Enhanced production of trehalose in Escherichia coli by homologous expression of otsBA in the presence of the trehalase inhibitor, validamycin A, at high osmolarity.

Trehalose production in Escherichia coli DH5α was explored by overexpressing otsBA operon encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. Production and subsequent degradation of trehalose resulted in low production of trehalose in engineered cells overexpressing otsBA, which was primarily due to the concomitant expression of endogenous trehalase. Through an in vitro enzyme assay and flask cultures of engineered cells, trehalase expression was shown to be directly related to the expression of otsBA rather than osmotic stress. Validamycin A effectively inhibited E. coli trehalase and the intracellular accumulation of trehalose was markedly enhanced in the presence of validamycin A at an optimal concentration in the medium. The trehalose production was further increased upon growth in a hypertonic medium in the presence of validamycin A, with most trehalose accumulating as an intracellular product. The highest titer was obtained when otsBA expression was induced by a medium-copy vector, ptrc99A, with 0.5mM of isopropyl ß-d-1-thiogalactopyranoside. Trehalose titer was 1.7 g/L in controlled bioreactor cultures using synthetic M9 medium supplemented with 40 g/L glycerol, 0.1mM validamycin A, and 300 mM NaCl.

Quantitative analysis of valiolamine through pre-column derivatization with phenylisocyanate using high-performance liquid chromatography with UV detection: selection of reagent, identification of derivative and optimization of derivatization conditions.

This report describes the improved quantitative determination of valiolamine in a medium for microbial culture using high-performance liquid chromatography with UV detection. Valiolamine aqueous solution was dried, dissolved in dimethyl sulfoxide and derivatization performances of phenylisocyanate (PHI), 1-fluoro-2,4-dinitrobenznene and 1-naphthylisothiocyanate were compared in the presence of triethylamine. The PHI was chosen as the most suitable derivatization reagent and the valiolamine-PHI derivative was identified by thin-layer chromatography and electrospray ionization mass spectrometry. The derivative eluted at 10.5 min on a reverse-phase column using a mobile phase composed of 10% acetonitrile in water containing 0.5 mM sodium octyl sulfate (pH 3.0), at a column flow rate of 1.0 mL/min with UV detection at 240 nm. The optimum conditions for derivatization were a reaction temperature of 30 degrees C, reaction time of 30 min, and PHI concentration higher than 33.6 mM. Calibration curves were linear in the range of 0.99-19.95 microg/mL for the standard solutions and 24.9-99.7 microg/mL for the spiked sample. The proposed method was validated and proven to be selective, accurate and precise and suitable for the quantitative analysis of valiolamine in medium for microbial cultures.