Trioxacarcin biosynthetic gene cluster and application thereof
A biosynthesis and gene cluster technology, applied in the field of microbial gene resources and genetic engineering, can solve problems such as lack of understanding of synthetic pathways
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[0145] Preparation of Sanxin Kaxin or its analogues
[0146] For a long time, one of the key goals of our research is to obtain new Sanxin Cassine structurally similar compounds with stable structure, better activity, and large-scale fermentation by microorganisms. Through genetic manipulation in vivo, including gene deletion, interruption, A series of mutant strains of Sanxin Cassine were finally obtained by means of substitution and other experiments. Through the fermentation analysis of some of them, it was found that Sanxin Cassine and its analogues were no longer produced; some of them had changed the yield of Sansin Cassine , there are also some mutant strains, through the isolation and purification of a large amount of fermentation, part of the intermediate compound of Sanxinkaxin is obtained (Fig. 8, Fig. 9).
[0147] In the present invention, the method for preparing Sanxinkaxin or its analogues is as follows:
[0148] (1) providing a mutant strain;
[0149] (2) Cul...
Embodiment 1
[0178] Extraction of Genomic DNA from Streptomyces bottropensis DO-45, which produces Sanxin Cassine:
[0179] 1) Collect mycelia
[0180] 100 μL 1 x 10 8 Streptomyces bottropensis DO-45 spore suspension per mL was inoculated into 3mL TSB liquid medium, cultured at 30°C, 230rpm for about 24hrs and reached the late logarithmic growth phase, and 2mL was inoculated into 50mL TSB (containing 25mM magnesium chloride), 30°C After culturing at 250rpm for about 36hrs, it reaches the early stage of the stable growth period, and it is milky yellow and turbid. Centrifuge the bacterial solution at 4°C and 3500rpm for 15min to collect mycelium, wash with lysis buffer, and collect 0.5mL of light yellow mycelium.
[0181] 2) Genomic DNA extraction
[0182] Add 10 mL of lysis buffer (containing 5 mg / mL lysozyme) to 1 mL of mycelia, vortex until uniform, and bathe in 37 °C water bath for 15 min. Add 0.1 mL of proteinase K solution (10 mg / mL, freshly prepared with lysis buffer), 1 mL of 10% ...
Embodiment 2
[0184] PCR cloning of the Sanxinkaxin synthetic gene:
[0185] Composition of 50μL PCR system:
[0186] Taq DNA Polymerase Buffer (10X) 5μL dNTP (10mM) 4μL (10mM) forward primer 1μM reverse primer 1μM template 0.1-1μL DMSO 4μL Taq DNA polymerase (2u / μL) 1-2u h 2 o Make up to 50μL
[0187] PCR program:
[0188] Taq enzyme
[0189] Cycle 1: 94°C, 3min, 1 cycle;
[0190] Cycle 2: 94°C, 30s; 55-65°C, 30s; 72°C, 1min / kb; 35 cycles;
[0191] Cycle 3: 72°C, 5-10 min; 1 cycle.
[0192] Primestar:
[0193] Step 1: 98°C, 10s
[0194] Step 2: 58-65°C, 15s
[0195] Step 3: 72°C, 1min-3min
[0196] Repeat steps 1-3 for 30 rounds
[0197] Step 4: 72°C, 10min
[0198] The PCR conditions of different primers were optimized based on the above conditions.
[0199] After PCR, the DNA band of the expected size was recovered and purified by low-melting point gel, and connected to the PCR cloning vector pGEM-TEasy vector, t...
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