WO2021238841A1 - 一种玫瑰孢链霉菌中提高癸酸利用率及耐受度的调控基因及应用 - Google Patents
一种玫瑰孢链霉菌中提高癸酸利用率及耐受度的调控基因及应用 Download PDFInfo
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- WO2021238841A1 WO2021238841A1 PCT/CN2021/095453 CN2021095453W WO2021238841A1 WO 2021238841 A1 WO2021238841 A1 WO 2021238841A1 CN 2021095453 W CN2021095453 W CN 2021095453W WO 2021238841 A1 WO2021238841 A1 WO 2021238841A1
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- streptomyces roseosporus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- the present invention relates to the technical field of genetic engineering, in particular to a regulatory gene for improving the utilization and tolerance of capric acid in Streptomyces roseosporus, its application and a method for obtaining Streptomyces with high antibiotic production.
- Daptomycin is a new type of cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. It interacts with cell membranes in a calcium ion-dependent manner and exerts bactericidal activity.
- the U.S. FDA approved a new dosing regimen for Cubicin, a daptomycin injection of Cubist Pharmaceutical Co., Ltd., as a 2min intravenous bolus once a day.
- Cubicin was first approved in the United States in 2003 for the treatment of complicated skin and skin tissue infections caused by certain gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, by intravenous infusion for 30 minutes once a day
- methicillin-resistant Staphylococcus aureus by intravenous infusion for 30 minutes once a day
- a number of studies have shown that the proportion of drug-resistant daptomycin has not increased significantly over the past 10 years after it has been on the market, demonstrating the unique advantages of daptomycin.
- Daptomycin is a microbial secondary metabolite with a complex structure and very low yield. Genetic engineering is an important strategy to increase the yield of daptomycin and reduce production costs.
- Decanoic acid is an exogenous precursor in the synthesis of daptomycin, which can change the direction of secondary metabolism of the bacteria.
- decanoic acid is an essential substance, but excessive decanoic acid will be toxic to Streptomyces roseosporus; on the contrary, if the concentration of decanoic acid is too low, it will lead to insufficient supply of the premise and make the fermentation broth The potency of the product is reduced.
- Cyclic Adenosine Receptor Protein (CRP) is a conservative metabolic regulator widely found in bacteria. It exists in both Gram-negative and positive bacteria, but does not exist in Bacillus and other Firmicutes. . CRP is the most extensively studied in E. coli. In E.
- CRP In actinomycetes, including Streptomyces, CRP also has an important global regulatory role. Numerous studies have shown that CRP is directly involved in the regulation of multiple antibiotics in Streptomyces coelicolor, indicating that it can affect the flux of precursors into the secondary metabolism, and Play a role in primary and secondary metabolic processes (Gao C, Hindra, Mulder D, et al. CRP Is a Global Regulator of Antibiotic Production in Streptomyces [J]. mBio, 2012, 3(6).).
- the nucleotide identity of the CRP of daptomycin and the CRP of Streptomyces coelicolor is 89.3%, and the identity of the amino acid sequence is 94.2%.
- CRP is widely present in Streptomyces and has a high Conservative, but its role in most Streptomyces is different. Some regulate the development of Streptomyces spores, and some only regulate secondary metabolites. However, it has not been reported that CRP gene can improve the tolerance of Streptomyces roseosporus to decanoic acid.
- the invention provides a regulatory gene for improving the utilization and tolerance of capric acid in Streptomyces roseosporus, and provides an application of the gene in the preparation of a high-yield daptomycin strain.
- the invention provides a regulating gene for the utilization and tolerance of capric acid in Streptomyces roseosporus, which can encode CRP protein.
- amino acid sequence of the CRP protein is shown in SEQ ID NO.3.
- the regulatory gene can be expressed in Streptomyces roseosporus.
- nucleotide sequence of the regulatory gene is shown in SEQ ID NO.1.
- the regulatory gene is obtained by subcloning the genome of Streptomyces roseosporus, and its length is 672 base pairs.
- the regulatory gene can improve the tolerance and utilization of decanoic acid by Streptomyces roseosporus, and the nucleotide sequence of the regulatory gene is shown in SEQ ID NO.1.
- the regulatory gene is the homology analysis of the CRP gene of Streptomyces coelicolor, and the gene with the highest similarity is found in the genome sequence of Streptomyces roseosporus, and its gene is named CDS- 7.
- the annotation is classified as CRP/Fnr family transcriptional regulator, but there is no related literature and research to report its function in daptomycin. Through further phylogenetic tree and amino acid sequence motif analysis, it is determined that it is a CRP homologous protein .
- the present invention also provides a recombinant plasmid that can express the protein of SEQ ID NO. 3 in Streptomyces roseosporus.
- the protein encoding gene is SEQ ID NO. 1.
- the plasmid backbone of the aforementioned recombinant plasmid includes a shuttle plasmid into which a strong promoter erme can be inserted, preferably pKC1139, pEST152, pOJ260 or pSOK804.
- nucleotide sequence of the recombinant plasmid using pSET152 as the backbone is SEQ ID NO.2.
- the method provided by the present invention for obtaining high antibiotic production Streptomyces using the regulatory gene CRP, which enhances the expression of Streptomyces roseosporus to increase the utilization rate of capric acid and increase the tolerance to capric acid comprises the following steps:
- step (e) Perform antibiotic determination on the recombinant bacteria obtained in step (d) to obtain a strain of Streptomyces roseosporus with high antibiotic production.
- the plasmid backbone of the expression vector described in step (b) includes a shuttle plasmid into which a strong promoter erme can be inserted, preferably pKC1139, pEST152, pOJ260 or pSOK804.
- Streptomyces roseosporus described in step (c) is Streptomyces roseosporus NO. CGMCC4.7231.
- the Streptomyces roseosporus described in step (e) has been submitted to the General Microbiology Center of the China Microbial Species Collection and Management Committee on July 26, 2019.
- the preservation information of the recombinant strain of the present invention is as follows:
- Streptomyces roseosporus Streptomyces roseosporus
- the present invention has the following beneficial effects:
- the present invention solves the problems of high workload and high blindness of traditional Streptomyces breeding, and obtains a high-yield daptomycin strain cultivated by genetic engineering.
- the application prospect is broad; using this method to transform the starting bacteria Streptomyces roseosporus, it was unexpectedly found that the CRP gene can increase the utilization of decanoic acid by Streptomyces roseosporus during the fermentation process and increase its tolerance to decanoic acid.
- Fig. 1 is a comparison diagram of the normalized fermentation yield of Streptomyces roseosporus and the existing Streptomyces roseosporus under different precursor concentrations in the culture medium;
- Figure 2 is a physical map of the recombinant plasmid pSET152CRP
- Figure 3 is an electrophoresis diagram of pSET152CRP digestion
- Figure 4 shows the results of pSET152CRP plasmid sequencing
- Figure 5 shows the two feeding processes of the constructed bacteria and the starting bacteria in the fermentation process, the daily supplement of the amount of capric acid in the normal production process and the daily supplement of twice the normal amount of capric acid, each Comparison chart after normalization of the titer of Zidatomycin;
- Fig. 6 is the two feeding processes of the constructed bacteria and the starting bacteria in the fermentation process, the daily supplement of the amount of capric acid in the normal production process and the daily supplement of twice the normal amount of capric acid, each Contrast chart of daily decanoic acid utilization (titer/accumulative added amount of decanoic acid).
- Example 1 Construction of pSET152CRP containing the recombinant plasmid of CRP gene:
- CRPS ATTTCTAGAAATACCTGACCGAGCACG;
- CRPA ATTGGATCCATCGCACTGTTTTACCGT.
- the pSET152 plasmid was treated with restriction enzymes, digested with DNA agarose gel electrophoresis, and the SanPerp column DNA gel recovery kit was used to recover the target band with a size of 858bp according to the instructions. Store the resulting DNA solution at -20°C or use it for subsequent experiments.
- the amplified CRP gene was ligated with the pSET152 plasmid fragment recovered by restriction enzyme digestion.
- the restriction enzyme digestion ligation reaction system vector 0.03pmol, fragment 0.03-0.3pmol, T4DNA ligase 1200U, buffer 5ul, and the remaining volume is made up with distilled water. Connect at 16°C for 16h to obtain the ligation product.
- the recombinant plasmid pSET152CRP is 7033 bp in size and carries an Ampra resistance gene. It can be screened in E. coli and Streptomyces roseosporus. Int ⁇ C31 is the integrase gene, attP is the integration site, and p*erme is the erythromycin promoter.
- LB medium formula tryptone 10g, yeast extract 10g, NaCl 10g, add deionized water to 1000ml, pH 7.0. Sterilize at 121°C for 30 minutes for culturing Escherichia coli. Add 2% agar powder to the solid medium.
- the Escherichia coli ET12567 containing the recombinant plasmid PSET152 CRP was conjugated and transferred with Streptomyces roseosporus NO.CGMCC4.7231, and the conjugant containing the PSET152 CRP plasmid was obtained by screening with ampramycin and nalidixic acid, and at the same time used to introduce an empty vector PSET152 empty plasmid vector as a control.
- the cells were resuspended twice with the same volume of LB medium, and finally resuspended in 0.1 times the volume of LB medium. While washing the cells, 10-8 spores required for each conjugation transfer are suspended in 500ul 2 ⁇ YT medium, heat shocked at 50°C for 10 minutes, and cooled to room temperature. Take 500ul of E. coli solution and Streptomyces roseosporus spore solution and mix thoroughly, centrifuge to remove most of the supernatant, and resuspend with the remaining liquid. Spread the mixed bacterial liquid on MS solid medium containing 10mM MgCl 2 and incubate at 29°C for 16-20h.
- Example 3 Fermentation process of recombinant bacteria
- Streptomyces roseosporus strain was cultured in R5 slant medium at 30°C for 5 days, it was transferred to a shaker flask containing 50ml YEME liquid medium, and cultured at 30°C with shaking (220rpm/min) for 25h, and once for fermentation Capric acid was used as the fermentation precursor, and the fermentation was cultured until the end of fermentation.
- the fermentation unit was determined by HPLC, and high-yielding strains were selected.
- the high antibiotic production Streptomyces roseosporus prepared in the present invention has a higher fermentation yield, that is, a better Dato The yield of mycin, the fermentation yield is increased by more than 200%.
- Trace element solution (per liter): ZnCl 2 40mg, FeCl 2 ⁇ 6H 2 O 200mg, CuCl 2 ⁇ 2H 2 O 10mg, MnCl 2 ⁇ 2H 2 O 10mg, Na 2 B 4 O 7 ⁇ 10H 2 O 10mg, (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O 10mg
- Yeast extract 1.5g, tryptone 5g, malt extract 3g, glucose 10g, sucrose 250g, add deionized water to 1000ml, and steam sterilize at 115°C for 15min.
- Chromatographic conditions Chromatographic column: Phenomenex IB-SIL C8 4.6 ⁇ 250mm 5um, flow rate: 1.0mL/min, detection wavelength: 223nm, injection volume: 25 ⁇ L, column temperature: 25°C, gradient elution mobile phase A: weigh 3.4 g Dihydrogen ammonium phosphate was dissolved in 1000 mL of distilled water, adjusted to pH 3.1 with phosphoric acid, mobile phase B: acetonitrile.
- Example 5 Tolerance test of the constructed strain to the concentration of precursor
- Example 3 Using the fermentation process of Example 3, the tolerance range of the starting strain and the constructed strain NO.CGMCC4.7231 to the concentration of the fermentation precursor in the medium was compared in a shake flask. The results of the experiment are shown in Figure 1 of the specification. The production of daptomycin was the highest when the precursor concentration of the constructed bacteria was 3% to 5% in the shake flask, and the yield of daptomycin was the highest when the concentration exceeded 5%; The body concentration is between 2% and 3%. After 3%, the yield will drop significantly. It shows that the CRP gene can improve the tolerance of Streptomyces roseosporus to the precursors in the culture medium, and can increase the amount of capric acid added in the fermentor during industrial production to obtain a higher yield per unit volume.
- Example 6 Comparison of pilot fermentation process with different starting bacteria and constructed bacteria
- the constructed Streptomyces roseosporus strain was cultured in the seed tank for 22-26 hours, then transferred to the fermentation tank, cultured at about 30°C for 25 hours, and continuously fed during fermentation.
- Decanoic acid is used as a fermentation precursor.
- the amount of decanoic acid added to the tank by the constructing bacteria is twice that of the normal process of the starting bacteria.
- the starting bacteria tried to use the same feeding process as the constructed bacteria that is, the supplementary amount of capric acid was twice that of the original process). Cultivation to the end of fermentation, HPLC determination of fermentation units, comparison.
- Seed pot formula potato starch 6%, glucose 1.5%, sugar cane molasses 0.72%, ferrous ammonium sulfate 0.08%, foam enemy 0.05%.
- Fermentation tank formula potato starch 7.2%, glucose 1%, sugar cane molasses 0.72%, yeast powder 1.2%, ferrous ammonium sulfate 0.086%, foam enemy 0.05%.
- the constructed bacteria significantly increased the expression of daptomycin on the tank and increased the yield of a single fermentation; secondly, the constructed bacteria significantly improved the utilization rate of capric acid, so that the same amount of daptomycin was added. After the capric acid, more daptomycin is produced.
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Abstract
Description
Claims (10)
- 一种能提高玫瑰孢链霉菌中达托霉素产量的CRP蛋白,其特征在于,所述蛋白序列如SEQ ID NO.3所示。
- 根据权利要求1所述的CRP蛋白,其特征在于,所述蛋白可在玫瑰孢链霉菌中表达。
- 根据权利要求2所述的CRP蛋白,其特征在于,所述蛋白的编码核苷酸序列如SEQ ID NO.1所示。
- 一种编码权利要求1-3所示的CRP蛋白的核苷酸片段,其特征在于,所述核苷酸序列如SEQ ID NO.1所示。
- 一种重组质粒,其特征在于,可在玫瑰孢链霉菌中表达SEQ ID NO.3所示的蛋白,优选的,所述蛋白的编码基因如SEQ ID NO.1所示。
- 根据权利要求5所述的重组质粒,其特征在于,质粒的骨架包括可插入强启动子erme的穿梭质粒,优选pKC1139、pEST152、pOJ260或pSOK804,更优选pSET152。
- 根据权利要求6所述的重组质粒,其特征在于,质粒的核苷酸序列如SEQ ID NO.2所示。
- 一种调控基因,其特征在于,可以提高玫瑰孢链霉菌对癸酸的耐受度和利用率。
- 根据权利要求9所述的调控基因,其特征在于所述调控基因的核苷酸序列如SEQ ID NO.1所示。
- 一种根据权利要求1-3中任一项所述CRP蛋白或权利要求4的核苷酸片段或权利要求5-7任一项所述的重组质粒或权利要求8-9任一项所述的调控基因在提高玫瑰孢链霉菌中达托霉素产量中的应用。
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CN116731134B (zh) * | 2023-04-28 | 2024-05-31 | 中国农业科学院植物保护研究所 | 一种糖转运蛋白tp6568及其在改造高产链霉菌中的应用 |
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