Oxazomycin high-yield engineering strain as well as construction method and application thereof

Abstract

The invention provides an oxazomycin high-yield engineering strain as well as a construction method and application thereof. The method for constructing the oxazomycin high-yield engineering strain comprises the following steps: driving high expression of a gene cluster by using a strong promoter in streptomyces longsheng, enhancing transcription of a key resistance gene module (ozmS-oxaA), and optimizing ribosome engineering of strain chassis cells and a culture medium. According to the invention, the rational synthetic biology and metabolic engineering technology is combined with the irrational ribosome engineering technology to gradually increase the yield of the streptomyces longsheng oxazomycin, and the established efficient biosynthesis system lays a foundation for further development of the oxazomycin.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology. Specifically, it relates to an oxazolomycin-high-yielding engineering strain, its construction method and application. Background Art

[0002] Streptomyces is a type of filamentous Gram-positive bacteria with a high G+C content, capable of synthesizing a large number of bioactive secondary metabolites, which are the main sources of natural drug molecules, including clinical antibacterial drugs, immunosuppressants, antitumor compounds, etc., and are a drug resource treasure house with great development potential. Activating and highly expressing the biosynthetic gene clusters of secondary metabolites has very important application value for microbial product mining and activity evaluation, and further expanding the drug lead compound resource library.

[0003] Oxazolomycin is a large class of polyketide-poly peptide antibiotics produced by Streptomyces, initially isolated from the liquid fermentation medium of Streptomyces KSM-2690, and almost all are composed of a left-side oxazole ring and a right-side β-spirolactone-γ-lactam structure connected in series by diene and triene chains. The complex structural characteristics of oxazolomycin compounds endow them with various biological activities. Currently reported members of the oxazolomycin family have antibacterial, antiviral and antitumor activities, etc. The structure of oxazolomycin is variable, including various combinations of different substituents and isomers, and these changes are closely related to its biological activity. Therefore, this type of compound shows high structural and activity plasticity and is a compound with great development potential. However, the current yield of oxazolomycin from microorganisms is very low. Although some structures of oxazolomycin can be obtained through chemical synthesis, due to its cumbersome synthesis steps, diverse spatial configurations and isomers, its yield is greatly reduced and the production cost is also very high. Both of them cannot meet the needs of further development, and it is particularly important to establish an efficient biosynthesis system for oxazolomycin. Summary of the Invention

[0004] The purpose of the present invention is to provide an oxazolomycin-high-yielding engineering strain, its construction method and application.

[0005] Oxazolomycin compounds have diversity in structure and biological activity, but their yields are low and cannot meet the requirements of their development and application, which is also one of the main reasons restricting their drug formation. Currently, no research reports on improving the biosynthesis of oxazolomycin have been found. Therefore, improving the biosynthesis ability of oxazolomycin in strains is the key to solving this bottleneck problem. The present invention gradually increases the yield of oxazolomycin in Streptomyces longshengensis through rational synthetic biology and metabolic engineering technologies combined with irrational ribosome engineering technologies. The established efficient biosynthesis system lays a foundation for the further development of oxazolomycin.

[0006] To achieve the object of the present invention, in a first aspect, the present invention provides a method for constructing a high-yield engineering strain of oxazolomycin, comprising the following steps:

[0007] (1) By means of genetic engineering, the sequence between two transcription units oxaB-G and oxaH-oxaQ (oxaBG and oxaHQ) in the oxazolomycin biosynthetic gene cluster in Streptomyces longshengensis is replaced with two strong promoters in opposite directions to obtain engineering strain I;

[0008] (2) An expression cassette of a resistance gene driven by a strong promoter is introduced into engineering strain I, and the resistance gene is composed of the oxaA gene derived from Streptomyces longshengensis and the ozmS gene derived from Streptomyces albus in series to obtain a high-yield engineering strain of oxazolomycin.

[0009] Further, the Streptomyces longshengensis has the number CGMCC 4.1101, and the public can obtain this strain from the China General Microbiological Culture Collection Center (CGMCC).

[0010] Further, the two strong promoters in opposite directions in step (1) are the P neo promoter and the P kasO* promoter, and the sequence is as shown in SEQ ID NO:2.

[0011] Further, the strong promoter in step (2) is a constitutive strong promoter, preferably the P hrdB promoter, the P neo promoter or the P kasO* promoter, and more preferably the P kasO* promoter.

[0012] Further, the sequence between the two transcription units oxaB-G and oxaH-oxaQ in step (1) is as shown in SEQ ID NO:1.

[0013] Further, the sequences of the oxaA gene and its encoded protein in step (2) are as shown in SEQ ID NO:3 and 4 respectively, and the sequences of the ozmS gene and its encoded protein are as shown in SEQ ID NO:5 and 6 respectively.

[0014] Further, between steps (1) and (2), there is also a step of mutagenizing engineering strain I with gentamicin to obtain a gentamicin-resistant mutant; correspondingly, an expression cassette of a resistance gene driven by a strong promoter is introduced into the gentamicin-resistant mutant.

[0015] In a second aspect, the present invention provides a high-yield engineering strain of oxazolomycin constructed according to the above method.

[0016] In a third aspect, the present invention provides a high-yield engineering strain SLROESA of oxazolomycin, and the strain is classified and named as Streptomyces longshengensis. It has been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a postal code of 100101. The deposit number is CGMCC No. 29320, and the deposit date is December 15, 2023.

[0017] In a fourth aspect, the present invention provides the application of the engineering strain in the fermentative production of oxazolomycin.

[0018] In a fifth aspect, the present invention provides a method for increasing the yield of oxazolomycin, and the method includes:

[0019] a) Fermentatively culturing the engineering strain to obtain a culture;

[0020] b) Collecting the produced oxazolomycin from the culture obtained in step a).

[0021] Furthermore, sodium acetate is added to the medium for fermentatively culturing the engineering strain.

[0022] Preferably, the addition amount of sodium acetate in the medium is 0.5 g / L.

[0023] For example, the fermentation medium is MS medium (20 g of mannitol, 20 g of soybean powder, made up to 1000 mL with water).

[0024] By virtue of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

[0025] (1) Compared with the strategies of individually highly expressing conventional transcription units or structural genes, or multi-gene assembly, as well as gene cluster cloning and duplication, etc. to enhance the transcription of the entire gene cluster, the present invention uses a bidirectional strong promoter to replace the intergenic region containing the original promoters of two transcription units, and can thus achieve enhanced expression of the transcription of the entire gene cluster, which is a simple and efficient research strategy.

[0026] (2) The present invention not only directionally highly expresses the oxazolomycin biosynthetic gene cluster, but also uses ribosome engineering technology to optimize the genetic background of the strain, which is a typical strategy that combines directional activation and non-rational modification to achieve the technical effect of advantageous integration.

[0027] (3) On the basis of increasing the synthesis of oxazolomycin by the strain, the present invention also enhances the expression of the transport gene, and further accelerates the efflux of the product from the cell, which can reduce the toxicity of the product to the producing bacteria and is conducive to the continuous synthesis and accumulation of the product.

[0028] (4) The obtained high-yield strain can increase the yield of oxazolomycin to 5.5 times that of the wild-type strain under suitable culture medium conditions; it is the engineering strain with the highest yield at present, laying a foundation for further drug development. Description of the Drawings

[0029] Figure 1 This is the oxazolomycin biosynthetic gene cluster in Streptomyces longshengensis and the results of its co-transcription analysis in the preferred embodiment of the present invention. Among them, A represents the oxazolomycin biosynthetic gene cluster, and B represents the co-transcription analysis of the oxazolomycin biosynthetic gene cluster.

[0030] Figure 2 This is the construction of the high-expression strain of the precursor synthesis gene high-expression module and the resistance gene module and the analysis of the oxazolomycin yield in the preferred embodiment of the present invention. Among them, A represents the schematic diagram of the construction of the pKC1139::P hrdB ::oxaB-G plasmid (left), plasmid construction (middle), and verification electrophoresis diagram of the recombinant strain SL BG (right). B represents the schematic diagram of the construction of the pSET156::P kasO* ::oxaSA plasmid (left), plasmid construction (middle), and verification electrophoresis diagram of the recombinant strain SL SA (right). C represents the schematic diagram of the construction of the pSET152::P hrdB ::ovmFGIH plasmid and the verification electrophoresis diagram of the recombinant strain SL FGIH D represents the HPLC yield analysis of the engineering strain oxazolomycin Toxa5.

[0031] Figure 3 This is the construction of the engineering strain SL OE in the preferred embodiment of the present invention and the analysis of the oxazolomycin yield therein. Among them, A represents the schematic diagram of the construction of the pKC1139::P nk plasmid. B represents the pKC1139::P nk plasmid construction and verification electrophoresis diagram of the recombinant strain SL OE .

[0032] Figure 4 This is the transcriptional analysis of the oxazolomycin biosynthetic genes in the engineering strain SL OE in the preferred embodiment of the present invention.

[0033] Figure 5 This is the ribosome engineering transformation of SL OE using gentamicin mutagenesis and the acquisition of the mutant strain SL ROE in the preferred embodiment of the present invention. Among them, A represents the determination of the minimum inhibitory concentration (MIC) of SL OE against gentamicin. B represents the three-round screening of the gentamicin-resistant mutant strain in SL OE .

[0034] Figure 6 Construction of SLROESA and analysis of oxazolomycin production in the preferred embodiment of the present invention. Among them, A represents the construction and verification of plasmid pSET156::oxaSA::oxaB-G. B represents the electrophoresis verification of recombinant engineering strains SL ROESA-BG 、SL ROEBG and SLROESA. C represents the HPLC analysis of oxazolomycin Toxa5 production in different recombinant engineering strains.

[0035] Figure 7 Effect of different culture conditions on oxazolomycin production by SLROESA in the preferred embodiment of the present invention. Among them, A represents the effect of different carbon sources, exogenous precursors, metal ions and zinc ion concentrations on the production of wild-type strain Toxa5. B represents the effect of different combinations of zinc sulfate and sodium acetate on the production of Toxa5 in high-yield strain SLROESA. Detailed implementation manners

[0036] The present invention aims to provide a new system and method for improving the production of streptomyces oxazolomycin, creating conditions for the large-scale preparation and in-depth development of this compound. The present invention uses strong promoter replacement and high expression of resistance genes to enhance the expression of the oxazolomycin gene cluster in a targeted manner, and optimizes the chassis cells through antibiotic pressure screening, increasing the production of oxazolomycin to more than 5 times that of the wild-type strain; indicating that these strategies have a synergistic effect on the biosynthesis of oxazolomycin.

[0037] The present invention adopts the following technical solutions:

[0038] 1. First, through co-transcriptional analysis, it was determined that the oxazolomycin biosynthetic gene cluster (oxa BGC) in Streptomyces longshengensis includes two transcription units (oxaBG and oxaHQ) with opposite directions, and the intergenic region between them is oxaG-oxaH.

[0039] 2. After highly expressing gene modules with different functions, it was found that the methoxypropanoyl-ACP biosynthetic genes oxaG-oxaB and the resistance gene module ozmS-oxaA have a significant enhancing effect on the production of oxazolomycin, and the production of the main component Toxa5 increased by 120% and 50% respectively.

[0040] 3. Then we designed a simple promoter replacement strategy, that is, using homologous double exchange technology to replace the self-promoter regions (P oxaG -P oxaH ) of the two presumed transcription units in oxa with strong promoters P neo and P kasO* respectively, to obtain engineering strain SL OE, where the yield of Toxa5 is 4 times that of the wild-type strain, and the transcription of related genes in oxa is increased to varying degrees.

[0041] 4. On this basis, sub-lethal dose of gentamicin was used to mutagenize SL OE , and then the high-expression modules of ozmS and oxaA were introduced into the resistant mutant strain SL ROE , and the high-yield engineering strain SLROESA was obtained. Combined with medium optimization, the yield of Toxa5 was finally increased to 182 mg / L, which was 5.5 times that of the wild-type strain.

[0042] The present invention also deposited the SLROESA strain as a biological material, and its deposit number is CGMCC No. 29320.

[0043] The recombinant strains and recombinant plasmids involved in the present invention are all within the scope claimed in the present invention. Those skilled in the art should understand that one or more amino acid mutations or modifications are made to the protein shown in the sequence in the sequence listing, such as replacing, adding, or deleting some amino acids, but still having the function of synthesizing oxazomycin and still being applicable to the present invention. The gene encoding the polypeptide or protein can also be used in the present invention to construct a recombinant bacterium that can increase the yield of oxazomycin. The promoter used in the present invention is not limited to the P hrdB promoter, and also includes other commonly used promoters P ermE* promoters (Bibb M J, White J, Ward J M, and Janssen G R. The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome-binding site. Mol. Microbiol., 1994, 14: 533-545), P kasO*Promoter (Wang W, Li X, Wang J, Xiang S, Feng X, and Yang K. An engineered strong promoter for streptomycetes. Appl. Environ. Microbiol., 2013, 79: 4484-4492). The gene overexpression plasmid in the present invention is not limited to pSET156 (Yuan F, Characterization of the catalase system of Streptomyces and improvement of oxytetracycline-producing strains. Institute of Microbiology, Chinese Academy of Sciences, 2021) and free high-copy plasmids such as pKC1139 (Kieser T, Bibb M J, Buttner M J, Chater K F, and Hopwood D A. Practical Streptomyces Genetics. Norwich, United Kingdom: John Innes Foundation, 2000), and can also be pSET152 (Bierman M, Logan R, O'Brien K, Seno E T, Rao R N, and Schoner B E. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 1992, 116: 43-49) or pIJ10500 (Gregory M A, Till R, and Smith M C. Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors. J. Bacteriol., 2003, 185: 5320-5323), etc. The above plasmids in the present invention can be integrated into the Streptomyces genome by techniques such as conjugal transfer technology, protoplast fusion technology, electroporation transformation, chemical transformation, etc. The present invention preferably uses conjugal transfer technology, but is still effective for other technologies. The recombinant technology used in the present invention can be any suitable method used in the art, such as knocking the target gene into any position of the strain genome, introducing a recombinant plasmid containing the target gene into the strain (such as the free high-copy plasmid pKC1139, etc.), integrating the recombinant plasmid containing the present target gene into the genome of the bacterial cell, etc.The longsheng Streptomyces used in the embodiment of the present invention is Streptomyces longshengensis CGMCC 4.1101, but those skilled in the art should understand that any suitable strain that can produce oxadiazine and the application of the method of the present invention to achieve the effect of increasing the yield of oxadiazine belong to the protection scope of the present invention.

[0044] Specifically, the purpose of the present invention can be further achieved by adopting the following technical measures.

[0045] The invention provides an engineered strain of Streptomyces longshengensis, which is a mutant strain with enhanced transcription of an oxazoline gene cluster (oxa) in Streptomyces longshengensis CGMCC 4.1101, high expression of a key resistance gene module ozmS-oxaA driven by a strong promoter, and modified by ribosome engineering.

[0046] Furthermore, the interregion between the two transcription units oxaB-G and oxaH-oxaQ in the oxazolidinone gene cluster is replaced by two strong promoters in opposite directions, and the resistance gene module ozmS-oxaA is operably connected to the strong promoter, and the ribosome engineering mutant strain refers to a mutant strain induced by gentamicin.

[0047] Optionally, the strong promoter is a strong constitutive promoter, such as P hrdB Promoter, P neo Promoter, P ermE* Promoter or P kasO* The promoter is preferably P hrdB Promoter, P neo Promoter or P kasO* Promoter.

[0048] Furthermore, the present invention provides a high-yield engineered strain SLROESA, whose deposit number is CGMCC No.29320.

[0049] The invention also provides use of the engineered strain in producing oxadiazine.

[0050] The present invention also provides a method for constructing a high-yield oxadiazine Streptomyces Longsheng engineering strain, the method comprising: introducing a strong promoter P containing two opposite directions neo and P kasO* Plasmid pKC1139::P nk Introduce Longsheng Streptomyces CGMCC4.1101 to obtain the engineered strain (SL OE ); the engineered strain was subjected to gentamicin mutagenesis to obtain a gentamicin-resistant mutant (SL ROE ); then the plasmid containing the expression ozmS-oxaA gene was introduced into the SL ROE .

[0051] Furthermore, the two strong promoters P neo and P kasO* sequences are operably linked to two transcription units oxaB-G and oxaH-oxaQ of the oxazolomycin gene cluster, and the oxaA and ozmS genes are operably linked to the strong promoter;

[0052] Optionally, the strong promoter is a strong constitutive promoter, such as P hrdB promoter, P neo promoter, P ermE* promoter or P kasO* promoter, preferably P hrdB promoter, P neo promoter or P kasO* promoter.

[0053] The present invention further provides a method for biosynthesis of oxazolomycin, the method comprising culturing the engineered strain under conditions sufficient to produce oxazolomycin.

[0054] Furthermore, the method further comprises isolating the oxazolomycin from the culture medium.

[0055] The intergenic region sequence between oxaB-G and oxaH-oxaQ is as shown in SEQ ID NO:1, and the nucleotide sequences of P neo and P kasO* are as shown in SEQ ID NO:2; the nucleotide sequence of the oxaA gene is as shown in SEQ ID NO:3, and the amino acid sequence is as shown in SEQ ID NO:4; the nucleotide sequence of the ozmS gene is as shown in SEQ ID NO:5, and the amino acid sequence is as shown in SEQ ID NO:6.

[0056] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as those described in the Molecular Cloning Laboratory Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0057] The plasmid, strain and primer information used in the following examples are shown in Tables 1 to 3 respectively:

[0058] Table 1 Plasmids used in the present invention and their descriptions

[0059]

[0060]

[0061] Table 2 Strains used in the present invention and their descriptions

[0062]

[0063] Table 3 Primer sequences

[0064]

[0065]

[0066]

[0067] Example 1 Co-transcriptional analysis of oxa in Streptomyces longshengensis

[0068] Previous studies found that there was an approximately 75-kb oxazolomycin biosynthetic gene cluster (oxa) in Streptomyces longshengensis CGMCC 4.1101, mainly composed of an approximately 65-kb PKS-NRPS module and other structural genes ( Figure 1 A), but no related regulatory genes were found. To better understand oxa, a genetic manipulation strategy was designed to construct a high-yield oxazolomycin strain. First, co-transcriptional analysis of oxa was performed. We extracted RNA and DNA from Streptomyces longshengensis fermented for 48 h, and reverse-transcribed the RNA to obtain cDNA. Using genomic DNA and cDNA as templates, the primer pairs orf(-1)F / oxaB F, oxaB R / oxaCF, oxaC R / oxaD F, oxaD R / oxaF F, oxaF R / oxaG F, oxaG R / oxaH R, oxaK R / oxaJ(d)F, oxaN R / oxaO(d)F, oxaO R / oxaP(d)F, oxaP R / oxaQ(d)F, oxaQ R / orf1 R were used to perform co-transcriptional analysis on partial gene segments of oxa respectively. The results showed that oxaB-G and oxaH-Q formed co-transcriptional units respectively ( Figure 1 B).

[0069] Example 2 Assembly and characterization of functional modules

[0070] High expression of key genes is one of the effective ways to improve antibiotic production. According to the antiSMASH analysis of oxa, it is speculated that Streptomyces longshengensis and Streptomyces albus produce the oxazomycin skeleton through a similar biosynthetic pathway, that is, using malonyl-CoA and methoxypropionyl-ACP as the main precursors, which are continuously loaded into the corresponding polyketide modules by acyltransferases OxaM and OxaC, and finally the oxazomycin end product is produced. To determine the effect of its key precursor synthesis genes on oxazomycin production, the malonyl-CoA synthesis gene and methoxypropionyl-ACP synthesis gene modules were first characterized. High expression of the former used the high-expression plasmid pSET152::P of the malonyl-CoA homologous gene ovmFGIH of Streptomyces anulatus hrdB ::ovmFGIH, while the latter was constructed based on the free plasmid pKC1139. First, the pKC1139 plasmid was double-digested with HindIII and EcoRI to obtain a linearized fragment, and then the genomic DNA of Streptomyces coelicolor M1146 was used as a template, and 1139-P hrdB -F / P hrdB -R was used as primers to obtain the promoter P hrdB target fragment by PCR amplification; using the genomic DNA of Streptomyces longshengensis CGMCC 4.1101 as a template, and using P hrdB -oxaG-F / oxaD-R, oxaD-F / oxaD(d)-R, oxaD(d)-F / 1139-oxaB-R as primers, the target fragments oxaB-G1, oxaB-G2, and oxaB-G3 were obtained by PCR amplification. Finally, the above 5 DNA fragments were ligated by Gibson assembly to obtain the methoxypropionyl-ACP plasmid pKC1139::P hrdB ::oxaB-G, and its correctness was verified by PCR and sequencing. Then the above plasmids were transferred into Streptomyces longshengensis by conjugation transfer respectively. After PCR verification, the engineering strains SL FGIH and SL BG ( Figure 2 , A-B) with high expression of the malonyl-CoA synthesis gene and methoxypropionyl-ACP synthesis gene were obtained respectively.

[0071] In addition, many studies have shown that transporters play an important role in enhancing the efflux ability to improve the strain's own resistance to antibiotics. According to the literature, at least two genes in Streptomyces albus are related to the efflux of oxazomycin, namely ozmA and ozmS. Through homologous alignment, a resistance gene oxaA highly homologous to ozmA was found outside the oxa cluster, while the latter was not found in the gene cluster and the genome. Therefore, the DNA sequence of the ozmS reading frame was obtained by gene synthesis, and the strong promoter P kasO*Co-assembled with it into plasmid pSET156 to obtain the high-expression plasmid pSET156::P kasO* ::ozmS. On this basis, pSET156::P was double-digested with BamHI and EcoRV kasO* ::ozmS plasmid to obtain a linearized fragment, and using Streptomyces longisporus genomic DNA as a template, 156-S-A-F / oxaA-R as primers for PCR amplification to obtain the oxaA target fragment. Finally, the above two DNA fragments were ligated by Gibson assembly, and the resulting recombinant plasmid was named pSET156::P kasO* ::oxaSA, and its correctness was verified by PCR amplification and sequencing. Then the plasmid was transferred into Streptomyces longisporus by conjugation transfer. After PCR verification, the engineered strain SL with high expression of the resistance gene was obtained SA ( Figure 2 C).

[0072] The wild-type strain, engineered SL FGIH , SL BG and SL SA were fermented using MS medium and analyzed by HPLC. The results showed that the yield of oxazomycin Toxa5 in the SL BG strain was increased to 2.2 times that of the wild-type strain, and the yield of oxazomycin Toxa5 in SL SA was increased to 1.5 times, while no obvious yield increase effect was seen in SL FGIH ( Figure 2 D), so oxaB-G and ozmS-oxaA can be used as elements for constructing high-yield strains.

[0073] Example 3 Expression of the NRPS-PKS module driven by a strong promoter

[0074] For the efficient synthesis of secondary metabolites, high expression of the entire gene cluster is often required. The oxazolomycin gene cluster is as long as 75 kb, especially containing large NRPS-PKS synthase genes, and it is difficult to clone and express these genes routinely and individually. Co-transcription analysis shows that oxa contains two main transcription units, and there may be two promoters with opposite directions between these two transcription units, which facilitates promoter modification. First, the pKC1139 plasmid was double-digested with HindIII and EcoRI to obtain a linearized fragment, and then using the pSET152::D-C plasmid (Li, Dong; Tian, Yuqing; Liu, Xiang; Wang, Wenxi; Li, Yue; Tan, Huarong; Zhang, Jihui. Reconstitution of a mini-genecluster combined with ribosome engineering led to effective enhancement ofsalinomycin production in Streptomyces albus. Microbial Biotechnology, 2021, 14(6): 2356-68) as a template, kan-P neo -F / P kasO* -R as primers, the target fragments of two promoters arranged bidirectionally were obtained by PCR amplification; using pET28a as a template, the target fragment of the kanR kanamycin resistance gene was obtained with oxaG-kan-F / kan-R as primers; using the genomic DNA of Streptomyces longshengensis as a template, with 1139-oxaG-F / oxaG-R and P neo -oxaH-F / oxaH-1139-R as primers, the target fragments of homologous arms F1 and F2 with a length of 2.5 kb were obtained. Finally, the above 5 DNA fragments were ligated by Gibson assembly, and the resulting recombinant plasmid was named pKC1139::P kasO* and P kasO* ( nk ( Figure 3 A), and its correctness was verified by PCR amplification and sequencing. Then, the pKC1139::P nk plasmid was introduced into Streptomyces longshengensis by conjugation transfer. The transconjugants with both apramycin and kanamycin resistance were selected and passaged onto a plate containing only kanamycin resistance. After culturing at 40 °C for 3-5 days, the resulting transformants were then passaged onto a double-resistant plate containing apramycin and kanamycin and a resistance plate containing only kanamycin. The strains that did not grow on the double-resistant plate but grew on the kanamycin resistance plate were selected as the promoter homologous double-exchange strain SL OE, and finally, the correctness of the strain was verified by PCR amplification ( Figure 3 B).

[0075] The wild-type strain and the engineered strain SL were fermented using MS medium OE and subjected to HPLC analysis. The results showed that OE the yield of oxazolomycin in SL increased by 4 times compared with the wild-type strain ( Figure 3 C). Subsequently, the RNA of the wild-type strain WT and the high-yield strain SL at fermentation times of 24 h, 48 h, and 72 h was extracted. Using the cDNA reverse-transcribed from the RNA as a template, the primer pairs q16S F / q16S R, qoxaB F / qoxaB R, qoxaG F / qoxaG R, qoxaH F / qoxaH R, qoxaL F / qoxaL R, qoxaMF / qoxaM R, qoxaN F / qoxaN R, qoxaO F / qoxaO R, qoxaQ F / qoxaQ R were used to perform fluorescence quantitative PCR on the internal reference gene 16S rRNA and the genes to be measured, oxaB, oxaG, oxaH, oxaL, oxaM, oxaN, oxaO, oxaQ. Through transcriptional analysis, it was found that after being driven by the strong promoter, the transcriptional levels of the key oxa genes in the strain SL OE all increased to varying degrees ( OE ), indicating that after the promoter replacement, the transcription of the entire gene cluster was enhanced, thereby increasing the yield of oxazolomycin. Figure 4 )

[0076] The above experimental results indicate that the methoxypropanoyl-ACP synthase gene module oxaB-G and the resistance gene module ozmS-oxaA can be used as high-yield functional elements for oxazolomycin, and the engineered strain SL OE has the most significant effect on increasing the production of oxazolomycin and can be used as a chassis cell for further optimization. These studies lay a foundation for the establishment of a subsequent high-efficiency expression system.

[0077] Example 4 Optimization of the chassis cell by sub-lethal dose antibiotic mutagenesis

[0078] Based on the high-yield strain SL OE , the genetic background of the chassis cell was further optimized by screening with a sub-lethal dose of antibiotic pressure to increase the yield of oxazolomycin. Through preliminary detection of different antibiotic sensitivities, it was found that Streptomyces longshengensis was sensitive to gentamicin. Therefore, this antibiotic was selected for mutagenesis of the strain. According to the minimum inhibitory concentration (Minimal Inhibitory Concentration, MIC) of gentamicin against Streptomyces longshengensis, its use concentration was determined to be 8 - 10 μg / mL. Figure 5A). Subsequently, Bacillus subtilis was used as the indicator bacterium for oxazomycin detection, and the agar block method was used to screen for gentamicin-resistant mutants of Streptomyces longshengensis. After two rounds of mutagenesis and screening, we found that the inhibition zone of strains No. 57 and No. 102 increased most significantly. To detect the stability of the resistant mutants, strains No. 57 and No. 102 were subcultured three times and then subjected to the third round of screening. It was found that the yield increase effect of strain No. 102 was the most prominent( Figure 5 B), and finally this mutant strain was selected for subsequent research and named SL ROE .

[0079] Based on the optimized chassis cells, the high-expression plasmids of the methoxypropanedioyl precursor synthesis module oxaB-G and the transport gene ozmS-oxaA module obtained in the previous functional module evaluation were assembled to further increase the yield of oxazomycin. The specific process was as follows: First, pSET156::P kasO* ::oxaSA plasmid was double-digested with SpeI and HindIII, and using pKC1139::P hrdB ::oxaB-G as the template, 156-oxaB-F / P kasO* -P hrdB -R as primers for PCR amplification to obtain the target fragment of oxaB-G driven by the strong promoter P hrdB . After the above two DNA fragments were ligated by Gibson assembly, and verified correct by PCR amplification and sequencing, the obtained recombinant plasmid was named pSET156::oxaSA::oxaB-G( Figure 6 A). Then pSET156::oxaSA::oxaB-G, the high-expression plasmid pKC1139::P hrdB ::oxaB-G of the methoxypropanedioyl precursor synthesis module oxaB-G constructed previously, and the high-expression plasmid pSET156::P kasO* ::oxaSA of the resistance gene ozmS-oxaA module were separately introduced into the above-mentioned chassis bacterium SL ROE by conjugation transfer, and the engineered strains SL ROEBG , SLROESA and SL ROESA-BG ( Figure 6 B) were obtained.

[0080] The wild-type strain WT, the chassis strain SL ROE and the above-mentioned engineered strains were fermented using MS medium, and HPLC analysis found that the yield of oxazomycin in the engineered strain SLROESA increased to 5.3 times that of the wild-type strain, with a yield of 176.6 mg / L. Finally, SLROESA was selected as the high-yield engineering strain for oxazomycin( Figure 6 C).

[0081] Example 5 Optimization of the culture medium to construct a high-efficiency expression system for oxazolomycin

[0082] The components of the fermentation medium MS of Streptomyces longshengensis mainly include soybean powder and mannitol. On the basis of the original MS medium, we optimized its formula and used the wild-type strain to detect the effects of different factors on the yield of oxazolomycin. First, the carbon source was improved, and mannitol was replaced with glucose, galactose, fructose, sucrose, maltose and potato starch at the same concentration. The wild-type Streptomyces longshengensis WT was fermented and analyzed by HPLC. The results showed that the yield of oxazolomycin did not increase significantly. Then we added different concentrations of exogenous factors, such as the essential amino acids serine (Ser), methionine (Met), glycine (Gly) for oxazolomycin biosynthesis and the precursor sodium acetate (CH3COONa), as well as betaine and dimethyl sulfoxide (DMSO). The results showed that the addition of 0.5 g / L of CH3COONa could increase the yield of oxazolomycin Toxa5 by 1.7 times. Since metal ions play an important global regulatory role in the metabolic network of Streptomyces, we externally added 1 g / L of different metal ions, including Ca 2+ 、Mn 2+ 、Zn 2+ and Mg 2+ . The results showed that the yield of oxazolomycin increased significantly in the presence of 1 g / L zinc ions. By further screening the optimal concentration of zinc ions, we found that both 2.5 g / L and 5 g / L zinc ions could increase the yield of oxazolomycin by 1.8 times ( Figure 7 A).

[0083] Based on the above optimization results of the culture medium components, two potentially effective factors were screened out: zinc sulfate and sodium acetate. Next, we used an orthogonal experiment to combine different concentrations of these two exogenous factors, and carried out fermentation of the high-yield strain SLROESA and HPLC analysis of the products. The results showed that the addition of zinc ions significantly reduced the yield of oxazolomycin in the high-yield strain, while in the medium with 0.5 g / L of CH3COONa added alone, the yield of Toxa5 could be increased by about 5% compared with the original fermentation conditions, and the final yield reached 182 mg / L ( Figure 7 B).

[0084] The specific fermentation and culture methods and the oxazolomycin detection process are as follows:

[0085] Seed culture medium: TSB medium (tryptone 17 g, soy peptone 3 g, glucose 2.5 g, sodium chloride 5.0 g, dipotassium hydrogen phosphate 2.5 g, dissolved in distilled water and made up to 1000 mL, adjusted to pH 7.3, sterilized at 121 °C for 15 min). Fermentation medium: MS medium (mannitol 20 g, soybean powder 20 g, made up to 1000 mL with water, 1.5 - 2% agar added for solid medium. Sterilized at 115 °C for 30 min).

[0086] Select MS medium as the seed culture medium, and inoculate the spores of Streptomyces longshengensis wild type or engineered strains into the seed culture medium respectively, and culture them at 28 °C with shaking at 220 rpm for 24 h; then transfer the seed liquid to the MS fermentation medium at an inoculation amount of 1%, and culture it on a shaker at 28 °C with shaking at 220 rpm for 3 d, then centrifuge to collect the supernatant of the fermentation broth, and filter it through a microporous filter membrane with a diameter of 0.22 μm, which can be used as a sample for activity detection or as a sample for HPLC analysis.

[0087] The HPLC analysis conditions for the above oxazolomycin are as follows: mobile phase A is H2O, mobile phase B is acetonitrile, eluted with 37% mobile phase B, running time 45 minutes, detection wavelength 280 nm.

[0088] The PCR reaction system used above is: 2×PCR Buffer KOD FX buffer 50 μL, 2 mM dNTPs 20 μL, KOD FX (1 U / μL) 2 μL (the above reagents are purchased from TOYOBO, Japan), 3 μL of each 10 μM primer, genomic template 5 μL (1 - 50 ng), ddH2O 17 μL, total reaction volume 100 μL. PCR cycling conditions: pre-denaturation: 94 °C, 3 min. Denaturation: 94 °C, 30 sec; annealing: 60 °C, 30 sec. Extension: 68 °C, 1 kb / min, 30 cycles; 68 °C, 5 min, hold at 4 °C. The corresponding primer pairs used for the amplified different DNA fragments are shown in Table 3.

[0089] The conjugation transfer used above is a conventional operation technique in the art, and the specific method is as follows:

[0090] The plasmid to be conjugated and transferred is first transformed into Escherichia coli ET12567 / pUZ8002 [see the strain information of Escherichia coli ET12567 / pUZ8002 in (Flett F, Mersinias V, and Smith C P. High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol. Lett., 1997, 155: 223-229)]. Single colonies of ET12567 / pUZ8002 containing the recombinant plasmid are selected and inoculated into 3 mL of liquid LB medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl sodium chloride, add distilled water to 1000 mL, sterilize at 121 °C for 30 min), and cultured overnight at 37 °C with shaking at 220 rpm; the overnight culture is transferred at an inoculation amount of 1% to 10 mL of liquid LB containing the corresponding antibiotics and 0.1% glucose, and cultured at 37 °C with shaking at 220 rpm until the OD 600 is approximately 0.4 - 0.6; the cells are collected by centrifugation at 5000 rpm, washed twice with an equal volume of liquid LB medium, and the cells are suspended in 500 μL of LB medium for standby; the spores of Streptomyces are collected in LB medium, centrifuged at 4500 rpm for 3 min, washed twice with LB, heat shocked in a water bath at 50 °C for 10 min, and cooled to room temperature; an equal amount of Escherichia coli cells and Streptomyces spores are mixed evenly, spread on an MS solid medium plate containing 10 mM MgCl2, cultured at 28 °C for 16 - 20 h, then nalidixic acid is spread to a final concentration of 25 μg / mL and other corresponding antibiotics to the corresponding concentrations, among which the use concentration of apramycin for Streptomyces longshengensis is 75 μg / mL and the use concentration of kanamycin is 50 μg / mL, and continue to culture at 28 °C for 3 - 5 days, and the correct conjugants are obtained through screening and PCR verification.

[0091] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Method for constructing oxazolomycin high-yield engineering strain, characterized in that, It includes the following steps: (1) By means of genetic engineering, the sequence between two transcription units oxaB-G and oxaH-oxaQ in the oxazolomycin biosynthetic gene cluster of Streptomyces longshengensis was replaced with two strong promoters in opposite directions to obtain engineering strain I; (2) A resistance gene expression cassette driven by a strong promoter was introduced into engineering strain I. The resistance gene was composed of the oxaA gene derived from Streptomyces longshengensis and the ozmS gene derived from Streptomyces albus in series to obtain a high-yield engineering strain of oxazolomycin.

2. The method according to claim 1, characterized in that The Streptomyces longshengensis has the accession number CGMCC 4.1101.

3. The method according to claim 1, wherein The two strong promoters with opposite directions described in step (1) are promoter P neo and promoter P kasO* .

4. The method according to claim 1, characterized in that, The strong promoter described in step (2) is a constitutive strong promoter, preferably the P hrdB promoter, the P neo promoter or the P kasO* promoter, more preferably the P kasO* promoter.

5. The method according to claim 1, characterized in that The sequence between the two transcription units oxaB-G and oxaH-oxaQ in step (1) is as shown in SEQ ID NO:1; The sequences of the two strong promoters in opposite directions are as shown in SEQ ID NO:2; In step (2), the sequences of the oxaA and ozmS genes are as shown in SEQ ID NO:3 and 5 respectively; the encoded protein sequences are as shown in SEQ ID NO:4 and 6 respectively.

6. The method according to any one of claims 1-5, characterized in that, Between steps (1) and (2), there is also a step of mutagenizing engineering strain I with gentamicin to obtain a gentamicin-resistant mutant; correspondingly, a resistance gene expression cassette driven by a strong promoter is introduced into the gentamicin-resistant mutant.

7. A high-yield engineering strain of oxazolomycin constructed by the method according to any one of claims 1-6.

8. Streptomyces longshengensis SLROESA, with the deposit number CGMCC No.29320.

9. Use of the engineering strain according to claim 7 or the Streptomyces longshengensis SLROESA according to claim 8 in the fermentation production of oxazolomycin.

10. A method for increasing the yield of oxazolomycin, characterized in that, The method includes: a) Fermentatively culturing the engineering strain according to claim 7 or the Streptomyces longshengensis SLROESA according to claim 8 to obtain a culture; b) Collecting the produced oxazolomycin from the culture obtained in step a); Sodium acetate is added to the medium for fermentatively culturing the engineering strain; Preferably, the addition amount of sodium acetate in the medium is 0.5 g / L.

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