CN110452865B - Recombinant escherichia coli for producing tyrosol as well as construction method and application thereof - Google Patents

Recombinant escherichia coli for producing tyrosol as well as construction method and application thereof Download PDF

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CN110452865B
CN110452865B CN201910754497.9A CN201910754497A CN110452865B CN 110452865 B CN110452865 B CN 110452865B CN 201910754497 A CN201910754497 A CN 201910754497A CN 110452865 B CN110452865 B CN 110452865B
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陈献忠
徐微
沈微
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Abstract

The invention discloses a recombinant escherichia coli for producing tyrosol, and a construction method and application thereof, and belongs to the technical field of biological engineering. The escherichia coli heterologously expresses a codon-optimized saccharomyces cerevisiae pyruvate decarboxylase gene ARO 10. The recombinant Escherichia coli is a strain containing multiple copies of ARO10 genes, which is obtained by deleting five sites of lacI site, trpE site, pabB site, pabA site and pykF site of Escherichia coli genome and integrating the ARO10 genes. On the basis of the recombinant bacteria, ARO10 gene integration is carried out at multiple sites randomly, and the ARO10 gene insertion at the yccX site is found to obtain a strain with high tyrosol yield. The strain is used for fermentation without an inducer or an antibiotic. The yield of tyrosol can reach 28mM after fermentation for 48 hours.

Description

Recombinant escherichia coli for producing tyrosol as well as construction method and application thereof
Technical Field
The invention relates to recombinant escherichia coli for producing tyrosol, a construction method and application thereof, and belongs to the technical field of biological engineering.
Background
Tyrosol (tyrosol) is a pharmacologically active phenolic compound, a derivative of phenethyl alcohol, a monophenolic antioxidant, of various natural sources, such as olive oil and green tea, etc. Tyrosol has many physiological activities, such as antioxidant, antifatigue, anti-anoxia, anti-stress, anti-cold, tranquilizing, cardiovascular disease, hypertension, etc. Tyrosol can also be used as flavoring agent of alcoholic beverages, and plays an important role in improving taste of alcoholic beverages, especially in sake, beer and wine. In addition, tyrosol is a precursor of hydroxytyrosol, 2- (3,4-dihydroxyphenyl) ethanol is an antioxidant beneficial to human health, has stronger oxidation resistance compared with tyrosol, and can synthesize many polymers. The research shows that it has many biological properties and can prevent cardiovascular and osteopenia. Tyrosol has therefore been of interest to researchers as a biologically active compound in the fine chemicals and pharmaceutical industries of the chemical industry.
The tyrosol synthesis method mainly comprises plant extraction, chemical synthesis and biosynthesis. At present, tyrosol is industrially produced mainly by a chemical synthesis method. The process method has many defects in the subsequent process of extracting the tyrosol, and the high-purity tyrosol is difficult to obtain. Tyrosol yields of up to 10.6mM have been reported. Therefore, a method for producing tyrosol with high yield is provided, and the method has important value for further application.
Disclosure of Invention
The first purpose of the invention is to provide a recombinant Escherichia coli, wherein five sites of lacI site, trpE site, pabB site, pabA site and pykF site of E.coli MG1655 genome are deleted, and simultaneously a Saccharomyces cerevisiae pyruvate decarboxylase gene ARO10 gene is integrated on each of the five sites, so as to obtain Escherichia coli YMGR 5A.
The Escherichia coli YMGR5A is preserved in the China center for type culture Collection in 24.5.2019, and the preservation number is CCTCC NO: m2019390, the preservation address is Wuhan, Wuhan university in China.
In one embodiment of the invention, the ARO10 gene has the nucleotide sequence shown in SEQ ID No. 1.
In one embodiment of the invention, the recombinant E.coli further deleted the yccX site and integrated the ARO 10: gene at this site to give Escherichia coli YMGR6A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR lacI: ARO 10: trpE: ARO 10: pabB: ARO 10: pabA: ARO 10: pykF: ARO 10: yccX: ARO 10).
The Escherichia coli YMGR6A is preserved in the China center for type culture Collection in 24/5 in 2019, and the preservation number is CCTCC NO: m2019391, the preservation address is Wuhan, Wuhan university in China.
In one embodiment of the present invention, the above gene editing is performed using CRISPR-cas9 technology or Red homologous recombination.
The second object of the present invention is to provide a method for producing tyrosol by fermentation using the above recombinant Escherichia coli.
In one embodiment of the invention, tyrosol is produced by fermentation using M9Y medium.
In one embodiment of the invention, the strain is streaked on a non-resistant LB plate and cultured; and selecting a single colony, inoculating the single colony in a liquid LB culture medium, performing seed liquid culture, and culturing for 8-10 h.
In one embodiment of the invention, the seed liquid is inoculated in the liquid LB culture medium according to the inoculation volume ratio of 1-5%, and is placed at 35-39 ℃ and 200-; collecting all thalli, removing supernatant after the thalli are collected, and cleaning the thalli once by using physiological saline; transferring the cleaned thallus to an M9Y fermentation medium, and then placing the thallus at 28-30 ℃ and shaking table fermentation at 200-220rpm for 40-60 h. Samples were taken every 12 h.
In one embodiment of the present invention, the seed solution is inoculated into the liquid LB medium at an inoculation volume ratio of 1-5%, the initial OD600 is controlled to be 0.05-0.06, and the liquid LB medium is subjected to shake cultivation at 35-39 ℃ and 200-220rpm, when the OD is reached600When the content of the glucose reaches 0.25-0.30 percent, inoculating the glucose into a fermentation tank which contains 40-45 percent of the culture medium M9Y, and supplementing the glucose and the yeast powder in the fermentation process.
The third purpose of the invention is to provide a method for constructing the recombinant escherichia coli, which is characterized in that five sites of lacI site, trpE site, pabB site, pabA site and pykF site of the E.coli MG1655 genome are deleted, simultaneously, a saccharomyces cerevisiae pyruvate decarboxylase gene ARO10 gene is integrated on each of the five sites, and the nucleotide sequence of the ARO10 gene is shown as SEQ ID NO. 1.
The fourth purpose of the invention is to provide the application of the recombinant escherichia coli in the fields of food, chemical engineering or pharmacy.
The invention has the beneficial effects that:
the invention constructs a strain for high-yield tyrosol, which is a strain containing ARO10 genes with multiple copies by deleting five sites of lacI site, trpE site, pabB site, pabA site and pykF site of escherichia coli genome and integrating the ARO10 genes. On the basis of the recombinant bacteria, ARO10 gene integration is carried out at multiple sites randomly, and the ARO10 gene insertion at the yccX site is found to obtain a strain with high tyrosol yield. The strain is used for fermentation without an inducer or an antibiotic. The yield of tyrosol can reach 28mM after fermentation for 48 hours.
Biological material preservation
An Escherichia coli (Escherichia coli) classified and named as Escherichia coli YMGR5A, which is preserved in China Center for Type Culture Collection (CCTCC) 24 months in 2019 with the preservation number of CCTCC NO: m2019390, the preservation address is Wuhan, Wuhan university in China.
An Escherichia coli (Escherichia coli) classified and named as Escherichia coli YMGR6A, which is preserved in China Center for Type Culture Collection (CCTCC) 24 months in 2019 with the preservation number of CCTCC NO: m2019391, the preservation address is Wuhan, Wuhan university in China.
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FIG. 1: the results of the yields of tyrosol from fermentation of the 9 strains (YMGRA; YMGEA, YMGR 2A; YMGB2A, YMGR 3A; YMGA3A, YMGR 4A; YMGF4A, YMGR5A) constructed by the present invention.
FIG. 2: the constructed YMGR5A fermentation tank fermentation tyrosol yield results.
Detailed Description
EXAMPLE 1 heterologous expression of the Saccharomyces cerevisiae pyruvate decarboxylase Gene in E.coli MG1655 for tyrosol production
(I) construction of plasmid pKK223-3-ARO10
The ARO10 gene sequence obtained by codon optimization was chemically synthesized by sco hong bio-corporation and inserted into EcoR I and Hind III sites of plasmid pKK223-3 to obtain recombinant plasmid pKK223-3-ARO 10.
(di) lacI: ARO 10: deletion expression cassette construction
Primers ARO10-L, LacIR (Table 1) were designed based on the sequence of pKK223-3 plasmid, and the expression fragment of tac-ARO 10-rrnB with promoter and terminator was inserted into pMD19-T simple plasmid to obtain recombinant plasmid 19Ts-tac-ARO 10-rrnB. Primers LacIL and PKDR are designed according to pKD13 to be used as a template for amplifying a Kana resistance fragment. Plasmid 19Ts-tac-ARO 10-rrnB was digested with Xho I and ligated to Kana resistant fragment to obtain recombinant plasmid 19Ts-Kana-tac-ARO 10-rrnB. The constructed plasmid 19Ts-Kana-tac-ARO 10-rrnB was used as a template to perform PCR amplification for lacIL and lacIR primers to obtain lacI: ARO10 deleted expression cassette.
TABLE 1 primers
Figure BDA0002168298980000031
Figure BDA0002168298980000041
(III) YMGRA (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR lacI: ARO 10:)
YMGR/pKD46(E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR/pKD46) was made electrically competent by Red homologous recombination, and the above deletion expression cassette of lacI: ARO10 was added to the competence and transformed. Transformants were picked and verified by colony PCR using primers YLACIL and YLACER, strain YMGR/pKD46 being used as a control. The plasmid pCP20 was used to transfer into the strain and eliminate kanamycin resistance. The plasmids pKD46 and pCP20 were eliminated by high temperature (42 ℃). Obtaining the strain YMGRA.
Example 2
YMGEA (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR. delta. trpE lacI: ARO 10. delta. trpE), YMGR2A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR lacI: ARO 10. delta. trpE: ARO 10) Strain construction
Construction of the trpE deletion cassette and the trpE: ARO 10: deletion expression cassette primers 700trpE-U-L, Δ trpE-U-R were designed based on the gene sequence of the trpE; Δ trpE-D-L, 700 trpE-D-R. Coli MG1655 genome as template PCR amplification to get fragments DtrpUP, DtrpeDown, 500trpE-U-L, 500trpE-D-R as primer, using nested PCR method amplification to get gene trpE delete box. Designing primers 700trpE-U-L and 700trpE-U-R according to the gene sequence of trpE and plasmid pKK223-ARO 10; trpE-ARO10-L, trpE-ARO 10-R; 700trpE-D-L, 700 trpE-D-R. Coli MG1655 and plasmid pKK223-ARO10 gene group as template are amplified separately to obtain trpUP, trpEWDown and ARO 10. The pTarget plasmid was digested with Xba I, and the fragment was collected. The four fragments were ligated using the one-step Vazyme cloning kit, transformed to obtain the correct plasmid, and subjected to PCR using 500trpE-U-L, 500trpE-D-R as primers to obtain the trpE: ARO 10: deleted expression cassette.
Construction of YMGEA (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR. delta. trpE lacI: ARO 10: trpE), YMGR2A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR lacI: ARO 10: trpE: ARO 10) strains Using the method of: CRISPR-cas9, YMGRA/pCas (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR lacI: ARO 10: pCas) was made electrically competent, and plasmid sg-pTarget-trpE with sgRNA and the above-mentioned deletion cassette or the deletion cassette of ARO 10: ARO10 were added to the transformant and transformed. Transformants were picked and verified by colony PCR using primers 700trpE-U-L and 700trpE-D-R, and strain YMGRA/pCas was used as a control. Induction with IPTG eliminated the sg-pTarget-trpE plasmid and the pCas plasmid was eliminated with the aid of high temperature (42 ℃). Strains YMGEA and YMGR2A were obtained.
Example 3
YMGB2A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR. delta. pabB lacI: ARO 10. delta. trpE: ARO 10) YMGR3A (E.coli MG 1655. delta. feaB. delta. pheB. delta. tyrB. delta. tyrR lacI: ARO 10. tre: ARO 10. delta. pabB: ARO 10) Strain construction
The pabB deletion cassette and pabB: ARO 10: deleted expression cassette were constructed similarly to the trpE deletion cassette and trpE: ARO 10: deleted expression cassette, and YMGR2A/pCas were made electropositive and transformed similarly to example 2. Strains YMGB2A and YMGR3A were obtained.
Example 4
YMGA3A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR. delta. pabA lacI: ARO 10. delta. trpE: ARO 10. delta. pabB: ARO 10), YMGR4A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR lacI: ARO 10. delta. trpE: ARO 10. pabB: ARO 10. pabA: ARO 10) construction of the strains
The pabA deletion cassette and pabA: ARO 10: deleted expression cassette were constructed similarly to the trpE deletion cassette and trpE: ARO 10: deleted expression cassette, and YMGR3A/pCas was made electropositive and transformed similarly to example 2. Strains YMGA3A and YMGR4A were obtained.
Example 5
YMGF4A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR. delta. pykF lacI: ARO 10. delta. trpE: ARO 10. delta. pabB: ARO 10. delta. pabA: ARO 10), YMGR5A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR lacI: ARO 10. delta. trpE: ARO 10. pabB: ARO 10. delta. pabA. jo. ARO 10. pykF: ARO 10) construction of the strain
Construction of the pykF deletion cassette and the pykF: ARO 10: deleted expression cassette similar to the trpE deletion cassette and the trpE: ARO 10: deleted expression cassette, YMGR4A/pCas was made electrically competent for transformation, in a manner similar to example 2. Strains YMGF4A and YMGR5A were obtained.
EXAMPLE 6 fermentation of a synthetic tyrosol microorganism
Streaking the strain on an nonresistant LB plate, selecting a single colony, inoculating the single colony in 20mL of liquid LB, performing seed liquid culture, and culturing for 8-10 h. Inoculating 500 μ L of seed liquid into 50mL of liquid LB, culturing at 37 deg.C for 200r min-1Shaking and culturing for 10 h. All the thalli are collected, the supernatant is removed after the thalli are collected, and the thalli are washed once by physiological saline. The washed cells were transferred to 50mL of M9Y fermentation medium, and then the medium was incubated at 30 ℃ for 200 r.min-1Fermenting for 48h by a shaking table. Samples were taken every 12 h. The tyrosol production was determined by High Performance Liquid Chromatography (HPLC). The results of tyrosol production are shown in fig. 1 and table 2, and the tyrosol production was increased in steps by knocking out the genes involved in the competitive pathway and increasing the copy number of ARO10 gene in an appropriate amount, and when the pykF gene was knocked out, the tyrosol production was 10.84mM, and when the pykF gene was knocked out and the ARO10 gene was integrated, the tyrosol production was 10.92mM, and we considered that the increase of ARO10 gene was not significant.
TABLE 2 tyrosol yields obtained by fermentation of different strains
Figure BDA0002168298980000061
EXAMPLE 7 production of tyrosol by fermenter culture YMGR5A
Culturing in a fermentation tank to produce tyrosol, streaking YMGR5A on an LB plate, and culturing; and selecting a single colony, inoculating the single colony in 20mL of liquid LB, performing seed liquid culture, and culturing for 8-10 h. Inoculating the seed liquid into 50mL liquid LB to control the initial OD600Is 0.05, and is placed at 37 ℃ for 200r min-1Shake culturing for 5h, and performing amplification culture when OD is reached600When the culture medium reaches 0.25, inoculating the culture medium into a 5L fermentation tank filled with 2L M9Y, sampling every 4h, and supplementing proper amount of glucose and yeast powder. The tyrosol production was determined by High Performance Liquid Chromatography (HPLC). The results of tyrosol production are shown in FIG. 2, and when fermented for 48h, the tyrosol production in the fermentor reached 27.96 mM.
Example 8 detection of tyrosol production by High Performance Liquid Chromatography (HPLC)
The chromatographic detection conditions are as follows: an Agela Innoval C18 column (4.6X 250mm, pore size 5 μm); the mobile phase is 80% of 0.1% formic acid and 20% of methanol in water; flow rate 1 mL/min-1(ii) a The sample volume is 10 mu L; an ultraviolet detector with the detection wavelength of 276 nm; the column temperature was 30 ℃.
EXAMPLE 9 YMGR6A (E.coli MG 1655. delta. feaB. delta. pheA. delta. tyrB. delta. tyrR lacI: ARO 10. delta. trpE: ARO 10. pabB: ARO 10. pabA: ARO 10. delta. pykF: ARO 10. yccx: ARO 10) Strain construction
yccx: ARO 10: ARO 8932: deletion cassette construction similar to trpE: ARO 10: deletion cassette construction, YMGR5A/pCas were made electrotranscompetent and transformed in a manner similar to example 2. The strain YMGR6A was obtained, and the tyrosol yield reached 11.74 mM.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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<211> 54
<212> DNA
<213> Artificial Synthesis
<400> 11
gagtcggtgc tttttttgaa ttctctagac caggtatttg cgctttttca agtc 54
<210> 12
<211> 43
<212> DNA
<213> Artificial Synthesis
<400> 12
atttacgacc tgcacagcca tcgggctggg tatctgattg ctt 43
<210> 13
<211> 43
<212> DNA
<213> Artificial Synthesis
<400> 13
aagcaatcag atacccagcc cgatggctgt gcaggtcgta aat 43
<210> 14
<211> 47
<212> DNA
<213> Artificial Synthesis
<400> 14
gaatgtcagc catcagaaag tctccgtttg tagaaacgca aaaaggc 47
<210> 15
<211> 47
<212> DNA
<213> Artificial Synthesis
<400> 15
gcctttttgc gtttctacaa acggagactt tctgatggct gacattc 47
<210> 16
<211> 53
<212> DNA
<213> Artificial Synthesis
<400> 16
ggtaatagat ctaagcttct gcaggtcgac gctgaaaaca gctggtggct ttc 53
<210> 17
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 17
ccagaccgtg gaaatttcca cg 22
<210> 18
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 18
gagaatggat tccggatgga actgg 25
<210> 19
<211> 47
<212> DNA
<213> Artificial Synthesis
<400> 19
gaatgtcagc catcagaaag tctcccgggc tgggtatctg attgctt 47
<210> 20
<211> 47
<212> DNA
<213> Artificial Synthesis
<400> 20
aagcaatcag atacccagcc cgggagactt tctgatggct gacattc 47
<210> 21
<211> 60
<212> DNA
<213> Artificial Synthesis
<400> 21
gtcctaggta taatactagt taaccggggc tccgaaagta gttttagagc tagaaatagc 60
<210> 22
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 22
taaccggggc tccgaaagta 20
<210> 23
<211> 49
<212> DNA
<213> Artificial Synthesis
<400> 23
gagtcggtgc tttttttgaa ttctctagac cctggatttc attggtgcc 49
<210> 24
<211> 46
<212> DNA
<213> Artificial Synthesis
<400> 24
atttacgacc tgcacagcca tcagtcctga ctctactggc tatgtg 46
<210> 25
<211> 46
<212> DNA
<213> Artificial Synthesis
<400> 25
cacatagcca gtagagtcag gactgatggc tgtgcaggtc gtaaat 46
<210> 26
<211> 42
<212> DNA
<213> Artificial Synthesis
<400> 26
aggctacggt attccacgtc gtttgtagaa acgcaaaaag gc 42
<210> 27
<211> 42
<212> DNA
<213> Artificial Synthesis
<400> 27
gcctttttgc gtttctacaa acgacgtgga ataccgtagc ct 42
<210> 28
<211> 50
<212> DNA
<213> Artificial Synthesis
<400> 28
ggtaatagat ctaagcttct gcaggtcgac cacgaattat gcctgcggtc 50
<210> 29
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 29
gcctgctgta atagataaag cc 22
<210> 30
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 30
ggcgactggc ttaactattc ac 22
<210> 31
<211> 44
<212> DNA
<213> Artificial Synthesis
<400> 31
caggctacgg tattccacgt ccagtcctga ctctactggc tatg 44
<210> 32
<211> 44
<212> DNA
<213> Artificial Synthesis
<400> 32
catagccagt agagtcagga ctggacgtgg aataccgtag cctg 44
<210> 33
<211> 60
<212> DNA
<213> Artificial Synthesis
<400> 33
gtcctaggta taatactagt acgttattcg ccactatgcc gttttagagc tagaaatagc 60
<210> 34
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 34
acgttattcg ccactatgcc 20
<210> 35
<211> 53
<212> DNA
<213> Artificial Synthesis
<400> 35
gagtcggtgc tttttttgaa ttctctagag cctttagtca ctcttactgc cgc 53
<210> 36
<211> 43
<212> DNA
<213> Artificial Synthesis
<400> 36
atttacgacc tgcacagcca tggcggctcc ggtacaaaag aac 43
<210> 37
<211> 43
<212> DNA
<213> Artificial Synthesis
<400> 37
gttcttttgt accggagccg ccatggctgt gcaggtcgta aat 43
<210> 38
<211> 44
<212> DNA
<213> Artificial Synthesis
<400> 38
gatcaccctg ttacgcataa acgtttgtag aaacgcaaaa aggc 44
<210> 39
<211> 44
<212> DNA
<213> Artificial Synthesis
<400> 39
gcctttttgc gtttctacaa acgtttatgc gtaacagggt gatc 44
<210> 40
<211> 49
<212> DNA
<213> Artificial Synthesis
<400> 40
ggtaatagat ctaagcttct gcaggtcgac tggatcggct caaccacca 49
<210> 41
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 41
gaccattgag cttggtccgc 20
<210> 42
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 42
ccacccaccg aaacggtaaa c 21
<210> 43
<211> 44
<212> DNA
<213> Artificial Synthesis
<400> 43
gatcaccctg ttacgcataa acggcggctc cggtacaaaa gaac 44
<210> 44
<211> 44
<212> DNA
<213> Artificial Synthesis
<400> 44
gttcttttgt accggagccg ccgtttatgc gtaacagggt gatc 44
<210> 45
<211> 60
<212> DNA
<213> Artificial Synthesis
<400> 45
gtcctaggta taatactagt atggttgcgg taacgtatga gttttagagc tagaaatagc 60
<210> 46
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 46
atggttgcgg taacgtatga 20
<210> 47
<211> 52
<212> DNA
<213> Artificial Synthesis
<400> 47
gagtcggtgc tttttttgaa ttctctagag gctaatgctg tacgtaatac gc 52
<210> 48
<211> 42
<212> DNA
<213> Artificial Synthesis
<400> 48
atttacgacc tgcacagcca tgttgagaag gatgggagaa ac 42
<210> 49
<211> 42
<212> DNA
<213> Artificial Synthesis
<400> 49
gtttctccca tccttctcaa catggctgtg caggtcgtaa at 42
<210> 50
<211> 43
<212> DNA
<213> Artificial Synthesis
<400> 50
catcagggcg cttcgatata cgtttgtaga aacgcaaaaa ggc 43
<210> 51
<211> 43
<212> DNA
<213> Artificial Synthesis
<400> 51
gcctttttgc gtttctacaa acgtatatcg aagcgccctg atg 43
<210> 52
<211> 50
<212> DNA
<213> Artificial Synthesis
<400> 52
ggtaatagat ctaagcttct gcaggtcgac cagcaatgcg ccttcagtag 50
<210> 53
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 53
ctgcacattt ctcggtacag ttc 23
<210> 54
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 54
cgcacaatgt gcgccattt 19
<210> 55
<211> 42
<212> DNA
<213> Artificial Synthesis
<400> 55
gtttctccca tccttctcaa cgtatatcga agcgccctga tg 42
<210> 56
<211> 42
<212> DNA
<213> Artificial Synthesis
<400> 56
catcagggcg cttcgatata cgttgagaag gatgggagaa ac 42
<210> 57
<211> 60
<212> DNA
<213> Artificial Synthesis
<400> 57
gtcctaggta taatactagt gaaagtctgc ataattgcct gttttagagc tagaaatagc 60
<210> 58
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 58
gaaagtctgc ataattgcct 20
<210> 59
<211> 51
<212> DNA
<213> Artificial Synthesis
<400> 59
gagtcggtgc tttttttgaa ttctctagag tgtccgtgct gaatatccac c 51
<210> 60
<211> 45
<212> DNA
<213> Artificial Synthesis
<400> 60
atttacgacc tgcacagcca ttgctgctct ccttatcctt aatgg 45
<210> 61
<211> 45
<212> DNA
<213> Artificial Synthesis
<400> 61
ccattaagga taaggagagc agcaatggct gtgcaggtcg taaat 45
<210> 62
<211> 46
<212> DNA
<213> Artificial Synthesis
<400> 62
cctgccaaaa ccggtaaaat gtatgtttgt agaaacgcaa aaaggc 46
<210> 63
<211> 46
<212> DNA
<213> Artificial Synthesis
<400> 63
gcctttttgc gtttctacaa acatacattt taccggtttt ggcagg 46
<210> 64
<211> 50
<212> DNA
<213> Artificial Synthesis
<400> 64
ggtaatagat ctaagcttct gcaggtcgac ccacccgcaa agatatgtcg 50
<210> 65
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 65
gatattctgc cccagcactc ag 22
<210> 66
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 66
gtgccacggt tagcctgtat 20

Claims (10)

1. Recombinant escherichia coli, wherein five sites of lacI site, trpE site, pabB site, pabA site and pykF site of E.coli MG1655 genome are deleted, and simultaneously, a saccharomyces cerevisiae pyruvate decarboxylase gene ARO10 gene is integrated on each of the five sites, and the nucleotide sequence of ARO10 gene is shown as SEQ ID NO. 1.
2. The recombinant escherichia coli of claim 1, wherein said recombinant escherichia coli further comprises a deletion of the yccX site with the ARO10 gene integrated at said site.
3. The recombinant escherichia coli of claim 1 or 2, wherein site deletion or gene integration is performed using CRISPR-cas9 technology or Red homologous recombination.
4. A method for producing tyrosol, characterized in that fermentation is carried out using the recombinant Escherichia coli according to any one of claims 1 to 3.
5. The method of claim 4, wherein M9Y medium is used as the fermentation medium.
6. The method according to claim 4, wherein the strain is streaked on LB plate; and selecting a single colony, inoculating the single colony in a liquid LB culture medium, performing seed liquid culture, and culturing for 8-10 h.
7. The method as claimed in claim 6, wherein the seed liquid is inoculated into the liquid LB medium at an inoculation volume ratio of 1-5%, and is subjected to shake cultivation at a temperature of 35-39 ℃ and a speed of 200-220rpm for 8-12 h; collecting all thalli, removing supernatant after the thalli are collected, and cleaning the thalli; transferring the cleaned thallus to an M9Y fermentation medium, and then placing the thallus at 28-30 ℃ and shaking table fermentation at 200-220rpm for 40-60 h.
8. The method of claim 6, wherein the initial OD is controlled by inoculating the seed solution into the liquid LB medium at a ratio of 1-5% of the inoculation volume6000.05-0.06, and shake culturing at 35-39 deg.C and 200 rpm and 220rpm when OD is reached600When the fermentation time reaches 0.25-0.30, inoculating into a fermentation tank filled with M9Y culture medium with liquid loading amount of 40-45%, and fermenting for 40-60 h.
9. The method for constructing recombinant Escherichia coli according to any one of claims 1 to 3, wherein a Saccharomyces cerevisiae pyruvate decarboxylase gene ARO10 gene is integrated at each of five sites while deleting five sites of lacI site, trpE site, pabB site, pabA site and pykF site of the E.coli MG1655 genome, and the nucleotide sequence of the ARO10 gene is shown in SEQ ID No. 1.
10. Use of the recombinant E.coli of any one of claims 1 to 3 in the food, chemical or pharmaceutical fields.
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