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The discovery that proteins, which would come to be known as luciferases, were responsible for bioluminescent production can be traced to early experiments by Raphael Dubois, who was able to produce bioluminescence in situ by mixing the contents of click beetle abdomens in cold water and extracting the components required for light production [ 2 ].

However, it was not until the late s that the first luciferase protein was successfully purified from fireflies [ 3 ]. Around that same time, bacterial luciferase was elucidated and successfully expressed in situ [ 4 ]. However, despite the progress made with these luciferases, it would be some time until biotechnology had advanced to the point where the genes responsible for their expression could be cloned and exogenously expressed, setting off the use of luciferases as tools for scientific discovery [ 5 , 6 ].

Following the exogenous expression of the previously described firefly and bacterial luciferases, Renilla luciferase was isolated from the sea pansy Renilla reniformis [ 7 ] and Oplophorus luciferase was isolated from the deep-sea shrimp, Oplophorus gracilirostris [ 8 ].

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Shortly thereafter, firefly luciferase was successfully expressed in mammalian cells [ 9 ] and it was demonstrated that different luciferases could be used in tandem within a single host if they utilized different luciferin compounds [ 10 ]. More recently, Gaussia luciferase has been isolated from the marine copepod, Gaussia princeps [ 11 ], which was a notable discovery because, unlike alternative luciferases, it is naturally secreted and thus could be monitored without needing to sacrifice the host cell during luciferin treatment.

Since the discovery of Gaussia luciferase there has been rapid development of these enzymes through genetic engineering, but little progress on the introduction of new systems. However, this was recently changed with the introduction of fungal luciferase as a novel luciferase system, which like bacterial luciferase is capable of genetically encoding both the luciferase and luciferin pathway genes to support autobioluminescent production [ 12 ]. The primary reasons for this are the lack of elucidated functional units, similarities in performance characteristics such as wavelength output relative to existing systems, the entrenchment of existing luciferase systems within the literature and as commercially-available products, and the relatively high monetary and time costs required to explore novel systems in depth relative to their ultimate utility as research tools.

As a result of these barriers, the luciferases available as research tools are generally limited to those listed in Table 1. Common luciferases available for biotechnological applications, their luciferin compound, and their output wavelength. Despite the variety of different luciferases available, it is impossible to identify just one that could fit the needs of every experimental design. Furthermore, it is unfortunately frequent that no luciferase can be found to fit the needs of a given experiment.

As a result, there has been significant effort to engineer the existing luciferase enzymes to improve their functionality, make them easier to use, and expand their utility. This is especially true as the prevalence of luciferase usage has increased in biomedical applications, which rely upon human cellular and small animal model systems that have significantly different physical and biochemical properties relative to the native host organisms from which these proteins were sourced.

These changes in physical properties and the constraints applied by the needs of biomedical research have necessitated that luciferases be modified to express at longer output wavelengths that better penetrate animal tissues or that can be co-expressed with alternative luciferases, to produce light upon exposure to alterative luciferin compounds, to produce altered signal output kinetics that are shorter or longer than their wild-type kinetics, to allow multimeric enzymatic structures to function as monomers, to stabilize or destabilize protein structure within the host, to make expression more efficient, and to increase output intensity so that it is easier to detect the signal.

Imparting these changes makes it possible to utilize specialized versions of each luciferase that better fit the experimental needs of the researcher. As the breadth of luciferase usage continues to grow, and as new luciferase systems have been introduced over the years, the lessons learned from these modifications are refined and re-applied in order to continuously unlock new applications and improved functionality. To support the need for continued luciferase improvement, a number of techniques have become commonplace for different engineering goals. The most commonly utilized approaches and their common engineering endpoints are shown in Table 2.

Examples of the use of these techniques can be found in each of the following sections. Firefly luciferase FLuc is perhaps the most well-known, well-studied, and widely-used of all the luciferases. The resulting oxyluciferin is initially produced in an excited state, and as it returns to its ground state energy is released in the form of light.

Although FLuc and click beetle luciferase were among the first luciferases to be studied [ 16 ], it was not until the mids that significant progress was made in understanding the system at a level where it could be experimentally useful. At this time, McElroy successfully extracted firefly luciferase from purified firefly lanterns and determined that ATP was required for bioluminescence [ 17 ]. With these pieces in place, chemists were able to isolate oxyluciferin as a purified product of the luminescence reaction and validate its mechanism of action [ 18 ]. This provided an alternative to the use of crude extracts of beetles as a source of the luciferase enzyme and opened the door for widespread use in biotechnological applications.

In its initial incarnation, FLuc was highly useful as a reporter in molecular biology and bioimaging studies and for assaying the presence and quantification of the metabolites that participate in or are connected to the light reaction. Further entrenching this technology was its exceptional sensitivity. This sensitivity for measuring ATP concentrations has been used in several applications including screening for microbial contamination in food industries, assessing cell viability [ 20 ], and assaying enzymes involving ATP generation or degradation [ 21 ].

The major limitation encountered during the use of FLuc or beetle luciferases has been the requirement that the luciferin substrate be exogenously provided for luminescence to occur. To date, there are no bacterial systems for generating luciferin de novo , which necessitates chemical synthesis and results in potential storage concerns due to the labile nature of the chemical [ 18 ].

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Furthermore, this often requires that the host cell harboring the luciferase be lysed to enable substrate uptake, which has prevented its use for reporting real-time expression. Applications of wild-type beetle luciferases can be limited due to structural and functional stability issues or variations in the specific activity of the enzyme under varying temperatures, pHs, ion concentrations, or inhibitors [ 22 ].

This required that more thermostable forms be developed to assay human and small animal model-relevant temperature conditions [ 23 ]. Site-directed mutagenesis experiments were then performed based on mutant sequences that produced increased luminescence. It was observed that the substitution of D with a non-bulky amino acid, I with a hydrophobic amino acid, and L with a positively charged amino acid all increased luminescence intensities relative to the wild-type enzyme. They further demonstrated that combining the mutations at I, D, and L resulted in an overall increase in affinity and turnover rate for the ATP and D-luciferin substrates that resulted in high amplification of luminescence intensity.

Studies like this represent an emerging trend of combining alterations to specific properties of firefly luciferases in order to enhance its overall practical utility. Engineering wavelength-shifted luciferases has become an intense area of study to enable multi-color assays and improve the efficiency of in vivo bioimaging. To overcome this limitation, mutagenic engineering approaches have been successfully used to generate a variety of red-shifted versions [ 26 , 27 ].

However, this is by no means the only option available.

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Today, the wide variety of available output wavelengths enables researchers to choose the variant most well suited to their needs, or multiple variants that can be simultaneously triggered upon exposure to D-luciferin. High or saturating concentrations produce flash-type kinetics that result in an intense initial signal followed by a rapid decay, while low concentrations produce glow-type kinetics with a relatively lower initial signal and a slower decay [ 18 ]. There are many possible inhibitors that could be responsible for these changes.

Under high substrate conditions, byproducts of the reaction such as oxyluciferin and L-AMP can act as tight active-site binding inhibitors preventing enzyme turnover, or inhibitor-based stabilization can increase activity when substrate levels are high enough to compete with the inhibitory compound [ 14 ]. These commercial reagents are now widely used to support different experimental needs [ 14 ]. Another strategy that has been applied to alter reaction kinetics is the modification of the luciferin substrate.

These synthetic analogues were designed to emit longer wavelength light by incorporating an aminoluciferin scaffold.

A more recent substrate modification strategy has been to conjugate the luciferin with distinctive functional groups. Derived from the sea pansy Renilla reniformis , RLuc is a decarboxylating oxidoreductase that uses coelenterazine as its substrate. The RLuc protein was first purified and characterized in the late s [ 7 ].

However, its cDNA sequence was not identified and cloned into Escherichia coli until [ 31 ]. Following that accomplishment, the recombinant RLuc protein was quickly expressed in other organisms, including yeast [ 32 ], plant [ 33 ], and mammalian cells [ 34 ] to serve as a gene expression reporter.

The successful detection of RLuc bioluminescence from mammalian cells was particularly important because it represented the proof-of-principle demonstration of this enzyme as a reporter target for in vivo animal imaging. And indeed, imaging of RLuc activity in living mice was successfully validated just several years later [ 35 ]. In this demonstration, Bhaumik and Gambhir showed that intraperitoneally implanted RLuc-expressing cells could be detected following the injection of coelenterazine into the tail-vein [ 35 ].

Similarly, when cells were injected via the tail-vein, bioluminescent signal could be used to visualize cell trafficking to the liver and lungs. This study also validated that D-luciferin could not be used as a substrate, opening the door for future studies to multiplex RLuc with FLuc as dual-reporters for in vivo applications. The initial limitation for using RLuc as a reporter was its less-than-optimal expression efficiency within mammalian cellular hosts. This limitation was overcome via a codon-optimization strategy that modified the RLuc gene sequence while maintaining the wild-type protein sequence.

A synthetic humanized version of the luciferase gene that utilizes this strategy, called hRLuc, is now commercially available and has been shown to produce up to several fold higher light output in many mammalian cell lines. Further hampering the expression of RLuc in cell culture and small animal imaging applications was its tendency to be rapidly inactivated upon exposure to animal serum. An early study by Liu and Escher showed that a single mutation from cysteine to alanine at amino acid RLucCA increased serum resistance, while simultaneously increasing overall light output [ 37 ].

Fortuitously, the RLuc8 mutant also exhibited a 4-fold improvement in brightness. The improved stability and light output characteristics of RLuc8 make it a more favorable reporter than wild-type RLuc for mammalian imaging applications. Unfortunately, these red-shifted mutants also possessed substantially reduced signal intensities. To restore light output, random mutagenesis was carried out on the red-shifted mutants. This process identified several residues where mutations increased light output or resulted in further red-shifting.

Based on these encouraging results, Loening and colleagues performed several more rounds of site-directed mutagenesis and successfully engineered three promising variants RLuc8. All three variants exhibited greater light output than wild-type RLuc, with the most improved, RLuc8.

In practice, this translated to roughly a 2. However, this substrate is currently not commercially available due to high background activity and difficulty in purification. Other analogs, such as coelenterazine- f , - h , and - e have been shown to increase signal intensity by 4- to 8-fold relative to coelenterazine in RLuc-expressing mammalian cells in vitro , but each has failed to compete with the native coelenterazine in living animal imaging [ 40 ].

This study also showed that the split RLuc reporter signal could be modulated by using an inducible promoter e. These types of split RLuc complementation assays have also been applied to profile protein-protein interactions in the Golgi apparatus in planta [ 49 ] and to study protein dynamics during chemotaxis in bacteria [ 50 ], making it a broadly applicable approach. In a follow-up study, Benedetti and colleagues 93 combined wake therapy, morning light therapy, lithium, and antidepressants for inpatients with bipolar depression.

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Although this study does not isolate light therapy as the critical intervention, it underscores the effectiveness of the chronotherapeutic combination with wake therapy in acutely reversing the depressive episode even in resistant cases. A case series of drug-resistant bipolar patients who continued light therapy at home with sustained benefit 86 points to the need for and utility of long-term maintenance treatment.

In an instructive pilot study of light therapy during depressed phases of rapid-cycling bipolar disorder, Leibenluft and colleagues 94 were struck by increasingly labile mood when 10,lux light was administered in the morning for up to 60 minutes, whereas midday presentation was effective.

Sit and colleagues 35 studied nine women with longstanding nonseasonal bipolar I or II disorder in which mood stabilizers controlled manic phases, but antidepressants did not relieve depressed phases.