Amino Acid Metabolic Profiles Analysis Coupled With RT-PCR Of Mutant Strain Streptomyces Hygroscopicus U3-SD For Overproducing Rapamycin

Rapamycin is an important natural macrolide antibiotic with all kinds of bioactivities, such as immunosuppressive, antifungal, anticancer, neuroprotective and anti-aging. In the clinical, rapamycin was applied to treat anti-rejection and autoimmune diseases. Compared to cyclosporine, rapamycin exhibits stronger clinical medicinal value and lower toxicity. Therefore, the research of rapamycin has attracted considerable attention from academia and pharmaceutical companies.According to the previous report[1], the addition of shikimic acid can improve the yield of rapamycin.Thus, a novel round of UV mutagenesis is performed in this study, and a mutant strain U3-SD was obtained and the rapamycin production increased to 389.7 mg/L by 20.3% compared to parent strain U2-3D9(324 mg / L).While a rapamycin overproducing strain was acquired by UV mutagenesis, but the mechanism of rapamycin overproduction is still unknown. Moreover, the comparative analysis in extracellular fermentation characteristics between the mutant strain U3-SD of Streptomyces hygroscopicus wan the wild-type strain ATCC 29253 could?t also provide a depth insights into the mechanism of rapamycin overproduction. To this end, to deeply understand the mechanism for strengthening rapamycin yield, amino acids metabolic profiling between the wild-type and mutant strain was performed. Firstly, a total of 86 metabolites were identified by gas chromatography–mass spectrometry. Pattern recognition methods, such as Principal Component Analysis and Partial Least Squares 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. Finally, on the basis of 22 bimarkers, metabolic pathway analysis revealed that amino acid metabolism played an important role in the synthesis of rapamycin, including lysine, valine, tryptophan, isoleucine, glutamate, arginine and ornithine.Subsequently, under the guidance of the amino acid metabolic profiling analysis, intracellular absolute amount of seven amino acids was detected by HPLC. And the result demonstrated that supply of amino acids was shortage in the mutant strain, namely “nitrogen starvation”, and the difference in the concentration of lysine between the mutant strain and wild-type strain is the most significant. In order to verify lysine metabolism is the most critical metabolic module for the synthesis of rapamycin. Thus real-time PCR and enzyme assays were conducted in this study. The result demonatrated that the key enzymes(dihydrodipicolinate synthase, pyruvate carboxylase and Lysine cyclase deaminase) were all improved in gene transcription and enzyme activity levels. Therefore, these three enzymes were closely related with rapamycin synthesis. Ultimately, the efficient fermentation of rapamycin was carried out by adding lysine with different concentration gradients. As a result, when 5g/L lysine was added in the fermentation medium at 24 h, rapamycin production of mutant strain increased to 435.7 mg/L form 389.7 mg/L.