Engineering escherichia coli pMki-nfss as well as construction method and application thereof

Abstract

The invention provides engineering escherichia coli pMki-nfss as well as a construction method and application thereof. The engineering escherichia coli comprises an mki gene and an nfss sequence. Wherein the nucleotide sequence of the mki gene is as shown in SEQ ID NO: 1, and the nucleotide sequence of the nfss sequence is as shown in SEQ ID NO: 2. According to the invention, a P-M recombinant plasmid containing an mki gene and a P-N recombinant plasmid containing an nfss sequence are co-transformed into E.coli BL21, such that engineering escherichia coli pMki-nfss is obtained. The engineering Escherichia coli can be used for producing sesterterpene fish oil alcohol through a whole-cell catalytic reaction and can be used for producing triterpenoid squalene through an enzyme catalytic reaction. The method disclosed by the invention is convenient to operate, high in efficiency and simple in equipment, has a better industrial application prospect in the field of biosynthesis of terpenoids, and is of great significance to research on biocatalysis of synthesis of terpenoids in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to an engineered Escherichia coli pMki-nfss and its construction method and application. Background Art

[0002] Terpenoids are one of the largest classes of natural products in nature, with thousands of known compounds. More than half of them are produced by terrestrial and marine plants, and they are crucial for the growth, development, and defense mechanisms of organisms against threats. Terpenoids are diverse in structure and function, and they also have a variety of pharmacological activities and are widely used in industries such as the pharmaceutical, cosmetic, and food industries.

[0003] Sesterterpenoids are important members of the terpenoid natural product family, with chemical skeletons ranging from acyclic, monocyclic to hexacyclic systems and extremely high structural diversity. These compounds exhibit a variety of biological properties, including anti-cancer, antibacterial, anti-inflammatory, immunosuppressive, anti-lipogenic, anti-protozoal, and antifeedant activities. In terms of anti-lipogenic activity, sesterfisherol can inhibit lipid content to 94.41% at 80 μmol / L. Squalene is a triterpenoid compound and is a unique active nutrient, docosahexaene, which has various physiological effects such as enhancing the activity of superoxide dismutase in the body, enhancing the immune capacity of the body, anti-fatigue, anti-aging, and anti-tumor. The main effects include the following aspects: First, it activates cells, supplies oxygen, and enhances physical strength. It can also improve the state of cell hypoxia and activate the body's functions, helping to eliminate fatigue. Second, it purifies the blood, promotes blood circulation, and improves the acidic constitution, which is beneficial for preventing or treating diseases caused by poor blood circulation, such as heart disease, hypertension, and stroke. Third, it strengthens the liver to protect the liver. Squalene can enhance liver function and bile secretion, reduce the liver enzyme level in hepatitis patients, and increase appetite, which has a certain effect on treating hepatitis. In addition, squalene can promote the metabolism of liver fat and assist in the treatment of fatty liver. Fourth, it enhances the body's immune function and has an anti-tumor effect. Squalene can significantly increase the level of superoxide dismutase, reduce hematopoietic tissue damage, accelerate the generation of hematopoietic cells, and prevent excessive destruction of white blood cells. At the same time, squalene can also enhance the metabolic function of white blood cells, which helps in the adjuvant treatment of various inflammations.

[0004] In recent years, terpenoids have received increasing attention and have been found in various sources including fungi, plants, bacteria, insects, and various marine invertebrates. Terpenoids are mainly obtained through plant biomass extraction methods, but the yields are often low, thus unable to meet industrial demands. Moreover, this production method may also be affected by seasonal and geographical variations. In addition, although chemical synthesis methods can also be used to produce terpenoids, these operations may be energy-intensive and generate a large amount of organic waste. The demand for alternative, efficient, sustainable, and eco-friendly supply routes for terpenoids has not been met. Nowadays, converting microorganisms into cell factories for the de novo biosynthesis of natural products is a very promising method. The two key precursors for terpenoid synthesis, IPP and DMAPP, are mainly produced through the methylerythritol-4-phosphate pathway (MEP) and the mevalonate pathway (MVA). However, these two pathways involve more than a dozen reactions, and the production process is complex and difficult to control. Therefore, constructing an ideal microbial host for producing terpenoids is crucial. Summary of the Invention

[0005] The purpose of the present invention is to provide an engineered Escherichia coli pMki-nfss and its construction method and application. By co-transforming the P-M recombinant plasmid containing the mki gene and the P-N recombinant plasmid containing the nfss sequence into E.coli BL21, the engineered Escherichia coli pMki-nfss is obtained. This engineered Escherichia coli can produce sesterterpenoid ichthyol using whole-cell catalytic reactions and can produce triterpenoid squalene using enzyme-catalyzed reactions, and has good industrial application prospects.

[0006] The present invention solves its technical problems by adopting the following technical solutions.

[0007] The present invention provides an engineered Escherichia coli pMki-nfss , comprising mki gene and nfss sequence. The nucleotide sequence of the mki gene is as shown in SEQ ID NO: 1, and the nucleotide sequence of the nfss sequence is as shown in SEQ ID NO: 2.

[0008] The present invention provides a construction method of an engineered Escherichia coli pMki-nfss , comprising the following steps: S1. After seamlessly connecting the gene sequences of Ec ThiM, Mj IPK, and IDI proteins, the mki gene is obtained. Then, using Eco R I and HinUse dIII as a restriction enzyme cleavage site to clone the mki gene into pCDFDuet-1 to construct the P-M recombinant plasmid; S2. Use Bam HI and Hin dIII as a restriction enzyme cleavage site to clone the nfss sequence into pRSFDuet-1 to construct the P-N recombinant plasmid; S3. Co-transform the P-M recombinant plasmid and the P-N recombinant plasmid into E.coli BL21 to obtain the engineered Escherichia coli pMki-nfss .

[0009] The present invention provides the application of the said engineered Escherichia coli pMki-nfss in the preparation of seseterfisherol.

[0010] The present invention provides the application of the said engineered Escherichia coli pMki-nfss in the preparation of squalene.

[0011] The beneficial effects of the engineered Escherichia coli of the embodiments of the present invention pMki-nfss and its construction method and application are as follows: (1) The strain of the present invention is simply modified in the ideal host Escherichia coli, that is, by co-transforming the recombinant plasmid containing the mki gene and the recombinant plasmid containing the nfss sequence, the engineered Escherichia coli pMki-nfss is successfully constructed. The present invention constructs the IUP pathway to replace the cumbersome MVA and MEP pathways, replaces more than a dozen steps of reactions with 4 genes in 3 steps, and uses the same engineered strain to produce scarce sesquiterpenes and triterpenoids through two different catalytic methods of whole cells and enzymes.

[0012] (2) The engineered Escherichia coli of the present invention pMki-nfss can produce complex sesquiterpenes and triterpenoids from the cheap substrate isoprenol after activation and strain culture, that is, it can produce seseterfisherol by whole cell catalytic reaction, and can produce squalene by enzyme catalytic reaction. The operation is convenient, the efficiency is high, and the equipment is simple, and it has good industrial application prospects in the field of terpene compound production. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0014] Figure 1 It is the GC-MS spectrum of the reaction product of Example 2 of the present invention; Figure 2 It is the GC-MS spectrum of the reaction product of Example 3 of the present invention; Figure 3 It is the GC-MS spectrum of the reaction product of Example 4 of the present invention; Figure 4 It is the GC-MS spectrum of the reaction product of Example 5 of the present invention; Figure 5 It is the GC-MS spectrum of the reaction product of Example 6 of the present invention; Figure 6 It is the GC-MS spectrum of the reaction product of Example 7 of the present invention. Detailed implementation manners

[0015] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0016] The following specifically describes the engineered Escherichia coli pMki-nfss of the embodiments of the present invention,

[0017] The present invention provides an engineered Escherichia coli pMki-nfss , comprising mki gene and nfss sequence, the nucleotide sequence of the mki gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the nfss sequence is shown in SEQ ID NO: 2. The engineered Escherichia coli of the present invention pMki-nfss contains two modules: the first module constructs the substrate isoprenol utilization pathway (IUP pathway) by direct splicing of three gene sequences (i.e., mki gene), and the second module contains the fungal bifunctional sesterterpene synthase nfss . The present invention successfully constructs the engineered Escherichia coli mki by co-transforming the recombinant plasmid containing the nfss sequence and the recombinant plasmid containing the pMki-nfss . An IUP pathway is constructed in this engineered Escherichia coli, and terpenoids can be obtained by replacing the MVA and MEP pathways with only three steps of reactions. Different types of terpenoids can be produced in two different environments of whole-cell catalysis and enzyme catalysis.

[0018] The present invention also provides a method for constructing an engineered Escherichia coli pMki-nfss , comprising the following steps: S1. After seamlessly ligating the gene sequences of ThiM, Ec IPK, and IDI proteins, the Mj gene is obtained. Then, using mki R I and Eco d III as restriction enzyme cutting sites, the Hin gene is cloned into pCDFDuet-1 to construct the P-M recombinant plasmid. mki

[0019] S2. Using Bam HI and Hin d III as restriction enzyme cutting sites, the nfss sequence is cloned into pRSFDuet-1 to construct the P-N recombinant plasmid.

[0020] S3. Co-transforming the P-M recombinant plasmid and the P-N recombinant plasmid into E.coli BL21 to obtain the engineered Escherichia coli pMki-nfss . It should be noted that the present invention can also separately construct the P-M recombinant plasmid and the P-N recombinant plasmid, and transform them into Escherichia coli BL21 respectively to obtain strains pCDF-mki and pRSF-nfss , and use the strains pCDF-mki and pRSF- nfss for the preparation of terpenoid compounds.

[0021] The present invention also provides the use of the above-mentioned engineered Escherichia coli pMki-nfss in the preparation of sesterterpenoid ichthyol.

[0022] Furthermore, in a preferred embodiment of the present invention, the above-mentioned engineered Escherichia coli pMki-nfss uses whole-cell catalysis to produce the sesterterpenoid ichthyol, comprising the following steps: Taking the bacterial liquid of the above-mentioned engineered Escherichia coli pMki-nfss , inoculating it into a TB medium containing double antibiotics and culturing it in a shake flask until the OD 600 reaches 0.6 - 0.8. Then, taking out the TB medium containing double antibiotics, adding IPTG for induction and simultaneously adding isopentenol, and after shake flask fermentation and extraction, the sesterterpenoid ichthyol is obtained.

[0023] Furthermore, in a preferred embodiment of the present invention, the temperature of the shake flask fermentation is 16 - 18 °C, and the time of the shake flask fermentation is 72 - 76 h.

[0024] The present invention also provides the above-mentioned engineered Escherichia coli pMki-nfssApplication in the preparation of triterpenoid squalene. The engineered Escherichia coli of the present invention pMki-nfss can use whole-cell catalytic reaction to produce the sesterterpene seseterfisherol, and use enzyme-catalyzed reaction to generate the triterpenoid squalene, which is convenient to operate and has good industrial application prospects in the field of terpene biosynthesis, and is of relatively important significance for the future research on the biosynthesis of terpene compounds by biocatalysis.

[0025] Furthermore, in a preferred embodiment of the present invention, the engineered Escherichia coli pMki-nfss uses enzyme catalysis to produce the triterpenoid squalene, including the following steps: Take the bacterial liquid of the engineered Escherichia coli pMki-nfss and inoculate it into a TB medium containing double antibiotics for shake flask culture until the OD 600 reaches 0.6 - 0.8. Take out the TB medium containing double antibiotics and add IPTG for induction. After shake flask culture, centrifuge and remove the supernatant, then resuspend the bacterial liquid with an imidazole solution. After ultrasonic treatment and centrifugation again, an enzyme solution is obtained; Mix ATP, MgCl 2 , the enzyme solution, isopentenol and Tris-HCl and react. After extraction, the triterpenoid squalene is obtained.

[0026] Furthermore, in a preferred embodiment of the present invention, the TB medium includes tryptone, yeast extract, glycerol, KH 2 PO 4 , K 2 HPO 4 and water. The mass-volume ratios of the tryptone, the yeast extract, the glycerol, the KH 2 PO 4 , the K 2 HPO 4 and the distilled water are 3:6:1:0.5775:3.135:250 (g / g / mL / g / g / mL).

[0027] Furthermore, in a preferred embodiment of the present invention, the imidazole solution includes NaCl, imidazole and distilled water. The mass-volume ratios of the NaCl, the imidazole and the distilled water are 4.25 - 4.35:1:734 - 736 (g / g / mL).

[0028] Furthermore, in a preferred embodiment of the present invention, the temperature of the shake flask culture is 16 - 18 °C, the shake flask culture time is 20 - 24 h, and the reaction time is 3 h.

[0029] The present invention passes through Ec ThiM from Escherichia coli MjAfter the gene sequences of IPK and IDI proteins are seamlessly ligated, the mki gene is obtained, and Eco R I and Hin d III are used as restriction enzyme cutting sites to mki clone the gene into pCDFDuet-1 to construct the P-M recombinant plasmid, and Bam H I and Hin d III are used as restriction enzyme cutting sites to clone the gene sequence of the protein Nfss from Novosphingobium aromaticivorans into pRSFDuet-1 to construct the P-N recombinant plasmid. Subsequently, the P-M recombinant plasmid and the P-N recombinant plasmid are co-transformed into E.coli BL21(DE3) to obtain the engineered Escherichia coli pMki-nfss . This engineered Escherichia coli pMki-nfss can be activated and cultured to obtain a functional strain, which can produce two types of terpenoid compounds by using only 4 genes when using the simple and inexpensive substrate isoprenol.

[0030] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0031] Example 1 This example provides an engineered Escherichia coli pMki-nfss . First, the target gene mki is screened and designed, and Eco R I and Hin d III are used as restriction enzyme cutting sites to mki clone the gene into the vector pCDFDuet-1 to construct the P-M recombinant plasmid; then Bam H I and Hin d III are used as restriction enzyme cutting sites to nfss clone the target sequence into the vector pRSFDuet-1 to construct the P-N recombinant plasmid. Finally, the P-M recombinant plasmid and the P-N recombinant plasmid are co-transformed into E.coli BL21(DE3), and the specific steps are as follows: Place E.col i BL21(DE3) competent cells on ice for 5 min, add 5 μL each of the target plasmids DNA P-M and P-N, pipette evenly, after ice-bathing for 30 min, heat-shock in a water bath at 42 °C for 90 s, quickly put it back on ice and let it stand for 3 min, add 800 μL of LB medium to the EP tube, place it at 37 °C, and culture it on a shaker at 200 rpm for 1 h. Take 100 μL and spread it on an LB solid culture dish with kanamycin and spectinomycin resistance, culture it at a constant temperature of 37 °C for 10 h, pick a single colony and culture it in 50 mL of LB liquid medium with the above two resistances at 37 °C and 200 rpm on a shaker for 10 h.

[0032] Example 2 This example uses the engineered Escherichia coli prepared in Example 1 pMki-nfss and produces sesterfisherol through a whole-cell catalytic reaction. The specific steps are as follows: (1) Preparation of engineered Escherichia coli pMki-nfss Bacterial liquid preparation: The engineered Escherichia coli strain in Example 1 pMki-nfss was screened on a double-antibiotic plate. After culturing on the plate for 12 - 16 hours, single colonies were picked and cultured in a shake flask with LB liquid medium for 8 - 12 hours to obtain the engineered Escherichia coli pMki-nfss bacterial liquid. Among them, the composition of the LB liquid medium is: 0.2 g of peptone, 0.1 g of yeast extract, 0.2 g of NaCl, and it is prepared with 20 mL of distilled water.

[0033] (2) Whole-cell catalytic reaction: Take 1% of the engineered Escherichia coli pMki-nfss bacterial liquid from step (1) and inoculate it into a shake flask with TB medium containing double antibiotics for culture until the OD 600 reaches 0.6 - 0.8. Then, take out the medium, add 0.1 mM IPTG for induction and simultaneously add 25 mM isopentenol, and continue shake flask fermentation at 18°C for 72 hours to obtain the fermentation broth. Among them, the composition of the TB medium is: 6 g of tryptone, 12 g of yeast extract, 2 mL of glycerol, 1.155 g of KH 2 PO 4 and 6.27 g of K 2 HPO 4 , and it is prepared with 500 mL of distilled water.

[0034] (3) Analysis and detection: After extracting the fermentation broth with an equal volume of n-hexane, take 1 microliter for GC-MS analysis. As Figure 1 shown is the GC-MS spectrum of the reaction product in Example 2. According to the analysis of the chromatogram and mass spectrum, it is found that the peak appearing at 21.972 min in the chromatogram corresponds to the substance at this elution position in the mass spectrum analysis, which has the characteristic ion peaks of 340 m / z and 358 m / z of sesterfisherol. By comparing the experimental GC-MS diagram with the GC-MS diagram of sesterfisherol in the literature, it is confirmed that the product at 21.972 min is sesterfisherol.

[0035] Example 3 This example uses the engineered Escherichia coli prepared in Example 1 pMki-nfss and produces triterpenoid squalene through an enzyme-catalyzed reaction. The specific steps are as follows: (1) Engineered Escherichia coli pMki-nfssPreparation of Bacterial Solution: The engineered Escherichia coli in Example 1 pMki-nfss strain was screened on a double-antibody plate. That is, after culturing on the plate for 12 - 16 hours, single colonies were picked and cultured in a shaking flask with LB liquid medium for 8 - 12 hours to obtain the engineered Escherichia coli pMki-nfss bacterial solution. Among them, the components of the LB liquid medium are: 0.2 g of peptone, 0.1 g of yeast extract, 0.2 g of NaCl, and it is prepared with 20 mL of distilled water.

[0036] (2) Preparation of Enzyme Sample: Take 1% of the engineered Escherichia coli pMki-nfss bacterial solution from step (1) and inoculate it into TB medium containing double antibodies for shaking flask culture until OD 600 reaches 0.6 - 0.8. Then, add 0.1 mM IPTG to the medium for induction and continue shaking flask culture at 18 °C for 20 hours. Take out the medium and centrifuge it at 4000 rpm for 20 min to remove the supernatant. Subsequently, resuspend the bacterial solution with an imidazole solution (20 mM, 20 mL), sonicate for 20 min, and then centrifuge at 10000 rpm for 30 minutes to retain the supernatant to obtain the enzyme solution. Among them, the components of the TB medium are: 6 g of tryptone, 12 g of yeast extract, 2 mL of glycerol, KH 2 PO 4 1.155 g, K 2 HPO 4 6.27 g, and it is prepared with 500 mL of distilled water; the components of the imidazole solution are: 1.46 g of NaCl, 0.34 g of imidazole, and it is prepared with 250 mL of distilled water.

[0037] (3) Enzyme-catalyzed Reaction: Mix 50 μL of ATP, 50 μL of MgCl 2 , 100 μL of enzyme solution, 200 μL of isopentenol, and 100 μL of Tris-HCl (25 mM, pH 7.5), and let it stand at room temperature for reaction for 3 hours to obtain the fermentation broth.

[0038] (4) Analysis and Detection: After extracting the fermentation broth with an equal volume of n-hexane, take 1 μL for GC-MS analysis. As Figure 2 shown, it is the GC-MS spectrum of the reaction product in Example 3. According to the analysis of the chromatogram and mass spectrum, it is found that the peak appearing at 22.559 min in the chromatogram corresponds to the substance at this elution position in the mass spectrum analysis, which has the characteristic ion peaks and fragment peaks of triterpene squalene. By comparing the experimental GC-MS diagram with the GC-MS diagram of triterpene squalene in the literature and predicting with the instrument's built-in database, it is confirmed that the product at 22.559 min is triterpene squalene.

[0039] Example 4 In this example, the engineered Escherichia coli prepared in Example 1 pMki-nfss was used to produce diplosqualenol through whole-cell catalytic reaction. The specific steps are as follows: (1)Engineered Escherichia coli pMki-nfss Bacterial liquid preparation: The engineered Escherichia coli of Example 1 pMki-nfss strain was screened on a double-antibiotic plate. That is, after plate cultivation for 12 - 16 hours, single colonies were picked and cultured in a shake flask with LB liquid medium for 8 - 12 hours to obtain the engineered Escherichia coli pMki-nfss bacterial liquid. Among them, the composition of the LB liquid medium is: peptone 0.2 g, yeast extract 0.1 g, NaCl 0.2 g, and it is prepared with 20 mL of distilled water.

[0040] (2)Whole-cell catalytic reaction: Take 1% of the engineered Escherichia coli pMki-nfss bacterial liquid from step (1) and inoculate it into a shake flask with TB medium containing double antibiotics for cultivation until the OD 600 reaches 0.6 - 0.8. Then, take out the medium, add 0.2 mM IPTG for induction and simultaneously add 25 mM isopentenol, and continue shake flask fermentation at 16 °C for 76 hours to obtain the fermentation broth. Among them, the composition of the TB medium is: tryptone 6 g, yeast extract 12 g, glycerol 2 mL, KH 2 PO 4 1.155 g, K 2 HPO 4 6.27 g, and it is prepared with 500 mL of distilled water.

[0041] (3)Analysis and detection: After extracting the fermentation broth with an equal volume of n-hexane, rotary evaporation and concentration are carried out to dissolve the residue in n-hexane, and 1 μL is taken for GC-MS analysis. As Figure 3 shown, it is the GC-MS spectrum of the reaction product of Example 4. According to the analysis of the chromatogram and mass spectrum, it is found that the peak appearing at 21.972 min in the chromatogram corresponds to the analysis of the mass spectrum, and the substance corresponding to this peak position has the fragment peak and characteristic ion peak of sesterfisherol, a sesquiterpenoid fish oil alcohol. By comparing the experimental GC-MS diagram with the sesterfisherol GC-MS diagram in the literature, it is confirmed that the product at 21.972 min is sesterfisherol, and its yield is increased compared with that in Example 2.

[0042] Example 5 In this example, the engineered Escherichia coli prepared in Example 1 pMki-nfss is used, and squalene, a triterpenoid, is produced through an enzymatic catalytic reaction. The specific steps are as follows: (1)Engineered Escherichia coli pMki-nfss Bacterial liquid preparation: The engineered Escherichia coli of Example 1 pMki-nfss strain was screened on a double-antibiotic plate. That is, after plate cultivation for 12 - 16 hours, single colonies were picked and cultured in a shake flask with LB liquid medium for 8 - 12 hours to obtain the engineered Escherichia coli pMki-nfssBacterial liquid. Among them, the components of the LB liquid medium are: 0.2 g of peptone, 0.1 g of yeast extract, 0.2 g of NaCl, and it is prepared with 20 mL of distilled water.

[0043] (2)Enzyme sample preparation: Take 1% of the engineered Escherichia coli in step (1) pMki-nfss bacterial liquid, inoculate it into a shake flask containing TB medium with double antibiotics and culture until OD 600 reaches 0.6 - 0.8. Take out the medium, add 0.2 mM IPTG for induction, and continue shake flask culture at 16 °C for 24 hours. Take out the medium, centrifuge at 4000 rpm for 20 min, remove the supernatant, then resuspend the bacterial liquid with imidazole solution (20 mM, 20 mL), sonicate for 20 min, and then centrifuge at 10000 rpm for 30 minutes to retain the supernatant. The supernatant is filtered and then subjected to Ni column affinity chromatography, and eluted with 250 mM imidazole to obtain the enzyme solution. Among them, the components of the TB medium are: 6 g of tryptone, 12 g of yeast extract, 2 mL of glycerol, KH 2 PO 4 1.155 g, K 2 HPO 4 6.27 g, and it is prepared with 500 mL of distilled water; the components of the imidazole solution are: 1.46 g of NaCl, 4.25 g of imidazole, and it is prepared with 250 mL of distilled water.

[0044] (3)Enzyme-catalyzed reaction: Mix 50 μL of ATP, 50 μL of MgCl 2 , 100 μL of enzyme solution, 200 μL of isopentenol, and 100 μL of Tris-HCl (25 mM, pH 8.3), and let it stand at room temperature for reaction for 3 hours to obtain the fermentation broth.

[0045] (4)Analysis and detection: After extracting the fermentation broth with an equal volume of n-hexane, take 1 μL for GC-MS analysis. As Figure 4 shown, it is the GC-MS spectrum of the reaction product of Example 5. According to the analysis of the chromatogram and mass spectrum, it is found that the peak appearing at 22.507 min in the chromatogram corresponds to the substance at this elution position in the mass spectrum analysis, which has the same characteristic ion peaks and fragment peaks as squalene triterpenoid in Case 3. By comparing the experimental GC-MS diagram with the GC-MS diagram of squalene triterpenoid in the literature and the prediction of the instrument's built-in database, it is confirmed that the product at 22.559 min is squalene triterpenoid, and its yield is slightly lower than that in Example 3.

[0046] Example 6 In this example, the P-M recombinant plasmid and the P-N recombinant plasmid were respectively transformed into Escherichia coli BL21, and squalene triterpenoid was produced through an enzyme-catalyzed reaction. The specific steps are as follows: (1)Strain sample preparation: The P-M recombinant plasmid constructed with the IUP pathway and containing nfssThe P-N recombinant plasmid of the gene was separately transformed into Escherichia coli BL21 to obtain strains pCDF-mki and pRSF-nfss . Then, positive clones were screened using plates with spectinomycin and plates with kanamycin respectively. After shaking flask culture in LB liquid medium for 8 - 12 hours, pCDF-mki bacterial solution and pRSF-nfss bacterial solution were obtained. Among them, the components of the LB medium were: peptone 0.2 g, yeast extract 0.1 g, NaCl 0.2 g, and it was prepared with 20 mL of distilled water.

[0047] (2)Enzyme sample preparation: Take 1% of the pCDF-mki bacterial solution in step (1) and 1% of the pRSF-nfss bacterial solution, and inoculate them into TB medium containing double antibiotics for shaking flask culture until OD 600 reaches 0.6 - 0.8. Then, add 0.1 mM IPTG to the medium for induction, and continue shaking flask culture at 18 °C for 20 hours. Take out the medium and centrifuge at 4000 rpm for 20 min to remove the supernatant. Subsequently, resuspend the bacterial solution with imidazole solution (20 mM, 20 mL), sonicate for 20 min, and then centrifuge at 10000 rpm for 30 minutes to retain the supernatant, thus obtaining pCDF - mki enzyme solution and pRSF - nfss enzyme solution. Among them, the components of the TB medium were: tryptone 6 g, yeast extract 12 g, glycerol 2 mL, KH 2 PO 4 1.155 g, K 2 HPO 4 6.27 g, and it was prepared with 500 mL of distilled water; the components of the imidazole solution were: NaCl 1.46 g, imidazole 4.25 g, and it was prepared with 250 mL of distilled water.

[0048] (3)Enzyme - catalyzed reaction: Mix 50 μL of ATP, 50 μL of MgCl 2 , 50 μL of pCDF - mki enzyme solution, 50 μL of pRSF - nfss enzyme solution, 200 μL of isopentenol, and 100 μL of Tris - HCl (25 mM, pH 8.3), and let it stand at room temperature for reaction for 3 hours to obtain the fermentation broth.

[0049] (4)Analysis and detection: After extracting the fermentation broth with an equal volume of n - hexane, take 1 μL for GC - MS analysis, as Figure 5The GC-MS spectrum of the reaction product of Example 6 is shown. According to the analysis of the chromatogram and mass spectrum, it is found that the peak appearing at 22.507 min in the chromatogram corresponds to the substance at the same elution position in the mass spectrum analysis, which has the same characteristic ion peaks and fragment peaks as squalene triterpene in Case 5. By comparing the obtained GC-MS diagram with the GC-MS diagram of squalene triterpene in the literature and predicting with the instrument's built-in database, it is confirmed that the product at 22.559 min is squalene triterpene.

[0050] Example 7 In this example, the P-M recombinant plasmid and the P-N recombinant plasmid were respectively transformed into Escherichia coli BL21, and squalene triterpene was produced through an enzyme-catalyzed reaction. The specific steps are as follows: (1)Strain sample preparation: The P-M recombinant plasmid containing the IUP pathway and the P-N recombinant plasmid containing the nfss gene were respectively transformed into Escherichia coli BL21 to obtain strains pCDF-mki and pRSF-nfss . Then, positive clones were screened with plates containing spectinomycin and plates containing kanamycin respectively. After shaking flask culture in LB liquid medium for 8 - 12 hours, pCDF-mki bacterial liquid and pRSF-nfss bacterial liquid were obtained. Among them, the components of the LB medium are: 0.2 g of peptone, 0.1 g of yeast extract, 0.2 g of NaCl, and it was prepared with 20 mL of distilled water.

[0051] (2)Enzyme sample preparation: Take 1% of the pCDF-mki bacterial liquid from step (1) and 1% of the pRSF-nfss bacterial liquid, and inoculate them into TB medium containing double antibiotics for shaking flask culture until OD 600 reaches 0.6 - 0.8. Then, add 0.2 mM IPTG to the medium for induction, and continue shaking flask culture at 16 °C for 24 hours. Take out the medium and centrifuge at 4000 rpm for 20 min to remove the supernatant. Subsequently, resuspend the bacterial liquid with imidazole solution (20 mM, 20 mL), sonicate for 20 min, and then centrifuge at 10000 rpm for 30 minutes to retain the supernatant. The supernatant was filtered and then subjected to Ni column affinity chromatography, and eluted with 250 mM imidazole to obtain pCDF-mki enzyme solution and pRSF-nfss enzyme solution. Among them, the components of the TB medium are: 6 g of tryptone, 12 g of yeast extract, 2 mL of glycerol, 1.155 g of KH 2 PO 4 , 6.27 g of K 2 HPO 4 , and it was prepared with 500 mL of distilled water; the components of the imidazole solution are: 1.46 g of NaCl, 4.25 g of imidazole, and it was prepared with 250 mL of distilled water.

[0052] (3) Enzymatic catalysis reaction: 50 μL of ATP, 50 μL of MgCl 2 , 50 μL of pCDF-mki enzyme solution, 50 μL of pRSF-nfss enzyme solution, 200 μL of isopentenol and 100 μL of Tris-HCl (25 mM, pH 7.5) were mixed well and allowed to react at room temperature for 3 hours to obtain the fermentation broth.

[0053] (4) Analysis and detection: After extracting the fermentation broth with an equal volume of n-hexane, 1 μL was taken for GC-MS analysis. As Figure 6 shown is the GC-MS spectrum of the reaction product of Example 7. According to the analysis of the chromatogram and mass spectrum, it was found that the peak appearing at 22.521 min in the chromatogram corresponded to the substance at the same elution position in the mass spectrum analysis, which had the same characteristic ion peaks and fragment peaks as squalene triterpenoid in Example 6. By comparing the experimental GC-MS graph with the GC-MS graph of squalene triterpenoid in the literature and predicting with the instrument's built-in database, it was confirmed that the product at 22.521 min was squalene triterpenoid, and its yield increased slightly compared to Example 6.

[0054] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

Claims

1. An engineered Escherichia coli pMki-nfss , characterized in that, include mki Genes and nfss sequence, the mki The nucleotide sequence of the gene is shown in SEQ ID NO:

1. nfss The nucleotide sequence of the sequence is shown in SEQ ID NO:

2.

2. An engineered Escherichia coli pMki-nfss The construction method is characterized in that The following steps are involved: S1. Ec ThiM, Mj After the gene sequences of IPK and IDI proteins are seamlessly connected, the mki Gene, then use Eco RI and Hin d III as a restriction enzyme cutting site mki The gene was cloned into pCDFDuet-1 to construct PM recombinant plasmid; S2. Use Bam HI and Hin d III as a restriction enzyme cutting site nfss The sequence was cloned into pRSFDuet-1 to construct the PN recombinant plasmid; S3, the PM recombinant plasmid and the PN recombinant plasmid were co-transformed into E. coli BL21, obtain engineered E. coli pMki-nfss .

3. The engineered Escherichia coli according to claim 1 pMki-nfss Application in the preparation of terpene iodine alcohol.

4. The use according to claim 3, characterized in that: The engineered Escherichia coli pMki-nfss The method of producing the terpene ichthyol by whole-cell catalytic reaction comprises the following steps: Take the engineered Escherichia coli pMki-nfss The bacterial suspension was inoculated into TB medium containing double antibodies and cultured in a shake flask until OD 600 When the concentration of terpenoids reaches 0.6-0.8, the TB culture medium containing the double antibodies is taken out, IPTG is added for induction, and isopentenol is added at the same time. After shake flask fermentation, the diterpenoid iodine alcohol is obtained by extraction.

5. The use according to claim 4, characterized in that: The temperature of the shake flask fermentation is 16-18° C., and the time of the shake flask fermentation is 72-76 hours.

6. The engineered Escherichia coli according to claim 1 pMki-nfss Application in the preparation of triterpene squalene.

7. The use according to claim 6, characterized in that: The engineered Escherichia coli pMki-nfss The triterpene squalene is produced by enzyme catalysis reaction, comprising the following steps: Take the engineered Escherichia coli pMki-nfss The bacterial suspension was inoculated into TB medium containing double antibodies and cultured in a shake flask until OD 600 When the concentration reaches 0.6-0.8, the TB medium containing the double antibodies is taken out and IPTG is added for induction. After flask culture, centrifugation is performed and the supernatant is removed. Then, the bacterial solution is resuspended with an imidazole solution, and the enzyme solution is obtained after ultrasonication and centrifugation again. ATP, MgCl2, the enzyme solution, isopentenol and Tris-HCl are mixed and reacted, and the triterpene squalene is obtained through extraction.

8. The use according to any one of claim 4 or claim 7, characterized in that: The TB culture medium includes peptone, yeast extract, glycerol, KH2PO4, K2HPO4 and water, and the mass volume ratio of the peptone, the yeast extract, the glycerol, the KH2PO4, the K2HPO4 and the distilled water is 3:6:1:0.5775:3.135:250 (g / g / mL / g / g / mL).

9. The use according to claim 7, characterized in that: The imidazole solution comprises NaCl, imidazole and distilled water, and the mass volume ratio of the NaCl, the imidazole and the distilled water is 4.25-4.35:1:734-736 (g / g / mL).

10. The use according to claim 7, characterized in that: The temperature of the shake flask culture is 16-18° C., the shake flask culture time is 20-24 h, and the reaction time is 3 h.

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