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 β-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.
Department of Chemical and Biological Engineering and ERI, GyeongSang National University, 900 Gajwadong, Jinju, Gyeongnam 660-701, Republic of Korea.
He
H
Li
Hong
H
Su
Sung Bae
SB
Kim
Yong Keun
YK
Chang
Soon-Kwang
SK
Hong
Yang-Gon
YG
Seo
Chang-Joon
CJ
Kim
D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D007294:Inositol / Q000031:analogs & derivatives / Q000494:pharmacology; D009876:Operon; D009994:Osmolar Concentration; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D014198:Trehalase / Q000037:antagonists & inhibitors*; D014199:Trehalose / Q000096:biosynthesis* / Q000378:metabolism
D004791:Enzyme Inhibitors; C030790:validamycin A; D007294:Inositol; D014199:Trehalose; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases; D014198:Trehalase