Biological synthesis gene cluster of nocathiacins
A nocathiazole and biosynthesis technology, applied in the field of microbial genetic resources and genetic engineering, can solve problems such as numerous transformation steps, high production costs, and complex chemical structures
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Embodiment 1
[0132] Nocardia sp. WW-12651 total DNA extraction of Nocardia sp.
[0133] 100μL of Nocardia sp.WW-12651 mycelium suspension was inoculated into 3mL TSB liquid medium at 30°C, 250rpm, shaking culture for about 36hr, reaching the late logarithmic growth phase. Take 3mL and inoculate into 50mL TSB (containing 5mM MgCl 2 , 0.5% Glycine), 30°C, 250rpm, shaking culture for about 25hrs to reach the early stage of stable growth phase, showing milky white turbidity, and there are a lot of hyphae suspension. The bacterial solution was centrifuged at 4° C., 3500 rpm for 15 min to collect the hyphae, and washed twice with lysis buffer to obtain about 2.5 mL of hyphae. Add 10 mL of lysis buffer (containing 5 mg / mL lysozyme) to 2.5 mL of mycelium, vortex until uniform, then add Achromopeptidase to 3 mg / ml, and mix well. 37°C water bath for 30 minutes. Add 0.1 mL of proteinase K (20 mg / mL, freshly prepared with lysis buffer), 1 mL of 10% SDS, mix and quickly put it in a 70°C water bath for 1...
Embodiment 2
[0135] Construction of Nocardia sp.WW-12651 Genome Library of Nocardia sp.
[0136] First, determine the amount of Sau3AI through a series of dilution experiments. On this basis, the DNA fragments obtained by a large number of enzyme digestion are slightly larger than 40kb, which is dephosphorized. The vector pOJ446 was first cut with HpaI from the middle of the two cos sequences and dephosphorized, and then cut with BamHI from the multiple cloning site to obtain two connecting arms, which were connected to the prepared DNA fragment with a length of about 40kb overnight. Take out the packaging kit (PromegaPackagene Extract) from -80°C and place it on ice. When it just melts, immediately add 5uL of the above-mentioned connection solution, flick and mix well, being careful not to generate bubbles, and leave it at room temperature (about 22°C) for 3hr. Add 445uL Phage buffer and mix upside down. Then add 25uL chloroform, mix upside down, make it slowly traverse the entire liquid to...
Embodiment 3
[0139] Fermentation, product separation, purification and identification of Nocardia sp. WW-12651, which produces nocardiac:
[0140] First, 300μL of Nocardia sp.WW-12651 mycelium suspension frozen at -80℃ was inoculated into 25mL seed culture medium (soluble starch 2%, glucose 0.5%, NZ Case 0.3%, yeast extract 0.2%, fish meat Extract 0.5%, calcium carbonate 0.3%), 32°C, 250rpm culture for 3 days. Take 2 mL from it and transfer it to 50 mL of fermentation medium (glucose 2%, HY-yeast 4121%, nutrient soybean 1%), culture at 30°C and 250 rpm for 4-5 days. Then the fermentation broth was combined, transferred to a centrifuge tube, centrifuged at 3800 rpm for 15 minutes, and the mycelial precipitate was discarded. The supernatant was extracted twice with an equal volume of ethyl acetate, and the organic phases were combined. Use anhydrous MgSO 4 Or anhydrous Na 2 SO 4 Dry, filter, and concentrate to dryness at 37°C under reduced pressure. The sample was dissolved in a mixed solven...
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