CN111849794B - A kind of Saccharomyces cerevisiae recombinant bacteria and its construction method and application - Google Patents

Abstract

The invention discloses a Saccharomyces cerevisiae recombinant bacterium, which integrates RgTAL gene, HpaB gene and HpaC gene in the genome. The invention also discloses the construction method and application of Saccharomyces cerevisiae recombinant bacteria. Finally, the invention also discloses a production method of caffeic acid. The present invention integrates three exogenous genes required for caffeic acid synthesis into the chromosome of Saccharomyces cerevisiae, and through the knockout of competition pathway genes, the elimination of feedback inhibition steps, and the overexpression of rate-limiting enzymes, the transition from glucose to For the total synthesis of caffeic acid, the shake flask yield reached about 760 mg/L, which is the highest level of yeast yield reported so far, which provides a new method for the industrial production of caffeic acid.

Description

A kind of Saccharomyces cerevisiae recombinant bacteria and its construction method and application

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a Saccharomyces cerevisiae recombinant bacterium and a construction method and application thereof.

Background technique

Caffeic acid is a secondary metabolite widely distributed in plants. It not only has biological activities such as antibacterial and disease resistance, but also is an important raw material and intermediate for medicine. At present, the main sources of caffeic acid are chemical synthesis and plant extraction. The chemical synthesis of caffeic acid has problems such as many synthesis steps, complex process, and unfriendly environment. However, the yield of plant extraction is low and the price is expensive. With the development of synthetic biology technology, the use of microbial fermentation to produce caffeic acid has become an important option.

The existing patent (CN 108949652 A) reported that the production of caffeic acid by glucose fermentation was achieved by constructing Escherichia coli genetic engineering bacteria. This method expresses L-lactate dehydrogenase in the Escherichia coli engineering bacteria, so that NAD in the bacteria is used as a coenzyme to convert caffeic acid. L-lactate is dehydrogenated to generate pyruvate and NADH, and the exogenously expressed tyrosine phenol lyase further catalyzes pyruvate, ammonia ions, and catechol to generate L-dopa, and L-dopa is introduced into tyrosine Ammonia lyase catalyzes the production of caffeic acid. In this method, the precursor substances lactic acid and catechol need to be added. Therefore, the cost is high and it is not economical. In addition, Escherichia coli is a non-food-safe strain and can produce endotoxin by itself, which is not conducive to the production of caffeic acid. As a biosafety strain, Saccharomyces cerevisiae has a clear genetic background and mature high-density fermentation technology, making it an ideal chassis cell for natural product synthesis. At present, a few literatures have achieved the production of caffeic acid by transforming Saccharomyces cerevisiae (ACS Synthetic Biology 2020, 9: 756-765; Engineering 2019, 5: 287-295), but the yield is relatively low, the highest only reaching 289.4 mg/L, And need to rely on the addition of the precursor L-tyrosine. Insufficient supply of precursors and low catalytic efficiency of exogenous pathway enzymes are the main reasons for limiting caffeic acid synthesis.

SUMMARY OF THE INVENTION

Purpose of the invention: In order to realize the construction of the above-mentioned caffeic acid production engineering strain, the starting strain involved in the present invention is YXWP-113 (from the research group of Professor Yu Hongwei of Zhejiang University, this strain is GAL80 by knocking out wild-type Saccharomyces cerevisiae BY4741. Obtained by gene construction, Metabolic Engineering, 2015, 30: 69-78), the technical problem to be solved by the present invention is to provide a recombinant Saccharomyces cerevisiae that produces caffeic acid at low cost.

The technical problem to be solved by the present invention is to provide a method for constructing the recombinant Saccharomyces cerevisiae.

The technical problem to be solved by the present invention is to provide the application of the Saccharomyces cerevisiae recombinant bacteria.

The final technical problem to be solved by the present invention is to provide a production method of caffeic acid.

In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is as follows: a recombinant Saccharomyces cerevisiae, the recombinant Saccharomyces cerevisiae has integrated the RgTAL gene, the HpaB gene and the HpaC gene in the genome.

Wherein, the base sequence of the RgTAL gene is shown in SEQ ID NO: 1, the base sequence of the HpaB gene is shown in SEQ ID NO: 2, and the base sequence of the HpaC gene is shown in SEQ ID NO: 3 shown.

The amino acid sequence corresponding to the RgTAL gene is shown in SEQ ID NO: 5, the amino acid sequence corresponding to the HpaB gene is shown in SEQ ID NO: 6, and the amino acid sequence corresponding to the HpaC gene is shown in SEQ ID NO: 7 shown.

Wherein, in order to improve the caffeic acid production of the recombinant strain of Saccharomyces cerevisiae, the recombinant strain of Saccharomyces cerevisiae also includes knocking out one or both of the Aro3 and Aro10 genes on the chromosome of Saccharomyces cerevisiae; and/or overexpression is not inhibited by tyrosine feedback One or a combination of the mutant Aro4 ^K229L and Aro7 ^G141S genes; and/or overexpressing the Zymomonas mobilis-derived prephenate dehydrogenase TyrC.

The above focus is to optimize the caffeic acid pathway in Saccharomyces cerevisiae and enhance the caffeic acid synthesis ability of Saccharomyces cerevisiae.

Wherein, the gene sequence encoding the prephenate dehydrogenase TyrC is shown in SEQ ID NO: 4, and the corresponding amino acid sequence thereof is shown in SEQ ID NO: 8. The TyrC gene of the present invention is obtained by whole gene synthesis after optimization according to the codon preference of Saccharomyces cerevisiae.

The content of the present invention also includes the construction method of described Saccharomyces cerevisiae recombinant bacteria, and the construction method of described recombinant bacteria comprises the following steps:

1) Obtaining the recombinant vectors pUMRI-13-HpaB-HpaC and pUMRI-11-RgTAL plasmids;

2) The recombinant vector pUMRI-13-HpaB-HpaC was introduced into Saccharomyces cerevisiae to obtain YCA113-1B;

2) The pUMRI-11-RgTAL plasmid obtained in step 1) was introduced into YCA113-1B to obtain a recombinant strain of Saccharomyces cerevisiae.

Wherein, the construction method of the recombinant bacteria includes, in addition to the construction method, knocking out the Aro3 and/or Aro10 genes on the chromosome of the obtained Saccharomyces cerevisiae recombinant bacteria; and/or overexpression of genes that are not inhibited by tyrosine feedback One or a combination of the mutant Aro4K229L and Aro7G141S genes; and/or overexpressing the Zymomonas mobilis-derived prephenate dehydrogenase TyrC.

The content of the present invention also includes the application of the recombinant Saccharomyces cerevisiae in the production of caffeic acid.

The content of the present invention also includes a method for producing caffeic acid, which comprises fermenting and culturing the recombinant Saccharomyces cerevisiae.

The present invention is based on the Saccharomyces cerevisiae aromatic amino acid pathway, and integrates three exogenous genes required for the synthesis of caffeic acid: tyrosine ammonia lyase gene, 4-hydroxyphenylacetic acid-3-monooxygenase (HpaB) gene and NADPH-flavin oxidoreductase (HpaC) gene, and regulated the steps of limiting the synthesis of precursors in cells, so as to obtain a stable and high-yielding Saccharomyces cerevisiae engineering bacteria, which realizes the one-step synthesis of glucose to caffeic acid without the need for Adding other exogenous precursor substances provides a reference for the green production of caffeic acid and a basis for industrial fermentation production.

Beneficial effects: the present invention integrates three exogenous genes required for the synthesis of caffeic acid into the chromosome of Saccharomyces cerevisiae, and through the knockout of the competitive pathway gene, the elimination of the feedback inhibition step, and the overexpression of the rate-limiting enzyme. The constructed pathway is shown in Figure 1), which realizes the total synthesis from glucose to caffeic acid, and the yield of the shake flask reaches about 760 mg/L, which is the highest level of yeast yield in the current report, and provides a new way for the industrial production of caffeic acid. Methods.

Description of drawings

Fig. 1 is a schematic diagram of the de novo biosynthesis of caffeic acid constructed by the present invention.

Figure 2. Liquid chromatograms of caffeic acid standards and fermentation products.

Figure 3. Gel electrophoresis of PCR products of RgTAL, HpaB and HpaC genes.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

The culture medium and storage solution used in the examples:

Luria-Bertani (LB) medium: purchased from Shanghai Bioengineering Co., Ltd., sterilized at 115° C. for 21 min.

Yeast Extract Peptone Dextrose (YPD) medium: 10g/L yeast extract powder, 20g/L peptone, 20g/L glucose, solid YPD medium with 1.5-2% agar powder, sterilized at 115°C for 21min.

Kanamycin stock solution (50 mg/mL): Dissolve 0.5 g of kanamycin in 10 mL of ddH ₂ O, sterilize by filtration, store at -20°C, and dilute 1000 times to make the final concentration 50 μg/mL when used.

Geneticin (G418) stock solution (20 mg/mL): Dissolve 0.2 g of G418 in 10 mL of ddH2O, filter sterilize, store at -20°C, and dilute 100 times to make the final concentration 200 μg/mL when used.

5-fluoroorotic acid (FOA) stock solution (100mg/mL): dissolve 0.1g of 5-FOA in 1mL of dimethyl sulfoxide, and directly add 1mL of mother liquor to 100mL of SD solid medium for use. Make SD-FOA plates.

10×YNB stock solution: Weigh 1.7% YNB and 5% (NH ₄ ) ₂ SO ₄ and dissolve in ddH ₂ O, filter and sterilize with a 0.22 μm sterile needle filter, store in a refrigerator at 4°C, and dilute before use 10 times.

10× Amino Acid Mixed Storage Solution: Weigh various amino acids and mix them in ddH ₂ O according to the following formula. The concentrations are: L-Adenine Sulfate 200mg/L, L-Arginine 200mg/L, L-Histidine Acid 200mg/L, L-Isoleucine 300 mg/L, L-Leucine 1000mg/L, L-Lysine 300mg/L, L-Methionine 200mg/L, L-Phenylalanine 500mg/L L, L-threonine 2000mg/L, L-tryptophan 200mg/L, L-tyrosine 300mg/L, L-uracil 200mg/L, L-valine 1500mg/L (Note: the preparation of the above When amino acid stock solution, the corresponding amino acid is deleted according to the difference of nutritional screening). Filter sterilize with a 0.22 μm sterile syringe filter and store in a refrigerator at 4°C. Dilute 10 times when using.

Synthetic Defined (SD) medium: 2% glucose, 10% (V/V) 10X YNB stock solution, 10% (V/V) 10X amino acid mixed stock solution. The specific process of configuring 100 mL SD medium is as follows: Dissolve 2 g of glucose in 80 mL of water, sterilize in 115 ℃ for 21 min by autoclaving, and after the medium is cooled to below 60 ℃, add 10 mL of 10×YNB mother liquor and 10 mL of 10× amino acid mixed mother liquor. Add 1.5-2% agar powder to solid SD medium. Wherein SD-URA- means SD medium deficient in uracil.

Examples of reagents used:

High-fidelity DNA polymerase (Prime STARTM HS DNA polymeras), DNA restriction endonuclease, and T4 DNA ligase were purchased from Dalian Bao Biological Company (Takara, Dalian); DNA marker (1kb DNA ladder) was purchased from Thermo Scientific; nucleic acid Electrophoresis-related reagents and yeast genome extraction kits were purchased from Shanghai Bioengineering Co., Ltd.; bacterial plasmid extraction kits, PCR product purification kits, and DNA gel purification kits were purchased from Hangzhou Axygen Company; amino-free yeast nitrogen base (Yeast nitrogen base without amino acids, YNB) was purchased from Shanghai Bioengineering Co., Ltd. for the preparation of synthetic medium; PCR primer synthesis and sequencing services were provided by Shanghai Bioengineering Co., Ltd. or Shanghai Boshang Biotechnology Co., Ltd.

The conventional technical method used in the embodiment:

E. coli competent preparation:

(1) Streak E.coli DH5α on the LB solid plate, and cultivate it overnight at 37 °C for about 15 hours; then pick a single colony and inoculate it into 5 mL of LB liquid medium, cultivate at 220 rpm and 37 °C overnight, and take 1 mL of the inoculum for transfer. It was then added to 100 mL of LB liquid medium, and cultured at 37 °C at 200 rpm to an OD ₆₀₀ of about 0.35-0.40.

(2) Dispense 25 mL of bacterial solution into a pre-cooled 50 mL centrifuge tube in each tube. After ice bathing for 10 min, centrifuge at 3000 rpm for 10 min at 4°C to collect the bacterial cells and discard the supernatant;

(3) Add 30 mL of pre-cooled CaCl ₂ -MgCl ₂ solution (80 mmol/L MgCl ₂ , 20 mmol/LCaCl ₂ ) to each tube of cells, resuspend the cells (operate on ice), and collect cells by low-temperature centrifugation at 3000 rpm for 10 min , discard the supernatant;

(4) Resuspend the cells with 2 mL of pre-cooled 0.1M CaCl ₂ -glycerol solution (containing 0.1 mol/L CaCl 2 and 15% glycerol), and after mixing, dispense 100 μL per tube into pre-cooled 1.5 mL centrifuge tube, stored at -80°C for later use.

Escherichia coli transformation method:

(1) Take out the E. coli competent cells from the -80°C refrigerator and thaw on ice.

(2) Add 10 μL of recombinant plasmid and place on ice for 20 min.

(3) Heat shock at 42°C for 90s, and immediately ice bath for 5min.

(4) Add 1 mL of LB and mix well, 37 shaker for recovery for 50 min.

(5) Centrifuge at 12,000 rpm for 1 min, remove the supernatant, leave 100 μL for resuspending, coat on LB plate containing the corresponding resistance, and culture at 37°C overnight.

Saccharomyces cerevisiae lithium acetate conversion method:

(1) Pick a single clone and inoculate it into a 5mL YPD test tube, inoculate at 30°C and 220rpm overnight, transfer 1mL of the inoculum to a 250mL conical flask containing 50mL YPD, incubate at 30°C, 220rpm for about 5h, and the OD ₆₀₀ is about 2 .

(2) Transfer the bacterial solution to a 50 mL sterilized centrifuge tube, centrifuge at 5000 × g for 5 min, and remove the supernatant.

(3) Add 30 mL of sterilized water to wash the cells, centrifuge at 5000 × g for 5 min, and then remove the supernatant.

(4) Add 800 μL of sterilized water to resuspend the bacterial cells, and after mixing, distribute 100 μL per tube into 1.5 mL EP tubes, centrifuge at 12,000 × g for 1 min, and remove the supernatant for later use.

(5) Add 240 μL of PEG MW3350 (50% w/v), 36 μL of 1.0 M lithium acetate, 30 μL of sterile water, and 50 μL of single-stranded DNA (Single-stranded carrier DNA, 2.0 mg/mL), 4 μL of the linearized plasmid fragment, and the rest with sterile water to a total volume of 360 μL, resuspend the cells, and mix well.

(6) Place the 1.5mL EP tube in a 42°C water bath for thermal shock for 40min.

(7) After the heat shock, centrifuge at 12,000 × g for 1 min, and remove the supernatant.

(8) Add 1 mL of YPD medium, mix the cells, place at 30°C, and recover with a shaker at 220 rpm for 1.5-2 hours.

(9) After the recovery of the cells, centrifuge at 12,000 × g for 1 min, remove the supernatant medium, wash the precipitate with 1 mL of sterilized purified water, centrifuge at 12,000 × g for 1 min, remove the supernatant, and finally add 1 mL of sterilized purified water to reselect the cells , 15μL was spread on the corresponding G418 plate, and cultured in a 30 ℃ incubator for 3 days.

Saccharomyces cerevisiae culture method:

Pick a single clone from the agar plate, inoculate it into a 5 mL fresh YPD test tube, and culture it in a constant temperature shaker at 30°C and 220 rpm for about 15 hours overnight. Then, it was transferred to a 250 mL Erlenmeyer flask containing 50 mL of YPD medium, the initial OD ₆₀₀ in the shake flask was 0.05, and it was placed in a constant temperature shaker at 30 °C and 220 rpm for 72 h.

Example 1 Construction of recombinant plasmids required for caffeic acid synthesis

First, the genome of Salmonella enteritidis C50336 (gifted by Mr. Gu Dan, School of Bioscience and Technology, Yangzhou University) was used as the template, and the primers were HpaC-F(EcoR I)(GCGGAATTCATGCAAGTAGATG AACAACGT) and HpaC-R(Bgl II)(CCTTAGATCTTTAAACAGGCGCTTCCATC TC) , the HpaC gene was amplified (Fig. 3), and then double digested with EcoR I and Bgl II, and the obtained fragment was ligated with the pUMRI-13 (GenBank: KM216415.1) plasmid that was double digested with the same EcoR I and Bgl II. After transformation, the pUMRI-13-HpaC plasmid was constructed.

The PCR reaction system is as follows:

The PCR procedure is as follows:

The plasmid or gene fragment restriction enzyme digestion system is as follows:

37℃, digested for 2h. The digested products were separated by 1.0% agarose gel electrophoresis, and the gel with gene fragments was excised, and purified and recovered with a gel recovery kit.

The connection system is as follows:

22°C, enzymatic ligation for 50 min, the ligation product was transformed into E. coli competent, and placed in a 37°C incubator overnight to obtain positive clones for plasmid extraction.

The gene HpaB synthesized after codon optimization was used as a template, and HpaB-F (BamH I) (GCGGGATCCATGAAGCCAGAAGACTTCAG) and HpaB-R (Sal I) (GGAAGTCGACTTATTGTCTGATTCTGTCCA) were used to amplify the two ends containing BamH I and Sal I restriction sites. The HpaB gene (Fig. 3) was then double digested with BamH I and Sal I, and the obtained fragment was ligated and transformed with the pUMRI-13-HpaC plasmid double digested by BamH I and Xho I according to the above method, and the pUMRI-13 was constructed to obtain pUMRI-13 -HpaB-HpaC plasmid.

The gene RgTAL synthesized after codon optimization was used as a template, and RgTAL-F(BamH I)(GCGGGATCCATGGCTCCAAGACCAACTTC) and RgTAL-R(Sal I)(GGAAGTCGACTTAAGCCAACATCTTCAACA) were used as primers to amplify the restriction sites containing BamH I and Sal I at both ends. RgTAL gene (Figure 3), then double-enzyme digestion with BamH I and Sal I, and the obtained fragment was ligated with the pUMRI-11 plasmid (GenBank: KM216413.1) double-digested with BamH I and Xho I as described above. After transformation, the pUMRI-11-RgTAL plasmid was constructed.

The construction of embodiment 2 caffeic acid producing strain

The recombinant vector pUMRI-13-HpaB-HpaC contains Sfi I site, and the vector was linearized by Sfi I digestion method for the first time.

Enzyme digestion system: total system 50 μL, plasmid 43.5 μL, 10×Quick Cut Buffer 5 μL, Quick cut SfiI enzyme 1.5 μL.

Enzyme digestion conditions: place at 50°C for digestion for 2-3 hours.

The linearized plasmid pUMRI-13-HpaB-HpaC was introduced into Saccharomyces cerevisiae YXWP-113, and the positive recombinant yeast was obtained by screening the YPD plate containing G418 resistance, which was named YCA113-1B. Since pUMRI-13-HpaB-HpaC contains URA3 and KanMX (encoding geneticin G418 resistance in yeast) dual selectable markers, URA3 encodes orotidine 5-phosphate decarboxylase, this The enzyme catalyzes a key reaction in the synthesis of yeast RNA pyrimidine nucleotides. When 5-fluoroorotic acid (5-FOA) is added to the medium, the orotidine 5-phosphate decarboxylase of normal prototrophic yeast cells can convert 5-FOA to cell-killing 5-fluorouracil (5-fluorouracil). fluorouracil). Saccharomyces cerevisiae strains are subcultured in YPD liquid medium, homologous recombination can occur between the forward repeats at both ends of URA3 and KanMX, URA3 and KanMX genes will be naturally lost during this process, so they can be screened on SD-FOA plates clones with lost resistance. Then, the selected clones were spotted on YPD plates without G418 resistance and YPD plates with G418 resistance respectively for verification, and the selected resistant strains were removed for the next round of gene integration. Further, pUMRI-11-ORgTAL was linearized with Sfi I in the same manner as above, and integrated into recombinant Saccharomyces cerevisiae YCA113-1B to obtain a caffeic acid-producing strain YCA113-2B.

Example 3 Analysis and detection of caffeic acid yield

The starting strain YXWP-113 and the engineering strain YCA113-2B were cultured in a YPD shake flask for 72 hours according to the Saccharomyces cerevisiae culture method. After the culture, 2 mL of the fermentation broth was placed in a 2 mL EP tube, centrifuged at 12,000 × g for 1 minute, and the supernatant was removed. Add 2 mL of purified water to wash the cells, and centrifuge at 12,000 × g for 1 minute. After removing the supernatant, place the centrifuge tube in a 100°C oven to dry to constant weight for dry weight measurement.

And follow the steps below for sample processing and yield analysis:

(1) Transfer 1 mL of fermentation broth to a 1.5 mL EP tube, and centrifuge at 12,000 × g for 1 min at room temperature.

(2) Take 50 μL of supernatant into a new 1.5 mL EP tube, add 950 μL of purified water to dilute 20 times.

(3) Filter the sample with a 0.22 μm water-based needle filter for the determination of caffeic acid.

Quantitative analysis of caffeic acid: The samples were analyzed using an Agilent 1200 high performance liquid chromatograph. The chromatographic conditions are: mobile phase A pump is 0.1% formic acid aqueous solution, B pump is 100% acetonitrile, and the gradient elution method is as follows:

QS-C18 Plus色谱柱(4.6×250mm，5μm)，流速1mL/min、柱温35℃、进样量20μL，检测波长320nm。结果如图2所示，在YCA113-2B发酵液中成功检测到了咖啡酸的积累，产量达到313.8mg/L。use

QS-C18 Plus chromatographic column (4.6×250mm, 5μm), flow rate 1mL/min, column temperature 35℃, injection volume 20μL, detection wavelength 320nm. The results are shown in Figure 2. The accumulation of caffeic acid was successfully detected in the YCA113-2B fermentation broth, and the yield reached 313.8 mg/L.

Example 4 Elimination of Feedback Inhibitory Step in Shikimic Acid Pathway to Improve Saccharomyces cerevisiae Caffeic Acid Production

In the precursor synthesis pathway of caffeic acid, two 3-deoxy-D-arabinose-hepturonic acid-7-phosphate synthases Aro3 and Aro4 in the shikimate pathway catalyze phosphoenolpyruvate (PEP) and Erythrose-4-phosphate (E4P) reacts to generate 3-deoxy-D-arabino-heptan-7-phosphate, an important precursor of caffeic acid, but these two enzymes are fed back by phenylalanine and tyrosine, respectively inhibition. In addition, the chorismate mutase Aro7 catalyzes the formation of prephenylpropionate from chorismate, which competes for the carbon flow of tryptophan biosynthesis and transfers it to the tyrosine biosynthesis pathway. However, the Aro7 enzyme activity is also inhibited by tyrosine feedback. If these feedback-inhibitory steps can be relieved, it is expected to increase the supply of caffeic acid precursors, which has great implications for caffeic acid production. In the previous stage, we obtained the Aro3 knockout plasmid pUMRI-ΔAro3 and pUMRI-ΔAro3-Aro4 ^K229L and pUMRI-ΔAro3-Aro4 ^K229L -Aro7 ^G141S plasmids with tyrosine feedback-insensitive mutant genes Aro4 ^K229L and Aro7 ^G141S (three Each plasmid was gifted by Professor Yu Hongwei from the School of Chemical Engineering and Bioengineering, Zhejiang University) (Shen Bin's research on the heterologous synthesis of vitamin E (tocotrienol) in Saccharomyces cerevisiae [D] Zhejiang University 2019).

According to Example 2, the pUMRI-ΔAro3 plasmid was linearized with Sfi I and introduced into the YCA113-2B strain. The constructed Saccharomyces cerevisiae was named YCA113-3B. The YCA113-3B strain with URA3 and KanMX tags removed was screened as in Example 2, The Sfi I-linearized pUMRI-ΔAro3-Aro4 ^K229L and pUMRI-ΔAro3-Aro4 ^K229L -Aro7 ^G141S plasmids were further integrated to obtain YCA113-4B and YCA113-5B strains, respectively. YCA113-2B, YCA113-3B, YCA113-4B, and YCA113-5B were cultured in shake flasks, and after completion, high-performance liquid chromatography was performed according to the method in Example 3, and the caffeic acid yield was determined. The results are shown in Table 1.

Table 1 shows the effect of eliminating tyrosine feedback inhibition step on caffeic acid production

Among them, YCA113-5B produced the highest caffeic acid, which was 1.76 times higher than that of YCA113-2B. Therefore, YCA113-5B was selected for further research.

Example 5 Knockout of competitive pathway and overexpression of rate-limiting enzyme improves caffeic acid production in Saccharomyces cerevisiae

In the caffeic acid synthesis pathway, the endogenous prephenate dehydrogenase Tyr1 of Saccharomyces cerevisiae catalyzes the production of 4-hydroxyphenylpyruvate (4-HPP), an important precursor of caffeic acid from prephenate, but in Saccharomyces cerevisiae it is affected by benzene Feedback inhibition by alanine thus limits 4-hydroxyphenylpyruvate synthesis. However, the prephenate dehydrogenase TyrC derived from Zymomonas mobilis is not subject to feedback inhibition by aromatic amino acids, so it can replace the function of Tyr1. In addition, the endogenous phenylpyruvate decarboxylase Aro10 in Saccharomyces cerevisiae competes with the caffeic acid synthesis pathway to convert 4-HPP into other by-products. Therefore, knocking out Aro10 gene fluid is expected to improve caffeic acid production. In the present invention, we obtained the Aro10 gene knockout plasmid pUMRI-ΔAro10 and the TyrC-expressing pUMRI-ΔAro10-TyrC plasmid (heterologous synthesis in S. Research on Vitamin E (Tocotrienols [D] Zhejiang University 2019). The linearized pUMRI-ΔAro10 and pUMRI-ΔAro10-TyrC plasmids of Sfi I were transformed into YCA113-5B strains with URA3 and KanMX tags removed respectively according to the method in Example 2, and the obtained Saccharomyces cerevisiae strains were named as YCA113-6B and YCA113-8B. The YCA113-6B and YCA113-8B strains were cultured in shake flasks according to the Saccharomyces cerevisiae culture method, and the production of caffeic acid was analyzed according to Example 3. The results are shown in Table 2:

Table 2 shows the effect of knockout Aro10 and overexpression of TyrC on caffeic acid production

By knocking out Aro10 and overexpressing TryC, the production of caffeic acid was significantly improved, reaching 769.3 mg/L, which is the highest level reported by yeast so far, laying the foundation for the industrial production of caffeic acid.

sequence listing

<110> Yangzhou University

<120> A Saccharomyces cerevisiae recombinant strain and its construction method and application

<160> 8

<170> SIPOSequenceListing 1.0

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<213> RgTAL gene (Artificial Sequence)

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caagttgaca tcgttgaaaa gatgttggct gctccaactg actctacttt ggaattggac 120

ggttactctt tgaacttggg tgacgttgtt tctgctgcta gaaagggtag accagttaga 180

gttaaggact ctgacgaaat cagatctaag atcgacaagt ctgttgaatt cttgagatct 240

caattgtcta tgtctgttta cggtgttact actggtttcg gtggttctgc tgacactaga 300

actgaagacg ctatctcgct ccaaaaggcg ttgttggaac accagctctg cggtgtcctc 360

ccatcttctt tcgactcttt cagattgggt agaggcctcg aaaacagctt gccattggaa 420

gttgttagag gtgctatgac tatcagagtt aactctttga ctagaggtca ctctgctgtt 480

agattggttg ttttggaagc tttgactaac ttcttgaacc acggtatcac tccaatcgtt 540

ccattgagag gtactatctc tgcttctggt gacttgtctc cattgtctta catcgctgct 600

gctatctctg gtcacccaga ctctaaggtt cacgttgttc acgaaggtaa ggaaaagatc 660

ttgtacgcta gagaagcgat ggcgttgttc aacctcgagc cagttgtcct cggtccaaag 720

gagggcttgg gcttggttaa cggtactgct gtttctgctt ctatggctac tctcgctctc 780

cacgacgctc atatgttgtc tttgttgtct caatctttga ctgctatgac tgttgaagct 840

atggttggtc acgctggttc tttccaccca ttcttgcacg acgttactag accacaccca 900

actcaaatcg aagttgctgg taacatcaga aagttgttgg aaggttctag attcgctgtt 960

caccacgaag aagaagttaa ggttaaggac gacgaaggta tcttgagaca agacagatac 1020

ccattgagaa cttctccaca gtggcttggt ccattggtaa gcgacttgat ccacgctcac 1080

gctgttttga ctatcgaagc tggtcaatct actactgaca acccattgat cgacgttgaa 1140

aacaagactt ctcaccacgg tggtaacttc caagctgctg ctgttgctaa cactatggaa 1200

aagactagat tgggtttggc tcaaatcggt aagttgaact tcactcaatt gactgaaatg 1260

ttgaacgctg gtatgaacag aggtttgcca tcttgtttgg ctgctgaaga cccatctttg 1320

tcttatcatt gtaagggtct cgacatcgct gcggctgctt acacttctga attgggtcac 1380

ttggctaacc cagttactac tcacgttcaa ccagctgaaa tggctaacca agctgttaac 1440

tctttggctt tgatctctgc tagaagaact actgaatcta acgacgttct cagcttgttg 1500

ctcgctactc acttgtactg tgttttgcaa gctatcgact tgagagctat cgaattcgaa 1560

ttcaagaagc aattcggtcc agctatcgtt tctttgatcg accaacactt cggttctgct 1620

atgactggtt ctaacttgag agacgaattg gttgaaaagg ttaacaagac tttggctaag 1680

agattggaac aaactaactc ttacgacttg gttccaagat ggcacgacgc tttctctttc 1740

gctgctggta ctgttgttga agttttgtct tctacttctt tgtctttggc tgctgttaac 1800

gcttggaagg ttgctgctgc tgaatctgct atctctttga ctagacaagt tcgtgaaaca 1860

ttctggtctg ctgcgtctac ttcttctcca gctttgtctt acttgtctcc aagaactcaa 1920

atcttgtacg ctttcgttag agaagaattg ggtgttaagg ctagaagagg tgacgttttc 1980

ttgggtaagc aagaagttac tatcggttct aacgtttcta agatctacga agctatcaag 2040

tctggtagaa tcaacaacgt tttgttgaag atgttggctt aa 2082

<210> 2

<211> 1563

<212> DNA

<213> HpaB gene (Artificial Sequence)

<400> 2

atgaagccag aagacttcag agcttctgct actagaccat tcactggtga agaatacttg 60

gcttctttga gagacgacag agaaatctac atctacggtg acagagttaa ggacgttact 120

tctcacccag ctttcagaaa cgctgctgct tctatggcta gattgtacga cgctttgcac 180

gacccacaat ctaaggaaaa gttgtgttgg gaaactgaca ctggtaacgg tggttacact 240

cacaagttct tcagatacgc tagatctgct gacgaattga gacaacaaag agacgctatc 300

gctgaatggt ctagattgac ttacggttgg atgggtagaa ctccagacta caaggctgct 360

ttcggttctg ctttgggtgc taacccaggt ttctacggta gattcgaaga caacgctaag 420

acttggtaca agagaatcca agaagcttgt ttgtacttga accacgctat cgttaaccca 480

ccaatcgaca gagacaagcc agttgaccaa gttaaggacg ttttcatctc tgttgacgaa 540

gaagttgacg gtggtatcgt tgtttctggt gctaaggttg ttgctactaa ctctgctttg 600

actcactaca acttcgttgg tcaaggttct gctcaattgt tgggtgacaa cactgacttc 660

gctttgatgt tcatcgctcc aatgaacact ccaggtatga agttgatctg tagaccatct 720

tacgaattgg ttgctggtat cgctggttct ccattcgact acccattgtc ttctagattc 780

gacgaaaacg acgctatctt ggttatggac aaggttttca tcccatggga aaacgttttg 840

atctacagag acttcgaaag atgtaagcaa tggttcccac aaggtggttt cggtagattg 900

ttcccaatgc aaggttgtac tagattggct gttaagttgg acttcatcac tggtgctttg 960

tacaaggctt tgcaatgtac tggttctttg gaattcagag gtgttcaagc tcaagttggt 1020

gaagttgttg cttggagaaa cttgttctgg tctttgactg acgctatgta cggtaacgct 1080

tctgaatggc acggtggtgc tttcttgcca tctgctgaag ctttgcaagc ttacagagtt 1140

ttggctccac aagcttaccc agaaatcaag aagactatcg aacaagttgt tgcttctggt 1200

ttgatctact tgccatctgg tgttagagac ttgcacaacc cacaattgga caagtacttg 1260

tctacttact gtagaggttc tggtggtatg ggtcacagag aaagaatcaa gatcttgaag 1320

ttgttgtggg acgctatcgg ttctgaattc ggtggtagac acgaattgta cgaaatcaac 1380

tacgctggtt ctcaagacga aatcagaatg caagctttga gacaagctat cggttctggt 1440

gctatgaagg gtatgttggg tatggttgaa caatgtatgg gtgactacga cgaaaacggt 1500

tggactgttc cacacttgca caacccagac gacatcaacg ttttggacag aatcagacaa 1560

taa 1563

<210> 3

<211> 513

<212> DNA

<213> HpaC gene (Artificial Sequence)

<400> 3

atgcaagtag atgaacaacg tctgcgtttt cgcgatgcga tggcaagtct ggcggcagcg 60

gtcaacatcg taaccacggc gggtcacgcc ggacgctgcg gtatcaccgc aacagcggtc 120

tgttccgtca ccgatacgcc gccctccgtg atggtatgta ttaatgccaa tagcgccatg 180

aaccccgtct ttcagggcaa cggcaagctg tgcattaatg tacttaacca tgagcaggag 240

ctgatggcgc gccactttgc cggtatgacg gggatggcga tggaagagcg ttttcaccag 300

ccatgttggc aaaacgggcc gctgggccag ccggtactta acggcgcgct ggccggtctt 360

gaaggcgaga tcagcgaggt acaaaccatt ggcacgcatc tggtgtatct ggtggcgatc 420

aaaaatatta ttcttagcca ggatgggcat ggcctgattt atttcaaacg ccgttttcat 480

ccggtcagac ttgagatgga agcgcctgtt taa 513

<210> 4

<211> 882

<212> DNA

<213> TyrC gene (Artificial Sequence)

<400> 4

atgacagttt ttaaacatat tgcaattatt ggtttgggtt tgattggttc ttcagcagct 60

agagctacta aagcatattg tccagatgtt acagtttctt tgtatgataa atctgaattt 120

gtttgtgata gggcaagggc tttgaattta ggtgataatg ttactgatga tattcaagat 180

gctgttagag aagctgattt agttttgttg tgtgttccag ttagagctat gggtattgtt 240

gcagcagcta tggctcctgc tttgaaaaaa gatgttatta tttgtgatac tggttctgtt 300

aaagtttcag ttattaaaac tttacaagat aatttaccta atcatattat tgttccttct 360

catccattag caggtacaga aaataatggt cctgatgcag gttttgcaga attgtttcaa 420

gatcatccag ttattttgac accagatgca catacacctg cacaagcaat tgcttattatt 480

gcagattatt gggaagaaat tggtggtagg attaatttaa tgtcagctga acatcatgat 540

catgttttag ctttgacttc acatttacca catgttattg catatcaatt aattggtatg 600

gtttctggtt atgaaaaaaa atctaggact ccaattatga ggtattcagc aggttctttt 660

agggatgcta ctagagttgc tgcatctgaa cctaggttgt ggcaagatat tatgttggaa 720

aatgctcctg cattgttacc agttttggat cattttattg ctgatttgaa aaaattaagg 780

acagcaattg cttctcaaga tgaagattat ttgttggaac attttaaaga atctcaaaaa 840

gcaaggttgg cattgaaaac agatcatgat attcatccat aa 882

<210> 5

<211> 693

<212> PRT

<213> RgTAL gene (Artificial Sequence)

<400> 5

Met Ala Pro Arg Pro Thr Ser Gln Ser Gln Ala Arg Thr Cys Pro Thr

1 5 10 15

Thr Gln Val Thr Gln Val Asp Ile Val Glu Lys Met Leu Ala Ala Pro

20 25 30

Thr Asp Ser Thr Leu Glu Leu Asp Gly Tyr Ser Leu Asn Leu Gly Asp

35 40 45

Val Val Ser Ala Ala Arg Lys Gly Arg Pro Val Arg Val Lys Asp Ser

50 55 60

Asp Glu Ile Arg Ser Lys Ile Asp Lys Ser Val Glu Phe Leu Arg Ser

65 70 75 80

Gln Leu Ser Met Ser Val Tyr Gly Val Thr Thr Gly Phe Gly Gly Ser

85 90 95

Ala Asp Thr Arg Thr Glu Asp Ala Ile Ser Leu Gln Lys Ala Leu Leu

100 105 110

Glu His Gln Leu Cys Gly Val Leu Pro Ser Ser Phe Asp Ser Phe Arg

115 120 125

Leu Gly Arg Gly Leu Glu Asn Ser Leu Pro Leu Glu Val Val Arg Gly

130 135 140

Ala Met Thr Ile Arg Val Asn Ser Leu Thr Arg Gly His Ser Ala Val

145 150 155 160

Arg Leu Val Val Leu Glu Ala Leu Thr Asn Phe Leu Asn His Gly Ile

165 170 175

Thr Pro Ile Val Pro Leu Arg Gly Thr Ile Ser Ala Ser Gly Asp Leu

180 185 190

Ser Pro Leu Ser Tyr Ile Ala Ala Ala Ile Ser Gly His Pro Asp Ser

195 200 205

Lys Val His Val Val His Glu Gly Lys Glu Lys Ile Leu Tyr Ala Arg

210 215 220

Glu Ala Met Ala Leu Phe Asn Leu Glu Pro Val Val Leu Gly Pro Lys

225 230 235 240

Glu Gly Leu Gly Leu Val Asn Gly Thr Ala Val Ser Ala Ser Met Ala

245 250 255

Thr Leu Ala Leu His Asp Ala His Met Leu Ser Leu Leu Ser Gln Ser

260 265 270

Leu Thr Ala Met Thr Val Glu Ala Met Val Gly His Ala Gly Ser Phe

275 280 285

His Pro Phe Leu His Asp Val Thr Arg Pro His Pro Thr Gln Ile Glu

290 295 300

Val Ala Gly Asn Ile Arg Lys Leu Leu Glu Gly Ser Arg Phe Ala Val

305 310 315 320

His His Glu Glu Glu Val Lys Val Lys Asp Asp Glu Gly Ile Leu Arg

325 330 335

Gln Asp Arg Tyr Pro Leu Arg Thr Ser Pro Gln Trp Leu Gly Pro Leu

340 345 350

Val Ser Asp Leu Ile His Ala His Ala Val Leu Thr Ile Glu Ala Gly

355 360 365

Gln Ser Thr Thr Asp Asn Pro Leu Ile Asp Val Glu Asn Lys Thr Ser

370 375 380

His His Gly Gly Asn Phe Gln Ala Ala Ala Val Ala Asn Thr Met Glu

385 390 395 400

Lys Thr Arg Leu Gly Leu Ala Gln Ile Gly Lys Leu Asn Phe Thr Gln

405 410 415

Leu Thr Glu Met Leu Asn Ala Gly Met Asn Arg Gly Leu Pro Ser Cys

420 425 430

Leu Ala Ala Glu Asp Pro Ser Leu Ser Tyr His Cys Lys Gly Leu Asp

435 440 445

Ile Ala Ala Ala Ala Tyr Thr Ser Glu Leu Gly His Leu Ala Asn Pro

450 455 460

Val Thr Thr His Val Gln Pro Ala Glu Met Ala Asn Gln Ala Val Asn

465 470 475 480

Ser Leu Ala Leu Ile Ser Ala Arg Arg Thr Thr Glu Ser Asn Asp Val

485 490 495

Leu Ser Leu Leu Leu Ala Thr His Leu Tyr Cys Val Leu Gln Ala Ile

500 505 510

Asp Leu Arg Ala Ile Glu Phe Glu Phe Lys Lys Lys Gln Phe Gly Pro Ala

515 520 525

Ile Val Ser Leu Ile Asp Gln His Phe Gly Ser Ala Met Thr Gly Ser

530 535 540

Asn Leu Arg Asp Glu Leu Val Glu Lys Val Asn Lys Thr Leu Ala Lys

545 550 555 560

Arg Leu Glu Gln Thr Asn Ser Tyr Asp Leu Val Pro Arg Trp His Asp

565 570 575

Ala Phe Ser Phe Ala Ala Gly Thr Val Val Glu Val Leu Ser Ser Thr

580 585 590

Ser Leu Ser Leu Ala Ala Val Asn Ala Trp Lys Val Ala Ala Ala Glu

595 600 605

Ser Ala Ile Ser Leu Thr Arg Gln Val Arg Glu Thr Phe Trp Ser Ala

610 615 620

Ala Ser Thr Ser Ser Pro Ala Leu Ser Tyr Leu Ser Pro Arg Thr Gln

625 630 635 640

Ile Leu Tyr Ala Phe Val Arg Glu Glu Leu Gly Val Lys Ala Arg Arg

645 650 655

Gly Asp Val Phe Leu Gly Lys Gln Glu Val Thr Ile Gly Ser Asn Val

660 665 670

Ser Lys Ile Tyr Glu Ala Ile Lys Ser Gly Arg Ile Asn Asn Val Leu

675 680 685

Leu Lys Met Leu Ala

690

<210> 6

<211> 520

<212> PRT

<213> HpaB gene (Artificial Sequence)

<400> 6

Met Lys Pro Glu Asp Phe Arg Ala Ser Ala Thr Arg Pro Phe Thr Gly

1 5 10 15

Glu Glu Tyr Leu Ala Ser Leu Arg Asp Asp Arg Glu Ile Tyr Ile Tyr

20 25 30

Gly Asp Arg Val Lys Asp Val Thr Ser His Pro Ala Phe Arg Asn Ala

35 40 45

Ala Ala Ser Met Ala Arg Leu Tyr Asp Ala Leu His Asp Pro Gln Ser

50 55 60

Lys Glu Lys Leu Cys Trp Glu Thr Asp Thr Gly Asn Gly Gly Tyr Thr

65 70 75 80

His Lys Phe Phe Arg Tyr Ala Arg Ser Ala Asp Glu Leu Arg Gln Gln

85 90 95

Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Thr Tyr Gly Trp Met Gly

100 105 110

Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Ser Ala Leu Gly Ala Asn

115 120 125

Pro Gly Phe Tyr Gly Arg Phe Glu Asp Asn Ala Lys Thr Trp Tyr Lys

130 135 140

Arg Ile Gln Glu Ala Cys Leu Tyr Leu Asn His Ala Ile Val Asn Pro

145 150 155 160

Pro Ile Asp Arg Asp Lys Pro Val Asp Gln Val Lys Asp Val Phe Ile

165 170 175

Ser Val Asp Glu Glu Val Asp Gly Gly Ile Val Val Ser Gly Ala Lys

180 185 190

Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Phe Val Gly Gln

195 200 205

Gly Ser Ala Gln Leu Leu Gly Asp Asn Thr Asp Phe Ala Leu Met Phe

210 215 220

Ile Ala Pro Met Asn Thr Pro Gly Met Lys Leu Ile Cys Arg Pro Ser

225 230 235 240

Tyr Glu Leu Val Ala Gly Ile Ala Gly Ser Pro Phe Asp Tyr Pro Leu

245 250 255

Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Lys Val

260 265 270

Phe Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Glu Arg Cys

275 280 285

Lys Gln Trp Phe Pro Gln Gly Gly Phe Gly Arg Leu Phe Pro Met Gln

290 295 300

Gly Cys Thr Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Gly Ala Leu

305 310 315 320

Tyr Lys Ala Leu Gln Cys Thr Gly Ser Leu Glu Phe Arg Gly Val Gln

325 330 335

Ala Gln Val Gly Glu Val Val Ala Trp Arg Asn Leu Phe Trp Ser Leu

340 345 350

Thr Asp Ala Met Tyr Gly Asn Ala Ser Glu Trp His Gly Gly Ala Phe

355 360 365

Leu Pro Ser Ala Glu Ala Leu Gln Ala Tyr Arg Val Leu Ala Pro Gln

370 375 380

Ala Tyr Pro Glu Ile Lys Lys Thr Ile Glu Gln Val Val Ala Ser Gly

385 390 395 400

Leu Ile Tyr Leu Pro Ser Gly Val Arg Asp Leu His Asn Pro Gln Leu

405 410 415

Asp Lys Tyr Leu Ser Thr Tyr Cys Arg Gly Ser Gly Gly Met Gly His

420 425 430

Arg Glu Arg Ile Lys Ile Leu Lys Leu Leu Trp Asp Ala Ile Gly Ser

435 440 445

Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ala Gly Ser

450 455 460

Gln Asp Glu Ile Arg Met Gln Ala Leu Arg Gln Ala Ile Gly Ser Gly

465 470 475 480

Ala Met Lys Gly Met Leu Gly Met Val Glu Gln Cys Met Gly Asp Tyr

485 490 495

Asp Glu Asn Gly Trp Thr Val Pro His Leu His Asn Pro Asp Asp Ile

500 505 510

Asn Val Leu Asp Arg Ile Arg Gln

515 520

<210> 7

<211> 170

<212> PRT

<213> HpaC gene (Artificial Sequence)

<400> 7

Met Gln Val Asp Glu Gln Arg Leu Arg Phe Arg Asp Ala Met Ala Ser

1 5 10 15

Leu Ala Ala Ala Val Asn Ile Val Thr Thr Ala Gly His Ala Gly Arg

20 25 30

Cys Gly Ile Thr Ala Thr Ala Val Cys Ser Val Thr Asp Thr Pro Pro

35 40 45

Ser Val Met Val Cys Ile Asn Ala Asn Ser Ala Met Asn Pro Val Phe

50 55 60

Gln Gly Asn Gly Lys Leu Cys Ile Asn Val Leu Asn His Glu Gln Glu

65 70 75 80

Leu Met Ala Arg His Phe Ala Gly Met Thr Gly Met Ala Met Glu Glu

85 90 95

Arg Phe His Gln Pro Cys Trp Gln Asn Gly Pro Leu Gly Gln Pro Val

100 105 110

Leu Asn Gly Ala Leu Ala Gly Leu Glu Gly Glu Ile Ser Glu Val Gln

115 120 125

Thr Ile Gly Thr His Leu Val Tyr Leu Val Ala Ile Lys Asn Ile Ile

130 135 140

Leu Ser Gln Asp Gly His Gly Leu Ile Tyr Phe Lys Arg Arg Phe His

145 150 155 160

Pro Val Arg Leu Glu Met Glu Ala Pro Val

165 170

<210> 8

<211> 293

<212> PRT

<213> TyrC gene (Artificial Sequence)

<400> 8

Met Thr Val Phe Lys His Ile Ala Ile Ile Gly Leu Gly Leu Ile Gly

1 5 10 15

Ser Ser Ala Ala Arg Ala Thr Lys Ala Tyr Cys Pro Asp Val Thr Val

20 25 30

Ser Leu Tyr Asp Lys Ser Glu Phe Val Cys Asp Arg Ala Arg Ala Leu

35 40 45

Asn Leu Gly Asp Asn Val Thr Asp Asp Ile Gln Asp Ala Val Arg Glu

50 55 60

Ala Asp Leu Val Leu Leu Cys Val Pro Val Arg Ala Met Gly Ile Val

65 70 75 80

Ala Ala Ala Met Ala Pro Ala Leu Lys Lys Asp Val Ile Ile Cys Asp

85 90 95

Thr Gly Ser Val Lys Val Ser Val Ile Lys Thr Leu Gln Asp Asn Leu

100 105 110

Pro Asn His Ile Ile Val Pro Ser His Pro Leu Ala Gly Thr Glu Asn

115 120 125

Asn Gly Pro Asp Ala Gly Phe Ala Glu Leu Phe Gln Asp His Pro Val

130 135 140

Ile Leu Thr Pro Asp Ala His Thr Pro Ala Gln Ala Ile Ala Tyr Ile

145 150 155 160

Ala Asp Tyr Trp Glu Glu Ile Gly Gly Arg Ile Asn Leu Met Ser Ala

165 170 175

Glu His His Asp His Val Leu Ala Leu Thr Ser His Leu Pro His Val

180 185 190

Ile Ala Tyr Gln Leu Ile Gly Met Val Ser Gly Tyr Glu Lys Lys Ser

195 200 205

Arg Thr Pro Ile Met Arg Tyr Ser Ala Gly Ser Phe Arg Asp Ala Thr

210 215 220

Arg Val Ala Ala Ser Glu Pro Arg Leu Trp Gln Asp Ile Met Leu Glu

225 230 235 240

Asn Ala Pro Ala Leu Leu Pro Val Leu Asp His Phe Ile Ala Asp Leu

245 250 255

Lys Lys Leu Arg Thr Ala Ile Ala Ser Gln Asp Glu Asp Tyr Leu Leu

260 265 270

Glu His Phe Lys Glu Ser Gln Lys Ala Arg Leu Ala Leu Lys Thr Asp

275 280 285

His Asp Ile His Pro

290

Claims (4)

1. The saccharomyces cerevisiae recombinant strain is characterized in that the saccharomyces cerevisiae recombinant strain integrates genomeRgTALGene, gene,HpaBGenes andHpaCa gene ofRgTALThe base sequence of the gene is shown as SEQ ID NO: 1, saidHpaBThe base sequence of the gene is shown as SEQ ID NO: 2, saidHpaCThe base sequence of the gene is shown as SEQ ID NO: 3, saidRgTALThe corresponding amino acid sequence of the gene is shown as SEQ ID NO: 5, saidHpaBThe corresponding amino acid sequence of the gene is shown as SEQ ID NO: 6, said HpaCThe corresponding amino acid sequence of the gene is shown as SEQ ID NO: 7, the saccharomyces cerevisiae recombinant strain also comprises a strain with a knockout on a saccharomyces cerevisiae chromosomeAro3AndAro10a gene; overexpression of mutants not subject to feedback inhibition by tyrosineAro4 ^K229L AndAro7 ^G141S a gene; the gene sequence of the preprenzoate dehydrogenase TyrC which is over-expressed and derived from zymomonas mobilis is shown as SEQ ID NO: 4, and the corresponding amino acid sequence is shown as SEQ ID NO: 8, the construction method of the saccharomyces cerevisiae recombinant bacteria comprises the following steps:

1) recombinant vector pUMRI-13-HpaB-HpaCAnd pUMRI-11-RgTALObtaining a plasmid;

2) recombinant vector pUMRI-13-HpaB-HpaCIntroducing Saccharomyces cerevisiae to obtain YCA 113-1B;

3) the pUMRI-11-one obtained in the step 1)RgTALIntroducing the plasmid into YCA113-1B to obtain Saccharomyces cerevisiae recombinant strain YCA 113-2B;

4) pUMRI-ΔAro3-Aro4 ^K229L -Aro7 ^G141S Introducing into Saccharomyces cerevisiae recombinant strain YCA113-2B to obtain YCA113-5B strain in the recombinant strain;

5) pUMRI-ΔAro10-TyrCThe strain was introduced into YCA113-5B to obtain a recombinant strain YCA 113-8B.

2. The construction method of the saccharomyces cerevisiae recombinant bacteria, which is characterized by comprising the following steps:

1) recombinant vector pUMRI-13-HpaB-HpaCAnd pUMRI-11- RgTALObtaining a plasmid;

2) recombinant vector pUMRI-13-HpaB-HpaCIntroducing Saccharomyces cerevisiae to obtain YCA 113-1B;

3) the pUMRI-11-one obtained in the step 1)RgTALIntroducing the plasmid into YCA113-1B to obtain Saccharomyces cerevisiae recombinant strain YCA 113-2B;

4) pUMRI-ΔAro3-Aro4 ^K229L -Aro7 ^G141S Introducing into Saccharomyces cerevisiae recombinant strain YCA113-2B to obtain YCA113-5B strain in the recombinant strain;

5) pUMRI-ΔAro10-TyrCThe strain was introduced into YCA113-5B to obtain a recombinant strain YCA 113-8B.

3. The recombinant Saccharomyces cerevisiae strain of claim 1 for use in the production of caffeic acid.

4. A production method of caffeic acid, which is characterized in that the method comprises the step of carrying out fermentation culture on the saccharomyces cerevisiae recombinant strain of claim 1.

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