Tyrosol-producing Recombinant Escherichia coli and Construction Method and Application Thereof

Abstract

The disclosure relates to a tyrosol-producing recombinant Escherichia coli and a construction method and application thereof and belongs to the technical field of bioengineering. The Escherichia coli undergoes heterologous expression of a codon-optimized Saccharomyces cerevisiae pyruvate decarboxylase gene ARO10*. According to the recombinant Escherichia coli, five sites of a lacI site, a trpE site, a pabB site, a pabA site and a pykF site of an Escherichia coli genome are deleted, and at the same time, the ARO10* gene is integrated at each site of the five sites to obtain a strain containing multiple copies of the ARO10* gene. On the basis of the above recombinant strain, the ARO10* gene is randomly integrated at multiple sites, and it is found that a strain with high-yield production of tyrosol can be obtained by inserting the ARO10* gene at a yccX site. Fermentation using this strain does not require inducers or antibiotics. After fermentation is carried out for 48 hours, the yield of tyrosol can reach 32.3 mM.

Claims

1. A recombinant Escherichia coli, wherein five sites of a lacI site, a trpE site, a pabB site, a pabA site and a pykF site are deleted from an Escherichia coli MG1655 genome, a Saccharomyces cerevisiae pyruvate decarboxylase gene ARO10* gene is integrated at each site of the five sites, and a nucleotide sequence of the ARO10* gene is set forth as SEQ ID NO:1.

2. The recombinant Escherichia coli according to claim 1, wherein a yccX site of the recombinant Escherichia coli is also deleted, and at the same time, the ARO10* gene is integrated at this site.

3. The recombinant Escherichia coli according to claim 2, wherein site deletion or gene integration is carried out by using a CRISPR-cas9 technology or Red homologous recombination.

4. A method for producing tyrosol, comprising using the recombinant Escherichia coli according to claim 1 to carry out fermentation.

5. The method according to claim 4, wherein an M9Y culture medium is used as a fermentation culture medium.

6. The method according to claim 4, further comprising: subjecting strains to streak culture on an LB plate; picking a single colony; inoculating a liquid LB culture medium with the single colony; and culturing a seed solution for 8-10 hours.

7. The method according to claim 6, further comprising: inoculating the liquid LB culture medium with the seed solution at an inoculation volume ratio of 1-5%, and then placing in a 200-220 rpm shaker for culturing at 35-39 C. for 8-12 hours; collecting all cells, removing a supernatant after all the cells are collected, and then cleaning the cells; and transferring the cleaned cells into the M9Y culture medium, and then placing in the 200-220 rpm shaker for fermentation at 28-30 C. for 40-60 hours.

8. The method according to claim 6, further comprising: taking the seed solution; inoculating the liquid LB culture medium with the seed solution at an inoculation volume ratio of 1-5%, controlling an initial OD.sub.600 to be 0.05-0.06; placing in a 200-220 rpm shaker for culturing at 35-39 C.; and when the OD.sub.600 reaches 0.25-0.30, inoculating a fermenter containing 40-45% of the M9Y culture medium with the seed solution for fermentation for 40-60 hours.

9. The method according to claim 8, wherein a formula of the M9Y culture medium comprises 17.1 g/L Na.sub.2HPO.sub.4.12H.sub.2O, 3 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 1 g/L NH.sub.4Cl, 20 g/L glucose and 0.25 g/L yeast powder, and MgSO4 is added at a final concentration of 5 mM after sterilization.

10. The method according to claim 4, wherein tyrosol obtained after production is used to prepare food or medicine.

11. A method for constructing the recombinant Escherichia coli according to claim 1, comprising: deleting five sites of a lacI site, a trpE site, a pabB site, a pabA site and a pykF site from an Escherichia coli MG1655 genome; and at the same time, integrating a Saccharomyces cerevisiae pyruvate decarboxylase gene ARO10* gene at each site of the five sites, wherein a nucleotide sequence of the ARO10* gene is set forth as SEQ ID NO:1.

12. The method according to claim 11, wherein a yccX site of the recombinant strain Escherichia coli is also deleted, and at the same time, the ARO10* gene is integrated at this site.

13. The method according to claim 11, wherein site deletion or gene integration is carried out by using a CRISPR-cas9 technology or Red homologous recombination.

Description

BRIEF DESCRIPTION OF FIGURES

[0026] FIG. 1: The yield result of fermented tyrosol of 9 strains (YMGRA; YMGEA, YMGR2A; YMGB2A, YMGR3A; YMGA3A, YMGR4A; YMGF4A, YMGR5A) constructed in the disclosure.

[0027] FIG. 2: The yield result of fermented tyrosol of YMGR5A constructed in the disclosure in a fermenter.

[0028] FIG. 3: The yield result of fermented tyrosol of YMGR6A constructed in the disclosure in a fermenter.

DETAILED DESCRIPTION

[0029] I. High Performance Liquid Chromatography (HPLC) is Used for Detecting the Yield of Tyrosol

[0030] Specific chromatographic detection conditions are as follows: An Agela Innoval C18 chromatographic column (4.6*250 mm, pore size 5 m); a mobile phase comprising 0.1% formic acid (80%) and methanol (20%); flow rate: 1 mL.Math.min.sup.1; sample injection volume: 10 L; a UV detector, detection wavelength: 276 nm; and column temperature: 30 C.

[0031] II. Culture Mediums

[0032] An M9Y culture medium: 17.1 g/L Na.sub.2HPO.sub.4.12H.sub.2O, 3 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 1 g/L NH.sub.4Cl, 20 g/L glucose and 0.25 g/L yeast powder; and MgSO.sub.4 is added at a final concentration of 5 mM after sterilization.

[0033] An LB culture medium: 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl.

Example 1 Heterologous Expression of a Saccharomyces cerevisiae Pyruvate Decarboxylase Gene in Escherichia coli MG1655 to Produce Tyrosol

[0034] I. Construction of a Plasmid pKK223-3-ARO10*

[0035] A codon-optimized ARO10* gene sequence is chemically synthesized by Suzhou Hongxun Biotechnologies CO., LTD. The synthesized gene sequence is inserted into the EcoR I and Hind III sites of a plasmid pKK223-3 to obtain a recombinant plasmid pKK223-3-ARO10*.

[0036] II. Construction of a lacI::ARO10* Deletion Expression Cassette

[0037] The plasmid pKK223-3 is used as a template, designed primers ARO10-L and LacIR (Table 1) are used for amplification to obtain an expression fragment of tac-ARO10*-rrnB with a promoter and a terminator, and the expression fragment is inserted into a pMD19-T simple plasmid to obtain a recombinant plasmid 19Ts-tac-ARO10*-rrnB. Primers LacIL and PKDR are designed, and pKD13 is used as a template for amplification to obtain a Kana resistant fragment. The plasmid 19Ts-tac-ARO10*-rrnB and the Kana resistant fragment are subjected to enzyme digestion and ligation with Xho I to obtain a recombinant plasmid 19Ts-Kana-tac-ARO10*-rrnB. The constructed plasmid 19Ts-Kana-tac-ARO10*-rrnB is used as a template, and lacIL and lacIR are used as primers for PCR amplification to obtain a lacI::ARO10* deletion expression cassette.

TABLE-US-00001 TABLE1 Primers Primername Sequence(5-3) Sequence ARO10-L GGCTCGAGATGGCTGTGCAGGTCGTAAAT SEQIDNO:2 IacIR GGGGTACCGTGAAACCAGTAACGTTATACGATG SEQIDNO:3 TCGCAGAGTTCA TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG TGCCAGCTGCATTAATGAATCGGCCAACGCGCG GGGAGAAGAGTTTGTAGAAACGC LacIL CCCTCGAGGTGAAACCAGTAACGTTATACGATG SEQIDNO:4 TCGCAGAGTATGCCGGTGTCTCTTATCAGACCG TTTGTGTAGGCTGGAGCTGCTTC PKDR CCCTCGAGattccggggatccgtcgacc SEQIDNO:5 YLACIL GAAGCGGCATGCATTTACGT SEQIDNO:6 YLACIR ACAACATACGAGCCGGAAGC SEQIDNO:7 pTarget-R CGGACTAGTATTATACCTAGGACTGAGC SEQIDNO:8 sg-trpE CGGACTAGTCCTGTTCTCTTATGACCTTGGTTTT SEQIDNO:9 AGAGCTAGAAATAGC sg-trpE-test CCTGTTCTCTTATGACCTTG SEQIDNO:10 700trpE-U-L gagtcggtgctttttttgaattctctagaCCAGGT SEQIDNO:11 ATTTGCGCTTTTTCAAGTC 700trpE-U-R ATTTACGACCTGCACAGCCA SEQIDNO:12 TCGGGCTGGGTATCTGATTGCTT trpE-ARO10-L AAGCAATCAGATACCCAGCCCGatggctgtgcaggt SEQIDNO:13 cgtaaat trpE-ARO10-R GAATGTCAGCCATCAGAAAGTCTCCGTTTGTAG SEQIDNO:14 AAACGCAAAAAGGC 700trpE-D-L gcctttttgcgtttctacaaacGGAGACTTTCTG SEQIDNO:15 ATGGCTGACATTC 700trpE-D-R GGTAATAGATCTAAGCTTCTGCAGGTCGACGCT SEQIDNO:16 GAAAACAGCTGGTGGCTTTC 500trpE-U-L CCAGACCGTGGAAATTTCCACG SEQIDNO:17 500trpE-D-R GAGAATGGATTCCGGATGGAACTGG SEQIDNO:18 trpE-U-R GAATGTCAGCCATCAGAAAGTCTCCCGGGCTG SEQIDNO:19 GGTATCTGATTGCTT trpE-D-L AAGCAATCAGATACCCAGCCCGGGAGACTTTCT SEQIDNO:20 GATGGCTGACATTC sg-pabB GTCCTAGGTATAATACTAGTTAACCGGGGCTC SEQIDNO:21 CGAAAGTAGITTIAGAGCTAGAAATAGC sg-pabB-test TAACCGGGGCTCCGAAAGTA SEQIDNO:22 700pabB-U-L gagtcggtgctttttttgaattctctagaCCCTG SEQIDNO:23 GATTTCATTGGTGCC 700pabB-U-R ATTTACGACCTGCACAGCCATCAGTCCTGACTCT SEQIDNO:24 ACTGGCTATGTG pabB-ARO10-L CACATAGCCAGTAGAGTCAGGACTGatggctgt SEQIDNO:25 gcaggtcgtaaat pabB-ARO10-R AGGCTACGGTATTCCACGTCGTTTGTAGAAACG SEQIDNO:26 CAAAAAGGC 700pabB-D-L gcctttttgcgtttctacaaacGACGTGGAATACC SEQIDNO:27 GCTAGCT 700pabB-D-R GGTAATAGATCTAAGCTTCTGCAGGTCGACCAC SEQIDNO:28 GAATTATGCCTGCGGTC 500pabB-U-L GCCTGCTGTAATAGATAAAGCC SEQIDNO:29 500pabB-D-R GGCGACTGGCTTAACTATTCAC SEQIDNO:30 pabB-U-R CAGGCTACGGTATTCCACGTCCAGTCCTGACTCT SEQIDNO:31 ACTGGCTATG pabB-D-L CATAGCCAGTAGAGTCAGGACTGGACGTGGAA SEQIDNO:32 TACCGTAGCCTG sg-pabA GTCCTAGGTATAATACTAGTACGTTATTCGCCACT SEQIDNO:33 ATGCCGTTTTAGAGCTAGAAATAGC sg-pabA-test ACGTTATTCGCCACTATGCC SEQIDNO:34 700pabA-U-L gagtcggtgctttttttgaattctctagaGCCTTT SEQIDNO:35 AGTCACTCTTACTGCCGC 700pabA-U-R ATTTACGACCTGCACAGCCATGGCGGCTCCGGT SEQIDNO:36 ACAAAAGAAC pabA-ARO10-L GTTCTTTIGIAC SEQIDNO:37 CGGAGCCGCCATGGCTGTGCAGGTCGTAAAT pabA-ARO10-R GATCACCCTGTTACGCATAAACGTTTGTAGAAA SEQIDNO:38 CGCAAAAAGGC 700pabA-D-L gcctttttgcgtttctacaaacGTTTATGCGTAACAGGG SEQIDNO:39 TGATC 700pabA-D-R GGTAATAGATCTAAGCTTCTGCAGGTCGACTGG SEQIDNO:40 ATCGGCTCAACCACCA 500pabA-U-L GACCATTGAGCTTGGTCCGC SEQIDNO:41 500pabA-D-R CCACCCACCGAAACGGTAAAC SEQIDNO:42 pabA-U-R GATCACCCTGTTACGCATAAACGGCGGCTCCGG SEQIDNO:43 TACAAAAGAAC pabA-D-L GTTCTTTTGTACCGGAGCCGCCGTTTATGCGTA SEQIDNO:44 ACAGGGTGATC sg-pykF GTCCTAGGTATAATACTAGTATGGTTGCGGTAAC SEQIDNO:45 GTATGAGTTTTAGAGCTAGAAATAGC sg-pykF-test ATGGTTGCGGTAACGTATGA SEQIDNO:46 700pykF-U-L gagtcggtgctttttttgaattctctagaGGCT SEQIDNO:47 AATGCTGTACGTAATACGC 700pykF-U-R ATTTACGACCTGCACAGCCATGTTGAGAAG SEQIDNO:48 GATGGGAGAAAC pykF-ARO10-L GTTTCTCCCATCCTTCTCAACATGGCTGTGCAGG SEQIDNO:49 TCGTAAAT pykF-ARO10-R CATCAGGGCGCTTCGATATACGTTTGTAGAAAC SEQIDNO:50 GCAAAAAGGC 700pykF-D-L gcctttttgcgtttctacaaacGTATATCGAAGCGCC SEQIDNO:51 CTGATG 700pykF-D-R GGTAATAGATCTAAGCTTCTGCAGGTCGACCAG SEQIDNO:52 CAATGCGCCTTCAGTAG 500pykF-U-L CTGCACATTTCTCGGTACAGTTC SEQIDNO:53 500pykF-D-R CGCACAATGTGCGCCATTT SEQIDNO:54 pykF-U-R GTTTCTCCCATCCTTCTCAACGTATATCGAAGCG SEQIDNO:55 CCCTGATG pykF-D-L CATCAGGGCGCTTCGATATACGTTGAGAAGGAT SEQIDNO:56 GGGAGAAAC sg-yccx GTCCTAGGTATAATACTAGTGAAAGTCTGCATAA SEQIDNO:57 TTGCCTGTTTTAGAGCTAGAAATAGC sg-yccx-test GAAAGTCTGCATAATTGCCT SEQIDNO:58 700yccx-U-L gagtcggtgctttttttgaattctctagaGTGTCC SEQIDNO:59 GTGCTGAATATCCACC 700pykF-U-R ATTTACGACCTGCACAGCCATTGCTGCTCT SEQIDNO:60 CCTTATCCTTAATGG yccx-ARO10-L ccattaaggataaggagagcagcaATGGCTGTGCAGG SEQIDNO:61 TCGTAAAT yccx-ARO10-R CCTGCCAAAACCGGTAAAATGTATGTTTGT SEQIDNO:62 AGAAACGCAAAAAGGC 700yccx-D-L gcctttttgcgtttctacaaacATACATTTTAC SEQIDNO:63 CGGTTTTGGCAGG 700yccx-D-R GGTAATAGATCTAAGCTTCTGCAGGTCGACCCA SEQIDNO:64 CCCGCAAAGATATGTCG 500yccx-U-L GATATTCTGCCCCAGCACTCAG SEQIDNO:65 500yccx-D-R GTGCCACGGTTAGCCTGTAT SEQIDNO:66

[0038] III. Construction of a Strain YMGRA (E. coli MG1655 feaB pheA tyrB tyrR lacI:ARO10*)

[0039] A Red homologous recombination method is adopted, YMGR/pKD46 (E. coli MG1655 feaB pheA tyrB tyrR/pKD46) is prepared into a competent cell, and the previously constructed lacI:ARO10* deletion expression cassette is transferred into the competent cell. A transformant is picked, colony PCR is carried out with primers YLACIL and YLACIR to verify the transformation situation, and the strain YMGR/pKD46 is used as a contrast. A plasmid pCP20 is transferred into the strain YMGR/pKD46 to eliminate kanamycin resistance. The high temperature of 42 C. is used for eliminating the plasmids pKD46 and pCP20. A strain YMGRA is obtained.

Example 2 Construction of a Strain YMGEA (E. coli MG1655 feaB pheA tyrB tyrR trpE lacI:ARO10* trpE) and a strain YMGR2A (E. coli MG1655 feaB pheA tyrB tyrR lacI:ARO10* trpE::ARO101

[0040] I. Construction of a trpE Deletion Cassette and a trpE::ARO10* Deletion Expression Cassette

[0041] Primers 700trpE-U-L, trpE-U-R, trpE-D-L and 700trpE-D-R are designed according to the gene sequence of trpE, an E. coli MG1655 genome is used as a template, and fragments DtrpEUP and DtrpEDown are obtained through respective PCR amplification. 500trpE-U-L and 500trpE-D-R are used as primers, and a nested PCR method is adopted for amplification to obtain a gene trpE deletion cassette. Primers 700trpE-U-L, 700trpE-U-R, trpE-ARO10-L, trpE-ARO10-R, 700trpE-D-L and 700trpE-D-R are designed according to the gene sequence of trpE and a plasmid pKK223-ARO10*; the E. coli MG1655 genome and the plasmid pKK223-ARO10* are used as templates respectively for amplification to obtain fragments trpEUP, trpEDown, and ARO10. A plasmid pTarget is subjected to enzyme digestion with Xba I, and fragments are recovered. The four fragments are ligated by using a Vazyme one-step cloning kit to obtain a correct plasmid, and 500trpE-U-L and 500trpE-D-R are used as primers for PCR amplification to obtain a trpE::ARO10* deletion expression cassette.

[0042] II. Construction of a Strain YMGEA (E. coli MG1655 feaB pheA tyrB tyrR trpE lacI:ARO10* trpE) and a Strain YMGR2A (E. coli MG1655 feaB pheA tyrB tyrR lacI:ARO10* trpE::ARO101

[0043] The CRISPR-cas9 method is adopted for preparing a YMGRA/pCas (E. coli MG1655 feaB pheA tyrB tyrR lacI:ARO10*/pCas) competent cell, and a sgRNA-containing plasmid sg-pTarget-trpE and the above trpE deletion cassette are transferred into the competent cell. A transformant is picked, colony PCR verification is carried out with primers 700trpE-U-L and 700trpE-D-R, and the strain YMGRA/pCas is used as a contrast. IPTG is adopted for induction, the plasmid sg-pTarget-trpE is eliminated, the high temperature of 42 C. is used for eliminating the plasmid pCas, and a strain YMGEA is obtained.

[0044] The sgRNA-containing plasmid sg-pTarget-trpE and the trpE::ARO10* deletion cassette are transferred into the competent cell. A transformant is picked, colony PCR verification is carried out with primers 700trpE-U-L and 700trpE-D-R, and the strain YMGRA/pCas is used as a contrast. IPTG is adopted for induction, the plasmid sg-pTarget-trpE is eliminated, the high temperature of 42 C. is used for eliminating the plasmid pCas, and a strain YMGR2A is obtained.

Example 3 Construction of a Strain YMGB2A (E. coli MG1655 feaB pheA tyrB tyrR pabB lacI:ARO10* trpE::ARO10*) and a Strain YMGR3A (E. coli MG1655 feaB pheA tyrB tyrR lacI:ARO10* trpE::ARO10* pabB::ARO10*)

[0045] A pabB deletion cassette and a pabB::ARO10* deletion expression cassette are constructed by using the strategy same as the construction of the deletion cassette and the trpE::ARO10* deletion expression cassette, YMGR2A/pCas is prepared into a competent cell by using the CRISPR-cas9 method, and a sgRNA-containing plasmid sg-pTarget-pabB and the constructed pabB deletion cassette are transferred into the competent cell for transformation. A transformant is picked, colony PCR verification is carried out with primers 700pabB-U-L and 700pabB-D-R, and the strain YMGR2A/pCas is used as a contrast. IPTG is adopted for induction, the plasmid sg-pTarget-pabB is eliminated, the high temperature of 42 C. is used for eliminating the plasmid pCas, the method is similar to that of Example 2, and a strain YMGB2A is obtained.

[0046] YMGR2A/pCas is prepared into the competent cell by using the CRISPR-cas9 method, and the sgRNA-containing plasmid sg-pTarget-pabB and the constructed pabB::ARO10* deletion expression cassette are added into the competent cell for transformation. A transformant is picked, colony PCR verification is carried out with primers 700pabB-U-L and 700pabB-D-R, and the strain YMGR2A/pCas is used as a contrast. IPTG is adopted for induction, the plasmid sg-pTarget-pabB is eliminated, the high temperature of 42 C. is used for eliminating the plasmid pCas, the method is similar to that of Example 2, and a strain YMGR3A is obtained.

Example 4 Construction of a Strain YMGA3A (E. coli MG1655 feaB pheA tyrB tyrR pabA lacI:ARO10* trpE::ARO10* pabB::ARO101 and a Strain YMGR4A (E. coli MG1655 feaB pheA tyrB tyrR lacI:ARO10* trpE::ARO10* pabB::ARO10* pabA::ARO10*)

[0047] A pabA deletion cassette and a pabA::ARO10* deletion expression cassette are constructed by using the strategy same as the construction of the trpE deletion cassette and the trpE::ARO10* deletion expression cassette, YMGR3A/pCas is prepared into an electrocompetent cell by using the CRISPR-cas9 method, and a sgRNA-containing plasmid sg-pTarget-pabA and the above pabA deletion cassette or the pabA::ARO10* deletion expression cassette are added into the competent cell for transformation. A transformant is picked, colony PCR verification is carried out with primers 700pabA-U-L and 700pabA-D-R, and the strain YMGR3A/pCas is used as a contrast. IPTG is adopted for induction, the plasmid sg-pTarget-pabA is eliminated, the high temperature of 42 C. is used for eliminating the plasmid pCas, and the method is similar to that of Example 2. Strains YMGA3A and YMGR4A are obtained.

Example 5 Construction of a Strain YMGF4A (E. coli MG1655 feaB pheA tyrB tyrR pykF lacI:ARO10* trpE::ARO10* pabB::ARO10* pabA::ARO10*) and a Strain YMGR5A (E. coli MG1655 feaB pheA tyrB tyrR lacI:ARO10* trpE::ARO10* pabB::ARO10* pabA::ARO10* pykF::ARO101

[0048] A pykF deletion cassette and a pykF::ARO10* deletion expression cassette are constructed by using the strategy same as the construction of the trpE deletion cassette and the trpE::ARO10* deletion expression cassette, YMGR4A/pCas is prepared into a competent cell by using the CRISPR-cas9 method, and a sgRNA-containing plasmid sg-pTarget-pykF and the above pykF deletion cassette or the pykF::ARO10* deletion expression cassette are added into the competent cell for transformation. A transformant is picked, colony PCR verification is carried out with primers 700pykF-U-L and 700pykF-D-R, and the strain YMGR4A/pCas is used as a contrast. IPTG is adopted for induction, the plasmid sg-pTarget-pykF is eliminated, the high temperature of 42 C. is used for eliminating the plasmid pCas, and the method is similar to that of Example 2. Strains YMGF4A and YMGR5A are obtained.

Example 6 Shake Flask Fermentation of Microorganisms for Synthetizing Tyrosol

[0049] Strains are subjected to streak culture on a non-resistant LB plate, a single colony is picked, 20 mL of a liquid LB culture medium is inoculated with the single colony, and a seed solution is cultured for 8-10 hours. 500 L of the seed solution is taken, and 50 mL of a liquid LB culture medium is inoculated with the 500 L of the seed solution for expanded culture, and then placed in a 200 r.Math.min.sup.1 shaker for culturing at 37 C. for 10 hours. All cells are collected, a supernatant is removed after the cells are collected, and then the cells are cleaned once with normal saline. The cleaned cells are transferred into 50 mL of M9Y fermentation culture medium to make the cell density in the culture medium reach 6*10.sup.9 CFU/mL when the OD.sub.600 is about 5, and the cells are fermented in the 200 r.Math.min.sup.1 shaker at 30 C. for 48 hours. Sampling is carried out every 12 hours. High performance liquid chromatography (HPLC) is used for detecting the yield of tyrosol. The yield result of tyrosol is shown as FIG. 1 and Table 2. The yield of tyrosol is gradually increased by knocking out relevant genes of competition pathways and appropriately increasing the copy number of the ARO10* gene. When the pykF gene is knocked out, the yield of tyrosol reaches 10.84. mM. When the pykF gene is knocked out and the ARO10* gene is integrated, the yield of tyrosol reaches 10.92 mM. It can be seen that continuous increase of the ARO10* gene has little effect on the yield of tyrosol.

TABLE-US-00002 TABLE 2 The yield of tyrosol obtained by fermenting different strains Strains YMGRA YMGEA YMGR2A YMGB2A YMGR3A YMGA3A YMGR4A YMGF4A YMGR5A Yield of 3.11 3.29 6.64 7.97 8.5 9.42 9.94 10.84 10.92 tyrosol (mM)

Example 7 Culture of YMGR5A in a Fermenter to Produce Tyrosol

[0050] YMGR5A is subjected to streak culture on an LB plate, a single colony is picked, 20 mL liquid LB culture medium is inoculated with the single colony, and a seed solution is cultured for 8-10 hours. The seed solution is taken, a 50 mL liquid LB culture medium is inoculated with the seed solution, the initial OD.sub.600 is controlled to be 0.05, and the liquid LB culture medium inoculated with the seed solution is placed in a 200 r.Math.min.sup.1 shaker for expanded culture at 37 C. for 5 hours. When the OD.sub.600 reaches 0.25, a 5 L fermenter containing 2 L of an M9Y culture medium is inoculated with the seed solution, sampling is carried out every 4 hours, and appropriate amounts of glucose and yeast powder are added. High performance liquid chromatography (HPLC) is used for detecting the yield of tyrosol. The yield result of tyrosol is shown as FIG. 2. When fermentation is carried out for 48 hours, the yield of tyrosol in the fermenter reaches 27.96 mM.

Example 8 Construction of a Strain YMGR6A (E. coli MG1655 feaB pheA tyrB tyrR lacI:ARO10* trpE::ARO10* pabB::ARO10* pabA::ARO10* pykF::ARO10* yccx::ARO10*)

[0051] A yccx::ARO10* deletion expression cassette is constructed by using the expression strategy same as the construction of the trpE::ARO10* deletion expression cassette, YMGR5A/pCas is prepared into a competent cell by using the CRISPR-cas9 method, and a sgRNA-containing plasmid sg-pTarget-yccx and the yccx::ARO10* deletion expression cassette are added into the competent cell for transformation. A transformant is picked, colony PCR verification is carried out with primers 700yccx-U-L and 700yccx-D-R, and the strain YMGR5A/pCas is used as a contrast. IPTG is adopted for induction, the plasmid sg-pTarget-yccx is eliminated, the high temperature of 42 C. is used for eliminating the plasmid pCas, and the method is similar to that of Example 2. A strain YMGR6A is obtained, the yield of tyrosol obtained after shake flask fermentation reaches 11.74 mM, and the fermentation method is the same as that of Example 6.

[0052] Culture in a fermenter to produce tyrosol: YMGR6A is subjected to streak culture on an LB plate, a single colony is picked, and 20 mL of a liquid LB culture medium is inoculated with the single colony, and a seed solution is cultured for 8-10 hours. The seed solution is taken, 50 mL of a liquid LB culture medium is inoculated with the seed solution, the initial OD.sub.600 is controlled to be 0.05, and the liquid LB culture medium inoculated with the seed solution is placed in a 200 r.Math.min.sup.1 shaker for expanded culture at 37 C. for 5 hours. When the OD.sub.600 reaches 0.25, a 5 L fermenter containing 2 L of an M9Y culture medium is inoculated with the seed solution, sampling is carried out every 4 hours, and appropriate amounts of glucose and yeast powder are added. High performance liquid chromatography (HPLC) is used for detecting the yield of tyrosol. The yield result of tyrosol is shown as FIG. 3. When fermentation is carried out for 48 hours, the yield of tyrosol reaches 32.3 mM.

[0053] Although the present disclosure has been disclosed as above as exemplary examples, it is not intended to limit the present disclosure. Any of those skilled in the art may make various alterations and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be as defined in the claims.