Genetically engineered bacterium for producing monensin and application of genetically engineered bacterium
By overexpressing phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase, and isobutyryl-CoA mutase in Streptomyces cinnamon, the problem of insufficient fermentation capacity of Streptomyces cinnamon in monensin production was solved, resulting in a significant increase in monensin yield and a reduction in cost.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-05-01
AI Technical Summary
Streptomyces cinnamon has poor fermentation ability in monensin production, limited primary and secondary metabolic pathways, and cannot efficiently produce monensin. In addition, the cost of adding precursor substances is high.
By constructing recombinant Streptomyces cinnamon and overexpressing phosphofructokinase (PFK), citrate synthase (CS), methylmalonyl-CoA isomerase (mutB), and isobutyryl-CoA mutase (ICM), the synthesis of acetyl-CoA and energy supply of monensin were improved, thus enhancing the supply capacity of synthetic precursors.
Without increasing the supply of precursor substances, it significantly increases monensin yield, reduces production costs, and achieves a yield of 20.4 g/L after 10 days of cultivation, thereby improving production efficiency.
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Figure CN121950647A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a genetically engineered bacterium that produces monensin and its applications, belonging to the field of metabolic engineering technology. Background Technology
[0002] Monensin is produced by *Streptomyces cinnamon* (…). Streptomyces cinnamonensis Monensin, a lipid-soluble antibiotic with a pentacyclic monocarboxylic acid polyether structure produced by fermentation, is also known as monensin or rumenin. In recent years, studies have investigated the in vitro anti-proliferation effects of monensin on malignant cells, finding that it can inhibit the proliferation of various human lymphoma and myeloma cell lines, thereby blocking cell circulation and inducing apoptosis. Therefore, increasing the yield of monensin has significant economic benefits. *Streptomyces cinnamon*, as an industrial production strain of monensin, possesses advantages such as genetic stability, substrate tolerance, and low byproduct production; however, it also has some drawbacks, primarily its poor fermentation capacity. The limited capacity of *Streptomyces*' natural primary and secondary metabolic pathways makes it unsuitable for efficient monensin production. Monensin, a typical type I polyketide antibiotic, is primarily mediated by a modular polyketide synthase system. One monensin molecule involves the linear elongation of the carbon chain using five malonyl-CoA, one ethylmalonyl-CoA, and seven methylmalonyl-CoA units. Adding precursors is a highly effective strategy to increase monensin yield during production, but it is costly.
[0003] Therefore, using metabolic engineering to enhance the capacity for primary and secondary metabolism is an effective strategy to improve the production of monensin by *Streptomyces cinnamon*. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides a recombinant *Streptomyces cinnamon* strain that increases monensin yield. This recombinant *Streptomyces cinnamon* strain simultaneously overexpresses phosphofructokinase (PFK), citrate synthase (CS), methylmalonyl-CoA isomerase (mutB), and isobutyryl-CoA mutase (ICM). Results show that the recombinant *Streptomyces cinnamon* strain overexpresses monensin acetyl-CoA synthesis and energy supply, increasing the synthesis of precursors (methylmalonyl-CoA and ethylmalonyl-CoA). This increases monensin yield without requiring additional precursor supply, thereby reducing production costs and improving production efficiency.
[0005] The first objective of this invention is to provide a recombinant Streptomyces cinnamonis that produces monensin, wherein the recombinant Streptomyces cinnamonis overexpresses phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase, and isobutyryl-CoA mutase.
[0006] In one embodiment of the present invention, the amino acid sequence of the phosphofructokinase is shown in SEQ ID NO.1; the amino acid sequence of the citrate synthase is shown in SEQ ID NO.3; the amino acid sequence of the methylmalonyl-CoA isomerase is shown in SEQ ID NO.5; and the amino acid sequence of the isobutyryl-CoA mutase is shown in SEQ ID NO.7.
[0007] In one embodiment of the present invention, using The promoter expresses phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase, and isobutyryl-CoA mutase. The nucleotide sequence of the promoter is shown in SEQ ID NO. 15.
[0008] In one embodiment of the present invention, the connection sequence of the promoter and the gene is as follows: -pfk- -cs- -mutB- -icm.
[0009] A second objective of this invention is to provide the application of the above-mentioned recombinant Streptomyces cinnamon in the preparation of monensin.
[0010] A third objective of this invention is to provide a method for producing monensin, which uses the aforementioned recombinant Streptomyces cinnamon to produce monensin through fermentation, comprising the steps of: (1) Inoculate the recombinant Streptomyces cinnamon onto Gao's No. 1 solid medium and culture at 30-33℃ for 5-7 days. Take out a piece of spores about 1 square centimeter and inoculate it into Streptomyces cinnamon seed medium. Culture at 30-33℃ and 220-260 rpm for 24-28 h to obtain seed liquid. (2) The seed liquid was inoculated into the fermentation medium of Streptomyces cinnamon and centrifuged to obtain the fermentation broth containing monensin.
[0011] In one embodiment of the present invention, the inoculation amount of seed liquid in step (2) is 18~22% (v / v).
[0012] In one embodiment of the present invention, the seed liquid inoculated into the fermentation medium of Streptomyces cinnamon in step (2) is cultured at 30~33℃, 220~260 rpm for 8~10 days.
[0013] The fourth objective of this invention is to provide a method for increasing the yield of monensin produced by *Streptomyces cinnamon*, wherein phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase, and isobutyryl-CoA mutase are simultaneously overexpressed in *Streptomyces cinnamon*. The amino acid sequence of the phosphofructokinase is shown in SEQ ID NO.1; the amino acid sequence of citrate synthase is shown in SEQ ID NO.3; the amino acid sequence of methylmalonyl-CoA isomerase is shown in SEQ ID NO.5; and the amino acid sequence of isobutyryl-CoA mutase is shown in SEQ ID NO.7.
[0014] In one embodiment of the present invention, using The promoter expresses phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase, and isobutyryl-CoA mutase. The nucleotide sequence of the promoter is shown in SEQ ID NO. 15, and the connection sequence of the promoter and the gene is as follows: -pfk- -cs- -mutB- -icm.
[0015] Beneficial effects of the present invention This invention provides a recombinant *Streptomyces cinnamon* strain that increases monensin production. This recombinant *Streptomyces cinnamon* strain simultaneously overexpresses phosphofructokinase (PFK), citrate synthase (CS), methylmalonyl-CoA isomerase (mutB), and isobutyryl-CoA mutase (ICM). The recombinant *Streptomyces cinnamon* strain constructed in this invention overexpresses monensin acetyl-CoA synthesis and energy supply, increasing the synthesis of precursors (methylmalonyl-CoA and ethylmalonyl-CoA). This increases monensin production without requiring additional precursor supply, achieving a monensin yield of 20.4 g / L after 10 days of cultivation, effectively reducing production costs and improving production efficiency. Attached Figure Description
[0016] Figure 1 PCR electrophoresis image for colony verification; Figure 2 This is a standard curve for monensin. Detailed Implementation
[0017] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0018] The construction method used in the example: (1) PCR amplification fragments and recovery Using the artificially synthesized sequence (pfk) shown in SEQ ID NO.2 as a template, PCR amplification was performed using pfk-F and pfk-R as primers to obtain the pfk fragment; Using the artificially synthesized sequence (cs) shown in SEQ ID NO.4 as a template, PCR amplification was performed using cs-F and cs-R as primers to obtain the cs fragment; Using the artificially synthesized sequence (mutB) shown in SEQ ID NO.6 as a template, PCR amplification was performed using mutB-F and mutB-R primers to obtain the mutB fragment; Using the artificially synthesized sequence (icm) shown in SEQ ID NO.8 as a template, PCR amplification was performed using icm-F and icm-R primers to obtain the icm fragment; The sequence shown in SEQ ID NO.15, which is artificially synthesized ( Using ) as a template, -F and PCR amplification was performed using primers -R, Pkasop-pfk-R, Pkasop-cs-R, Pkasop-mutB-R, and Pkasop-icm-R to obtain... Fragments; primer sequences are shown in Table 1.
[0019] Table 1. Nucleotide sequences of primers
[0020] The PCR amplification reaction conditions were as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 10 s, 67℃ annealing for 15 s, 72℃ extension for 25 s, 30 cycles; 72℃ final extension for 5-10 min. The PCR amplification reaction system was as follows: 25 μL 2×Primerstar maximum buffer, 1.5 μL forward primer (10 μM), 1.5 μL reverse primer (10 μM), 50 ng template, and ddH2O to a final volume of 50 μL.
[0021] After the PCR reaction, the reaction products were subjected to agarose gel electrophoresis, irradiated under UV light, and the gel block containing the target band was cut off. The gel block was weighed, and DNA was recovered according to the instructions of the kit (Shanghai Sangon Biotech General Agarose Gel DNA Recovery Kit). After recovery, the DNA concentration was measured and stored at -20℃ for later use.
[0022] (2) Gibson assembly The pSET152-BX and the PCR recovered product pSET152-BX were then combined. - pfk, pfk was subjected to a Gibson assembly reaction using 2×SeamlessCloning Mix to obtain Pset152- PFK recombinant vector; reaction conditions: 50℃, 30 min; reaction system: Seamless Cloning Mix 5 μL, pSET152-BX 2 μL (0.02 poml), -pfk 0.5 μL (0.04 poml), pfk1 μL (0.04 pmol).
[0023] The pSET152-BX and the PCR recovered product pSET152-BX were then combined. -cs and cs were subjected to a Gibson assembly reaction using 2×SeamlessCloning Mix to obtain Pset152- -cs recombinant vector; reaction conditions were 50℃, 30 min. The reaction system consisted of: Seamless Cloning Mix 5 μL, pSET152-BX 2 μL (0.02 poml). 0.5μL (0.04 poml), cs 1 μL (0.04 pmol).
[0024] The pSET152-BX and the PCR recovered product pSET152-BX were then combined. -mutB and mutB were subjected to a Gibson assembly reaction using 2×SeamlessCloning Mix to obtain Pset152- -mutB recombinant vector; reaction conditions: 50℃, 30 min; reaction system: Seamless Cloning Mix 5 μL, pSET152-BX 2 μL (0.02 poml) -mutB 0.5 μL (0.04 poml), mutB 1 μL (0.04 pmol).
[0025] The pSET152-BX and the PCR recovered product pSET152-BX were then combined. -icm and icm were subjected to Gibson assembly reaction using 2×SeamlessCloning Mix to obtain Pset152- -icm recombinant vector; reaction conditions: 50℃, 30 min; reaction system: Seamless Cloning Mix 5 μL, pSET152-BX 2 μL (0.02 poml), -icm 0.5 μL (0.04poml), icm 1 μL (0.04pmol).
[0026] The pSET152-BX and the PCR recovered product pSET152-BX were then combined. - pfk, pfk, -cs, cs, -mutB、mutB、 -icm and icm were subjected to a Gibson assembly reaction using 2×Seamless Cloning Mix to obtain Pset152- -pfk- -cs- -mutB- -icm recombinant vector; reaction conditions: 50℃, 90 min; reaction system: Seamless Cloning Mix 5 μL, pSET152-BX 2 μL (0.02 poml), - pfk 0.5 μL (0.04 poml), -cs 0.5 μL (0.04 poml), -mutB 0.5 μL (0.04 poml), -icm 0.5 μL (0.04 poml), pfk 1 μL (0.04 pmol), cs 1 μL (0.04 pmol), mutB 1 μL (0.04 pmol), icm 1 μL (0.04 pmol).
[0027] (3) Bacterial transformation and colony PCR identification DH5α competent cells were removed from a -80°C freezer and placed on ice. After thawing naturally, five reaction products (Pset152-) obtained from the Gibson assembly step were added to each cell. - pfk, Pset152 - -cs、Pset152- -icm、Pset152- -mutB、Pset152- - pfk - -cs- -mutB- Mix the -icm recombinant vector gently. Incubate on ice for 30 min, then heat-shock in a 42°C water bath for 1.5 min, followed by incubation on ice for 2-3 min. Add 600 μL of antibiotic-free LB broth to a clean bench and incubate at 37°C with shaking for 30 min. Centrifuge at 3000 rpm for 5 min at room temperature, discard most of the supernatant, resuspend the cells in 100 μL of supernatant, and plate onto LB plates with the appropriate antibiotic. Incubate upside down at 37°C for 24 h. Once several appropriately sized single colonies have grown, remove them for colony PCR identification and streak the single-colony plates for preservation.
[0028] Recombinant vector Pset152- pfk, Pset152- -cs、Pset152- -icm、Pset152- -mutB、Pset152- -pfk- -cs- -mutB- -icm colonies were identified using the Arp-F and Arp-R primers listed in Table 1. After the PCR reaction, agarose gel electrophoresis was performed, and the images were photographed under UV light to identify positive colonies based on the recorded electrophoretic patterns.
[0029] (4) Plasmid extraction The above positive transformants were cultured overnight in LB medium. The 6 mL of overnight culture was collected by centrifugation in a 2 mL centrifuge tube. Plasmid extraction was performed according to the instructions of the kit (Vazyme Plasmid Mini-Prep Kit). Further sequencing was used to verify whether the required plasmid was constructed correctly.
[0030] (5) ET competence transformation and conjugation transfer The recombinant vector pPset152- - pfk, Pset152 - -cs、Pset152- -icm、Pset152- -mutB、Pset152- - pfk - -cs- -mutB- -icm was transferred into ET12567 (pUZ8002) competent cells, and the transformation method was the same as bacterial transformation, resulting in 5 types of ET competent bacteria containing recombinant vectors.
[0031] Five ET colonies containing the recombinant vector were picked from plates and transferred to 5 mL of LB broth containing apramycin (Apr, 50 μg / mL), chloramphenicol (cm, 25 μg / mL), and kanamycin (kana, 50 μg / mL), respectively. The cultures were incubated overnight at 37°C and 220 rpm. The overnight cultures were then transferred 1:100 to LB broth containing apramycin (Apr, 50 μg / mL), chloramphenicol (cm, 25 μg / mL), and kanamycin (kana, 50 μg / mL), and cultured at 37°C and 220 rpm until the OD value was reached. 600When the concentration was 0.4, 10 mL of bacterial culture was collected in a centrifuge tube, centrifuged at 5000 rpm for 2 min, washed twice with 20 mL of 2×YT (10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride), and resuspended in 500 μL of 2×YT to obtain the recombinant vector Pset152-. - pfk, Pset152 - -cs、Pset152- -icm、Pset152- -mutB、Pset152- -pfk- -cs- -mutB- -icm has 5 donor bacteria.
[0032] Take an appropriate amount of Streptomyces cinnamon from the glycerin tube. S. cinnamonensis The spores of 2110 were suspended in a shake flask containing 30 mL of TSB liquid medium and cultured at 30℃ and 220 rpm for 30 h. After culturing, 10 mL of the culture was transferred to a centrifuge tube and centrifuged at 8000 rpm for 2 min. The culture was then washed once with 20 mL of water and twice with 20 mL of 2×YT. The culture was then resuspended in 2-4 mL of 2×YT to obtain the recipient bacterial culture.
[0033] Transfer 100 μL of recipient bacterial culture to a centrifuge tube containing resuspended donor bacteria, mix thoroughly, spread onto a plate containing 10 mM MgCl2MS medium, and incubate at 30°C for 16 h.
[0034] Remove the plate from the incubator and cover it with 1 mL of sterile water containing Apr (50 μg / mL) and sodium naphthiopicrylate (25 μg / mL). Dry the plate in a laminar flow hood, rotating it once every 15–30 minutes. Once completely dry, return it to the incubator for further incubation.
[0035] (6) Identification of engineered bacteria After 5-7 days, single colony zygotes were observed growing on the plate. The zygotes were verified by mycelial PCR and antibiotic resistance testing. The primers Arp in Table 1 were used. F and Arp R was used for PCR verification, and the fragment length was 777bp. The system and colony PCR identification were shown, with the annealing temperature at 65℃ and the extension time at 1 min.
[0036] The results are as follows Figure 1 As shown, this embodiment successfully inserted the target gene into the strain, resulting in the engineered strain Pset152- containing the recombinant vector. - pfk is denoted as S1, containing the recombinant vector Pset152- The engineered bacteria of type -cs are designated S2, containing the recombinant vector Pset152- The engineered bacteria of -icm are denoted as S3, Pset152- The engineered strain of -mutB is denoted as S4, Pset152- -pfk- -cs- -mutB- The engineered bacteria of -icm are denoted as S5.
[0037] Test method: Fermentation method using engineered bacteria: After verification, the correct engineered strains S1-S5 and the empty vector control strain (transformed only into the pSET152 empty vector, without the target gene sequence, named S0) were inoculated onto Gao's No. 1 solid medium and cultured at 30℃ for 7 days. A spore fragment of approximately 1 square centimeter was then inoculated into *Streptomyces cinnamon* seed culture medium and cultured at 30℃ and 220 rpm for 30 h. Then, at a 5% inoculation rate, the inoculation was carried out into *Streptomyces cinnamon* fermentation medium and cultured at 30℃ and 220 rpm for 10 days, yielding fermentation broths for strains S0, S1, S2, S3, S4, and S5, respectively.
[0038] Methods for detecting monensin potency: Preparation of monensin standard solution: Accurately weigh an appropriate amount of monensin standard into a 100 mL volumetric flask, and dilute to the mark with anhydrous methanol to prepare a solution of 1000 U / mL. -1 Monensin standard solution can be stored at 4°C for 10 days.
[0039] To prepare a 3% vanillin solution: accurately weigh 3 g of vanillin into a beaker, add 100 mL of anhydrous methanol, and after it dissolves, add 1 mL of concentrated sulfuric acid, mix well, and transfer to a brown bottle for later use. It can be stored at 4℃ for 7 days.
[0040] Accurately measure 5 mL of monensin standard solution into a 25 mL volumetric flask, and dilute to the mark with anhydrous methanol to prepare a solution of 200 U / mL. -1 The solution was diluted according to Table 2 to prepare solutions of 0, 20, 40, 60, 80, 100, and 120 U·mL, respectively. -1 The standard solution was thoroughly mixed and incubated in a 50°C water bath for 30 min. After cooling to room temperature, the absorbance at 520 nm was measured. The absorbance was calculated using monensin potency (U·mL⁻¹). -1 The x-axis represents the absorbance value (OD) at 520 nm. 520 Plot a standard curve using the ordinate as the vertical axis. Figure 2 Monensin standard solution concentration ranges from 20 to 120 U / mL.-1 At that time, the absorbance and concentration showed a good linear relationship.
[0041] Table 2 Preparation of monensin standard curve
[0042] Raw materials used in the examples: Streptomyces cinnamonensis 2110 has been disclosed in the literature “Enhancing the biosynthesis of monensin from Streptomyces cinnamonis using metabolic engineering technology; DOI:10.13343 / j.cnki.wsxb.20240178”.
[0043] Gao's medium: 20 g / L soluble starch, 1 g / L potassium nitrate, 0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate heptahydrate, 0.5 g / L sodium chloride, 30 g / L agar. (Dissolve the soluble starch in a small amount of cold water first, then add it to 80℃ hot water.) Used for the culture of Streptomyces.
[0044] Seed culture medium: 5 g / L glucose, 15 g / L soybean meal, 20 g / L dextrin, 2.5 g / L yeast extract, pH adjusted to approximately 6.8 with sodium hydroxide, then add 1 g / L calcium carbonate. The volume is 100 mL / 500 mL. (Dissolve the soybean meal in a small amount of water first, then boil three times in a microwave oven.) 33℃, 220 rpm, 24 h.
[0045] Fermentation medium: glucose 36 g / L, soybean meal 33 g / L, anhydrous sodium sulfate 2.2 g / L, sodium nitrate 2.2 g / L, dipotassium hydrogen phosphate 0.075 g / L, manganese chloride 0.33 g / L, ferrous sulfate heptahydrate 0.12 g / L, aluminum sulfate 0.7 g / L. Adjust the pH to approximately 7.2 with sodium hydroxide, then add 2.5 g / L calcium carbonate, mix well, and add 45 g / L soybean oil. The volume is 50 mL / 500 mL. (Dissolve the soybean meal in a small amount of water first, then boil it three times in a microwave oven.) 33℃, 220 rpm, 7-8 days.
[0046] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar (solid medium), pH 7.2. SM mycelial culture medium: glucose 10 g / L, peptone 4 g / L, yeast extract 4 g / L, potassium dihydrogen phosphate 0.2 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate 0.05 g / L, pH 7.2, volume 25 mL / 250 mL.
[0047] MS medium: mannitol 10 g / L, soybean meal 20 g / L, agar 20 g / L, pH 7.2. 2×YT medium: tryptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L, pH 7.2.
[0048] 2×YT: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.
[0049] Gao's No. 1 culture medium: soluble starch 20 g / L, KNO3 1 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.01 g / L, NaCl 0.5 g / L, agar powder 30 g / L, sterilized at 121℃ for 30 min, pH=8.
[0050] Seed culture medium for *Streptomyces cinnamonis*: 15 g / L soybean meal, 5 g / L glucose, 20 g / L dextrin, 2.5 g / L yeast extract, 1 g / L light calcium carbonate, adjusted to pH 6.8-6.9 with NaOH, and sterilized at 121℃ for 30 min.
[0051] Fermentation medium for *Streptomyces cinnamon*: 30 g / L soybean meal, 30 g / L glucose, 2.2 g / L Na₂SO₄, 2.2 g / L NaNO₃, 0.075 g / L K₂HPO₄, 0.3 g / L MnCl₂, 0.3 g / L FeSO₄·7H₂O, 0.7 g / L Al₂(SO₄)₃, 2.5 g / L light calcium carbonate, 45 g / L soybean oil. Adjust the pH to 7.2 with NaOH and sterilize at 121℃ for 30 min. Example 1: Construction of a genetically engineered strain producing monensin 1. Constructing recombinant bacteria S1 Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO.15) and the pfk gene (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) were inserted into the pSET152 vector between the Nsi I and Xba I restriction sites via Gibson assembly to construct the plasmid pSET152- -pfk; Plasmid pSET152- -pfk transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -pfk, named S1.
[0052] 2. Construct S2 Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO.15) and the cs gene (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4) were inserted into the pSET152 vector between the Nsi I and Xba I restriction sites via Gibson assembly to construct the plasmid pSET152- -cs; Plasmid pSET152- -cs transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -cs, named S2.
[0053] 3. Construct S3 Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO. 15) and the mutB gene (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6) were inserted into the pSET152 vector between the Nsi I and Xba I restriction sites via Gibson assembly to construct the plasmid pSET152- -mutB; Plasmid pSET152- -mutB transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -cs, named S3.
[0054] 4. Build S4 Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO.15) and the icm gene (amino acid sequence as shown in SEQ ID NO.7, nucleotide sequence as shown in SEQ ID NO.8) were inserted into the pSET152 vector between the Nsi I and Xba I restriction sites via Gibson assembly to construct the plasmid pSET152- -icm; Plasmid pSET152- -mutB transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -icm, named S4.
[0055] 5. Constructing recombinant bacteria S5 Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO. 15), pfk gene (amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 2), cs gene (amino acid sequence as shown in SEQ ID NO. 3, nucleotide sequence as shown in SEQ ID NO. 4), mutB gene (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6), and icm gene (amino acid sequence as shown in SEQ ID NO. 7, nucleotide sequence as shown in SEQ ID NO. 8) were assembled in the following order via Gibson assembly ( -pfk- -cs- -mutB- The plasmid Pset152- was constructed by inserting the -icm) between the Nsi I and Xba I restriction sites in the pSET152 vector. -pfk- -cs- -mutB- -icm.
[0056] Plasmid Pset152- -pfk- -cs- -mutB- -icm transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -pfk- -cs- -mutB- -icm, named S5.
[0057] Comparative Example 1: Overexpression of only mutB and icm Construction of recombinant bacteria S6 Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO. 15), the mutB gene (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6), and the icm gene (amino acid sequence as shown in SEQ ID NO. 7, nucleotide sequence as shown in SEQ ID NO. 8) were assembled sequentially via Gibson assembly. -mutB- The plasmid Pset152- was constructed by inserting the -icm) between the Nsi I and Xba I restriction sites in the pSET152 vector. -mutB- -icm.
[0058] Plasmid Pset152- -mutB- -icm transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152-- -mutB- -icm, named S6.
[0059] Comparative Example 2: Using different genes 1. Construct recombinant strain S7 (replace pfk with glk) Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO. 15), glk gene (amino acid sequence as shown in SEQ ID NO. 9, nucleotide sequence as shown in SEQ ID NO. 10), cs gene (amino acid sequence as shown in SEQ ID NO. 3, nucleotide sequence as shown in SEQ ID NO. 4), mutB gene (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6), and icm gene (amino acid sequence as shown in SEQ ID NO. 7, nucleotide sequence as shown in SEQ ID NO. 8) were assembled sequentially via Gibson assembly. -glk- -cs- -mutB- The plasmid Pset152- was constructed by inserting the -icm) between the Nsi I and Xba I restriction sites in the pSET152 vector. -glk- -cs- -mutB- -icm.
[0060] Plasmid Pset152- -glk- -cs- -mutB- -icm transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -glk- -cs- -mutB- -icm, named S7.
[0061] 2. Construct recombinant bacteria S8 (replace cs with idh) Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO. 15), pfk gene (amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 2), idh gene (amino acid sequence as shown in SEQ ID NO. 11, nucleotide sequence as shown in SEQ ID NO. 12), mutB gene (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6), and icm gene (sequence as shown in SEQ ID NO. 7, nucleotide sequence as shown in SEQ ID NO. 8) were assembled sequentially via Gibson assembly. -pfk- -idh- -mutB- The plasmid Pset152- was constructed by inserting the -icm) between the Nsi I and Xba I restriction sites in the pSET152 vector. -pfk- -idh- -mutB- -icm.
[0062] Plasmid Pset152- -pfk- -idh- -mutB- -icm transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -pfk- -idh- -mutB- -icm, named S8.
[0063] 3. Construct recombinant bacteria S9 (ICM with different sequences) Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO. 15), pfk gene (amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 2), cs gene (amino acid sequence as shown in SEQ ID NO. 3, nucleotide sequence as shown in SEQ ID NO. 4), mutB gene (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6), and icm' gene (amino acid sequence as shown in SEQ ID NO. 13, nucleotide sequence as shown in SEQ ID NO. 14) were assembled sequentially via Gibson assembly. -pfk- -cs- -mutB- The '-icm' restriction enzyme was inserted between the Nsi I and Xba I restriction sites in the pSET152 vector to construct the plasmid Pset152- -pfk- -cs- -mutB- -icm'.
[0064] Plasmid Pset152- -pfk- -cs- -mutB- -icm' transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -pfk- -cs- -mutB- -icm', named S9.
[0065] 4. Construct recombinant bacteria S10 (replace pfk with glk and cs with idh) Using pSET152 as the starting carrier, The promoter (nucleotide sequence as shown in SEQ ID NO. 15), glk gene (amino acid sequence as shown in SEQ ID NO. 9, nucleotide sequence as shown in SEQ ID NO. 10), idh gene (amino acid sequence as shown in SEQ ID NO. 11, nucleotide sequence as shown in SEQ ID NO. 12), mutB gene (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6), and icm gene (amino acid sequence as shown in SEQ ID NO. 7, nucleotide sequence as shown in SEQ ID NO. 8) were assembled sequentially via Gibson assembly. -glk- -idh- -mutB- The plasmid Pset152- was constructed by inserting the -icm) between the Nsi I and Xba I restriction sites in the pSET152 vector. -glk- -idh- -mutB- -icm'.
[0066] Plasmid Pset152- -glk- -idh- -mutB- -icm' transfer Streptomyces cinnamonensis 2110. Screening, verification, and construction of recombinant bacteria. Streptomyces cinnamonensis 2110-pSET152- -glk- -idh- -mutB- -icm, named S10.
[0067] Example 2: Monensin production The empty vector pSET152, which does not contain the target gene sequence, was transferred and named S0.
[0068] Strains S0-S9 were inoculated onto Gao's No. 1 solid medium and cultured at 30°C for 7 days. A spore fragment of about 1 square centimeter was then inoculated into Streptomyces cinnamon seed medium and cultured at 30°C and 220 rpm for 30 h to obtain the seed liquid.
[0069] The seed culture was inoculated into Streptomyces cinnamon fermentation medium at an inoculation rate of 5% (v / v), and cultured at 30°C and 220 rpm for 10 days. The fermentation broth was then obtained by centrifugation.
[0070] Monensin production was determined, and the results are shown in Table 3.
[0071] Table 3. Monensin Production
[0072] The sequences involved in this invention are as follows: Phosphofructokinase (PFK) amino acid sequence SEQ ID NO.1: MRIGILTAGGDCPGLNAVIRSVVHRAVTHYGDEVIGFEDGYAGLLEGRYRALDLNAVSGILARGGTILGSSRLERDRFRAACHNAKELIEKSGFDALIPIGGEGTLTAAGMLADAGVPVVGVPKTIDNDISSTDRTFGFDTAVGVATEAMDRLKTTAESHQRVMVVEVMG RHAGWIALESGMAGGAHGICLPERPFDPADLVALVEERFSRGKKFAVICVAEGAHPQDGTMNYGKGAIDQFGHERFQGIGTALAYELEARLGKEAKPVILGHVQRGGTPTAYDRVLATRFGWHAVEAVHRGEYGKMTALRGTDITMVPLAEAVTELKTVPKDRMDEAESVF Citrate synthase (cs) amino acid SEQ ID NO.3: MSTNPFDDPDGRFLVLVNDEGQHSLWPSFAEVPGGWTIAFEENTRDACLAYIETNWTDLRPRSLAASVDG The amino acid sequence of methylmalonyl-CoA isomerase (mutB) is SEQ ID NO. 5: MTVLPDDGLSLAAEFPDATHEQWHRLVEGVLRKSGKDVSGTAAEEALSTTLEDGLTTRPLYTARDAAPDAGFPGFAPFVRGSVPEGNTPGGWDVRQRYASADPARTNEAVLTDLENGVTSLWLTLGSAGLPVTGLERALDGVYLDLVPVALDAGSEAATAARELLRLYEAAGVADDAVRGTLGADPLGHEARTGEKSTSFAAVAELARLCGERYPGLRALTVDALPYHEAGASAAQELGASLATGVEYLRALHDKGLGVEKAFAQLEFRFAATADQFLTIAKLRAARRLWARVAEVSGVPAAGAQRQHAVTSPVMMTRRDPWVNMLRTTVACLGAGVGGADAVTVLPFDHELGLPDAFARRIARNTSTILLEESHLARVIDPAGGSWYVERLTDELAHAAWDFFKEIERADGQVAALRSGLVGDRIAATWAERRKKLARRREPITGVSEFPLLTERPVEREPAPAAPPGGLPRVRRDEAYEELRGRSDAHLEATGARPKVFIAALGPAAAHTARATFAANLFMAGGVEPVHDPVSVDAETAAEAFAASGATVACLCSSDVLYAEQAEAVARALKSAGALRVFLAGRGEFADIDEYVFAGCDAVAVLTSTLDRMGVA Amino acid sequence of isobutyryl-CoA mutase (icm) SEQ ID NO.7: MGVAAGPIRVVVAKPGLDGHDRGAKVIARALRDAGMEVIYTGLHQTPEQVVDTAIQEDADAIGLSILSGAHNTLFARVLELLKERDAEDIKVFGGGIIPEADIAPLKEKGVAEIFTPGATTTSIVEWVRGNVRQAV Amino acid sequence of glucokinase (glk) SEQ ID NO.9: MSTYRDLAHRGSARATVLRTVGTRERRSHLTAPRVNTVGIDIGGTKVMAGVVDPDGNILETLRTETPDKSKSPRVVEDTIIELVLDLSDRHDVHAVGIGAAGWVDADRNRVLFAPHLSWRNEPLRDRISGRLAVPVMVDNDANTAAWAEWRFGAGRGEDHLVMITLGTGIGGAILEDGQVKRGKFGVAGEFGHMQVVPGGHRCPCGNRGCWEQYSSGNALVREARELAAADSPVAYGIIERVKGNVPDITGPLITELAREGDAMCVELLQDIGQWLGVGIANLAAALDPSCFVIGGGVSAADDLLIGPARDAFRRHLTGRGYRPEARIARAQLGPEAGMVGAADLARLVARRFRRANRRRVERYERYERYAEARRTNDRTSQGTQ Amino acid sequence of isocitrate dehydrogenase (idh) SEQ ID NO.11: VTDSTIIYTHTDEAPALATHSFLPVVQAYASTAGVGVETRDISLAGRIIASFPERLEESQRISDALAELGELAKTPGANIIKLPNISASIPQLKAAIAELQGQGYALPDYPDDPKTDEERDIRARYDKVKGSAVNPVLREGNSDRRAPASVKNYAKAHPHRMGAWTADSKTNVATMGENDFRSTEKSAVITEAGSLRIELVGDDGSTTVLRESVPVLAGEVVDASVMRVAALREFLKQQVARAKSQGVLFSVHLKATMMKVSDPIVFGHVVRAFFPKTFAAHGETLAAAGLTPNDGLGGIFKGLESLPEGDKIKASFEAELAEGPELAMVDSDKGITNLHVPSDVIVDASMPAMIRTSGHMWGPDGEEHDTLAVLPDSSYAGIYQVVIDDCRANGAFDPATMGSVPNVGLMAQKAEEYGSHDKTFEVPTTGTVRVVDTAGNVVLEQTVAAGDIFRMCQTKDAPVQDWVKLAVTRARATGTPAVFWLDETRAHDAVLIGKVRQYLADHDTEGLDIRIMSPVEATAFSLERIRRGEDTISVTGNVLRDYLTDLFPILELGTSAKMLSVVPLMNGGGLFETGAGGSAPKHVQQLVKENYLRWDSLGEFLALAVSFEHLATTTGNARAQILADTLDRATATFLENDKSPSRRLGGIDNRGSHFYLALYWAQELAKQTEDAQLAEAFAALAKTLAEQEQTIVDELIAVQGSPADIGGYYQPDAAKASAVMRPSATFNQALSTLG Amino acid sequence of methylmalonyl-CoA isomerase (icm') SEQ ID NO.13: MDADAIEEGRRRWQARYDKARKRDADFTTLSGDPVDPVYGPRPGDTYDGFERIGWPGEYPFTRGLYATGYRGRTWTIRQFAGFGNAEQTNERYKMILANGGGGLSVAFDMPTLMGRDSDDPRSLGEVGHCGVAIDSAADME VLFKDIPLGDVTTSMTISGPAVPVFCMYLVAAERQGVDPAVLNGTLQTDIFKEYIAQKEWLFQPEPHLRLIGDLMEHCARDIPAYKPLSVSGYHIREAGATAAQELAYTLADGFGYVELGLSRGLDVDVFAPGLSFFFDAHV DFFEEIAKFRAARRIWARWLRDEYGAKTEKAQWLRFHTQTAGVSLTAQQPYNNVVRTAVEALAAVLGGTNSLHTNALDETLALPSEQAAEIALRTQQVLMEETGVANVADPLGGSWYIEQLTDRIEADAEKIFEQIRERGR RACPDGQHPIGPITSGILRGIEDGWFTGEIAESAFQYQRSLEKGDKRVVGVNCLEGSVTGDLEILRVSHEVEREQVRELAGRKGRRDDARVRASLDAMLAAARDGSNMIAPMLEAVRAEATLGEICGVLRDEWGVYVEPPGF promoter Nucleotide sequence SEQ ID No. 15: TGTTCACATTCGAACCGTCTCTGCTTTGACAACATGCTGTGCGGTGTTGTAAAGTCGTGGCC Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A recombinant Streptomyces cinnamonis producing monensin, characterized in that, The recombinant Streptomyces cinnamon simultaneously overexpressed phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase, and isobutyryl-CoA mutase.
2. The recombinant Streptomyces cinnamon according to claim 1, characterized in that, The amino acid sequence of the phosphofructokinase is shown in SEQ ID NO.1; the amino acid sequence of citrate synthase is shown in SEQ ID NO.3; the amino acid sequence of methylmalonyl-CoA isomerase is shown in SEQ ID NO.5; and the amino acid sequence of isobutyryl-CoA mutase is shown in SEQ ID NO.
7.
3. The recombinant Streptomyces cinnamon according to claim 1 or 2, characterized in that, use The promoter expresses phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase, and isobutyryl-CoA mutase. The nucleotide sequence of the promoter is shown in SEQ ID NO.
15.
4. The recombinant Streptomyces cinnamon according to claim 3, characterized in that, The connection sequence of promoter and gene is as follows -pfk- -cs- -mutB- -icm.
5. The use of the recombinant Streptomyces cinnamon according to any one of claims 1 to 4 in the preparation of monensin.
6. A method for producing monensin, characterized in that, The production of monensin by fermentation using the recombinant Streptomyces cinnamon according to any one of claims 1 to 4 includes the following steps: (1) Inoculate the recombinant Streptomyces cinnamon onto Gao's No. 1 solid medium and culture at 30-33℃ for 5-7 days. Take out a piece of spores about 1 square centimeter and inoculate it into Streptomyces cinnamon seed medium. Culture at 30-33℃ and 220-260 rpm for 24-28 h to obtain seed liquid. (2) The seed liquid was inoculated into the fermentation medium of Streptomyces cinnamon and centrifuged to obtain the fermentation broth containing monensin.
7. The method according to claim 6, characterized in that, In step (2), the inoculation amount of seed liquid is 18~22% (v / v).
8. The method according to claim 6, characterized in that, In step (2), the seed culture liquid is inoculated into the fermentation medium of Streptomyces cinnamon and cultured at 30-33℃, 220-260 rpm for 8-10 days.
9. A method for increasing the yield of monensin produced by *Streptomyces cinnamon*, characterized in that, Phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase and isobutyryl-CoA mutase were simultaneously overexpressed in Streptomyces cinnamon. The amino acid sequence of the phosphofructokinase is shown in SEQ ID NO.1; the amino acid sequence of citrate synthase is shown in SEQ ID NO.3; the amino acid sequence of methylmalonyl-CoA isomerase is shown in SEQ ID NO.5; and the amino acid sequence of isobutyryl-CoA mutase is shown in SEQ ID NO.
7.
10. The method according to claim 9, characterized in that, use The promoter expresses phosphofructokinase, citrate synthase, methylmalonyl-CoA isomerase, and isobutyryl-CoA mutase; The nucleotide sequence of the promoter is shown in SEQ ID NO.15, and the connection sequence of the promoter and the gene is as follows: -pfk- -cs- -mutB- -icm.