State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology and Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240, China.
State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology and Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240, China.
Chin J Nat Med. 2017 Oct;15(10):794-800. doi: 10.1016/S1875-5364(17)30111-5.
Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhydrazine (DNPH) in 10 mmol·L HPO at 37 °C for 45 min and the derivatives were separated on Eclipse XDB-C (5 μm, 4.6 mm × 150 mm) column at 30 °C eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5-2 000 μg·mL. The intra- and inter-day precisions were 1.1%-2.7% and 1.7%-2.2%, respectively. The absolute recovery of the spiked samples was 97.2%-102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.
Valienone 是 C7-环糖醇家族中重要的天然碳环糖成员，是糖苷酶抑制剂药物的有价值前体。它是 validamycin A 生物合成途径的中间体，在天然链霉菌生产菌的发酵液中积累很少。定量分析方法对于开发克服天然代谢通量消耗的突破性微生物工艺至关重要。本研究旨在开发一种用于 valienone 定量的柱前衍生高效液相色谱法，并帮助建立一种通过代谢工程化吸水链霉菌 5008 生产 valienone 的简单发酵工艺。Valienone 在 37°C 下用 10 mmol·L HPO 中的 2,4-二硝基苯肼 (DNPH) 衍生化 45 min，衍生产物在 Eclipse XDB-C（5 μm，4.6 mm×150 mm）柱上在 30°C 下以 50%乙腈洗脱 18 min 进行分离。衍生物在 380nm 处用二极管阵列检测器检测，衍生物的构型通过计算研究确定。该方法有效、灵敏且可靠。在 5-2000μg·mL 范围内发现良好的线性关系。日内和日间精密度分别为 1.1%-2.7%和 1.7%-2.2%。加标样品的绝对回收率为 97.2%-102.6%。到目前为止，这是微生物培养基中 valienone 的第一个反相高效液相色谱检测方法。该方法成功地帮助评估了工程化吸水链霉菌 5008 的 valienone 生产能力，并且结合代谢组学方法，对于不同工程化突变体的 C7-环糖醇分析可能具有广阔的前景。
