Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

Tomomi Kaku,Kenji Osabe,Takeharu Nagai,Kazunori Sugiura,Tetsuyuki Entani

doi:10.1038/s41598-021-94551-4

Abstract

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. 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.

Highlights

Using the lux operon of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells
Comparison of the gene constructs for expression of luxA and luxB in Escherichia coli
Our results showed that the optimization of the bioluminescence resonance energy transfer (BRET) between the bacterial luciferase and yellow fluorescent protein Venus can enhance bioluminescence

Summary

Introduction

Using the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. 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). To further improve on the signal of the bacterial bioluminescence, we attempted to increase the luminescence of bacterial luciferase by fusion to a fluorescent protein. This approach is based on the phenomenon of bioluminescence resonance energy transfer (BRET) between the luciferase (as a donor) and fluorescent protein (as an acceptor)[12,13].

Methods

Results

Conclusion