Common luciferases available for biotechnological applications, their luciferin compound, and their output wavelength.
This chapter explores the history of the bioengineering advances that have been applied to common luciferase enzymes and the improvements that have been accomplished by this work. The primary focus is placed on firefly luciferase (FLuc), Gaussia luciferase (GLuc), Renilla luciferase (RLuc), Oplophorus luciferase (OLuc; NanoLuc), and bacterial luciferase (Lux). Beginning with the cloning and exogenous expression of each enzyme, their step-wise modifications are presented and the new capabilities endowed by each incremental advancement are highlighted. Using the historical basis of this information, the chapter concludes with a prospective on the overall impact these advances have had on scientific research and provides an outlook on what capabilities future advances could unlock.
- firefly luciferase (FLuc)
- Gaussia luciferase (GLuc)
- Renilla luciferase (RLuc)
- Oplophorus luciferase (OLuc; NanoLuc)
- bacterial luciferase (Lux)
1.1 Historical perspective on the discovery of luciferase enzymes
The bioluminescent phenotype, which is spread across a variety of different insects, bacteria, fungi, and marine animals, has intrigued mankind since before the dawn of the modern scientific era . 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
Following the exogenous expression of the previously described firefly and bacterial luciferases,
1.2 Available luciferase systems for biotechnological applications
Of the ~40 different bioluminescent systems known to exist in nature , relatively few are available for biotechnological applications. 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.
|Luciferase||Luciferin compound||Output wavelength (nm)|
|Firefly luciferase (FLuc)||D-luciferin||560|
|Bacterial luciferase (Lux)||Tetradecanal||490|
1.3 The necessity of engineering luciferase proteins
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.
1.4 Common methods for engineering improvements
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.
|Mutagenic PCR||Wavelength shifting, thermostability improvement, improve signal output intensity|
|Rational sequence mutation||Wavelength shifting, altering luciferin compatibility, altering signal output kinetics|
|Synthetic recapitulation||Enable functionality in alternative hosts, improve expression efficiency, improve ease of use|
|Codon optimization||Improve expression efficiency|
|Circular permutation||Thermostability improvement, improve expression efficiency, expand reporter functionality|
|Alternative luciferin supplementation||Wavelength shifting, altering signal output kinetics|
|Split luciferase complementation||Alter signal output kinetics, expand reporter functionality|
2. Firefly and click beetle luciferases
Firefly luciferase (FLuc) is perhaps the most well-known, well-studied, and widely-used of all the luciferases. It, and its close relatives from click beetles, both function through the ATP-dependent oxidation of reduced D-luciferin (2-(4-hydroxybenzothiazol-2-yl)-2-thiazoline acid) in the presence of magnesium (Mg2+) and molecular oxygen (O2) to yield carbon dioxide (CO2), AMP, inorganic pyrophosphate (PPi), and oxyluciferin. 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. The naturally occurring peak emission wavelength for FLuc (as commonly derived from
Although FLuc and click beetle luciferase were among the first luciferases to be studied , it was not until the mid-1900s 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 . This led to the determination of D-luciferin’s structure as 2-(4-hydroxybenzothiazol-2-yl)-2-thiazoline acid and its eventual chemical synthesis . 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 . In 1985, FLuc cDNA was cloned by DeLuca et al. . 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.
2.2 Initial application and limitations
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. The early discovery that ATP concentration was proportional to light intensity in beetle luciferase reactions made this assay the primary method for monitoring the cell’s main source of energy. Further entrenching this technology was its exceptional sensitivity. FLuc-based bioluminescent ATP assays display detection capabilities down to 10−17 mol . This sensitivity for measuring ATP concentrations has been used in several applications including screening for microbial contamination in food industries, assessing cell viability , and assaying enzymes involving ATP generation or degradation . However, ATP concentrations found in living cells (1–10 mM) are generally saturating for FLuc and therefore it cannot be routinely used to assay intracellular ATP content . In a similar vein, FLuc has also been used to assay for the other metabolites that participate in its bioluminescence reaction: CoA, AMP, and PPi .
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
2.3 Engineering improved expression and output
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 . For instance, wild-type FLuc protein has a half-life of only 15 minutes at 37°C. This required that more thermostable forms be developed to assay human and small animal model-relevant temperature conditions . Pozzo et al. sought to address this issue by combining amino acid mutations shown to enhance thermostability with other mutations reported to enhance catalytic activity, resulting in an eight amino acid FLuc mutant that exhibited both improved thermostability and brighter luminescence at low luciferin concentrations .
Similarly, Fujii et al. produced variants capable of producing 10-fold higher luminescence than the wild-type enzyme by screening a mutant library of FLuc proteins generated by random mutagenesis . Site-directed mutagenesis experiments were then performed based on mutant sequences that produced increased luminescence. It was observed that the substitution of D436 with a non-bulky amino acid, I423 with a hydrophobic amino acid, and L530 with a positively charged amino acid all increased luminescence intensities relative to the wild-type enzyme. They further demonstrated that combining the mutations at I423, D436, and L530 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.
2.4 Engineering alternative output wavelengths
Engineering wavelength-shifted luciferases has become an intense area of study to enable multi-color assays and improve the efficiency of
2.5 Engineering alternative signal kinetics
It has been demonstrated that varying the concentrations of FLuc’s substrates (D-luciferin, ATP, etc.) can alter its reaction kinetics. 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 . 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 . Commercial reagents containing micromolar concentrations of components such as pyrophosphate and/or CoASH have been shown to convert FLuc reactions from flash- to glow-type kinetics, possibly due to the breakdown of oxidized luciferin-AMP
Another strategy that has been applied to alter reaction kinetics is the modification of the luciferin substrate. Mofford et al. demonstrated that near-infrared light emission can be increased >10-fold from wild-type FLuc by replacing D-luciferin with synthetic analogues . These synthetic analogues were designed to emit longer wavelength light by incorporating an aminoluciferin scaffold. Nearly all the aminoluciferins tested in their studies resulted in higher total near-IR (695–770 nm) photon flux from live cells under both high- and low-dose conditions. A more recent substrate modification strategy has been to conjugate the luciferin with distinctive functional groups. These so-called “caged” luciferins react when they are cleaved by enzymes or bioactive molecules and subsequently freed . This strategy allows for specified monitoring of biological processes by linking light output to the activity and/or concentration of enzymes or molecules reacting to cleave the caged luciferins.
The RLuc protein was first purified and characterized in the late 1970s . However, its cDNA sequence was not identified and cloned into
3.2 Engineering improved expression and output
The initial limitation for using RLuc as a reporter was its less-than-optimal expression efficiency within mammalian cellular hosts. This limitation was overcome
3.3 Engineering alternative output wavelengths
Despite the improvements made to increase expression efficiency and output, RLuc’s 480 nm output maximum remained problematic for
In addition to engineering the protein itself, synthetic coelenterazine substrate analogs have also been created to improve light output and/or yield red-shifted emission spectra. The analog coelenterazine-
3.4 Engineering split luciferase applications
Due to its small size (311 amino acids, ~36 kDa) and monomeric orientation, the RLuc protein is an attractive option for use in split luciferase complementation assays aimed at monitoring real-time protein-protein interaction. In an early study attempting to achieve this goal, Paulmurugan and Gambhir  created RLuc fragment pairs at two split sites (I223/P224 and G229/K230) and fused the individual fragments to either the MyoD or Id proteins. They then successfully demonstrated that RLuc could properly re-fold and restore luciferase activity upon complementation during MyoD/Id interaction. This study also showed that the split RLuc reporter signal could be modulated by using an inducible promoter (e.g., NFκB promoter/enhancer) to regulate the expression level of one of the two fragments. The fragment pair based on the G229/K230 split site was later used to characterize interactions between heat shock protein 90 (Hsp90) and the co-chaperone protein Cdc37 , between Hsp90 and the Epstein-Barr virus protein kinase GBLF4 , and to visualize androgen receptor translocation in the brains of living mice . Kaihara et al. similarly leveraged a variant of RLuc split between S91 and Y92 to demonstrate the recovery of bioluminescent activity during insulin-stimulated protein-protein interactions , and Stefen et al. created a split variant using fragments separated between residues 110 and 111 fused to protein kinase A (PKA) regulatory and catalytic subunits to quantify G protein-coupled receptor (GPCR)-induced disassembly of the PKA complex in living cells . These types of split RLuc complementation assays have also been applied to profile protein-protein interactions in the Golgi apparatus
Isolated from the marine copepod
4.2 Engineering improved expression and output
To enable improved expression efficiency in biomedical applications, a humanized version of GLuc, hGLuc, was generated
Building on this codon optimization-based approach, which enhances light output by improving protein expression in the host organism without modifying the peptide sequence, mutagenetic approaches have similarly been successfully applied to engineer variants that produce greater signal intensities than the wild-type protein. In one such example, Kim et al. performed site-directed mutagenesis to the hydrophilic core region of GLuc and identified that changing the isoleucine at position 90 to leucine (I90L) was the major contributing factor for improved signal intensity . The I90L variant produced six times higher light output than the wild-type protein in mammalian cells. Using a directed molecular evolution approach, Degeling et al. also identified a variant (S16K/M43V/V159M) that showed 2-fold enhanced luciferase activity .
4.3 Engineering alternative output wavelengths
One limitation of the native GLuc protein is that its relatively blue-shifted emission wavelength is easily absorbed and scattered by pigmented molecules in animal tissues. This limits its utility in
4.4 Engineering alternative signal kinetics
Wild-type GLuc catalyzes a flash-type bioluminescent reaction, meaning that the light signal decays rapidly following luciferin exposure. Practically, this necessitates immediate signal reading after substrate addition and thus makes GLuc unsuitable for the majority of high-throughput applications. To overcome this rapid signal decay, researchers have successfully engineered mutants that emit more stable bioluminescence [61, 62, 63]. Noticeably, a L30S/L40P/M43V variant has been shown to exhibit glow-type kinetics with only a 20% loss in signal intensity over 10 minutes, compared to the >90% loss in signal intensity after 1 minute from the wild-type enzyme . GLuc mutants such as these have been demonstrated to function in 96- and 384-well plate formats, which effectively allows them to overcome the wild-type kinetic limitations and enables their use in high-throughput assay formats.
4.5 Engineering split luciferase applications
Like RLuc, GLuc’s small size (185 amino acids, 19.9 kDa) makes it a good candidate for split luciferase complementation assays. In an early attempt at developing this functionality, Remy and Michnick evaluated the ability of fragment pairs generated from cut sites between amino acids 65–109 of a truncated hGLuc sequence exclusive of the secretion signal to reconstitute luciferase activity upon rejoining . By fusing the respective 5′ and 3′ sequences of the split hGLuc gene to a GCN4 leucine zipper-coding sequence and co-expressing the resulting fusions in HEK293 cells they were able to show that hGLuc activity could be successfully reconstituted by leucine zipper-induced complementation of the split fragments. Their study determined that the optimal split site for complementation was between G93 and E94. This fragment pair has since been further demonstrated to be inducible and reversible, which allows it to function as a highly sensitive tool for quantifying protein-protein interactions in cells and living mice . Similarly, Kim and colleagues also developed a split GLuc variant dissected at Q105 and demonstrated its utility to monitor calcium-induced calmodulin and M13 peptide interaction, phosphorylation of the estrogen receptor, and steroid-receptor binding in living cells .
OLuc was first discovered in 1975 , and shortly after in 1978 the mechanics of its bioluminescent reaction were identified . Inouye et al. were the first to clone the OLuc cDNAs encoding the 35 and 19 kDa subunit proteins, which led to their discovery that the 19 kDa protein was responsible for catalyzing the luminescent oxidation of coelenterazine. Although this 19 kDa protein was found to be the smallest known protein capable of catalyzing bioluminescence, it was also found to be poorly expressed and unstable without the support of its 35 kDa partner .
5.2 Engineering improved expression and output
The need to co-express the 19 and 35 kDa subunits of OLuc made it problematic for routine reporter usage. To overcome this, Hall et al. performed three rounds of mutagenesis on the 19 kDa subunit to produce a novel variant, which they termed NanoLuc (NLuc). This variant showed improved structural stability as well as increased bioluminescent activity and glow-type kinetics with a peak emission wavelength of 460 nm. Furthermore, it was shown that this variant could oxidize an alternative luciferin, furimazine, which resulted in greater light intensity and lower background autoluminescence than when coelenterazine was used. NLuc’s 19 kDa size and absence of post-translational modifications made it more agile than FLuc, while its naturally high tolerance to temperature and pH made it more robust. In practice, this NLuc variant was shown to poses 150-fold greater specific activity than either FLuc or RLuc . However, these improvements proved to be a double-edged sword. The high stability and glow-type kinetics made it difficult to employ NLuc for transient reporting activities, while its highly blue-shifted output limited its signal penetration in mammalian cellular applications.
Nonetheless, NLuc’s small size and efficient expression make it an excellent choice for studying low-dynamic activities. In one such example, Chen et al. developed a sensitive assay in which NLuc was used to study the activity of deubiquitinating enzymes. In this work, NLuc was fused to the C-terminus of His-tagged ubiquitin that was attached to Ni2+ agarose beads. This allowed NLuc to be released as the α-peptide linkages were cleaved so that deubiquitination could be monitored
5.3 Engineering split and paired luciferase applications
Zhao et al. showed that a split luciferase-based system could be used to monitor protein stability by tracking protein aggregation with NLuc-based luminescence . To accomplish this, they broke NLuc into two fragments, termed N65 and 66C, and demonstrated that, upon interaction, luminescence was modulated by the solubility of the protein fused to the N65 fragment. This property was maintained in both bacterial and mammalian systems, confirming its utility for sensitive detection of protein solubility in a straightforward, high-throughput assay format in living cells.
In addition to these traditional split luciferase applications, NLuc has also been employed for paired luciferase applications that utilize an unfused variant to provide the highest possible light intensity and sensitivity, a destabilized variant with an appended degradation signal (e.g., NLuc-PEST) that allows rapid response to dynamic changes in environment, and a secreted variant (e.g., secNLuc) .
6. Bacterial luciferase
Unlike the monomeric luciferases discussed above, bacterial luciferase (Lux) is a heterodimer of two genes,
Although this process has been most well-studied in marine bacteria from the
6.2 Initial uses and limitations
Because Lux emits its bioluminescent signal without the need for external stimulation, it quickly became a valuable tool for optical imaging. The low hanging fruit for this system was the real-time monitoring of gene expression. This was first demonstrated by Enbreghet et al. , who fused Lux to inducible promoters to study the mechanics of IPTG and arabinose induction in
Despite the advantages offered by avoiding the need for external stimulation concurrent with visualization, Lux was significantly handicapped by its inability to function within eukaryotic cells. Because of this, it was not originally applicable to most modern biotechnological and biomedical applications outside of tracking bacterial infections . Furthermore, as a consequence of encoding both the luciferase and luciferin generation pathways this system required significantly more foreign DNA to be introduced in order to function exogenously. This made the system more difficult to work with at the molecular level; especially before the advent of today’s more efficient genetic assembly tools. Similarly, the heterodimeric nature of the luciferase enzyme is more cumbersome than the monomeric orientation of its counterparts. Nonetheless, given its relative advantages over the other systems, it continues to be engineered to overcome these detriments and expand its utility.
6.3 Engineering eukaryotic expression
Although several early attempts were made to enable Lux functionality within eukaryotic hosts, none of these achieved significant success [81, 82, 83]. The first major breakthrough came with the expression of the luciferase in
Despite this success in
6.4 Engineering increased light output
To overcome Lux’s low level of bioluminescent output in human cells the orientation of the cassette was subjected to further engineering. It was determined that the use of multiple plasmids was detrimental to achieving high level expression, and that the use of IRES elements was inefficiently expressing the downstream genes in the paired orientation. Therefore, the IRES elements were replaced with viral 2A linker sequences. These sequences were significantly shorter than the IRES sequences they replaced and allowed for each linker region to have a unique genetic code that reduced the chance for unintended recombination events. As a result, the full bacterial luciferase cassette, inclusive of the flavin oxidoreductase component, could be placed under the control of a single promoter and expressed from a single plasmid. This new orientation made it possible to express bacterial luciferase as a single genetic construct similar to what was commonly done with the alternative monomeric, luciferin-requiring luciferase systems. As a result, the bacterial luciferase system could be expressed more easily across a larger number of cell types and was capable of producing an enhanced level of signal output relative to its previous incarnation .
In addition to engineering increased expression
6.5 Engineering improved bioreporter functionality
Just as it has been used extensively as a bioreporter in bacterial species, the engineering of bacterial luciferase to function in eukaryotic cells opened the door to this same functionality under much broader applications. The transition of the Lux cassette to function as a single open reading frame made it possible to replace the constitutive promoter with an inducible promoter and regulate its expression in response to compound bioavailability . However, computational modeling aimed at calculating the metabolism of the required substrates and cofactors for the reaction relative to their intracellular availably suggested that that control of the system should be imparted at the level of the aldehyde recycling pathway, with
7. Fungal luciferase
Fungal luciferase is the most recent luciferase system to be functionally elucidated and made available for biotechnological applications. At the core of this system is a monomeric luciferase gene,
7.2 Initial uses and limitations
Unlike the previous luciferases that have been discussed, fungal luciferase has only recently been elucidated as of the time of this chapter. As a result, there have yet to be any reports of its functionality outside of its initial validation . Regardless, the initial characterization of the system provides valuable insights into its functionality and potential limitations. From a practical standpoint, it has been demonstrated that the system can be fully recapitulated in yeast to achieve autobioluminescent signal production. At this time only one luciferin synthesis pathway has been demonstrated, but because genes sourced from alternative organisms are used to enable caffeic acid synthesis in hosts that do not natively support these reactions, it is likely that alterative genes could be substituted for these parts of the pathway.
For more complex hosts, such as human cells, the functionality of the system has been demonstrated only under non-autobioluminescent conditions. In this case, only the luciferase was genetically encoded and the luciferin was exogenously applied. Using this strategy, it has been possible to observe luminescence in cultured human cells,
It is currently unknown if the lack of demonstrated autobioluminescent production in hosts outside of yeast is incidental, or if it is the result of metabolic or molecular limitations on the expression of the full cassette within these organisms. One possible explanation is that the required culture temperatures were not compatible with full cassette functionality. It has been shown that fungal luciferase is temperature sensitive and begins to decrease its output signal at temperatures >18°C. Relative light output is halved at room temperature (26°C) and is abolished above 30°C. This is detrimental to the use of this luciferase in human cell culture and small animal model systems, as they will require the maintenance of temperatures above 30°C to avoid the introduction of secondary environmental effects. Similarly, the luciferase is only ~50% efficient at pH 7, which could be detrimental to some experimental designs. The optimal pH is 8, with improved retention of performance at increased pH relative to decreased pH.
7.3 Potential future engineering goals
There are ~100 fungal species that use this luciferase/luciferin pathway for bioluminescent production . It is believed that fungal bioluminescence evolved only once, but that evolutionary pressure led to uneven distribution of the phenotype among species. While this simplifies the system by allowing development to focus on only a single incarnation, it is also potentially limiting in that there are fewer evolutionary cues that can be leveraged as starting points for biotechnological advancement. Nonetheless, this system is clearly in its infancy and will benefit from the copious knowledgebase developed through the engineering of alternative luciferases. It is likely that the primary development target will be overcoming the thermostability issues present in the current incarnation of the system. Beyond this, and similar to Lux, it is likely that investigators will seek to streamline expression of the relatively large cassette size to make it more manageable from a molecular biology standpoint. Once these efforts are achieved, the autobioluminescent nature and somewhat red-shifted output of the fungal system will make it a welcome addition for real-time imaging applications that currently rely on only the bacterial luciferase system.
8. Outlook for future developments
There are ~40 different bioluminescent systems known to exist in nature . However, only seven different families have been well described and only five of the six detailed in this chapter enjoy widespread use . Despite the relative wealth of unexplored systems, relatively few new systems have become available in recent history. Within the last 10 years, the most notable advancements have been the engineering of the bacterial luciferase system to function in eukaryotic organisms and the elucidation of the fungal luciferase genetic pathway. Despite this, the considerable progress of incremental engineering for firefly luciferase and the development of NanoLuc from
There are a variety of different luciferase systems available for biotechnological applications that can help investigators achieve their experimental goals. The high utility afforded by these enzymes is the result of a rich history of engineering that has enabled them to become versatile research tools. Historically, significant shifts in utility have occurred with the elucidation and introduction of new luciferases, followed by slower, but steady, incremental improvements as they are iteratively engineered to improve their ease of use and expand their functionality. In the context of the historical achievements that have been made with firefly,
Research funding was provided by the U.S. National Institutes of Health under award numbers NIGMS-1R43GM112241, NIGMS-1R41GM116622, NIEHS-2R44ES022567, NIEHS-1R43ES026269, and NIMH-1R43MH118186, and the U.S. National Science Foundation under award number CBET-1530953.
Conflict of interest
S.R., G.S., and D.C. are board members in the for-profit entity 490 BioTech.
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