Bacterial strain for de novo synthesis of hydroxytyrosol and application of bacterial strain

Abstract

The invention discloses a strain for de novo synthesis of hydroxytyrosol and application of the strain, and belongs to the technical field of biology. The recombinant escherichia coli E.coli HT delta-glk provided by the invention can realize conversion of glucose and glycerol into hydroxytyrosol, the yield of produced hydroxytyrosol can reach 4.27 g / L, which is 3.62 times of the yield of hydroxytyrosol produced by YMGRH1D1 fermentation, and an inducer does not need to be added in the fermentation process; therefore, the raw materials are cheap and easy to obtain, additives, heavy metals and antibiotics which are harmful to human health are avoided, a good foundation is laid for later product purification, and the requirements of commercial application can be better met.

Description

Technical Field

[0001] The invention relates to a strain for synthesizing hydroxytyrosol from scratch and application thereof, belonging to the field of biotechnology. Background Art

[0002] Hydroxytyrosol, also known as dihydroxyphenylethanol, is a polyphenolic compound that is mainly found in olive oil. Hydroxytyrosol has a variety of pharmacological activities, such as antioxidant, antiviral, anticancer, neuroprotective and cardioprotective. Due to its antioxidant and free radical scavenging abilities, hydroxytyrosol is widely used in beauty products to protect the skin from external factors such as ultraviolet rays and delay skin aging. In addition, hydroxytyrosol has obtained GRAS (Generally Recognized as Saf) certification from the FDA, and its safety is widely recognized and widely used in a variety of foods as an antioxidant or antibacterial agent. The broad application potential of hydroxytyrosol in food, medicine, chemical industry, cosmetics and other fields has attracted great attention. In 2024, the global hydroxytyrosol market is estimated to be worth $751.2 million. By 2034, the total market valuation may reach $1.4255 billion.

[0003] Hydroxytyrosol can be obtained through a variety of channels. In the past, people often relied on extraction from plants. Although plant extraction can utilize natural resources, the extraction process is complicated and the product quality is easily restricted by geography, climate, environment, and product content. In addition, chemical synthesis can also synthesize hydroxytyrosol, but the starting materials and catalysts of the reaction are expensive, and may involve high temperature, high pressure, and the use of hazardous chemicals, so it is not the preferred method. In recent years, the development of synthetic biology has provided a strong impetus for the construction of cell factories, and through sophisticated system transformation, microorganisms can efficiently synthesize target products. Compared with the traditional fossil-based production model, this green biomanufacturing process embodies environmental friendliness and sustainability, and has great potential.

[0004] Currently, hydroxytyrosol is known to have a variety of effects: (1) Antioxidant effect: It can scavenge free radicals, protect cells from oxidative stress damage, and enhance skin elasticity and moisture. It has been used in products such as face creams and sunscreens, and can also be used as a food additive to prevent food spoilage; (2) Protect cardiovascular health: It shows potential in the prevention and treatment of cardiovascular diseases, including anti-atherosclerosis, anti-hypertension and anti-thrombosis; (3) Anti-cancer and cancer prevention: It can prevent and treat lung cancer, breast cancer, uterine cancer, prostate cancer, etc., and promote late cancer recovery and improve chemotherapy effects; (4) Anti-inflammatory effect: It can fight inflammatory diseases; (5) Neuroprotection: It has a potential preventive effect on neurodegenerative diseases and can protect nerve cells; (6) Antibacterial and antiviral: It can be used as a natural antibacterial and antiviral product in agriculture and pest control.

[0005] In existing reports, although there is a safe and efficient biological method for synthesizing tyrosol, for example, by expressing genes such as HpaBC, LAAD, aro10, and adh6 in Escherichia coli, the shake flask yield of hydroxytyrosol reached 3.12 g / L (article "Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydrotyrosol from Glycerol in Escherichia coli"), the yield needs to be further improved. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for biosynthesizing hydroxytyrosol. Utilizing the technology of modern biological genetic engineering, a method for high-yield hydroxytyrosol is constructed on the basis of existing tyrosol high-yield strains (the transformation method of metabolic pathways such as Figure 3 As shown), and the fermentation does not require an inducer, the purpose is to solve the technical problem that the output of tyrosol synthesized by biological method in the prior art needs to be further improved.

[0007] The first technical solution provided by the present invention is a recombinant Escherichia coli, which uses Escherichia coli YMGRH1D1 as a host cell, knocks out the ptsG gene and expresses the glucokinase gene glk from Escherichia coli.

[0008] In certain embodiments, the strain accession number of the Escherichia coli YMGRH1D1 is CCTCC NO: M2019390, and the Escherichia coli is recorded in the Chinese patent application with publication number CN112813013A and is named E. coli YMGRH1D1.

[0009] In certain embodiments, the amino acid sequence of the glucokinase is shown as SEQ ID NO.1.

[0010] In certain embodiments, the nucleotide sequence of the glucokinase gene glk is shown as SEQ ID NO.2.

[0011] The second technical solution provided by the present invention is a method for constructing the recombinant Escherichia coli described in the first technical solution, wherein the glucokinase gene glk is inserted into the plasmid pkk223-3, and transformed into the Escherichia coli YMGRH1D1 in which the gene ptsG is knocked out to obtain the recombinant Escherichia coli.

[0012] In certain embodiments, the method comprises the following steps:

[0013] (1) Construction of recombinant plasmid pkk223-glk: The genome of E. coli MG1655 was used as a template; primers glk-L and glk-R were used to perform PCR to obtain the glk (Gene ID: 946858) gene, which was then inserted into plasmid pkk223-3 to obtain the recombinant plasmid pkk223-glk;

[0014] (2) Construction of ptsG knockout expression cassette: Design primers, use PCR amplification to obtain fragments ptsG-U and ptsG-D, and connect the above fragments to obtain a ptsG deletion expression cassette;

[0015] (3) Using the CRISPR-cas9 method, the pCas plasmid was first transformed into the E. coli YMGRH1D1 competent cells by electroporation, and then the plasmid sg-pTarget with sgRNA and the deletion expression cassette of ptsG were electroporated into the E. coli YMGRH1D1 / pCas competent cells; the transformants were selected for colony PCR verification, and the verified correct strains were induced with IPTG to eliminate the sg-pTarget plasmid, and the recombinant E. coli was constructed and named E. coli HTΔ;

[0016] (4) Transformation of recombinant plasmid pkk223-glk: The recombinant plasmid pkk223-glk of step (1) was transferred into E. coli HTΔ by chemical transformation to obtain engineered bacteria E. coli HTΔ-glk.

[0017] In certain embodiments, in step (1), the glk gene is obtained from the genome of Escherichia coli MG1655 by PCR and inserted into the EcoR I and Pst I sites of plasmid pkk223-3 to obtain the recombinant plasmid pkk223-glk.

[0018] The third technical solution provided by the present invention is a method for producing hydroxytyrosol, wherein the method is to produce hydroxytyrosol by fermentation using the recombinant Escherichia coli described in the first technical solution.

[0019] In certain embodiments, the method comprises inoculating the recombinant Escherichia coli into a seed culture medium for cultivation to prepare a seed solution; and inoculating the seed solution into a fermentation culture medium to ferment and prepare hydroxytyrosol.

[0020] In certain embodiments, the recombinant Escherichia coli is inoculated onto LB solid culture medium to obtain a single colony, and the single colony is picked and inoculated into LB liquid culture medium, and cultured at 36-38°C and 180-220r / min for 10-12h to prepare a seed solution.

[0021] In certain embodiments, the recombinant Escherichia coli is inoculated onto LB solid culture medium and streaked, a single colony is picked and inoculated into a 100 mL conical flask containing 20 mL LB liquid culture medium, and cultured at 36-38° C. and 180-220 r / min for 10-12 h to prepare a seed solution.

[0022] In certain embodiments, the prepared seed liquid is inoculated into LB liquid culture medium, and the bacteria are collected after culturing at 36-38° C. and 180-220 r / min for 8-10 hours; the collected bacteria are inoculated into M9Y* liquid culture medium for fermentation culture to prepare hydroxytyrosol.

[0023] In some embodiments, the seed solution is inoculated into a 500 mL conical flask containing 100 mL LB liquid medium at an inoculation rate of 1% (v / v), and the cells are collected after being cultured at 37°C and 200 r / min for 10 hours, and the cells are washed once with physiological saline. The cells are transferred into a 250 mL conical flask containing 50 mL M9Y* liquid medium and cultured at 30°C and 200 r / min for 48 hours.

[0024] The fourth technical solution provided by the present invention is the use of the recombinant Escherichia coli described in the first technical solution, the method described in the second technical solution or the method described in the third technical solution in the preparation of hydroxytyrosol and products containing hydroxytyrosol.

[0025] The technical effects of the present invention are as follows:

[0026] (1) The recombinant E. coli HTΔ-glk provided by the present invention can realize the conversion of glucose and glycerol into hydroxytyrosol, and no inducer needs to be added during the fermentation process; therefore, the raw materials of the present invention are cheap and easy to obtain, and do not contain additives, heavy metals and antibiotics that are harmful to human health, which lays a good foundation for the subsequent product purification and can meet the requirements of commercial application.

[0027] (2) The yield of hydroxytyrosol produced by fermentation of the recombinant E. coli HTΔ-glk provided by the present invention in a medium containing glucose and glycerol can be as high as 4.27 g / L, which is higher than the results of most current research teams.

[0028] (3) The recombinant strain E. coli HTΔ-glk constructed by the present invention was used to ferment and produce hydroxytyrosol. The yield of hydroxytyrosol in 84 h was 3.62 times that of the hydroxytyrosol produced by YMGRH1D1 fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The tyrosol production results of the fermentation of the strain E. coli HTΔ-glk constructed in the present invention and the strain E. coli YMGRH1D1 are shown.

[0030] Figure 2 The hydroxytyrosol production results of the strain E. coli HTΔ-glk constructed in the present invention were obtained by fermentation.

[0031] Figure 3 The main metabolic pathway of the strain E. coli HTΔ-glk constructed by the present invention is modified. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0033] The culture medium involved in the following examples is as follows:

[0034] LB medium formula (g / L): yeast powder 5, peptone 10, NaCl 10, solid medium plus 1.5%-2.0% agar powder.

[0035] M9Y medium formula (g / L): Na2HPO4·12H2O 17.1, KH2PO4 3, NaCl 0.5, NH4Cl 1, glucose 20, yeast powder 0.25, supplemented with final concentration of MgSO4 5mmol·L -1 .

[0036] The plasmid pTarget, plasmid pCas, plasmid pkk223-3 and E. coli JM109 mentioned below are all commercial plasmids and strains.

[0037] The detection methods involved in the following embodiments are as follows:

[0038] Tyrosol detection method: high performance liquid chromatography (HPLC) detection was adopted. The specific chromatographic detection conditions were as follows: AgelaInnoval C18 chromatographic column (4.6×250 mm, pore size 5 μm); mobile phase was 80% 0.1% formic acid aqueous solution, 20% methanol; flow rate was 1 mL / min; injection volume was 10 μL; UV detector, detection wavelength was 280 nm; column temperature was 28°C.

[0039] The primers involved in the following examples are shown in Table 1:

[0040] Table 1: Primer sequences

[0041]

[0042] Table 2: Preparation of PCR system

[0043]

[0044] Example 1: Construction of recombinant E. coli HTΔ

[0045] The specific steps are as follows:

[0046] (1) Construction of ptsG deletion expression cassette: Primers sg-ptsG-D, U-ptsG-L, U-ptsG-R, D-ptsG-L, D-ptsG-R, ptsG-U, ptsG-D (as shown in Table 1) were designed based on the ptsG (Gene ID: 945651) gene sequence; the E. coli MG1655 genome was used as a template for amplification to obtain fragments ptsG-U and ptsG-D; the two fragments were connected by nested PCR using U-ptsG-L and D-ptsG-R as primers to obtain the ptsG deletion expression cassette.

[0047] (2) Using the CRISPR-cas9 method, E. coli YMGRH1D1 was prepared into an electroporation competent state, and the plasmid pCas carrying the transporter gene was electroporated into the competent cells and spread on a kanamycin-resistant LB solid culture medium plate. The single colony that grew was YMGRH1D1 / pCas.

[0048] (3) Design ptsG site sgRNA primers sg-ptsG-D and sgRNA-U, perform whole plasmid PCR using pTarget plasmid as template, purify the obtained gene fragment, digest the template plasmid with DpnⅠ and then transfer it into E.coli JM109 competent cells by chemical transformation to obtain transformation products, spread the transformation products on LB solid culture medium plates containing spectinomycin, pick out single colonies for plasmid extraction and sequencing, and the correct ones are the correct transformants.

[0049] (4) The pTarget-ptsG plasmid and the above-mentioned ptsG deletion expression cassette were co-transformed into YMGRH1D1 / pCas competent cells by electroporation, spread on LB solid culture medium plates containing dual resistance to kanamycin and spectinomycin, and cultured at 30°C for 24 h. The grown transformants were verified by colony PCR using primers U-ptsG-L and D-ptsG-R, and the strain YMGRH1D1 / pCas was used as a control.

[0050] (5) Pick the correct strain and induce it with IPTG, then spread it on a LB solid medium plate containing kanamycin monoclonal resistance and culture it at 30°C. Pick a single colony that has grown for spot plate verification. The colony that only grows on the kanamycin monoclonal resistance plate and does not grow on the double resistance plate is considered to be a strain that has eliminated the pTarget-ptsG plasmid. Streak the strain on a plate of LB solid medium without resistance and culture it at 42°C until a single colony grows. Spot plate verification is also performed. The colony that only grows on the plate without resistance and does not grow on the kanamycin resistance plate is considered to be a strain that has eliminated the pCas plasmid, that is, a stable strain E. coli HTΔ without resistance.

[0051] Referring to the above method, the crr gene (Gene ID: 946880) in the strain E. coli HTΔ was knocked out using the primers in Table 1 to obtain the strain E. coli HT-ΔptsGΔcrr.

[0052] Example 2: Construction of a glucokinase gene ptsG free expression strain

[0053] The specific steps are as follows:

[0054] (1) Preparation of E. coli JM109 competent cells: Inoculate the E. coli JM109 strain onto LB solid medium and streak culture to obtain a single colony, pick a single colony and inoculate it into a 100 mL conical flask containing 20 mL LB liquid medium, and activate and culture it at 37°C and 200 r / min for 12 h to prepare a seed solution. Inoculate the prepared seed solution into a 250 mL conical flask containing 50 mL LB liquid medium at an inoculum of 1% (v / v), and culture it at 37°C and 200 r / min until the OD reaches 0. 600 =0.5 to obtain fermentation liquid, ice-bath the fermentation liquid for 30 minutes, centrifuge at 6000r / min for 5 minutes at 4°C to collect the bacterial cells, wash the obtained bacterial cells once with precooled ultrapure water, wash them 3 times with 0.1mol / L precooled CaCl2 solution, add 2mL of 0.1mol / L CaCl2 solution and 2mL of 30% glycerol to the collected bacterial cells. Each 100μL portion is packaged and stored in a 1.5mL EP tube at -70°C for future use, to prepare E.coli JM109 competent cells.

[0055] (2) Construction of recombinant plasmid pkk223-glk: The genome of E. coli MG1655 was used as a template; primers glk-L and glk-R were used to perform PCR to obtain the glk (Gene ID: 946858) gene, which was then inserted into the EcoR I and Pst I sites of plasmid pkk223-3 to obtain the recombinant plasmid pkk223-glk.

[0056] (3) The recombinant plasmid pkk223-glk prepared in step (2) is introduced into the competent cells of E. coli JM109 obtained in step (1) by chemical method to obtain a transformation product, and the transformation product is applied to LB solid medium containing spectinomycin, and cultured at 37° C. until transformants grow. The transformants are cultured for plasmid extraction, and the plasmid is verified by EcoR I and HindIII restriction enzyme digestion. After the correct strain is cultured, the recombinant plasmid pkk223-glk is extracted.

[0057] (4) Construction of recombinant E. coli: Prepare E. coli HTΔ competent cells according to the method of step (1), and chemically introduce the recombinant plasmid pkk223-glk extracted in step (3) into E. coli HTΔ competent cells to obtain free recombinant E. coli E. coli HTΔ-glk.

[0058] Referring to the above method, the empty plasmid pkk223-3 was transformed into E. coli HTΔ to obtain recombinant E. coli HTΔ-3.

[0059] Example 3: Production of Hydroxytyrosol by Shake Flask Fermentation

[0060] The specific steps are as follows:

[0061] (1) The recombinant E. coli HTΔ and E. coli HT-ΔptsGΔcrr prepared in Example 1 and the E. coli HTΔ-glk and E. coli HTΔ-3 strains prepared in Example 2 were streaked on LB solid culture medium to obtain single colonies, and single colonies were picked and inoculated into a 100-mL conical flask containing 20 mL of LB liquid culture medium, and cultured at 37° C. and 200 r / min for 12 h to prepare a seed solution.

[0062] (2) The seed solution prepared in step (1) was inoculated into a 500 mL conical flask containing 100 mL LB liquid culture medium at a 1% (v / v) inoculation rate, and the culture was carried out at 37° C. and 200 r / min for 10 h, and then the bacteria were collected.

[0063] (3) The bacterial cells obtained in step (2) were washed once with physiological saline, and then transferred into a 250 mL conical flask containing 50 mL of M9Y* liquid culture medium, and cultured at 30°C and 200 r / min for 84 h. Samples were taken every 12 h to detect the product content. The results are shown in Tables 2-3 and Figures 1-2 Among them, the tyrosol production results are shown in Figure 1 The hydroxytyrosol production results are shown in Figure 2 shown.

[0064] Table 3 Tyrosol production of different recombinant Escherichia coli

[0065]

[0066] Table 4 Hydroxytyrosol production of different recombinant Escherichia coli

[0067]

[0068] It can be seen from Tables 3 and 4 that knocking out the ptsG gene alone has a better effect and higher yield than knocking out the ptsG and crr genes. The recombinant strain E. coli HTΔ-glk constructed by the present invention is used to ferment and produce hydroxytyrosol. The yield of hydroxytyrosol in 84 hours is 3.62 times that of the hydroxytyrosol produced by fermenting E. coli YMGRH1D1.

[0069] As shown in Table 4, the yield of hydroxytyrosol produced by the recombinant E. coli HTΔ-glk provided by the present invention in a shake flask fermentation for 60 hours can reach 3.97 g / L, and the yield of hydroxytyrosol produced by the fermentation for 84 hours can reach 4.27 g / L. Therefore, the technical solution of the present invention is more suitable for industrial production.

[0070] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli, characterized in that Escherichia coli YMGRH1D1 was used as a host cell, and the glucokinase glk derived from Escherichia coli was expressed after the gene ptsG was knocked out. The strain collection number of the Escherichia coli YMGRH1D1 is CCTCC NO: M2019390.

2. The recombinant Escherichia coli according to claim 1, characterized in that The amino acid sequence of the glucokinase is shown in SEQ ID NO.

1.

3. A method for constructing the recombinant Escherichia coli according to claim 1 or 2, characterized in that: The method comprises inserting the glucokinase gene glk into an expression vector, and transforming the vector into Escherichia coli YMGRH1D1 in which the gene ptsG is knocked out, so as to obtain the recombinant Escherichia coli.

4. The method according to claim 3, characterized in that The following steps are involved: (1) Construction of recombinant plasmid pkk223-glk: Using the genome of E. coli MG1655 as a template, the glk gene was obtained by primer PCR and inserted into the plasmid pkk223-3 to obtain the recombinant plasmid pkk223-glk; (2) Construction of ptsG knockout expression cassette: Design primers, use PCR amplification to obtain fragments ptsG-U and ptsG-D, and connect the above fragments to obtain a ptsG deletion expression cassette; (3) Using the CRISPR-cas9 method, the pCas plasmid was first transformed into the E. coli YMGRH1D1 competent cells by electroporation, and then the plasmid sg-pTarget with sgRNA and the ptsG deletion expression cassette were electroporated into the E. coli YMGRH1D1 / pCas competent cells; IPTG was used for induction to eliminate the sg-pTarget plasmid and construct a recombinant E. coli named E. coli HTΔ; (4) Transformation of recombinant plasmid pkk223-glk: The recombinant plasmid pkk223-glk of step (1) was transferred into E. coli HTΔ to obtain engineered bacteria E. coli HTΔ-glk.

5. The method according to claim 3 or 4, characterized in that: The Gene ID of the glk gene is 946858.

6. A method for producing hydroxytyrosol, characterized in that The method comprises the step of using the recombinant Escherichia coli described in claim 1 or 2 to ferment and produce hydroxytyrosol.

7. The method according to claim 6, characterized in that The method comprises the following steps: inoculating the recombinant Escherichia coli into a seed culture medium for cultivation to prepare a seed solution; and inoculating the seed solution into a fermentation culture medium to ferment and prepare hydroxytyrosol.

8. The method according to claim 7, characterized in that The recombinant Escherichia coli was inoculated on LB solid culture medium to obtain a single colony, and the single colony was picked and inoculated into LB liquid culture medium, and cultured at 36-38° C. and 180-220 r / min for 10-12 hours to prepare a seed solution.

9. The method according to claim 7 or 8, characterized in that: The seed liquid is inoculated into LB liquid culture medium, and the bacteria are collected after being cultured at 36-38° C. and 180-220 r / min for 8-10 hours; the collected bacteria are inoculated into M9Y liquid culture medium for fermentation culture to prepare hydroxytyrosol.

10. Use of the recombinant Escherichia coli according to claim 1 or 2, the method according to any one of claims 3 to 5, or the method according to any one of claims 6 to 9 in the preparation of hydroxytyrosol and products containing hydroxytyrosol.

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