Regulation Of Osmotic Pressure On Lincomycin Production By Streptomyces Lincolnensis And Its Mechanism

Lincomycin is one of the lincosamide antibiotics,which is widely used in clinic.In this paper,with the goal of decreasing the content of lincomycin B by Streptomyces lincolnensis in fermentation broth,osmotic pressure was optimized in flask and 15 L bioreactor fermentation,and the mechanism of osmotic pressure regulation on lincomycin fermentation process was studied.First of all,the flask experiment was conducted to determine the optimal osmotic pressure for lincomycin fermentation,the results showed in the range of 0.860-1.355 Osmol/kg.The central composite design was used to optimize the fermentation medium by adding osmotic pressure regulator(sodium chloride,potassium chloride and calcium chloride),osmotic protective agent(glycerin,mannitol,betaine,glycine and proline)or antioxidant(cysteine).The optimal condition was as follows:sodium chloride 10.93 g/L,glycerol 2.25 g/L,proline 0.057 g/L.The flask experiment results showed that the content of lincomycin B decreased to 1.42%,which was 60.33%lower than that of the control(3.58%);the titer of lincomycin A is 5795 U/mL,much higher than that of the control(4326 U/mL),33.96%increased.Secondly,based on the experiment of flask,the effect of osmotic pressure on the fermentation of lincomycin was studied in 15 L bioreactor.The content of lincomycin B in the broth was decreased to 5.78%when adding 10 g/L NaCl,while the content was 8.20%in the control(29.5%decreased).Then,a method to determine the intracellular sulfur metabolites such as cysteine,homocysteine,ergothioneine of S.lincolnensis was established.During the fermentation process,the cysteine concentration was lower and the homocysteine concentration was higher when adding NaCl.The proportion of homocysteine was higher than that of the control.It was suggested that more flux flows to the synthesis of homocysteine.The increased concentration of ergothioneine in the growth phase maybe support the synthesis of lincomycin during fermentation.The concentration of intracellular S-adenosylmethionine was higher than that of the control,which can provide more methyl donor for lincomycin production,then the content of lincomycin B was reduced.Finally,high-throughput mRNA-seq technology was used to explore the global transcriptome changes during lincomycin production process.After adding NaCl,60 and 103 genes were up-regulated while 124 and 325 genes were down-regulated at 41 h and 89 h,respectively.The results showed that transcription of 23 genes in lincomycin biosynthesis cluster were significantly up-regulated at 41 h,which was in agreement with the increase of lincomycin A production.However,genes involved in the synthesis of methylthio-lincosamide were down-regulated at 89 h,which may hinder lincomycin A production during the late fermentation process.Under the osmotic stimulation,the transcription of key enzymes in glycolysis and HMP was increased,while the genes in TCA cycle were decreased,which may be beneficial to provide more precursors for lincomycin biosynthesis.The genes involved in sulfur assimilation and mycothiol,ergothioneine,cysteine and S-adenosylmethionine production were up-regulated,which can provide more methyl donor(S-adenosylmethionine)and sulfur donor(mycothiol and ergothioneine)during lincomycin fermentation.This might be the reason of increased lincomycin A production and decreased lincomycin B content.