Comparative metabolic profiling reveals the key role of amino acids metabolism in the rapamycin overproduction by Streptomyces hygroscopicus

Abstract

Rapamycin is an important natural macrolide antibiotic with antifungal, immunosuppressive and anticancer activity produced by Streptomyces hygroscopicus. In this study, a mutant strain obtained by ultraviolet mutagenesis displayed higher rapamycin production capacity compared to the wild-type S. hygroscopicus ATCC 29253. To gain insights into the mechanism of rapamycin overproduction, comparative metabolic profiling between the wild-type and mutant strain was performed. A total of 86 metabolites were identified by gas chromatography-mass spectrometry. Pattern recognition methods, including principal component analysis, partial least squares and partial least squares discriminant analysis, were employed to determine the key biomarkers. The results showed that 22 potential biomarkers were closely associated with the increase of rapamycin production and the tremendous metabolic difference was observed between the two strains. Furthermore, metabolic pathway analysis revealed that amino acids metabolism played an important role in the synthesis of rapamycin, especially lysine, valine, tryptophan, isoleucine, glutamate, arginine and ornithine. The inadequate supply of amino acids, or namely "nitrogen starvation" occurred in the mutant strain. Subsequently, the exogenous addition of amino acids into the fermentation medium of the mutant strain confirmed the above conclusion, and rapamycin production of the mutant strain increased to 426.7 mg/L after adding lysine, approximately 5.8-fold of that in the wild-type strain. Finally, the results of real-time PCR and enzyme activity assays demonstrated that dihydrodipicolinate synthase involved with lysine metabolism played vital role in the biosynthesis of rapamycin. These findings will provide a theoretical basis for further improving production of rapamycin.

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus

“…The culture samples for enzyme activity analysis in vitro were harvested at the end of exponential phase (72 h) by centrifugation at 8000× g for 10 min at 4 °C. Cell pellets were washed 3 times by 100 mM Tris·HCl (pH 7.0) containing 5 mM MnSO 4 , 20 mM KCl, 2 mM DTT and 0.1 mM EDTA, and then resuspended in the same buffer [ 44 ]. Cell suspension was treated by ultrasonication for 10 min at 250 W for 5 cycles of 1 min each on ice bath.…”

Increasing Ascomycin Yield in Streptomyces Hygroscopicus var. Ascomyceticus by Using Polyhydroxybutyrate as an Intracellular Carbon Supply Station

“…ascomyceticus, so enhancing the supply of precursors is an e cient way to increase the production of this antibiotic [45]. It is well-known that the exogenous feeding of important metabolites is an effective method to increase the biosynthesis of precursors [46][47][48]. In our previous study, metabolic pro ling analysis showed that lysine (Lys), used for the synthesis of precursors pipecolic, and valine (Val), used for the synthesis of precursors methylmalonyl-CoA, were the key primary metabolites in the production of FK520 under the condition of adding resin HP20.…”

Generation of Streptomyces hygroscopicus cell factories with enhanced ascomycin production by combined elicitation and pathway‐engineering strategies

“…The final high ascomycin production strain ( S. hygroscopicus var. ascomyceticus H16) was identified using a previously reported high‐throughput screening method (B. Wang, Liu, Liu, Huang, & Wen, ). All engineered strains of Streptomyces are derivatives of S. hygroscopicus var.…”