Temperature-induced escherichia coli with high yield of tyrosol and hydroxytyrosol as well as construction method and application of escherichia coli

Abstract

The invention discloses temperature-induced Escherichia coli for high yield of tyrosol and hydroxytyrosol as well as a construction method and application of the temperature-induced Escherichia coli, and belongs to the technical field of microbial synthesis. On the basis of escherichia coli with high tyrosine yield, a temperature induction system is utilized for the first time, AroGfbr, ARO10, ADHs and TyrAfbr genes are over-expressed, and de novo synthesis of tyrosol is achieved; and screening ARO10 and ADHs genes to obtain the strain with high yield of tyrosol. On the basis, an HpaB / C gene is over-expressed, meanwhile, pykA, tyrB and mhpB genes are knocked out on a genome, glf and glk genes are knocked into a ptsG site, the yield of hydroxytyrosol is remarkably increased, the shake flask yield reaches 1.28 g / L, and the yield of a 5L reactor reaches 4.97 g / L. The method lays a foundation for low-cost and high-efficiency industrial production of tyrosol and hydroxytyrosol, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial synthesis, and particularly to an Escherichia coli with temperature-induced high production of tyrosol and hydroxytyrosol, a construction method thereof, and an application thereof. Background Art

[0002] Tyrosol and its derivative hydroxytyrosol are phenyl ethanol compounds existing in plants such as olives, and have pharmacological effects such as antioxidant, antibacterial, and antiviral effects. They are widely used in fields such as food, chemical industry, and medicine, and have great commercial value. In particular, hydroxytyrosol has strong antioxidant properties, can effectively scavenge free radicals in the body, helps delay cell aging, and has multiple benefits for human health. At present, tyrosol and hydroxytyrosol are mainly obtained through plant extraction and chemical synthesis, but both methods have problems of high cost and environmental unfriendliness. With the continuous development of synthetic biology technology, the method of microbial fermentation is expected to solve these problems and achieve the green and efficient synthesis of tyrosol and hydroxytyrosol.

[0003] Microorganisms can utilize simple carbon sources such as glucose and glycerol to achieve the de novo synthesis of tyrosol and its derivative hydroxytyrosol through the shikimate pathway. There are mainly three synthesis pathways of tyrosol in Escherichia coli: one is that 4-hydroxyphenylpyruvic acid (4-HPP) is decarboxylated by phenylpyruvate decarboxylase (ARO10) to generate 4-hydroxyphenylacetaldehyde (4-HPAA), and the latter is reduced by alcohol dehydrogenase (ADHs) to generate tyrosol; the second is that tyrosine is catalyzed by aromatic aldehyde synthase (AAS) to generate 4-HPAA, and then reduced by ADHs to generate tyrosol; the third is that tyrosine is catalyzed by tyrosine decarboxylase (TDC) to generate tyramine, and the latter is then oxidized by tyramine oxidase (TYO) to generate 4-HPAA, and then catalyzed by ADHs to generate tyrosol. Among them, the metabolic pathway of the first pathway is the shortest, and then tyrosol is catalyzed by hydroxylase (HpaB / C) to generate hydroxytyrosol. At present, the vast majority of induction methods of engineering bacteria for producing tyrosol and hydroxytyrosol are induction with isopropyl-β-D-thiogalactoside (IPTG), but the price of IPTG is high and it is environmentally unfriendly. In addition, IPTG also has certain toxicity, which may limit large-scale fermentation production. Therefore, there is an urgent need to provide a construction method for high-yield Escherichia coli that produces tyrosol and its derivative hydroxytyrosol at low cost and efficiently. Summary of the Invention

[0004] The purpose of the present invention is to provide an Escherichia coli with temperature-induced high production of tyrosol and hydroxytyrosol, a construction method thereof, and an application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a method for constructing Escherichia coli with high production of tyrosol or high production of hydroxytyrosol, which lays a foundation for the low-cost and efficient industrial production of tyrosol and hydroxytyrosol, and has broad application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for constructing Escherichia coli with high tyrosol production, comprising the following steps:

[0007] Using the temperature-inducible expression vector pBV220 as the expression plasmid, overexpressing the AroG fbr gene, TyrA fbr gene, ARO10 gene and PAR4 gene in the Escherichia coli starting strain;

[0008] Wherein, the nucleotide sequence of the AroG fbr gene is as shown in SEQ ID NO.63;

[0009] The nucleotide sequence of the TyrA fbr gene is as shown in SEQ ID NO.64;

[0010] The nucleotide sequence of the ARO10 gene is as shown in SEQ ID NO.66;

[0011] The nucleotide sequence of the PAR4 gene is as shown in SEQ ID NO.74.

[0012] Preferably, the Escherichia coli starting strain is a high-tyrosine-producing strain.

[0013] The present invention also provides a recombinant Escherichia coli obtained according to the above construction method. It is named EcoTyl09.

[0014] The present invention also provides an application of the above recombinant Escherichia coli in constructing an engineering bacterium with high hydroxytyrosol production.

[0015] The present invention also provides a method for constructing an engineering bacterium with high hydroxytyrosol production, comprising the following steps:

[0016] Using the above recombinant Escherichia coli as the starting strain, overexpressing the HpaB / C gene, knocking out the pykA gene, tyrB gene and mhpB gene, and knocking in the glf gene and glk gene;

[0017] Wherein, the nucleotide sequence of the HpaB / C gene is as shown in SEQ ID NO.76;

[0018] The nucleotide sequence of the glf gene is as shown in SEQ ID NO.77;

[0019] The nucleotide sequence of the glk gene is as shown in SEQ ID NO.78;

[0020] The nucleotide sequence of the mhpB gene is as shown in SEQ ID NO.79;

[0021] The knockout vector used for knocking out the pykA gene was obtained by amplifying with primers having nucleotide sequences as shown in SEQ ID NO. 29-34;

[0022] The knockout vector used for knocking out the tyrB gene was obtained by amplifying with primers having nucleotide sequences as shown in SEQ ID NO. 45-50.

[0023] Preferably, the target site for knock-in is the ptsG site.

[0024] The present invention also provides an engineered bacterium obtained according to the above construction method, named EcoHT05.

[0025] The present invention also provides an application of the above recombinant Escherichia coli EcoTyl09 or the above engineered bacterium EcoHT05 in the production of tyrosol and / or hydroxytyrosol.

[0026] The present invention also provides a method for producing tyrosol, comprising the step of inoculating the above recombinant Escherichia coli EcoTyl09 and performing fermentation.

[0027] The present invention also provides a method for producing hydroxytyrosol, comprising the step of inoculating the above engineered bacterium EcoHT05 and performing fermentation.

[0028] The present invention discloses the following technical effects:

[0029] The present invention for the first time utilizes a temperature induction system in Escherichia coli to overexpress AroG fbr , ARO10, ADHs, TyrA fbr genes to achieve de novo synthesis of tyrosol; screening ARO10 and ADHs from different sources improves the yield of tyrosol, making the shake flask yield of tyrosol reach 4.05 g / L and the yield in a 5 L reactor reach 6.18 g / L; on this basis, introducing HpaB / C to achieve de novo synthesis of hydroxytyrosol, verifying the degradation effect of mhpB on hydroxytyrosol and simultaneously knocking out pykA, tyrB, mhpB on the genome and knocking in glf and glk at the ptsG site, significantly improving the yield of hydroxytyrosol, with the shake flask yield reaching 1.28 g / L and the yield in a 5 L reactor reaching 4.97 g / L.

[0030] The present invention provides new ideas for the industrial application of using Escherichia coli to improve the biosynthesis of tyrosol and hydroxytyrosol. Description of the Drawings

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0032] Figure 1 Fermentation experiment diagram of recombinant bacteria constructed for de novo synthesis of tyrosol;

[0033] Figure 2 Screening result diagram of tyrosol synthesis key enzyme ARO10 from different sources;

[0034] Figure 3 Screening result diagram of tyrosol synthesis key enzymes ADHs from different sources;

[0035] Figure 4 Fermentation experiment diagram of recombinant bacteria constructed for de novo synthesis of hydroxytyrosol;

[0036] Figure 5 Functional verification of the gene mhpB related to hydroxytyrosol degradation;

[0037] Figure 6 Experimental diagrams related to genome editing to improve the yield of hydroxytyrosol; among them, A is a schematic diagram of the strategy for genome editing to increase the content of 4-HPP; B is a comparison diagram of the hydroxytyrosol yields during the shake-flask fermentation of strains EcoHT01 - EcoHT05. Detailed implementation manners

[0038] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0041] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the description of this invention, which will be apparent to those skilled in the art. Other embodiments obtained from the description of this invention will be apparent to those skilled in the art. The description of this invention and the examples are merely exemplary.

[0042] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0043] The biological materials related to this invention:

[0044] Escherichia coli 23Tyr has been disclosed in the patent document "An Escherichia coli engineering bacterium with high tyrosine production and its application CN109554325B", and it is the HGPP strain in this patent document, which is preserved in the inventor's laboratory. The pBV220 and pET28a plasmids are commercial plasmids and can be purchased through any commercial channels. The pBV220 and pET28a plasmids used in the examples are the plasmid skeletons purchased and preserved by the inventor's laboratory. The applicant undertakes to make the above biological materials available to the public within 20 years from the filing date of this invention.

[0045] The sequences and operation methods related to this invention:

[0046] AroG fbr and TyrA fbr are preserved in the inventor's laboratory. EcoyahK, EcoYqhD, EcoYeaE and EcodkgB are amplified from the genome of E. coli; SceARO10, SceADH2, SceADH6 are amplified from the genome of S. cerevisiae; YliARO10, YliPAR4, YliADH2 are amplified from the genome of Y. lipolytica; PpaARO10 is amplified from the genome of P. pastoris; the hydroxylase genes HpaB / C are amplified from the genome of E. coli BL21; glf and glk are synthesized by Shanghai Qingke Biotechnology Co., Ltd.

[0047] AroG fbrThe gene sequence is as shown in SEQ ID NO.63 and expresses 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase.

[0048] TyrA fbr The gene sequence is as shown in SEQ ID NO.64 and expresses chorismate mutase / prephenate dehydrogenase.

[0049] The SceARO10 gene sequence is as shown in SEQ ID NO.65, the YliARO10 gene sequence is as shown in SEQ ID NO.66, and the PpaARO10 gene sequence is as shown in SEQ ID NO.67; the above 3 ARO10 genes all express phenylpyruvate decarboxylase.

[0050] The EcoyahK gene sequence is as shown in SEQ ID NO.68, the EcoYqhD gene sequence is as shown in SEQ ID NO.69, the EcoYeaE gene sequence is as shown in SEQ ID NO.70, the EcodkgB gene sequence is as shown in SEQ ID NO.71, the SceADH2 gene sequence is as shown in SEQ ID NO.72, the SceADH6 gene sequence is as shown in SEQ ID NO.73, the YliPAR4 gene sequence is as shown in SEQ ID NO.74, and the YliADH2 gene sequence is as shown in SEQ ID NO.75; the above 8 ADHs genes all express alcohol dehydrogenase.

[0051] The HpaB / C gene sequence is as shown in SEQ ID NO.76 and expresses hydroxylase.

[0052] The glf gene sequence is as shown in SEQ ID NO.77 and expresses glucose facilitator.

[0053] The glk gene sequence is as shown in SEQ ID NO.78 and expresses glucokinase.

[0054] The mhpB gene sequence is as shown in SEQ ID NO.79 and expresses a protein related to hydroxytyrosol degradation;

[0055] Methods related to gene knockout / knock-in and the backbone plasmids for constructing knock-in / knockout plasmids can be found in the non-patent literature "A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli"; the design of sgRNA is from the website CHOPCHOP (http: / / chopchop.cbu.uib.no / ); the procedures related to PCR refer to the instructions in the manual of Phanta Super-Fidelity DNA polymerase (Vazyme Biotech, Nanjing, China, catalog no. P501-d1 / d2 / d3).

[0056] The primer sequences used in the present invention are shown in Table 1:

[0057] Table 1 Primers used in the examples of the present invention

[0058]

[0059]

[0060]

[0061] The construction process of the plasmids of the present invention is as follows:

[0062] The construction process of pBV220 plasmid is as follows: Using pBV220-AroG fbr -TyrA fbr related plasmid as a template, use primers F1F and F1R to amplify the backbone containing pBV220-AroG fbr -TyrA fbr ; Using S. cerevisiae genome as a template, use primers F2F and F2R to amplify the SceARO10 gene fragment; Using E. coli genome as a template, use primers F3F and F3R to amplify the EcoyahK gene fragment. By homologous recombination, connect the SceARO10 and EcoyahK fragments with the pBV220-AroG fbr -TyrA fbr backbone to form plasmid pBV220-SceARO10-EcoyahK. The construction methods of the related plasmids with pBV220 and pET28a backbones only differ in the use of primers from the above steps.

[0063] The construction process of the pTarget-pykA plasmid is as follows: The primers F15F and F15R are placed in a PCR instrument for annealing. The ligation product after the annealing reaction is ligated with the pTarget backbone (treated with Bsa I enzyme) through T4 ligase to form the plasmid pTarget-sgRNA. Subsequently, using strain 23Tyr as a template, the upper homologous arm is amplified with F16F and F16R, and the lower homologous arm is amplified with F17F and F17R, and they are assembled through homologous recombination with the pTarget-sgRNA backbone (digested with Kpn I and Nsi I) to form the plasmid pTarget-pykA. The plasmid construction method of the pTarget backbone only differs in the use of primers from the above steps.

[0064] The construction process of the strain of the present invention is as follows:

[0065] The process of constructing the strain by chemical transformation method is as follows: According to an inoculation amount of 1%-2%, transfer the bacterial liquid into a 250 mL Erlenmeyer flask containing 50 mL of LB, and culture at 37 °C for several hours until the OD 600 reaches 0.4 - 0.6. Ice bath for 20 - 30 min, and at the same time pre-cool 0.1 M CaCl2, 0.1 M CaCl2·MgCl2, 15% CaCl2·glycerol, 50 mL sterile centrifuge tubes and the centrifuge; Transfer the pre-cooled bacterial cells into a 50 mL centrifuge tube, centrifuge at 4 °C, 5000 rpm for 10 min to collect the bacterial cells. Pour off the supernatant, resuspend with 0.1 M CaCl2·MgCl2, centrifuge at 4 °C, 5000 rpm for 10 min to collect the bacterial cells. Then pour off the supernatant again, resuspend with 0.1 M CaCl2, centrifuge at 4 °C, 5000 rpm for 10 min to collect the bacterial cells. Pour off the supernatant, resuspend with 1 - 2 mL of 15% CaCl2·glycerol, aliquot into sterile EP tubes to complete the preparation of chemically competent cells; Mix the ligation product or plasmid with the competent cells, and immediately ice bath for 20 min. Then heat shock in a 42 °C water bath for 90 s, ice bath for 2 min, add 500 μL of sterile LB liquid medium, culture at 37 °C, 220 rpm for 45 - 60 min; Finally, centrifuge at 12000 rpm for 1 min. Pour off part of the supernatant, mix the remaining bacterial liquid with a pipette, and evenly spread it on an LB solid plate with the corresponding antibiotic. Invert the plate and incubate at 37 °C overnight.

[0066] The process of constructing the strain by electrotransformation method is as follows: According to an inoculation amount of 1%-2%, transfer the bacterial liquid into a 250 mL Erlenmeyer flask containing 50 mL of LB, and culture at 37 °C for several hours until the OD 600When it reaches 0.6 - 0.8, perform an ice bath for 20 - 30 min. At the same time, pre - cool 10% glycerol, 50 mL sterile centrifuge tubes, and the centrifuge; transfer the pre - cooled bacterial cells to a 50 mL centrifuge tube, centrifuge at 4°C, 5000 rpm for 10 min, and collect the bacterial cells. Pour off the supernatant, resuspend with 10% glycerol, centrifuge at 4°C, 5000 rpm for 10 min, and repeat once. Pour off the supernatant, resuspend with 1 - 2 mL of 10% glycerol, aliquot into sterile EP tubes to complete the preparation of electro - competent cells; mix the plasmid with the competent cells, perform an ice bath for 5 min, then transfer the mixed solution to a pre - cooled electroporation cuvette, avoiding the generation of bubbles. Dry the outer wall of the electroporation cuvette with a tissue paper, place it in the card slot of the electroporator, and perform electroporation at a voltage of 1.8 kv or 2.5 kv; quickly pipette 1 mL of liquid LB medium into the electroporation cuvette, mix well and transfer the liquid to an EP tube, and culture at 30°C, 220 rpm for 60 - 90 min. Then centrifuge and spread on an LB solid plate containing the corresponding antibiotic, and incubate overnight at 37°C.

[0067] The conditions for the fermentation culture of Escherichia coli in the present invention are as follows:

[0068] The fermentation medium formula is divided into four components. Component 1: Weigh 5 g of (NH4)2SO4, 3 g of K2HPO4·3H2O, 1.5 g of sodium citrate, 1 g of NaCl, 4 g of peptone, and 2 g of yeast extract, dissolve in 800 mL of water, the liquid loading volume in a 250 mL shake flask is 40 mL, and add 0.6 g of CaCO3 to each flask; Component 2: Weigh 35 g of glucose and dissolve in 100 mL of water; Component 3: Weigh 3 g of MgSO4·7H2O and dissolve in 100 mL of water; Component 4: Weigh 0.075 g of vitamin B1, 0.1125 g of FeSO4·7H2O, 1.5 mL of TES solution (containing 2 g / L of Al2(SO4)3·18H2O, 0.75 g / L of CoSO4·7H2O, 2.5 g / L of CuSO4·5H2O, 0.5 g / L of H3BO3, 24 g / L of MnSO4·H2O, 2.5 g / L of NiSO4·6H2O, 15 g / L of ZnSO4·7H2O) and dissolve in water, make up the volume to 10 mL. Components 1, 2, and 3 are sterilized by high - pressure steam at 115°C for 30 min, and Component 4 is sterilized by filtration through a 0.22 μm sterile filter head. Add 5 mL of Component 2, 5 mL of Component 3, and 500 μL of Component 4 to 40 mL of Component 1 to form a complete fermentation medium, and add the corresponding antibiotic before fermentation. Vitamin C (sterilized by filtration) and betaine are added to the fermentation medium as needed.

[0069] Shake - flask fermentation: All Escherichia coli strains are cultured in 250 mL shake flasks containing 50 mL of fermentation medium. The seed liquid (OD 600Inoculate at an inoculum size of about 5%, culture at 30 °C for 3 h before induction at a rotation speed of 220 rpm, then change the temperature to 37 °C and start fermentation timing. The fermentation cycle is 60 h.

[0070] Fermentation in a 5 L reactor: In addition to the above fermentation medium components, 2 g / L VC and 0.5 g / L betaine need to be additionally added. The initial fermentation conditions of the tyrosol engineering bacteria are: 32 °C, 300 rpm, using 4 M NaOH to control the pH to maintain at 6.8, DO is 2, and the dissolved oxygen is controlled at 30%, with the dissolved oxygen and rotation speed linked. When OD 600 reaches 20, change the temperature to 38 °C, sample 10 mL at regular intervals, and detect the content of tyrosol, the biomass of the bacteria, and the residual amount of glucose in the sample.

[0071] The hydroxytyrosol engineering bacteria are fermented by a constant rotation speed method, and the other conditions are the same as those for the tyrosol engineering bacteria fermentation.

[0072] Example 1 De novo synthesis of tyrosol

[0073] Using the pBV220 plasmid as a vector, overexpress SceARO10, EcoyahK, AroG fbr and TyrA fbr four genes to construct a temperature-induced synthesis module to obtain the plasmid pBV220-SceARO10-EcoyahK. Use the chemical transformation method to transfer the plasmid into Escherichia coli 23Tyr to obtain the strain EcoTyl01.

[0074] Use HPLC to detect the fermentation samples of the strain EcoTyl01. The detection results show that the samples contain tyrosol. After 48 h of fermentation, the tyrosol yield reaches the highest, which is 207.43 mg / L, 7.57 times that without temperature induction (27.39 mg / L) ( Figure 1 ). At 60 h, the OD 600 induced by temperature exceeds 10, and the OD 600 without temperature induction is 2. The biomass of the strain is also significantly increased after temperature induction.

[0075] Thus, it can be seen that the constructed temperature-induced synthesis module plays a role.

[0076] Example 2 Screening of the key enzyme ARO10 for tyrosol synthesis

[0077] In the present invention, YliARO10 from Y. lipolytica and PpaARO10 from P. pastoris are respectively used to replace SceARO10 to obtain the strains EcoTyl02 and EcoTyl03.

[0078] As Figure 2As shown, the strain EcoTyl02 overexpressing YliARO10 can effectively synthesize tyrosol under the induction condition at 37°C, and the yield reaches 1.50 g / L at 48 h. Compared with the strains EcoTyl01 (207.43 mg / L) and EcoTyl03 (45.68 mg / L), EcoTyl02 has the best effect in tyrosol synthesis. At the same time, the yield of the control group remains at a low level, indicating that the temperature-induced expression module can be used normally. In addition, compared with EcoTyl01, when EcoTyl02 is cultured at 30°C and 37°C, its biomass OD 600 is about 5, showing no significant difference.

[0079] Example 3 Screening of Key Enzymes ADHs for Tyrosol Synthesis

[0080] In the present invention, the alcohol dehydrogenases EcoyqhD, EcoyaeE, and EcodkgB from E. coli, SceADH2 and SceADH6 from S. cerevisiae, and YliPAR4 and YliADH2 from Y. lipolytica were used as screening targets respectively, and the strains EcoTyl04 - EcoTyl10 were obtained.

[0081] The strains EcoTyl02, EcoTyl04 - EcoTyl10 were subjected to shake-flask fermentation. As Figure 3 shown, the yields were as follows: EcoTyl02: 1.57 g / L; EcoTyl04: 233.78 mg / L; EcoTyl05: 166.53 mg / L; EcoTyl06: 1.79 g / L; EcoTyl07: 6.32 mg / L; EcoTyl08: 92.03 mg / L; EcoTyl09: 4.05 g / L; EcoTyl10: 49.47 mg / L. Among them, EcoTyl09 had the highest tyrosol yield, reaching 4.05 g / L after 60 h of fermentation, and the final biomass OD 600 was maintained at about 5.

[0082] Subsequently, the strain EcoTyl09 was fermented in a 5 L bioreactor, and the tyrosol yield reached 6.18 g / L, and the biomass OD 600 was maintained at 30.

[0083] Therefore, in the construction of microbial cell factories, it is crucial to select enzymes from suitable host sources to improve the titer of target products.

[0084] Example 4 De Novo Synthesis of Hydroxytyrosol

[0085] To verify the ability of HpaB / C to catalyze the synthesis of hydroxytyrosol, plasmid pET28a-HpaB / C was constructed. The results showed that in LB medium with tyrosol concentrations of 0.5, 1.0, and 2.0 g / L as substrates, it was able to catalyze the production of approximately 150 mg / L of hydroxytyrosol respectively. The HpaB / C gene was introduced into the tyrosol synthesis module to obtain strain EcoHT01.

[0086] Strain EcoHT01 was subjected to shake-flask fermentation, and the results were as Figure 4 shown. The final yield of hydroxytyrosol reached 358.56 mg / L, and the biomass OD 600 was maintained at around 5. The results indicated that HpaB / C has the ability to produce hydroxytyrosol.

[0087] Example 5

[0088] In the Escherichia coli genome, the 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (EC: 1.13.11.16 enzyme) encoded by the gene mhpB can open-loop degrade the compound 2,3-dihydroxyphenylpropionate containing ortho-dihydroxy groups. Therefore, it was speculated that hydroxytyrosol containing ortho-dihydroxy groups might also be catalytically degraded by the mhpB enzyme.

[0089] To verify this hypothesis, the mhpB was expressed as a protein using the pET28a vector, and the degradation ability of mhpB towards hydroxytyrosol was detected. The results of SDS-PAGE showed that the mhpB protein was successfully expressed. Subsequently, the crude enzyme solution after induced expression was used to verify the degradation ability of mhpB towards HT. As Figure 5 shown, the results indicated that under the action of the mhpB enzyme, the degradation rate of hydroxytyrosol was significantly higher than that of the control group, and the concentration of hydroxytyrosol decreased by approximately 300 mg / L within 4 h.

[0090] Therefore, subsequent research should focus on knocking out the mhpB gene to determine whether the yield of hydroxytyrosol increases. The discovery of this gene also provides a reference for the biosynthesis of other similar compounds.

[0091] Example 6 Enhancing the supply of precursor 4-HPP and knocking out mhpB to increase the production of hydroxytyrosol

[0092] To further increase the yield of hydroxytyrosol, enhancing the flux of 4-HPP towards the synthesis of hydroxytyrosol became the focus of the experiment ( Figure 6In Escherichia coli, glucose is transported through the phosphoenolpyruvate-dependent phosphotransferase system (PTS), and the PTS transport system consumes phosphoenolpyruvate (PEP). PEP and E4P are precursors of 4-HPP. Reducing the consumption of PEP in the sugar transport system can increase the influx of PEP into the shikimate pathway. PtsG (encoding the IIBC component specific for glucose in the PTS system) is involved in the PTS transport system and plays a role in the conversion of PEP to pyruvate; pykA (encoding pyruvate kinase) catalyzes the conversion of PEP to pyruvate through glycolysis.

[0093] Using the CRISPR-Cas9 technology, ptsG and pykA were knocked out. At the same time, in order to reduce the impact on sugar transport after the knockout of ptsG, the genes glf and glk were knocked into the ptsG locus to enhance sugar transport of the non-PTS system. Finally, strain EcoHT02 was obtained, and its yield increased to 768.62 mg / L, which was 2.14 times higher than that of EcoHT01.

[0094] Subsequently, in order to verify whether the knockout of the mhpB gene was beneficial to the improvement of hydroxytyrosol production, the mhpB gene was further knocked out on the genome to obtain strain EcoHT03, and its yield reached 1.15 g / L at 60 h. Compared with strain EcoHT02, the yield increased by nearly 1.49 times. Therefore, knocking out mphB significantly improved the production of hydroxytyrosol.

[0095] To eliminate the competing pathway, the tyrB gene was knocked out to obtain strain EcoHT04, and its final yield in shake-flask fermentation reached 1.17 g / L at 60 h, and the yield was 1.52 times higher than that of EcoHT02. It can be seen that the knockout of tyrB caused more 4-HPP to flow into the synthesis of hydroxytyrosol. Subsequently, after knocking out tyrB and mhpB simultaneously, the yield of hydroxytyrosol of strain EcoHT05 reached 1.28 g / L, and the final biomass OD 600 was maintained at about 7, and the yield was 1.09 times higher than that of EcoHT04.

[0096] By appropriately editing the genomes of the strains, the production of hydroxytyrosol was significantly improved. The yield of the strain EcoHT05 with the highest yield was 3.57 times higher than that of the strain EcoHT01 without genome editing ( Figure 6 in B). It can be seen that genome integration of the microbial chassis strain is an important strategy to improve the yield of target compounds.

[0097] Finally, the strain EcoHT05 was fermented in a 5 L reactor. A constant rotation speed was adopted and 2 g / L of VC was added to reduce the oxidation of hydroxytyrosol, and 0.5 g / L of betaine was added to improve the osmotic tolerance of the cells. Finally, the yield of hydroxytyrosol reached 4.97 g / L, and the biomass OD 600 reached a maximum of 36, but decreased with the accumulation of hydroxytyrosol, and finally the biomass was maintained at about 27.

[0098] In summary, the present invention uses a temperature induction system to achieve the de novo synthesis of tyrosol and hydroxytyrosol. Phenylpyruvate decarboxylases ARO10 and alcohol dehydrogenases ADHs from different sources were screened. Among them, YliARO10 and YliPAR4 had the highest efficiency, and the tyrosol content in the shake flask reached 4.05 g / L. By increasing the precursor supply, improving the sugar transport system, and knocking out the hydroxytyrosol degradation-related gene mhpB, the shake flask yield of hydroxytyrosol reached 1.28 g / L. In a 5 L bioreactor, the final titers of tyrosol and hydroxytyrosol reached 6.18 and 4.97 g / L, respectively. The present invention lays a foundation for the industrial production of tyrosol and hydroxytyrosol and provides a reference for reducing the degradation of hydroxytyrosol analogs.

[0099] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing Escherichia coli with high tyrosol production, characterized in that, Comprising the following steps: Using the temperature-inducible expression vector pBV220 as the expression plasmid, overexpress the AroG fbr gene, TyrA fbr gene, ARO10 gene and PAR4 gene in the Escherichia coli starting strain; Among them, the nucleotide sequence of the AroG fbr gene is as shown in SEQ ID NO.63; The TyrA fbr gene has a nucleotide sequence as shown in SEQ ID NO.64; The nucleotide sequence of the ARO10 gene is shown as SEQ ID NO.66; The nucleotide sequence of the PAR4 gene is shown as SEQ ID NO.

74.

2. The construction method according to claim 1, wherein The starting strain of Escherichia coli is a high-tyrosine-producing strain.

3. A recombinant Escherichia coli obtained by the construction method according to claim 1 or 2.

4. Use of the recombinant Escherichia coli according to claim 3 in constructing an engineering bacterium with high hydroxytyrosol production.

5. A method for constructing an engineered bacterium with high yield of hydroxytyrosol, characterized in that, Comprising the following steps: Using the recombinant Escherichia coli according to claim 3 as the starting strain, overexpressing the HpaB / C gene, knocking out the pykA gene, tyrB gene and mhpB gene, and knocking in the glf gene and glk gene; Among them, the nucleotide sequence of the HpaB / C gene is shown as SEQ ID NO.76; The nucleotide sequence of the glf gene is shown as SEQ ID NO.77; The nucleotide sequence of the glk gene is shown as SEQ ID NO.78; The nucleotide sequence of the mhpB gene is shown as SEQ ID NO.79; The knockout vector used for knocking out the pykA gene is amplified using primers with nucleotide sequences shown as SEQ ID NO.29-34; The knockout vector used for knocking out the tyrB gene is amplified using primers with nucleotide sequences shown as SEQ ID NO.45-50.

6. The construction method according to claim 5, characterized in that The target site for knocking in is the ptsG site.

7. An engineering bacterium obtained by the construction method according to claim 5 or 6.

8. Use of the recombinant Escherichia coli according to claim 3 or the engineering bacterium according to claim 7 in the production of tyrosol and / or hydroxytyrosol.

9. A method for producing tyrosol, characterized in that, Comprising the step of inoculating the recombinant Escherichia coli according to claim 3 and performing fermentation.

10. A method for producing hydroxytyrosol, characterized in that, Comprising the step of inoculating the engineering bacterium according to claim 7 and performing fermentation.

Images