A multi-module collaborative optimization of brown carotenoid and brevifolin production of saccharomyces cerevisiae engineering bacteria and its construction method and application

Abstract

The present application relates to the technical field of synthetic biology, in particular to a multi-module synergistic optimization of brown cyanidin and brevifolin production of saccharomyces cerevisiae engineering bacteria and its construction method and application. The saccharomyces cerevisiae engineering bacteria includes naringenin synthesis pathway, optimization pathway of shikimic acid, synergistic pathway of phenylalanine, 6-OH luteolin synthesis pathway, brown cyanidin and brevifolin synthesis pathway, multiple copies of SbF6H and MpalOMT2, NADPH optimization pathway, and SAM cycle optimization pathway. The saccharomyces cerevisiae engineering bacteria is used for producing brown cyanidin and brevifolin, and improving the yield of the two.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, specifically to a multi-module synergistically optimized Saccharomyces cerevisiae strain that produces cyanidin and euphratin, its construction method, and its application. Background Technology

[0002] Brown cyanidin (4′,5,7-trihydroxy-3,6-dimethoxyflavone) and zeularin (5,7-dihydroxy-3′,4′,6-trimethoxyflavone) belong to the polyoxymethyl flavonoid class of compounds, mainly derived from plants in the Asteraceae family such as Artemisia argyi, and have certain medicinal development value. Currently, there is still a significant shortage of stable and large-scale availability of these compounds.

[0003] In existing technologies, cyanidin and eupatorium are mainly obtained through plant extraction or chemical synthesis. Plant extraction is limited by the low content of the target product in the plant, and the quality of the raw material is easily affected by climate, region, and harvest batch, making it difficult to guarantee yield and quality stability, and thus failing to meet the needs of continuous, large-scale supply. Chemical synthesis, due to the polymethoxylated structure of the product, usually involves multiple steps and selective control, resulting in complex routes, unsatisfactory yields, high reagent consumption, and environmental burden, which is also unfavorable for industrial-scale promotion. Therefore, utilizing microbial cell factories to prepare cyanidin and eupatorium using low-cost carbon sources has become an important direction to replace traditional acquisition methods. In previous research (Chinese patent application number: 202311644882.0), the applicant disclosed the construction of a flavonoid synthesis route starting from aromatic amino acids in Saccharomyces cerevisiae, and the synthesis of cyanidin and eupatorium by introducing enzymes related to hydroxylation and methylation modification; however, the yield of cyanidin and eupatorium produced by this engineered strain is low, making it difficult to meet the needs of further development and scale-up production. The shortcomings of the existing technology can be summarized as follows: (1) When achieving de novo synthesis of brown cyanidin and zellin in Saccharomyces cerevisiae, the upstream precursor supply capacity is insufficient, which easily leads to substrate limitation; (2) The expression and function of key enzymes in the pathway (including plant-derived flavonoid synthase, hydroxylase and oxymethyltransferase) in yeast are limited, resulting in low overall catalytic efficiency; (3) The post-modification steps are significantly dependent on cofactors, of which hydroxylation depends on NADPH and methylation depends on SAM. Insufficient cofactor supply and regeneration capacity will further limit the formation of the final product.

[0004] Therefore, there is an urgent need for a technical solution to improve the synthesis efficiency of brown cyanidin and eupatorium in Saccharomyces cerevisiae. Improvements should be made at least in areas such as enhanced precursor supply, enhanced expression / multiple copy enhancement of key rate-limiting enzymes, and improved intracellular supply and circulation capacity related to NADPH and SAM. This aims to address the low efficiency of existing engineered strains and enhance the industrial potential of product synthesis. Based on the original patent (Chinese Patent Application No.: 202311644882.0), this invention reconstructs a high-yield engineered Saccharomyces cerevisiae strain of brown cyanidin and eupatorium (its biosynthetic pathway diagram is shown in...). Figure 1 The contents described in Chinese Patent Application No. 202311644882.0 are incorporated herein by reference in their entirety. Summary of the Invention

[0005] This invention provides a high-yield brewer's yeast engineered strain of brown cyanidin and eupatorium, characterized in that the engineered brewer's yeast includes a synthetic pathway for naringenin, an optimized pathway for shikimic acid, a synergistic pathway for phenylalanine, a synthetic pathway for 6-OH luteolin, a synthetic pathway for brown cyanidin and eupatorium, multiple copies of SbF6H and MpalOMT2, an optimized pathway for NADPH, and an optimized pathway for the SAM cycle.

[0006] The synthetic pathway of naringenin involves expressing FjTAL (tyrosine ammonia-lyase), Pc4CL (coumaroyl-CoA ligase), PhCHS (chalcone synthase), and MsCHI (chalcone isomerase) in the Saccharomyces cerevisiae strain WAT11; the optimized pathway of shikimic acid involves expressing ARO1 (multifunctional aromatic amino acid synthase), ARO2 (branched acid synthase), ARO3, ARO4, ARO7 (branched acid mutase), and EcA in the Saccharomyces cerevisiae that synthesizes naringenin. ROL (dehydroquinic acid synthase) and knockout of ARO10 (aromatic amino acid decarboxylase) and PDC5 (pyruvate decarboxylase); the phenylalanine co-synergistic pathway is the expression of AtPAL (phenylalanine ammonia-lyase) and AtC4H (cinnamic acid-4-hydroxylase) in shikimic acid-optimized Saccharomyces cerevisiae; the 6-OH luteolin synthesis pathway is the expression of SbFNSII-1 (flavonoid synthase) and AtF3'H (flavonoid 3'-hydroxylase) in phenylalanine-co-synergistic Saccharomyces cerevisiae. The synthesis pathways of brown cyanidin and eupatorium are as follows: MpalOMT2 (3',4'-oxymethyltransferase) and ObFOMT4 (6'-oxymethyltransferase) are expressed in *Saccharomyces cerevisiae* that synthesizes 6-OH luteolin; the multiple copies of SbF6H and MpalOMT2 are SbF6H expressed 2-3 times and MpalOMT2 expressed 2-4 times; the optimized pathway for NADPH is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] The yeast expresses BDH1 (3-hydroxybutyrate dehydrogenase), IDH1 (isocitrate dehydrogenase), TYR1 (prephenylate dehydrogenase), and ARO8 (aromatic amino acid transaminase); the optimized SAM cycle pathway is to express MET6 (methionine synthase), SAH1 (S-adenosyl homocysteine ​​hydrolase), ADO1 (S-adenosylmethionine synthase), and ScMET13 (methylenetetrahydrofolate reductase) in Saccharomyces cerevisiae with NADPH pathway optimization.

[0007] The DNA sequence of FjTAL is SEQ ID NO:1, the DNA sequence of Pc4CL is SEQ ID NO:2, the DNA sequence of PhCHS is SEQ ID NO:3, and the DNA sequence of MsCHI is SEQ ID NO:4; the DNA sequence of ARO1 is SEQ ID NO:5, the DNA sequence of ARO2 is SEQ ID NO:6, the DNA sequence of ARO3 is SEQ ID NO:7, the DNA sequence of ARO4 is SEQ ID NO:8, the DNA sequence of ARO7 is SEQ ID NO:9, the DNA sequence of EcAROL is SEQ ID NO:10, the DNA sequence of ARO10 is SEQ ID NO:11, the DNA sequence of PDC5 is SEQ ID NO:12, the DNA sequence of AtPAL is SEQ ID NO:13, the DNA sequence of AtC4H is SEQ ID NO:14, the DNA sequence of SbFNSII-1 is SEQ ID NO:15, and the DNA sequence of AtF3'H is SEQ ID NO:15. The DNA sequence of SbF6H is SEQ ID NO:17, the DNA sequence of MpalOMT2 is SEQ ID NO:18, the DNA sequence of ObFOMT4 is SEQ ID NO:19, the DNA sequence of BDH1 is SEQ ID NO:20, the DNA sequence of IDH1 is SEQ ID NO:21, the DNA sequence of TYR1 is SEQ ID NO:22, the DNA sequence of ARO8 is SEQ ID NO:23, the DNA sequence of MET6 is SEQ ID NO:24, the DNA sequence of SAH1 is SEQ ID NO:25, the DNA sequence of ADO1 is SEQ ID NO:26, and the DNA sequence of ScMET13 is SEQ ID NO:27.

[0008] Another embodiment of the present invention provides a brewer's yeast strain that produces high yields of cyanidin and zeylanin, characterized in that the method for constructing the brewer's yeast strain includes the following steps:

[0009] (1) A Saccharomyces cerevisiae strain for naringenin synthesis was constructed by expressing FjTAL, Pc4CL, PhCHS, and MsCHI in Saccharomyces cerevisiae WAT11.

[0010] (2) ARO1, ARO2, ARO3, ARO4, ARO7, and EcAROL were expressed on the Saccharomyces cerevisiae obtained in step (1), and ARO10 and PDC5 were knocked out to obtain Saccharomyces cerevisiae with optimized shikimic acid pathway;

[0011] (3) AtPAL and AtC4H were expressed on the Saccharomyces cerevisiae obtained in step (2) to obtain Saccharomyces cerevisiae with optimized phenylalanine co-pathway;

[0012] (4) SbFNSII-1, AtF3'H and SbF6H were expressed on the Saccharomyces cerevisiae obtained in step (3) to obtain Saccharomyces cerevisiae that synthesizes 6-OH luteolin;

[0013] (5) MpalOMT2 and ObFOMT4 were expressed on the Saccharomyces cerevisiae obtained in step (4) to obtain Saccharomyces cerevisiae that synthesizes brown cyanidin and zellin;

[0014] (6) The Saccharomyces cerevisiae obtained in step (5) expresses SbF6H 2-3 times and MpalOMT2 2-4 times to obtain the Saccharomyces cerevisiae with optimized rate-limiting steps;

[0015] (7) The Saccharomyces cerevisiae obtained in step (6) express BDH1, IDH1, TYR1, and ARO8 to obtain Saccharomyces cerevisiae with optimized NADPH pathway;

[0016] (8) MET6, SAH1, ADO1, and ScMET13 are expressed on the Saccharomyces cerevisiae obtained in step (7) to obtain SAM cycle pathway optimized Saccharomyces cerevisiae, which is the Saccharomyces cerevisiae engineered strain that produces high levels of brown cyanidin and zeylanin.

[0017] Another embodiment of the present invention provides the application of the engineered Saccharomyces cerevisiae constructed by the above method of the present invention in the production of brown cyanidin and / or zellin.

[0018] Compared with the prior art, the advantages of the present invention are as follows: (1) The brewer's yeast cells for efficient production of cyanidin and zelandrin directly obtain cyanidin and zelandrin products from carbon sources or tyrosine, reducing production costs and alleviating the shortage of cyanidin and zelandrin in the market. (2) The present invention constructs an efficient production pathway for cyanidin and zelandrin in brewer's yeast cells, which can efficiently produce active flavonoid compounds cyanidin and zelandrin (the main active ingredients of the Korean marketed drug Stella tablets), providing a new way to obtain cyanidin and zelandrin. (3) The brewer's yeast cells of the present invention can produce high-purity cyanidin and zelandrin in large quantities through scale-up and optimization, without being limited by environmental and land resources. Attached Figure Description

[0019] Figure 1 This is a biosynthetic pathway diagram of cyanidin and zellin.

[0020] Figure 2This is a comparison chart of naringin production.

[0021] Figure 3 This is a comparison chart of brown cyanidin production.

[0022] Figure 4 This is a comparison chart of the yield of Zelanlin.

[0023] Figure 5 This is a graph showing the yield of shake-flask fermentation.

[0024] Figure 6 This is a diagram showing the production output of the fermenter during batch fermentation with feeding. Detailed Implementation

[0025] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be specifically noted that: unless otherwise specified in the following embodiments, conditions performed were based on conventional conditions or conditions recommended by the manufacturer; the enzymes and kits used in the following embodiments are all commercially available products. The PCR amplification methods, fusion methods of different fragments, gene knockout and overexpression methods used in the following embodiments can employ techniques commonly used in the art, such as fusion PCR, homologous recombination, and CRISPR-Cas9 technology.

[0026] Experimental methods:

[0027] Transformation was performed using lithium acetate / PEG3350. The transformation method used in the following examples was as follows: The host strain was first activated in 2×YPD medium and cultured overnight at 30°C and 200 rpm. Then, it was inoculated into fresh 2×YPD medium with an initial OD value of 0.2, and cultured at 30°C for another 4-4.5 h. 5 OD of the bacterial culture was then centrifuged at 5000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were washed twice with sterile ultrapure water to obtain yeast cells. A DNA mixture was prepared by taking 5 OD of the bacterial culture for each construction and mixing it with 50 μL of the DNA mixture to resuspend the cells. The 50 μL DNA mixture consisted of 2 μg of the inserted fragment, 250 ng of the tool plasmid, and sufficient ddH2O. The lithium acetate transformation mixture was added to the suspended cells, and after culturing, cells were obtained, plated on selection plates, and single colonies were obtained, which were the recombinant Saccharomyces cerevisiae. After sequencing verification of successful transformation, the recombinant Saccharomyces cerevisiae was preserved.

[0028] Colony PCR and sequencing verification: After single colonies grow on the screening plates, colony PCR and sequencing verification are performed. The specific steps are as follows: A small amount of cells is picked up with a pipette tip and placed in 20 μL of 20 mmol / L NaOH solution, vortexed, and incubated in a metal bath at 95℃ for 20 min. After vortexing, 1 μL of the bacterial solution is used as a template for colony PCR reaction. The size of the clonal bands is compared with that of the negative clonal bands. The bacterial solutions of positive colony PCR clones are selected and sent to the company for sequencing verification. Stranded strains with correct sequencing are streaked and cryopreserved in glycerol.

[0029] Culture of recombinant Saccharomyces cerevisiae strain: Single colonies were placed in 3 mL of 1×YPD 24-well plates and cultured overnight in a shaker for 16 h (30 ℃, 200 rpm). The overnight culture was diluted 10-fold with 1×YPD and the OD of the culture was measured using a UV spectrophotometer at 600 nm. The culture was then transferred to 3 mL of 1×YPG medium with an initial OD of 0.2 and cultured at 30 ℃, 800 rpm. After transfer, 300 μL of 20% galactose was added 12 h later, and another 300 μL of 20% galactose was added 24 h later, followed by culturing for 96 h.

[0030] Extraction of recombinant Saccharomyces cerevisiae products: Take a sample, add chromatographic grade ethyl acetate for extraction (extract twice), vortex for 1 min, centrifuge at 12000 rpm for 2 min, transfer the supernatant to a clean EP tube (capacity 1.5 mL), evaporate to dryness, add 140 μL of methanol to the EP tube, vortex for 1 min, centrifuge for 1 min, transfer to a liquid chromatography vial, and wait for analysis.

[0031] Shake-flask fermentation: Pick bacteria from YPD solid plates, add 3 ml of 1×YPD, and incubate at 30℃ and 220 rpm for 24 h. Take 1% of the bacterial solution and add it to 500 mL of 1×YPD medium, and incubate at 30℃ and 220 rpm for 120 h. During this period, take a sample every 12 h and add carbon source once.

[0032] Fed-batch fermentation: Pick bacteria from YPD solid plates, add 3 ml of 1×YPD, and incubate at 30℃ and 220 rpm for 24 h. Take 1% of the bacterial culture and add it to 200 mL of 1×YPD medium, and incubate at 30℃ and 220 rpm for 24 h. Inoculate the seed culture into the fermenter with 2 L of 1×YPD medium, incubate at 30℃, dissolved oxygen 30%, pH 5.5 for 120 h. From 12 h to 120 h, continuously add 20% glucose, and take samples every 12 h.

[0033] Product Analysis and Identification: The product was analyzed by HPLC using an Agilent 1290 Infinity II-6470A instrument with an Inertsil ODS-35um, 4.6×250 mm (GL Science) column and UV single-wavelength scanning as the detection mode. Chromatographic analysis was performed using gradient elution: mobile phase A was 0.05% formic acid acetonitrile, mobile phase B was 0.05% formic acid in water, and the flow rate was constant at 1.0 mL / min. The gradient program was as follows: A / B = 25 / 75 at 0.00 min; then linearly to A / B = 40 / 60 at 5.00 min; continued linearly to A / B = 70 / 30 at 15.00 min; and returned to A / B = 25 / 75 at 16.00 min; finally, the run was terminated at A / B = 25 / 75 at 20.00 min.

[0034] Preparation of main reagents: (1) 1×YPD liquid culture medium: Yeast extract (10g), Peptone (20g), D-(+)-Glucose (20g), add ddH2O to make up to 1000 mL, mix well and filter to sterilize.

[0035] (2) 20% D-(+)-Glucose: Add a small amount of ddH2O to D-(+)-Glucose (40g) and heat in a microwave oven to dissolve it. Then add ddH2O to make up to 200 mL. Mix well and filter to sterilize.

[0036] (3) 10×YNB solution: yeast nitrogen source (without amino acids and ammonium sulfate) (8.5g), ammonium sulfate (25g), add ddH2O to make up to 500 mL, filter to sterilize.

[0037] (4) Nutritional deficiency culture medium: 5×SC-Leu-Trp-Ura-His (3.5 g / 500 mL), 10×Ura (0.38 g / 500 mL), 10×Trp (0.38 g / 500 mL), 10×His (0.38 g / 500 mL), 10×Leu (1.8 g / 500 mL).

[0038] SC-Ura (200 mL): 5×Sc-leu-trp-ura-his (20 mL), 10×Leu (20 mL), 10×(YNB+ammonium sulfate) (20 mL), 5% agar powder solution (100 mL), 20% glucose (20 mL), ddH2O (20 mL).

[0039] Example 1: Method for constructing a high-yield (high-yield) *Saccharomyces cerevisiae* strain of cyanidin and zebulinerin.

[0040] A high-yield brewer's yeast engineered strain of brown cyanidin and eupatorium, characterized in that the engineered brewer's yeast includes a synthetic pathway for naringenin, an optimized pathway for shikimic acid, a synergistic pathway for phenylalanine, a synthetic pathway for 6-OH luteolin, a synthetic pathway for brown cyanidin and eupatorium, multiple copies of SbF6H and MpalOMT2, an optimized pathway for NADPH, and an optimized pathway for the SAM cycle.

[0041] (1) Synthetic pathway of naringenin: A naringenin synthetic pathway was constructed by expressing FjTAL (tyrosine ammonia-lyase), Pc4CL (coumaroyl-CoA ligase), PhCHS (chalcone synthase), and MsCHI (chalcone isomerase) in Saccharomyces cerevisiae WAT11. The DNA sequence of FjTAL is SEQ ID NO:1, the DNA sequence of Pc4CL is SEQ ID NO:2, the DNA sequence of PhCHS is SEQ ID NO:3, and the DNA sequence of MsCHI is SEQ ID NO:4.

[0042] (2) The shikimic acid pathway was optimized by expressing ARO1, ARO2, ARO3, ARO4, ARO7, and EcAROL on a strain that synthesizes naringenin, and knocking out ARO10 and PDC5; the DNA sequence of ARO1 is SEQ ID NO:5, the DNA sequence of ARO2 is SEQ ID NO:6, the DNA sequence of ARO3 is SEQ ID NO:7, the DNA sequence of ARO4 is SEQ ID NO:8, the DNA sequence of ARO7 is SEQ ID NO:9, the DNA sequence of EcAROL is SEQ ID NO:10, the DNA sequence of ARO10 is SEQ ID NO:11, and the DNA sequence of PDC5 is SEQ ID NO:12.

[0043] By adopting the above technical solution, ARO1, ARO2, ARO3, ARO4, ARO7 and EcAROL were expressed in the naringenin synthesizing strain, and ARO10 and PDC5 were knocked out to obtain the shikimic acid pathway optimized Saccharomyces cerevisiae strain EU34.

[0044] (3) The phenylalanine co-pathway was constructed by expressing AtPAL and AtC4H in EU34. The DNA sequence of AtPAL is SEQ ID NO:13, and the DNA sequence of AtC4H is SEQ ID NO:14.

[0045] By adopting the above technical solution, AtPAL and AtC4H were expressed in Saccharomyces cerevisiae strain EU34, resulting in Saccharomyces cerevisiae strain EU35 with optimized phenylalanine co-pathway.

[0046] (4) The 6-OH luteolin synthesis pathway was constructed by expressing SbFNSII-1 (flavonoid synthase), AtF3'H (flavonoid 3'-hydroxylase), and SbF6H (flavonoid 6-hydroxylase) in Saccharomyces cerevisiae strain EU35. The DNA sequence of SbFNSII-1 is SEQ ID NO:15, the DNA sequence of AtF3'H is SEQ ID NO:16, and the DNA sequence of SbF6H is SEQ ID NO:17.

[0047] By adopting the above technical solution, SbFNSII-1, AtF3'H and SbF6H were expressed in the Saccharomyces cerevisiae strain EU35 to construct the 6-OH luteolin synthesis pathway, and the Saccharomyces cerevisiae strain EU43 synthesizing 6-OH luteolin was obtained.

[0048] (5) Synthetic pathways of brown cyanidin and eupatorium: The oxygen methyltransferases MpalOMT2 (3',4'-oxymethyltransferase) and ObFOMT4 (6'-oxymethyltransferase) were continued to be expressed in Saccharomyces cerevisiae strain EU43 to construct the synthetic pathways of brown cyanidin and eupatorium. The DNA sequence of MpalOMT2 is SEQ ID NO:18, and the DNA sequence of ObFOMT4 is SEQ ID NO:19.

[0049] By adopting the above technical solution, the Saccharomyces cerevisiae strain EU44, which expresses MpalOMT2 and ObFOMT4 in the 6-OH luteolin synthase strain, was obtained.

[0050] (6) The multiple copies of the flavonoid 6-hydroxylase SbF6H and oxygen methyltransferase MpalOMT2 genes are expressed 2-3 times (preferably 2 times) of SbF6H and 2-4 times (preferably 3 times) of MpalOMT2 in Saccharomyces cerevisiae strain EU44.

[0051] By adopting the above technical solution, SbF6H and MpalOMT2 were expressed on the strain that synthesizes zeylanin, the rate-limiting step was optimized, and the synthesis of the key intermediate 6-OH luteolin was increased, while the synthesis of brown cyanidin and zeylanin was further improved, resulting in the Saccharomyces cerevisiae strain EU46.

[0052] (7) The NADPH pathway is optimized to express BDH1, IDH1, TYR1, and ARO8 in Saccharomyces cerevisiae EU46. The DNA sequence of BDH1 is SEQ ID NO:20, the DNA sequence of IDH1 is SEQ ID NO:21, the DNA sequence of TYR1 is SEQ ID NO:22, and the DNA sequence of ARO8 is SEQ ID NO:23.

[0053] By adopting the above technical solution, expressing BDH1, IDH1, TYR1, and ARO8 in Saccharomyces cerevisiae that synthesizes naringenin can yield Saccharomyces cerevisiae strain EU47 with optimized NADPH pathway.

[0054] (8) The SAM cycle pathway was optimized by expressing MET6, SAH1, ADO1, and ScMET13 in Saccharomyces cerevisiae EU47. The DNA sequence of MET6 is SEQ ID NO:24, the DNA sequence of SAH1 is SEQ ID NO:25, the DNA sequence of ADO1 is SEQ ID NO:26, and the DNA sequence of ScMET13 is SEQ ID NO:27.

[0055] By adopting the above technical solution, MET6, SAH1, ADO1, and ScMET13 were expressed in the Saccharomyces cerevisiae strain EU47, which is highly efficient at synthesizing brown cyanidin and zellin, resulting in the SAM cycle optimized Saccharomyces cerevisiae strain EU49.

[0056] Example 2: Specific Construction Method Used in this Invention

[0057] A high-yield brewer's yeast engineered strain of brown cyanidin and eupatorium, characterized in that the engineered brewer's yeast includes a synthetic pathway for naringenin, an optimized pathway for shikimic acid, a synergistic pathway for phenylalanine, a synthetic pathway for 6-OH luteolin, a synthetic pathway for brown cyanidin and eupatorium, multiple copies of SbF6H and MpalOMT2, an optimized pathway for NADPH, and an optimized pathway for the SAM cycle.

[0058] The method for constructing Saccharomyces cerevisiae cells for efficient production of cyanidin and euphratin specifically includes the following steps:

[0059] S1. Using the Saccharomyces cerevisiae strain WAT11 as the starting strain, the yeast synthesis pathway of naringenin was first constructed to form EU24.

[0060] First, the 911b-Up-ADH1t-FjTAL-SeGAL2p-PTDH3p-Pc4CL-CYC1t-911b-Down expression cassette was integrated into the WAT11 genome. The integrated gene fragment was amplified by PCR using DNA polymerase: using the genome of *Saccharomyces cerevisiae* WAT11 as a template, the 911b-Up fragment (1000 bp) was amplified using primers P1 / P2, and the 911b-Down fragment (1000 bp) was amplified using primers P3 / P4, yielding the upstream homologous arm 911b-UP fragment and the downstream homologous arm 911b-Down fragment. Using the genome of a strain possessing ADH1t-FjTAL-SeGAL2p as a template, ADH1t-FjTAL-SeGAL2p (2477 bp) was amplified using primers P5 / P6. Using the genome of a strain possessing PTDH3p-Pc4CL-CYC1t as a template, the PTDH3p-Pc4CL-CYC1t fragment (2438 bp) was amplified using primers P7 / P8. The final expression cassette, 911b-Up-ADH1t-FjTAL-SeGAL2p-PTDH3p-Pc4CL-CYC1t-911b-Down, was then transformed into *Saccharomyces cerevisiae* WAT11 to obtain strain EU14.

[0061] Table 1 Primer sequences for constructing strain EU14

[0062]

[0063] The next step is to integrate the 1414a-Up-ENO1t-PhCHS-GAL1p-SeGAL2p-MsCHI-TDH1t-1414a-Down expression cassette into the EU14 genome. Using the genome of Saccharomyces cerevisiae WAT11 as a template, the 1414a-Up fragment (1000 bp) was amplified using primers P9 / P10, and the 1414a-Down fragment (1000 bp) was amplified using primers P11 / P12, yielding the 1414a-Up fragment upstream of the integration site and the 1414a-Down fragment downstream of the integration site. Using the genome of the ENO1t-PhCHS-GAL1p strain as a template, the ENO1t-PhCHS-GAL1p fragment (1930 bp) was amplified using primers P13 / P14. Using the genome of the SeGAL2p-MsCHI-TDH1t strain as a template, the SeGAL2p-MsCHI-TDH1t fragment (1963 bp) was amplified using primers P15 / P16. The final expression cassette, 1414a-Up-ENO1t-PhCHS-GAL1p-SeGAL2p-MsCHI-TDH1t-1414a-Down, was obtained. This cassette fragment was then transformed into *Saccharomyces cerevisiae* EU14 to obtain strain EU24. Strains with correctly sequenced genes were then used for further culturing.

[0064] Table 2 Primer sequences for constructing strain EU24

[0065]

[0066] S2. On the naringenin synthesizing strain EU24, the shikimic acid pathway was optimized and the phenylalanine synergistic pathway was constructed to improve the yield of naringenin, resulting in the high-yielding Saccharomyces cerevisiae strain EU35.

[0067] First, the PDC5-Up-ScENO1t-ARO2-GAL1p-PTDH3p-ARO3-TDH1t-TRP ORF-PDC5-Down expression cassette was integrated into the EU24 genome. Using the genome of *Saccharomyces cerevisiae* WAT11 as a template, the PDC5-Up fragment (1000 bp) was amplified using primers P17 / P18, and the PDC5-down fragment (1000 bp) was amplified using primers P19 / P20, yielding the upstream homologous arm PDC5-Up fragment and the downstream homologous arm PDC5-down fragment of the integration site. Using the genome of a strain with the ScENO1t-ARO2-GAL1p as a template, the ScENO1t-ARO2-GAL1p fragment (1956 bp) was amplified using primers P21 / P22. Using the genome of a strain with the PTDH3p-ARO3-TDH1t as a template, the PTDH3p-ARO3-TDH1t fragment (1963 bp) was amplified using primers P23 / P24. Using the genome of a strain with the Trp ORF as a template, the TrpORF fragment (1758 bp) was amplified using primers P25 / P26. The final expression cassette, PDC5-Up-ScENO1t-ARO2-GAL1p-PTDH3p-ARO3-TDH1t-TRP ORF-PDC5-Down, was obtained. This PDC5-Up-ScENO1t-ARO2-GAL1p-PTDH3p-ARO3-TDH1t-TRP ORF-PDC5-Down, was then transformed into *Saccharomyces cerevisiae* EU24 to obtain strain EU32.

[0068] Table 3 Primer sequences for constructing strain EU32

[0069]

[0070] The next step is to integrate the ARO10-Up-ScENO1t-AROL-GAL1t-PTDH3p-ARO1-TDH1t-HIS ORF-ARO10-Down expression cassette into the EU32 genome. Using the genome of *Saccharomyces cerevisiae* WAT11 as a template, the ARO10-Up fragment (1000 bp) was amplified using primers P27 / P28, and the ARO10-down fragment (1000 bp) was amplified using primers P29 / P30, yielding the upstream homologous arm ARO10-Up fragment and the downstream homologous arm ARO10-down fragment of the integration site. Using the genome of a strain with the ScENO1t-AROL-GAL1t as a template, the ScENO1t-AROL-GAL1t fragment (1350 bp) was amplified using primers P31 / P32. Using the genome of a strain with the PTDH3p-ARO1-TDH1t as a template, the PTDH3p-ARO1-TDH1t fragment (5617 bp) was amplified using primers P33 / P34. Using the genome of a strain with the HIS ORF as a template, the HIS ORF fragment (1185 bp) was amplified using primers P35 / P36. The ARO10-Up-ScENO1t-AROL-GAL1t-PTDH3p-ARO1-TDH1t-HIS ORF-ARO10-Down expression cassette was finally obtained. This ARO10-Up-ScENO1t-AROL-GAL1t-PTDH3p-ARO1-TDH1t-HIS ORF-ARO10-Down expression cassette fragment was then transformed into *Saccharomyces cerevisiae* EU32 to obtain strain EU33. Strains with correctly sequenced genes were then used for further culture.

[0071] Table 4 Primer sequences for constructing strain EU33

[0072]

[0073] Next, the Gal80-Up-TDH1t-ARO4-GAL1p-GAL10p-ARO7-PGK1t-Gal80-Down expression cassette was integrated into the EU33 genome. Using the genome of a strain possessing Gal80-Up-TDH1t-ARO4 as a template, the Gal80-Up-TDH1t-ARO4 fragment (1863 bp) was amplified using primers P37 / P38; using the genome of a strain possessing GAL1p-GAL10p as a template, GAL1p-GAL10p (812 bp) was amplified using primers P39 / P40; and using the genome of a strain possessing ARO7-PGK1t-Gal80-Down as a template, the ARO7-PGK1t-Gal80-Down fragment (1556 bp) was amplified using primers P41 / P42. The Gal80-Up-TDH1t-ARO4-GAL1p-GAL10p-ARO7-PGK1t-Gal80-Down expression cassette was finally obtained. Then, the Gal80-Up-TDH1t-ARO4-GAL1p-GAL10p-ARO7-PGK1t-Gal80-Down expression cassette fragment was transformed into *Saccharomyces cerevisiae* EU33 to obtain strain EU34.

[0074] Table 5 Primer sequences for constructing strain EU34

[0075]

[0076] The next step is to integrate the 1114a-Up-GAL1p-AtC4H-ADH1t-CYC1t-AtPAL-PGK1p-1114a-Down expression cassette into the EU34 genome. Using the genome of Saccharomyces cerevisiae WAT11 as a template, the 1114a-Up fragment (1000 bp) was amplified using primers P43 / P44, and the 1114a-Down fragment (1000 bp) was amplified using primers P45 / P46, yielding the 1114a-Up fragment upstream of the integration site and the 1114a-Down fragment downstream of the integration site. Using the genome of a strain possessing GAL1p-AtC4H as a template, the GAL1p-AtC4H fragment (1315 bp) was amplified using primers P47 / P48. Using the genome of a strain possessing ADH1t-CYC1t-AtPAL-PGK1p as a template, the fragment ADH1t-CYC1t-AtPAL-PGK1p (2877 bp) was amplified using primers P49 / P50. The final expression cassette, 1114a-Up-GAL1p-AtC4H-ADH1t-CYC1t-AtPAL-PGK1p-1114a-Down, was obtained. This cassette fragment was then transformed into *Saccharomyces cerevisiae* EU34 to obtain strain EU35. Strains with correctly sequenced genes were then used for further culturing.

[0077] Table 6 Primer sequences for constructing strain EU35

[0078]

[0079] S3. The hexahydroxyluteolin synthesis pathway and the eupatorium synthesis pathway were constructed in the high-yielding naringenin strain EU35 to obtain the total synthesis strain EU44 of brown cyanidin and eupatorium.

[0080] The YPRCd15C-Up-SkGAL2p-FNS2-ENO1t-ADH1t-AtF3'H-PTDH3p-YPRCd15C-Down expression cassette was integrated into the EU35 genome. Using the genome of a strain possessing YPRCd15C-Up-SkGAL2p-FNS2 as a template, the YPRCd15C-Up-SkGAL2p-FNS2 fragment (3209 bp) was amplified using primers P51 / P52; using the genome of a strain possessing ENO1t-ADH1t as a template, the ENO1t-ADH1t fragment (475 bp) was amplified using primers P53 / P54; and using the genome of a strain possessing AtF3'H-PTDH3p-YPRCd15C-Down as a template, the AtF3'H-PTDH3p-YPRCd15C-Down fragment (3142 bp) was amplified using primers P55 / P56. The final expression cassette, YPRCd15C-Up-SkGAL2p-FNS2-ENO1t-ADH1t-AtF3'H-PTDH3p-YPRCd15C-Down, was obtained. The YPRCd15C-Up-SkGAL2p-FNS2-ENO1t-ADH1t-AtF3'H-PTDH3p-YPRCd15C-Down expression cassette fragment was then transformed into Saccharomyces cerevisiae EU35 to obtain strain EU43.

[0081] Table 7 Primer sequences for constructing strain EU43

[0082]

[0083] Next, we continued to integrate the 416d-Up-ADH1t-MpalOMT2-PTDH3p-pSeGal2-SbF6H-ENO1t-CYC1t-ObFOMT4-GAL1p-416d-down expression cassette. Using the genome of Saccharomyces cerevisiae WAT11 as a template, we amplified the 416d-Up fragment using primers P57 / P58, obtaining the 416d-UP fragment (1000 ppm) upstream homologous arm of the integration site. Using the genome of a strain possessing the ADH1t-MpalOMT2-PTDH3p-pSeGal2-SbF6H-ENO1t as a template, the ADH1t-MpalOMT2-PTDH3p-pSeGal2-SbF6H-ENO1t fragment (4185 bp) was amplified using primers P59 / P60; using the genome of a strain possessing the CYC1t terminator as a template, the terminator fragment CYC1t (203 bp) was amplified using primers P61 / P62. Using the genome of a strain possessing ObFOMT4-GAL1p-416d-down as a template, the ObFOMT4-GAL1p-416d-down fragment (2611 bp) was amplified using primers P63 / P64; the final expression cassette, 416d-Up-ADH1t-MpalOMT2-PTDH3p-pSeGal2-SbF6H-ENO1t-CYC1t-ObFOMT4-GAL1p-416d-down, was then transformed into Saccharomyces cerevisiae EU43 to obtain strain EU44.

[0084] Table 8 Primer sequences for constructing strain EU44

[0085]

[0086] S4. Multiple copies of SbF6H and MpalOMT2 were made on the strain EU44, which is a complete synthesizer of brown cyanidin and euphratin, to initially obtain the Saccharomyces cerevisiae strain EU46, which is highly efficient in producing brown cyanidin and euphratin.

[0087] First, the 607b-Up-SeGal2p-SbF6H-ENOT1t-607b-Down expression cassette was integrated. Using the genome of *Saccharomyces cerevisiae* WAT11 as a template, the 607b-Up fragment was amplified using primers P65 / P66, and the 607b-Down fragment (1000 bp) was amplified using primers P67 / P68, yielding the upstream and downstream homologous arms of the 607b-Up and 607b-Down fragments at the integration site. Using the genome of a strain possessing SeGal2p-SbF6H-ENOT1t as a template, the SeGal2p-SbF6H-ENOT1t fragment (2485 bp) was amplified using primers P69 / P70. The final 607b-Up-SeGal2p-SbF6H-ENOT1t-607b-Down expression cassette was obtained. The 607b-Up-SeGal2p-SbF6H-ENOT1t-607b-Down expression cassette fragment was then transformed into Saccharomyces cerevisiae EU44 to obtain strain EU45.

[0088] Table 9 Primer sequences for constructing strain EU45

[0089]

[0090] Next, we continued to integrate the 308a-Up-ScGAL1p-MpalOMT2R1-ADH1t-CYC1t-MpalOMT2R2-PGK1p-308a-Down expression cassette. Using the genome of a strain possessing 308a-Up-ScGAL1p-MpalOMT2R1 as a template, we amplified the 308a-Up-ScGAL1p-MpalOMT2R1 fragment (1950 bp) using primers P71 / P72; using the genome of a strain possessing ADH1t-CYC1t as a template, we amplified the ADH1t-CYC1t fragment (452 ​​bp) using primers P73 / P74; and using the genome of a strain possessing MpalOMT2R2-PGK1p-308a-Down as a template, we amplified the MpalOMT2R2-PGK1p-308a-Down fragment (2171 bp) using primers P75 / P76. The final expression cassette, 308a-Up-ScGAL1p-MpalOMT2R1-ADH1t-CYC1t-MpalOMT2R2-PGK1p-308a-Down, was obtained. This cassette fragment was then transformed into *Saccharomyces cerevisiae* EU45 to obtain strain EU46. Strains with correctly sequenced genes were then used for further culturing.

[0091] Table 10 Primer sequences for constructing strain EU46

[0092]

[0093] S5. The NADPH pathway was optimized on the Saccharomyces cerevisiae EU46 yeast strain to further obtain the Saccharomyces cerevisiae EU47 strain which produces brown cyanidin and zellin efficiently.

[0094] Integrate the 511b-Up-GAL1p-BDH1-ENO1t-ADH1t-IDH1-PTDH3p-SeGAL2p-TYR1-PGK1t-TDH1t-ARO8-Gal10p-511b-Down expression box. Using the genome of *Saccharomyces cerevisiae* WAT11 as a template, the 511b-Up fragment (500 bp) was amplified using primers P77 / P78, and the 511b-Down fragment (500 bp) was amplified using primers P79 / P80, yielding the 511b-Up fragment upstream of the integration site and the 511b-Down fragment downstream of the integration site. Using the genome of a strain possessing the promoter GAL1p as a template, the promoter GAL1p fragment (529 bp) was amplified using primers P81 / P82. Using the genome of WAT11 as a template, the BDH1 fragment (1149 bp) was amplified using primers P83 / P84 / P85 / P86. Using the genome of the terminator ENO1t as a template, the terminator ENO1t fragment (225 bp) was amplified using primers P87 / P88. Using the genome of a strain possessing ADH1t-IDH1-PTDH3p as a template, the ADH1t-IDH1-PTDH3p fragment (1933 bp) was amplified using primers P89 / P90; using the genome of a strain possessing SeGAL2p-TYR1-PGK1t as a template, the SeGAL2p-TYR1-PGK1t fragment (2809 bp) was amplified using primers P91 / P92; using the genome of a strain possessing TDH1t-ARO8-Gal10p as a template, the TDH1t-ARO8-Gal10p fragment (1965 bp) was amplified using primers P93 / P94. The final expression cassette fragment, 511b-Up-GAL1p-BDH1-ENO1t-ADH1t-IDH1-PTDH3p-SeGAL2p-TYR1-PGK1t-TDH1t-ARO8-Gal10p-511b-Down, was obtained. This fragment was then transformed into *Saccharomyces cerevisiae* EU46 to obtain strain EU47.

[0095] Table 11 Primer sequences for constructing strain EU47

[0096]

[0097] S6. Using the strain constructed in the previous step as the chassis, the SAM cycle pathway was optimized to obtain a Saccharomyces cerevisiae strain that efficiently produces brown cyanidin and eupatorium acetonide.

[0098] Integrate the 1021b-Up-GAL1p-MET6-ENO1t-ADH1t-SAH1-SeGAL2p-1021b-Down expression box. Using the genome of *Saccharomyces cerevisiae* WAT11 as a template, the 1021b-Up fragment (1000 bp) was amplified using primers P95 / P96, and the 1021b-Down fragment (1000 bp) was amplified using primers P97 / P98, yielding the 1021b-Up fragment upstream of the integration site and the 1021b-Down fragment downstream of the integration site. Using the genome of a strain possessing GAL1p-MET6 as a template, the GAL1p-MET6 fragment (2833 bp) was amplified using primers P99 / P100. Using the genome of a strain possessing ENO1t-ADH1t as a template, the ENO1t-ADH1t fragment (475 bp) was amplified using primers P101 / P102. Using the genome of a strain possessing SAH1-SeGAL2p as a template, the SAH1-SeGAL2p fragment (2050 bp) was amplified using primers P103 / P104. The final expression cassette, 1021b-Up-GAL1p-MET6-ENO1t-ADH1t-SAH1-SeGAL2p-1021b-Down, was obtained. This cassette fragment was then transformed into *Saccharomyces cerevisiae* EU47 to obtain strain EU48.

[0099] Table 12 Primer sequences for constructing strain EU48

[0100]

[0101] The next step is to continue integrating the HIS3b-Up-PTDH3p-ADO1-CYC1t-TDH1t-ScMET13-PGK1p-HIS3b-Down expression cassette. Using the genome of Saccharomyces cerevisiae WAT11 as a template, the HIS3b-Up fragment (1000 bp) was amplified using primers P105 / P106, and the HIS3b-Down fragment (1000 bp) was amplified using primers P107 / P108, yielding the upstream homologous arm HIS3b-Up fragment and the downstream homologous arm HIS3b-Down fragment of the integration site. Using the genome of a strain possessing PTDH3p-ADO1-CYC1t as a template, the PTDH3p-ADO1-CYC1t fragment (1826 bp) was amplified using primers P109 / P110. Using the genome of a strain possessing TDH1t-ScMET13-PGK1p as a template, the TDH1t-ScMET13-PGK1p fragment (2797 bp) was amplified using primers P111 / P112. The HIS3b-Up-PTDH3p-ADO1-CYC1t-TDH1t-ScMET13-PGK1p-HIS3b-Down expression cassette was finally obtained. This HIS3b-Up-PTDH3p-ADO1-CYC1t-TDH1t-ScMET13-PGK1p-HIS3b-Down expression cassette fragment was then transformed into *Saccharomyces cerevisiae* EU48 to obtain strain EU49.

[0102] Table 13 Primer sequences for constructing strain EU49

[0103]

[0104] In summary, using *Saccharomyces cerevisiae* strain WAT11 as a chassis, the naringenin synthesis pathway was first constructed by introducing FjTAL, Pc4CL, PhCHS, and MsCHI, resulting in *Saccharomyces cerevisiae* EU24. The pathway was then modified by introducing PDC5-Up-ScENO1t-ARO2-GAL1p-PTDH3p-ARO3-TDH1t-TRP ORF-PDC5-Down and ARO10-Up-ScENO1t-EcAROL-GAL1t-PTDH3p-ARO1-TDH1t-HIS. Transforming the three expression cassettes ORF-ARO10-Down, Gal80-Up-TDH1t-ARO4-GAL1p-GAL10p-ARO7-PGK1t-Gal80-Down into the Saccharomyces cerevisiae host EU24 optimizes the shikimic acid pathway, increases naringenin production, and forms yeast cell EU34. The next step is to transform the expression cassette 1114a-up-GAL1p-AtC4H-ADH1t-CYC1t-AtPAL-PGK1p-1114a-down into Saccharomyces cerevisiae EU34 to construct the phenylalanine co-transport pathway, further increasing naringenin production and forming yeast cell EU35. The next step involves expressing SbFNSII-1, AtF3'H, and SbF6H in the Saccharomyces cerevisiae host EU35 to construct the 6-OH luteolin synthesis pathway on EU35. This will then lead to the expression of the oxygen methyltransferases MpalOMT2 and ObFOMT4, resulting in the total synthesis strain EU44 of cyanidin and euphratinoin. The next step involves transferring an expression cassette, 607b-Up-SeGal2p-F6H-ENOT1t-607b-Down, into Saccharomyces cerevisiae EU44 to optimize the rate-limiting step, promoting the conversion of luteolin to the key intermediate 6-OH luteolin, and further increasing the yield of cyanidin and euphratinoin, thus obtaining Saccharomyces cerevisiae strain EU45. Then, an expression cassette, 308a-Up-ScGAL1p-MpalOMT2R1-ADH1t-CYC1t-MpalOMT2R2-PGK1p-308a-Down, was transformed into *Saccharomyces cerevisiae* EU45 to increase the yield of brown cyanidin and euphratin, thus initially constructing *Saccharomyces cerevisiae* strain EU46, which efficiently synthesizes brown cyanidin and euphratin. Next, an expression cassette, 511b-up-GAL1p-BDH1-ENO1t-ADH1t-IDH1-PTDH3p-SeGAL2p-TYR1-PGK1t-TDH1t-ARO8-Gal10p-511b-down, was transformed into *Saccharomyces cerevisiae* strain EU46 to obtain *Saccharomyces cerevisiae* strain EU47 with optimized NADPH pathway.Finally, the two expression cassettes, 1021b-Up-GAL1p-MET6-ENO1t-ADH1t-SAH1-SeGAL2p-1021b-Down and HIS3b-Up-PTDH3p-ADO1-CYC1t-TDH1t-ScMET13-PGK1p-HIS3b-Down, were transformed into EU47 to obtain a Saccharomyces cerevisiae strain with optimized SAM cycling pathway, namely the Saccharomyces cerevisiae strain EU49, which produces high levels of brown cyanidin and euphratin.

[0105] Table 14 Strain Construction Information

[0106]

[0107] Example 3: Application of a yeast cell for the efficient production of cyanidin and zellin.

[0108] Application of a yeast cell for the efficient production of brown cyanidin and euphratin, wherein the yeast cell for the efficient production of brown cyanidin and euphratin produces brown cyanidin and euphratin from a carbon source or tyrosine.

[0109] Performance testing

[0110] 1. Comparison of naringin production

[0111] Saccharomyces cerevisiae strains EU24, EU33, EU34, and EU35 were cultured, and the bacterial cultures were collected and extracted. HPLC analysis was then performed to detect and quantify naringenin production. Results are as follows: Figure 2 As shown, strain EU24 produced 27.88 mg / L of naringenin, while the Saccharomyces cerevisiae strains EU33 and EU34, which were optimized for the shikimic acid pathway using EU24 as the chassis, produced 38.14 mg / L and 46.82 mg / L of naringenin, respectively. The Saccharomyces cerevisiae strain EU35, which constructed a phenylalanine co-pathway using EU34 as the chassis, produced 65.15 mg / L of naringenin.

[0112] 2. Analysis of cornflower seed extract yield

[0113] EU44, EU45, EU46, EU47, and EU49 were cultured, bacterial cultures were collected and extracted, and then analyzed by HPLC to detect the production of brown cyanidin and to quantify brown cyanidin. The results are as follows: Figure 3As shown, the brown cyanidin yield of EU44 was 7.64 mg / L, the brown cyanidin yield of strain EU45 (optimized by SbF6H multiple copies (2 times)) was 12.81 mg / L, the brown cyanidin yield of strain EU46 (optimized by MpalOMT2 multiple copies (3 times) using EU45 as the chassis) was 14.79 mg / L, the brown cyanidin yield of strain EU47 (optimized by NADPH pathway using EU46 as the chassis) was 28.96 mg / L, and the brown cyanidin yield of strain EU49 (optimized by SAM cycling pathway using EU47 as the chassis) was 42.16 mg / L.

[0114] 3. Analysis of the yield of eupatorium fortunei

[0115] EU44, EU45, EU46, EU47, and EU49 were cultured, bacterial cultures were collected and extracted, and then analyzed by HPLC to detect the production of zebulin and to quantify zebulin. The results are as follows: Figure 4 As shown, the yield of zebulin from EU44 was 5.3 mg / L, the yield of zebulin from strain EU45 (which underwent SbF6H multicopy (2 times) optimization) was 8.63 mg / L, the yield of zebulin from strain EU46 (which underwent MpalOMT2 multicopy (3 times) optimization using EU45 as the chassis) was 12.28 mg / L, the yield of zebulin from strain EU47 (which underwent NADPH pathway optimization using EU46 as the chassis) was 19.28 mg / L, and the yield of zebulin from strain EU49 (which underwent SAM cycling pathway optimization using EU47 as the chassis) was 27.31 mg / L.

[0116] 4. Synthesis of brown cyanidin and eupatorium by shake-flask fermentation

[0117] Saccharomyces cerevisiae strain EU49 was cultured in shake flasks for 120 h. The bacterial culture was collected and extracted every 12 h, followed by HPLC analysis to detect and quantify the production of cyanidin and euphratin. Results are as follows: Figure 5 As shown, the yields of zeylanin and brown cyanidin reached 29.09 mg / L and the yield of zeylanin reached 35.01 mg / L after 120 h.

[0118] 5. Fed-feed batch fermentation to synthesize brown cyanidin and eupatorium phosphate.

[0119] Saccharomyces cerevisiae strain EU49 was cultured in shake flasks for 144 h. The bacterial culture was collected every 12 h and extracted, then analyzed by HPLC to detect the production of cyanidin and euphratin, and the quantification of cyanidin and euphratin was performed. Results are as follows: Figure 6As shown, the yield of brown cyanidin reached 40.87 mg / L and the yield of zeylanin reached 45.56 mg / L after 144 hours.

[0120] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A brewer's yeast strain that produces cyanidin and zeylanin, characterized in that... The engineered Saccharomyces cerevisiae includes the following pathways: naringenin synthesis pathway, shikimic acid optimization pathway, phenylalanine synergistic pathway, 6-OH luteolin synthesis pathway, brown cyanidin and zellin synthesis pathway, multiple copies of SbF6H and MpalOMT2, NADPH optimization pathway, and SAM cycle optimization pathway. The synthetic pathway for naringenin involves expressing FjTAL, Pc4CL, PhCHS, and MsCHI in the Saccharomyces cerevisiae strain WAT11; the optimized pathway for shikimic acid involves expressing ARO1, ARO2, ARO3, ARO4, ARO7, and EcAROL in the Saccharomyces cerevisiae strain that synthesizes naringenin, while knocking out ARO10 and PDC5; the synergistic pathway for phenylalanine involves expressing AtPAL and AtC4H in the Saccharomyces cerevisiae strain that optimizes the shikimic acid pathway; and the synthetic pathway for 6-OH luteolin involves expressing SbFNSII-1, AtF3'H, and SbF6 in the Saccharomyces cerevisiae strain that is synergistic with phenylalanine. H; The synthetic pathway of brown cyanidin and eupatorium is to express MpalOMT2 and ObFOMT4 in Saccharomyces cerevisiae synthesizing 6-OH luteolin; The multiple copies of SbF6H and MpalOMT2 are SbF6H expressed 2-3 times and MpalOMT2 expressed 2-4 times; The optimized pathway of NADPH is to express BDH1, IDH1, TYR1, and ARO8 in Saccharomyces cerevisiae synthesizing brown cyanidin and eupatorium; The optimized pathway of SAM cycle is to express MET6, SAH1, ADO1, and ScMET13 in Saccharomyces cerevisiae with optimized NADPH pathway; The DNA sequence of FjTAL is SEQ ID NO:1, the DNA sequence of Pc4CL is SEQ ID NO:2, the DNA sequence of PhCHS is SEQ ID NO:3, and the DNA sequence of MsCHI is SEQ ID NO:4; the DNA sequence of ARO1 is SEQ ID NO:5, the DNA sequence of ARO2 is SEQ ID NO:6, the DNA sequence of ARO3 is SEQ ID NO:7, the DNA sequence of ARO4 is SEQ ID NO:8, the DNA sequence of ARO7 is SEQ ID NO:9, the DNA sequence of EcAROL is SEQ ID NO:10, the DNA sequence of ARO10 is SEQ ID NO:11, the DNA sequence of PDC5 is SEQ ID NO:12, the DNA sequence of AtPAL is SEQ ID NO:13, the DNA sequence of AtC4H is SEQ ID NO:14, the DNA sequence of SbFNSII-1 is SEQ ID NO:15, and the DNA sequence of AtF3'H is SEQ ID NO:

15. The DNA sequence of SbF6H is SEQ ID NO:17, the DNA sequence of MpalOMT2 is SEQ ID NO:18, the DNA sequence of ObFOMT4 is SEQ ID NO:19, the DNA sequence of BDH1 is SEQ ID NO:20, the DNA sequence of IDH1 is SEQ ID NO:21, the DNA sequence of TYR1 is SEQ ID NO:22, the DNA sequence of ARO8 is SEQ ID NO:23, the DNA sequence of MET6 is SEQ ID NO:24, the DNA sequence of SAH1 is SEQ ID NO:25, the DNA sequence of ADO1 is SEQ ID NO:26, and the DNA sequence of ScMET13 is SEQ ID NO:

27.

2. The engineered brewer's yeast strain according to claim 1, characterized in that... The multiple copies of SbF6H and MpalOMT2 are SbF6H expressed twice and MpalOMT2 expressed three times.

3. The method for constructing the engineered Saccharomyces cerevisiae according to any one of claims 1-2, characterized in that... Includes the following steps: (1) A Saccharomyces cerevisiae strain for naringenin synthesis was constructed by expressing FjTAL, Pc4CL, PhCHS, and MsCHI in Saccharomyces cerevisiae WAT11. (2) ARO1, ARO2, ARO3, ARO4, ARO7, and EcAROL were expressed on the Saccharomyces cerevisiae obtained in step (1), and ARO10 and PDC5 were knocked out to obtain Saccharomyces cerevisiae with optimized shikimic acid pathway; (3) AtPAL and AtC4H were expressed on the Saccharomyces cerevisiae obtained in step (2) to obtain Saccharomyces cerevisiae with optimized phenylalanine co-pathway; (4) SbFNSII-1, AtF3'H and SbF6H were expressed on the Saccharomyces cerevisiae obtained in step (3) to obtain Saccharomyces cerevisiae that synthesizes 6-OH luteolin; (5) MpalOMT2 and ObFOMT4 were expressed on the Saccharomyces cerevisiae obtained in step (4) to obtain Saccharomyces cerevisiae that synthesizes brown cyanidin and zellin; (6) The Saccharomyces cerevisiae obtained in step (5) expresses SbF6H 2-3 times and MpalOMT2 2-4 times to obtain the Saccharomyces cerevisiae with optimized rate-limiting steps; (7) The Saccharomyces cerevisiae obtained in step (6) express BDH1, IDH1, TYR1, and ARO8 to obtain Saccharomyces cerevisiae with optimized NADPH pathway; (8) MET6, SAH1, ADO1, and ScMET13 are expressed on the Saccharomyces cerevisiae obtained in step (7) to obtain SAM cycle pathway optimized Saccharomyces cerevisiae, which is the Saccharomyces cerevisiae engineered strain that produces brown cyanidin and zellin.

4. The use of the engineered Saccharomyces cerevisiae according to any one of claims 1-2 in the production of brown cyanidin and / or zeularin.

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