleomycin resistance gene, and is used for screening positive strains. MPC2 homology arms were PCR amplified using yeast genomic DNA as template. Primers used to construct the genomic assembly strain WN16 are shown in Table 9, with the overlap underlined.

TABLE 9 knockout MPC2 genome Whole primer

The left arm of MPC2, the BleoR expression cassette, the ADA expression cassette and the right arm of MPC2 obtained by PCR amplification are transformed into a saccharomyces cerevisiae WN1 competent cell together, SD-His-Ura-Trp-Leu-BleoR solid culture medium is used for screening, positive engineering bacteria WN16 are verified by colony PCR, and WN16 is fermented for 5 days in a 100mL shake flask containing 10mLSD culture medium with the temperature of 30 ℃ and the pH value of 6.2. After fermentation is finished, detection is carried out by GC-MS, WN16 can synthesize 36.80 mg/L ursolic acid.

The pRS42K-KanMX-ALD6 plasmid was transformed into Saccharomyces cerevisiae WN16 competent cells by lithium acetate to obtain recombinant yeast WN 29. WN29 was fermented for 5 days at 30 ℃ in a 100mL shake flask containing 10mLSD medium at pH 6.2. After fermentation is finished, detection is carried out by GC-MS, WN29 can synthesize 64.75 mg/L ursolic acid.

Example 3 preparation of high expression plasmid for promoting cofactor equilibrium Gene and Strain synthesizing Ursolic acid

Assembling metabolic pathway key gene expression cassette P by the same method as constructing plasmid gene expression cassette_PYK1-GaPC-T_ENO2Expression cassette P amplified from the genome of laboratory Strain BA05_TEF2-rHMGR-T_TPI1The primers are shown in Table 10, and GaPC is derived from Kluyveromyces lactis (A), (B)Kluyveromyces lactis) The glyceraldehyde-3-phosphate dehydrogenase of (1), rHMGR is derived from Bacillus silicate (A), (B)Silicibacterpomeroyi) NADH-dependent HMG 1. The empty vector pRS42K was digested with restriction enzymes and recovered, and the digestion system is shown in Table 11. The amplified two expression cassettes and the linear vector were assembled using the Gibson assembly method, the Gibson system is shown in Table 5. Mu.l of the reaction mixture was transformed into 50. mu.l of E.coli competent cells, and plasmid pRS42K-KanMX-rHMGR-GaPC was constructed by large intestine transformation, and the procedure of E.coli heat shock transformation was described in example 1.

pRS42K-KanMX-rHMGR-GaPC plasmid was transformed into Saccharomyces cerevisiae WN7 competent cells by lithium acetate to obtain recombinant yeast WN 85.

TABLE 10 PCR primers

TABLE 11 plasmid cleavage System

The recombinant yeast WN85 obtained was fermented at 30 ℃ for 5 days in a 100mL shake flask containing 10mLSD medium with pH 6.2. After fermentation is finished, detection is carried out by GC-MS, WN85 can synthesize 60.78 mg/L ursolic acid.

Example 4 fermentation optimization and product detection

The 1XSD glucose medium contains 6.7 g/L of amino acid-free yeast nitrogen source, 20g/L of glucose and 0.8g/L of abscisic acid mixture. The 1XSD galactose culture medium contains 6.7 g/L of amino acid-free yeast nitrogen source, 20g/L of galactose and 0.8g/L of abscisic acid mixture. 2X, 3X, 4X medium was multiplied by 2, 3, 4 according to the above recipe. The following was performed in a 100mL shake flask fermentation: WN29 and WN85 single colonies were inoculated in 3-5mL of liquid 1XSD glucose medium, after shaking culture at 30 ℃ for 16 hours, WN29 and WN85 were inoculated in 10mL of 3X glucose medium and 4X glucose medium at pH =6.2, respectively, in the initial OD =0.1 inoculum size, and after shaking culture at 30 ℃ for 48 hours, WN29 and WN85 were replaced in 3X galactose medium and 4X galactose medium, respectively, under the same conditions, and continuously cultured for 3 days.

The 5L fermentor fed-batch fermentation experiments were performed using the WN85 recombinant strain. The operation is as follows: WN85 single colony was inoculated into 3-5mL of liquid 1XSD glucose medium, after shaking culture at 30 ℃ for 16 hours, WN85 was inoculated into 4X glucose medium with pH =6.2 in the initial OD =0.1 inoculum size and cultured for 48 hours, then changed to 4X galactose medium under the same conditions, and culture was continued for 96 hours under 30 ℃ dissolved oxygen 35%, during which time 5g/L ethanol was supplemented at 120 hours of fermentation. After the fermentation is finished, 200 mu L of bacterial liquid is taken, 500 mu L of saturated sodium chloride solution is added, a proper amount of acid-washed glass beads with the diameter of 0.45-0.55 mm are added, and the beads are crushed for 40 minutes. Adding 500 μ L ethyl acetate into the disrupted cell mixture, extracting for 10min by vortex oscillation, centrifuging for 5min at 12000rpm, absorbing the upper layer of ethyl acetate, and repeating for three times. Concentrating the extract with ethyl acetate in the upper layer to 200 μ L, transferring to liquid phase vial, evaporating to dryness with vacuum concentrator, adding 100 μ L pyridine and 200 μ L N, O-bis (methylsilyl) trifluoroacetamide, dissolving with vortex shaking for 1min, and dissolving in 80 deg.C water bath for 35 min. Transferring into a liner tube, and analyzing by using a gas chromatography-mass spectrometer.

The analysis procedure of the gas chromatograph-mass spectrometer is as follows: injection temperature 250 ℃, flow rate of carrier gas helium 1.0 mL/min: the injection inlet temperature is 300 ℃, the split injection is carried out, the injection amount is 1 mu L, and the split ratio is 1: 30. The GC program sets up: the initial temperature of the temperature rise is 80 ℃, the temperature is raised to 320 ℃ at the speed of 20 ℃/min for 1min, and the temperature is maintained for 28 min; the mass spectrum scanning range is 45-750 mz.

Through detection, in a 100mL shake flask, the highest yield of the ursolic acid of the WN29 recombinant strain can reach 79.56 mg/L under the condition of a 3X galactose medium with the pH value of 6.2; the WN85 recombinant strain is fermented for 120h under the condition of 4X galactose medium with pH 6.2, and the highest yield of ursolic acid can reach 89.53 +/-1.53 mg/L (figure 4). In a 5L fermenter, the highest yield of ursolic acid produced by WN85 recombinant strain through 144h fed-batch fermentation was 264.44 + -13.42 mg/L (FIG. 5).

Sequence listing

<110> Beijing university of science and engineering, Qinghua university

<120> recombinant yeast for synthesizing ursolic acid and construction method thereof

<130> P0102022030221Y

<160> 27

<170> SIPOSequenceListing 1.0

<210> 1

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tctaatccgt acttcaatat agcaatgagc ag 32

<210> 2

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

taccgggccc cccctcgagg tcgacgagcg acctcatgct atag 44

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atattgaagt acggattaga agccgcc 27

<210> 4

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cggccgctct agaactagtg gatcccttcg agcgtcccaa aac 43

<210> 5

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cctgcagccc gggggatcca ctagtcgatt tgggcgcgaa tcc 43

<210> 6

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtcaaagtgt agcttagtca ttgttttata tttgttgtaa aaagtag 47

<210> 7

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttttacaaca aatataaaac aatgactaag ctacactttg acac 44

<210> 8

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccttcctttt cggttagagc ggatttacaa cttaattctg acagctttta c 51

<210> 9

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtaaaagctg tcagaattaa gttgtaaatc cgctctaacc gaaaag 46

<210> 10

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agggaacaaa agctggagct ccttcgagcg tcccaaaac 39

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cattggagcc acgtatgttt tc 22

<210> 12

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ctatggtgtg tgggggatct tttcttttct ttaatctttt tctgtataac 50

<210> 13

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gaaaaagatt aaagaaaaga aaagatcccc cacacaccat ag 42

<210> 14

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gaagcccgat gtctgggtgt acattttggc gaaaagccaa ttagtg 46

<210> 15

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gtatcacact aattggcttt tcgccaaaat gtacacccag acatc 45

<210> 16

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

caaagtttcg agttgtaaat tcggcgtagg tatcatctcc atctccc 47

<210> 17

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gatatgcata tgggagatgg agatgatacc tacgccgaat ttacaactcg 50

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gttcgaaagc tcgcatgtg 19

<210> 19

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ctgttatata aaaatgtaca cccagacatc 30

<210> 20

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ctcgttggtc atatcgggca ttgtgatgat gttttatttg ttttgattg 49

<210> 21

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

caatcaaaac aaataaaaca tcatcacaat gcccgatatg accaac 46

<210> 22

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ctaataattc ttagttaaaa gcactttaca cgccggcctc g 41

<210> 23

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gacttcgagg ccggcgtgta aagtgctttt aactaagaat tattagtctt ttc 53

<210> 24

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cggccgctct agaactagtg gatccaggta tcatctccat ctc 43

<210> 25

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

taccgggccc cccctcgagg tcgacgggcg ccataaccaa ggtatc 46

<210> 26

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tgtacatttt tatataacag ttgaaatttg gataagaaca tcttctc 47

<210> 27

<211> 1455

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

atggaacact tctacttgac tttgttgttg ggtttcgttt ctttcatcac tttgtctttg 60

tctgttttgt tctacagaca cagagctcaa ttcgttggta ctaacttgcc accaggtaag 120

gttggttacc cagttatcgg tgaaacttac caattcttgg ctactggttg gaagggtcac 180

ccagaaaagt tcatcttcga cagaatgact aagtactctt ctgaagtttt caagacttct 240

ttgatgggtg aaaaggctgc tatcttctgt ggtgctgctt gtaacaagtt cttgttctct 300

aacgaaaaca agttggttac tgcttggtgg ccatcttctg ttaacaaggt tttcccatct 360

tctttggaaa cttctgctaa ggaagaagct aagaagatga gaaagatgtt gccaaacttc 420

atgaagccag aagctttgca aagatacatc ggtatcatgg acactgttgc tagaagacac 480

ttcgctgaag gttgggaaaa caagaaggaa gttgaagttt tcccattggc taagaactac 540

actttctggt tggctgctag attgttcgtt tctttggaag actctgttga aatcgctaag 600

ttgggtgacc cattcgctgt tttggcttct ggtatcatct ctatgccatt ggacttccca 660

ggtactccat tctacaaggc tatcaaggct tctaacttca tcagagaaga attgactaag 720

atcatcaagc aaagaaagat cgacttggct gaaggtaagg cttctccaac tcaagacatc 780

ttgtctcaca tgttgttgtt gtgtgacgaa cacggttctc acatgaagga acacgacatc 840

gctgacaaga tcttgggttt gttgatcggt ggtcacgaca ctgcttctgc tacttgtact 900

ttcatcgtta agtacttggc tgaattgcca cacatctacg acgaagttta caaggaacaa 960

atggaagttt tgtctgctaa ggctccaggt gaattgttga actgggacga cttgcaaaag 1020

atgaagtact cttggaacgt tgctcaagaa gttttgagat tggctccacc attgcaaggt 1080

gctttcagag aagctttgtc tgacttcgtt ttcaacggtt tcactatccc aaagggttgg 1140

aagttgtact ggtctgctaa ctctactcac aagaacgctg cttacttccc agaaccattc 1200

aagttcgacc caactagatt cgaaggtaac ggtccagctc catacacttt cgttccattc 1260

ggtggtggtc caagaatgtg tccaggtaag gaatacgcta gattggaaat cttggttttc 1320

atgcacaact tggttaagag attcaagtgg gaaaaggttt tgccagacga acaaatcgtt 1380

gttgacccat tgccaatgcc agctaagggt ttgccagtta gattgttctc tcacccaaag 1440

actgctactg cttaa 1455

Claims

1. A recombinant yeast for synthesizing ursolic acid is characterized in that the recombinant yeast expresses CYP450 enzyme and CPR enzyme, and the CYP450 enzyme is derived from loquat.

2. The recombinant yeast of claim 1, wherein the CPR enzyme is derived from alfalfa, licorice, or lotus japonicus.

3. The recombinant yeast according to claim 2, wherein the recombinant yeast overexpresses genes of the acetyl-coa precursor synthesis pathway, overexpresses cofactor supply genes, and/or reduces expression of genes of the acetyl-coa precursor consumption pathway;

the genes of the synthesis pathway of the acetyl-CoA precursor are selected from coding genes of acetyl-CoA synthase, alcohol dehydrogenase and/or acetaldehyde dehydrogenase;

the cofactor supply gene is selected from isocitrate dehydrogenase, malate dehydrogenase, acetaldehyde dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and/or NADH dependent hydroxymethyl glutarate reductase encoding gene;

the genes of the acetyl-CoA precursor consumption pathway are selected from encoding genes of mitochondrial porin, mitochondrial transport protein and/or citrate synthase.

4. The recombinant yeast according to any one of claims 1 to 3, wherein the recombinant yeast is a yeast for synthesizing α -amynol, and the recombinant yeast is selected from one of Saccharomyces cerevisiae, Pichia pastoris, Candida lipolytica, yarrowia lipolytica, Hansenula anomala, Schizosaccharomyces pombe, Rhodotorula glutinis, Candida tropicalis, and Candida utilis.

5. A plasmid for constructing the recombinant yeast of claim 1, wherein the plasmid comprises genes encoding CYP450 enzyme and CPR enzyme, and the CYP450 enzyme is derived from Eriobotrya japonica.

6. A construction method of recombinant yeast is characterized in that the construction method comprises the step of introducing CYP450 enzyme and CPR enzyme coding genes into the recombinant yeast, wherein the CYP450 enzyme is derived from loquat.

7. The method according to claim 6, wherein the method comprises introducing a gene in an acetyl-CoA precursor synthesis pathway, and/or introducing a cofactor supply gene, and/or knocking out a gene in an acetyl-CoA precursor consumption pathway into the recombinant yeast.

8. The method according to claim 6 or 7, wherein each of the introduced genes is a single copy or multiple copies.

9. A method for synthesizing ursolic acid, which comprises fermenting and culturing the recombinant yeast of any one of claims 1 to 4.

10. The method of claim 9, wherein the fermentation medium comprises galactose.