A method for obtaining structural analogs of sansanmycin by mutational synthesis
A technology of compound and structural formula, applied in the field of microbial catalytic synthesis, can solve the problem of not being applied to obtain uridine peptide antibiotic analogs and the like
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Embodiment 1
[0077] Example 1. In-frame knockout and recovery of ssaX
[0078] To investigate the role of ssaX in sansanmycin biosynthesis, we utilized the PCR-targeting method[ 21 ] Successfully constructed a mutant strain with in-frame deletion of ssaX. In order to promote homologous recombination, the cosmid 13R-1 (full sequence / structural information of the plasmid is disclosed in reference 11, 13R-1 uses pOJ446 as the vector and contains most of the sansanmycin biosynthesis genes including ssaX)[ 11 The Streptomyces plasmid replicon SCP2* on ] was replaced with the Amp resistance gene bla, resulting in the cosmid 13R-1-SCP2KO. Then, the ssaX on 13R-1-SCP2KO was knocked out in the same frame, and the obtained 13R-1-SCP2KO-XKO was introduced into the wild-type strain by conjugative transfer (isolated from a soil sample in Anshun, Guizhou, China in 1965, China Microorganism Culture Collection The culture collection number of the General Microbiology Center of the Management Committee i...
Embodiment 2
[0114] Example 2 Isolation and structural identification of sansanmycin analogues in ssaX blocking strains
[0115] 1. The fermentation and cultivation method of the ssaX blocking strain is the same as in Example 1. Combine 50 L of fermentation broth, centrifuge, take the supernatant and put it on the macroporous adsorption resin D4006, elute the target components with 30% acetone aqueous solution, and concentrate under reduced pressure , vacuum freeze-drying to obtain the crude product.
[0116] 2. Dissolve the crude product in a buffer solution of 0.02M Tris (pH8.5), put it on a DEAE-sephadex-A25 column, and elute it with different concentrations of NaCl solutions, and detect components with a purity greater than 50% by HPLC Collect, collect the eluate, concentrate under reduced pressure, and freeze-dry to obtain subpure products of each component.
[0117] 3. Then purified by preparative liquid phase, preparation conditions: chromatographic column: XBridge BEH C 18 OBD Pr...
Embodiment 3
[0124] Example 3. Production of structurally diverse sansanmycin analogs using ssaX blocking strains
[0125] First feed 20 kinds of amino acids including Tyr, Phe and Met to explore the feasibility of mutation synthesis. When feeding Tyr (3mM), the output of sansanmycin MX-2 whose N-terminal amino acid is Tyr increases to 187% ( image 3 ). When feeding Phe (3mM), the N-terminal amino acid is the production of sansanmycin MX-4 of Phe doubled ( image 3 ). However, when fed other amino acids including Met, the peak shape of HPLC did not change significantly. This may be due to the substrate preference of NRPS, which favors Phe and Tyr over other amino acids. This result is consistent with the yield of sansanmycin analogs in SS / XKO. Simultaneously, the increase of production of sansanmycin MX-2 and MX-4 suggested that our mutant synthesis may be suitable for producing sansanmycin analogues whose N-terminus was substituted.
[0126] The incorporation of halogen atoms into ...
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