Novel luminescent reporters from marine environmental bacteria for prokaryotic and eukaryotic systems

Bioluminescent reporters are commonly used as reporters in molecular biology. Luciferases from various sources have been cloned and exploited as reporters due to their fast time dynamics and easily detectable and quantifiable outputs. While all eukaryotic luciferases require the addition of an exogenous substrate to produce light, bacterial bioluminescence is generated by a single operon and has the benefit of autonomous luminescence. Yet, compared to fluorescent protein reporters, little engineering has been applied to improve and optimize the lux operon on a protein level. In this work, we have sequenced the genomes of 19 luminescent marine bacteria from the genus Vibrio, Aliivibrio and Photobacterium isolated from shore seawater around the world. After identifying the lux operons, we cloned both the full operons and LuxAB genes separately into an E. coli for characterization in a common background, where they exhibited a wide range of luminescence. Using this pool of homologous luminescent lux operon sequences from diverse sources, we used a DNA-shuffling directed evolution technique to evolve luxAB proteins specific to an exogenous decanal substrate 1.5 times brighter than the best wild-type construct. The whole operon was also restructured into a reporter component and substrate pathway component, which were co-evolved to generate an autonomous luminescent construct 25 times brighter than the best wild-type construct. The evolved luciferase proteins were further fused into a single protein and also fused with a yellow fluorescent protein to create a spectrally distinct luminescent reporter. Furthermore, the evolved reporter proteins and substrate pathway were refactored for use in the plant Marchantia polymorpha, where they exhibit autonomous luminescence.