An improved pathway for autonomous bioluminescence imaging in eukaryotes

The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.

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Reviewers' Comments:
Reviewer #1: Remarks to the Author: Shakhova et al. describe the optimization of the fungal bioluminescent pathway in different eukaryotic expression systems (plant, yeast and mammalian).By combining mutagenesis of nnLuz and nnH3H, screening of HispS from different fungi and coexpression of NpgA, bioluminescence emission is improved by 1-2 orders of magnitude.A particular focus of the work is on the generation of different plant species with enhanced luminescence which largely seems to originate from the incorporation of NpgA (e.g. Figure 1b and Extended Data Figure 2), as also described in a very recent publication (doi: 10.1111/pbi.14068).In mammalian cells, luminescence emission still depends on the addition of caffeic acid as a luciferin precursor so that fully autonomous bioluminescence emission is not yet achieved.Nevertheless, the described improvements of several enzymes of the fungal bioluminescent system represent a significant progress towards this goal and will therefore be of great interest to a broad readership.The manuscript is clearly written and contains comprehensive and technically sound data that describe the obtained improvements in detail using appropriate methodology and statistics.The following points should be addressed before publication: 1.The influence of nnLuz_v4 and nnH3H_v2, the incorporation of NpgA and the replacement of nnHispS by mcitHispS on the overall light emission in the different expression systems is not fully clear.To allow a better evaluation, in each of Figures 1b-d the following variants should be compared (indicating the fold improvements in luminescence relative to FBP1 in each case): FPB1, FBP1+nnH3H_v2/nnLuz_v4, FBP2 and FBP3.Numbers indicating the fold change in luminescence between FBP1 and FBP1+nnH3H_v2/nnLuz_v4 should also be included in other figures (Figure 2a, Extended Data Figure 5, Supplementary Figures 9a,c and 12b).
2. A major advantage of the fungal bioluminescent system is that it potentially enables autonomous bioluminescence emission in different eukaryotic hosts also other than plants, most importantly mammalian cells.Although addition of caffeic acid still seems to be a requirement here, a direct comparison of the luminescence from FBP3 (+caffeic acid) to the bacterial bioluminescent system which already allows fully autonomous bioluminescence emission would be of interest (described for instance in FEMS Yeast Res 4, 305-313 (2003) for yeast and in PNAS 116, 26491-26496 (2019) for mammalian cells).In this way, the autonomous luminescence intensity that could ultimately be achieved with the developed FBP3 enzymes (once cellular caffeic acid synthesis is established) can be better evaluated.8 and 9, the luciferins and their concentrations should be given for each luciferase.

In Extended Data Figures
4. In the plasmids for the creation of transgenic plant lines, the genes nnLuz, nnCPH and nnH3H have been rearranged between FBP1 and FBP1+nnLuz_v4/nnH3H_v2 (Extended Data Figure 4).Does this rearrangement affect the luminescence emission? 5. Why are different designations for the luminescence signal used in the figures throughout the manuscript (e.g. Figure 1b-d)?Where possible, axes should be labeled consistently.
6. Page 1, line 23: Bacterial bioluminescence is not restricted to marine bacteria, but is also found in some terrestrial and freshwater bacteria.
7. Figure 1: The concentration of caffeic acid should be stated in the legend (where added). 1 and 2: At what temperature were the measurements performed?9. Supplementary Figure 7: Why is the ratio of hispidin RLU / luciferin RLU shown (instead of hispidin RLU only)?Coexpression of the nnLuz variant should be stated in the legend.10.Supplementary Figure 8: y-axis should be labeled with at least two numbers to determine the scale.

Supplementary Figures
Reviewer #2: Remarks to the Author: Shakhowa et al. describe new bioluminescence imaging tools by engineering enzymes in fungal bioluminescence pathway.They focused on enzymes that catalyzes light-emitting caffeic acid cycle in the fungus Neonothopanus nambi, which has recently attracted attention for developing reporter tools.fortransient expression assays in plants.By introducing substitution mutations in the enzymes, they successfully increased enzyme stability and achieved brighter luminescence by 2 sets of mutated enzyme combination: FBP2 and FBP3.Surprisingly, the authors also showed that the luminescence of plants can be observed using a consumer camera and smartphone cameras.These sets of enzymes can broaden the applicability of self-sustained luminescence for plant biology.Furthermore, caffeic acid was able to produce luminescence using FBP3 in HEK293 cells although its signal intensity was much less than those produced by luciferin-firefly luciferase and luciferin-NanoLuc reactions.This study on the use of fungal bioluminescence pathway is of potential interest and seems to be useful for developing bioluminescence reporter tools.However, the reviewer felt a lack of information on superiority and versatility compared to existing imaging systems.The reviewer is particularly concerned regarding the following points The newly constructed high-intensity reporter system can be highly evaluated for realizing simple and highly sensitive in-vivo imaging in plants.On the other hand, the usefulness of the constructed new system has not been sufficiently demonstrated: 1) The authors focused on the light-emitting caffeic acid cycle in the fungus Neonothopanus nambi, an improved version of the recently described luminescence system.not their original.In addition, methods for improvement include the introduction of substitutional mutations using existing information and the introduction of random mutations, and methods newly developed by them are not used.
2) Highly sensitive optical in vivo imaging has been achieved by fluorescence imaging.The multiplicity of fluorescence makes it possible to visualize many molecules simultaneously, which is useful for elucidating their roles and interactions.From a methodology point of view, they need to demonstrate the superiority of this system by showing results that can only be observed using their luminescence system.
3) The high luminescence intensity has unfortunately not been obtained except for plant.Although they certainly observed luminescence in human cells, any images were provided.Fluorescence provides images with better resolution than luminescence.Therefore, this reviewer cannot come up with a methodology in which luminescence outperforms fluorescence in observations at the level of cultured cells.If this system can be applied to small animals, it will be very valuable as a new optical imaging system.
Minor comment; 1) The authors use uM instead of µM throughout the manuscript.It should be revised.
2) Fig. S9d does not seem to reflect the result of Fig. S9c (all combinations are p < 0.001).Is Fig. S9d correct?
Reviewer #3: Remarks to the Author: Bioluminescence had been used for imaging, and the authors have reported modifications to the fungal bioluminescent system (FBP2 and FBP3) that increased the autoluminescence intensity by up to 1-2 orders of magnitude compared to the wild-type.The study extends from the authors previous report using the fungal autoluminescence system in multiple hosts (Ref), and enhancing the luminescence intensity is an important aspect to use this system as a tool for biological studies.The data seems to point to the inclusion of NpgA as the most substantial contributor to enhanced luminescence, and I felt that this should be discussed more, including the mechanisms of how the mutation of Luz and H3H contributed to enhanced luminescence.In addition, I have several concerns that would be better explained for the readers to understand the study.
(1) The authors describe that the newly developed bioluminescence (FBP2 and FBP3) has much higher luminescence intensity than the wild-type (FBP1), however, the data does not always include all three systems depending on the experiment host, and this makes it difficult to understand the relative luminescence intensity against FBP1.
(2) When luminescence intensity is compared for the Luz mutants using E.coli, how was the luminescence normalized?I suspect that the protein expression level and possibly the characteristics of the protein (e.g.solubility, codon preference) may be affected by the mutation, which will influence the luminescence intensity.
(3) In Fig. 1b, the fold-change of FBP2 vs. FBP3 is not stated.From a glance, this fold-change seems to be much greater than the difference of nnHispS vs. mcitHispS in Ext.data 1a, despite both data compared nnHispS and mcitHispS in BY-2 cells.Is there a reason for this difference in luminescence fold-change?(4) The authors compare FBP luminescence with Nanoluc and Fluc (Extended data Fig. 8 and 9).However, the conversion efficiency of caffeic acid to luciferin and the actual cellular concentration of the substrates are not described.In addition, each system uses a substrate with different chemical structure/properties, and I am unsure whether the cellular permeability is the same (caffeic acid for FBP).Similarly, the kinetics depends on the concentration of the luciferase and luciferin.Taken together, I feel it is difficult to compare the luminescent intensity by this method, and would like to suggest in vitro comparison using purified proteins, or a similar approach that can be normalized to the quantity of the luciferase.
(5) The fold-increase achieved in this study is remarkable, however, there are no insights into the mechanism of the enhanced luminescence.In relation to the mutagenesis of Luz, H3H, or the activity of mcitHispS, or NpgA it would be interesting to see whether the enzymatic properties (e.g.kcat, quantum yield) caused the enhancement.(6) From the transient assay of NpgA in plant cells (Extended Fig. 2), the inclusion of NpgA seemed to have the greatest effect (up to 280-fold increase in petunia) on the luminescence intensity among all of the modifications presented in this study.In stable transgenic plants, 8-and 54-fold increase was reported for N. benthamiana and N. tabacum, respectively.I feel that this was the largest contributor that enhanced luminescence and is worth discussing about the possible reasons.For example, was there a difference in transgene expression level?FBPs protein activity?Endogenous caffeic acid level?(7) When comparing the different FBP systems (FBP1,FBP2, FBP3), can the transgene expression level of each gene influence the luminescence intensity?Can the transgene expression level change depend on the order of the transgenes within the multiple expression cassette (e.g.possible transgene silencing triggered depending on the position of the transgene in the cassette)?Perhaps, a RT-qPCR to check whether there are any differences in transgene expression level of the different constructs may help.Or confirming the expression of each protein may also help.
(8) FBP3 was not brighter than FBP2 in stable transgenic N. benthamiana lines, which is different to the BY-2, mammalian, yeast cells.Is there any reason for this? (9) When comparing FBP1 to FBP2 or FBP3 in luminescent plants (Figure 2, Extended data Fig. 6, Supplementary Fig. 12, Supplementary Fig. 14-15, Supplementary Fig. 17-18), which generation of the plant was used?And if different generations were compared, was the luminescence intensity stable over generations?(10) nnLuz v4 did not show any enhancement of luminescence intensity in yeast (Supplementary Fig. 8).Is there any reason for this, and I feel it would help the readers if this was discussed.(11) For the HEK293 and pichia assay comparing FBP1 and FBP3 (Supplementary Fig. 10 ad 11), was the FBP1 co-transfected with NpgA and FBP3 in a single vector?If this is the case, could the co-transfection efficiency affect the luminescence intensity in these data?and the data would be inconclusive about how much NpgA or H3H_v2 contributed to the luminescence intensity?(12) The effect of NpgA was negligible in N. benthamiana transgenic lines (Supplementary Fig. 14), but large in N. tabacum.Is there any reason for this?

Minor points
Page 1: In the sentence, " Low enzymatic activity and limited stability of enzymes at physiologically relevant temperatures 2 resulted in modest light output…" Should this be quantitative?It may be better to briefly describe the limitations of the FBP1 in relation to its application (e.g.imaging) for the readers to understand the benefit of the new system easier.
Page 2: In the sentence "Similarly to nnHispS, mcitHispS was efficiently activated by phosphopantetheinyl transferase NpgA from Aspergillus nidulans, which we confirmed to be a necessary component for bioluminescence in most plant species", the FBP system has been tested mainly on dicot species and it may be an overstatement to say "most plant species".Otherwise, if it can be explained that most plant species do not have phosphopanteteinyl transferase, it may clarify the point.
Page 2: For the sentence "When stably expressed from a genomic copy, the wild-type fungal pathway FBP1 performed well in tobacco species, however, in our hands it did not yield sufficiently bright luminescence in other species.",depending on the imaging system, and its purpose, dim luminescence can still be useful.I was not able to follow what would be the required brightness for what application.
Page 2: For the sentence "Furthermore, the brightest tissues -petunia flower buds -could be recorded on modern smartphone cameras.",I could not find the image data taken using smartphone cameras.
Tested on benthamiana, petunia BY-2.Maybe overstatement to say most plant species, especially without demonstrating on monocots.
Figure 1 caption: The comparison of FBP2 is lacking in mammalian and yeast cells.Was there any reason for this, and if FBP2 is to be omitted, the sentence should be rephrased to clarify this. Figure 1e: It would help the readers to see the difference of luminescence intensity between FBP1, FBP2 and FBP3 as an image for comparison of brightness.This would be important as the comparison in the graph has used tobacco BY-2 cells, which may be different to petnuia. Figure 2: Because the ISO range that was used is large, it would help the readers to understand the relative brightness of each plant by adding the details about the imaging conditions for each species (e.g.ISO, distance to the subject (if they were different for each species)).
Extended Fig. 3: Please add the fold-difference compared to the wild-type for understanding the contribution of the different HispS and H3H to the luminescence intensity.
Extended Fig. 7: It would also be nice to show the wild-type (non-transgenic) plants for phenotypic analysis.Supplementary Fig. 4b (right-side of the graph): How was the "Expression" determined?Gene expression or protein expression level?Supplementary Fig. 5: Was there any reason the Supplementary Fig. 3 used E. coli and Supplementary Fig 5 .used Pichia?Also, if there are no particular reasons, consistent presentation style may be easier for viewing (i.e.line graph or box and whisker plot).Supplementary Fig. 17 and 18: The two figures are somewhat redundant in the sense that the only difference is the age of the plant.Perhaps it may be better to combine the graphs so it is more obvious that the luminescence intensity has gained over development.
Graphs in general: I felt that the font size relative to the graph was small, and sometimes difficult to read.

Author Rebuttal to Initial comments Decision Letter, first revision:
Dear Karen, I hope all has been well since we met at Janelia.Thank you for submitting your revised manuscript "An improved pathway for autonomous bioluminescence imaging in eukaryotes" (NMETH-BC51937B).It has now been seen by the original referees and their comments are below.The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Methods, pending minor revisions to satisfy the referees' final requests and to comply with our editorial and formatting guidelines.
In response to the remaining referee comments we ask that you complete the following: (1) Address the minor suggestions regarding clarifications or corrections.
(2) Better discuss how the method will enable biological discovery in plants and fungi.
(3) Clearly discuss the need for caffeic acid for the system to work.
We do not ask you to make more stable cell lines to compare FBP2 and FBP3 as requested by Reviewer 3.While we understand their point and agree that these experiments would strengthen your conclusions regarding differences in their performance in mammalian cells, we do not think it would change the overall impact of the paper as a whole.
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Reviewer #1 (Remarks to the Author): The following points should be addressed before publication: -In Figure 1b and c, the fold changes between FBP1 and FBP3 should be added.
-Unlike stated in the rebuttal letter, the temperature in Supplementary Figures 1 and 2 is not given and should be added.
All other points have been satisfactorily addressed.
Reviewer #2 (Remarks to the Author): Shakhowa et al. developed new bioluminescence imaging tools using a genetically engineered fungal bioluminescence pathway.The novelty of constructing a strong light emitting system using a fungal bioluminescence pathway and the efforts of the authors are worthy of praise.On the other hand, the bioluminescence pathway used in this study has already been reported, and methods for increasing luminescence intensity by modifying existing related genes have also been established (e.g.SCIENCE, 359:6378, 935-939, 2018.DOI: 10.1126/science.aaq1067),so there is no methodological novelty or originality to be found in this research.Furthermore, many examples have already been reported for simply acquiring photos and videos that do not require quantitative analysis using non-CCD cameras, such as iPhone and digital cameras, have been reported, including the aforementioned paper.Therefore, this reviewer's comments to the original manuscript was to ask the authors to clarify the superiority of this system by showing an example where imaging is not possible without using this system.Unfortunately, the authors' responses to my comments were not what I expected.
1) This journal focuses on methodology, and if improving performance in identifying versions of the fungal bioluminescent pathway is a methodological superiority of this study, it should be clearly stated.
2) In response to my comments, the authors described the disadvantages of fluorescence imaging.This reviewer is not looking to see how it differs from existing imaging technologies, and also has hope that "improved version of the pathway reported in this manuscript will enable broader applications of autoluminescence across plant and likely fungal species".The results presented in this manuscript, however, do not seem to represent much of advantages in terms of advances in imaging technology.For example, GFP gene-modified plants allow us to image the plant's growth over time without using special equipment such as a fluorescence microscope (e.g.PNAS 99 (6) 4103-4108, https://doi.org/10.1073/pnas.052484099.).I hoped that the authors' method, which was established by optimizing the fungal bioluminescence pathway, would demonstrate the superiority of the imaging method by showing examples of imaging that cannot be obtained with GFP gene-modified plants, or by showing applications that enables new imaging when combined with other imaging methods.
3) This reviewer was impressed the strong intensity of the luminescence shown in the revised manuscript (fig.S26 and Video S1) obtained by this system.However, it is unclear how much better it is compared to existing methods as it has not been compared to other imaging systems.
Minor comment; 1) Fig. S1, Fig. S2, and Fig. S3 still include "uM".In Figs.S13cd and S13ef, the concentration of caffeic acid are 100 mM and 220 mM, respectively.Are they correct?The concentrations are thought to be considerably higher than the physiological concentration.
(8) I understand the author's explanation that there were limitations on the number of lines that were used to compare FBP2 to FBP3.I am concerned whether the comparison of FBP2 and FBP3 in stable transgenic lines can be concluded with these results as there was only one transgenic line used for the comparison of both experiments (one line of FBP3 for Fig. 2 and one line of FBP2 for Ext.Fig. 5), which produced a different outcome.I feel multiple independent stable transgenic lines are needed for comparing FBP2 and FBP3.(9) I appreciate the authors for providing further information about the transgenic generations used in this study.I have no further comment on this point.(10) The authors repeated the experiment with additional replicates and obtained different results, which show increased luminescence using nnLuz_v4 compared to its wild type.I have no further comment on this point.(11) I appreciate the explanation and additional information in the manuscript.I have no further comments on this point.( 12) As point (6), I would appreciate an indication of where the information was added in the manuscript.

Minor points
My apologies for the comment in Extended Data Fig. 7 about adding the wild-type plants, which was already shown in Supplementary Figure 23 and described in previous work.The authors have revised and responded to all minor comments appropriately.I have no further comments on these points.

Author Rebuttal, first revision:
Final Decision Letter: Dear Karen, I am pleased to inform you that your Brief Communication, "An improved pathway for autonomous bioluminescence imaging in eukaryotes", has now been accepted for publication in Nature Methods.The received and accepted dates will be March 17, 2023 andDec 13, 2023.This note is intended to let you know what to expect from us over the next month or so, and to let you know where to address any further questions.
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