Patent Grant
11286475
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.
Tyrosol is a phenolic compound with pharmacological activity, a derivative of phenethyl alcohol and a monophenolic antioxidant and has a variety of natural sources, such as olive oil and green tea. Tyrosol has many physiological active functions, such as an anti-oxidation function, an anti-fatigue function, an anti-anoxia function, an anti-stress function, an anti-cold function, a sedation function, a cardiovascular disease treatment function and a high blood pressure treatment function. Tyrosol can also be used as a flavoring agent for liquors and alcoholic beverages and plays an important role in enhancing the taste of alcoholic beverages, especially in sake, beer and wine. In addition, tyrosol is the precursor of 2-(3,4-dihydroxyphenyl)ethanol, which is an antioxidant beneficial to human health. Compared with tyrosol, 2-(3,4-dihydroxyphenyl)ethanol has a higher anti-oxidation property, and at the same time, 2-(3,4-dihydroxyphenyl)ethanol can also be used for synthesizing many polymers. Researches show that tyrosol has many biological properties and can be used for preventing cardiovascular diseases, osteohalsiteresis and other diseases. Therefore, tyrosol, as a fine chemical in the chemical industry and a biologically active compound in the pharmaceutical industry, has always attracted the attention of researchers.
Tyrosol synthesis methods mainly comprise plant extraction, chemical synthesis and biosynthesis. At present, the industrial production of tyrosol is mainly completed through chemical synthesis. This process has many drawbacks in subsequent extraction of tyrosol, and it is difficult to obtain high-purity tyrosol. It has been reported that the highest yield of tyrosol is 10.6 mM. Therefore, it is of great value to provide a high-yield production method of tyrosol for further application.
The disclosure provides a recombinant Escherichia coli, five sites of a lacI site, a trpE site, a pabB site, a pabA site and a pykF site are deleted from an E. coli MG1655 genome, and at the same time, a Saccharomyces cerevisiae pyruvate decarboxylase gene ARO10* gene is integrated at each site of the five sites to obtain Escherichia coli YMGR5A.
The Escherichia coli YMGR5A is preserved at China Center for Type Culture Collection on May 24, 2019, the preservation number is CCTCC NO: M2019390, and the preservation address is Wuhan University, Wuhan, China.
In an embodiment of the disclosure, a nucleotide sequence of the ARO10* gene is shown as SEQ ID NO:1.
In an embodiment of the disclosure, a yccX site of the recombinant Escherichia coli is also deleted, and at the same time, the ARO10* gene is integrate at this site to obtain Escherichia coli YMGR6A (E. coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR lacI::ARO10* trpE::ARO10* pabB::ARO10* pabA::ARO10* pykF::ARO10* yccx::ARO10*).
The Escherichia coli YMGR6A is preserved at China Center for Type Culture Collection on May 24, 2019, the preservation number is CCTCC NO: M2019391, and the preservation address is Wuhan University, Wuhan, China.
In an embodiment of the disclosure, gene editing is carried out by using a CRISPR-cas9 technology or Red homologous recombination.
The disclosure provides a method for producing tyrosol, and the above recombinant Escherichia coli is used for fermentation.
In an embodiment of the disclosure, an M9Y culture medium is used for fermentation to produce tyrosol.
In an embodiment of the disclosure, the method comprises: subjecting strains to streak culture on a non-resistant LB plate, picking a single colony, inoculating a liquid LB culture medium the single colony, and culturing a seed solution for 8-10 hours.
In an embodiment of the disclosure, the method comprises: inoculating the liquid LB culture medium with the seed solution at an inoculation volume ratio of 1-5%, 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 the cells are collected, and then cleaning the cells once with normal saline; and transferring the cleaned cells into the M9Y culture medium and then placing in the 200-220 rpm shaker for fermention at 28-30° C. for 40-60 hours. Sampling is carried out every 12 hours.
In an embodiment of the disclosure, the method comprises: taking the seed solution, inoculating the liquid LB culture medium with the seed solution at the inoculation volume ratio of 1-5%, controlling an initial OD600 to be 0.05-0.06, placing in the 200-220 rpm shaker for culturing at 35-39° C., and when the OD600 reaches 0.25-0.30, inoculating a fermenter containing 40-45% of the M9Y culture medium with the seed solution. Glucose and yeast powder are added in the fermentation process.
In an embodiment of the disclosure, a formula of the M9Y culture medium comprises 17.1 g/L Na2HPO4.12H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 20 g/L glucose and 0.25 g/L yeast powder, and MgSO4 is added at a final concentration of 5 mM after sterilization.
The disclosure provides a method for constructing the recombinant Escherichia coli, five sites of a lacI site, a trpE site, a pabB site, a pabA site and a pykF site are deleted from an E. coli MG1655 genome, at the same time, 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 shown as SEQ ID NO:1.
In an embodiment of the disclosure, a yccX site of the recombinant Escherichia coli is also deleted, and at the same time, the ARO10* gene is integrated at this site.
In an embodiment of the disclosure, gene editing is carried out by using a CRISPR-cas9 technology or Red homologous recombination.
The disclosure provides application of the recombinant Escherichia coli in the fields of food, chemical engineering or pharmacy.
The disclosure provides application of the method for producing tyrosol in the fields of food, chemical engineering or pharmacy.
Beneficial Effects of the Disclosure
The disclosure constructs a strain with high-yield production of tyrosol, the five sites of the lacI site, the trpE site, the pabB site, the pabA site and the pykF site of the 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 recombinant strain, the ARO10* gene is randomly integrated at multiple sites, and it is found that the strain with high-yield production of tyrosol can be obtained by inserting the ARO10* gene at the 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.
Preservation of Biological Materials
An Escherichia coli, classified and named as Escherichia coli YMGR5A, is preserved at China Center for Type Culture Collection on May 24, 2019, the preservation number is CCTCC NO: M2019390, and the preservation address is Wuhan University, Wuhan, China.
An Escherichia coli, classified and named as Escherichia coli YMGR6A, is preserved at China Center for Type Culture Collection on May 24, 2019, the preservation number is CCTCC NO: M2019391, and the preservation address is Wuhan University, Wuhan, China.
I. High Performance Liquid Chromatography (HPLC) is Used for Detecting the Yield of Tyrosol
Specific chromatographic detection conditions are as follows: An Agela Innoval C18 chromatographic column (4.6*250 mm, pore size 5 μm); a mobile phase comprising 0.1% formic acid (80%) and methanol (20%); flow rate: 1 mL·min−1; sample injection volume: 10 μL; a UV detector, detection wavelength: 276 nm; and column temperature: 30° C.
II. Culture Mediums
An M9Y culture medium: 17.1 g/L Na2HPO4.12H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 20 g/L glucose and 0.25 g/L yeast powder; and MgSO4 is added at a final concentration of 5 mM after sterilization.
An LB culture medium: 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl.
I. Construction of a Plasmid pKK223-3-ARO10*
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*.
II. Construction of a lacI::ARO10* Deletion Expression Cassette
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.
III. Construction of a Strain YMGRA (E. coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR lacI:ARO10*)
A Red homologous recombination method is adopted, YMGR/pKD46 (E. coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR/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.
I. Construction of a trpE Deletion Cassette and a trpE::ARO10* Deletion Expression Cassette
Primers 700trpE-U-L, ΔtrpE-U-R, ΔtrpE-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.
II. Construction of a Strain YMGEA (E. coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR ΔtrpE lacI:ARO10* trpE) and a Strain YMGR2A (E. coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR lacI:ARO10* trpE::ARO10*)
The CRISPR-cas9 method is adopted for preparing a YMGRA/pCas (E. coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR 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.
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.
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.
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.
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.
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.
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 μL of the seed solution is taken, and 50 mL of a liquid LB culture medium is inoculated with the 500 μL of the seed solution for expanded culture, and then placed in a 200 r·min−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*109 CFU/mL when the OD600 is about 5, and the cells are fermented in the 200 r·min−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
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 OD600 is controlled to be 0.05, and the liquid LB culture medium inoculated with the seed solution is placed in a 200 r·min−1 shaker for expanded culture at 37° C. for 5 hours. When the OD600 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
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.
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 OD600 is controlled to be 0.05, and the liquid LB culture medium inoculated with the seed solution is placed in a 200 r·min−1 shaker for expanded culture at 37° C. for 5 hours. When the OD600 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
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019107544979 | Aug 2019 | CN | national |
| Number | Date | Country |
|---|---|---|
| 104099379 | Oct 2014 | CN |
| 104946575 | Sep 2015 | CN |
| 106566794 | Apr 2017 | CN |
| 106754607 | May 2017 | CN |
| 107435049 | Dec 2017 | CN |
| 110452865 | Nov 2019 | CN |
| 2011088425 | Jul 2011 | WO |
| Entry |
|---|
| Bai et al., Scientific Reports 4:6640, pp. 1-8, published Oct. 17, 2014. |
| Number | Date | Country | |
|---|---|---|---|
| 20210115429 A1 | Apr 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2019/012014 | Nov 2019 | US |
| Child | 17134633 | US |
