Using the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB.
Fig: Comparison of the expression constructs of luxA and luxB genes in E. coli. (a) Schematic representation of expression constructs of luxA and luxB genes in pRSET B vector. (b) Luminescence intensities in the whole cell suspensions of JM109(DE3) expressing the recombinant proteins from each construct. The luminescence reaction was initiated by the addition of 1% decanal. Data are means ± SD of three different clones.
Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.
Kaku, T., Sugiura, K., Entani, T. et al. Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer. Sci Rep 11,14994 (2021). https://doi.org/10.1038/s41598-021-94551-4