Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42°C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.

Insufficient thermal stability of firefly luciferases often limits their application in a wide range of fields. The substitution A217L is known to greatly increase thermal stability of many firefly luciferases. However, for Hotaria parvulafirefly luciferase, that shares 98% degree of homology with Luciola mingrelicaluciferase, the A217L mutation is known to dramatically decrease catalytic activity. We analyzed the environment of A217 in the 3D-structure of L. mingrelicaluciferase with the purpose of identifying possible additional mutations that would allow retention of the high thermal stability of the A217L mutant while preserving the high level of activity. The G216N/A217L double mutant of L. mingrelicaluciferase demonstrated significantly improved stability at 42 and 45 °C but retained only 10% of activity; the loss in activity was accompanied by a large red shift of bioluminescence emission maximum from 566 to 611 nm compared with the wild-type enzyme. The triple mutant G216N/A217L/S398M exhibited high thermal stability of the double mutant together with the high activity and bioluminescence spectra close to that of the wild-type luciferase.

Approaches to engineer stability of beetle luciferases

Bioluminescence spectra of firefly luciferases demonstrate highly pH-sensitive spectra changing the color from green to red light when pH is lowered from alkaline to acidic. This reflects a change of ratio of the green and red emitters in the bimodal spectra of bioluminescence. We show that the mutations strongly stabilizing green (Y35N) or red (H433Y) emission compensate each other leading to the WT color of firefly luciferase. We further used this compensating ability of Y35N to search for strong red-shifting mutations in the C-domain of firefly luciferase by random mutagenesis. The discovered mutation E457K substantially increased the contribution of the red emitter and caused a 12 nm red shift of the green emitter as well. E457 is highly conservative not only in beetle luciferases but also in a whole ANL superfamily of adenylating enzymes and forms a conservative structural hydrogen bond with V471. Our results suggest that the removal of this hydrogen bond only mildly affects luciferase properties and that most of the effect of E457K is caused by the introduction of positive charge. E457 forms a salt bridge with R534 in most ANL enzymes including pH-insensitive luciferases which is absent in pH-sensitive firefly luciferases. The mutant A534R shows that this salt bridge is not important for pH-sensitivity but considerably improves in vivo thermostability. Although E457 is located far from the oxyluciferin-binding site, the properties of the mutant E457K suggest that it affects color by influencing the AMP binding.