Method for producing phloretin by using saccharomyces cerevisiae

Abstract

The invention discloses a method for producing phloretin by using saccharomyces cerevisiae, and belongs to the technical field of microbial genetic engineering. A natural metabolic phloretin path in a plant is successfully constructed in the saccharomyces cerevisiae BY4741, synthesis of phloretin in microorganisms is achieved, synthesis of the phloretin in the microorganisms is enhanced by enhancing synthesis path genes of tyrosine and phenylalanine in the saccharomyces cerevisiae, including elimination of tyrosine feedback inhibition and introduction of exogenous genes, synthesis of prerequisite tyrosine and phenylalanine is enhanced, and the yield of the phloretin is increased. And the yield of the phloretin is further improved by enhancing the expression of a key gene in a phloretin synthesis pathway. The yield of phloretin synthesized from the beginning by the engineering yeast can reach 246.21 mg / L, the synthesis problem of phloretin serving as a carbohydrase inhibitor is effectively solved, and a foundation is laid for synthesizing phloretin through efficient fermentation of microorganisms.

Description

Technical Field

[0001] The invention relates to a method for producing phloretin by utilizing saccharomyces cerevisiae, and belongs to the technical field of microbial genetic engineering. Background Art

[0002] Phloretin, 2,4,6-trihydroxy-3 (4-hydroxyphenyl) propiophenone, is a natural dihydrochalcone substance derived from apples. It has excellent anti-sugar, antioxidant, antibacterial, anti-inflammatory, anti-allergic and anti-thrombotic properties. For decades, α-glucosidase has become the main target for the prevention and treatment of type 2 diabetes. Phloretin exhibits excellent inhibitory activity against α-glucosidase. Studies have shown that the addition of phloretin to food can delay starch hydrolysis and may reduce postprandial blood glucose concentration, and has good prospects for food and medical use. At present, the main source of phloretin is plant extraction. The process is complicated and affected by other similar flavonoids in plants, resulting in certain difficulties in separation and purification, and to a certain extent, it is limited by space and time, which affects the progress of its industrial production. Chemical synthesis methods are prone to environmental pollution. Therefore, it is necessary to develop new methods for synthesizing plant natural products using microorganisms. Summary of the invention

[0003] The present invention provides a method for producing phloretin by using Saccharomyces cerevisiae. By editing the gene of Saccharomyces cerevisiae and introducing the relevant metabolic pathways for phloretin synthesis, the constructed Saccharomyces cerevisiae engineered bacteria can produce phloretin using glucose as a substrate.

[0004] The present invention provides an engineered yeast strain for producing phloretin, which overexpresses the tyrosine ammonia lyase gene FjTAL, the coumaroyl-CoA ligase gene At4CL, the chalcone synthase gene HaCHS and the enoyl-CoA reductase TSC13 on the basis of the starting strain, and performs one or more of the following improvements:

[0005] (1) Integrate and express the phenylalanine ammonia lyase gene AtPAL2, the cinnamate hydroxylase gene AtC4H, the cytochrome P450 reductase gene AtATR2, and the cytochrome B5 gene CYB5;

[0006] (2) Overexpression of endogenous gene ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8 and PHA2 and / or expressing the exogenous genes EcAroL and Bbxfpk opt ;

[0007] (3) Use plasmids to enhance the expression of genes FjTAL, At4CL, TSC13, and HaCHS.

[0008] In one embodiment, the starting strain includes but is not limited to Saccharomyces cerevisiae BY4741.

[0009] In one embodiment, the engineered Saccharomyces cerevisiae has FjTAL gene, At4CL gene, HaCHS gene and TSC13 gene integrated into its genome.

[0010] In one embodiment, the genes FjTAL, At4CL, HaCHS, TSC13, AtPAL2, AtC4H, AtATR2, and CYB5 are expressed by constitutive strong promoters derived from Saccharomyces cerevisiae, respectively. The constitutive strong promoters include but are not limited to P THD3 , P TEF1 , P PGK1 , P FBA1 .

[0011] In one embodiment, the gene ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL and Bbxfpk opt The expression is regulated by a constitutive strong promoter derived from Saccharomyces cerevisiae, including but not limited to P ccw14 , P PGK1 , P HXT7 , P FBA1 , P THD3 , P TEF1 , P ADH1 .

[0012] In one embodiment, the gene integration operation of Saccharomyces cerevisiae is achieved by Crispr / Cas9 technology, with the purpose of achieving gene editing in Saccharomyces cerevisiae to enhance the synthesis of phloretin.

[0013] In one embodiment, the sequences of the genes FjTAL, At4CL, HaCHS, TSC13, AtPAL2, AtC4H, AtATR2, and CYB5 are shown in SEQ ID NO.1 to SEQ ID NO.8; the sequences of the gene ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL, Bbxfpk opt The nucleotide sequences are shown in SEQ ID NO.9 to SEQ ID NO.18.

[0014] The present invention also provides a method for increasing the yield of phloretin in Saccharomyces cerevisiae, the method comprising optimizing the expression of genes in the tyrosine and phenylalanine metabolic pathways; specifically comprising: overexpressing the anti-feedback inhibition gene 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene mutant ARO4 derived from Saccharomyces cerevisiae K229L , chorismate mutase gene mutant ARO7 G141S , prephenate dehydrogenase TYR1, pentafunctional aromatic protein ARO1, branching synthase ARO2, 3-dehydroquinate synthase ARO3, aromatic aminotransferase ARO8 and prephenate dehydratase PHA2; as well as knocking out the endogenous genes of Saccharomyces cerevisiae pyruvate decarboxylase PDC5 and phenylpyruvate decarboxylase ARO10; overexpressing the codon-optimized phosphoketolase gene Bbxfpk from Bifidobacterium breve opt and overexpression of the codon-optimized shikimate kinase EcAROL from Escherichia coli.

[0015] In one embodiment, the method is to modify the gene ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL and Bbxfpk opt to the Saccharomyces cerevisiae genome and knocked out the genes PDC5 and ARO10.

[0016] In one embodiment, the gene integration operation of Saccharomyces cerevisiae is achieved by Crispr / Cas9 technology, with the aim of achieving gene editing in Saccharomyces cerevisiae to enhance tyrosine and phenylalanine synthesis.

[0017] The present invention also provides a method for preparing phloretin by fermentation, which comprises culturing the engineered yeast Saccharomyces cerevisiae in a culture medium for a period of time and collecting the phloretin in the fermentation liquid.

[0018] In one embodiment, the engineered Saccharomyces cerevisiae is cultured in YPD medium.

[0019] In one embodiment, the pH of the culture medium during the fermentation process is controlled to be 5.2-6.4.

[0020] In one embodiment, the method is to culture at 28-32°C, 180-240 rpm until the initial OD 600 =5-10 as seed liquid, and then transfer the seed liquid to the fermentation medium for fermentation.

[0021] In one embodiment, the fermentation medium is YPD medium.

[0022] In one embodiment, the initial OD of the fermentation 600=0.05~0.1, during the fermentation process, the pH was controlled at 5.2~6.4, 28~32℃, 180-300rpm, and the fermentation was carried out for 72~120h.

[0023] The invention also provides application of the engineered yeast Saccharomyces cerevisiae in preparing phloretin or a product containing phloretin.

[0024] Beneficial effects:

[0025] The present invention successfully constructs an engineered yeast of Saccharomyces cerevisiae capable of synthesizing phloretin, comprising:

[0026] (1) Integrate the constitutive strong promoter P into the genome of Saccharomyces cerevisiae BY4741 THD3 , P TEF1 , P PGK1 , P FBA1 The regulated FjTAL, At4CL, HaCHS, TSC13 genes were used to construct the Saccharomyces cerevisiae engineered strain Y-1 that can utilize tyrosine to synthesize phloretin;

[0027] (2) Based on the engineered yeast Y-1, the expression of the constitutive strong promoter P THD3 , P TEF1 , P PGK1 , P FBA1 The genes AtPAL2, AtC4H, AtATR2, and CYB5 are regulated respectively, and the engineered yeast Y-2 with enhanced phenylalanine utilization ability is constructed;

[0028] (3) Based on the engineered yeast Y-2, the genes PDC5 and ARO10 were knocked out, and a constitutive strong promoter was used to increase the expression of the endogenous gene ARO4. K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8 and PHA2 and the exogenous genes EcAroL and Bbxfpk opt The yeast engineering strain Y-5 was constructed by expression of

[0029] (4) Yeast engineered strain Y-6 was obtained by using plasmids to enhance the expression of genes FjTAL, At4CL, TSC13, and HaCHS in yeast engineered strain Y-5.

[0030] The yeast engineered bacteria Y-6 constructed by the present invention can synthesize phloretin de novo using glucose as a substrate, and the phloretin yield at the shake flask level can reach 246.21 mg / L. The cost-effectiveness of converting glucose into phloretin is extremely high, which effectively solves the problem that wild brewer's yeast cannot synthesize phloretin de novo, provides new ideas and methods for the efficient production of phloretin by microorganisms, and is of great significance for promoting the green and efficient synthesis of phloretin. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention provides a gene map of the engineered yeast Saccharomyces cerevisiae BY4741 that can produce phloretin after iterative genetic modification.

[0032] Figure 2 It is the yield of phloretin synthesized by all the engineered yeasts of the present invention using glucose as a substrate. DETAILED DESCRIPTION

[0033] (I) Biochemical materials

[0034] Saccharomyces cerevisiae BY4741 (our laboratory); phloretin standard (Solarbio, China); restriction endonucleases, T4 DNA ligase (Novozyme Biotech Co., Ltd.); Prime STAR Max DNA polymerase, loading buffer, LE agarose and S4 nucleic acid electrophoresis dye (Takara); seamless cloning kit (Wuhan Aibote Biotechnology Co., Ltd.); antibiotics, yeast whole genome extraction kit and SanPrep column plasmid DNA small-scale extraction kit, DNA5000ladder and DNA10000ladder (Shanghai Shenggong Biotechnology Co., Ltd.); tryptone and yeast extract (Oxoid, UK); yeast amino acid nitrogen base and anhydrous sodium glutamate (Solarbio, China); yeast amino acid premix powder (Shanghai Weidi Biotechnology Co., Ltd.); tyrosine ammonia lyase gene FjTAL, coumaroyl-CoA ligase gene At4CL, chalcone synthase gene HaCHS, phenylalanine ammonia lyase gene AtPAL2, cinnamate hydroxylase gene AtC4H cytochrome P450 reductase gene AtATR2 and phosphotransketolase gene Bbxfpk in Bifidobacterium breve opt Codon optimization and synthesis (Suzhou Jinweizhi Co., Ltd.).

[0035] (ii) Culture medium:

[0036] Preparation of YPD liquid culture medium: 20 g / L glucose, 20 g / L trypsin, 10 g / L yeast extract, sterilized at 115°C for 30 min.

[0037] Preparation of YPD agar medium: Add 15g agar powder to 1L of YPD liquid medium, sterilize with high-pressure steam and pour into sterile plates to obtain antibiotic-free YPD solid medium; if antibiotics are added, the corresponding antibiotic-selective YPD solid medium will be obtained. After condensation, place in a 4°C refrigerator for later use.

[0038] LB liquid culture medium preparation: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, sterilized at 120°C for 20 min.

[0039] Preparation of LB agar medium: Add 15 g agar powder to 1 L of LB liquid medium. The sterilization method is the same as that of YPD agar medium.

[0040] Preparation of SD-Ura+G418 agar medium: 1.7 g / L yeast nitrogen base without amino acids, 5 g / L anhydrous sodium glutamate, 1.31 g / L Ura-deficient yeast amino acid premix, 20 g / L glucose and 15 g / L agar powder, 500 mg / L geneticin G418, sterilized at 115°C for 30 min.

[0041] 3. Strains, plasmids and primers

[0042] The strain information is shown in Table 1, the plasmid is shown in Table 2, and the primer information is shown in Table 3.

[0043] Table 1 Strains and their genotypes

[0044]

[0045] Table 2 Plasmids and their genotypes

[0046]

[0047] Table 3 Primers

[0048]

[0049]

[0050] (IV) Sequence information

[0051] The nucleotide sequences of genes FjTAL, At4CL, HaCHS, TSC13, AtPAL2, AtC4H, AtATR2, and CYB5 involved in the embodiment are shown in SEQ ID NOs. 1 to 8, respectively; K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL, Bbxfpk opt The nucleotide sequences are shown in SEQ ID NO.9 to SEQ ID NO.18 respectively.

[0052] The promoter sequence is shown in Table 4.

[0053] Table 4 Promoter and terminator

[0054]

[0055]

[0056] (V) Basic operations of molecular biology:

[0057] (1) E. coli plasmid extraction, genome extraction, PCR product purification, and gel recovery were performed according to the instructions of the corresponding kit unless otherwise specified.

[0058] (2) PCR and overlap extension PCR were used to amplify DNA fragments and perform site-directed mutation of genes, respectively, and different reaction systems and procedures were used according to different experimental requirements. The reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

[0059] Table 5 PCR reaction system

[0060]

[0061] Table 6 PCR reaction program

[0062]

[0063]

[0064] (3) Construction of gRNA plasmid: BsaI digestion and T4 ligase reaction system and reaction conditions were performed according to the instructions on the website of Novozymes.

[0065] (4) Transformation of competent E. coli by heat shock method: First, take 5ul of the ligation product and add 50ul of freeze-thawed competent cells, and place on ice for 20min; then place the competent cells in a 42℃ metal bath for heat shock for 90s, quickly place on ice, and place on ice for 5min; then add 1mL of fresh LB medium and shake at 37℃ for 60min to recover; finally, take 200ul of the bacterial solution and spread it on a solid LB plate with the corresponding resistance and culture it at 37℃ overnight.

[0066] (5) Preparation of competent cells of Saccharomyces cerevisiae and chemical transformation:

[0067] 1) Pick a single colony after streaking and culture it in YPD medium overnight, inoculate 10% in fresh YPD medium, and wait for it to grow to OD 600 = about 2.0, take 1 mL of brewer's yeast solution, place it in a 1.5 mL EP tube, and use a refrigerated centrifuge to centrifuge at 4°C, 1 min, 4000 rpm.

[0068] 2) Pour off the supernatant and add 1 mL of 1×TE buffer to the centrifuge tube. Gently pipette to resuspend, and centrifuge at 4000 rpm for 1 min at 4°C.

[0069] 3) Pour off the supernatant, add 30 μL of 1×LiAC solution to the centrifuge tube, pipette gently to resuspend, and let stand for 10 minutes. Prepare a mixed solution of 10×LiAc, 10×TE, and 50% PEG3350 in a ratio of 1:1:8.

[0070] 4) Add plasmid or DNA fragment to the bacterial solution and gently shake to mix.

[0071] 5) Add 250 μL of a mixed solution of 10×LiAc, 10×TE, and 50% PEG3350, and mix by pipetting.

[0072] 6) After mixing, place the centrifuge tube in a constant temperature water bath and incubate at 30°C for 30 minutes.

[0073] 7) After incubation, add 30 μL of dimethyl sulfoxide (DMSO) and mix by gently inverting the tube up and down 7 times.

[0074] 8) Heat shock the centrifuge tube in a constant temperature water bath at 42°C for 7 min.

[0075] 9) After heat shock, centrifuge at 4000 rpm for 1 min at 4°C.

[0076] 10) Pour off the supernatant, add 1 mL of 1×TE buffer, pipette to mix, and centrifuge at 4°C, 1 min, 4000 rpm. Pour off the supernatant, leaving about 200 μL of liquid, mix and apply to the corresponding solid culture medium.

[0077] Strain culture and fermentation method: Pick a single colony from the agar plate and inoculate it into 5 mL of YPD liquid medium. The yeast is placed in a constant temperature shaker at 220 rpm and 30°C for overnight culture. Then 1 mL of bacterial liquid is taken from the overnight culture and transferred to 50 mL of YPD medium for expansion culture. Add corresponding antibiotics according to the strain requirements, such as G418, with a final working concentration of 200 ug / mL.

[0078] (VI) Detection method of phloretin:

[0079] Phloretin in the supernatant of the fermentation broth was detected using an HPLC-UV system (Waters 2695). After fermentation, 2 mL of the fermentation broth was centrifuged at a speed of 10,000 g for 5 min, and the supernatant was filtered with a 0.22 μm water membrane, and then detected with a C18 column (250×4.6 mm id, 5 μm; Agilent). The injection volume was 10 μL, the detection wavelength was 280 nm, the column temperature was 30°C, and the flow rate was 0.3 mL / min. The mobile phases were 0.1% formic acid in A and 100% acetonitrile in B. The liquid phase elution program adopted gradient elution: 0-1min, 90%-80%A, 1-3min, 80%-80%A, 3-4min, 80%75%A, 4-8min, 75%-30%A, 8-9min, 30%-5%A, 9-12min, 5%-5%A, 12-12.2min, 5%-90%A, 12.2-15.2min, 90%-90%A, detection wavelength 280nm; column temperature 30C, flow rate 0.5mL / min, injection volume 10ul.

[0080] Example 1: Genetically engineered wild-type Saccharomyces cerevisiae BY4741 and tested for its de novo phloretin production

[0081] (1) Construction of yeast engineering strain Y-1:

[0082] The genes FjTAL, At4CL, HaCHS, TSC13 and the corresponding promoters and terminators were cloned into the vector pCEV-G1-Km by seamless cloning to obtain the plasmid pCEV-TTCC. The gene fragment TTCC (T ENO2 -TSC13-P FBA1 -P THD3 -FjTAL-T CYC1- T ADH1 -At4CL-P TEF -P PGK1 -HaCHS-T HIS3 ), and a high-purity fragment TTCC was obtained after gel electrophoresis verification and gel recovery. The fragment TTCC was integrated into the XI-2 site on the chromosome of Saccharomyces cerevisiae BY4741 through Crispr / Cas9 technology, and the resulting yeast engineering strain was named Y-1.

[0083] The engineered yeast Y-1 was fermented in YPD medium at 30°C for 72 hours, and the phloretin production in the fermentation supernatant was determined using an HPLC-UV system. Figure 2 As shown, through the chromosomal integration of the phloretin exogenous pathway gene, phloretin could be detected in the fermentation broth with a yield of 21.87 mg / mL.

[0084] (2) Construction of yeast engineering strain Y-2:

[0085] Genes AtPAL2, AtC4H, AtATR2, CYB5 and the corresponding promoters and terminators were cloned into the vector pCEV-G1-Km by seamless cloning to obtain plasmid pCEV-AAAC. Using pCEV-AAAC as a template, gene fragment AAAC (T ENO2 -AtPAL2-P FBA1 -P THD3 -AtC4H-T CYC1 -T ADH1 -AtATR2-P TEF -P PGK1 -CYB5-T HIS3 ), and the high-purity fragment AAAC was obtained by gel electrophoresis verification and gel recovery. The fragment AAAC was integrated into the chromosome XI-4 site of the yeast engineering strain Y-1 by Crispr / Cas9 technology to obtain the yeast engineering strain Y-2.

[0086] The engineered yeast Y-2 was fermented in YPD medium at 30°C for 72 hours, and the phloretin production in the fermentation supernatant was determined using an HPLC-UV system. Figure 2 As shown, phloretin production was increased to 90.22 mg / mL by chromosomal integration of pathway genes that enhance phenylalanine utilization.

[0087] Example 2: Optimizing the metabolic pathways of tyrosine and phenylalanine in Saccharomyces cerevisiae to increase phloretin production

[0088] (1) The genes PDC5 (Gene ID: 850825) and ARO10 (Gene ID: 851987) of the yeast engineered strain Y-2 were knocked out by Crispr / Cas9 technology to obtain the yeast engineered strain Y-3. The constructed Saccharomyces cerevisiae engineered strain Y-3 was fermented in YPD medium for 72 hours, and the phloretin production in the fermentation supernatant was determined by HPLC-UV system. The results are shown in Figure 2 As shown in Figure 2, by optimizing the tyrosine and phenylalanine metabolic pathways, the phloretin production can be increased to 90.22 mg / mL.

[0089] (2) Gene ARO4 K229L , ARO7 G141S , ARO1, ARO2, ARO3 and the corresponding promoters and terminators were cloned into the vector PUC19 by seamless cloning to obtain the plasmid pCEV-AAAA; using PUC-AAAA as a template, the gene fragment AAAAA (PTEF1 -ARO4 K229L -T CYC1 -T ADH1 -ARO7 G141S -P ccw14 -P PGK1 -ARO1-T CYC1 -T ADH1 -ARO2-P HXT7 -P FBA1 -AR O3-T CYC1 ), after gel electrophoresis verification and gel recovery, a high-purity fragment AAAAA was obtained, and the fragment AAAAA was integrated into the chromosome of Saccharomyces cerevisiae Y-3 by Crispr / Cas9 technology to obtain the yeast engineering strain Y-4, with a genotype such as Figure 1 Y-4 was fermented in YPD medium for 72 hours, and the phloretin production in the fermentation supernatant was determined using an HPLC-UV system. Figure 2 As shown, by optimizing the tyrosine and phenylalanine metabolic pathways, the phloretin production was increased to 140.37 mg / mL.

[0090] (3) Genes ARO8, TYR1, PHA2, EcAroL, Bbxfpk and the corresponding promoters and terminators were cloned into the vector PUC by seamless cloning to obtain the plasmid pCEV-ATPAB. Using PUC-ATPAB as a template, the gene fragment ATPAB (T CYC1 -ARO8-P THD3 -P TEF1 -TYR1-T ADH1 -T CYC1 -EcAroL-P PGK1 -P ccw14 -Bbxfpk-T ADH1 -P FBA1 -PHA2-T CY C1 ), after gel electrophoresis verification and gel recovery, a high-purity fragment ATPAB was obtained, and the fragment ATPAB was integrated into the chromosome of Saccharomyces cerevisiae Y-4 by Crispr / Cas9 technology to obtain the yeast engineering strain Y-5, the genotype of which is as follows Figure 1 Y-5 was fermented in YPD medium for 72 hours, and the phloretin production in the fermentation supernatant was determined using an HPLC-UV system. Figure 2 As shown in Figure 2, by optimizing the tyrosine and phenylalanine metabolic pathways, the phloretin production was increased to 170.37 mg / mL.

[0091] Example 3: Overexpression of phloretin synthesis pathway genes in engineered Saccharomyces cerevisiae to increase phloretin production

[0092] The plasmid pCEV-TTCC constructed in Example 1 was transformed into the yeast engineered strain Y-5 constructed in Example 2 to obtain the yeast engineered strain Y-6, which was fermented in YPD medium supplemented with geneticin at a final concentration of 200 ug / mL for 72 hours, and the phloretin production in the fermentation supernatant was determined using an HPLC-UV system. The results are shown in FIG. Figure 2 As shown, by enhancing the expression of exogenous rate-limiting enzymes 4CL and CHS, the phloretin production reached a maximum of 246.21 mg / mL.

[0093] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An engineered yeast of phloretin-producing Saccharomyces cerevisiae, characterized in that: Based on the starting strain, the tyrosine ammonia lyase gene FjTAL, the coumaroyl-CoA ligase gene At4CL, the chalcone synthase gene HaCHS and the enoyl-CoA reductase TSC13 are overexpressed, and one or more of the following improvements are made: (1) Integrate and express the phenylalanine ammonia lyase gene AtPAL2, the cinnamate hydroxylase gene AtC4H, the cytochrome P450 reductase gene AtATR2, and the cytochrome B5 gene CYB5; (2) Overexpression of endogenous gene ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8 and PHA2 and / or expressing the exogenous genes EcAroL and Bbxfpk opt ; (3) Use plasmids to enhance the expression of genes FjTAL, At4CL, TSC13, and HaCHS.

2. The brewer's yeast engineering bacterium according to claim 1, characterized in that The FjTAL gene, At4CL gene, HaCHS gene and TSC13 gene were integrated into the genome.

3. The engineered yeast of claim 1, characterized in that: The genes FjTAL, At4CL, HaCHS, TSC13, AtPAL2, AtC4H, AtATR2, and CYB5 are expressed by constitutive strong promoters derived from Saccharomyces cerevisiae, respectively. The constitutive strong promoters include but are not limited to P THD3 , P TEF1 , P PGK1 , P FBA1 .

4. The engineered yeast of claim 1, characterized in that: The gene ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL and Bbxfpk opt The expression is regulated by a constitutive strong promoter derived from Saccharomyces cerevisiae, including but not limited to P ccw14 , P PGK1 , P HXT7 , P FBA1 , P THD3 , P TEF1 , P ADH1 .

5. The engineered yeast of any one of claims 1 to 4, characterized in that: The starting strain includes but is not limited to Saccharomyces cerevisiae BY4741.

6. A method for increasing the yield of phloretin in Saccharomyces cerevisiae, characterized in that: Enhance the expression of genes in the tyrosine and phenylalanine metabolic pathways of Saccharomyces cerevisiae; specifically, overexpression of the anti-feedback inhibition gene 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene mutant ARO4 from Saccharomyces cerevisiae K229L , chorismate mutase gene mutant ARO7 G141S , prephenate dehydrogenase TYR1, pentafunctional aromatic protein ARO1, branching synthase ARO2, 3-dehydroquinate synthase ARO3, aromatic aminotransferase ARO8 and prephenate dehydratase PHA2; as well as knocking out the endogenous genes pyruvate decarboxylase PDC5 and phenylpyruvate decarboxylase ARO10 in Saccharomyces cerevisiae; overexpressing the phosphoketolase gene Bbxfpk opt and the shikimate kinase EcAROL.

7. The method according to claim 6, characterized in that ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL and Bbxfpk opt to the Saccharomyces cerevisiae genome and knocked out the genes PDC5 and ARO10.

8. A method for preparing phloretin by fermentation, characterized in that: The engineered yeast of any one of claims 1 to 5 is cultured in a culture medium for a period of time, and phloretin in the fermentation liquid is collected.

9. The method according to claim 8, characterized in that Initial fermentation OD 600 =0.05~0.1, during the fermentation process, the pH was controlled at 5.2~6.4, 28~32℃, and the fermentation time was 72~120h.

10. Use of the engineered yeast of Saccharomyces cerevisiae according to any one of claims 1 to 5, or the method according to any one of claims 6 to 9 in the preparation of phloretin or a product containing phloretin.

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