Improved method for the preparative synthesis of labeled trehalose of high specific activity by Escherichia coli.

Abstract

We report an improvement of a published procedure using Escherichia coli to synthesize 14C-labeled trehalose from [14C]glucose (B. Brand and W. Boos, Appl. Environ. Microbiol. 55:2414-2415, 1989). Instead of inducing the expression of the trehalose-synthesizing enzymes encoded by the chromosomal genes otsAB by high osmolarity, we now induce their expression from a plasmid under normal growth conditions by the addition of IPTG (isopropyl-beta-D-thiogalactopyranoside). Instead of using a pgi zwf double mutant to prevent glucose utilization, we use a pgi::Tn10 insertion only. In addition to being defective in treA, which encodes a periplasmic trehalase, the strain is now also defective in treF, which encodes a newly discovered cytoplasmic trehalase. This strain is genetically stable; it has no growth defects; and after induction with IPTG, it will transform [14C]glucose to [14C]trehalose in minimal medium without any carbon source under aerobic conditions at a rate of 3 nmol/min/10(9) cells. With the improved method, the overall yield of trehalose from glucose is about 80% and the process takes place without dilution of the specific radioactivity of the glucose residues. The accumulated trehalose is extracted from the bacteria by 70% hot ethanol and can easily be purified radiochemically by chromatographic techniques.