Engineering streptomycete strain for producing neohypericin and construction method of engineering streptomycete strain

Abstract

The invention discloses an engineering streptomycete strain for producing neohypericin and a construction method of the engineering streptomycete strain, the engineering streptomycete strain is obtained by modifying a neohypericin biosynthetic gene cluster from streptomyces spectaculum CGMCC (China General Microbiological Culture Collection Center) 4.6311, and integrating the modified neohypericin biosynthetic gene cluster and a selection marker gene into a genome of streptomyces albus J1074, and the engineering streptomycete strain is obtained by constructing the engineering streptomycete strain. The average shake flask fermentation level can reach 153 mg / L. And the method has wide application value for heterologous expression and industrial production of the neomeicin.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an engineering streptomyces strain producing neoaureosin and a construction method thereof. Background Art

[0002] Spectinabilin is derived from Streptomyces and is a rare polyketide compound containing nitrophenyl, which has anticancer and antiviral activities. Its molecular structure includes a (nitro) aryl substituent, a tetrahydrofuran moiety and an O-methylated pyrone group connected by a polyene chain (Busch B, Hertweck C. Evolution of metabolic diversity in polyketide-derived pyrones: using the non-colinear aureothinassembly line as a model system [J]. Phytochemistry. 2009; 70 (15-16): 1833-40). At present, there are very few such compounds found in nature, only about 150. Spectinabilin has rich biological activity and is a potential new drug with broad application prospects in clinical medicine.

[0003] The spectinabilin biosynthetic gene cluster is about 44 kb long and contains fourteen coding genes (Choi YS, et al., Cloning and heterologous expression of the spectinabilin biosynthetic gene cluster from Streptomyces spectabilis [J]. Mol Biosyst. 2010, 6 (2): 336-8.). The biosynthetic starting unit of spectinabilin is a molecule of p-nitrobenzoate (PNBA), which is catalyzed by the four proteins SpnE / F / G / K encoded by the genes in the cluster to produce branched acid. The extended precursor of spectinabilin is 1 molecule of M-CoA and 6 molecules of MM-CoA, which are condensed to form a linear polyketide molecule under the action of type I polyketide synthase (SpnA / A' / B / C). SpnI is used to methylate the pyrone group in spectinabilin, and SpnH is related to the formation of two CO bonds in the furan part. The spnD gene encodes a pathway-specific positive regulator responsible for activating the biosynthesis of neopyroxithrin. There are some shortcomings in the production of neopyroxithrin using Streptomyces spectabilis, such as the need to explore the fermentation culture conditions, the lack of in-depth research on the genetic background of the strain, and the lack of efficient genetic manipulation tools. These factors limit the yield optimization and application of neopyroxithrin. In recent years, with the development of genetic engineering technology, heterologous expression strategies have been widely used to obtain target natural products. This not only circumvents the problems of culturing and genetic modification of native strains, but also makes it easier and more efficient to modify biosynthetic gene clusters. Summary of the invention

[0004] The purpose of the embodiments of the present application is to address the deficiencies of the prior art and provide an engineered Streptomyces strain that produces neoaureus and a method for constructing the same, integrate the complete neoaureus biosynthetic gene cluster into Streptomyces albus, and amplify the expression of positive regulatory factor genes related to neoaureus biosynthesis to achieve efficient heterologous synthesis of neoaureus.

[0005] According to the first aspect of the embodiment of the present application, an engineered Streptomyces strain producing neoaureusin is provided, with Streptomyces albus as the host strain, expressing the neoaureusin biosynthetic gene cluster, the nucleotide sequence of the biosynthetic gene cluster is shown in SEQ ID NO.1-5.

[0006] Furthermore, the albicans Streptomyces is albicans Streptomyces J1074.

[0007] Furthermore, the novel aureothricin biosynthetic gene cluster originates from Streptomyces spectabilis CGMCC 4.6311.

[0008] Furthermore, the engineered Streptomyces strain is named Streptomyces albus SKD, and the strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.31721 and a deposit date of August 22, 2024.

[0009] According to a second aspect of the embodiment of the present application, a method for constructing the engineered Streptomyces strain according to the first aspect is provided, comprising the following steps:

[0010] (1) Using large genomic fragment in vivo recombination technology, the new aureomycin biosynthetic gene cluster in Streptomyces spectabilis CGMCC 4.6311 was fished out, and a recombinant vector pSpec containing the biosynthetic gene cluster was constructed;

[0011] (2) replacing the promoter of the pathway-specific positive regulator gene spnD in the biosynthetic gene cluster on the recombinant vector pSpec with a promoter suitable for the host strain to obtain the recombinant vector pSKD;

[0012] (3) The recombinant vector pSKD was introduced into Streptomyces albus J1074 by conjugation transfer and inserted into the genome under the action of integrase to obtain an engineered Streptomyces strain that produces neoaureusin.

[0013] Further, step (1) comprises:

[0014] (1.1) Extracting genomic DNA of Streptomyces spectabilis CGMCC 4.6311;

[0015] (1.2) Using plasmid pBE48 as a template, amplify the p48 fragment, which carries the downstream homology arm of the neoauretin biosynthetic gene cluster and the BAC origin; using plasmid pBE45 as a template, amplify the p45 fragment, which carries the upstream homology arm of the neoauretin biosynthetic gene cluster, the Apolla resistance gene, the Streptomyces conjugative transfer-related element and the integrase;

[0016] (1.3) using restriction endonuclease EcoRV to digest the genomic DNA of Streptomyces spectabilis CGMCC 4.6311, and purifying and recovering the genomic DNA after digestion;

[0017] (1.4) After taking an appropriate amount of the above-recovered genomic DNA, p45 fragment and p48 fragment for in vitro ligation by DNA polymerase and ligase, the obtained receptor fragment mixture is filtered and desalted and electroporated into Escherichia coli NEB10-β competent cells. After intracellular Cre-lox recombination, the plasmid pSpec containing the neoaureusin biosynthetic gene cluster is obtained.

[0018] Furthermore, in step (1.2), the Streptomyces conjugative transfer-related elements include traJ protein, oriT, and integration site.

[0019] Furthermore, in step (2), the promoter applicable to the host strain is kasOp* or ermEp*.

[0020] Furthermore, the integrase in step (3) is Integrase or VWB integrase.

[0021] Collection Instructions

[0022] Classification and nomenclature of biological materials: Streptomyces albus.

[0023] Name of the depository unit of biological materials: General Microbiology Center of China Microbiological Culture Collection Administration.

[0024] The abbreviation of the depository of biological materials is: CGMCC.

[0025] The address of the depository of biological materials is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China 100101.

[0026] Deposit date of biological material: August 22, 2024.

[0027] The registration number of the Collection Center for Biological Materials is: CGMCC No.31721.

[0028] The technical solution provided by the embodiments of the present application may have the following beneficial effects:

[0029] The present application constructs a genetically engineered Streptomyces strain capable of efficiently synthesizing neogoldenmycin, and realizes heterologous synthesis of neogoldenmycin in S.albus J1074 for the first time. The biosynthetic gene cluster of neogoldenmycin is obtained based on the Cre-lox in vivo recombination technology assisted by enzyme cutting, which is convenient for its heterologous expression; by constitutively expressing the positive regulatory factor gene spnD of the biosynthetic pathway of neogoldenmycin, the expression of the gene cluster can be maintained at a high level, and finally the white Streptomyces engineered strain SKD capable of efficiently synthesizing neogoldenmycin is obtained. S.albus SKD is fermented, and the fermentation product is detected by high-performance liquid chromatography (HPLC). The results show that the shake flask fermentation level of neogoldenmycin is an average of 153 mg / L, which is significantly higher than the fermentation level of the original strain spectacular Streptomyces CGMCC 4.6311. The obtained strain can also further improve the fermentation level of neogoldenmycin by optimizing the fermentation conditions, the separation and purification steps of the target compound, etc., to achieve the purpose of industrial production, so the present invention has a wide range of application value.

[0030] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0032] Figure 1 This is the pSKD plasmid map of the present invention.

[0033] Figure 2 The agarose gel electrophoresis diagram of the genotype analysis of the S.albus SKD strain of the present invention. a: 616bp target band amplified by C31-F and C31-R primers; b: 685bp target band amplified by EF and ER primers; c: 443bp target band amplified by BF and BR primers. Wherein, M represents 1kb DNA Marker; K1, K2, K3 are target fragments amplified by SKD.

[0034] Figure 3 The fermentation results of S.albus SKD and S.spectabilis in the universal medium of S.spectabilis of the present invention. a: HPLC analysis results of fermentation products. In the figure, (i) is the spectrum of the standard product of neopyroxithrin, (ii) is the product detection spectrum of the S.spectabilis strain fermentation for 5 days, (iii) is the product detection spectrum of the S.albus SKD strain fermentation for 5 days, and (iv) is the product detection spectrum of the S.albus J1074 strain fermentation for 5 days. b: shake flask fermentation level of neopyroxithrin in S.spectabilis, c: shake flask fermentation level of neopyroxithrin in S.albus SKD.

[0035] Figure 4 The fermentation results of S.albus SKD of the present invention in YEME-CaCO3 liquid medium. a: HPLC analysis results of S.albusSKD fermentation products. In the figure, (i) is the spectrum of the standard product of neoaureusin, and (ii) is the product detection spectrum of the S.albus SKD strain fermentation for 8 days. b: Shake flask fermentation level of neoaureusin in S.albus SKD. c: Growth curves of S.albus SKD and S.albus J1074. DETAILED DESCRIPTION

[0036] Here, exemplary embodiments are described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application.

[0037] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0038] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The sources and trade names of the reagents used and their components, if necessary, are indicated when they first appear. The same reagents used thereafter are the same as those first indicated unless otherwise specified.

[0039] Among various heterologous expression strains, Streptomyces has the natural advantage of providing abundant precursors for the synthesis of secondary metabolites and reducing power, and is an excellent host bacteria. Among them, the model strain of Streptomyces albus J1074 is safe and non-toxic, simple to genetically manipulate, and has a clear background of secondary metabolites, and has strong tolerance or resistance to biologically active natural products. Therefore, heterologous expression and specific modification of biosynthetic gene clusters in J1074 have great application potential.

[0040] Example 1 Cloning of the new aureomycin synthesis gene cluster

[0041] The present invention combines the enzyme cutting technology with the Cre-lox in vivo recombination technology to clone the new aureusin biosynthetic gene cluster (SEQ ID NO.1-5).

[0042] 1. Extraction of small amount of genomic DNA from S. spectabilis CGMCC 4.6311 strain

[0043] The aerial hyphae of Streptomyces cultured on ISP4 solid medium for 4-6 days were transferred to TSB medium and cultured at 30°C with shaking at 220rpm for about 24h; an appropriate amount of cells were collected, resuspended with 1mL 1×TE buffer, centrifuged at 12000rpm for 1min at room temperature, and the supernatant was discarded; the cells were resuspended with 500μL 1×TE (containing 5mg / mL lysozyme) buffer, and 2μL RNase A was added, and incubated at 37°C constant temperature shaker for 1h; 30μL proteinase K (20mg / mL) was added, and inverted to mix; then 50μL 10% SDS solution was added, inverted to mix, and incubated at 55°C for 1-2h; 250μL 5M NaCl solution was added, inverted to mix, and then 500μL 25 phenol: 24 chloroform: 1 isoamyl alcohol (volume ratio) solution, gently invert to mix, centrifuge at 12000rpm for 10min; use a 1mL pipette tip with a scissor tip to draw 500μL of supernatant into a 2mL EP tube; add 35μL NaAc solution (pH=7.5), gently invert to mix; then add 1.2mL anhydrous ethanol, gently invert to mix, to form a white flocculent precipitate; use a 200μL pipette tip to pick up the floccules and put them into a 1.5mL EP tube containing 1mL 70% ethanol, centrifuge at 12000rpm for 5min, discard the supernatant, and place at 45℃ for 15min to evaporate ethanol and water; finally, add 100μL deionized water to dissolve the obtained S.spectabilis genomic DNA.

[0044] ISP4 solid culture medium composition: dipotassium hydrogen phosphate 1g / L, sodium chloride 1g / L, ammonium sulfate 2g / L, soluble starch 10g / L, calcium carbonate 2g / L, magnesium sulfate 1g / L, ferrous sulfate 0.001g / L, magnesium chloride 0.001g / L, zinc sulfate 0.001g / L, agar powder 20g / L.

[0045] TSB liquid culture medium composition: tryptone 17g / L, sodium chloride 5g / L, soy peptone 3g / L, dipotassium hydrogen phosphate 2.5g / L, glucose 2.5g / L.

[0046] 2. Preparation of the New Aureobacterium Biosynthetic Gene Cluster Receptor

[0047] (2.1) Using plasmid pBE48 as a template, primers pBE48-F and pBE48-R were used to amplify the plasmid to obtain a p48 fragment carrying the 39 bp downstream homology arm of the neoauretin biosynthetic gene cluster and the BAC origin;

[0048] (2.2) Plasmid pBE45 was used as a template and primers pBE45-F and pBE48-R were used to amplify the fragment p45, which carried the upstream homology arm of the 39-bp neoauretin biosynthesis gene cluster, the abola resistance gene, and elements related to Streptomyces conjugative transfer (including traJ protein, oriT, integration site) and Integrase.

[0049] In a specific implementation, VWB integrase and the like may also be used.

[0050] The sequences of the primers pBE48-F, pBE48-R, pBE45-F, and pBE45-R are shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

[0051] (2.3) The S. spectabilis genomic DNA was digested with restriction endonuclease EcoRV, and the digested genomic DNA was purified and recovered.

[0052] 3. Preparation of Competent E. coli Cells for Cre-lox Recombination in Vivo

[0053] (3.1) Inoculate a single clone of E. coli NEB10-β containing the pBE14 plasmid into 5 mL SOB medium, add 8 μg / mL tetracycline, and culture at 30°C with shaking at 220 rpm for 12 h. Transfer 100 μL of the cultured bacterial solution to 10 mL fresh SOB medium, add 8 μg / mL tetracycline, and culture again with shaking until OD 600 About 0.2.

[0054] (3.2) Add 100 μL 1M arabinose and culture at 30°C with shaking at 220 rpm until OD 600 About 0.45-0.55.

[0055] (3.3) The culture medium was transferred into a 10 mL centrifuge tube, and centrifuged at 12000 rpm for 30 s at 4°C, and the supernatant was removed. The cells were washed three times with 10% glycerol in an ice bath to obtain competent E. coli NEB10-β cells, and finally suspended in 70 μL 10% glycerol and kept in an ice bath for later use.

[0056] SOB medium composition: tryptone 20 g / L, yeast extract 1 g / L, sodium chloride 10 mM, potassium chloride 2.5 mM.

[0057] 4. Recombinant cloning of the new aureomycin biosynthetic gene cluster

[0058] 3-3.75 μg of recovered S. spectabilis CGMCC 4.6311 genomic DNA, 35 ng p45, 44 ng p48 and 1.5 μL T4 DNA Buffer were added to a PCR tube, incubated at 65°C for 10 min without mixing, and then 0.75 U of T4 DNA polymerase was added and gently mixed; the mixture was incubated at 25°C for 60 min, 75°C for 20 min, and 50°C for 30 min, and stored at 10°C; 1 μL of 1 mM NAD was added to the incubated mixture. + , 0.4 μL 10 mM dNTPs, 3UT4 DNA polymerase, 400 U T4 DNA ligase, gently mix, incubate at 37 °C for 60 min, incubate at 75 °C for 20 min, and store at 10 °C before transformation; filter the incubated mixture in ddH2O for 30 min using a 0.025 μm filter membrane for desalting.

[0059] The obtained receptor fragment mixture was added to the prepared Escherichia coli NEB10-β competent cells, and electrotransformed at 1250V. The electrotransformed bacteria were spread on LB plates containing 50μg / mL abola antibiotics and cultured at 37°C for 16-24h. The obtained clone (i.e., Escherichia coli NEB10-β competent cells containing the new abola antibiotics) was inoculated into an LB solution containing 50μg / mL abola antibiotics and cultured at 37°C and 220rpm for 12h before extracting the plasmid. The integrity of the gene cluster was verified by enzyme digestion (EcoRI / BglII) to obtain the plasmid pSpec containing the new abola biosynthetic gene cluster.

[0060] LB medium composition: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 15g / L (added when preparing solid plate medium).

[0061] Example 2: Transformation of the new aureusin biosynthetic gene cluster

[0062] Gene spnD is a positive regulatory factor gene in the neopyroxithrin biosynthesis gene cluster. Enhancing its expression is beneficial to the high expression of the entire gene cluster.

[0063] 1. Replace the native promoter of spnD

[0064] The PCR-Targeting method was used to replace the native promoter of spnD in the neopyroxithromycin biosynthesis gene cluster. Since spnD is the first gene in the neopyroxithromycin gene cluster, the native promoter and the redundant fragment in front of it on the pSpec plasmid can be replaced with AmpR-kasOp*.

[0065] Alternatively, the native promoter of spnD may be replaced with a promoter such as ermEp*.

[0066] (1.1) Using plasmid pBE48 as a template, primers amp-kF and amp-R were used to amplify the ampicillin resistance gene and the upstream region of the spnD gene (bases -9575 to -9536) as the homology arm; using plasmid pIJ8660-kasOp*-GFP as a template, primers kF and amp-kR were used to amplify the kasOp* promoter sequence and the front end (initial 39 bases) of the spnD gene as the homology arm.

[0067] The sequences of the primers amp-kF, amp-R, kF, and amp-kR are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13.

[0068] (1.2) Use primers amp-kF and amp-kR to perform fusion PCR on the two fragments amplified in (1.1) to obtain the DNA fragment AmpR-kasOp* containing the recombinant homology arms.

[0069] (1.3) The pSpec plasmid was electroporated into competent E. coli BW25113 / pIJ790 (Gust B., PCR targeting system in Streptomyces coelicolor A3 (2).) cells, and selected with chloramphenicol and apramycin to obtain the E. coli BW25113 / pIJ790 strain containing the pSpec plasmid. Then, 1 μg of the target DNA AmpR-kasOp* was introduced for linear-loop recombination, and selected with ampicillin resistance to obtain the recombinant plasmid pSKD ( Figure 1 ).

[0070] Example 3 Heterologous expression of the new aureusin biosynthetic gene cluster

[0071] 1. Transformation of pSKD into S.albus J1074 by three-parent conjugation transfer

[0072] S.albus J1074 was used as the host strain, E. coli ET12567 / pUB307 was used as the auxiliary transfer strain, and E. coli DH10B / pSKD was used as the strain containing the DNA to be transformed (pSKD plasmid). The three strains were operated by the three-parent combination transfer method, screened by apramycin resistance, and finally the pSKD plasmid was integrated into the S.albus J1074 genome. attB site.

[0073] 2. Verification of Genotype

[0074] Three possible mutant strains of S.albus J1074 integrated with pSKD plasmid were randomly selected, and their genomic DNA was used as template to clone the mutant strains of S.albus J1074 using primers C31-F and C31-R, EF and ER, and BF and BR, respectively. PCR verification was performed at the attB integration site, spnE gene, and spnB gene. K1, K2, and K3 are the target fragments amplified from the mutant strain, which are completely consistent with expectations (the amplification product of primers C31-F and C31-R: 616bp; the amplification product of primers EF and ER: 685bp; the amplification product of primers BF and BR: 443bp). Finally, the strain with the correct genotype was named S.albus SKD.

[0075] The sequences of the primers C31-F, C31-R, EF, ER, BF, and BR are shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19.

[0076] The results of PCR verification of the genotype of S.albus SKD mutant strain are as follows Figure 2 shown.

[0077] Example 4 Fermentation of S. albus SKD and S. Spectabilis CGMCC 4.6311 strains in S. spectabilis universal liquid medium

[0078] In this example, S. albus SKD and S. spectabilis CGMCC 4.6311 strains were fermented in a universal medium for Streptomyces spectabilis to compare the ability of S. albus SKD and S. spectabilis to produce neoaureosin.

[0079] 1. Fermentation of S.albus SKD and S.spectabilis CGMCC 4.6311

[0080] After the frozen spores were thawed, 10 μL of the spore suspension was streaked onto MS solid medium for single colonies, cultured at 30°C for 3-5 days, and single colonies were streaked onto blocks, cultured at 30°C for 3-5 days. The above blocks were inoculated into 20 mL of TSB liquid medium and cultured at 30°C for 24 hours so that the bacteria were in the logarithmic growth phase. The OD 600 The range is 5-15. 10 OD of the bacterial solution was transferred to 50 mL of S. spectabilis general fermentation medium and cultured at 30°C and 220 rpm for 5-8 days.

[0081] MS solid culture medium composition: soybean powder 20g / L, mannitol 20g / L, agar powder 20g / L.

[0082] The general liquid culture medium of S. spectabilis is composed of: 10 g / L soybean powder, 2 g / L peptone, 20 g / L glucose, 5 g / L soluble starch, 2 g / L yeast extract, 4 g / L sodium chloride, 0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and 2 g / L calcium carbonate (Gao X, et al., The antiproliferative effect of spectinabilins from Streptomyces spectabilis on hepatocellular carcinoma cells in vitro and invivo[J]. Bioorg Chem. 2019, 93: 103311.).

[0083] 2. Detection of Neostylosin by HPLC

[0084] Take 500 μL of fermentation culture medium into a 10 mL centrifuge tube containing 4.5 mL of ethyl acetate, ultrasonicate for 10 min, and then stand for extraction; after 12 h, centrifuge at 9000 rpm for 15 min, take 2 mL of supernatant and divide it into new 2 mL EP tubes; freeze-dry the supernatant, dissolve it again with 500 μL of methanol, and then filter the supernatant with a 0.22 μm filter membrane for high performance liquid chromatography detection.

[0085] The high performance liquid chromatography detection conditions of the new aureusin are as follows: aqueous mobile phase: 1% acetic acid; organic mobile phase: chromatographically pure acetonitrile. Elution conditions: 0-30min, the organic phase ratio increases from 25% to 90%; 30-35min, the organic phase ratio increases from 90% to 100%; 35-40min, the organic phase ratio decreases from 100% to 25%; 40-42min, the organic phase ratio is maintained at 25%. The detection wavelength is 378nm.

[0086] 3. Fermentation biomass detection

[0087] Take 1 mL of the fermentation liquid at the time point to be tested into a weighed 1.5 mL EP tube, centrifuge at 12000 rpm for 10 min, discard the supernatant, then place the EP tube in a 65°C oven, dry to constant weight, and weigh using an electronic balance.

[0088] 4. Analysis of fermentation results of S.albus SKD and S.spectabilis

[0089] The results are as follows Figure 3 As shown, Figure 3 a in the figure is the HPLC analysis result of the fermentation broth extracts of each strain, which, from top to bottom, represent the fermentation extracts of the new aureusin standard (concentration of 10 mg / L), S. spectabilis CGMCC 4.6311, S. albus SKD and S. albus J1074, respectively. Figure 3 Figures b and c are the fermentation levels of neuraminidase of S. spectabilis CGMCC 4.6311 and S. albus SKD calculated from the standard curve. The native bacteria S. spectabilis CGMCC 4.6311 had the highest fermentation level of about 53 mg / L on the 5th day, and S. albus SKD had the highest fermentation level of about 57 mg / L on the 4th day.

[0090] Example 5 Fermentation of S. albus SKD in YEME-CaCO3 liquid medium

[0091] The culture medium is crucial for the growth of bacteria and the synthesis of secondary metabolites. In order to further improve the fermentation level of neoauretin in S.albus SKD, it is necessary to select a suitable culture medium. After repeated screening, YEME medium was finally selected as the fermentation medium for SKD, and calcium carbonate was added on this basis to maintain the pH value of the culture environment.

[0092] 1. S.albus SKD fermentation

[0093] The fermentation process was the same as in Example 4. After the frozen spores of S.albus SKD and control S.albus J1074 were thawed, 10 μL of the spore suspension was streaked on MS solid medium for single colonies, cultured at 30°C for 3-5 days, and single colonies were picked for streaking and cultured at 30°C for 3-5 days. The above bacterial blocks were inoculated into 20 mL of TSB liquid medium and cultured at 30°C for 24 hours so that the bacteria were in the logarithmic growth phase. The OD 600 The range was 5-15. 10OD of the bacterial solution was transferred to 50mL YEME-CaCO3 fermentation medium and cultured at 30℃ and 220rpm for 10d.

[0094] YEME-CaCO3 liquid culture medium composition: glucose 40g / L, yeast extract 3g / L, malt extract powder 3g / L, tryptone 5g / L, calcium carbonate 1g / L.

[0095] 2. Detection of new aureosin and fermentation biomass

[0096] Same as Example 4.

[0097] 3. Analysis of fermentation results of S.albus SKD strain

[0098] The results are as follows Figure 4 As shown. Among them, Figure 4 a in the table is the result of HPLC analysis. Figure 4 b in the figure is the shake flask fermentation level of neoauretin in S.albus SKD strain calculated based on the standard curve. Figure 4 Figure c is the growth curve of S.albus SKD and S.albus J1074. The growth curve of the engineered strain S.albus SKD is significantly different from that of S.albus J1074. The fermentation biomass of the J1074 strain increases over time until it reaches a plateau, while the fermentation biomass of S.albus SKD drops sharply after the first day and then rises slowly. The shake flask fermentation level of neoauretin in the S.albus SKD strain can reach an average of 153 mg / L, which is significantly higher than that of the native strain.

[0099] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the contents disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art that are not disclosed in the present application.

Claims

1. An engineered Streptomyces strain producing neoauretin, characterized in that: Streptomyces albus is used as a host strain to express a novel aureothricin biosynthetic gene cluster, the nucleotide sequence of which is shown in SEQ ID NO. 1-5.

2. The engineered Streptomyces strain according to claim 1, characterized in that The albicans Streptomyces is albicans Streptomyces J1074.

3. The engineered Streptomyces strain according to claim 1, characterized in that The novel aureothricin biosynthetic gene cluster is derived from Streptomyces spectabilis CGMCC 4.6311.

4. The engineered Streptomyces strain according to claim 1, characterized in that The name of the engineered Streptomyces strain is Streptomyces albus SKD, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31721 and a deposit date of August 22, 2024.

5. A method for constructing the engineered Streptomyces strain according to claim 1, characterized in that: The following steps are involved: (1) Using large genomic fragment in vivo recombination technology, the new aureomycin biosynthetic gene cluster in Streptomyces spectabilis CGMCC 4.6311 was fished out, and a recombinant vector pSpec containing the biosynthetic gene cluster was constructed; (2) replacing the promoter of the pathway-specific positive regulator gene spnD in the biosynthetic gene cluster on the recombinant vector pSpec with a promoter suitable for the host strain to obtain the recombinant vector pSKD; (3) The recombinant vector pSKD was introduced into Streptomyces albus J1074 by conjugation transfer and inserted into the genome under the action of integrase to obtain an engineered Streptomyces strain that produces neoaureusin.

6. The method according to claim 5, characterized in that Step (1) comprises: (1.1) Extracting genomic DNA of Streptomyces spectabilis CGMCC 4.6311; (1.2) Using plasmid pBE48 as a template, amplify the p48 fragment, which carries the downstream homology arm of the neoauretin biosynthetic gene cluster and the BAC origin; using plasmid pBE45 as a template, amplify the p45 fragment, which carries the upstream homology arm of the neoauretin biosynthetic gene cluster, the Apolla resistance gene, the Streptomyces conjugative transfer-related element and the integrase; (1.3) using restriction endonuclease EcoRV to digest the genomic DNA of Streptomyces spectabilis CGMCC 4.6311, and purifying and recovering the genomic DNA after digestion; (1.4) After taking an appropriate amount of the above-recovered genomic DNA, p45 fragment and p48 fragment for in vitro ligation by DNA polymerase and ligase, the obtained receptor fragment mixture is filtered and desalted and electroporated into Escherichia coli NEB10-β competent cells. After intracellular Cre-lox recombination, the plasmid pSpec containing the neoaureusin biosynthetic gene cluster is obtained.

7. The method according to claim 6, characterized in that In step (1.2), the Streptomyces conjugative transfer-related elements include traJ protein, oriT, and integration site.

8. The method according to claim 5, characterized in that In step (2), the promoter suitable for the host strain is kasOp* or ermEp*.

9. The method according to claim 5, characterized in that The integrase in step (3) is Integrase or VWB integrase.

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