Transcriptomics Analysis And Optimization Of Spiramycin Fermentation Process

Spiramycin is a 16-membered macrolide antibiotic produced by Streptomyces ambofaciens,and it is widely used in the clinical treatment.The production of spiramycin with higher yield and less impurity components has important industrial value.In this study,RNA-seq was used to analyze the fermentation process regulation mechanism of spiramycin which was produced by S.ambofaciens in a 15 L bioreactor,and the goal of increasing spiramycin yield and reducing impurity components was achieved based on the transcriptomics data analysis.We selected six time series samples which from bacterial growth period and antibiotic production period during spiramycin fermentation process for high-throughput sequencing of mRNA(RNA-seq)experiments.Results showed that a total of 926 genes were significantly differentially transcribed during the fermentation process,and they were divided into 9 clusters according to the transcription patterns.These genes were mainly related to nutrients transport and metabolism,response to oxidative and osmotic stress,lysis and auto lysis of bacteria,efflux of spiramycin,biosynthesis of acyl-CoAs precursor,and biosynthesis of spiramycin.From the results of genes transcription and spiramycin synthesis,it is inferred that a sequential variation was present between gene transcription and spiramycin synthesis.The results also showed that the transcription of acyl coenzymes(acyl-CoA)biosynthesis genes was decreased during the late fermentation process,and the insufficient supply of acyl-CoAs may be the main reason for the decrease of spiramycin synthesis.Acyl-CoAs synthesis precursors were added to the spiramycin fermentation medium.The adding time was chosen at the beginning of fermentation and 48 h of fermentation.The result showed that addition of acyl-CoAs synthesis precursors increased the production of spiramycin.Among them,adding malonate at the beginning of fermentation improved spiramycin production to 26483 U/mL,which was 31.5%higher than the control(20137 U/mL);adding crotonic acid at the beginning of the fermentation makes the spiramycin titer reach to 26097 U/mL,which is an increase of 21.4%compared with the control(21497 U/mL).The addition of succinate at 48 h of fermentation resulted in a titer of 25728 U/mL,which was 28.8%higher than the control(19975 U/mL);the addition of pyruvate at 48 h of fermentation resulted in a spiramycin titer of 27340 U/mL,which was 31.4%higher than the control(20802 U/mL).Using UPLC-MS to determine the concentration of acyl-CoAs and RT-PCR to determine the transcription level of acyl-CoAs synthesis genes,it was found that the concentration of acyl-CoA in cells was increased significantly after the addition of precursors of acyl-CoA synthesis,and the gene transcription level was also increased.The srm21 gene was overexpressed in the spiramycin industrial strain to study the function of srw21 in the N-demethylation of forosamine which may be related to the synthesis of impurity F.The fermentation results showed that the titer of spiramycin I produced by the srm21 gene overexpression strain S.ambofaciens-21 was 17.6%higher than the S.ambofaciens-152 strain which contains the plasmid pSET152,and the impurity component F was reduced by 38.9%.These results indicated that the srm21 overexpression can reduce the production of impurity component F and increase the production of spiramycin I.In addition,our study showed that the introduction of exogenous plasmid has a great impact on the production of spiramycin in industrial strians.The reasons why the introduction of exogenous plasmids has greatly reduced the production of spiramycin in industrial strains has been explored.It was found that the S.ambofaciens-152 strain had different fermentation parameters such as PMV,pH,and reducing sugar compared with the original strain S.ambofaciens.It is speculated that the glucose metablism of the S.ambofaciens-152 strain may be altered which produced more neospiramycin(impurity A),and reduced the production of spiramycin.The transcription level of genes which related to mycarose biosynthesis and regulatory genes was decreased compared with S.ambofaciens strain.By adjusting the composition of spiramycin fermentation medium,it was found that adding glucose at 48 h,reducing K+concentration,and increasing Mg2+concentration can increase the spiramycin production.