Method for synthesizing glucose transport inhibitor phlorizin or trilobatin by using saccharomyces cerevisiae

Abstract

The invention discloses a method for synthesizing a glucose transport inhibitor phlorizin or trilobatin by using saccharomyces cerevisiae, and belongs to the technical field of microbial genetic engineering. According to the invention, natural metabolic phlorizin or trilobatin pathway genes in plants are expressed in the brewer's yeast engineering bacteria for producing phlorizin, so that the phlorizin or trilobatin is synthesized from the beginning in the yeast; the expression of a key gene in a phlorizin or trilobatin synthesis pathway is further enhanced; the yield of phlorizin or trilobatin is improved. The yield of phlorizin and the yield of trilobatin synthesized by the engineering yeast from the beginning are 340.41 ml / mL and 269.73 ml / mL respectively, the synthesis problem of phlorizin and trilobatin serving as a hypoglycemic agent and a sweetening agent is effectively solved, and a foundation is laid for synthesizing phlorizin and trilobatin through efficient fermentation of microorganisms.

Description

Technical Field

[0001] The invention relates to a method for brewer's yeast to synthesize glucose transport inhibitors phlorizin or trilobatin, belonging to the technical field of microbial genetic engineering. Background Art

[0002] Phlorizin and trilobatin are dihydrochalcone glycosides, which are important sweeteners and anti-sugar agents. Phlorizin, which was first identified in apples in 1835, is the first dihydrochalcone glycoside. It regulates glucose metabolism and lowers blood sugar by inhibiting glucose transporters SGLT1 and SGLT2 involved in intestinal glucose absorption and renal glucose reabsorption, and has an important influence on the development of many synthetic anti-diabetic drugs. Trilobatin is an isomer of phlorizin. By activating the Nrf2 / ARE signaling pathway, it can effectively improve the abnormal signal conduction of Type II diabetes. It also has outstanding effects in anti-oxidation and Alzheimer's treatment, and has good prospects for food and medical use.

[0003] At present, the production methods of phlorizin and trilobatin are mainly plant extraction and chemical synthesis. Since phlorizin and trilobatin are mainly present in the roots and leaves of some plants, the natural extraction yield is low and the purification is complicated, which is limited by space and time to a certain extent, affecting the process of industrial production. Chemical synthesis is not only costly, but also pollutes the environment. Therefore, it is necessary to develop new methods for synthesizing plant natural products by microorganisms. Summary of the invention

[0004] The present invention provides a method for producing phlorizin or trilobatin using Saccharomyces cerevisiae. The method comprises the following steps: by editing the gene of Saccharomyces cerevisiae, introducing the relevant metabolic pathways for phlorizin synthesis, and expressing exogenous glycosyltransferase, the constructed Saccharomyces cerevisiae engineered bacteria can produce phlorizin or trilobatin using glucose as a substrate.

[0005] The invention provides an engineered yeast of cerevisiae, which overexpresses 2'-O-glucosyltransferase genes MdUTG and PcUGT, or overexpresses 4'-O-glucosyltransferase AtUGT on the basis of a starting strain; the starting strain is a cerevisiae with the ability to synthesize phloretin.

[0006] In one embodiment, the overexpression comprises integrated expression and / or plasmid expression.

[0007] In one embodiment, the nucleotide sequence of the 2'-O-glucosyltransferase gene MdUTG is shown as SEQ ID NO.19; the nucleotide sequence of the 2'-O-glucosyltransferase gene PcUGT is shown as SEQ ID NO.20; and the gene sequence encoding the 4'-O-glucosyltransferase AtUGT is shown as SEQ ID NO.21.

[0008] In one embodiment, the starting strain is a strain that integrates and expresses the tyrosine ammonia lyase gene FjTAL, coumaroyl-CoA ligase gene At4CL, chalcone synthase gene HaCHS, enoyl-CoA reductase ScTSC13, phenylalanine ammonia lyase gene AtPAL2, cinnamate hydroxylase gene AtC4H, cytochrome P450 reductase gene AtATR2, and cytochrome B5 gene CYB5 in the genome of Saccharomyces cerevisiae BY4741.

[0009] In one embodiment, the starting strain also strengthens the tyrosine and phenylalanine synthesis pathway genes, including: ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL and Bbxfpk opt

[0010] In one embodiment, the promoter P THD3 Regulate the expression of the gene MdUTG, using the promoter P TEF1 Regulate the expression of the gene PcUTG.

[0011] In one embodiment, the gene integration operation of Saccharomyces cerevisiae is achieved by Crispr / Cas9 technology.

[0012] In one embodiment, the genes FjTAL, At4CL, HaCHS, TSC13, AtPAL2, AtC4H, AtATR2, CYB5, ARO4 K229L , ARO7 G141S ,TYR1,ARO1,ARO2,ARO3,ARO8,PHA2,EcAROL,Bbxfpk opt The nucleotide sequences of MdUTG and PcUTG are shown in SEQ ID NO.1 to SEQ ID NO.20, respectively.

[0013] In one embodiment, a strong constitutive promoter P THD3 Regulate the expression of the gene AtUTG.

[0014] In one embodiment, plasmid pCEV-G1-Ph is used to express phloretin 2'-O-glucosyltransferase gene MdUTG and phloretin 2'-O-glucosyltransferase gene PcUTG; the constructed expression vector is named pCEV-UU (pCEV-Km-MdUTG-PcUTG).

[0015] In one embodiment, the plasmid pCEV-G1-Km is used to express the genes FjTAL, At4CL, HaCHS and TSC13, and the constructed expression vector is named pCEV-TTCC (pCEV-Km-FjTAL-At4CL-TSC13-HaCHS)

[0016] The present invention also provides a method for increasing the yield of phlorizin or trilobatin in Saccharomyces cerevisiae, which comprises expressing the gene of (a) or (b) in Saccharomyces cerevisiae;

[0017] (a) 2'-O-glucosyltransferase genes MdUTG and PcUGT,

[0018] (b) 4'-O-glucosyltransferase AtUGT.

[0019] In one embodiment, the method is to transform pCEV-UU (pCEV-Km-MdUTG-PcUTG) and pCEV-TTCC (pCEV-Km-FjTAL-At4CL-TSC13-HaCHS) into the starting strain.

[0020] In one embodiment, the starting strain is a genetically engineered strain Y-11 in which the genes of tyrosine and phenylalanine synthesis pathways are integrated and expressed in the genome of Saccharomyces cerevisiae BY4741, having "△PDC5, △ARO10, XI-2::T ENO2 -TSC13-P FBA1 -P THD3 -FjTAL-T CYC1 -T ADH1 -At4CL-P TEF -P PGK1 -HaCHS-T HIS , XI-4::CYB5-T ENO2 -AtPAL2-P FBA1 -P THD3 -AtC4H-T CYC1 -T ADH1 -AtATR2-P TEF -P PGK1 -CYB5-T HIS3 , XII-2::P TEF1 -ARO4 K229L -T CYC1 -T ADH1 -ARO7 G141S -P ccw14 -P PGK1 -ARO1-T CYC1 -T ADH1 -ARO2-P HXT7 -P FBA1-ARO3-T CYC1 , XII-4::T CYC1 -ARO8-P THD3 -P TEF1 -TYR1-T ADH1 -T CYC1 -EcAroL-P PGK1 -P ccw14 -Bbxfpk-T ADH1 -P FBA1 -PHA2-T CYC1 ” improvements.

[0021] The invention also provides a method for producing trilobatin or phlorizin by fermentation using the engineered yeast of saccharomyces cerevisiae.

[0022] In one embodiment, the method is to culture the engineered yeast Saccharomyces cerevisiae in a medium containing glucose for a period of time, and collect trilobatin or phlorizin in the fermentation broth.

[0023] In one embodiment, the seed solution of the engineered yeast Saccharomyces cerevisiae is inoculated into the fermentation medium at an inoculation rate of 0.5-1%, and the initial OD value of the fermentation is controlled. 600 =0.05~0.1, at 28~32℃, 180-300rpm, ferment for 72~120h.

[0024] In one embodiment, the seed solution is prepared by culturing in YPD medium at 28-32°C, 180-240 rpm until the initial OD 600 =5~10, as seed liquid.

[0025] In one embodiment, the fermentation medium is YPD medium.

[0026] The present invention also provides application of the engineered yeast Saccharomyces cerevisiae in preparing phlorizin or products containing phlorizin.

[0027] The present invention also provides application of the engineered yeast Saccharomyces cerevisiae in preparing trilobatin or products containing trilobatin.

[0028] Beneficial effects:

[0029] (1) The present invention overexpresses 2'-O-glucosyltransferase genes MdUTG and PcUGT in a strain capable of synthesizing phlorizin, thereby achieving the production of phlorizin in microorganisms, with the yield reaching 198.21 ml / mL; the present invention further increases the phlorizin yield to 340.41 mg / mL by overexpressing genes related to the phlorizin synthesis pathway through a plasmid.

[0030] (2) The present invention integrates the 4'-O-glucosyltransferase gene AtUTG into the genome of Saccharomyces cerevisiae that has the ability to synthesize phloretin, thereby achieving the de novo synthesis of trilobatin with a yield of 143.22 ml / mL. The present invention also overexpresses genes related to the phloretin synthesis pathway through a plasmid, thereby increasing the trilobatin yield to 269.73 ml / mL.

[0031] (3) The present invention realizes the preparation of phlorizin and trilobatin using glucose as a precursor by microbial fermentation. The fermentation cost is low and the process is simple. It provides a new idea and method for the efficient production of phlorizin and trilobatin by microorganisms, and provides a reference for the synthesis and industrial production of dihydrochalcone glycosides in cell factories. It is of great significance to the development of the green synthetic biology industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention provides a gene map of an engineered strain of Saccharomyces cerevisiae BY4741 that can produce phlorizin or trilobatin after iterative genetic modification.

[0033] Figure 2 It is the yield of phloretin, phloretin and trilobatin synthesized by all the engineered yeasts of the present invention using glucose as a substrate. DETAILED DESCRIPTION

[0034] (I) Biochemical materials

[0035] Saccharomyces cerevisiae BY4741 (our laboratory); phloretin, phloretin and trilobatin standards (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, DNA 5000ladder and DNA 10000ladder (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 and phosphotransketolase gene Bbxfpk in Bifidobacterium breve opt Codon optimization and synthesis (Suzhou Jinweizhi Co., Ltd.).

[0036] (ii) Culture medium:

[0037] YPD liquid medium: glucose 20 g / L, trypsin 20 g / L, yeast extract 10 g / L, sterilized at 115°C for 30 min.

[0038] 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.

[0039] LB liquid medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, sterilized at 120°C for 20 min.

[0040] 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.

[0041] SD-Ura+G418 agar medium configuration: yeast nitrogen base without amino acids 1.7 g / L, anhydrous sodium glutamate 5 g / L, Ura-deficient yeast amino acid premix powder 1.31 g / L, glucose 20 g / L and agar powder 15 g / L, Geneticin G418 500 mg / L; sterilize at 115°C for 30 min.

[0042] (iii) Strains, plasmids, primers and promoter sequences.

[0043] Table 1 Strains and their genotypes

[0044]

[0045] Table 2 Plasmids and their genotypes

[0046]

[0047]

[0048] Table 3 Primers

[0049]

[0050] Table 4 Promoter sequence

[0051]

[0052]

[0053] (IV) Basic operations of molecular biology:

[0054] (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.

[0055] (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.

[0056] Table 5 PCR reaction system

[0057]

[0058]

[0059] Table 6 PCR reaction program

[0060]

[0061] (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.

[0062] (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.

[0063] (5) Preparation of competent cells of Saccharomyces cerevisiae and chemical transformation:

[0064] 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.

[0065] 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.

[0066] 3) Pour off the supernatant, add 30 μL of 1×LiAC solution to the centrifuge tube, and resuspend by slight pipetting; let stand for 10 minutes. Prepare a mixed solution of 10×LiAc, 10×TE, and 50% PEG3350 in a ratio of 1:1:8.

[0067] 4) Add plasmid or DNA fragment to the bacterial solution and gently shake to mix.

[0068] 5) Add 250 μL of a mixed solution of 10×LiAc, 10×TE, and 50% PEG3350, and mix by pipetting.

[0069] 6) After mixing, place the centrifuge tube in a constant temperature water bath and incubate at 30°C for 30 minutes.

[0070] 7) After incubation, add 30 μL of dimethyl sulfoxide (DMSO) and mix by gently inverting the tube up and down 7 times.

[0071] 8) Heat shock the centrifuge tube in a constant temperature water bath at 42°C for 7 min.

[0072] 9) After heat shock, centrifuge at 4000 rpm for 1 min at 4°C.

[0073] 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.

[0074] 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.

[0075] (V) Detection methods of phlorizin and trilobatin:

[0076] Phloretin, phloretin and trilobatin in the supernatant of the fermentation broth were 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 aqueous membrane, and then detected using 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 A was 0.1% formic acid and B was 100% acetonitrile. 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.

[0077] Example 1: Genetically engineered wild-type Saccharomyces cerevisiae BY4741 and tested for its de novo synthesis of phlorizin

[0078] The Crispr / Cas9 technology was used to integrate the genes ScTSC13, FjTAL, At4CL, and HaCHS into the XI-2 locus of the Saccharomyces cerevisiae BY4741 genome, and the genes CYB5, AtPAL2, AtC4H, and AtATR2 were integrated into the XI-4 locus of the Saccharomyces cerevisiae BY4741 genome, and the gene ARO4 K229L 、ARO7 G141S , ARO1, ARO2, and ARO3 were integrated into the XII-2 locus of the Saccharomyces cerevisiae BY4741 genome, and genes ARO8, TYR1, PHA2, EcAROL, and Bbxfpk opt The gene was integrated into the XII-4 site of the chromosome of Saccharomyces cerevisiae BY4741 to obtain the yeast engineered strain Y-11.

[0079] The engineered yeast Y-11 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 phloretin exogenous pathway genes and the optimization of amino acid biosynthesis metabolic pathways, phloretin could be detected in the fermentation broth with a yield of 170.37 mg / mL.

[0080] Example 2: Genetically engineered Saccharomyces cerevisiae and tested its yield of de novo phlorizin synthesis

[0081] The genes MdUGT, PcUGT and the corresponding promoters and terminators were cloned into the vector pCEV-G1-Ph by seamless cloning to obtain the plasmid pCEV-UU. The gene fragment UU (T CYC1 -MdUTG-P TDH3 -P TEF1 -PcUTG-T ADH1 ), and a high-purity fragment UU was obtained by gel electrophoresis verification and gel recovery. The fragment UU was integrated into the genome X-2 site of the engineered yeast strain Y-11 constructed in Example 1 by Crispr / Cas9 technology to obtain the engineered yeast strain Y-12.

[0082] The engineered yeast Y-12 was fermented in YPD medium at 30°C for 72 hours, and the phloridzin production in the fermentation supernatant was determined using an HPLC-UV system. Figure 2 As shown, through the chromosomal integration of the phlorizin exogenous pathway gene, phlorizin could be detected in the fermentation broth with a yield of 198.2 mg / mL.

[0083] Example 3: Overexpression of glycosyltransferase gene in engineered yeast Saccharomyces cerevisiae to increase the yield of phloridzin, a glycosylated product of phloridzin

[0084] Genes FjTAL, At4CL, HaCHS, TSC13 and the corresponding promoters and terminators (T ENO2 -TSC13-P FBA1 -P THD3 -FjTAL-T CYC1 -T ADH1 -At4CL-P TEF -P PGK1 -HaCHS-T HIS ) was cloned into the vector pCEV-G1-Km by seamless cloning to obtain plasmid pCEV-TTCC; plasmid pCEV-TTCC and plasmid pCEV-UU constructed in Example 2 were transformed into yeast engineering strain Y-12 constructed in Example 2 to obtain yeast engineering strain Y-14, and Y-14 was fermented at 30°C for 72 hours in YPD medium supplemented with a final concentration of 200ug / mL geneticin and 40ug / mL, and the phlorizin production in the fermentation broth supernatant was determined by HPLC-UV system. The results are shown in Figure 2 As shown, by enhancing the expression of exogenous rate-limiting enzymes MdUGT and PcUGT, the yield of phloridzin was increased compared with Y-12, reaching 340.41 mg / mL.

[0085] Example 4: Genetically engineering Saccharomyces cerevisiae and detecting its yield of de novo synthesis of trilobatin

[0086] The gene AtUGT and the corresponding promoter P TDH3 and terminator T CYC1 The plasmid pCEV-U was obtained by seamless cloning into the vector pCEV-G1-Ph. The gene fragment U (T CYC1 -AtUTG-P TDH1 ), and a high-purity fragment U was obtained by gel electrophoresis verification and gel recovery. Fragment U was integrated into the genome of the engineered yeast strain Y-11 constructed in Example 2 by Crispr / Cas9 technology to obtain the engineered yeast strain Y-13, with a genotype as shown in FIG. Figure 1 Integration at the X-4 site is shown.

[0087] The engineered yeast Y-13 was fermented in YPD medium at 30°C for 72 hours, and the trilobatin production in the fermentation supernatant was determined using an HPLC-UV system. Figure 2 As shown, through the chromosomal integration of the trilobatin exogenous pathway gene, trilobatin could be detected in the fermentation broth with a yield of 143.22 mg / mL.

[0088] Example 5: Overexpression of glycosyltransferase gene in engineered yeast Saccharomyces cerevisiae to increase the yield of trilobatin, a glycosylated product of phloretin

[0089] The plasmid pCEV-TTCC constructed in Example 3 and the plasmid pCEV-UU constructed in Example 2 were co-transformed into the yeast engineering strain Y-13 constructed in Example 3 to obtain the yeast engineering strain Y-15. The strain Y-15 was fermented at 30°C for 72 hours in a YPD medium supplemented with a final concentration of 200 ug / mL geneticin and 40 ug / mL, and the trilobatin 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 enzyme AtUGT, the yield of trilobatin was increased compared with Y-13, reaching 269.73 mg / mL.

[0090] 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 strain of Saccharomyces cerevisiae, characterized in that: On the basis of the starting strain, 2'-O-glucosyltransferase genes MdUTG and PcUGT are overexpressed, or 4'-O-glucosyltransferase AtUGT is overexpressed; the starting strain is Saccharomyces cerevisiae with the ability to synthesize phloretin.

2. The brewer's yeast engineering bacterium according to claim 1, characterized in that The starting strain is an engineered bacterium obtained by integrating and expressing the tyrosine ammonia lyase gene FjTAL, coumaroyl-CoA ligase gene At4CL, chalcone synthase gene HaCHS, enoyl-CoA reductase ScTSC13, phenylalanine ammonia lyase gene AtPAL2, cinnamate hydroxylase gene AtC4H, cytochrome P450 reductase gene AtATR2, and cytochrome B5 gene CYB5 in the genome of Saccharomyces cerevisiae BY4741.

3. The engineered yeast of claim 1 or 2, characterized in that: The starting strain also strengthens the tyrosine and phenylalanine synthesis pathway genes, including: ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL and Bbxfpk opt .

4. The engineered yeast of any one of claims 1 to 3, characterized in that: With promoter P THD3 Regulate the expression of the gene MdUTG, using the promoter P TEF1 Regulate the expression of the gene PcUTG.

5. A method for increasing the yield of phlorizin in Saccharomyces cerevisiae, characterized in that: The 2'-O-glucosyltransferase genes MdUTG and PcUGT were expressed in Saccharomyces cerevisiae capable of synthesizing phloretin.

6. A method for increasing the yield of trilobatin in Saccharomyces cerevisiae, characterized in that: The 4'-O-glucosyltransferase AtUGT was expressed in Saccharomyces cerevisiae capable of synthesizing phloretin.

7. The method according to claim 5 or 5, characterized in that: The cerevisiae yeast having the ability to synthesize phloretin is obtained by integrating and expressing the genes (a) and / or (b) in the genome of cerevisiae BY4741: (a) Tyrosine ammonia lyase gene FjTAL, coumaroyl-CoA ligase gene At4CL, chalcone synthase gene HaCHS, enoyl-CoA reductase gene ScTSC13, phenylalanine ammonia lyase gene AtPAL2, cinnamate hydroxylase gene AtC4H, cytochrome P450 reductase gene AtATR2, cytochrome B5 gene CYB5; (b) Tyrosine and phenylalanine biosynthesis pathway gene ARO4 K229L , ARO7 G141S , TYR1, ARO1, ARO2, ARO3, ARO8, PHA2, EcAROL and Bbxfpk opt .

8. A method for producing trilobatin or phlorizin by fermentation, characterized in that: The engineered yeast of any one of 1 to 4 is cultured in a medium containing glucose for a period of time, and trilobatin or phlorizin in the fermentation broth is collected.

9. Use of the engineered yeast of Saccharomyces cerevisiae according to any one of claims 1 to 4, or the method according to any one of claims 6 to 8 in the preparation of phlorizin or a product containing phlorizin.

10. Use of the engineered yeast of any one of claims 1 to 4, or the method of any one of claims 6 to 8 in the preparation of trilobatin or a product containing trilobatin.

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