Studies On The Breeding Of Production Strains Of Josamycin

A stable and high-yielding strain was chosen through JM6,JM8,JM14,JM30 and JM310 strains of josamycin producer Streptomyces narbonensis var.Josamyceticus which preserved in our laboratory by stable experimentation.Firstly,JOS8 strain was mutated by LiCl-UV,and UV90 and UV50 strain which both had the higher fermentation ability were obtained.Secondly,UV90 strain was mutated by ⁶⁰Co-γirradiation,and no higher fermentation ability strain was obtained.Thirdly,a stable and high-yielding strain UV50-20 was obtained from UV50 strain by stable experimentation.Fourthly,the condition of the enzyme for protoplast forming and regenerating was studied using UV50-20 strain.The optimal conditions were 34-hour-age seeds hydrolyzing in the enzyme concentration of 4 mg/mL at 32℃for 90 min.Fifthly, we filtrated the protoplast regeneration strains of UV50-20,and obtained the higher fermentation ability strain of Y13 and the zero fermentation ability strain of Y19.Then, we treated Y13 strain by protoplast-UV,and obtained YU15 strain.Finally,we fused the protoplast of strains Y19 and YU15 which was UV-inactivated,and then obtained a strain R29.The results showed that those mutation methods could enhance the fermentation ability of trains.The chemical titer of the five high-yielding mutant strains UV50, UV50-20,Y13,YU15,and R19 were increased by 634.41%,3.51%,122.49%,15.89%, 17.72%respectively compared with their parent strains.Subculture test indicated that JM8,UV50,UV50-20,Y19,Y13 and R29 had relatively stable hereditary characteristics.SAS system was used to optimize the fermentation medium components for josamycin production by R19.Firstly,the prime factors affecting production josamycin were selected by means of Plackett-Burman design;the selected ones that affected the fermentation were cottonseed protein powder and soybean oil.Then,the steepest ascent experiment was adopted to approach the optimal region of the medium composition. Lastly,a central composite design was used to optimize the prime factors and the results were shown in the response surface polts.When the medium comprised cottonseed cake 1.9%and soybean oil 0.41%,the biological fermentation titer of josamycin could reach the highest at 550μg/mL,which was 10.66%higher than the initial medium components before optimization.Lastly,we checked pH,the filtration rate,the titer,and the concentration of glucose and NH₄⁺ in the fermentation fluid at different fermentation time.They could provide the reference for large-scale fermentation of josamycin.