ABSTRACT
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.
Subject(s)
Bacterial Proteins , Genetics , Metabolism , Biosynthetic Pathways , Chromatography, Reverse-Phase , Methods , Cyclohexenes , Hexosamines , Metabolic Engineering , Streptomyces , Genetics , MetabolismABSTRACT
Conjugal plasmid pGH112 has been developed based on the replicons of Streptomyces coelicolor plasmid SCP2 and E. coli ColE. The plasmid contains ampicilin resistance gene(amp) for selection in E. coli and thiostrepton resistance gene (tsr) for selection in Streptomycetes, and a 0.76 kb oriT fragment of (IncP) RK2. Conjugal transfer of pGH112 was performed from E. coli to S. coelicolor A3(2), S. avermitilis, S. lividans TK54, S. toxytricini NNRL15443, S. venezuelae ISP5230 and Sacc. erythraea by conjugation, results show that the plasmid was able to transfer efficenctly from E. coli to Streptomycetes, was stably inherited in the recipients. pGH113 was constructed from pGH112 by combining the constitutive ermE promoter with green fluorescent protein gene(gfp).