| Bitespiramycin(BT),which is produced by Streptomyces spiramyceticus F21 WSJ-195,is a hybrid 16-membered macrolide antibiotic developed in a new drug discovery program in China for antibacterial infections.It showed excellent therapeutic effects in both phase ? and? clinical trials.However,there are still existing problems for its application and industrialization due to its low titer.This article studied the effects of exogenous precusors and streptomyces signaling molecule analogue addition trying to solve the problems of low fermentation titer and contents of isovalerylspiramycin III and total isovalerylspiramycin.The mechanism of exogenous A-Factor analogue 1,4-Butyrolactone stimulating BT biosynthesis was preliminarily investigated by the analyses of key enzymes activities,intracellular metabolite profiling and metabolic flux distribution.Firstly,an improved BT chemical titer determination method,which was suitable for high throughput determination with more efficiency,was developed on the basis of the existing method.Compared with conventional chemical titer determination,high throughput determination method was higher in linearity and parallelism with a relative coefficient of 0.999 on standard curve.Additionally,according to BT biological antimicrobial properties,a biological titer detection system based on Bacillus subtilis also with a linearly relative coefficient of 0.999 was established.The comparison of the results from two chemical titer determination methods showed the similar overall trends,even though the results of high throughput determination method was lower.Due to the complexity of BT,biological titer in follow-up experiment with a supplementary of chemical titer was adopted to truly reflect the titer and quality of BT.Secondly,the effects of exogenous 3C-substance(n-propanol,glycerol,sodium lactate and sodium propionate)addition on BT biosynthesis was investigated.The results showed when 3 g/L n-propanol was added,BT biological titer improved 8%,but the addition of n-propanol had a negative effect on contents of isovalerylspiramycin ? and total isovalerylspiramycin.In contrast,9 g/L glycerol addiction could enhance 8%biological titer,3%isovalerylspiramycin ? and 4%total isovalerylspiramycin at the same time.As for sodium lactate,the results revealed the addition of sodium lactate did not have significant effects on BT biological titer and contents of isovalerylspiramycin ? and total isovalerylspiramycin.Notably,the best fermentation performance was achieved with the supplement of 0.4 g/L sodium propionate at 18 h.Though the BT biological titer and total isovalerylspiramycin content were similar to the control,the isovalerylspiramycin ? content was significantly enhanced by 30%.Thirdly,it was found that the addition of 1 mM 1,4-butyrolactone(1,4-BL)at 12 h,as an analogue of A factor,could improve the BT biological titer over 20%with a tiny difference in the contents of isovalerylspiramycin ? and total isovalerylspiramycin.Due to the above results,a combined strategy with the addition of 1 mM 1,4-BL at 12 h and 0.4 g/L sodium propionate at 18 h was carried out.Unexpectedly,although there was an improvement on the content of isovalerylspiramycin ?,BT biological had a dramatic decline,revealing there was no synergetic roles between 1,4-BL and sodium propionate.Finally,the mechanism of 1,4-BL stimulating BT biosynthesis was explored by the analyses of the enzyme activities of EMP key enzyme pyruvate kinase(PK),TCA key enzyme citrate synthase(CS)and replenishment pathway key enzyme pyruvate carboxylase(PC)as well as intracellular metabolite profile and metabolic flux distribution.During the early phase of the fermentation,the activity of PK with 1,4-BL addition was much higher than the control,which could also be proved by the analysis of intracellular metabolites to some extent,as the intracellular concentrations of intermediate metabolites in PP pathway as well as in the upstream of glycolysis were remarkably higher than the control,but the intracellular concentrations of intermediate metabolites in the downstream of glycolysis pathway were lower.All the results mentioned above indicated that the higher activity of PK led to enhancing glycolytic flux.In accordance with the low concentrations of TCA intermediate metabolites,the activity of CS was obviously lower than the control.The continuous low level of PC activity further clarified TCA flux drop off.Pyruvic acid was mainly metabolized into acetyl-CoA for BT biosynthesis.On the other side,during the period of BT rapid biosynthesis,intracellular metabolic flux distribution showed glycolysis and PP pathway were enhanced,while TCA flux was weakened.Simultaneously,the synthetic fluxes of methylmalonyl-CoA and malonyl-CoA,which were immediate precursors in BT macrolide biosynthesis,were 2.28 and 1.73 folds of control,respectively.In summary,one of the mechanism of 1,4-BL stimulating BT biosynthesis was the regulation of TCA flux providing more acetyl-CoA for BT biosynthesis,another was the enhancements on glycolysis and PP pathway flux,resulting in more coenzyme materials and NADPH synthesis,which finally raised BT production.This study would provide theoretical and technical bases for the application of precursor and 1,4-BL addition strategy to industrial bitespiramycin production. |
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