Genetically engineered bacteria capable of high-yielding azamycin F, and construction method and application thereof

Abstract

The present invention discloses a genetically engineered bacterium that produces a high yield of azamycin F, a construction method thereof, and an application thereof, and belongs to the field of genetic engineering and microbial technology. The genetically engineered bacterium that produces a high yield of azamycin F of the present invention is a Streptomyces that overexpresses the azl4 gene (SEQ ID NO.1) and / or the azl6 gene (SEQ ID NO.2). The construction method thereof comprises the following steps: constructing an overexpression plasmid that drives the expression of the target gene with a strong promoter element, and transferring the constructed plasmid into a Streptomyces host that produces azamycin F by a conjugation transfer method. The present invention overexpresses the endogenous genes azl4 and azl6 related to the biosynthesis of azamycin F through genetic engineering means, thereby significantly increasing the yield of azamycin F. The present invention lays a foundation for increasing the yield of azamycin F and reducing the production cost of azamycin F.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and microbial technology, and particularly relates to a genetically engineered bacterium with high azamycin F production, a construction method thereof, and an application thereof. Background Art

[0002] Azamycin F is a 36-membered macrolide compound isolated from the liquid culture medium of Streptomyces hygroscopicus var. azalomyceticus. Its main components include azamycin F5a, F4a and F3a. It is reported that Streptomyces such as S. hygroscopicus MSU / MN-4-75B and S. malaysiensis MJM1968 can metabolize and produce azamycin F-type macrolides. Later, Yuan Ganjun et al. also isolated 12 36-membered macrolides including azamycin F5a, F4a, F3a and 9 new analogues from the fermentation broth of marine Streptomyces sp. 211726. Based on the research of Chandra and Iwasaki et al., they revised and improved the planar structure and 13C and 1H nuclear magnetic resonance signal assignments of azamycin F5a, F4a, F3a, and reported the relative configurations of azamycin F5a, F4a, F3a and their 7 new structural analogues for the first time.

[0003] Azamycin F compounds are reported to have broad-spectrum antibacterial activity, particularly against Gram-positive bacteria and fungi. These compounds primarily bind to bacterial and fungal cell membranes, altering their permeability and leading to leakage of intracellular substances and cell death. Yuan Ganjun reported that azamycin F5a, F4a, and F3a exhibit significant activity against methicillin-resistant Staphylococcus aureus (MRSA). Azamycin F5a targets lipoteichoic acid by binding to the polar head groups of cell membrane phospholipids, killing MRSA. Furthermore, their research results demonstrated that azamycin F exhibited moderate cytotoxic activity against human colon cancer cells, HCT-116. Yang Peiwen et al. also reported that azamycin F compounds exhibited significant protective effects against tomato gray mold, rice blast, tobacco brown spot, and pepper anthracnose. This suggests that azamycin F has the potential to be developed as a highly effective biogenic fungicide.

[0004] Xu Wei et al. scanned the entire genome of Streptomyces 211726 and, combined with experimental data, located a possible biosynthetic gene cluster for azamycin F, covering 23 open reading frames. These clusters contain eight polyketide synthase genes, azlBCDEFGHA, and a total of 19 polyketide synthase modules that catalyze the formation of the polyketide backbone. Fifteen non-PKS genes, azl1-15, are flanked by the polyketide synthase genes and involved in the biosynthesis of the azamycin F series. Researchers believe that the biosynthesis of azamycin F begins with 4-guanidinobutyramide. The amidase Azl13 hydrolyzes the amide group on the precursor to form a carboxyl group, generating the compound 4-guanidinobutyric acid. This is then activated by the ligase Azl4 to form 4-guanidinobutyric acid-CoA. This is then esterified by the acyltransferase Azl5 and attached to the PKS. The PKS then catalyzes carbon chain extension, followed by cyclization and release by the thioesterase domain of the final polyketide synthase module, forming the polyene macrolide backbone. Finally, post-modification enzymes are involved to produce the azamycin F series of compounds.

[0005] Currently, the main method used for breeding high-yielding strains of actinomycetes is genetic breeding. There are two main genetic breeding methods: one is traditional (physical / chemical) mutagenesis breeding technology, which uses ultraviolet mutagenesis, chemical mutagenesis, microwave mutagenesis, space mutagenesis, and ion beam mutagenesis to mutagenize the production strains, and then obtains mutant strains with better production performance through large-scale screening. This method has made great contributions to the breeding of high-yielding antibiotic strains, and many excellent strains have been successfully bred. However, this method has great blindness when screening for excellent mutant strains, and the screening workload is very large, which often makes it difficult to achieve the desired effect in a short period of time. Another genetic breeding method for actinomycetes is modern molecular biology genetic breeding technology, which further improves the production performance of strains by genetically modifying specific genes. This method can directly genetically modify genes that affect the level of antibiotic production in the strain, which is more targeted and the genetic manipulation is also very simple. Summary of the Invention

[0006] The present invention aims to provide a genetically engineered bacterium with high yield of azamycin F and a construction method and application thereof, for preparing azamycin F compounds.

[0007] The first object of the present invention is to provide a genetically engineered bacterium that produces azamycin F in high yield. The genetically engineered bacterium is obtained by overexpressing at least one of the following genes in a Streptomyces host that produces azamycin F:

[0008] (a) azl4 gene (4-guanidinobutanoate:CoA ligase), the nucleotide sequence of which is shown in SEQ ID NO. 1;

[0009] (b) azl6 gene (TetR-family transcriptional regulator), the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0010] In one embodiment, the Streptomyces that produces azamycin F is Streptomyces TKPJ3039 with a deposit number of CCTCC NO: M2013221. Streptomyces TKPJ3039 has been disclosed in Chinese patent CN103232964A "A high-yield azamycin F compound strain Streptomyces TKPJ3039 and its application", which was deposited in the China Center for Type Culture Collection on May 20, 2013.

[0011] In one embodiment, the azl4 and / or azl6 genes are overexpressed in a Streptomyces host using a site-specific integrating vector containing a strong promoter. The site-specific integrating vector is preferably a pSET152 or pIB139 plasmid. The strong promoter is preferably the constitutive promoter hrdBp, the nucleotide sequence of which is shown in SEQ ID NO. 3.

[0012] A second objective of the present invention is to provide a method for constructing a genetically engineered bacterium that produces high levels of azamycin F. The method comprises the following steps: constructing an overexpression plasmid in which a strong promoter element drives expression of a target gene (azl4 gene and / or azl6 gene), and transferring the constructed plasmid into a Streptomyces host that produces azamycin F by conjugation to obtain a genetically engineered bacterium that produces high levels of azamycin F. The overexpression plasmid is preferably carried by pSET152 or pIB139 plasmids. The strong promoter is preferably the constitutive promoter hrdBp.

[0013] Furthermore, the construction method comprises the following steps:

[0014] (1) Characterize and screen the strength of different promoters in Streptomyces host bacteria that produce azamycin F to obtain promoters with strong activity in the host bacteria;

[0015] (2) constructing an overexpression plasmid containing the target gene (azl4 gene and / or azl6 gene), wherein the plasmid contains the strong promoter element selected in step (1) and uses the apramycin resistance gene aac(3)IV as a marker;

[0016] Preferably, the overexpression plasmid uses pSET152 or pIB139 plasmid as a vector;

[0017] In one embodiment, the strong promoter element of the overexpression plasmid of the azl4 gene and / or the azl6 gene is the constitutive promoter hrdBp;

[0018] (3) transferring the plasmid constructed in step (2) into a Streptomyces host strain producing azamycin F;

[0019] (4) Screening: After selecting the conjugates, they are subcultured to screen for strains resistant to apramycin and perform colony PCR verification to obtain strains with complete integration, that is, genetically engineered bacteria with high production of apramycin F.

[0020] As an embodiment of the present invention, in step (1), the strength of different promoters in the host bacteria is characterized and screened, comprising the following steps:

[0021] ① Transfer the plasmid containing the promoter and kanamycin resistance gene neo into Streptomyces

[0022] Plasmids containing different promoters and the kanamycin resistance gene neo were transformed into Escherichia coli ET12567 / pUZ8002 competent cells, and the correct E. coli transformants were selected for conjugation transfer with Streptomyces host bacteria that produce azamycin F. The plasmids were transferred to Streptomyces host bacteria that produce azamycin F by conjugation transfer.

[0023] ② Screening

[0024] After the conjugative transfer products were subcultured on SFM medium at 28°C, colonies growing on apramycin-resistant plates were picked and verified by colony PCR to screen for strains in which the target plasmid was successfully integrated into the host bacterial genome.

[0025] Collect spores of each strain and suspend them in sterile water, adjust OD 600 To the same level, perform a gradient dilution, and evenly spot 10 μL of the spore suspension on a kanamycin gradient plate to test the resistance. The strength of the kanamycin resistance exhibited by the strain can directly indicate the activity of its corresponding promoter.

[0026] Furthermore, as an embodiment of the present invention, the method for constructing the above-mentioned genetically engineered bacteria that produces high-yield azamycin F specifically comprises the following steps:

[0027] (1) Construction of a conjugative transfer plasmid containing the target gene

[0028] The promoter hrdBp was inserted into the plasmid pSET152 to generate the pWHU1288 vector. The specific steps included designing primers hrdB-pF and hrdB-pR within the hrdB gene of Streptomyces coelicolor M145, adding an NdeI restriction site at the start codon ATG, amplifying the hrdB promoter (hrdBp) of Streptomyces coelicolor M145 by PCR, as shown in SEQ ID NO. 3, and digesting the amplified 451 bp DNA fragment containing hrdBp with XbaI and BamHI, and inserting the fragment into the XbaI and BamHI sites of the integrative vector pSET152 (commercial vector, GenBank sequence number: AJ414670.1), to generate the plasmid pWHU1288.

[0029] The gene azl4 related to the biosynthesis precursor of azamycin F was connected to the pWHU1288 vector by enzyme digestion and ligation to construct the overexpression plasmid pMX301.

[0030] The azamycin F biosynthesis-related regulatory gene azl6 was ligated with the pWHU1288 vector by enzyme digestion and ligation to construct the target gene overexpression plasmid pMX303.

[0031] After transformation into E. coli DH5α competent cells, the plasmid was extracted and digested with enzymes for verification to obtain a conjugative transfer plasmid containing the target gene;

[0032] (2) Conjugation transfer to Streptomyces

[0033] The plasmid in step (1) is transferred into Escherichia coli ET12567 / pUZ8002 competent cells, and the correct Escherichia coli transformants are selected for conjugation transfer with azamycin F-producing Streptomyces. The plasmid is transferred into azamycin F-producing Streptomyces by conjugation transfer to achieve overexpression of the target gene;

[0034] (3) Screening of genetically engineered strains

[0035] After the conjugates were picked and subcultured on SFM medium at 28°C, colonies grown on Abraxine-resistant plates were picked for colony PCR verification, and strains with successful integration of the target gene were screened, namely, genetically engineered bacteria with high production of azamycin F were obtained.

[0036] The third object of the present invention is to provide the use of the genetically engineered bacteria in the preparation of azamycin F.

[0037] A method for preparing azamycin F comprises the following steps: inoculating the seed liquid of the genetically engineered bacteria into a fermentation medium for fermentation.

[0038] In one embodiment, the fermentation medium consists of 10 g of glucose, 35 g of soluble starch, 2 g of yeast powder, 4 g of casein, and 1 L of deionized water, and the pH is adjusted to 7.2-7.4.

[0039] In one embodiment, the seed liquid is a bacterial liquid inoculated with genetically engineered bacterial spore liquid in TSBY liquid culture medium and cultured at 28° C. and 220 rpm for 48-72 hours.

[0040] In one embodiment, the fermentation conditions are 28° C., 220 rpm, 10% container filling volume, and a fermentation period of 10-11 days.

[0041] Advantages and beneficial effects of the present invention: By genetically engineering the endogenous genes azl4 and azl6 involved in the biosynthesis of azamycin F, the resulting engineered strains achieve 136.5% and 94.3% higher azamycin F yields, respectively, compared to control strains. This invention provides a new approach to the industrial production of azamycin F and lays the foundation for increasing azamycin F yield and reducing azamycin F production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Schematic diagram of the promoter strength in the host bacteria after culturing on basic medium at 28°C incubator for 6 days;

[0043] Figure 2 : Schematic diagram of the promoter strength in the host bacteria after culturing on fermentation medium at 28°C in an incubator for 6 days;

[0044] Figure 3 : Schematic diagram of the recombinant plasmid containing the strong promoter hrdBp for overexpressing the target gene;

[0045] Figure 4 : Agarose gel electrophoresis of PCR verification of recombinant strains; wherein, lane M is a DNA molecular weight standard; lane 1 and lane 2 are the products of PCR amplification using chromosomal DNA of recombinant strains TKPJ3039::pMX301 and TKPJ3039::pMX303 as templates and primers M13F and M13R, respectively;

[0046] Figure 5 : HPLC detection of fermentation products of wild-type strain TKPJ3039; among them, those with retention times of 22.975 minutes and 23.296 minutes were azamycin F homologues;

[0047] Figure 6 : Comparison of relative yields of azamycin F between genetically engineered strains and wild-type strains. DETAILED DESCRIPTION

[0048] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, rather than all of them. It should be noted that all other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0049] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] The molecular cloning tool E. coli DH5α and the two-parent conjugative transfer tool E. coli ET12567 / pUZ8002 are commercial products, and their competent cells were prepared according to the "Molecular Cloning Experimental Guide." The deposit number of the azamycin F-producing strain Streptomyces TKPJ3039 is CCTCC NO: M2013221, which is disclosed in Chinese patent CN103232964A, "A High-yield Azamycin F-Class Compound Strain Streptomyces TKPJ3039 and Its Application."

[0051] DNA sequencing was performed by Wuhan Qingke Biotechnology Co., Ltd.

[0052] The primers involved in the experimental process of the present invention are shown in Table 1 below. The primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0053] Table 1 Primers involved in the experiment of the present invention

[0054]

[0055]

[0056] The culture medium and reagents involved in the experimental process of the present invention are as follows:

[0057] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, add distilled water to 1000 mL, sterilize at 115°C for 30 min.

[0058] LA medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, add distilled water to 1000 mL, sterilize at 115°C for 30 min.

[0059] 2×YT medium: 16 g tryptone, 10 g yeast extract, 5 g sodium chloride, add distilled water to 1000 mL, sterilize at 115°C for 30 min.

[0060] AZ medium: 20 g soybean cake powder, 15 g bran, 0.02 g asparagine, 7.5 g soluble starch, 0.3 g K2HPO4, 1 g KNO3, 0.5 g NaCl, 1 g CaCO3, 15 g agar, add distilled water to 1000 mL, pH 7.5 (adjust with NaOH), 115°C, sterilize for 30 min, add sterilized 5 M CaCl2 to a final concentration of 120 mM when used.

[0061] SFM culture medium: 20 g soybean cake powder, 20 g mannitol, 20 g agar, add distilled water to 1000 mL, 115°C, and sterilize for 30 min.

[0062] TSBY medium: 103 g sucrose, 30 g Oxoid tryptic soy broth powder, 5 g yeast extract, add distilled water to 1000 mL, sterilize at 115°C for 30 min.

[0063] MM medium: L-asparagine 0.5 g, K2HPO4 0.5 g, magnesium sulfate heptahydrate 0.2 g, ferrous sulfate heptahydrate 0.01 g, mannitol 5 g, agar 10 g, add deionized water to 1000 mL, sterilize at 115°C for 30 min.

[0064] Azamycin F fermentation medium: 10 g glucose, 35 g soluble starch, 2 g yeast extract, 4 g casein, add deionized water to 1000 mL, pH 7.2-7.4, 115°C, and sterilize for 30 min.

[0065] Spore pre-germination medium: 1% yeast extract, 1% casamino acids, 0.01 M CaCl2.

[0066] TES solution: Weigh 2.292 g of tris(hydroxymethyl)aminoethanesulfonic acid (TES) powder, add deionized water to 200 mL, adjust the pH to 8.0, and sterilize at 121°C for 20 min.

[0067] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0068] Example 1: Construction of tool plasmids

[0069] Using the kanamycin resistance gene neo as a reporter gene, reporter plasmids pLXY37, pLXY38, pLXY39, pLXY40, and pLXY41 derived from the site-specific integration vector pIB139 or pSET152 were constructed to detect the expression activities of the constitutive strong promoters ermEp*, ermEpA, hrdBp, SCO5768p, and kasOp* in actinomycete host cells, respectively.

[0070] The construction method includes the following steps:

[0071] 1. Primers DQ195F and DQ195R were designed and used as a template to amplify an 809-bp DNA fragment containing the kanamycin-resistance gene neo as a reporter gene from the commercial plasmid pHZ1358 (GenBank: AY667410.1). This fragment was then cloned into the commercial blunt-end vector pEasy-Blunt Zero Vector to obtain the intermediate plasmid pLXY36, which was verified by restriction enzyme digestion and sequencing. pLXY36 was digested with NdeI and EcoRI to recover a 795-bp DNA fragment containing the kanamycin-resistance gene neo. This fragment was then inserted into the commercial vector pIB139 between the NdeI and EcoRI restriction sites to generate plasmid pLXY37. The neo reporter gene on this plasmid is under the control of the promoter ermEp*, which can be used to characterize the strength of the promoter ermEp* in different hosts and culture conditions.

[0072] 2. Design primers LYL25F and LYL25R to amplify a 75-bp DNA fragment containing the ribosome binding region of ermEp* using the vector pIB139 as a template. Primer LYL25F introduces a mutation site, altering the original sequence of the ribosome binding region. The amplified product was cloned into the commercial blunt-end vector pEasy-Blunt Zero Vector to obtain an intermediate plasmid, which was verified by enzyme digestion and sequencing. A 71bp DNA fragment containing the mutated ribosome binding region was recovered by digestion with BglII and EcoRI, and replaced the original ribosome binding region between BamHI and EcoRI of the commercial vector pIB139 to obtain plasmid pIB139A. The promoter of this plasmid has a different DNA sequence in the ribosome binding region compared to the promoter ermEp* on pIB139, so the altered promoter was named ermEpA to distinguish it. pLXY36 was digested with NdeI and EcoRI to recover a 795bp DNA fragment containing the kanamycin resistance gene neo, which was inserted between the NdeI and EcoRI restriction sites of pIB139A to obtain plasmid pLXY38. The neo reporter gene of this plasmid is under the control of the promoter ermEpA, which can be used to characterize the strength of the promoter ermEpA in different hosts and different culture conditions.

[0073] 3. Primers hrdB-pF and hrdB-pR were designed, and the promoter hrdBp of the gene hrdB (i.e., SCO5820) was amplified using chromosomal DNA of Streptomyces coelicolor (GenBank: AL645882.2) as a template. The amplified promoter was then cloned into the pSET152 vector between the XbaI and BamHI restriction sites to construct the overexpression vector pWHU1288 containing hrdBp. The overexpression vector was verified by enzyme digestion and sequencing. pLXY36 was digested with NdeI and EcoRI to recover a 795 bp DNA fragment containing the kanamycin resistance gene neo. This fragment was then inserted into the NdeI and EcoRI restriction sites of pWHU1288 to generate the plasmid pLXY39. The neo reporter gene in this plasmid is under the control of the promoter hrdBp, which can be used to characterize the strength of the promoter hrdBp in different hosts and culture conditions.

[0074] 4. Primers DQ200F and DQ200R were designed, and the promoter of SCO5768 was amplified using chromosomal DNA of Streptomyces coelicolor as a template. The promoter was then cloned into the XbaI and BamHI restriction sites of the vector pSET152 to construct the overexpression vector pWHU1289 containing SCO5768p. The expression was confirmed by enzyme digestion and sequencing. pLXY36 was digested with NdeI and EcoRI to recover a 795 bp DNA fragment containing the kanamycin resistance gene neo. The fragment was then inserted between the NdeI and EcoRI restriction sites of pWHU1289 to obtain plasmid pLXY40. The neo reporter gene of this plasmid is under the control of the promoter SCO5768p, which can be used to characterize the strength of the promoter SCO5768p in different hosts and culture conditions.

[0075] 5. Primers DQ222F and DQ222R were designed to amplify the promoter of the Streptomyces coelicolor gene kasO, which was then cloned into the vector pSET152 between the XbaI and BamHI restriction sites to construct the overexpression vector pWHU1290 containing kasOp*. Enzyme digestion and sequencing confirmed its correctness. pLXY36 was digested with NdeI and EcoRI to recover a 795-bp DNA fragment containing the kanamycin-resistance gene neo. This fragment was inserted between the NdeI and EcoRI restriction sites of pWHU1290 to generate plasmid pLXY41, which contains the neo reporter gene under the control of the kasOp* promoter and can be used to characterize the strength of the kasOp* promoter in different hosts and culture conditions.

[0076] Example 2: Characterization and screening of the strength of different promoters in host bacteria

[0077] The pLXY37, pLXY38, pLXY39, pLXY40 and pLXY41 constructed in Example 1 were respectively transformed into Streptomyces TKPJ3039 to detect the expression intensity of the constitutive strong promoters ermEp*, ermEpA, hrdBp, SCO5768p and kasOp* in the host cells.

[0078] 1. Transformation of E. coli ET12567 / pUZ8002

[0079] According to the "Molecular Cloning Experiment Guide", the plasmid was transformed into Escherichia coli ET12567 / pUZ8002 competent cells, as briefly described below: 1 μL of the target plasmid was added to the Escherichia coli ET12567 / pUZ8002 competent cells, mixed gently, and placed in an ice bath for 30 minutes; heat shock at 42°C for 90 seconds; ice bath for 3 minutes; 1 mL of LB medium was added; incubated on a shaker at 37°C for 45 minutes; centrifuged at 6000 rpm for 3 minutes; discarded the supernatant, and 300 μL of LB medium was added to suspend the precipitate; spread on LA plates containing apramycin, kanamycin, and chloramphenicol at final concentrations of 50 μg / mL, 50 μg / mL, and 25 μg / mL, respectively, and air-dried; and incubated in a 37°C incubator overnight. Single colonies were picked and transferred to LB medium containing apramycin, kanamycin, and chloramphenicol at final concentrations of 50 μg / mL, 50 μg / mL, and 25 μg / mL, respectively, and cultured in a shaking incubator at 37°C overnight; plasmids were extracted and detected by agarose gel electrophoresis.

[0080] 2. Indirect transfer of Escherichia coli ET12567 / pUZ8002 into Streptomyces TKPJ3039

[0081] Referring to the "Streptomyces Experiment Manual" and Wuhan University doctoral thesis "Cloning and Functional Analysis of Azamycin F3a Biosynthesis-Related Genes" by Ma Yanling, the brief process is as follows:

[0082] Escherichia coli ET12567 / pUZ8002 containing the target plasmid was inoculated into 5 mL of LB medium containing 50 μg / mL apramycin, 50 μg / mL kanamycin, and 25 μg / mL chloramphenicol, and cultured overnight at 37°C in a shaker. The overnight cultured Escherichia coli was transferred to 5 mL of fresh LB medium containing 50 μg / mL apramycin, 25 μg / mL chloramphenicol, and 50 μg / mL kanamycin at a ratio of 1:20, and cultured at 37°C in a shaker until the OD 600 Collect 1 mL of the culture medium by centrifugation and discard the supernatant. Wash the cells twice with fresh LB medium without antibiotics and suspend them in 500 μL of 2×YT medium for later use.

[0083] Suspend Streptomyces TKPJ3039 spores in 1 mL of 0.05 M TES solution (pH 8.0), shake to mix, centrifuge at 10,000 rpm for 1 minute, and discard the supernatant. Repeat this step once. Resuspend the spores in 700 μL of fresh TES. Heat shock at 50°C for 10 minutes. After cooling to room temperature, add 500 μL of spore pre-germination medium and incubate at 37°C on a shaker for 1-2 hours before use.

[0084] Mix the prepared E. coli suspension with the pre-germinated spore suspension, incubate on a shaker at 28°C for 1 hour, and then spread onto AZ medium plates. After 17 hours of incubation at 28°C, overlay with 1 mL of sterile water containing apramycin (final concentration 75 μg / mL) and nalidixic acid (final concentration 25 μg / mL). Incubate at 28°C for 5-7 days and observe for the presence of conjugative transfer.

[0085] 3. Conjugative transfer verification

[0086] Single conjugant colonies from the conjugation transfer plate were inoculated into SFM medium containing apramycin (50 μg / mL) and cultured at 28°C for 5-7 days. The culture was then transferred to TSBY liquid medium containing apramycin (25 μg / mL) and cultured on a shaker at 28°C for 48 hours. The cells were then harvested for total DNA extraction. Chromosomal DNA extraction was performed according to the "Streptomyces Experimental Manual." The following is a brief procedure: 500 μL of mycelium was placed in a sterile 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 2 minutes, discarding the supernatant. The cells were washed twice with sterile water and centrifuged, discarding the supernatant. 500 μL of lysozyme (2 mg / mL) was added, and the tube was incubated at 37°C for 30 minutes, mixing by inversion every 2 minutes. 4% SDS was added. 25μL, in a 55℃ oven for 5-10min, until transparent; add 300μL of phenol-chloroform, vortex to mix, and centrifuge at 12000rpm for 5min; take 700μL of the upper liquid, add 70μL of sodium acetate (3M), mix well, then add 770μL of isopropanol, gently turn upside down 5-6 times, and place at -40℃ for 20min; centrifuge at 12000rpm for 5min, discard the supernatant, add 1mL of 75% ethanol, let stand at room temperature for 1min, centrifuge at 12000rpm for 1min, discard the upper layer, and repeat this step once; dry in a 55℃ oven, add 40-60μL of sterile ultrapure water to dissolve, and store at -40℃ for use.

[0087] The reporter plasmids pLXY37, pLXY38, pLXY39, pLXY40, and pLXY41 were derived from the vectors pIB139 or pSET152. The binding regions for the universal primers M13F and M13R are located upstream and downstream of the multiple cloning site. Therefore, primers M13F and M13R were synthesized and used to verify the correctness of the conjugates by PCR. Their sequences are shown in Table 1. PCR was performed using the 2× Taq Master Mix (Dye Plus) kit from Nanjing Novozymes Biotech Co., Ltd. A 20 μL PCR system consisted of: template, 1 μL; primer F, 1 μL; primer R, 1 μL; sterile deionized water, 7 μL; and 2× Taq Master Mix, 10 μL. The PCR reaction program was as follows: 95°C for 3 min; 95°C for 15 s; 60°C for 15 s; 72°C for 60 s, 30 cycles; and 72°C for 5 min. PCR products were analyzed by agarose gel electrophoresis.

[0088] 4. Promoter Screening

[0089] Collect spores of each strain and suspend them in sterile water, adjust the spore concentration to the same as the mother solution, and then dilute it in a gradient. -2 -10 -5 10 μL of spore suspension with different dilution multiples was evenly spotted on kanamycin gradient concentration plates (culture medium was basic medium MM or azamycin F fermentation medium) for resistance strength test, and cultured at 28°C for 6 days. The results are shown in the table. Figure 1 The kanamycin resistance exhibited by the strain can directly indicate the activity of its corresponding promoter. The final kanamycin concentrations on the MM medium plates were: 0μg / mL, 2.5μg / mL, 5.0μg / mL, 10μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, and 250μg / mL; the final kanamycin concentrations on the fermentation medium plates were: 0μg / mL, 10μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, 250μg / mL, 300μg / mL, 400μg / mL, 500μg / mL, and 650μg / mL.

[0090] Depend on Figure 1 The results show that on the MM culture medium plate, the promoter expression intensity is as follows: hrdBp, KasOp*>SCO5768p, ermEpA>ermEp*. Figure 2 The results showed that under the growth environment of fermentation medium, the promoter expression strength was hrdBp>KasOp*, SCO5768p>ermEpA, ermEp*.

[0091] The results showed that the hrdBp promoter had the strongest activity in the strain TKPJ3039 producing azamycin F. Overexpression of azamycin F biosynthesis genes will be carried out using the plasmid pWHU1288 containing the hrdBp promoter as a vector to construct the target gene overexpression plasmid.

[0092] The construction of the plasmid pWHU1288 as a vector includes PCR amplification of the hrdB promoter in Streptomyces coelicolor M145. The specific steps include: designing primers hrdB-pF and hrdB-pR within the hrdB gene in Streptomyces coelicolor M145, adding an NdeI restriction site at the start codon ATG, PCR amplifying the hrdB promoter (hrdBp) in Streptomyces coelicolor M145, as shown in SEQ ID NO. 3, and digesting the amplified 451 bp DNA fragment containing hrdBp with XbaI and BamHI, and inserting it into the XbaI and BamHI sites of the integrative vector pSET152 (commercial vector, GenBank sequence number: AJ414670.1) to obtain plasmid pWHU1288.

[0093] Example 3: Azamycin F high-yielding strain and its construction method

[0094] In order to obtain a genetically engineered bacterium with high production of azamycin F, it is planned to overexpress the azamycin F starting unit synthesis-related genes azl4, azl5, azl13 and azl14, the azamycin F synthesis regulation-related genes azl6 and azl12, and the transport-related gene azl10.

[0095] 1. Construction of target gene overexpression plasmid

[0096] Using the total DNA of azamycin F-producing strain TKPJ3039 as a template, primers DQ201F and DQ201R were used for PCR amplification to obtain a 2596 bp DNA fragment containing azl4. The PCR was performed using 2×Hieff Canace from Shanghai Yisheng Company. TM PCRMaster Mix high-fidelity enzyme premix system. 20μL PCR system includes: template, 1μL; primer F, 1μL; primer R, 1μL; DMSO, 1μL; sterile deionized water, 6μL; 2× Hieff Canace TMPCR Master Mix, 10 μL. PCR reaction program: 98°C, 3 min; 98°C, 10 s, 60°C, 20 s, 72°C, 90 s (30 s / kb), 30 cycles; 72°C, 5 min. The DNA fragment obtained by PCR amplification was isolated and purified using agarose gel electrophoresis and an Omega Bio-Tek gel recovery kit. PCR amplification was performed using the purified DNA fragment as a template with primers MX01F and MX01R. A 1467 bp DNA fragment containing the target gene azl4 (SEQ ID NO. 1) was obtained. This fragment was reisolated and purified, digested with NdeI and EcoRI, and isolated and purified again. The vector pWHU1288 was digested with NdeI and EcoRI, isolated and purified. The exogenous fragment and the vector fragment were ligated using T4 ligase. The ligated fragment was transformed into DH5α competent cells, and the plasmid was extracted using an Omega Bio-Tek plasmid extraction kit. Enzyme digestion was verified and DNA sequencing was performed. The correct plasmid was named pMX301, the azl4 overexpression plasmid, and the bacterial strain and plasmid DNA were preserved.

[0097] The same method was used to construct overexpression plasmids of azl5, azl6, azl10, azl12, azl13, azl14 and adpA (the plasmid schematic diagram is shown in Figure 3 The bacterial strain and plasmid DNA were saved for future use. The construction process is briefly described as follows:

[0098] Total DNA from the azamycin F-producing strain TKPJ3039 was used as a template for PCR amplification using primers DQ201F and DQ201R, yielding a 2596 bp DNA fragment containing the azl5 gene. The purified DNA fragment was then used as a template for PCR amplification using primers MX02F and MX02R, yielding a 1058 bp DNA fragment containing the target gene azl5 (SEQ ID NO. 4). The fragment was digested with NdeI and EcoRI, ligated with the pWHU1288 vector treated with the same enzymes, and transformed into Escherichia coli DH5α. Transformants were cultured, plasmids were extracted, and enzyme digestion and sequencing were performed to verify the correct plasmid. The resulting plasmid was designated pMX302, the azl5 overexpression plasmid.

[0099] Total DNA from the azamycin F-producing strain TKPJ3039 was used as a template for PCR amplification using primers DQ202F and DQ202R, yielding an 891 bp DNA fragment containing the azl6 gene. The purified DNA fragment was then used as a template for PCR amplification using primers MX03F and MX03R, yielding a 633 bp DNA fragment containing the target gene azl6 (SEQ ID NO. 2). The fragment was digested with NdeI and EcoRI, ligated with the pWHU1288 vector treated with the same enzymes, and transformed into Escherichia coli DH5α. The transformants were cultured, plasmids were extracted, and enzyme digestion and sequencing were performed to verify the correct plasmid. The resulting plasmid was designated pMX303, the azl6 overexpression plasmid.

[0100] Total DNA from the azamycin F-producing strain TKPJ3039 was used as a template for PCR amplification using primers DQ204F and DQ204R to obtain a 1003 bp DNA fragment containing the azl10 gene. The purified DNA fragment was used as a template for PCR amplification using primers MX05F and MX05R to obtain an 812 bp DNA fragment containing the target gene azl10 (SEQ ID NO. 5). The fragment was digested with NdeI and EcoRI, ligated with the vector pWHU1288 treated with the same enzymes, and transformed into Escherichia coli DH5α. The transformants were transferred and cultured, and plasmids were extracted, verified by enzyme digestion, and sequenced. The correct plasmid was designated pMX305, the azl10 overexpression plasmid.

[0101] Using total DNA from the azamycin F-producing strain TKPJ3039 as a template, PCR amplification was performed with primers DQ206F and DQ206R to obtain a 977-bp DNA fragment containing the azl12 gene. This DNA fragment was purified and used as a template for PCR amplification using primers MX07F and MX07R. An 827-bp DNA fragment containing the target gene azl12 (SEQ ID NO. 6) was obtained. The fragment was digested with NdeI and EcoRI, ligated with the pWHU1288 vector treated with the same enzymes, and transformed into Escherichia coli DH5α. Transformants were cultured, plasmids were extracted, and enzyme digestion and sequencing were performed to verify the correct plasmid. The resulting plasmid was designated pMX307, the azl12 overexpression plasmid.

[0102] Total DNA from the azamycin F-producing bacterium TKPJ3039 was used as a template for PCR amplification using primers DQ207F and DQ207R to obtain a 2589 bp DNA fragment containing the azl13 gene. The purified DNA fragment was used as a template for PCR amplification using primers MX08F and MX08R to obtain an 829 bp DNA fragment containing the target gene azl13 (SEQ ID NO. 7). The fragment was digested with NdeI and EcoRI, ligated with the vector pWHU1288 treated with the same enzymes, and transformed into Escherichia coli DH5α. The transformants were transferred and cultured, and plasmids were extracted, verified by enzyme digestion, and sequenced. The correct plasmid was designated pMX308, the azl13 overexpression plasmid.

[0103] Using total DNA from the azamycin F-producing strain TKPJ3039 as a template, PCR amplification was performed with primers DQ207F and DQ207R to obtain a 2589 bp DNA fragment containing the azl14 gene. This DNA fragment was purified and used as a template for PCR amplification using primers MX09F and MX09R. A 1470 bp DNA fragment containing the target gene azl14 (SEQ ID NO. 8) was obtained. This fragment was digested with NdeI and EcoRI, ligated with the pWHU1288 vector treated with the same enzymes, and transformed into Escherichia coli DH5α. Transformants were cultured, plasmids were extracted, and enzyme digestion and sequencing were performed to verify the correct plasmid. The resulting plasmid was designated pMX309, the azl14 overexpression plasmid.

[0104] Separately, using total DNA from Streptomyces coelicolor M145 as a template, primers adpA-F and adpA-R were used to amplify a 1229-bp DNA fragment containing the 1197-bp gene adpA, encoding a functionally defined global transcriptional regulator. The relevant sequence is listed in the NCBI database as GenBank ID 1098226. After isolation and purification, the fragment was digested with NdeI and PmeI and ligated with the pWHU1288 vector, which had been digested and purified with NdeI and EcoRV. The fragment was then transformed into Escherichia coli DH5α. Transformants were then cultured and the plasmid was isolated. After verification of enzyme digestion, DNA sequencing confirmed the fragment to be consistent with the original sequence. The correct plasmid was designated pLXY55, the adpA overexpression plasmid.

[0105] 2. Indirect transfer of the target gene overexpression plasmid from Escherichia coli to Streptomyces TKPJ3039

[0106] The above-mentioned overexpression plasmids were transformed into Escherichia coli ET12567 / pUZ8002 competent cells, and the transformants were picked and cultured. The correct transformants were selected and transferred to Streptomyces TKPJ3039 for biparental conjugation.

[0107] For the specific method of this step, please refer to step 2 in Example 2.

[0108] 3. Phenotypic Screening and Genotypic Verification of Gene Overexpression Strains

[0109] Single apramycin-resistant conjugant colonies were picked and transferred to SFM plates containing apramycin (50 μg / mL) for culture; colonies that grew well on apramycin-resistant plates were selected and transferred to TSBY liquid medium containing apramycin (25 μg / mL) for culture, the bacteria were collected, and total DNA was extracted.

[0110] Using total DNA from the overexpressing strain as a template, PCR amplification with primers M13F and M13R revealed that the theoretically generated DNA fragment sizes for the correct Streptomyces conjugants, TKPJ3039::pMX301, TKPJ3039::pMX302, TKPJ3039::pMX303, TKPJ3039::pMX305, TKPJ3039::pMX307, TKPJ3039::pMX308, TKPJ3039::pMX309, and TKPJ3039::pLXY55, were 1956 bp, 1548 bp, 1122 bp, 1305 bp, 1317 bp, 1317 bp, 1959 bp, and 1718 bp, respectively. Agarose gel electrophoresis analysis confirmed the correct conjugants and preserved the strains for future use. The agarose gel electrophoresis analysis of TKPJ3039::pMX301 and TKPJ3039::pMX303 was as follows: Figure 4 Lanes 1 and 2 are shown.

[0111] For the specific method of this step, please refer to step 3 in Example 2.

[0112] Example 4: Fermentation of overexpression strains and detection of fermentation products

[0113] 1. Seed culture: The overexpression strain obtained in step 3 of Example 3 was activated on an SFM plate containing apramycin (50 μg / mL). An appropriate amount of spore liquid was collected and inoculated into 5 mL of TSBY liquid medium containing apramycin (25 μg / mL). The culture was carried out at 28°C and 220 rpm for 2-3 days to obtain a seed liquid.

[0114] 2. Shake flask fermentation: The seed solution obtained in step 1 was inoculated into a 250 mL shake flask containing 25 mL of azamycin F fermentation medium at a 10% inoculation rate and fermented at 28°C and 220 rpm for 10 days.

[0115] 3. Fermentation Broth Treatment: Centrifuge the fermentation broth from step 2 at 3500 rpm for 15 minutes to separate the cells and supernatant. Soak the cells in an equal volume of methanol. Collect the organic phase by centrifugation, combine it with the supernatant, and adjust the volume to 45 mL with methanol. Mix thoroughly. Take 1 mL of the sample and centrifuge at 12000 rpm for 2 minutes. Collect the supernatant and filter through a 0.22 μm organic microporous filter.

[0116] 4.HPLC detection: Azamycin F was detected using Shimadzu SPD-M20A / LC-20AT chromatograph. C18 chromatographic column: Shim-pack VP-ODS, 250×4.6mm, 5μm; column oven: 25°C; pump mode: binary high pressure gradient; HPLC total flow rate: 1.0mL / min; set the injection volume to 20μL; full wavelength detection, total detection time 30min. Mobile phase A is deionized water and phase B is methanol. Elution was performed according to the following conditions: 0-2min; mobile phase B was maintained at 70%, 2-25min, mobile phase B increased from 70% to 90%; 25-28min; mobile phase B decreased from 90% to 70%, 28-30min, mobile phase B was maintained at 70%. HPLC detection results are as follows: Figure 5 As shown, according to the standard, the peaks with retention times of 22.975min and 23.296min are various homologues of azamycin F. The peak areas were integrated, and the yields of the wild-type strain and the overexpression strain were converted according to the standard curve. The changes in the yield of azamycin F in the overexpression strain were calculated. This was repeated three times, and the statistical results are shown in Figure 2. Figure 6 As shown in the figure, the wild-type strain produced 5.01 g / L of azamycin F, which is 100%. The strains overexpressing the azl4 gene, TKPJ3039::pMX301, and overexpressing the azl6 gene, TKPJ3039::pMX303, increased their azamycin F production by 136% and 94%, respectively. No significant increase in azamycin F production was observed in the other strains.

[0117] The foregoing description is a description of a specific embodiment of the present invention. After reading the above description of the present invention, any person skilled in the art may make various changes and modifications within the technical scope disclosed by the present invention, and all such changes and modifications shall be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope of protection of the claims.

Claims

1. A genetically engineered bacterium that produces a high yield of azamycin F, characterized by: The genetically engineered bacteria are obtained by overexpressing at least one of the following genes in a Streptomyces host that produces azamycin F: the azl4 gene with a nucleotide sequence as shown in SEQ ID NO.1, and the azl6 gene with a nucleotide sequence as shown in SEQ ID NO.

2.

2. The genetically engineered bacterium for high production of azamycin F according to claim 1, characterized in that: The Streptomyces producing azamycin F is Streptomyces TKPJ3039 with a deposit number of CCTCC NO: M2013221.

3. The genetically engineered bacterium for high production of azamycin F according to claim 1, characterized in that: The azl4 gene and / or azl6 gene are overexpressed in a Streptomyces host using a site-specific integration vector containing a strong promoter.

4. The genetically engineered bacterium for high production of azamycin F according to claim 3, characterized in that: The site-specific integration vector is a pSET152 or pIB139 plasmid.

5. The genetically engineered bacterium for high production of azamycin F according to claim 3, characterized in that: The strong promoter is the constitutive promoter hrdBp, and its nucleotide sequence is shown in SEQ ID NO.

3.

6. The method for constructing a genetically engineered bacterium capable of producing azamycin F in a high yield according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: constructing an overexpression plasmid for driving the expression of a target gene with a strong promoter element, and transferring the constructed plasmid into a Streptomyces host that produces azamycin F by a conjugation transfer method to obtain a genetically engineered bacterium with high azamycin F production.

7. The construction method according to claim 6, characterized in that: The steps include: (1) Characterize and screen the strength of different promoters in Streptomyces host bacteria that produce azamycin F to obtain promoters with strong activity in the host bacteria; (2) constructing an overexpression plasmid containing the target gene, wherein the plasmid contains the strong promoter element selected in step (1) and uses the apramycin resistance gene as a marker; (3) transferring the plasmid constructed in step (2) into a Streptomyces host strain producing azamycin F; (4) Screening: After selecting the conjugates, they are subcultured to screen for strains resistant to apramycin and perform colony PCR verification to obtain strains with complete integration, that is, genetically engineered bacteria with high production of apramycin F.

8. The construction method according to claim 7, wherein: In step (1), the strength of different promoters in the host bacteria is characterized and screened, including the following steps: ① Transfer the plasmid containing the promoter and kanamycin resistance gene neo into Streptomyces Plasmids containing different promoters and the kanamycin resistance gene neo were transformed into Escherichia coli ET12567 / pUZ8002 competent cells, and the correct E. coli transformants were selected for conjugation transfer with Streptomyces host bacteria that produce azamycin F. The plasmids were transferred to Streptomyces host bacteria that produce azamycin F by conjugation transfer. ② Screening After the conjugative transfer products were subcultured on SFM medium at 28°C, colonies growing on apramycin-resistant plates were picked and verified by colony PCR to screen for strains in which the target plasmid was successfully integrated into the host bacterial genome; Collect spores of each strain and suspend them in sterile water, adjust OD 600 To the same level, gradient dilution was performed, and 10 μL of spore suspension was evenly spotted on kanamycin gradient concentration plates for resistance strength test. The strength of kanamycin resistance shown by the strain represents the activity of its corresponding promoter.

9. Use of the genetically engineered bacterium capable of producing azamycin F according to any one of claims 1 to 5 in the preparation of azamycin F.

10. A method for preparing azamycin F, characterized in that: The method comprises the following steps: inoculating the seed liquid of the genetically engineered bacteria with high yield of azamycin F according to any one of claims 1 to 5 into a fermentation medium for fermentation.

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