Optimization Of Lincomycin Fermentation Process And Metabolic Analysis By Streptomyces Lincolnensis

Lincomycin is a kind of lincosamides,which is widely used in the field of medicine.This paper is focused on increasing of lincomycin A production and decreasing lincomycin B content by optimizing the fermentation process using Streptomyces lincolnensis as the producer microorgnism.Response surface method was used to optimize the content of fermentation medium components such as sulfur donors(sodium sulfate,methionine,and cysteine),precursor of HMP(calcium gluconate,sodium gluconate)and related alcohol&ions(inositol,cobalt chloride).The optimal fermentation conditions were determined as follows:cobalt chloride 7.97 mg/L,calcium gluconate 6.0 g/L,and inositol 0.42 g/L.In 15 L bioreactor fermentation,titer of lincomycin A was 7705 U/mL,35%higher than that of the control(5715 U/mL).Content of lincomycin B was decreased to 2.4%at 90 h,50%lower than that of the control(4.8%).After 100 h fermentaion,the content of impurity component B maintained at about 5.0%,lower than that of the control(7.0%).The decrease of lincomycin B content is related to the concentration of cobalt ions.In this paper,the adding manner of calcium gluconate was studied to promote the lincomycin A production.By changing the start concentration,adding time,and changing from standard single shot to continuous addition(v=0.0638 g/L/h,t=111-158 h),the titer of lincomycin A(9160 U/mL)was maximized,41.3%higher than that of the control(6480 U/mL).In a 1 L bioreactor,the mechanism of adding calcium gluconate to increase lincomycin A production was studied from enzymology,metabolite analysis,and redox balance.Through determination of enzyme activity of the center metabolic pathways,intermediate accumulation and redox potentials(NADPH,NADH),it was found that the activity of glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase was 7-fold and 4-fold higher respectively than that of the control.At the same time,the intracellular accumulation of a-ketoglutarate,succinate,fumarate and malate was reduced.The intracellular accumulation of ribose-5-phosphate and sedoheptose-7-phosphate to form the MTL moiety of lincomycin A were almost zero.Change of NADH and NADPH concentration revealed that the level of NADPH was 1.27 times higher than that of the control,suggesting the flux of HMP and TCA pathway was enhanced to provide more precursor to synthesize lincomycin A.The results also suggested that the HMP pathway may be a rate-limiting pathways for lincomycin A synthesis.