Trehalose phosphate synthases O ts A 1 and O ts A 2 of R hodococcus opacus 1 CP

R hodococcus opacus 1 CP produces trehalose dinocardiomycolates during growth on long‐chained n ‐alkanes. Trehalose and trehalose‐6‐phosphate, which are synthesized via the O ts AB pathway, are probable intermediates in the biosynthesis of these biosurfactants. By molecular genetic screening for trehalose‐6‐phosphate synthases ( TPS s and O ts A s), two chromosomal fragments of strain 1 CP were obtained. Each contained an ORF whose amino acid sequence showed high similarity to TPS s. To prove the activity of the otsA1 and otsA2 gene product and to detect catalytic differences, both were expressed as H is‐tagged fusion proteins. Enzyme kinetics of the enriched proteins using several potential glucosyl acceptors showed an exclusive preference for glucose‐6‐phosphate. In contrast, both enzymes were shown to differ significantly from each other in their activity with different glucosyl nucleotides as glucosyl donors. O ts A 1‐ H is 10 showed highest activity with ADP ‐glucose and UDP ‐glucose, whereas O ts A 2‐ H is 10 preferred UDP ‐glucose. In addition, the wild‐type O ts A activity of R . opacus 1 CP was investigated and compared with recombinant enzymes. Results indicate that O st A 2 mainly contributes to the trehalose pool of strain 1 CP . O ts A 1 seems to be involved in the overproduction of trehalose lipids. For the first time, a physiological role of two different O ts A s obtained of a single R hodococcus strain was presumed.