Study On Fermentation Conditions And Genetic Operating System Of Streptomyces Spiramyceticus

Streptomyces spiramyceticus is a domestic production strain of spiramycin(SPM).The main components of spiramycin are SPM Ⅱ and SPM Ⅲ,and SPM Ⅰ content in spiramycin is desired to be less than 12%.Therefore,the fermentation conditions of S.spiramyceticus were optimized and an efficient genetic operating system was established for S.spiramyceticus in order to increase the yield of spiramycin and reduce SPMI content.After iterative screen from wild S.spiramyceticus,the strain can be produced stably with the titer about 3000 U/mL.To explore the fermentation conditions of S.spiramyceticus,we first tested the effects of supplementing the precursors required for spiramycin production at around 48 h of fermentation,including valine,threonine,isoleucine,and leucine.It was found that the addition of isoleucine could reduce the production of SPM I and increase the production of SPM III significantly.Further experiments suggested the optimal isoleucine addition timing and addition amount were 36 h of cultivation and 1.25 g/L isoleucine.Secondly,the pH value was adjusted in the fermentation process,which affected the total yield of spiramycin falling.But no SPM I component was detected when using NH3·H2O to adjust the pH.For the establishment of the genetic operating system of S.spiramyceticus,the bacterial solution of S.spiramycreticus was directed mixed with the solution.S.spiramyceticus conjugation was established where the transfer efficiency was 1.93×10-4 when selecting ISP4 medium as the optimal medium,using pCRISPRcas9 plasmid,the ratio of donor to recipient was 103:1,and 22 h was the resistance coverage time.Using this method,the sspA gene was successfully knocked out in S.spiramyceticus,and the fermentation verification indicated that the precursors of spiramycin decreased by knocking out sspA.