Engineering uroporphyrinogen III methyltransferase as a red fluorescent reporter in E. coli.

Uroporphyrinogen III methyltransferase (UMT) is a novel reporter owing to the catalytic products in the cells that emit strong red fluorescence under UV light. Here, we engineered the gene encoding the functional barley UMT (bUMT) by error-prone PCR and broadened the application UMT as a red fluorescent reporter in Escherichia coli. A variant, termed mbUMT, was selected and emitted stronger cell fluorescence than the wild type bUMT expressed in different E. coli strains, under different promoters and induction conditions respectively. The constructed mbUMT with a C-terminal ssrA tag was degraded in cells by the protease ClpXP encoded by E. coli chromosome, whereas the bUMT was expressed as active aggregates. Before they are exported to the periplasm, both proteins catalyze the substrate in the cytoplasm and emit cell fluorescence. The results suggested that the evolved bUMT is a better candidate to monitor in vivo degradation by E. coli ClpXP.