Optimization And Mechanism Analysis Of Spiramycin ? Fermentation Process By Streptomyces Ambofaciens

Spiramycin,a 16-membered macrolide antibiotic,is synthesized by Streptomyces ambofaciens and used in the field of medicine.In industrial production,higher content of impurity D will seriously reduce the extraction yield of spiramycin and affect the quality of the product.This project optimized the fermentation process of spiramycin to increase the yield of spiramycin I and reduce the content of impurity D,and analyzed the mechanism of fermentation process.Firstly,we studied the effect of adding glycerol on the fermentation of spiramycin I in the early stage of fermentation.It was found that adding glycerol with a final concentration of 0.4%at 24 h of shake flask fermentation increased the titer of spiramycin I by 17.12%,reduced the content of imputity D by 21.39%.The fermentation process in a 15 L bioreactor showed that adding glycerol with a final concentration of 0.4%at 15 h of fermentation resulted in a final titer of 23314 U/mL,which was 27.20%higher than the control(18329 U/mL),the content of imputity D was 8.84%,which was 26.27%lower than that of the control(11.99%).The genes involved in spiramycin biosynthesis were selected for transcriptional level studies by real-time fluorescence quantitative PCR.The results showed that the addition of glycerol could effectively increase the transcription level of the polyketide synthase gene srm10(a lactone ring synthesis-related gene)at 48 h,and the transcription of aminotransferase gene srm20(a gene for the synthesis of forosamine)and the methyltransferase gene srm33(a gene related to mycarose synthesis)were significantly promoted at 72 h.Thereby,the spiromycin I titer was increased,and the content of the impurity D was reduced to some extent.Secondly,in order to furtherly reduce the biosynthesis of impurity D and improve the product quality of spiramycin,we studied the effects of different oxygen supply levels on the fermentation of spiramycin I.It was found that increasing the oxygen supply level in the late fermentation stage can greatly reduce the content of the impurity D.The relative transcript level of related genes showed that the increase of oxygen supply level in the late stage of fermentation could effectively promote the relative transcription level of srm21(a gene for the synthesis of forosamine)and srm39(a gene for the synthesis of mycarose),and the promotion of srm21 was greater,and the synthesis ratio of the forosamine was relatively increased,resulting in a decrease in the content of the impurity D.Then,we combined the conditions for the addition of glycerol and increasing the level of oxygen supply,compared with the control,the titer of spiramycin I was increased by 40.24%,and the content of impurity D was decreased by 39.12%.Finally,the effect of raw materials on the fermentation of spiramycin I was investigated.At first,the content of related indicators in soybean cake powder and corn steep liquor was detected.Combined with the fermentation data,the key indicators affecting the fermentation titer were selected through cluster analysis,and the content range is determined to provide guidance and basis for the selection of raw materials in actual production.The results showed that the content of alkali(water)soluble protein and ammonia nitrogen in soybean cake powder had a great influence on the fermentation titer,and the suitable level of ammonia nitrogen(1900?2200 mg/100ml)in corn steep liquor was beneficial to the increase of fermentation titer.