A method for constructing a genetically engineered bacterium producing ectoine and its application

Abstract

The present invention discloses a construction method and application of a genetically engineered bacterium producing ectoine, and belongs to the field of biosynthesis. The present invention uses Escherichia coli BL21 as a starting strain, knocks out the lacI Gene, construct tryptophan-regulated expression system, can regulate the synthesis of product ectoine without adding inducer. The present invention transforms the phosphotransferase system of Escherichia coli BL21. ptsG Glucose facilitator protein encoding gene Zmglf , relieving the inhibitory effect of carbon catabolism and reducing the cost of culture medium. ppc and aspartate semialdehyde dehydrogenase encoding genes asd , which enhanced the accumulation of precursor substances oxaloacetate and L-aspartate-β-semialdehyde during growth metabolism and increased the synthesis level of the product ectoine.

Description

Technical Field

[0001] The invention belongs to the field of biosynthesis, and specifically relates to a construction method of an ectoine-producing genetic engineering bacterium and its application. Background Art

[0002] Tetrahydropyrimidine, also known as ectoine, is a small molecule amino acid derivative with the appearance of white crystals or powder. It originally originated from halophilic bacteria living in the salt lake of the Egyptian desert and was first discovered by German scientists in 1985. Tetrahydropyrimidine has very good water solubility, and its solubility in water at 25°C can reach 550g / L. It has a good protective effect on biological macromolecules such as proteins, nucleic acids, and lipids, and helps to cope with adverse external environmental factors. For example, ectoine can form a complex with water molecules in an aqueous solution, increase the number of water molecules around protein molecules, and enhance hydrophobic interactions, thereby maintaining the structural stability of the entire protein molecule. In addition, ectoine is also a compatible solute. When the cell is in a hypertonic environment, it can increase the intracellular water activity and help maintain the osmotic pressure balance inside and outside the cell.

[0003] Studies on ectoine have shown that it has a variety of effects that are beneficial to the skin: ① It forms an isolation layer around skin cells, blocks or reduces the stimulation of external stimuli to skin cells, relieves adverse reaction symptoms, and makes skin cells have better "resistance to impact"; ② It protects the important immune cells of the skin, Langerhans cells, from damage by external stimuli and enhances skin immunity; ③ When skin cells are subjected to environmental stress, it produces emergency protection and regeneration reactions to accelerate cell repair; ④ It prevents the chain reaction caused by UVA irradiation, reduces the expression of inflammatory factors, reduces and repairs ultraviolet damage, and resists photoaging; ⑤ It improves the skin's moisturizing ability, penetrates deep into the epidermis, and repairs the skin's water-locking system. ⑥ It improves the surface condition of the skin and resists aging and wrinkles. Ectoine has been widely used as a moisturizer and conditioner in cosmetics.

[0004] The main method for producing ectoine at present is biological fermentation, and the fermentation strains mainly used include wild-type or genetically modified Halomonas, Corynebacterium glutamicum, Escherichia coli, etc. Escherichia coli is the main production strain of ectoine because of its fast growth rate, short fermentation cycle, and relatively simple genetic engineering operation. For example, CN 105018403 discloses an Escherichia coli containing the ectABC gene of Halomonas elongatus, and the ectoine production can reach 12~18g / L after fermentation for 20~28h. The current ectoine fermentation uses glucose as the main carbon source, and the expensive inducer isopropyl-β-D-thiogalactoside needs to be added during the fermentation process. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention proposes a method for constructing a genetically engineered bacterium producing ectoine and its application. The present invention recombinantly expresses the ectoine synthesis gene cluster from Halobacillus halophilus DSM 2266, knocks out the lacI gene of the starting strain Escherichia coli BL21, and transforms the phosphotransferase system to construct an engineered bacterium that can produce ectoine using agricultural waste corncob hydrolyzate rich in glucose and xylose as a carbon source. The fermentation process does not require the addition of an inducer, and has high economic efficiency. By separating and purifying the fermentation broth, an ectoine finished product with a purity of more than 99%, good crystal form, and uniform particle size can be obtained, which can be quickly applied to industrial production.

[0006] The present invention first provides a method for constructing a genetically engineered bacterium producing ectoine, which comprises the following steps:

[0007] S1: synthesizing the trpR-Ptrp-T7 polymerase gene fragment shown in SEQ ID NO.1, and replacing the lacI gene of the plasmid pRSFDuet with the trpR-Ptrp-T7 polymerase gene fragment through a recombination reaction to obtain a recombinant plasmid pRSFDuet*;

[0008] S2: synthesize the ectoine synthesis gene cluster fragment HhectABC shown in SEQ ID NO.2, and connect it to the downstream of the first T7 promoter of plasmid pRSFDuet* through recombination reaction to obtain the recombinant plasmid pRSFDuet*-HhectABC;

[0009] S3: Using the genome of Escherichia coli BL21 as a template, PCR amplification was performed to obtain the phosphoenolpyruvate carboxylase ppc gene fragment (GenBank accession number: NZ_CP170101, location=4027427..4030078) and the aspartate semialdehyde dehydrogenase asd gene fragment shown in SEQ ID NO.3, and then overlapping PCR amplification was performed to obtain the ppc-asd gene fragment, which was connected to the downstream of the second T7 promoter of the recombinant plasmid pRSFDuet*-HhectABC through a recombination reaction to obtain the recombinant plasmid pRSFDuet*-HhectABC-ppc-asd;

[0010] S4: knocking out the lacI gene of Escherichia coli BL21 to obtain Escherichia coli BL21ΔlacI;

[0011] The glucose facilitator protein encoding gene Zmglf shown in SEQ ID NO.4 was synthesized; the Zmglf gene was integrated into the ptsG gene site of the Escherichia coli BL21ΔlacI genome to obtain the Escherichia coli BL21ΔlacIΔptsG::Zmglf modified with the phosphotransferase system;

[0012] S5: Prepare electroporation competent cells of Escherichia coli BL21ΔlacIΔptsG::Zmglf, transform the competent cells with the recombinant plasmid pRSFDuet*-HhectABC-ppc-asd to obtain the ectoine-producing engineered bacteria BL21ΔlacIΔptsG::Zmglf / pRSFDuet*-HhectABC-ppc-asd.

[0013] The present invention also provides an ectoine-producing engineered bacterium constructed according to the above method.

[0014] The present invention further provides a method for the biosynthesis of ectoine, which uses the ectoine-producing genetically engineered bacteria of the present invention to carry out the biosynthesis of ectoine; add tryptophan to the basic fermentation medium, inhibit the synthesis of ectoine products through tryptophan, and make the bacteria grow and reproduce; when the carbon source in the basic fermentation medium is exhausted and the dissolved oxygen rises, start to continuously add a feed medium, the feed medium does not contain tryptophan, and the synthesis of ectoine is started to produce ectoine. Therefore, the present invention can regulate the synthesis of the product ectoine without adding an inducer, effectively reducing the cost of fermentation materials.

[0015] Furthermore, the carbon source in the basic fermentation medium and feed medium of the present invention can be a conventional carbon source (such as glucose and xylose) or an agricultural waste rich in glucose and xylose such as corn cob hydrolyzate.

[0016] Corncob hydrolyzate can be prepared by any known method, and the present invention does not limit this. It is only necessary to convert the total reducing sugar amount to meet the set requirement of the culture medium. Typically, but not limiting, the corncob hydrolyzate can be prepared by the following method: crush the corncob and dry it to constant weight; weigh the crushed corncob, add 1% sulfuric acid solution according to a solid-liquid ratio of 1:9, and high-pressure treat it at 115°C for 1h; filter after cooling to room temperature, centrifuge the filtrate at 4000rpm for 20min, and collect the supernatant. Add 1000U / g of cellulase to the filter residue at a solid-liquid ratio of 1:10, react at 50°C for 24h, filter, centrifuge the filtrate at 4000rpm for 20min, and collect the supernatant. Combine the two parts of the supernatant, add excess calcium hydroxide, mix well, centrifuge at 4000rpm for 20min, collect the supernatant, adjust the pH to 6.0 with dilute sulfuric acid, add 2% activated carbon and keep warm at 60°C for 1h. The activated carbon was removed by filtration, and the clear liquid was concentrated to a total reducing sugar concentration of 600 g / L.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention knocks out the lacI gene of Escherichia coli BL21 and constructs a tryptophan-regulated expression system. When tryptophan exists in the culture medium, product synthesis is inhibited and the bacteria can grow and reproduce rapidly. When tryptophan in the culture medium is exhausted, product synthesis starts. The synthesis of the product ectoine can be regulated without adding an inducer, effectively reducing the cost of fermentation materials.

[0019] (2) The present invention transforms the phosphotransferase system of Escherichia coli BL21 and integrates the glucose facilitator protein encoding gene Zmglf from Zymomonas mobilis at the ptsG gene locus, thereby relieving the inhibitory effect of carbon catabolism, allowing the strain to use corn cob hydrolysate, an agricultural waste that is cheap and rich in glucose and xylose, as a carbon source to synthesize the product ectoine. This not only reduces the cost of the culture medium and has high economic efficiency, but also realizes the recycling of waste resources.

[0020] (3) The present invention enhances the accumulation of precursor substances oxaloacetate and L-aspartate-β-semialdehyde in the growth metabolism process and improves the synthesis level of the product ectoine by overexpressing the phosphoenolpyruvate carboxylase encoding gene ppc and the aspartate semialdehyde dehydrogenase encoding gene asd.

[0021] (4) The present invention separates and purifies the fermentation broth containing ectoine to obtain an ectoine product with a purity of more than 99%, good crystal form and uniform particle size, which can be quickly applied to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Schematic diagram of the synthesis pathway of ectoine by the engineering bacteria of the present invention;

[0023] Figure 2 Schematic diagram of expression of the ectoine synthesis gene cluster regulated by tryptophan in the present invention;

[0024] Figure 3 This is the fermentation process curve of tetrahydropyrimidine;

[0025] Figure 4 It is the finished product of tetrahydropyrimidine;

[0026] Figure 5 This is the liquid chromatogram of the finished product of tetrahydropyrimidine. DETAILED DESCRIPTION

[0027] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0028] The PCR method used in the embodiment of the present invention is described as follows:

[0029] PCR reaction system (gene fragment amplification): 5× PrimeSTAR buffer 10μL, dNTP 4μL, upstream primer and downstream primer 1μL each, DNA template (genomic or plasmid DNA containing the target gene) 0.5μL, HS PrimeStar DNA polymerase 0.5μL, and fill with ddH2O to a total volume of 50μL.

[0030] PCR reaction conditions (gene fragment amplification): pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec; annealing at 55°C for 30 sec; extension at 72°C (extension time calculated as 1 kb / min), 32 cycles; extension at 72°C for 10 min; storage at 4°C.

[0031] PCR reaction system (colony PCR verification): 2× Rapid Taq Master Mix 10μL, upstream primer and downstream primer 0.4μL each, ddH2O 9.2μL, appropriate amount of DNA template, total volume 20μL.

[0032] PCR reaction conditions (colony PCR verification): pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec; annealing at 58°C for 15 sec; extension at 72°C (extension time calculated at 4 kb / min), 28 cycles; extension at 72°C for 2 min; storage at 4°C.

[0033] The recombination reaction method used in the embodiment of the present invention is:

[0034] Recombination reaction system: 2×GenRec Assembly Master Mix 10 μL, linearized plasmid 0.03 pmol, target gene fragment 0.09 pmol, ddH2O to make up to a total volume of 20 μL.

[0035] Recombination reaction conditions: Place the prepared reaction solution in a 50°C water bath for 50 min.

[0036] The culture medium formula used in the embodiment of the present invention is:

[0037] LB liquid culture medium formula: OXOID yeast extract 5 g / L, OXOID tryptone 10 g / L, sodium chloride 10 g / L.

[0038] LB solid medium formula: OXOID yeast extract 5 g / L, OXOID tryptone 10 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0039] Fermentation medium formula: Angel yeast extract powder FM802 7~10g / L, ammonium sulfate 5~8g / L, Liwei corn steep liquor powder CS001 5~8g / L, corn cob hydrolyzate equivalent to total reducing sugar 13~20g / L, potassium dihydrogen phosphate 2~3g / L, dipotassium hydrogen phosphate 2~3g / L, magnesium sulfate 0.5~1.5g / L, citric acid 1~1.5g / L, ferrous sulfate 0.01~0.02g / L, manganese sulfate 0.01~0.02g / L, L-tryptophan 0.1~0.2g / L.

[0040] Feed medium formula: Angel yeast extract powder FM802 5~7g / L, corn cob hydrolyzate equivalent to total reducing sugar 500~600g / L, magnesium sulfate 2~3g / L.

[0041] Preparation method of corncob hydrolyzate in culture medium: crush the corncob to 30 mesh and dry at 70℃ to constant weight. Weigh the crushed corncob, add 1% sulfuric acid solution according to the solid-liquid ratio of 1:9, and treat under high pressure at 115℃ for 1h. Cool to room temperature and filter, centrifuge the filtrate at 4000rpm for 20min, and collect the supernatant. Add 1000U / g cellulase to the residue at a solid-liquid ratio of 1:10 and react at 50℃ for 24h. After filtering, centrifuge the filtrate at 4000rpm for 20min and collect the supernatant. Combine the two parts of the supernatant, add excess calcium hydroxide, mix well, centrifuge at 4000rpm for 20min, collect the supernatant, adjust the pH to 6.0 with dilute sulfuric acid, add 2% activated carbon and keep warm at 60℃ for 1h. Filter to remove the activated carbon, and concentrate the clear liquid to a total reducing sugar concentration of 600g / L.

[0042] The ectoine high performance liquid chromatography detection method used in the embodiment of the present invention is:

[0043] (1) Instrument: Shimadzu LC-20A high performance liquid chromatograph, with ultraviolet detector as detector.

[0044] (2) Chromatographic column model: Diamonsil C18 (250×4.6mm, 5μm).

[0045] (3) Method parameters: The mobile phase was 5% acetonitrile in water, the flow rate was 0.6 mL / min, the column temperature was 35 °C, the injection volume was 5 μL, and the detection wavelength was 210 nm.

[0046] (4) Method for establishing a standard curve: Weigh 0.2 g of tetrahydropyrimidine standard (accurate to 0.0001 g), dissolve it in ultrapure water, and dilute to 100 mL as the standard stock solution. Take 1.25 mL, 2.5 mL, 5 mL, 10 mL, and 20 mL of the standard stock solution, dilute to mL with ultrapure water, and prepare standard sample solutions with concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL. Filter through a 0.22 μm needle filter and perform liquid chromatography analysis under the above conditions. Use the standard sample concentration as the horizontal axis and the peak area as the vertical axis to draw a standard curve and calculate the regression equation. The correlation coefficient should be greater than 0.99.

[0047] (5) Method for detecting the concentration of ectoine in liquid samples: dilute the sample with pure water by an appropriate multiple so that the concentration of ectoine in the sample falls within the range of the standard curve, filter with a 0.22 μm filter membrane, and inject and detect according to the above method to obtain the peak area of ​​the sample. Calculate the concentration of ectoine in the sample using the standard curve and the dilution multiple.

[0048] (6) Content detection method of ectoine finished product:

[0049] ① Preparation of reference solution: Take an appropriate amount of tetrahydropyrimidine reference substance, place it in a 105℃ oven for constant temperature drying for 3 hours, cool to room temperature, accurately weigh 0.02g (accurate to 0.0001g), place it in a 100mL volumetric flask, add pure water to dissolve and dilute to the scale, shake well, and filter with a 0.22μm filter membrane.

[0050] ②Preparation of sample solution: Take an appropriate amount of ectoine finished product sample, place it in a 105℃ oven for constant temperature drying for 3 hours, cool it to room temperature, accurately weigh 0.02g (accurate to 0.0001g), place it in a 100mL volumetric flask, add pure water to dissolve and dilute to the scale, shake well, and filter with a 0.22μm filter membrane. Prepare two copies in parallel.

[0051] ③Detection: Take the reference solution and sample solution, inject them according to the chromatographic conditions respectively, record the chromatogram, and calculate the content of ectoine in the sample by the external standard one-point method.

[0052] ④Result calculation:

[0053] The content of ectoine in the sample = 100% × (As × mr × Pr) / (Ar × ms);

[0054] Where As is the peak area of ​​ectoine in the sample solution, Ar is the peak area of ​​ectoine in the reference solution, ms is the sample weight of ectoine (g), mr is the sample weight of ectoine reference (g), and Pr is the content of ectoine reference. The arithmetic mean of the results of two parallel determinations is used as the reported value, and the absolute difference between the two parallel determinations shall not exceed 2% of the arithmetic mean.

[0055] (7) Purity test method for finished ectoine:

[0056] ① Sample solution preparation: Take an appropriate amount of ectoine finished product sample, place it in a 105℃ oven for constant temperature drying for 3 hours, cool it to room temperature, accurately weigh 0.02g (accurate to 0.0001g), place it in a 100mL volumetric flask, add pure water to dissolve and dilute to the scale, shake well, and filter with a 0.22μm filter membrane. Prepare two copies in parallel.

[0057] ② Detection and result calculation: Take the sample solution and inject it according to the chromatographic conditions, record the chromatogram, and calculate the purity of ectoine in the sample by the peak area percentage according to the area normalization method. The arithmetic mean of the results of two parallel determinations shall be the reported value, and the absolute difference between the two parallel determination results shall not exceed 2% of the arithmetic mean.

[0058] The construction process and fermentation application of the engineering bacteria of the present invention are described below in conjunction with specific examples.

[0059] Example 1 Construction of tryptophan regulated expression system plasmid pRSFDuet*

[0060] The gene fragment of trpR-Ptrp-T7 polymerase (SEQ ID NO.1) was obtained by total gene synthesis. The gene fragment was used as template DNA, P1 and P2 were used as primers, and PCR amplification was performed to obtain the target gene fragment of trpR-Ptrp-T7 polymerase with the homologous sequence of plasmid pRSFDuet. The commercial plasmid pRSFDuet was used as template DNA, P3 and P4 were used as primers, and PCR amplification was performed to obtain the plasmid fragment with the lacI gene deleted. The target gene fragment of trpR-Ptrp-T7 polymerase with the homologous sequence of plasmid pRSFDuet and the plasmid fragment with the lacI gene deleted were subjected to recombination reaction, so that the target gene fragment of trpR-Ptrp-T7 polymerase replaced the lacI gene of plasmid pRSFDuet; the reaction product was transformed into Escherichia coli JM109 competent cells, coated with LB solid medium containing 50 mg / L kanamycin sulfate, and cultured at 37°C overnight. The transformants were verified by colony PCR using primers P1 and P2, and the correct transformants were expanded and cultured to extract plasmids, which were further sequenced for verification. The verified correct plasmid was named pRSFDuet*. The primer sequences in Example 1 are shown in Table 1.

[0061] Table 1 Primer names and sequences used in Example 1

[0062]

[0063] Example 2 Construction of recombinant plasmid pRSFDuet*-HhectABC-ppc-asd

[0064] The ectoine synthesis gene cluster fragment HhectABC (SEQ ID NO.2) derived from Halobacillus halophilus DSM 2266 and optimized for Escherichia coli codons was obtained by total gene synthesis. The ectoine synthesis gene cluster fragment HhectABC with a homologous sequence of plasmid pRSFDuet* was obtained by PCR amplification using the gene cluster fragment as template DNA and P5 and P6 as primers. The linearized plasmid pRSFDuet* was obtained by PCR amplification using the plasmid pRSFDuet* constructed in Example 1 as template DNA and P7 and P8 as primers. The ectoine synthesis gene cluster fragment HhectABC with the homologous sequence of plasmid pRSFDuet* and the linearized plasmid pRSFDuet* were recombined, and the gene cluster fragment HhectABC was connected to the downstream of the first T7 promoter of plasmid pRSFDuet* through the recombination reaction. The reaction product was transformed into Escherichia coli JM109 competent cells, coated with LB solid medium containing 50 mg / L kanamycin sulfate, and cultured at 37°C overnight. The transformants were verified by colony PCR using primers P5 and P6, and the correct transformants were expanded and cultured to extract plasmids, which were further sequenced for verification. The verified correct plasmid was named pRSFDuet*-HhectABC.

[0065] The genomic DNA of Escherichia coli BL21 was extracted using a bacterial genome extraction kit according to the instructions. Using the genomic DNA of Escherichia coli BL21 as a template and P9 and P10 as primers, the phosphoenolpyruvate carboxylase ppc gene fragment with a homologous sequence of plasmid pRSFDuet* was obtained by PCR amplification (GenBank accession number: NZ_CP170101, location=4027427..4030078); using P11 and P12 as primers, the aspartate semialdehyde dehydrogenase asd gene fragment with a homologous sequence of plasmid pRSFDuet* was obtained by PCR amplification (SEQ ID NO.3). Further, using the phosphoenolpyruvate carboxylase ppc gene fragment with a homologous sequence of plasmid pRSFDuet* and the aspartate semialdehyde dehydrogenase asd gene fragment with a homologous sequence of plasmid pRSFDuet* as template DNA, P9 and P12 as primers, the ppc-asd gene fragment was obtained by overlapping PCR amplification. Using plasmid pRSFDuet*-HhectABC as template DNA and P13 and P14 as primers, linearized plasmid pRSFDuet*-HhectABC was obtained by PCR amplification. The ppc-asd gene fragment and the linearized plasmid pRSFDuet*-HhectABC were recombined, and the ppc-asd gene fragment was connected to the downstream of the second T7 promoter of the recombinant plasmid pRSFDuet*-HhectABC. The reaction product was transformed into Escherichia coli JM109 competent cells, coated with LB solid medium containing 50 mg / L kanamycin sulfate, and cultured overnight at 37°C. The transformants were verified by colony PCR using primers P9 and P12, and the correct transformants were expanded and cultured to extract plasmids, and further sequenced for verification. The verified correct plasmid was named pRSFDuet*-HhectABC-ppc-asd. The sequences of the primers in Example 2 are shown in Table 2.

[0066] Table 2 Primer names and sequences used in Example 2

[0067]

[0068] Example 3 Knockout of the lacI gene of Escherichia coli BL21 and modification of the phosphotransferase system

[0069] The idea of ​​knocking out the lacI gene of E. coli BL21 and modifying the phosphotransferase system is as follows: the lacI gene of E. coli BL21 (GenBank accession number: NZ_CP170101, location=337260..338342) was knocked out using CRISPR / Cas9 technology to obtain E. coli BL21ΔlacI. The glucose facilitator protein encoding gene Zmglf (SEQ ID NO.4) derived from Zymomonas mobilis, containing the constitutive promoter PJ23119 and having been codon-optimized for E. coli, was obtained by total gene synthesis. The above gene was integrated into the ptsG gene site of the E. coli BL21ΔlacI genome (GenBank accession number: NZ_CP170101, location=1128713..1130146) using CRISPR / Cas9 technology to obtain E. coli BL21ΔlacIΔptsG::Zmglf with modified phosphotransferase system.

[0070] Example 3 illustrates an optional and specific modification operation method, which is as follows: using the E. coli BL21 genome as template DNA, using P15 and P16 as primers, PCR amplification is performed to obtain the gene fragment lacI-U; using P17 and P18 as primers, PCR amplification is performed to obtain the gene fragment lacI-D. Further, using the lacI-U gene fragment and the lacI-D gene fragment as template DNA, using P15 and P18 as primers, overlapping PCR amplification is performed to obtain the lacI gene deletion cassette.

[0071] The commercial plasmid pTarget was used as a template, and P19 (lacI site sgRNA primer) and P20 were used as primers for full plasmid PCR amplification. The obtained PCR product was digested, purified and ligated, and transformed into Escherichia coli JM109 competent cells by chemical transformation. The cells were coated with LB solid medium plates containing 30 mg / L spectinomycin and cultured at 37°C to obtain single colonies. Primers P20 and P21 were used for colony PCR verification, and the correct transformants were expanded and cultured to extract plasmids, which were further sequenced for verification. The verified correct plasmid was named pTargetF-sglacI.

[0072] E. coli BL21 was activated on LB solid medium and inoculated into LB liquid medium for overnight culture. The seed solution was transferred to 25 mL LB liquid medium at 1% and cultured at 30°C and 200 rpm until OD 600=0.5, ice bath for 30 min; 4°C, 4000 rpm centrifugation for 10 min to collect the bacteria, wash the bacteria three times with pre-cooled 10% glycerol solution; add 300 μL 10% glycerol solution to resuspend the bacteria, and distribute them into sterile 1.5 mL centrifuge tubes, 80 μL / tube, to prepare Escherichia coli BL21 electrotransformation competent cells.

[0073] The commercial plasmid pCas was electrotransformed into the electrocompetent cells of Escherichia coli BL21, and the cells were coated with LB solid medium containing 30 mg / L kanamycin, and cultured at 37°C to obtain a single colony BL21 / pCas. The electrocompetent cells of Escherichia coli BL21 / pCas were prepared, and the lacI gene deletion box and pTargetF-sglacI plasmid were electrotransformed into the competent cells at the same time, and the cells were coated with LB solid medium plates containing 30 mg / L kanamycin and spectinomycin, and cultured at 30°C to obtain a single colony. Colony PCR verification was performed using primers P15 and P18 to verify that the correct strain was Escherichia coli BL21ΔlacI / pCas / pTargetF-sglacI.

[0074] E. coli BL21ΔlacI / pCas / pTargetF-sglacI was inoculated into LB liquid medium containing kanamycin, IPTG with a final concentration of 0.5 mM was added, and cultured at 30°C and 200 rpm for 2 h, and then coated with LB solid medium plates containing 30 mg / L kanamycin and cultured at 30°C. Single colonies without spectinomycin resistance were screened to obtain E. coli BL21ΔlacI / pCas without the pTargetF-sglacI plasmid.

[0075] The glucose facilitator protein encoding gene Zmglf (SEQ ID NO. 4) derived from Zymomonas mobilis, containing the constitutive promoter PJ23119 and having been codon-optimized for Escherichia coli, was obtained by total gene synthesis. The gene fragment ptsG-U was obtained by PCR amplification using the Escherichia coli BL21 genome as template DNA and P22 and P23 as primers; the gene fragment ptsG-D was obtained by PCR amplification using P24 and P25 as primers. Furthermore, the Zmglf gene expression cassette was obtained by overlapping PCR amplification using lacI-U, lacI-D and Zmglf gene fragments as template DNA and P22 and P25 as primers.

[0076] The commercial plasmid pTarget was used as a template, and P26 (ptsG site sgRNA primer) and P20 were used as primers for full plasmid PCR amplification. The resulting PCR product was digested, purified and ligated, and transformed into Escherichia coli JM109 competent cells by chemical transformation. The cells were coated with LB solid medium plates containing 30 mg / L spectinomycin and cultured at 37°C to obtain single colonies. Primers P20 and P27 were used for colony PCR verification, and the correct transformants were expanded and cultured to extract plasmids, which were further sequenced for verification. The verified correct plasmid was named pTargetF-sgptsG.

[0077] E. coli BL21ΔlacI / pCas was used to prepare electroporation competent cells, and the Zmglf gene expression cassette and pTargetF-sgptsG plasmid were electroporated into the competent cells at the same time, and the cells were spread on LB solid medium plates containing 30 mg / L kanamycin and spectinomycin, and cultured at 30°C to obtain single colonies. Colony PCR verification was performed using primers P22 and P25, and the correct strain was verified to be E. coli BL21ΔlacIΔptsG::Zmglf / pCas / pTargetF-sgptsG.

[0078] E. coli BL21ΔlacIΔptsG::Zmglf / pCas / pTargetF-sgptsG was inoculated into LB liquid medium containing kanamycin, IPTG with a final concentration of 0.5 mM was added, and cultured at 30°C and 200 rpm for 2 h, and then coated with LB solid medium plates containing 30 mg / L kanamycin and cultured at 30°C. Single colonies without spectinomycin resistance were screened to obtain E. coli BL21ΔlacIΔptsG::Zmglf / pCas with the pTargetF-sgptsG plasmid removed.

[0079] E. coli BL21ΔlacIΔptsG::Zmglf / pCas was inoculated into an LB liquid medium without resistance, cultured at 42°C and 200 rpm for 2 hours, and then coated onto an LB solid medium plate without resistance, and cultured at 42°C. Single colonies without kanamycin resistance were screened to obtain E. coli BL21ΔlacIΔptsG::Zmglf without the pCas plasmid.

[0080] Table 3 Primer names and sequences used in Example 3

[0081]

[0082] Example 4 Construction of engineered bacteria producing tetrahydropyrimidine

[0083] The Escherichia coli BL21ΔlacIΔptsG::Zmglf of Example 3 was used to prepare electroporation competent cells, and the recombinant plasmid pRSFDuet*-HhectABC-ppc-asd of Example 2 was used to transform competent cells, and LB solid medium supplemented with 50 mg / L kanamycin sulfate was applied. The positive transformants were verified by PCR using P5 and P12 as primers, and the correct transformants were verified to be the ectoine-producing engineered bacteria BL21ΔlacIΔptsG::Zmglf / pRSFDuet*-HhectABC-ppc-asd. The ectoine synthesis pathway of the ectoine-producing engineered bacteria constructed by the present invention is as follows: Figure 1 As shown, the engineered bacteria knocked out the lacI gene of Escherichia coli BL21 and constructed a tryptophan-regulated expression system. When tryptophan exists in the culture medium, product synthesis is inhibited and the bacteria can grow and reproduce rapidly. When tryptophan in the culture medium is exhausted, product synthesis starts ( Figure 2 ). The synthesis of the product ectoine can be regulated without adding inducers, effectively reducing the cost of fermentation materials. In addition, the phosphotransferase system of Escherichia coli BL21 was modified, and the glucose facilitator protein encoding gene Zmglf from Zymomonas mobilis was integrated at the ptsG gene locus to relieve the inhibition of carbon catabolism, so that the strain can use cheap agricultural waste rich in glucose and xylose as a carbon source to synthesize the product ectoine; the engineered bacteria enhanced the accumulation of precursor substances oxaloacetate and L-aspartate-β-semialdehyde during growth metabolism by overexpressing the phosphoenolpyruvate carboxylase encoding gene ppc and the aspartate semialdehyde dehydrogenase encoding gene asd, thereby improving the synthesis level of the product ectoine.

[0084] Example 5 5L tank fermentation to produce tetrahydropyrimidine

[0085] The tetrahydropyrimidine-producing engineered bacteria BL21ΔlacIΔptsG::Zmglf / pRSFDuet*-HhectABC-ppc-asd obtained in Example 4 was streaked and activated on LB solid medium supplemented with 50 mg / L kanamycin sulfate and cultured at 37°C for 15 h. The activated colonies were inoculated into shake flask fermentation medium and cultured at 37°C and 220 rpm for 7.5 h. OD 600=3.2, prepare seed liquid. Inoculate the cultured seed liquid at a 4% inoculation rate into a 5L fermenter with an initial liquid volume of 2L fermentation medium. Control the fermentation temperature to 37°C, control the pH to 6.8-7.2 by adding ammonia water, set the initial ventilation volume to 1vvm, the initial stirring speed to 300rpm, and control the dissolved oxygen to 30-40% by adjusting the ventilation volume and stirring speed. When the carbon source in the basal culture medium is exhausted, the dissolved oxygen rises to 45g / h constant flow feed medium for 30h of fermentation, and 20g / h constant flow feed medium is added after 30h until the end of fermentation. The yield of tetrahydropyrimidine is 86.9g / L at 72h of fermentation.

[0086] Example 6 50L tank fermentation to produce tetrahydropyrimidine

[0087] The tetrahydropyrimidine-producing engineered bacteria BL21ΔlacIΔptsG::Zmglf / pRSFDuet*-HhectABC-ppc-asd obtained in Example 4 was streaked and activated on LB solid medium supplemented with 50 mg / L kanamycin sulfate and cultured at 37°C for 15 h. The activated colonies were inoculated into shake flask fermentation medium and cultured at 37°C and 220 rpm for 8 h. OD 600 =3.7, prepare the first-level seed solution. Inoculate the shake flask fermentation medium with the first-level seed solution at a 1.5% inoculation rate, and culture at 37°C and 220 rpm for 5.5 h. 600 =3.3, prepare the first-level seed liquid. The cultured second-level seed liquid was inoculated at a 6% inoculation rate into a 50L fermenter with an initial liquid volume of 15L fermentation medium. The fermentation temperature was controlled at 37°C, and the pH was controlled at 6.8-7.2 by adding ammonia water. The initial ventilation volume was set to 1vvm, the initial stirring speed was 200rpm, and the dissolved oxygen was controlled to 30-40% by adjusting the ventilation volume and stirring speed. When the carbon source in the basal culture medium is exhausted and the dissolved oxygen recovers, feeding begins. From the start of feeding to 30h of fermentation, the feed medium is added at a constant rate of 350g / h. After 30h until the end of fermentation, the feed medium is added at a constant rate of 160g / h. The maximum yield of tetrahydropyrimidine is 94.5g / L ( Figure 3 ).

[0088] Example 7 Separation and purification of tetrahydropyrimidine in fermentation broth

[0089] 28L of tetrahydropyrimidine fermentation broth obtained in Example 6 was filtered through a 50nm ceramic membrane, the pressure was controlled to be 0.1~0.4MPa, and 1 volume of water was added to collect the clear liquid. The collected ceramic membrane clear liquid was added with activated carbon YL-303 at a ratio of 1.5%, stirred at 65°C for 1h, and the activated carbon was removed using a 0.45μm pore size filter membrane or filter, and the decolorized liquid was collected. The decolorized liquid was adsorbed by a 732 type resin, the feed rate was controlled to be 1BV / h, and after the feed was completed, 1BV of purified water was used for top washing, and then 0.2M ammonia water was used as an eluent for product elution, the elution rate was controlled to be 1BV / h, and the eluate with an outlet product content of >0.5% was collected. The eluate was concentrated under reduced pressure until the product concentration reached 550g / L, cooled to 25°C, anhydrous ethanol of the same volume as the concentrate was added, stirred at 50rpm until crystals precipitated, and then cooled to 5°C and stirred for 2h. The crystals were separated from the mother liquor by filtration, and an equal volume of anhydrous ethanol was added to the crystals for washing, and the crystals were collected. The crystals were dried at 75°C for 6 hours to constant weight to obtain a finished tetrahydropyrimidine product with good properties ( Figure 4 ), liquid chromatography purity is 99.787% ( Figure 5 ).

[0090] Comparative Example 1 5L tank fermentation to produce tetrahydropyrimidine

[0091] The 5L tank fermentation of the tetrahydropyrimidine engineering bacteria was carried out under the same conditions as in Example 5, except that the Escherichia coli used in this comparative example was BL21ΔlacI / pRSFDuet*-HhectABC-ppc-asd, and the phosphotransferase system was not modified compared with the Escherichia coli BL21ΔlacIΔptsG::Zmglf / pRSFDuet*-HhectABC-ppc-asd used in Example 5. The tetrahydropyrimidine yield of this comparative example fermented to 72h was 56.3g / L. It shows that the modification of the phosphotransferase system of the engineering bacteria in the present invention effectively improves the utilization of the carbon source by the strain and the synthesis of the product tetrahydropyrimidine.

[0092] Comparative Example 2: Production of tetrahydropyrimidine by fermentation in 5L tank

[0093] The 5L tank fermentation of tetrahydropyrimidine engineering bacteria was carried out under the same conditions as in Example 5, except that the Escherichia coli used in this comparative example was BL21ΔlacIΔptsG::Zmglf / pRSFDuet*-HhectABC, and compared with the Escherichia coli BL21ΔlacIΔptsG::Zmglf / pRSFDuet*-HhectABC-ppc-asd used in Example 5, the overexpression of the phosphoenolpyruvate carboxylase encoding gene ppc and the aspartate semialdehyde dehydrogenase encoding gene asd was not performed. The tetrahydropyrimidine yield of this comparative example fermented to 72h was 70.4g / L. It shows that the overexpression of key genes in the tetrahydropyrimidine synthesis pathway of the engineering bacteria in the present invention strengthens the synthesis of precursor substances and effectively improves the synthesis of the product tetrahydropyrimidine.

[0094] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for biosynthesis of tetrahydropyrimidine, characterized in that: The method comprises the following steps: using ectoine-producing genetically engineered bacteria to carry out biosynthesis of ectoine; adding tryptophan to a basic fermentation medium to inhibit the synthesis of ectoine products through tryptophan, thereby causing the bacteria to grow and reproduce; when the carbon source in the basic fermentation medium is exhausted and the dissolved oxygen rises, continuously adding a feed medium, wherein the feed medium does not contain tryptophan, and ectoine synthesis is initiated to produce ectoine; The basic fermentation medium formula includes: yeast extract powder 7-10 g / L, ammonium sulfate 5-8 g / L, corn steep liquor powder 5-8 g / L, corn cob hydrolyzate equivalent to total reducing sugar 13-20 g / L, potassium dihydrogen phosphate 2-3 g / L, dipotassium hydrogen phosphate 2-3 g / L, magnesium sulfate 0.5-1.5 g / L, citric acid 1-1.5 g / L, ferrous sulfate 0.01-0.02 g / L, manganese sulfate 0.01-0.02 g / L, and L-tryptophan 0.1-0.2 g / L. The feed medium formula is: yeast extract powder 5-7 g / L, corn cob hydrolyzate equivalent to total reducing sugar 500-600 g / L, magnesium sulfate 2-3 g / L; The ectoine-producing genetically engineered bacteria are constructed by the following method: S1: Synthesize the SEQ ID NO. trpR -Ptrp-T7 polymerase gene fragment, plasmid pRSFDuet lacI Gene use trpR -Ptrp-T7 polymerase gene fragment was replaced to obtain the recombinant plasmid pRSFDuet*; S2: Synthesize the ectoine synthesis gene cluster fragment HhectABC shown in SEQ ID NO.2, and connect it to the downstream of the first T7 promoter of plasmid pRSFDuet* to obtain the recombinant plasmid pRSFDuet*- HhectABC ; S3: Using the genome of Escherichia coli BL21 as a template, obtain phosphoenolpyruvate carboxylase ppc Gene fragment and aspartate semialdehyde dehydrogenase shown in SEQ ID NO.3 asd Gene fragments were obtained by overlapping PCR amplification ppc - asd The gene fragment was connected to the recombinant plasmid pRSFDuet*- HhectABC The second T7 promoter was downstream of the recombinant plasmid pRSFDuet*- HhectABC - ppc - asd ; S4: Escherichia coli BL21 lacI The gene was knocked out to obtain Escherichia coli BL21Δ lacI ; Synthesize the gene encoding the glucose facilitator protein shown in SEQ ID NO.4 Zgf ;Will Zgf Gene integration into E. coli BL21Δ lacI Genomic ptsG Gene locus, and the phosphotransferase system-modified Escherichia coli BL21Δ lacI Δ ptsG :: Zgf ; S5: Escherichia coli BL21Δ lacI Δ ptsG :: Zgf Prepare electroporation competent cells and transfer the recombinant plasmid pRSFDuet*- HhectABC - ppc - asd The competent cells were transformed to obtain the ectoine-producing engineered bacteria.

2. The method for biosynthesis of ectoine according to claim 1, characterized in that: S1, the plasmid pRSFDuet lacI Gene use trpR -Ptrp-T7 polymerase gene fragment is replaced, comprising: using plasmid pRSFDuet as a template DNA, using primer sequences shown in SEQ ID NO.7 and SEQ ID NO.8, obtaining a plasmid fragment with a lacI gene deleted by PCR amplification, and performing a recombination reaction on the trpR-Ptrp-T7 polymerase target gene fragment and the plasmid fragment with a lacI gene deleted to obtain a recombinant plasmid pRSFDuet*.

3. The method for biosynthesis of ectoine according to claim 1, characterized in that: In S3, the phosphoenolpyruvate carboxylase is obtained by PCR amplification using the genomic DNA of Escherichia coli BL21 as a template and the primer sequences shown in SEQ ID NO.13 and SEQ ID NO.

14. ppc Gene fragment; using Escherichia coli BL21 genomic DNA as a template and the primer sequences shown in SEQ ID NO.15 and SEQ ID NO.16, the aspartate semialdehyde dehydrogenase was obtained by PCR amplification asd Gene fragments; Phosphoenolpyruvate carboxylase ppc Aspartate semialdehyde dehydrogenase asd The gene fragment was used as template DNA, and the primer sequences shown in SEQ ID NO.13 and SEQ ID NO.16 were used to obtain the ppc - asd Gene fragment.

4. The method for biosynthesis of ectoine according to claim 1, characterized in that: In S4, the Escherichia coli BL21 lacI The GenBank accession number of the gene is NZ_CP170101, location = 337260..338342; CRISPR / Cas9 technology was used to generate the lacI Gene knockout, lacI The sgRNA primer of the site is shown as SEQ ID NO.

23.

5. The method for biosynthesis of ectoine according to claim 1, characterized in that: Said ptsG The GenBank accession number of the gene is: NZ_CP170101, location = 1128713..1130146. The CRISPR / Cas9 technology was used to clone the gene encoding the glucose facilitator protein. Zgf Integration into E. coli BL21Δ lacI Genomic ptsG Genetic loci, used by the integration process ptsG The sgRNA primer of the gene is shown in SEQ ID NO.

30.

6. The method for biosynthesis of ectoine according to claim 1, characterized in that: The glucose facilitator protein encoding gene Zgf From Zymomonas mobilis , and contains constitutive promoter PJ23119 and has been codon-optimized for E. coli; the glucose facilitator protein encoding gene Zgf The sequence is shown in SEQ ID NO.4.

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