Wang Baohua, Liu Jiao, Liu Huanhuan, Huang Di, Wen Jianping
Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, People's Republic of China.
J Ind Microbiol Biotechnol. 2015 Jun;42(6):949-63. doi: 10.1007/s10295-015-1611-z. Epub 2015 Apr 4.
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.
雷帕霉素是一种由吸水链霉菌产生的具有抗真菌、免疫抑制和抗癌活性的重要天然大环内酯类抗生素。在本研究中，通过紫外线诱变获得的突变株与野生型吸水链霉菌ATCC 29253相比，表现出更高的雷帕霉素生产能力。为深入了解雷帕霉素过量生产的机制，对野生型和突变株进行了比较代谢谱分析。通过气相色谱 - 质谱法共鉴定出86种代谢物。采用主成分分析、偏最小二乘法和偏最小二乘判别分析等模式识别方法来确定关键生物标志物。结果表明，22种潜在生物标志物与雷帕霉素产量的增加密切相关，并且在两株菌之间观察到了巨大的代谢差异。此外，代谢途径分析表明，氨基酸代谢在雷帕霉素的合成中起重要作用，尤其是赖氨酸、缬氨酸、色氨酸、异亮氨酸、谷氨酸、精氨酸和鸟氨酸。突变株中发生了氨基酸供应不足，即“氮饥饿”。随后，向突变株的发酵培养基中外源添加氨基酸证实了上述结论，添加赖氨酸后突变株的雷帕霉素产量增加到426.7 mg/L，约为野生型菌株的5.8倍。最后，实时定量PCR和酶活性测定结果表明，参与赖氨酸代谢的二氢吡啶二羧酸合酶在雷帕霉素的生物合成中起关键作用。这些发现将为进一步提高雷帕霉素产量提供理论依据。
