Streptomyces autolyticus with high yield of autolytimycin
A technology for dissolving streptomyces and autolyzing mycin, applied in the field of microorganisms, can solve the problems of difficult chemical synthesis of autolyzing mycin, low yield of autolyzing mycin, hindering research and development, etc.
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Embodiment 1
[0023] Autolytic Streptomyces alm M Construction of gene knockout mutants: The cloned and sequenced geldanamycin gene cluster was analyzed with the help of tools such as ORF finder, glimmer and BLAST on the NCBI website, and the function of each open reading frame (ORF) was speculated. Then, use the Red / ET one-step PCR method to construct alm M Insertion mutation of gene (SEQ ID NO: 1) ( figure 1 ). In this method, first use pHY773 as a template, and use PCR to amplify alm M aac(3)IV gene insertion cassette for gene homologous sequence. The primers for PCR amplification were: 5′-GTTCCAGGACACGGTCGATCTGATCCTGGCGGGCGAATGATTCCGGGGATCCGTCGACC-3′ (SEQ ID NO: 2) and 5′-GAAGGGGACCAGTTCGTGTTCGGGTGGGGGAGTGCCTCATGTAGGCTGGAGCTGCTTC-3′ (SEQ ID NO: 3). The PCR amplification conditions were: 95°C preheating for 5 minutes, 1 cycle; denaturation at 94°C for 1 minute, renaturation at 50°C for 45 seconds, amplification at 72°C for 2.5 minutes, 10 cycles; denaturation at 94°C for 1 minute...
Embodiment 2
[0025] Autolytic Streptomyces wild-type strain and alm M Detection of fermentation products of gene knockout mutants: autolytic Streptomyces wild-type strain and alm M Fresh spores of gene knockout mutant strains were inoculated into 50 mL TSB medium respectively, and at 28 o C. Cultivate at 200 rpm for 72 hours to prepare a fermented seed solution, and then inoculate 0.5 mL of the seed solution into the fermentation medium (2% soybean flour, 0.2% peptone, 2% glucose, 0.5% soluble starch, 0.2% Yeast extract, 0.4 % NaCl, 0.05 % K 2 HPO 4 , 0.05% MgSO 4 , 0.2% CaCO 3 ), at 28o C, 250 rpm fermentation culture for 7 days. After the fermentation broth was centrifuged (3500 g, 30 minutes), the supernatant was taken, extracted twice with ethyl acetate, and then evaporated to dryness under reduced pressure at 45°C by a rotary evaporator. The fermentation products were respectively dissolved in appropriate amount of methanol and analyzed by HPLC ( image 3 ), autolysomycin was...
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