丙三醇对螺旋霉素Ⅰ发酵的影响及机理分析

Effect and Mechanism of Glycerol Addition on Spiramycin I Fermentation

以提高螺旋霉素Ⅰ产量及降低杂质D(Spiramycin V)组分质量分数为目标,研究添加丙三醇对螺旋霉素I产生菌生二素链霉菌发酵的影响,并对其影响机理进行分析。摇瓶添加最终体积分数为0.4%的丙三醇使螺旋霉素I效价提高17.12%,杂质D质量分数降低21.39%。15 L发酵罐的放大实验表明,添加最终体积分数为0.4%的丙三醇,螺旋霉素I最终效价达到23314 U/mL,相比对照组(18329 U/mL)提高27.20%,最终杂质D质量分数为8.84%,与对照组(11.99%)相比降低了26.27%。相关基因转录水平研究发现,聚酮合酶基因srm10(内酯环合成相关基因),氨基转移酶基因srm20(福洛氨糖合成相关基因)与甲基转移酶基因srm33(碳霉糖合成相关基因)的转录水平提高。结果表明,在发酵15 h添加最终体积分数为0.4%的丙三醇,能提高内酯环合成相关基因及糖基合成相关基因的转录水平,从而提高螺旋霉素I效价,降低杂质D组分的质量分数。

Spiramycin is a 16-membered macrolide antibiotic which is produced by Streptomyces ambofaciens.It is widely used in human medicine because of its activity against bacteria.Several impurities were easily accumulated in spiramycin-fermentation broth,especially impurity D,in which the forosamine sugar is replaced by mycarose.To improve the yield of spiramycin I and reduce the content of impurity D,the effect of glycerol addition on the fermentation of Streptomyces ambofaciens was studied and the possible mechanism was analyzed.In this study,HPLC was used to determine the fermentation titer of spiramycin I and the content of impurity.The adding of glycerol with a final concentration of 0.4%at 24 h can increase the spiramycin I titer by 17.12%,and decrease the content of impurity D by 21.39%in shake flask fermentation.The results of fermentation obtanined in a 15 L bioreactor showed that the addition of glycerol at 15 h could promote spiramycin I production with a final titer of 23314 U/mL,27.20%higher than control(18329 U/mL),and the final impurity D content was 8.84%,26.27%lower than control(11.99%).The level of genes transcription related to synthesis of spiramycin I was investigated by real-time PCR.The transcription analysis showed that the polyketide synthase gene srm10(for lactone ring synthesis),aminotransferase gene srm20(for forosamine synthesis)and methyltransferase gene srm33(for mycarose synthesis)were increased for almost 3−5 folds in the early stage of fermentation.This study indicates that the addition of glycerol in the early stage of fermentation can promote the transcription of genes involved in lactone ring and glycosyl synthesis,leading to an increase in the titer of spiramycin I and a decrease in content of impurity D.