The Breeding Of Rapamycin Producing Strain And Fermentation Process Optimization

Rapamycin(RAPA),also known as Sirolimus is a kind of a triene macrolide antibiotic with nitrogen,which produced by Str.hygroscopicus or Actinoplanes.It can not only resist fungi,but also has anti-proliferative and anti-tumor effect,it is the most promising new potent immunosuppressant of the current world.It can not only be used for anti-rejection of the organ transplantation,but also for the treatment of rheumatoid arthritis and other autoimmune diseases,and it shows some activity in extending the life cycle of mammals and preparing a potent influenza vaccine.These characteristics determine the rapamycin must have broad application prospects in the pharmaceutical industry.Some research was did on Actinoplanes,mainly including two aspects of mutation breeding of the strains and optimization of fermentation process.First,a mutagenesis of atmospheric and room temperature plasma was used to deal with the starting strain Actinoplanes BCLP-016,and some single colonies were picked out according to the strain colony morphology,after fermentation screening,a high-yielding strain of rapamycin ARTP-039 was screened out,its rapamycin production can reach 369.39 mg/L,compared with the yield of the original strain BCLP-016 256.86 mg/L,increased by 43.81%,the colony morphology of it was isbasin uplift.Then choosing the strain ARTP-039 as the starting strain,the traditional UV mutagenesis was conducted on it,and five kinds of resistant material of streptomycin,gentamicin,rifampicin,chloramphenicol and erythromycin were selected to conduct the resistance screening,based on the theory of the ribosome engineering.After fermentation rescreening,a high-yielding strain StB+Gen6+Rif9+Ch13+Er4-015 was screened out,it has five kinds of resistance.The strain fermentation test showed that after 7 d fermentation,the rapamycin output can reach 589.79 mg/L,compared with the original strain BCLP-016 yield increased by 129.61%,and its genetic stability is good.The strain St8+Gen6+Rif9+Chl3+Er4-015 was took as the research object,and its fermentation process was optimized.First of all,the fermentation medium was optimized through the single factor experiment and the method of response surface optimization,and the best medium formula is:glucose 32.71 g/L,corn starch 12.33 g/L,soybean meal 37.69 g/L,corn steep liquor 3.94 g/L,yeast extract powder 3 g/L,FeSO4 1.9 g/L,MgSO4 2.5 g/L,L-lysine hydrochloride 7 g/L,tyrosine 2 g/L,CaCO3 4 g/L,pH 7.0-72.After fermentation verification the rapamycin production can reach 999.52 mg/L,improved 69.47%compared with the yield 589.79 mg/L which obtained before the optimization.Then on the basis of the medium,the shaker fermentation process was optimized,and the optimum culture conditions obtained for fermentation were:liquid volume 70 mL,inoculum size 9%,adding amino acids material at 0 h of the fermentation.At last the fermentation conditions of 5 L fermentor were preliminarily explored,on the conditions of liquid filling factor 60%,ventilation ratio 1:1(vvm),tank pressure 0.03 MPa,the following fermentation conditions were preliminarily determined as following,the dissolved oxygen was set at 30%connecting with the rotational speed(200 r/min-500 r/min),during the middle stage of fermentation,the pH was stabilized at 6.5 when the natural pH dropped to 6.5.Under the optimized fermentation conditions,the yield of rapamycin can reach 992.56 mg/L,although slightly lower than the yield of shaker,it increased by 46.64%,compared with the yield of 676.89 mg/L that uncontrolled pH.