Small secreted luciferase from copepod Metridia longa (MLuc) catalyzes simple reaction of oxidation of coelenterazine with molecular oxygen without any additional cofactors resulting in the blue light emission (lmax~485 nm). MLuc is represented by multiple isoforms that were distributed into four paralogous groups according to sequence features. Despite differences in length (16.5–22.0 kDa), sequence and some properties, all studied isoforms showed a high bioluminescent activity and great potential for use as reporter proteins. To date, the first cloned isoform of M. longa luciferase, MLuc164, has been successfully applied as a sensitive bioluminescent reporter in vivo. However, some of its properties such as low temperature optimum of bioluminescent reaction (12-20°С), rapid decrease of the emission intensity after substrate addition and blue emission spectrum need improvement so to expand the range of use, including bioimaging that requires red-shifted luciferases.

In this study, we tried to improve the reporter properties of MLuc164. In the absence of structural information, we applied random mutagenesis (error-prone PCR) to generate MLuc164 mutant variants and further select those with desired properties. The mutagenesis library was created in E. coli using the expression construct for luciferase as a fusion with YebF protein permitting extracellular secretion. A high-throughput visual screening assays were performed on bacterial colonies with a cooled CCD camera. This approach was successful in producing MLuc164 mutants with elevated activity, prolonged luminescence, and changed temperature optimum of bioluminescent reaction (20-28°С) that are more suitable for reporter application. The advantageous properties of mutant luciferases were demonstrated in noninvasive monitoring of metabolic activity of human melanoma cells.

These studies were funded by RFBR, Government of Krasnoyarsk Territory, Krasnoyarsk Region Science and Technology Support Fund to the research project 16-44-242099 and the state budget allocated to the fundamental research at the Russian Academy of Sciences (project 0356-2016-0712).