Breeding of High-Yield Nosiheptide Strain and Studying of Its Formation Condition
|School||Anhui University of Engineering|
|Keywords||nosiheptide Streptomyces actuosus mutagenesis breeding protoplast medium optimization pilot|
In order to obtain the high-yield nosiheptide strain applied in the industrial production. The high-yield nosiheptide strain was obtained from Streptomyces actuosus CB040517 by the traditional mutation, protoplast regeneration, physical mutation and rational mutation. The properties of fermentation of the strain for producing nosiheptide were investigated.A relatively stable and high-yield nosiheptide strain NXT-UV19 (754.16μg/mL) was obtained from the starting strain by UV Mutagenesis.The potency of the strain NXT-UV19 was increased 43.91% than the original strain (524.06μg/mL). Then the NXT-UV19 was treated by microwave, laser and NTG. The relationship between mutation dose and mortality was studied in order to determine the best mutation dose. The results showed that laser was a better way to select mutant which had a high positive mutation rate. A high-yield nosiheptide strain NXT-UV19-J2 was obtained when NXT-UV19 was treated by laser for 2 h. The yield of nosiheptide was 905.22μg/mL. Then NXT-UV19-J2 was treated by 1000μg/ml NTG for 2 h and then cultured in a medium supplemented with compound amino acid(L-Glu1500μg/ml、L-Thr300μg/ml、L-Ser500μg/ml、L-Cys300μg/ml、L-Trp1500μg/ml). Three high-yield nosiheptide strains were screened. They were NXT-UV19-J2-NA10、NXT-UV19-J2-NA36 and NXT-UV19-J2-NA38. The potency of the strain NXT-UV19-J2-NA10 was increased by 54.18% which reached 1110.56μg/ml, after cultivation for 118 h, compared with the starting strain(720.30μg/ml). The genetic steady of three strains was investigated. The results showed that the ability to produce nosiheptide was 89.8% of that of the first generation after five generation of NXT-UV19-J2-NA10. Its genetic feature was steady.The preparation and regeneration of protoplasts from nosiheptide strain- Streptomyces actuosus were explored. The effects of inoculation amount, lysozyme concentration and enzyme time on the preparation and regeneration of protoplasts were studied. The optimal conditions for preparation and regeneration of protoplasts were as follows:12ml inoculation amount,2.0mg/ml lysozyme concentration and 60min enzyme time. NXT-UV19 was used as an original strain, the effects of UV, microwave and UV-microwave compound to Streptomyces actuosus protoplast were investigated. The results showed that compound mutagenesis had obvious lethal effect on Streptomyces actuosus protoplast. A good strain was obtained by UV - microwave compound mutation named PUW-13. The production of nosiheptide reached 886.67μg/mL in shaking flasks, which was increased by 17.43% than that of the original strain.Response surface methodology was applied to optimize the fermentation medium for producing nosiheptide. The optimal medium for producing nosiheptide is composed of starch 7.0%、glucose 0.5%、yeast 0.31%、soybean powder 6.42%、NaCl 0.41%、(NH4)2SO4 0.1%、KNO3 0.05%、CaCO30.4%、inoculation amount 5.5%、liquid amount 40ml/500ml.The potency of the fermentation was increased by 10.95% than before, which reached 1123.714μg/ml.The strain NXT-UV19-J2-NA10 was verified by pilot. To increase the fermentation level, nutrients were added in the medium during the fermentation process and the fermentation time was prolonged. The results showed that altering the temperature of the fermentation, controlling the ventilation flow in different phases and cultivation for 257 hours, the yield of nosiheptide reached 2201μg/mL. It has provided the reference condition for the industrial production.