A saccharomyces cerevisiae engineered bacterium for producing glycosyl donor UDP-arabinose and its construction method and application

Abstract

The present invention belongs to the field of biosynthesis, and specifically relates to an engineered yeast for producing glycosyl donor UDP-arabinose, and a construction method and application thereof. The present invention introduces the synthesis pathway of UDP-Ara in Saccharomyces cerevisiae for the first time by sequentially expressing UDP-Glc dehydrogenase, UDP-GlcA decarboxylase, UDP-Xyl-4-epimerase or UDP-Glc-epimerase in WAT11; the de novo synthesis of Ara-oleanolic acid is achieved by introducing arabinosyltransferase and oleanolic acid synthesis pathway; yeast containing UDP-Ara synthesis pathway and arabinosyltransferase genes can also generate Ara-betulinic acid from betulinic acid. The present invention solves the yeast total synthesis of arabinosyl donors, provides a new biotechnology platform for the production of natural glycoside compounds, and has important theoretical value and broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biosynthesis, and specifically relates to an engineering yeast of saccharide donor UDP-arabinose for producing saccharide donor UDP-arabinose, and a construction method and application thereof. Background Art

[0002] Glycoside compounds are important natural products, which have been widely used in medicine, cosmetics, health care products and other fields due to their significant biological activities. For example, ginsenoside, as a type of triterpenoid saponin, has attracted much attention due to its various pharmacological activities; various glycoside forms of flavonoid compounds are also widely used due to their antioxidant and anti-inflammatory activities.

[0003] Glycoside compounds are derived from natural product aglycones through glycosylation. Glycosylation is an important modification of natural products, which improves their bioavailability and pharmacological activity by changing their physicochemical properties. Common modified sugars include D-glucose (Glc), D-galactose (Gal), D-glucuronic acid (GlcA), L-rhamnose (Rha), D-xylose (Xyl), D-apiose and L-arabinose (Ara). Among them, L-Ara is an important component of plant cell wall formation. Arabinosylation modification is relatively rare in natural products, but as a key small molecule glycosylation unit, it plays a unique role in regulating the activity of natural products. However, a major challenge in achieving arabinosylation modification is the high cost of its sugar donor, UDP-β-L-arabinose (UDP-Ara) (about $90 / mg). This high cost is mainly attributed to its complex chemical synthesis steps and low yield, which greatly limits its application in large-scale production of glycoside compounds.

[0004] Using microorganisms such as Escherichia coli and yeast as heterologous hosts to introduce and express plant-derived natural product synthesis pathways is a robust and sustainable alternative. Saccharomyces cerevisiae has the advantages of a clear genetic background, strong genetic operability, and superior fermentation performance, making it an ideal host for constructing heterologous metabolic pathways. Unlike prokaryotic hosts such as Escherichia coli, yeast cells contain endoplasmic reticulum, which is a cell structure necessary for membrane-bound enzymes such as CYP450 enzymes involved in the biosynthesis of plant glycoside natural products. A large amount of UDP-Glc exists in Saccharomyces cerevisiae cells, providing an important precursor for the production of various glycosyl donors such as UDP-Ara. In addition, yeast cells can also be appropriately engineered in metabolic pathways to increase the yield of target products. Therefore, yeast has significant advantages in the fermentation production of complex glycoside compounds.

[0005] At present, there is no report on the construction of UDP-Ara synthesis pathway in Saccharomyces cerevisiae. The synthesis pathway of UDP-Ara is longer and more complicated than that of other UDP sugars. First, UDP-Glc is converted into UDP-GlcA under the catalysis of UDP-Glc dehydrogenase (UGDH); then, UDP-GlcA is further converted into UDP-Xyl under the catalysis of UDP-GlcA decarboxylase (UXS); finally, UDP-Xyl is converted into UDP-Ara under the action of UDP-Xyl-4-epimerase (UXE) or UDP-Glc-4-epimerase (UGE). The present invention uses synthetic biology technology, takes Saccharomyces cerevisiae as the starting strain, introduces and expresses the above-mentioned key enzymes (UGDH, UXS, UXE or UGE), constructs a complete UDP-Ara synthesis pathway, realizes the biosynthesis of UDP-Ara in yeast for the first time, and successfully prepares arabinosylated oleanolic acid and betulinic acid, which has important research value and broad application prospects in the industrial production of arabinosylated glycoside compounds. Summary of the invention

[0006] In order to solve the problem of fermentation production of arabinosylated complex glycoside compounds in yeast, the present invention provides an engineering strain of Saccharomyces cerevisiae for producing glycosyl donor UDP-arabinose, and a construction method and application thereof.

[0007] The present invention provides an engineered yeast of UDP-Ara, characterized in that the engineered yeast of UDP-Ara is constructed by sequentially expressing UDP-Glc dehydrogenase (UGDH), UDP-GlcA decarboxylase (UXS), UDP-Xyl-4-epimerase (UXE) or UDP-Glc-4-epimerase (UGE) on a saccharomyces cerevisiae strain WAT11; the UGDH is selected from AtUGDH3, the UXS is selected from AtUXS3 or GuUXS2, the UXE is selected from GuUXE1, and the UGE is selected from PsUGE2;

[0008] The DNA sequence of AtUGDH3 is SEQ ID NO: 1, which contains the amino acid sequence of SEQ ID NO: 2;

[0009] The DNA sequence of AtUXS3 is SEQ ID NO: 3, which contains the amino acid sequence of SEQ ID NO: 4;

[0010] The DNA sequence of GuUXS2 is SEQ ID NO:5, which contains the amino acid sequence of SEQ ID NO:6;

[0011] The DNA sequence of PsUGE2 is SEQ ID NO:7, which contains the amino acid sequence of SEQ ID NO:8;

[0012] The DNA sequence of GuUXE1 is SEQ ID NO:9, which contains the amino acid sequence of SEQ ID NO:10.

[0013] Another embodiment of the present invention provides the above-mentioned engineered Saccharomyces cerevisiae for producing UDP-Ara, characterized in that the UXS is selected from AtUXS3, and the UGE is selected from PsUGE2; or the UXS is selected from GuUXS2, and the UXE is selected from GuUXE1.

[0014] Another embodiment of the present invention provides a method for constructing the above-mentioned UDP-Ara-producing Saccharomyces cerevisiae engineered bacteria, characterized by comprising the following steps:

[0015] (1) Expressing AtUGDH3 in the Saccharomyces cerevisiae strain WAT11 to construct the UDP-GlcA synthesis pathway and form the WA1 strain;

[0016] (2) expressing AtUXS3 in the WA1 strain to construct a synthetic pathway for UDP-Xyl; continuing to express PsUGE2 to construct a synthetic pathway for UDP-Ara, thereby forming the WA2 strain, which is the engineered yeast for producing UDP-Ara;

[0017] Or (3) expressing GuUXS2 in the WA1 strain to construct a synthetic pathway for UDP-Xyl; continuing to express GuUXE1 to construct a synthetic pathway for UDP-Ara, forming the WA3 strain, which is the engineered Saccharomyces cerevisiae strain producing UDP-Ara.

[0018] By adopting the above technical scheme, the synthesis pathway of UDP-Ara is designed, and an engineered Saccharomyces cerevisiae strain that produces UDP-Ara is constructed, that is, the synthesis route of UDP-Ara is constructed in the engineered Saccharomyces cerevisiae strain, and the three sugar donors UDP-GlcA, UDP-Xyl and UDP-Ara on the UDP-Ara synthesis pathway can be obtained at the same time. After further amplification and optimization, it is expected to promote the industrial production of arabinosylated glycoside compounds in yeast.

[0019] Another aspect of the present invention provides the use of the above-mentioned UDP-Ara-producing Saccharomyces cerevisiae engineered bacteria (WA2 and / or WA3) in the preparation of Ara-oleanolic acid. The use preferably comprises the following steps:

[0020] (1) Squalene epoxidase (ERG1), squalene synthase (ERG9), β-amyrin synthase (CqbAS1), and oxidase (CqCYP716A78) were sequentially expressed in the above two engineered UDP-Ara-producing Saccharomyces cerevisiae strains WA2 and / or WA3 to complete the construction of the de novo oleanolic acid synthesis pathway, thereby forming strains WA6 and / or WA7.

[0021] The DNA sequence of ERG1 is SEQ ID NO: 11, which contains the amino acid sequence of SEQ ID NO: 12;

[0022] The DNA sequence of ERG9 is SEQ ID NO: 13, which contains the amino acid sequence of SEQ ID NO: 14;

[0023] The DNA sequence of CqbAS1 is SEQ ID NO: 15, which contains the amino acid sequence of SEQ ID NO: 16;

[0024] The DNA sequence of CqCYP716A78 is SEQ ID NO:17, which contains the amino acid sequence of SEQ ID NO:18.

[0025] (2) Introducing the arabinose transferase gene UGT99D1 into strain WA6 and / or WA7 to complete the construction of the de novo synthesis pathway of Ara-oleanolic acid to form strain WA8 and / or WA9;

[0026] The DNA sequence of the UGT99D1 is SEQ ID NO:19, which contains the amino acid sequence of SEQ ID NO:20.

[0027] Another embodiment of the present invention provides an engineered yeast strain for producing Ara-oleanolic acid, characterized in that the construction method of the engineered yeast strain is as follows:

[0028] (1) In the above two UDP-Ara-producing Saccharomyces cerevisiae engineered strains WA2 and / or WA3, squalene epoxidase (ERG1), squalene synthase (ERG9), β-amyrin synthase (CqbAS1), and oxidase (CqCYP716A78) were sequentially expressed to complete the construction of the de novo synthesis pathway of oleanolic acid, thereby forming strains WA6 and / or WA7;

[0029] (2) Introducing the arabinose transferase gene UGT99D1 into strain WA6 and / or WA7 to complete the construction of the de novo synthesis pathway of Ara-oleanolic acid, thereby forming strain WA8 and / or WA9, which is the engineered Saccharomyces cerevisiae strain producing Ara-oleanolic acid.

[0030] Another aspect of the present invention provides the use of the above-mentioned UDP-Ara-producing Saccharomyces cerevisiae engineered bacteria (WA2 and / or WA3) in the preparation of Ara-betulinic acid. The use preferably comprises the following steps:

[0031] (1) Introducing the arabinose transferase gene UGT99D1 into the above two engineered strains of Saccharomyces cerevisiae producing UDP-Ara, WA2 and / or WA3, to form strains WA12 and / or WA13;

[0032] The DNA sequence of the UGT99D1 is SEQ ID NO: 19, which contains the amino acid sequence of SEQ ID NO: 20;

[0033] (2) Feeding betulinic acid culture strain WA12 and / or WA13 to prepare Ara-betulinic acid.

[0034] The brewer's yeast cell line described in the present invention is WAT11 (refer to the applicant's previous Chinese invention patent application number: 202311644882.0), which is an engineered strain constructed by replacing the endogenous CPR of yeast with the CPR1 of Arabidopsis thaliana, which improves the electron transfer system and enables efficient expression of plant CYP450 enzymes. When using this yeast cell line, the prepared microbial cells are more likely to express related enzymes in the glycoside synthesis pathway, which is conducive to the construction of the synthesis pathway and further amplification of production, thereby obtaining high-purity and high-yield glycoside products.

[0035] Compared with the prior art, the advantages of the present invention are as follows: (1) The present invention introduces the synthesis pathway of UDP-Ara into the cerevisiae yeast strain WAT11 for the first time, constructs an engineered cerevisiae yeast that produces UDP-Ara; further introduces the oleanolic acid synthesis pathway and arabinosyltransferase to produce Ara-oleanolic acid (or Ara-betulinic acid after feeding betulinic acid), which provides a basis for the production of rare arabinosylated glycoside compounds in engineered yeast. (2) The engineered cerevisiae yeast of the present invention can simultaneously produce three glycosyl donors, UDP-GlcA, UDP-Xyl and UDP-Ara, and can be used to produce various types of natural glycoside compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 :The biosynthesis pathway of Ara-oleanolic acid in Saccharomyces cerevisiae;

[0037] Figure 2 : Analysis of oleanolic acid products in WA11 strain (target ion currents of three experimental groups and two control groups were extracted, and the difference peaks were the target products);

[0038] Figure 3: Analysis of Ara-oleanolic acid products in WA8 strain (target ion currents of three experimental groups and two control groups were extracted, and the difference peaks were the target products);

[0039] Figure 4 : Analysis of Ara-oleanolic acid products in WA9 strain (target ion currents of three experimental groups and two control groups were extracted, and the difference peaks were the target products);

[0040] Figure 5 : Analysis of Ara-betulinic acid products in WA12 strain (target ion currents of three experimental groups and two control groups were extracted, and the difference peaks were the target products);

[0041] Figure 6 : Analysis diagram of Ara-betulinic acid products in WA13 strain (target ion currents of three experimental groups and two control groups were extracted, and the difference peaks were the target products). DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. It is specially noted that the following examples without specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer, and the enzymes and kits used in the following examples are commercially available products that can be purchased. The PCR amplification method, the fusion method of different fragments, the gene knockout and overexpression method used in the following examples can adopt common technical means in the art, such as fusion PCR, homologous recombination, and CRISPR-Cas9 technology.

[0043] Experimental methods:

[0044] The following examples were transformed using the lithium acetate transformation method: first, the host strain was activated in 2×YPD medium and cultured overnight at 30°C and 200 rpm. Then, it was inoculated into a new 2×YPD medium to make the initial OD value 0.2. After continuing to culture at 30°C for 4-4.5 h, 5OD bacterial solution was taken, centrifuged at room temperature 3000 rcf for 5 min, the supernatant was discarded, and the yeast cells were washed twice with sterilized ultrapure water; a DNA mixture was prepared, 5 OD bacterial solution was taken for each construction to obtain cells, and mixed with 50 μL DNA mixture to resuspend the cells. The 50 μL DNA mixture was mixed with 2 g of the insert fragment, 250 ng of the tool plasmid and sufficient ddH2O. The lithium acetate transformation mixture was added to the suspended cells, and the cells were obtained after culture, and spread on the screening plate to obtain a single colony, which was the recombinant Saccharomyces cerevisiae. After sequencing to verify the successful transformation, the recombinant Saccharomyces cerevisiae was preserved.

[0045] Colony PCR and sequencing verification: After a monoclonal colony grows on the screening plate, colony PCR and sequencing verification are performed. The specific steps are: pick a small amount of cells with a pipette tip and place them in 20 μL 20 mml / L NaOH solution, vortex mix, incubate in a metal bath at 95°C for 20 min, vortex mix, take 1 μL of bacterial solution as a template for colony PCR reaction, compare the size of the clone band with the negative clone band, and select the bacterial solution of the colony PCR positive clone to be sent to the company for sequencing verification. Strains with correct sequencing are streaked and frozen in glycerol.

[0046] Cultivation of recombinant Saccharomyces cerevisiae strains: A single colony was placed in a 24-well plate with 3 mL 1×YPD. After overnight culture in a shaker for 16 h (conditions were 30 ℃, 200 rpm), the overnight bacterial solution was diluted 10 times with 1×YPD and the OD of the bacterial solution was detected by a UV spectrophotometer with a wavelength set to 600 nm. Then the starting OD was 0.2 and transferred to 3 mL 1×YPG culture medium for culture. The culture method was: after transfer, 1 μL substrate (concentration of 1 M) and 300 μL 20% galactose were added every 24 h. After 72 h of culture, 200 μL of bacterial solution was collected as a sample.

[0047] Extraction of recombinant Saccharomyces cerevisiae product: take a sample, add chromatography-grade ethyl acetate for extraction (extract twice), vortex for 1 min, centrifuge at 12,000 rpm for 2 min, aspirate the supernatant into a clean EP tube (capacity 1.5 mL), spin dry, add 140 μL of methanol to the EP tube, vortex for 1 min, centrifuge for 1 min, transfer to a liquid phase vial, and wait for analysis.

[0048] Product analysis and identification: The product was analyzed by LC-MS, the instrument model was Agilent 1290 Infinity II-6470A, the chromatographic column model was: Poroshell 120 EC-C18, 2.7 µm, 3×100 mm (Agilent), and the mass spectrometry detection mode was negative ion full scan mode. The chromatographic analysis conditions are shown in Table 1:

[0049] Table 1: Chromatographic conditions

[0050] Time / time(min) Mobile phase A (acetonitrile) Mobile phase B (0.01% ammonium acetate in water) Flow rate (mL / min) 0.00 35% 65% 0.3 9.00 65% 35% 0.3 9.01 95% 5% 0.3 12.00 95% 5% 0.3 12.01 35% 65% 0.3 15.00 35% 65% 0.3

[0051] Preparation of main reagents:

[0052] (1) 1×YPD liquid medium

[0053] Yeast extract 10 g

[0054] Peptone 20 g

[0055] D-(+)-Glucose 4 g

[0056] Add ddH2O to make up to 1000 mL, mix well and filter to sterilize.

[0057] (2) 40% D-(+)-Glucose

[0058] D-(+)-Glucose 40 g

[0059] Add a small amount of ddH2O and heat in a microwave oven to dissolve, then add more ddH2O to make the volume up to 100 mL, mix well, and filter to sterilize.

[0060] (3) 10×YNB solution

[0061] Yeast nitrogen source (without amino acids and ammonium sulfate) 8.5g

[0062] Ammonium sulfate 25g

[0063] Add ddH2O to make up to 500 mL and filter sterilize;

[0064] (4) Nutrient-deficient culture medium

[0065] 5×SC-Leu-Trp-Ura-His 3.5 g / 500 mL

[0066] 10×Ura 0.38 g / 500 mL

[0067] 10×Trp 0.38 g / 500 mL

[0068] 10×His 0.38 g / 500 mL

[0069] 10×Leu 1.8 g / 500 mL

[0070] SC-Ura (100 mL):

[0071] 5×Sc-leu-trp-ura-his 20 mL

[0072] 10×Trp 10 mL

[0073] 10×Leu 10 mL

[0074] 10×His 10 mL

[0075] 10×(YNB+ammonium sulfate) 10 mL

[0076] 40 mL 5% agar powder solution

[0077] 20% glucose 10 mL

[0078] Example Construction of an engineered strain of Saccharomyces cerevisiae producing UDP-Ara

[0079] A UDP-Ara producing yeast engineered bacterium is obtained by expressing UGDH, UXS, UXE or UGE on a WAT11 UDP-Ara strain, and provides a construction method for two UDP-Ara yeast synthesis pathways, wherein the products include UDP-GlcA, UDP-Xyl and UDP-Ara. The invention obtains two UDP-Ara synthesis pathways by sequentially expressing AtUGDH3, AtUXS3 and PsUGE2 or sequentially expressing AtUGDH3, GuUXS2 and GuUXE1 in WAT11.

[0080] The method for constructing an engineered yeast strain of Saccharomyces cerevisiae that produces UDP-Ara comprises the following steps:

[0081] Using the Saccharomyces cerevisiae strain WAT11 as the starting strain, AtUGDH3 was first expressed to construct the UDP-GlcA synthesis pathway to form the WA1 strain:

[0082] DNA polymerase was used to amplify the integrated gene fragment by PCR: the genome of Saccharomyces cerevisiae strain WAT11 was used as a template, primers P1 / P2 were used to amplify the 911b-up-SeGAL1 fragment (1529 bp), and Arabidopsis cDNA was used as a template, primers P3 / P4 were used to amplify the AtUGDH3 fragment (1395 bp); primers P5 / P6 were used to amplify the ScADH1-911b-down fragment (1250 bp), and the 911b-up-SeGAL1 fragment with the homology arm upstream of the integration site connected to the promoter and the ScADH1-911b-down fragment with the homology arm downstream of the integration site connected to the terminator were obtained; finally, the 911b-up-SeGAL1-AtUGDH3-ScADH1-911b-down expression cassette was obtained. Then the 911b-up-SeGAL1-AtUGDH3-ScADH1-911b-down expression cassette fragment was transformed into Saccharomyces cerevisiae WAT11 to obtain strain WA1.

[0083] Table 2 Primer sequences for constructing strain WA1

[0084] Primer number Primer sequences 1 SEQ ID NO: 21 2 SEQ ID NO: 22 3 SEQ ID NO: 23 4 SEQ ID NO: 24 5 SEQ ID NO: 25 6 SEQ ID NO: 26

[0085] Next, the 607b-up-ScADH1-AtUXS3-PTDH3-pSeGal2-PsUGE2-ScENO1-607b-down expression cassette and the 607b-up-ScADH1-GuUXS2-PTDH3-pSeGal2-GuUXE1-ScENO1-607b-down expression cassette were integrated into the WA1 genome, respectively.Using the genome of the strain with 607b-up- ScADH1 as a template, primers P7 / P8 were used to amplify the 607b-up- ScADH1 fragment (1192 bp), and using Arabidopsis as a template, primers P9 / P10 were used to amplify the AtUXS3 fragment (999 bp); using the genome of the strain with PTDH3-pSeGal2 as a template, primers P11 / P12 were used to amplify the PTDH3-pSeGal2 fragment (1300 bp); using pea cDNA as a template, primers P13 / P14 were used to amplify the PsUGE2 fragment (1308 bp); using the genome of the strain with ScENO1-607b-down as a template, primers P15 / P16 were used to amplify the ScENO1-607b-down fragment (1252 bp), and the 607b-up-ScADH1 with the homology arm upstream of the integration site connected to the promoter was obtained. The fragment and the homology arm downstream of the integration site were connected to the terminator of the ScENO1-607b-down fragment; the 607b-up-ScADH1-AtUXS3-PTDH3-pSeGal2-PsUGE2-ScENO1-607b-down expression cassette was obtained; the 607b-up-ScADH1 fragment (1192 bp) was amplified using primers P17 / P18 with the genome of the strain with 607b-up-ScADH1 as a template, and the GuUXS2 fragment (1308 bp) was amplified using primers P19 / P20 with licorice cDNA as a template; the PTDH3-pSeGal2 fragment (1300 bp) was amplified using primers P21 / P22 with the genome of the strain with PTDH3-pSeGal2 as a template; the GuUXE1 fragment (1248 bp); using the genome of the strain with ScENO1-607b-down as a template, primers P25 / P26 were used to amplify the ScENO1-607b-down fragment (1252bp), and the 607b-up-ScADH1 fragment with the homology arm upstream of the integration site connected to the promoter and the ScENO1-607b-down fragment with the homology arm downstream of the integration site connected to the terminator were obtained; 607b-up-ScADH1-GuUXS2-PTDH3-pSeGal2-GuUXE1-ScENO1-607b-down were obtained; then these two expression cassettes were respectively transferred into the genome of the WA1 strain to construct the WA2 and WA3 strains, i.e., two yeast cells producing UDP-Ara. The strain with the correct gene sequencing was used for subsequent culture.

[0086] Table 3 Primer sequences for constructing strains WA2 and WA3

[0087] Primer number Primer sequences 7 SEQ ID NO: 27 8 SEQ ID NO: 28 9 SEQ ID NO: 29 10 SEQ ID NO: 30 11 SEQ ID NO: 31 12 SEQ ID NO: 32 13 SEQ ID NO: 33 14 SEQ ID NO: 34 15 SEQ ID NO: 35 16 SEQ ID NO: 36 17 SEQ ID NO: 37 18 SEQ ID NO: 38 19 SEQ ID NO: 39 20 SEQ ID NO: 40 21 SEQ ID NO: 41 22 SEQ ID NO: 42 23 SEQ ID NO: 43 24 SEQ ID NO: 44 25 SEQ ID NO: 45 26 SEQ ID NO: 46

[0088] Application Example 1 Application of a yeast cell producing Ara-oleanolic acid

[0089] The invention discloses an application of a yeast cell producing Ara-oleanolic acid, wherein the yeast cell producing Ara-oleanolic acid produces Ara-oleanolic acid from scratch.

[0090] Method for constructing yeast cells producing Ara-oleanolic acid

[0091] An engineered yeast strain for producing Ara-oleanolic acid is obtained by introducing a synthetic pathway for oleanolic acid and arabinosyl transferase into UDP-Ara-producing yeast. The construction method thereof comprises the following steps:

[0092] (1) Squalene epoxidase (ERG1), squalene synthase (ERG9), β-amyrin synthase (CqbAS1), and oxidase (CqCYP716A78) were expressed in the two UDP-Ara-producing Saccharomyces cerevisiae engineered strains WA2 and WA3 and the starting strain WAT11, respectively, to introduce the synthesis pathway of oleanolic acid:

[0093] The squalene epoxidase (ERG1) and squalene synthase (ERG9) gene expression cassettes were integrated into the WA2 and WA3 strains, respectively, to construct the chassis strains WA4 and WA5 with high production of 2,3-oxidosqualene: the genome of the strain with 511b-up-GAL10p was used as a template, and the primers P27 / P28 were used to amplify the 511b-up-GAL10p fragment (712 bp); the ERG1 fragment (1491 bp) was amplified using the primers P29 / P30 using the Saccharomyces cerevisiae WAT11 as a template; the PGK1t-ScADH1 fragment (610 bp) was amplified using the primers P31 / P32 using the genome of the strain with PGK1t-ScADH1; the ERG9 fragment (1335 bp) was amplified using the primers P33 / P34 using the Saccharomyces cerevisiae WAT11 as a template. bp); using the genome of the CCW12p-511b-down strain as a template, primers P35 / P36 were used to amplify the CCW12p-511b-down fragment (1193 bp), and the 511b-up-GAL10p fragment with the homology arm upstream of the integration site connected to the promoter and the CCW12p-511b-down fragment with the homology arm downstream of the integration site connected to the promoter were obtained; finally, the 511b-up-GAL10p-ERG1-PGK1t-ScADH1-ERG9-CCW12p-511b-down expression cassette was obtained. Then the 511b-up-GAL10p-ERG1-PGK1t-ScADH1-ERG9-CCW12p-511b-down expression cassette fragment was transformed into Saccharomyces cerevisiae WA2 and WA3 to obtain strains WA4 and WA5. The 511b-up- GAL10p-ERG1-PGK1t-ScADH1-ERG9-CCW12p-511b-down expression cassette was transferred into the WAT11 chassis to construct WA10 in order to verify whether it can catalyze the production of oleanolic acid.

[0094] Table 4 Primer sequences for constructing strains WA4, WA5 and WA10

[0095] Primer number Primer sequences 27 SEQ ID NO: 47 28 SEQ ID NO: 48 29 SEQ ID NO: 49 30 SEQ ID NO: 50 31 SEQ ID NO: 51 32 SEQ ID NO: 52 33 SEQ ID NO: 53 34 SEQ ID NO: 54 35 SEQ ID NO: 55 36 SEQ ID NO: 56

[0096] Integrate β-amyrin synthase (CqbAS1) and oxidase (CqCYP716A78) gene expression cassettes into strains WA4, WA5, and WA10 to construct chassis that produce both UDP-Ara and oleanolic acid and chassis that produce only oleanolic acid:

[0097] Using the genome of the strain with 1414a-up-ScADH1 as a template, primers P37 / P38 were used to amplify the 1414a-up-ScADH1 fragment (1250 bp); using the cDNA of quinoa as a template, primers P39 / P40 were used to amplify the CqbAS1 fragment (2292 bp); using the genome of the strain with pSeGal2-pTDH3 as a template, primers P41 / P42 were used to amplify the pSeGal2-pTDH3 fragment (1300 bp); using the cDNA of quinoa as a template, primers P43 / P44 were used to amplify the CqCYP716A78 fragment (1452 bp); using the genome of the strain with ScADH1-1414a-down as a template, primers P45 / P46 were used to amplify the ScADH1-1414a-down fragment (1179 bp), and obtained the 1414a-up-ScADH1 fragment with the homology arm upstream of the integration site connected to the terminator and the ScADH1-1414a-down fragment with the homology arm downstream of the integration site connected to the terminator; finally, the 1414a-up-ScADH1- CqbAS1-pSeGal2-pTDH3-CqCYP716A78-ScADH1-1414a-down expression cassette was obtained. Then, the 1414a-up-ScADH1-CqbAS1- pSeGal2-pTDH3- CqCYP716A78-ScADH1-1414a-down expression cassette fragment was transformed into Saccharomyces cerevisiae WA4, WA5 and WA10, and strains WA6 and WA7 containing both the UDP-Ara synthesis pathway and the oleanolic acid synthesis pathway were obtained, as well as the strain WA11 containing only the oleanolic acid synthesis pathway.

[0098] Table 5 Primer sequences for constructing strains WA6, WA7 and WA11

[0099] Primer number Primer sequences 37 SEQ ID NO: 57 38 SEQ ID NO: 58 39 SEQ ID NO: 59 40 SEQ ID NO: 60 41 SEQ ID NO: 61 42 SEQ ID NO: 62 43 SEQ ID NO: 63 44 SEQ ID NO: 64 45 SEQ ID NO: 65 46 SEQ ID NO: 66

[0100] (2) Integrate the arabinosyltransferase (UGT99D1) gene expression cassette into strains WA6 and WA7 to construct strains WA8 and WA9 containing the Ara-oleanolic acid synthesis pathway:

[0101] Using the genome of the strain with 1114a-up-ScGAL2 as a template, primers P47 / P48 were used to amplify the 1114a-up-ScGAL2 fragment (1700 bp), and using oat cDNA as a template, primers P49 / P50 were used to amplify the UGT99D1 fragment (1470 bp); using the genome of the strain with TDH1-1114a-down as a template, primers P51 / P52 were used to amplify the TDH1-1114a-down fragment (1250 bp), and the 1114a-up-ScGAL2 fragment with the homologous arm upstream of the integration site connected to the promoter and the TDH1-1114a-down fragment with the homologous arm downstream of the integration site connected to the terminator were obtained; finally, the 1114a-up-ScGAL2-UGT99D1-TDH1-1114a-down expression cassette was obtained. Then the 1114a-up-ScGAL2- UGT99D1-TDH1-1114a-down expression cassette fragment was transformed into Saccharomyces cerevisiae WA6 and WA7 to obtain strains WA8 and WA9 containing the Ara-oleanolic acid synthesis pathway.

[0102] Table 6 Primer sequences for constructing strains WA8 and WA9

[0103] Primer number Primer sequences 47 SEQ ID NO: 67 48 SEQ ID NO: 68 49 SEQ ID NO: 69 50 SEQ ID NO: 70 51 SEQ ID NO: 71 52 SEQ ID NO: 72

[0104] Performance testing

[0105] 1. Analysis of oleanolic acid products WA11 and the starting strain WAT11 were cultured, the bacterial liquid was collected and extracted, and then LC-MS analysis was performed to detect the production of oleanolic acid. The mass / charge ratio of oleanolic acid in negative ion mode is 455. Using oleanolic acid standards as a control, the characteristic ion peak with a mass / charge ratio of 455 was extracted to identify whether the sample contained oleanolic acid products. The results are shown in Figure 1. Figure 2 As shown, the original strain WAT11 did not produce oleanolic acid, while the recombinant strain WA11 expressing the oleanolic acid biosynthesis pathway detected the production of oleanolic acid ( Figure 2 ). 2. Analysis of Ara-oleanolic acid products WA8, WA9 and WAT11 were cultured, the bacterial liquid was collected and extracted, and then LC-MS analysis was performed to detect the production of Ara-oleanolic acid. The mass / charge ratio of Ara-oleanolic acid in negative ion mode is 588. The characteristic ion peak with a mass / charge ratio of 588 was extracted to identify whether the sample contained Ara-oleanolic acid products. The results are shown in Figures 3 and 4. Ara-oleanolic acid products were detected in WA8 and WA9, respectively, while there was no obvious difference peak in the control WAT11, indicating that the recombinant yeast WA8 and WA9 with the Ara-oleanolic acid synthesis pathway successfully produced Ara-oleanolic acid ( Figure 3-Figure 4 ).

[0106] Application Example 2 Application of a Yeast Cell Producing Ara-betulinic Acid

[0107] The invention discloses an application of a yeast cell producing Ara-betulinic acid. The yeast cell producing Ara-betulinic acid produces Ara-betulinic acid from betulinic acid.

[0108] Method for constructing yeast cells producing Ara-betulinic acid

[0109] An engineered yeast strain for producing Ara-betulinic acid is obtained by introducing arabinosyltransferase into the UDP-Ara-producing yeast strain by feeding the substrate betulinic acid. The construction method comprises the following steps:

[0110] Using the genome of the strain with 1114a-up-ScGAL2 as a template, primers P47 / P48 were used to amplify the 1114a-up-ScGAL2 fragment (1700 bp), and using oat cDNA as a template, primers P49 / P50 were used to amplify the UGT99D1 fragment (1470 bp); using the genome of the strain with TDH1-1114a-down as a template, primers P51 / P52 were used to amplify the TDH1-1114a-down fragment (1250 bp), and the 1114a-up-ScGAL2 fragment with the homologous arm upstream of the integration site connected to the promoter and the TDH1-1114a-down fragment with the homologous arm downstream of the integration site connected to the terminator were obtained; finally, the 1114a-up-ScGAL2-UGT99D1-TDH1-1114a-down expression cassette was obtained. Then the 1114a-up-ScGAL2- UGT99D1-TDH1-1114a-down expression cassette fragment was transformed into Saccharomyces cerevisiae WA2 and WA3 to obtain strains WA12 and WA13.

[0111] Table 7 Primer sequences for constructing strains WA12 and WA13

[0112] Primer number Primer sequences 47 SEQ ID NO: 67 48 SEQ ID NO: 68 49 SEQ ID NO: 69 50 SEQ ID NO: 70 51 SEQ ID NO: 71 52 SEQ ID NO: 72

[0113] Performance testing

[0114] The production of Ara-betulinic acid was verified by feeding another pentacyclic triterpenoid compound, betulinic acid, to strains WA12 and WA13.

[0115] 1. Analysis of Ara-betulinic acid products

[0116] After feeding betulinic acid, WA12, WA13 and WAT11 were cultured, the bacterial liquid was collected and extracted, and then LC-MS analysis was performed to detect the production of Ara-betulinic acid. The mass / charge ratio of Ara-betulinic acid in negative ion mode is 588. The characteristic ion peak with a mass / charge ratio of 588 was extracted to identify whether the sample contained Ara-betulinic acid products. The results are shown in Figures 5 and 6. Ara-betulinic acid products were detected in WA12 and WA13, respectively, while there was no obvious difference peak in the control WAT11, indicating that the recombinant yeast WA8 and WA9 with the Ara-betulinic acid synthesis pathway successfully produced Ara-betulinic acid ( Figure 5-Figure 6 ).

[0117] In summary, by transforming the 911b-up-SeGAL1-AtUGDH3-ScADH1-911b-down expression cassette and the 607b-up-ScADH1-AtUXS3-PTDH3-pSeGal2-PsUGE2-ScENO1-607b-down expression cassette or the 607b-up-ScADH1-GuUXS2-PTDH3-pSeGal2-GuUXE1-ScENO1-607b-down expression cassette into the Saccharomyces cerevisiae strain WAT11, the synthesis pathway of UDP-Ara can be constructed on the Saccharomyces cerevisiae strain WAT11 to form the Saccharomyces cerevisiae engineered strains WA2 and WA3 that produce UDP-Ara. Then, the 511b-up-GAL10p-ERG1-PGK1t-ScADH1-ERG9-CCW12p-911b-down expression cassette and the 1414a-up-ScADH1-CqbAS1-pSeGal2-pTDH3-CqCYP716A78-ScADH1-1414a-down expression cassette were transformed into the Saccharomyces cerevisiae engineering strains WA2 and WA3, and the synthesis pathway of UDP-Ara and oleanolic acid was constructed on Saccharomyces cerevisiae to form strains WA6 and WA7. Finally, the 1114a-up-ScGAL2-UGT99D1-TDH1-1114a-down expression cassette fragment was transformed into strains WA6 and WA7 to construct the synthesis pathway of Ara-oleanolic acid, forming the Saccharomyces cerevisiae engineering strains WA8 and WA9 that produce Ara-oleanolic acid, realizing the application of the UDP-Ara chassis in the production of Ara-oleanolic acid. Finally, the inventors also fed strains WA12 and WA13 with other pentacyclic triterpenoid compounds (betulinic acid) and found the production of Ara-betulinic acid, and applied the UDP-Ara chassis to the production of Ara-betulinic acid. The construction of the arabinose donor yeast chassis provides a new biotechnology platform for the synthesis of natural glycoside compounds, which has important theoretical value and broad application prospects.

[0118] Table 8 Strain construction information

[0119] Yeast cells Chassis Gene information WA1 WAT11 911b-up-SeGAL1-AtUGDH3-ScADH1-911b-down WA2 WA1 607b-up-ScADH1-AtUXS3-PTDH3-pSeGal2-PsUGE2-ScENO1-607b-down WA3 WA1 607b-up-ScADH1-GuUXS2-PTDH3-pSeGal2-GuUXE1-ScENO1-607b-down WA4 WA2 511b-up- GAL10p-ERG1-PGK1t-ScADH1-ERG9-CCW12p-511b-down WA5 WA3 511b-up- GAL10p-ERG1-PGK1t-ScADH1-ERG9-CCW12p-511b-down WA6 WA4 1414a-up-ScADH1- CqbAS1- pSeGal2-pTDH3- CqCYP716A78-ScADH1-1414a -down WA7 WA5 1414a-up-ScADH1- CqbAS1- pSeGal2-pTDH3- CqCYP716A78-ScADH1-1414a -down WA8 WA6 1114a-up-ScGAL2- UGT99D1-TDH1-1114a -down WA9 WA7 1114a-up-ScGAL2- UGT99D1-TDH1-1114a -down WA10 WAT11 511b-up- GAL10p-ERG1-PGK1t-ScADH1-ERG9-CCW12p-511b-down WA11 WA10 1414a-up-ScADH1- CqbAS1- pSeGal2-pTDH3- CqCYP716A78-ScADH1-1414a -down WA12 WA2 1114a-up-ScGAL2- UGT99D1-TDH1-1114a -down WA13 WA3 1114a-up-ScGAL2- UGT99D1-TDH1-1114a -down

[0120] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A UDP-Ara producing yeast, characterized in that The cerevisiae engineered bacteria is constructed by sequentially expressing UDP-Glc dehydrogenase AtUGDH3, UDP-GlcA decarboxylase AtUXS3, and UDP-Glc-4-epimerase PsUGE2 on the cerevisiae strain WAT11 or sequentially expressing UDP-Glc dehydrogenase AtUGDH3, UDP-GlcA decarboxylase GuUXS2, and UDP-Xyl-4-epimerase GuUXE1 on the cerevisiae strain WAT11; The DNA sequence of AtUGDH3 is SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2; The DNA sequence of AtUXS3 is SEQ ID NO:3, which encodes the amino acid sequence of SEQ ID NO:4; The DNA sequence of GuUXS2 is SEQ ID NO:5, which encodes the amino acid sequence of SEQ ID NO:6; The DNA sequence of PsUGE2 is SEQ ID NO:7, which encodes the amino acid sequence of SEQ ID NO:8; The DNA sequence of GuUXE1 is SEQ ID NO:9, which encodes the amino acid sequence of SEQ ID NO:

10.

2. The method for constructing the engineered yeast of Saccharomyces cerevisiae according to claim 1, characterized in that The steps include: (1) Expressing AtUGDH3 in the Saccharomyces cerevisiae strain WAT11 to construct the UDP-GlcA synthesis pathway and form the WA1 strain; (2) expressing AtUXS3 in the WA1 strain to construct a synthetic pathway for UDP-Xyl; continuing to express PsUGE2 to construct a synthetic pathway for UDP-Ara, thereby forming the WA2 strain, which is the engineered yeast of Saccharomyces cerevisiae; Or (3) expressing GuUXS2 in the WA1 strain to construct a synthetic pathway for UDP-Xyl; and continuing to express GuUXE1 to construct a synthetic pathway for UDP-Ara to form a WA3 strain, which is the engineered yeast of Saccharomyces cerevisiae.

3. Use of the engineered yeast Saccharomyces cerevisiae according to claim 1 in the preparation of Ara-oleanolic acid.

4. The use according to claim 3, characterized in that The steps include: (1) In the two UDP-Ara-producing Saccharomyces cerevisiae engineered strains WA2 and / or WA3 constructed in claim 2, squalene epoxidase (ERG1), squalene synthase (ERG9), β-amyrin synthase (CqbAS1), and oxidase (CqCYP716A78) are sequentially expressed to complete the construction of the de novo synthesis pathway of oleanolic acid, thereby forming strains WA6 and / or WA7; The DNA sequence of ERG1 is SEQ ID NO: 11, which encodes the amino acid sequence of SEQ ID NO: 12; The DNA sequence of ERG9 is SEQ ID NO: 13, which encodes the amino acid sequence of SEQ ID NO: 14; The DNA sequence of CqbAS1 is SEQ ID NO: 15, which encodes the amino acid sequence of SEQ ID NO: 16; The DNA sequence of CqCYP716A78 is SEQ ID NO: 17, which encodes the amino acid sequence of SEQ ID NO: 18; (2) Introducing the arabinose transferase gene (UGT99D1) into strain WA6 and / or WA7 to complete the construction of the de novo synthesis pathway of Ara-oleanolic acid to form strain WA8 and / or WA9; The DNA sequence of UGT99D1 is SEQ ID NO:19, which encodes the amino acid sequence of SEQ ID NO:

20.

5. An engineered strain of Saccharomyces cerevisiae for producing Ara-oleanolic acid, characterized in that The construction method of the Saccharomyces cerevisiae engineering bacteria is as follows: (1) In the two UDP-Ara-producing Saccharomyces cerevisiae engineered strains WA2 and / or WA3 constructed in claim 2, squalene epoxidase (ERG1), squalene synthase (ERG9), β-amyrin synthase (CqbAS1), and oxidase (CqCYP716A78) are sequentially expressed to complete the construction of the de novo synthesis pathway of oleanolic acid, thereby forming strains WA6 and / or WA7; (2) Introducing the arabinose transferase gene (UGT99D1) into strain WA6 and / or WA7 to complete the construction of the de novo synthesis pathway of Ara-oleanolic acid, thereby forming strain WA8 and / or WA9, which are the engineered Saccharomyces cerevisiae strains producing Ara-oleanolic acid; The DNA sequence of ERG1 is SEQ ID NO: 11, which encodes the amino acid sequence of SEQ ID NO: 12; The DNA sequence of ERG9 is SEQ ID NO: 13, which encodes the amino acid sequence of SEQ ID NO: 14; The DNA sequence of CqbAS1 is SEQ ID NO: 15, which encodes the amino acid sequence of SEQ ID NO: 16; The DNA sequence of CqCYP716A78 is SEQ ID NO: 17, which encodes the amino acid sequence of SEQ ID NO: 18; The DNA sequence of UGT99D1 is SEQ ID NO:19, which encodes the amino acid sequence of SEQ ID NO:

20.

6. Use of the engineered yeast Saccharomyces cerevisiae according to claim 1 in the preparation of Ara-betulinic acid.

7. The use according to claim 6, characterized in that The steps include: (1) Introducing the arabinose transferase gene UGT99D1 into the two UDP-Ara producing Saccharomyces cerevisiae engineered strains WA2 and / or WA3 constructed according to claim 2 to form strains WA12 and / or WA13; The DNA sequence of UGT99D1 is SEQ ID NO: 19, which encodes the amino acid sequence of SEQ ID NO: 20; (2) Feeding betulinic acid culture strain WA12 and / or WA13 to prepare Ara-betulinic acid.

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