Decarboxylated FR-008 derivative polyketone antibiotic and use thereof
A technology of FR-008 and antibiotics, applied in the field of biomedicine, can solve problems such as unbearable for patients, cumbersome production process, and high price
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Embodiment 1
[0044] Example 1: Construction of gene replacement plasmid for cytochrome P450 monooxygenase gene fscP
[0045] Based on the sequence information obtained from the FR-008 gene cluster, a gene replacement plasmid for the cytochrome P450 monooxygenase gene fscP was constructed. pJTU28 is a derivative clone of pIJ2925 carrying the 2.3 kb PstI-KpnI fragment containing the fscP gene. pJTU28 was obtained by self-ligating pJTU26 after digestion with PstI. pJTU26 is a clone in which the 6.6 kb Kpn I fragment from pHZ145 was inserted into the pIJ2925 Kpn I cloning site. There are two BssH II restriction sites 165 and 561 bases downstream of the start codon of fscP in the cytochrome P450 monooxygenase gene. After pJTU28 was cut with BssH II, the 396 bp sequence inside the fscP gene was deleted, and the 1.4 kb fragment containing the apramycin resistance gene excised from pJTU33 was inserted to obtain pJTU37. pJTU33 is a derivative plasmid with the BamH I fragment inserted from pHZ107...
Embodiment 2
[0046] Example 2: Screening of deletion mutants of fscP gene
[0047] The gene replacement plasmid pJTU50 was introduced into Streptomyces FR-008 by conjugative transfer of two parents, and after two rounds of relaxation culture without antibiotics, a mutant strain CS103 resistant to apramycin and sensitive to thiostrepton was obtained by counter selection .
Embodiment 3
[0048] Example 3: Fermentation of the deletion mutant strain CS103 of the fscP gene
[0049] Medium (m / v): glucose 2%, starch 1%, glycerol 1.389%, yeast powder 0.3%, industrial amino acid powder 0.5%, peptone 0.101%, NaCl 1%, FeSO 4 ·7H 2 O 0.05%, CuSO 4 0.00428%. Before sterilization, the pH was adjusted to 6.90 with 5 mol / l NaOH solution, the inoculation time was 30 hours, the inoculation amount (v / v) was 10%, and the shaker was cultivated at 28 degrees and 200 rpm for 4 days.
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