Screening And Fermentation Iptimization Of A High Demeclocycline-producting Strain

Demeclocycline is not only a widely used antibiotic,but also a synthetic precursor of minocycline,tegacycline and PTK0796,which are important antibiotics for the treatment of drug-resistant bacterial infections.A demeclocycline-producing strain,Streptomyces aureofaciens HCCB 10088,was selected as the starting strain for demeclocycline biosynthesis under different conditions.The starting strain of S.aureofaciens HCCB 10088 was mutated by EMS,LiCl&UV and ARTP,respectively.Then mutants were screened under certain concentration of demeclocycline and malonic acid.A mutant strain UM2-16 with good genetic stability and 23.5%higher titer was finally obtained.Then,influence of seeds on the mutant strain UM2-16 fermentation was studied.The results showed that slant preservation time could not exceed half a month,considering that long preservation time would affect spore germination;inoculation amount of 1 slant strain per 100 L seeding tank was enough and fermentation level within 4 stage had the same effect on fermentation titer;under normal inoculation amount and culture temperature,inoculum age of primary seeding tank was determined to be 50 hours since too short or too long inoculum age would cause metabolic disorder;the optimum subcultivation amount of fermentation tank was 8-10%,under which the lag phase of microorganism was shortened and the growth of strain was not excessively rapid or overrich.The fermentation medium of UM2-16 was further explored.The optimized medium composition obtained after testing different kinds and concentrations of nitrogen source,carbon source and mineral salts was:starch 70 g/L,soybean flour 25 g/L,lysine hydrochloride 8 g/L,sodium chloride 3 g/L,ammonium sulfate 2 g/L,corn steep liquor 3 g/L,calcium carbonate 10 g/L,soybean oil 9.3 g/L,and a-amylase 0.07%.The initial concentration of reducing sugar in the basal culture medium was generally controlled within 1%to prevent the abnormal growth of bacteria caused by tachytrophism.Using the optimized medium composition,the fermentation titer of UM2-16 reached to 6524?g/mL.In addition,the feeding process of UM2-16 fermentation was investigated.The fed-batch of starch hydrolysate was initiated when the concentration of total sugar in fermentation broth reduced to 4.5%,the total sugar concentration was controlled at the range of 2.5?3.5%by adjusting fed-batch speed,and the carbohydrate supplementation was stopped 1 day before the fermentation finished;the optimum pH range for product synthesis was 5.9-6.1,which was realized by supplementing ammonium hydroxide-and the defoamer(mixture of soybean oil and GPE at ratio of 3:1)was started to supplement at about 30 h according to the bubble conditions.Using the optimized medium composition and feeding technics,the fermentation titer of UM2-16 is about 9000 ?g/mL.Ultimately,the blade type of the agitator and stirring method used in the fermentation process of UM2-16 were studied.The results suggested that:the combination of CD-6 and lightnin A-315 as the bottom and upper-middle layers of agitator could lower shearing action and achieve stronger mixture.Using this blend strategy,the morphology of mycelium was mainly spherical and the viscosity of fermentation broth was moderate,which were conducive to the biosynthesis of demeclocycline.The stirring speed was controlled according to the dissolved oxygen value.At the beginning of fermentation,the motor frequency was set to 30 hz.When the dissolved oxygen was less than 40%,the motor frequency increased by 5 hz until the stirring is fully opened.Using the optimized production process,the fermentation titer of demeclocycline of the mutagenic strain UM2-16 reached to 9000 ?g/mL,which was 70%higher than that before the optimization.