Recombinant streptomyces nodosus for producing 8-deoxyampholactone as well as construction method and application of recombinant streptomyces nodosus

Abstract

The invention discloses recombinant streptomyces nodosus for producing 8-deoxyampholactone as well as a construction method and application of the recombinant streptomyces nodosus. A primer D3U.F with a sequence as shown in SEQ ID NO1 and a primer D3U.R with a sequence as shown in SEQ ID NO2 are adopted to amplify an amph DIII gene upstream homologous arm, and a primer D3D.F with a sequence as shown in SEQ ID NO3 and a primer D3D.R with a sequence as shown in SEQ ID NO4 are adopted to amplify an amph DIII gene downstream homologous arm; the method comprises the following steps: amplifying a kanamycin resistance gene by adopting a primer kan.F as shown in a sequence SEQ ID NO5 and a primer kan.R as shown in a sequence SEQ ID NO6; inserting the fragment into a vector by using a homologous recombination technology to construct a recombinant plasmid; and introducing the recombinant plasmid into streptomyces nodosus through conjugational transfer, and screening a double-exchange mutant strain. According to the method, an amphDIII gene is knocked out through a homologous recombination technology, and glycosylation modification of trehalose amine at a C19 site is blocked, so that a mutant strain delta amphDIII with high yield of 8-deoxyampholactone is obtained; the product has clear nuclear magnetic resonance and mass spectrum characteristics, and can be used as a low-toxicity antifungal drug precursor.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and microbial fermentation, in particular to a recombinant Streptomyces nodosa for producing 8-deoxyamphoteric lactone, a construction method and an application thereof. Background Art

[0002] Amphotericin B (AmB) is a polyene macrolide antibiotic synthesized by Streptomyces nodosus. AmB is a widely used drug for deep fungal infections. It exhibits broad-spectrum, potent antifungal activity, and resistance is rare. Currently, there are no alternatives, making it the "gold standard" for the clinical treatment of deep fungal infections. Structurally, AmB belongs to the heptaene macrolide class of antibiotics, characterized by its seven conjugated polyene macrolides. In Streptomyces nodularis, three propionate and 16 acetate precursors undergo 18 elongation steps to cyclize the polyketide backbone, which is then subjected to three post-modification steps to ultimately form AmB. These post-modifications primarily involve glycosylation of trehalosamine at position C19, controlled by the genes amph DIII (GDP-aD-mannose-4,6-dehydratase), amph DII (GDP-3-keto-6-deoxy-mannose-3-aminotransferase), and amph DⅠ (trehalose transferase); oxidative carboxylation at position C16, controlled by amph N and amph M; and hydroxylation at position C8, mediated by amph L. In the AmB-producing strain, GDP-mannose is synthesized as a precursor for C19 trehalosamine. GDP-trehalosamine is synthesized under the sequential catalysis of amph DIII and amph DII, and then transferred by amph DI. Of these three enzymes, amphDⅢ is the key enzyme in its synthesis. The synthesis of the modifying groups at positions C8, C16, and C19 follows a sequential order, likely synthesizing the C16 carboxyl group first, followed by glycosylation of trehalose amine at C19, and finally hydroxylation at C8. The antifungal mechanism of AmB is binding to ergosterol on the fungal cell membrane, forming micropores in the membrane, ultimately leading to the loss of intracellular substances such as nucleotides and amino acids, causing bacterial death. However, AmB also interacts with cholesterol on mammalian cell membranes during treatment, albeit to a lesser extent, but with certain side effects, which significantly limits the drug's application.

[0003] Amphotericin B (AmB) is a core drug for treating deep fungal infections, but its production process is plagued by impurity accumulation (e.g., byproducts with a relative retention time (RRT) of 1.35), compromising purification efficiency and drug safety. The synthesis of AmB requires polyketide chain elongation and multiple post-modification steps (including trehalose aminoglycosylation at C19 and hydroxylation at C8). However, existing technologies lack a clear understanding of the function of the amph D III gene, hindering targeted optimization of metabolic pathways. Conventional genetic modification techniques for AmB production strains are inefficient, resulting in low homologous recombination success rates and a lack of precise verification of metabolite structures.

[0004] To reduce toxicity and improve drug utilization efficiency, much research has been conducted on the delivery forms of AmB. For example, liposome drugs and protein nanoformulations such as Abelcet and Ambisome appeared in the 1990s, such as the patent "CN202010420654.5 Amphotericin B Albumin Nanoformulation, Preparation Method, and Application thereof." However, these have not fundamentally resolved the product's problems. At the same time, many researchers have conducted detailed research on the structural modification of this product. For example, Borowski, Matsumori, and Burke have fundamentally resolved the product's binding problem with cholesterol by modifying the product's structure. Although some similar structures with almost no toxicity have been screened (C2'deAMB), this is the only highly effective and non-toxic AmB derivative discovered to date. Preclinical studies of this compound are currently underway, and it is expected to develop into a drug that can replace AmB.

[0005] Although there are many obstacles to the chemical modification of AMB, as prodrug research has become an important part of new drug design, the strategy of connecting AMB with high molecular weight polymers to prepare water-soluble prodrugs has shown broad application prospects. Pharmacists have studied prodrugs such as AmB-AG and AMBPEG, and proved that they have good drugability, effectively improving the defects of the prototype drug such as low bioavailability, poor stability, unsatisfactory distribution and absorption in the body, and large adverse reactions. Therefore, chemical modification based on the parent nucleus of AmB has gradually become the future research direction of pharmacists. With the rapid development and progress of molecular biology, some important intermediates, such as 8-deoxyamphotericin B lactone (8-deoxyamphotericin B lactone, structural formula C 41 H 62 O 13 , 8-deoxyamphoteronolides B), can be produced by metabolically engineered recombinant bacteria. These important intermediates serve as the mother nucleus for new structural modifications and provide important basic support for the subsequent chemical modification of AmB. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a recombinant Streptomyces nodosa that can produce 8-deoxyamphoteric lactone in high yield; the present invention also provides a method for constructing the recombinant Streptomyces nodosa that produces 8-deoxyamphoteric lactone; the present invention also provides an application of the recombinant Streptomyces nodosa that produces 8-deoxyamphoteric lactone.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is: using the primer D3U.F shown in SEQ ID NO1 and the primer D3U.R shown in SEQ ID NO2 to amplify the upstream homology arm of the amph DⅢ gene, and using the primer D3D.F shown in SEQ ID NO3 and the primer D3D.R shown in SEQ ID NO4 to amplify the downstream homology arm of the amph DⅢ gene; using the primer kan.F shown in SEQ ID NO5 and the primer kan.R shown in SEQ ID NO6 to amplify the kanamycin resistance gene; using homologous recombination technology to insert the above fragments into a vector to construct a recombinant plasmid; introducing the recombinant plasmid into Streptomyces nodosa by conjugation transfer, screening double-exchange mutants, and thus obtaining a recombinant Streptomyces nodosa strain Δamph DⅢ.

[0008] Furthermore, the amplified length of the upstream homology arm of the amph DIII gene was 1883 bp, and the amplified length of the downstream homology arm of the amph DIII gene was 1938 bp; and the amplified length of the kanamycin resistance gene was 795 bp.

[0009] Furthermore, the amplified fragment of Δamph DⅢ is 1416 bp in length.

[0010] In order to solve the above technical problems, the technical solution adopted by the method of the present invention includes the following steps: 1) amplifying the upstream homology arm of the amphDⅢ gene and the downstream homology arm of the amphDⅢ gene; 2) Using the pSET152-kan plasmid as a template, amplify the kanamycin resistance gene; 3) Using multi-fragment homologous recombination technology, the gene fragments from steps 1) and 2) are inserted into the vector to construct a recombinant plasmid; 4) introducing the recombinant plasmid into Streptomyces nodosa by conjugation; 5) The introduced Streptomyces nodosa is cultured on a culture medium and double-crossover mutants are screened to obtain the recombinant Streptomyces nodosa strain.

[0011] Furthermore, in step 3), the pKC1139 vector is used.

[0012] Furthermore, in step 4), the recombinant plasmid is transformed into Escherichia coli and then introduced into Streptomyces nodosa by conjugation transfer.

[0013] Furthermore, in step 5), MS medium containing kanamycin and nalidixic acid is used.

[0014] In order to solve the above technical problems, the technical solution adopted by the present invention is: the recombinant Streptomyces nodosa is fermented and cultured to produce 8-deoxyamphoteric lactone.

[0015] Furthermore, a fermentation medium with the following formula is used: 1.5% to 2.0% peanut cake powder, 3.0% to 5.0% starch, 1.6% to 2.4% glucose, and the balance is water.

[0016] The beneficial effects of the above technical solution are as follows: the present invention aims to knock out the amph DⅢ gene of industrial Streptomyces nodosa, block the glycosylation modification of trehalose amine at position C19, thereby accumulating 8-deoxyamphoteric lactone, and reveal the regulatory effect of the gene on C8 hydroxylation, providing technical support for the development of new antifungal drugs.

[0017] The method presented here utilizes homologous recombination to knock out the amphDⅢ gene, blocking glycosylation of trehalose at position C19. This results in a mutant strain, ΔamphDⅢ, that produces a high yield of 8-deoxyamphidolactone. This product exhibits clear nuclear magnetic resonance (1H-NMR) and mass spectrometry (HRMS m / z = 761.4104) characteristics and is suitable for use as a low-toxic antifungal drug precursor. This method provides key technical support for optimizing the synthesis pathway of amphotericin B and developing novel polyketide antibiotics.

[0018] The present invention uses recombinant Streptomyces tuberosum to produce 8-deoxyamphotericin B lactone. By constructing this recombinant Streptomyces tuberosum, quantitative production of 8-deoxyamphotericin B lactone can be achieved. It also serves as the parent nucleus of AmB, providing important product support for subsequent chemical transformation of AmB. The resulting 8-deoxyamphotericin lactone can be used as an AmB prodrug or a novel antifungal compound, reducing the nephrotoxicity of existing drugs and providing a general technical model for the targeted metabolic engineering of polyketide antibiotics. Furthermore, this research can clarify the metabolic pathway of this impurity, thereby controlling the generation of this impurity in existing products and effectively improving the quality of existing products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Figure 1 Schematic diagram of the gene knockout principle of the present invention; Figure 2 Schematic diagram of screening of strain ΔamphDⅢ described in the examples of the present invention; Figure 3 This is a photo of PCR verification of the genome of the strain ΔamphDⅢ obtained in the example of the present invention; Figure 4is a liquid chromatogram of 8-deoxyampholactone obtained in an embodiment of the present invention; Figure 5 This is an infrared absorption spectrum of 8-deoxyampholactone obtained in an embodiment of the present invention; Figure 6 This is a nuclear magnetic resonance spectrum (NMR) of the 8-deoxyampholactone obtained in the example of the present invention; Figure 7 yes Figure 6 A partial enlarged view of Figure 8 yes Figure 6 A partial enlarged view of Figure 9 This is a high-resolution mass spectrum of the 8-deoxyampholactone obtained in the examples of the present invention. DETAILED DESCRIPTION

[0021] The recombinant Streptomyces nodosus strain producing 8-deoxyamphoteric lactone was used to amplify the upstream homology arm of the amph DIII gene using primers D3U.F as shown in SEQ ID NO. 1 and D3U.R as shown in SEQ ID NO. 2, with an amplified length of 1883 bp. The downstream homology arm of the amph DIII gene was amplified using primers D3D.F as shown in SEQ ID NO. 3 and D3D.R as shown in SEQ ID NO. 4, with an amplified length of 1938 bp. The amph DIII gene is a Streptomyces nodosus gene accessed by NCBI (GenBank: AAK73500.1). The kanamycin resistance gene was amplified using primers kan.F as shown in SEQ ID NO. 5 and kan.R as shown in SEQ ID NO. 6, with an amplified length of 795 bp. The amplified kanamycin resistance gene (kan gene) was obtained. The gene fragments were inserted into a vector using homologous recombination technology to construct a recombinant plasmid. The recombinant plasmid was introduced into Streptomyces nodosa by conjugation, and double-crossover mutants were screened to obtain the recombinant Streptomyces nodosa Δamph DIII. Information on the amplification primers is shown in Table 1. Table 1: Amplification primer information

[0022] The method for constructing a recombinant Streptomyces nodosa that produces 8-deoxyamphoteric lactone comprises the following steps: 1) Construction of gene knockout vector: 1.1) Figure 1As shown, the upstream homology arm of the amph DⅢ gene was amplified using the primer D3U.F shown in SEQ ID NO1 and the primer D3U.R shown in SEQ ID NO2, with an amplified length of 1883 bp; the downstream homology arm of the amph DⅢ gene was amplified using the primer D3D.F shown in SEQ ID NO3 and the primer D3D.R shown in SEQ ID NO4, with an amplified length of 1938 bp; and the amplified homology arm of the amph DⅢ gene was obtained; 1.2) Using the pSET152-kan plasmid as a template, primer kan.F having the sequence shown in SEQ ID NO: 5 and primer kan.R having the sequence shown in SEQ ID NO: 6 were used to amplify the kanamycin resistance gene, with an amplified length of 795 bp to obtain the amplified kan gene; 1.3) Using multi-fragment homologous recombination technology, the amph D III gene homology arms amplified in step 1) and the kan gene fragment amplified in step 2) were inserted into the pKC1139 vector to construct the recombinant plasmid pKC1139-amphD III-kan.

[0023] 2) Conjugative transfer and mutant screening: 2.1) Transform the recombinant plasmid pKC1139-amphDIII-kan into Escherichia coli, preferably E. coli ET12567 / pUZ8002 (E. coli ET12567(pUZ8002) Strain), and then introduce it into Streptomyces nodosa by conjugation. 2.2) The introduced Streptomyces nodosa was cultured on MS medium containing 50-75 μg / mL kanamycin and 25-50 μg / mL nalidixic acid, and double-crossover mutants were screened to obtain the recombinant Streptomyces nodosa strain Δamph DIII. The culture medium was 28-32° C. for 3-6 days. Apr-sensitive and Kan-resistant transformants were screened to obtain the recombinant Streptomyces nodosa strain Δamph DIII. The amplified fragment of Δamph DIII was 1416 bp in length.

[0024] 3) Fermentation and product purification: The ΔamphDIII strain is cultivated using a fermentation process. The fermented mycelium is extracted and purified under alkaline conditions to obtain the 8-deoxyamphidolactone. The fermentation medium comprises the following composition (by weight): 1.5% to 2.0% peanut meal, 3.0% to 5.0% starch, 1.6% to 2.4% glucose, and the balance water. The fermentation process parameters are: 25-30°C, pH 4.5-6.0, aeration rate of 1:1.0 to 1.2 V / V / min, and a fermentation cycle of 72 to 96 hours. Methanol extraction is preferred, and the extract is purified by preparative liquid chromatography using an acetonitrile-acetic acid aqueous solution mobile phase with gradient elution.

[0025] 4) Structural confirmation: The structure of the 8-deoxyamphoteronolides (8-deoxyamphoteronolides B) is shown in the following formula (A):

[0026] (A) By nuclear magnetic resonance (1H-NMR), high resolution mass spectrometry (HRMS) [MH] - m / z=761.4104 and characteristic peaks of infrared spectrum (IR) verified the structure of the product. Example

[0027] 1) Amplify the homology arms and kan gene using the above primers and ligate them into the pKC1139 vector using the Novizan One-Step Cloning Kit to construct the recombinant plasmid pKC1139-amphDⅢ-kan.

[0028] 2) Transform the recombinant plasmid pKC1139-amphDⅢ-kan into Escherichia coli ET12567 / pUZ8002 and then introduce it into Streptomyces nodosa by conjugation. Spread the plasmid on MS medium containing 50 μg / mL kanamycin and 25 μg / mL nalidixic acid, culture at 30°C for 5 days, and select Apr-sensitive / Kan-resistant transformants. Figure 2 As shown, Figure 2 In the middle, the two upper left figures are the growth of strains 1-16 at 5μg / mL and 10μg / mL concentrations of Apr; the two lower left figures are the growth of strains 1-16 at 5μg / mL and 10μg / mL concentrations of Kan; the right figure is a schematic diagram of the location of strains 1-16; according to Figure 2 As shown, transformants resistant to Kan but sensitive to Apr, namely transformants 6, 7, 8, 11, 12, and 13, were selected to obtain the strain ΔamphDⅢ.

[0029] Figure 3As shown, the screened strain ΔamphDⅢ was verified by PCR using primers amphDⅢ-Test.F / R, and a 1416 bp specific band was obtained, which was completely different from the 1165 bp specific band of the wild-type amph DⅢ, proving that the correct amphDⅢ genetic mutant strain was obtained.

[0030] 3) The ΔamphDⅢ strain was cultured using a fermentation process: a 50-L fermentor was used with a 10% inoculum, a stirring rate of 400 rpm, and a dissolved oxygen concentration of 20%–30%. The fermentation medium (wt%) consisted of 1.8% peanut meal, 4.0% starch, 2.0% glucose, and the balance water. Fermentation parameters included 28°C, a pH of 4.5–6.0, an aeration rate of 1:1.2 V / V / min, and a fermentation time of 84 h. The fermented mycelium was extracted using a methanol-water system at pH 11, and then the pH was adjusted to 3.5 for crystallization. The crystalline product was purified using a preparative liquid phase with a mobile phase of acetonitrile: acetic acid water = 45:55 (v / v) to obtain the 8-deoxyampholactone.

[0031] 4) Detection: The liquid chromatogram of 8-deoxyampholactone obtained in this example is shown in Figure 4 、Infrared absorption spectrum Figure 5 、NMR diagram Figure 6-Figure 8 、HRMS diagram Figure 9 ,Depend on Figure 4-9 It can be seen that the product is determined to be 8-deoxyampholactone; the yield of the 8-deoxyampholactone obtained in this example is ≥85%, and the purity is ≥90%. The HPLC detection conditions are: Waters XBridge® Shield RP18 column, λ=383 nm.

Claims

1. A recombinant Streptomyces nodosa that produces 8-deoxyamphoteric lactone, characterized in that: The upstream homology arm of the amph DⅢ gene is amplified using the primer D3U.F shown in SEQ ID NO. 1 and the primer D3U.R shown in SEQ ID NO. 2, and the downstream homology arm of the amph DⅢ gene is amplified using the primer D3D.F shown in SEQ ID NO. 3 and the primer D3D.R shown in SEQ ID NO. 4; the kanamycin resistance gene is amplified using the primer kan.F shown in SEQ ID NO. 5 and the primer kan.R shown in SEQ ID NO. 6; the above fragments are inserted into a vector using homologous recombination technology to construct a recombinant plasmid; the recombinant plasmid is introduced into Streptomyces nodosa by conjugation transfer, and double-exchange mutants are screened to obtain a recombinant Streptomyces nodosa strain Δamph DⅢ.

2. The recombinant Streptomyces nodosa producing 8-deoxyamphoteric lactone according to claim 1, characterized in that: The amplified length of the upstream homology arm of the amphDⅢ gene is 1883 bp, and the amplified length of the downstream homology arm of the amphDⅢ gene is 1938 bp; the amplified length of the kanamycin resistance gene is 795 bp.

3. The recombinant Streptomyces nodosa producing 8-deoxyamphoteric lactone according to claim 1 or 2, characterized in that: The amplified fragment of Δamph DⅢ is 1416 bp in length.

4. A method for constructing the recombinant Streptomyces nodosa producing 8-deoxyamphoteric lactone according to claim 1, characterized in that: The method comprises the following steps: 1) amplifying an upstream homology arm of the amph D III gene and a downstream homology arm of the amph D III gene; 2) Using the pSET152-kan plasmid as a template, amplify the kanamycin resistance gene; 3) Using multi-fragment homologous recombination technology, the gene fragments from steps 1) and 2) are inserted into the vector to construct a recombinant plasmid; 4) introducing the recombinant plasmid into Streptomyces nodosa by conjugation; 5) The introduced Streptomyces nodosa is cultured on a culture medium and double-crossover mutants are screened to obtain the recombinant Streptomyces nodosa strain Δamph DⅢ.

5. The method for constructing a recombinant Streptomyces nodosa producing 8-deoxyamphoteric lactone according to claim 4, characterized in that: In step 3), pKC1139 vector is used.

6. The method for constructing a recombinant Streptomyces nodosa producing 8-deoxyamphoteric lactone according to claim 4, characterized in that: In step 4), the recombinant plasmid is transformed into Escherichia coli and then introduced into Streptomyces nodosa by conjugation transfer.

7. The method for constructing a recombinant Streptomyces nodosa producing 8-deoxyamphoteric lactone according to claim 4, 5 or 6, characterized in that: In the step 5), MS medium containing kanamycin and nalidixic acid is used.

8. A use of the recombinant Streptomyces nodosa producing 8-deoxyamphoteric lactone according to claim 1, characterized in that: The recombinant Streptomyces tuberosum ferments and cultures 8-deoxyamphoteric lactone.

9. The use of the recombinant Streptomyces nodosa producing 8-deoxyamphoteric lactone according to claim 8, characterized in that: The fermentation medium has the following formula: 1.5% to 2.0% peanut powder, 3.0% to 5.0% starch, 1.6% to 2.4% glucose, and the balance water.

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