Novel ll-d49194α1 analogue, preparation method and application thereof
A technology for LL-D49194 and analogues, applied in the field of novel LL-D49194α1 analogues, which can solve problems that have not yet been developed
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Embodiment 1
[0093] Example 1. Construction of mutant strains sDL05030, sDL05021 and sDL05027
[0094] 1. Construction of deletion plasmids for lldO10, lldB3 and lldO2 genes
[0095] The primer sequences for cloning the left arm of the in-frame deletion of lldO10 are as follows:
[0096] lldO10 left arm forward primer (SEQ ID NO: 1)
[0097] 5'-ATAGAATTCACGAGGGTGCGGTCGTCT-3'
[0098] lldO10 left arm reverse primer (SEQ ID NO:2)
[0099] 5'-ATATCTAGAATCGGCGACCAGGGCCTC-3'
[0100] The primer sequences for cloning the right arm of the in-frame deletion of lldO10 are as follows:
[0101] lldO10 right arm forward primer (SEQ ID NO:3)
[0102] 5'-ATATCTAGACAACGCCTGCCGGTCCGGTTC-3'
[0103] lldO10 right arm reverse primer (SEQ ID NO:4)
[0104] 5'-ATAAAGCTTACGTTCGCGCTGCCCCGGTTC-3'
[0105] The primer sequences for cloning the left arm of the in-frame deletion of lldB3 are as follows:
[0106] lldB3 left arm forward primer (SEQ ID NO:5)
[0107] 5'-ATAGAATTCCCTGTTCCAGCTCCGCCTGG-3'
[010...
Embodiment 2
[0131] Example 2. Fermentation, detection, separation, purification and structural identification of LL-D49194α1 analogs
[0132] The mutant strains sDL05030, sDL05021 and sDL05027 were respectively inoculated in 100 mL seed medium (containing TSB 30 g / L) for shaking culture at 220 rpm and 30°C for 36 hours. Inoculate 5-10mL seed shake flask bacterial liquid into 100mL fermentation medium (containing soluble starch 60g / L, glucose 10g / L, yeast extract 10g / L, sodium chloride 3g / L, dipotassium hydrogen phosphate 1g / L , magnesium sulfate heptahydrate 1g / L, calcium carbonate 2g / L, trace salt solution 0.1mL (1000 times storage solution: copper sulfate pentahydrate 70g / L, ferrous sulfate heptahydrate 10g / L, manganese chloride tetrahydrate 8g / L L, zinc sulfate heptahydrate 2g / L, cobalt chloride heptahydrate 0.06g / L), pH=7.3±0.2), 30°C, 220rpm culture, add HP20 resin (3g / 100mL) after inoculation 2-3 days, continue The fermentation broth was processed after culturing until day 6 after ...
Embodiment 3
[0161] Embodiment 3. The fermentation product HPLC analysis of departure bacterial strain and modified bacterial strain
[0162] HPLC ( Figure 20 ) detection conditions are:
[0163] Instrument: Dionex Ultimate 3000 system
[0164] Detection wavelength: UV=400nm
[0165] Column: Acclaim PolarAdvantage II, C18, 4.6×250mm, 5μm, (ThermoScientific Company);
[0166] Mobile phase: A=H 2 O (including 1‰HCOOH); B=CH 3 CN (including 1‰HCOOH)
[0167] Flow rate: 1mL / min
[0168] The mobile phase gradient ratio is shown in Table 6.
[0169] Table 6
[0170] time (min) A％ B％ 0 10 90 5 10 90 24 90 10 26 90 10 27 10 90 31 10 90
[0171] The fermentation products were analyzed by HPLC respectively, and the results were as follows:
[0172] Figure 20 Indicates the HPLC analysis results of novel LL-D49194α1 analogs obtained by optimizing the fermentation conditions of the mutant strain, wherein, Figure 20 i is the fermentati...
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