Firefly Blue (Chapter 16 Book 2)
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Publisher Summary This chapter discusses purification and properties of firefly luciferase. GLuc mutants such as these have been demonstrated to function in and well plate formats, which effectively allows them to overcome the wild-type kinetic limitations and enables their use in high-throughput assay formats. 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— of a truncated hGLuc sequence exclusive of the secretion signal to reconstitute luciferase activity upon rejoining [ 64 ].
Their study determined that the optimal split site for complementation was between G93 and E 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 [ 65 ]. Similarly, Kim and colleagues also developed a split GLuc variant dissected at Q 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 [ 60 ].
Oplophorus luciferase OLuc is a naturally-secreted luciferase isolated from the decapod Oplophorus gracilorostris , a deep-sea shrimp that ejects OLuc from the base of an antennae in a brightly luminous cloud when stimulated. Even in its wild-type form, OLuc possesses robust biochemical and physical characteristics relative to alternative luciferases. OLuc was first discovered in [ 66 ], and shortly after in the mechanics of its bioluminescent reaction were identified [ 8 ].
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. 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.
This allowed cytokine-induced upregulation to be measured in HAP1 cells. Under this design, they were able to show that NLuc luminescence correlated strongly with quantitative PCR data, demonstrating that NLuc could reliably be used to monitor gene expression. 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.
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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.
Unlike the monomeric luciferases discussed above, bacterial luciferase Lux is a heterodimer of two genes, luxA and luxB , that must join together to form a functional unit. It is also only one of two systems, along with the fungal system discussed below, that additionally has a known genetic pathway for luciferin synthesis.
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In the case of bacterial luciferase, this pathway consists of three additional genes, luxC , luxD , and luxE , that work together to produce a long chain fatty aldehyde [ 72 ]. In this process, luxD transfers an activated fatty acyl group to water, forming a fatty acid. The luxE gene finally reduces this fatty acyl-AMP to an aldehyde [ 72 ]. The natural aldehyde for this reaction is tetradecanal, however, the luciferase is also capable of functioning with alternative aldehydes as substrates [ 72 ].
Along with these genetic components, the system requires two cofactors: oxygen and reduced riboflavin phosphate. Although this process has been most well-studied in marine bacteria from the Vibrio genus, the genetic organization and biochemical underpinnings of the system are consistent across all known bacterial phyla [ 18 ]. Due to the complexity of this system relative to its monomeric counterparts, it was not exogenously expressed until the early s. Even then, it was initially utilized through expression of the luxA and luxB genes as a standalone luciferase [ 5 ] before subsequently being employed as a fully functional cassette that was capable of functioning in an autonomous fashion [ 73 ].
Shortly after these demonstrations the crystal structure of the bacterial luciferase heterodimer was determined [ 74 ], however, this structural knowledge has yet to be leveraged as a means for engineering improved functionality. 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 proved to be a valuable approach because it allowed samples to be continuously monitored in order to track gene expression dynamics over time.
Building upon this work, a variety of instances have been described where Lux has been placed under the control of a promoter with a known inducer to track compound bioavailability. At a higher level, it has been used for in situ bacterial monitoring, such as the visualization of bacterial invasion of leaf [ 77 ] and root structures [ 78 ]. Further, due to the absence of light production from non-bioluminescent species, it was also used to track specific populations of bacteria within mixed communities within unperturbed environments [ 79 ].
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 [ 80 ].
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. 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. 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 S. This achievement was made possible by using luciferase genes from the terrestrial bacterium, Photorhabdus luminescens , which showed higher thermal stability than those of marine bacteria, and expressing the individual heterodimer genes from a single promoter using an internal ribosomal entry site IRES to link them together.
Under this orientation the luciferase was able to properly express within the cell and produce light upon exposure to an n-decanal substrate. This same strategy was then expanded to incorporate the expression of IRES-linked luciferin synthesis pathway genes from dual promoters.
Red-shifting the optical response of firefly oxyluciferin with group 15/16 substitutions
When expressed concurrently with the luciferase genes, the cell produced a bioluminescent signal without external stimulation. The functionality of the system was then further improved by shifting the intracellular redox balance to a more reduced state through the introduction of a flavin oxidoreductase gene, frp. Despite this success in S. To achieve this, the genes were codon optimized for the human genome and mammalian-optimized IRES elements were employed to improve expression of the downstream genes in human cells [ 85 ].
It was also determined that the full pathway could not be expressed from a single promoter using IRES elements, so the luciferin synthesis pathway was encoded on a separate plasmid. This approached allowed for functionality in human cells, but the overall level of bioluminescent production was several orders of magnitude lower than that of alterative bioluminescent systems such as firefly luciferase [ 86 ].
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. 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 [ 87 ].
In addition to engineering increased expression via improved expression efficiency, work has also been performed to alter the peptide sequence of the bacterial luciferase genes to make light output more efficient. Of these mutations, six were within the luciferase genes three each in luxA and luxB , six were in the luciferin synthesis pathway with all six located in the luxC gene , and three were located in the oxidoreductase gene, frp. 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 [ 87 ].
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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 luxA and luxB expressed continuously, and luxC , luxD , and luxE placed under the control of the inducible promoter [ 89 ].
This model was later proven to be correct when direct comparisons were performed using either single open reading frame constructs where the full cassette was controlled by the inducible promoter, or split cassettes where the luciferase and luciferin pathway genes were switched between inducible and constitutive promoters [ 90 ]. Together, these results significantly improved the functionality of the bacterial luciferase system as a bioreporter despite its relative complexity compared the other luciferases. 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, luz.
In addition to the luciferase, two luciferin synthesis genes: hisps and h3h , work together as a polyketide synthase and a 3-hydroxybenzoate 6-monooxygenase to supply the required luciferin, 3-hydroxyhispidin. In addition to these genetic components, the reaction also requires molecular oxygen and NAD P H as co-factors [ 91 , 92 ]. Like Lux, fungal luciferase is notable in that the genetic sequence of all components required for bioluminescent production is characterized.
This allows the fungal luciferase cassette to be genetically encoded and exogenously expressed to produce an autobioluminescent phenotype [ 12 ]. However, for this to occur the host organism must either be capable of naturally synthesizing caffeic acid to act as a precursor for luciferin synthesis, or the necessary genes for caffeic acid synthesis must be co-expressed. 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 [ 12 ].
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, Xenopus laevis embryos, and small animal models subcutaneously injected with labeled cells. These demonstrations bode well for the use of the fungal luciferase in the types of experimental designs most commonly associated with traditional luciferase reporters and provide researchers with a novel imaging tool that can be differentiated from alternative luciferases based on its luciferin specificity.
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.
The optimal pH is 8, with improved retention of performance at increased pH relative to decreased pH. 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.