Influence Of Osmotic Stress And Taurine Addition On Lincomycin Production In Streptomyces Lincolnensis

Lincomycin,a kind of lincosamide antibiotics,is effective on gram positive bacterium and has been widely applied in clinical medicine.Its biosynthesis is mainly obtained by fermentation in Streptomyces lincolnensis.This thesis connected osmotic stress and sulfur source metabolism,studied the influence sulfur source on lincomycin production and identified taurine’s utilization and positive effect on lincomycin production.Previous research had revealed that adding sodium chloride can lower the concentration of by-product lincomycin B.Based on this idea,this thesis further studied the influence of osmotic stress on lincomycin A production.The result showed adding 1 70 mmol/L NaCl could raise lincomycin titer by 28%.Quantification on small-molecular weight thiols found that the relative amount of mycothiol was significantly higher in condition group,leading to the fact that mycothiol played a key role in osmoregulation.Gene transcription levels showed osmotic stress could induced the genes involved in the biosynthesis of thiols and PPL,a component of lincomycin biosynthesis.Re-analysis of GEO(Gene Expression Omnibus)database revealed osmotic stress had an even wider effect on primary metabolism such as carbon metabolism,amino acid metabolism and secondary metabolism such as antibiotic metabolism in Streptomyces coelicolor and Mycobacterium tuberculosis.In both strains,taurine biosynthesis and mycothiol biosynthesis have been enriched,indicating a potential relationship between them.Further study on transcriptome data in S.lincolnensis found taurine transport gene cluster tau and sulfonate transport gene cluster ssu is upregulated under osmotic stress,illustrating that osmotic stress has a positive effect on sulfur source transport and taurine could function as a potential sulfur source.Flask fermentation was conducted to study the influence of different sulfur source(taurine and ammonium sulfate),while no sulfur source was added as a negative control.The result showed taurine can be utilized as a sulfur source in the absence of sulfate.Lincomycin titer reached 2566 U/ml(60%of sulfate group)in contrast to 732 U/ml of negative control.Results showed both taurine and ammonium sulfate could induce the transcription level of tau and ssu and taurine had a more significant induction on tau(1.5 fold).Additional 6 mmol/L taurine was added to flask fermentation medium to study the combination,resulting a 14%increase in lincomycin titer,reaching to 3716 U/ml.To optimize taurine addition,fermentation in 15 L bioreactor was conducted.The result showed lincomycin titer of condition group was higher than control during 0-120 h and lincomycin synthesis rate was 36%higher in the early and middle phase of fermentation.However,in the late phase,synthesis rate decreased.LC-MS/MS was used to quantify extracellular taurine to study the difference of taurine consumption rate between flask and 15 L bioreactor,causing the accumulation of taurine during the late fermentation phase,which may be a possible reason for the decrease of production rate.