A proposal for a 'photobiology research' group

One of the most important aspects of a scientist's CV is the amount and size of grants or fellowships that we have obtained throughout our careers. However, the CV omits all the grant and fellowship applications that were not successful for any reason. I have decided to post here some of those research proposals that did not make it. Clearly, their panel of reviewers didn't know a thing ;)

This is my research proposal to an application I submitted to Institut Pasteur for the creation of new junior research groups. Enjoy!

The Photobiology Group

I propose here three multidisciplinary research modules that will be the research core of the Photobiology Group:

1.    Functional and structural characterization of  photochemical reaction centers

2.    Design, remodeling, and enhancement of photosynthetic systems

3.   Origin and evolution of photosynthesis and bioenergetics systems

Figure 1. Photochemical reaction centers from Cyanobacteria. Both are transmembrane multiprotein complexes carrying hundreds of cofactors, included chlorophylls, carotenoids, lipids, hemes, FeS clusters, among others. Photosystem II is unique because of the water oxidizing complex, a Mn₄CaO₅ cluster where oxygen evolution occurs.

Photochemical reaction centers are distributed in seven phyla of bacteria: Cyanobacteria, Chloroflexi, Firmicutes, Chlorobi, Proteobacteria, Acidobacteria, and Gemmatimonadetes. Despite such great biodiversity, today 95% of research in photosynthesis is done in the Cyanobacteria/plant system, and less than 5% in the remaining types of phototrophic systems (estimated by the total number of publications in the last five years mentioning model organisms from each group). From this 5%, 3.5% is research performed in a single strain of Alphaproteobacteria, Rhodobacter spheroides, probably best known for the 1988 Nobel Prize in Chemistry. We know virtually nothing about most of the diversity of photosynthetic organisms currently inhabiting Earth.

Even though the least studied groups of phototrophs represent, at best, less than 1.5% of the total photosyn­thesis research carried out in the world, there is a lot to be learnt from them. For example, quantum coherence in living systems was first discovered in the FMO light-harvesting complex of the Chlorobi¹, and potential applications of this quality may provide some insight to develop more efficient solar cells². Strains of the phylum Chloroflexi have a unique car­bon fixation pathway, unlike those in plants and Cyanobacteria, which is thought to be more energy efficient under specific conditions³. Efforts to engineer this pathway in a model cyanobacterium have been attempted recently⁴. Heliobacteria are ubiquitous and powerful nitrogen fixers, commonly found in rice paddies around the world⁵. They are some of the fastest growing photoheterotrophic bacteria in nature; however, their ecological importance has not been determined and their biotechnological potential has not even been acknowledged in the literature. In conclusion, the entire diversity of photosynthetic bacteria represents a new frontier of research that if pursued, will certainly have a far-reaching societal and technological impact.

Functional and structural characterization of photochemical reaction centers

As mentioned above, reaction centers are distributed in at least seven groups of distantly related bacteria. They come in two forms distinguished by the primary photochemical steps and known as Type I and Type II reaction centers (Figure 1). Today, there are crystal structures available for reaction centers in Cyanobcateria, plants, and in Proteo­bacteria, but none in the other phototrophic systems. The Photobiology Group will therefore obtain high-resolution crystal structures of reaction centers from strains that are very poorly understood; namely, Heliobacterium modesticaldum (Heliobacteria), Roseiflexus castenholzii (Chlor­oflexi), and Chlorobium tepidum (Chlorobi). Nonetheless, I already have in my laboratory 8 additional strains selected because of their remarkable reaction centers that I will bring with me to France.

I have selected those three targets because their function and structure still remains to be elucidated. Moreover, they contain immense evolutionary information. For example, the Type I reaction center from Heliobacteria and the Chlorobi are the simplest in nature and might be structurally similar to the earliest evolving systems. However, their fundamental chemistry still remains a puzzle because the role of quinones in electron transfer has not been conclusively demonstrated. It is possible that under certain conditions these Type I reaction centers may behave like Type II instead. Demonstrating that these simple reaction centers could have a dual function would be a fantastic discovery that could change the way we think about photosynthesis. On the other hand, the reaction center from Roseiflexus has a unique protein domain not seen in any other proteins that could give clues on how water oxidation catalysis evolved in Cyanobacteria⁶.

Experimentally the reaction centers will be studied in vivo, in isolated membranes, and in the purified enzyme. For example, excitation energy and electron transfer under different conditions will be measured; as well as any alterations to the energetics of cofactors, protein composition, or oligomeric forms. Changes to the photosynthetic machinery under stress conditions (e.g. high light intensity, iron or nitrogen starva­tion) will be monitored too. The results will be compared to those in Cyanobacteria for which extensive data is available. The key objective is to have a clear picture of the function and dynamics of the reaction center in the target strains, a picture that is not yet available to a satisfactory level of detail, if at all. Simultaneously, crystal trials will be initiated as purified enzymes become available. I have experience purifying the reaction center from H. modesticaldum, Photosystem II, and Photosystem I. I also have experience with various spectroscopic methods such as, absorption, fluorescence, and electron paramagnetic resonance (EPR) spectroscopy; in addition to gel-based and gel-free proteomic approaches and in the application of electrochemical methods to reaction centers. These techniques will be applied judiciously in order to study function as deemed necessary.

Currently, I am optimizing crystallization conditions for the reaction center from H. modesticaldum; pre­liminary data suggests promising conditions. These conditions and those available for Photosystem I⁷ could be used as a starting base for the structure of Chlorobium. An attempt at crystallizing the reaction center from Chlor­o­flexus aurantiacus was published 20 years ago, but a structure was never re­leased⁸. This protocol could be further improved for the structure of Roseiflexus. Alternatively, modifications to available meth­ods to crystallize Photosystem II⁹ or the proteobacterial reaction center¹⁰ could be tried as well. Another possibility is to obtain structural models using electron microscopy. The complete characterization and structural determination of one or two of the reaction center shall make for a very exciting PhD project or postdoctoral position, which should pro­vide extensive results for multiple high-impact publications.

Design, remodeling, and enhancement of photosynthetic systems

We require a new source of energy. Biofuels from photosynthetic organisms have been considered to be part of the solution to the energy crisis, but one of the grand challenges is that overall, the efficiency of photosynthesis is low¹¹. This is because the solar to biomass energy conversion efficiency is around 1% or less (in real life, not under optimal laboratory conditions). In other words, the ratio of en­ergy returned in the biofuel relative to the energy invested to produce it is currently quite unfavorable, even in the best case scenarios. As a result, it has been hypothesized that the natural limits of photosynthesis could be enhanced or overcome¹², but no experimental validation of such hypotheses has been provided yet. All of the approaches proposed to improve photosynthesis in living system require genetic engineering. For example, it has been suggested that a reaction center could be engineered to absorb light in the far-red region beyond the photosyn­thetically active radiation, and this could potentially double its photosynthetic efficiency. Such approach requires: 1) the expression of new pigment synthesis pathways in parallel to the native ones, 2) the expression of a new reaction center from a distinct organism into the host strain, or 3) both 1) and 2) at the same time. However, we still do not completely understand pigment synthesis and reaction center biogenesis. Although great advances have been made in the past decades, still some of the steps, enzymes in the pathway, and assembly factors, have not been identified or are very poorly characterized.

          I propose here a novel strategy―not yet discussed in the literature―to get great insight into how correctly engineer a photosynthetic system and consequently, how to improve it. The first stage of this module is to engineer photosynthesis in a heterotrophic bacterium; or in other words, to reverse engineer photosynthesis from scratch. My group will transfer a photosynthetic gene cluster from a phototrophic gammaproteobacterium to Escherichia coli, which is also a gammaproteobacterium. Genetically, they should be somewhat alike. Similar approaches have been attempted before to engineer N₂-fixation in E. coli successfully¹³. To do this, a nitrogenase gene cluster from Klebsiella oxytoca containing about 20 genes was refactored and then transferred into E. coli. K. oxytoca is also a gammaproteobacterium. The photosynthetic gene cluster varies in size from organism to organism ranging from 15 to 25 genes. Therefore, this technology could be used as a starting foundation. We will take it several steps further.

In addition, photosynthetic gammaproteobacteria are known to transfer genes in nature: for exam­ple, Cyanobacteria of the marine Synechococcus and Prochlorococcus clade have obtained numerous photosynthetic genes from Gammaproteobacteria, including circadian clock components, carboxysome components, Ru­bisco, chlorophyll synthesis genes, among many others. Gammaproteobacteria have also been shown to donate a photosynthetic gene cluster to strains of the rare phylum Gemmatimondetes, and these have been demonstrated to be able to express functional reaction centers¹⁴. Horizontal gene transfer (HGT) events between organisms of different phylum should be much more difficult to occur than within more closely related bacteria, therefore I think inserting a photosynthesis gene cluster into E. coli is quite feasible. Furthermore, several phototrophic gammaproteobacte­rial genomes are publicly available and some strains are amenable to cultivation and genetic engineering. I will start with the photosynthesis gene cluster of Thiocapsa roseopercisina; an anoxygenic photosynthetic gammapro­teobacterium, which has been of particular interests because of its O₂-tolerant hydrogenase.

There are other alternatives that can be tried in parallel. For example, the photosynthesis gene cluster of a heliobacaterium (a Firmicutes) could be transferred to a strain of clostridia, their closest non-phototrophic relative. Clostridia are a type of bacteria with immense potential in the production of chemicals (e.g. ethanol, butanol, acetone, etc.) and making them phototrophic may give current strains a technological and renewable edge.

If a functional reaction center can be engineered as a proof-of-concept in a non-phototrophic bacterium, the possibilities to follow this up are limitless. First, selected genes in the cluster could be removed or new ones added, in order to find the minimum necessary genetic requirements for phototrophy and photoautotrophy. A mix of genes from different organisms could be put together into novel gene clusters using high throughput methods for generating combinatorial libraries (e.g. through Golden Gate cloning) and for screening. Then it will be possible to test whether functionality or activity yields could be improved or not. In addition, the gene cluster could be inserted into strains of E. coli that have already been engineered to produce diverse biofuels or compounds of interest to test if production is coupled or enhanced with light utilization. A step farther would consist in expressing a photosynthetic gene cluster in a yeast model. However, the ultimate goal is to transfer a photosynthetic gene cluster encoding the capacity for oxygenic photosynthesis from Cyanobacteria. A gene cluster for oxygenic photosynthesis does not exist in nature, so it would be 100% artificially designed. In this case, the engineered strain would use water and light as the main energy source and thus would be completely photoautrotrophic. This project, though risky, will provide invaluable insight into the nature of photosynthesis and teach us immeasurably on the creation of novel life forms.

           

Origin and evolution of photosynthesis and bioenergetic systems

Another one of my personal scientific interests is evolution. How photosynthesis originated and diversified remains one of the greatest puzzles in the history of life. I have set myself the personal goal to reconstruct the most detailed evolutionary scenario yet for the origin and diversification of photosynthesis. I have made good progress towards this with some of my publications in the past four years^6,15,16. Earlier this year, I published a major reassessment of the evolution of reaction centers⁶. In addition, I led and published an exhaustive phylogenetic study of the D1 protein of Photosystem II, which provided for the first time, a clear picture of how the water oxidizing complex of oxygenic photosynthesis evolved and the dramatic transitions Photosystem II underwent in its path to acquiring water oxidation catalysis¹⁶. My work demonstrated how the structural and functional data available for Photosystem II can be used to gain evolutionary information at an unprecedented level of detail, if integrated with powerful phylogenetic analysis. As structural and functional information from the studied photochemical reaction centers become available in my group, these will be used to create even more precise molecular evolutionary models.

       At Institut Pasteur I also plan to extend these evolutionary studies to the evolution of several major cofactor synthesis pathways relevant to photosynthesis: namely, the chlorophyll, heme, quinone, and carotenoid biosynthesis pathways. Understanding the evolution and extent of the current diversity of cofactor biosynthetic pathway could come in handy when redesigning and refactoring the photosynthetic gene clusters. It could inform us on what genes or strains could be most promising. This would be harder to achieve if a good understanding of the diversity and evolution of phototrophy is lacking.

References

1 Engel, G. S. et al. Evidence for wavelike energy transfer through quantum coherence in photosynthetic systems. Nature (2007) 446, 782-786.

2 Park, H. et al. Enhanced energy transport in genetically engineered excitonic networks. Nat Mater (2015) doi: 10.1038/nmat4448.

3 Zarzycki, J., Brecht, V., Muller, M. & Fuchs, G. Identifying the missing steps of the autotrophic 3-hydroxypropionate CO2 fixation cycle in Chloroflexus aurantiacus. PNAS (2009) 106, 21317-21322.

4 Shih, P. M., Zarzycki, J., Niyogi, K. K. & Kerfeld, C. A. Introduction of a synthetic CO2-fixing photorespiratory bypass into a cyanobacterium. J Biol Chem (2014) 289, 9493-9500.

5 Asao, M. & Madigan, M. T. Taxonomy, phylogeny, and ecology of the heliobacteria. Photosynth res (2010) 104, 103-111.

6 Cardona, T. A fresh look at the evolution and diversification of photochemical reaction centers. Photosynth res (2015) 126, 111-134.

7 Jordan, P. et al. Three-dimensional structure of cyanobacterial Photosystem I at 2.5 Å resolution. Nature (2001) 411, 909-917.

8 Feick, R., Ertlmaier, A. & Ermler, U. Crystallization and X-ray analysis of the reaction center from the thermophilic green bacterium Chloroflexus aurantiacus. FEBS lett (1996) 396, 161-164.

9 Umena, Y., Kawakami, K., Shen, J. R. & Kamiya, N. Crystal structure of oxygen-evolving Photosystem II at a resolution of 1.9 Å. Nature (2011) 473, 55-60.

10 Xu, Q. et al. X-Ray structure determination of three mutants of the bacterial photosynthetic reaction centers from Rb. sphaeroides; altered proton transfer pathways. Structure (2004) 12, 703-715.

11 Cotton, C. A., Douglas J. S., De Causmaecker, S., Brinkert, K., Cardona T., et al. Photosynthetic constraints on fuel from microbes. Front Bioeng Biotechnol (2015) 3, 36, doi: 10.3389/fbioe.2015.00036.

12 Ort, D. R. et al. Redesigning photosynthesis to sustainably meet global food and bioenergy demand. PNAS (2015) 112, 8529-8536.

13 Smanski, M. J. et al. Functional optimization of gene clusters by combinatorial design and assembly. Nat Biotechnol (2014) 32, 1241-U1104.

14 Zeng, Y. H., Feng, F. Y., Medova, H., Dean, J. & Koblizek, M. Functional Type 2 photosynthetic reaction centers found in the rare bacterial phylum Gemmatimonadetes. PNAS (2014) 111, 7795-7800.

15 Cardona, T., Sedoud, A., Cox, N. & Rutherford, A. W. Charge separation in Photosystem II: A comparative and evolutionary overview. BBA-Bioenergetics (2012) 1817, 26-43.

16 Cardona, T., Murray, J. W. & Rutherford, A. W. Origin and evolution of water oxidation before the last common ancestor of the cyanobacteria. Mol Biol Evol (2015) 32, 1310-1328.

Reconstructing the origin of oxygenic photosynthesis

I want to share with you a link to my most recent article where I describe in detail, how photosynthesis and water oxidation evolved.

http://journal.frontiersin.org/article/10.3389/fpls.2016.00257/abstract


Abstract

Due to the great abundance of genomes and protein structures that today span a broad diversity of organisms, now more than ever before, it is possible to reconstruct the molecular evolution of protein complexes at an incredible level of detail. Here, I recount the story of oxygenic photosynthesis or how an ancestral reaction center was transformed into a sophisticated photochemical machine capable of water oxidation. First, I review the evolution of all reaction center proteins in order to highlight that Photosystem II and Photosystem I, today only found in the phylum Cyanobacteria, branched out very early in the history of photosynthesis. Therefore, it is very unlikely that they were acquired via horizontal gene transfer from any of the described phyla of anoxygenic phototrophic bacteria. Second, I present a new evolutionary scenario for the origin of the CP43 and CP47 antenna of Photosystem II. I suggest that the antenna proteins originated from the remodeling of an entire Type I reaction center protein and not from the partial gene duplication of a Type I reaction center gene. Third, I highlight how Photosystem II and Photosystem I reaction center proteins interact with small peripheral subunits in remarkably similar patterns and hypothesize that some of this complexity may be traced back to the most ancestral reaction center. Fourth, I outline the sequence of events that led to the origin of the Mn4CaO5 cluster and show that the most ancestral Type II reaction center had some of the basic structural components that would become essential in the coordination of the water-oxidizing complex. Finally, I collect all these ideas, starting at the origin of the first reaction center proteins and ending with the emergence of the water-oxidizing cluster, to hypothesize that the complex and well-organized process of assembly and photoactivation of Photosystem II recapitulate evolutionary transitions in the path to oxygenic photosynthesis.

I will update this soon!

Relations of phototrophic bacteria and the evolution of photosynthesis

I am very interested in the evolution of photosynthesis. From my research, I have come to conclude that photosynthesis evolved near the root or at the root of the tree of life of the domain Bacteria. In other words, the core components of photosynthesis such as reaction center proteins or the enzymatic components of the chlorophyll and bacteriochlorophyll synthesis pathway, originated before most of the known phyla of bacteria appeared for the first time.

A way to understand how and when photosynthesis originated we need to understand the phylogenetic relations of bacteria. Known phototrophic bacteria are found in seven phyla of bacteria: Cyanobacteria, Firmicutes, Chloroflexi, Chlorobi, Proteobacteria, Acidobacteria, and Gemmatimonadetes. In addition to this, it has been shown that the phylum Actinobacteria might have been ancestrally capable of phototrophy, because some of the strains in this phylum seem to have a vestigial chlorophyll synthesis pathway.

An interesting aspect about the evolution of photosynthesis is that all the phyla containing phototrophic bacteria are clustered within non-phototrophic groups.

Within the phylum Chlorobi there are at least two classes, Chlorobea and Ignavibacteria. All described members of the Ignavibacteria class lack phototrophy. The phylum Chlorobi is very closely related to the phylum Bacteroidetes and no phototrophic Bacteroidetes have been described yet.

Within the phylum Firmicutes, phototrophy is only found in the family Heliobacteraceae (Heliobacteria). Heliobacteria is closely related to the family Peptococcaceae, with the closest strains to Heliobacterium modesticaldum (the only fully sequenced strain of Heliobacteria) being Desulfitobacterium and Synthrophobotulus, which are not phototrophic. What is more, the phylum Firmicutes is subdivided in several classes including, Clostridia, Bacilli, and others. The Heliobacteraceae family belongs to the class Clostridia.

Within the phylum Proteobacteria, only the Alpha-, Beta-, and Gammaproteobacteria are phototrophic, but this is not a universal trait among them. There are many representative of these classes that are not phototrophic. Currently there are no described strains in the class Delta- and Epsilonproteobacteria  with phototrophy.

Only a single strain in the phylum Acidobacteria has been described with phototrophy. Chloracidobacterium thermophilum, but it is probably not the only one. All other strains described in this phylum are non-phototrophic. It should be said though, that only very few strains in this phylum have been characterized. Acidobacteria is closely related to the Proteobacteria as demonstrated by many phylogenomic analysis, sometimes the phylum actually clusters within the Deltaproteobacteria. It is very likely that Acidobacteria and Proteobacteria shared a common ancestor.

Cyanobacteria were thought to be made of only phototrophic strains, however the discovery of Melainabacteria has shown that also Cyanobacteria have very close non-phototrophic relatives. It has been suggested that Melainabacteria should be classified within the phylum Cyanobacteria, and the known photosynthetic Cyanobacteria should be downgraded to class level.

The phylum Chloroflexi is made of about eight different classes, only two of them, the Chloroflexia and the Anaerolineae, have been shown to contain phototrophic strains.

Only one strain in the phylum Gemmatimonadetes has been described to be capable of phototrophy. It appears that this strain obtained photosynthesis via horizontal gene transfer from proteobacteria. However, metagenomic analysis has demonstrated that there are more strains in this phylum with phototrophy.

It is usually considered that the scattered distribution of phototrophy in the tree of life of bacteria is the outcome of horizontal gene transfer, but this is probably not completely correct. So it is suggested for example, that phototrophy could have evolved in the phylum Chlorobi and then via horizontal gene transfer it seeded all other branches. In the case of Cyanobacteria, it is common to suggest that the phylum obtained both reaction centers from two distinct phyla of anoxygenic phototrophic bacteria. This is just one example but almost all possible origins have been considered, including Heliobcteria, Chloroflexi, Cyanobacteria, and Proteobacteria as the original innovators of photosynthesis.

There is absolutely no data or piece of evidence to suggest that photosynthesis originated in any of the described phototrophic groups. All reaction center proteins and all core proteins of the chlorophyll synthesis pathway (e.g. Mg-chelatase, Mg-protoporphyrin IX methyl transferase, protochlorophyllide a oxidoredutase, chlorophyllide a oxidoreductase) share a common origin. They should have descended from ancestral forms that existed more than 3.5 billion years ago. Therefore these ancestral forms should have existed before they diversified into the forms commonly found in the extant groups that we have right now. None of the groups of phototrophs we know today carry these ancestral forms of reaction center proteins or chlorophyll synthesis genes. So for example, suggesting that photosynthesis evolved in the Chlorobi implies that the phylum Chlorobi already existed 3.5 billion years ago; not only that, but it implies that the divergence of Ignavibacteria and Chlorbia had already occurred then, and the divergence of Bacteroidetes and Chlorbi too, and in consequence it has the ultimate implication that the vast majority of groups of bacteria had already differentiated at that time: which is virtually impossible. The same applies if we select any of the phototrophic groups as possible birth places of photosynthesis. In fact, it is very likely that the divergence events that caused the diversification of the group of bacteria mentioned above: Chlorobi/Bacteroidetes, Cyanobacteria/Melainabacteria, Acidobacteria/Proteobacteria, Chlostridia/Bacilli occurred around the Great Oxygenation Event or after. Most likely, photosynthesis is an ancestral trait of the domain Bacteria that has been lost as the different groups of microbes adapted to an oxygenic world, to heterotrophic lifestyles (due to global primary production being relegated to oxygenic photosynthesis), and to live in symbiotic or parasitic relationships with all eukaryotes on Earth. This is valid even if horizontal gene transfer has occurred among ancestor of today’s phyla at some point in time. The only convincing and unambiguous case of horizontal gene transfer of phototrophy is the case of Gemmatimonas and this probably occurred only recently.

Can rubisco be used in a technology for carbon capture?

How much rubisco is needed to capture 37 Gigatonnes of CO₂ in one year? 37 Gigatonnes is the current amount of human emissions per year.

Assuming that 1 single rubisco enzyme fixes 3 CO₂ molecules per second, then in a year 1 mole of rubisco, weighing about 0.49 tonnes, could fix 4163.69 tonnes of CO₂. To fix 37 Gigatonnes of CO₂ in one year, it will be necessary 4.35 Megatonnes of rubisco. To produce this amount of rubisco in one year, the rate of production needed is 0.138 tonnes of rubisco per second. I found somewhere that in nature about 1000 tonnes of rubisco are produced per second, so the rate of production would be equivalent to 0.01% of nature’s.

In comparison 300 Megatonnes of plastic are produced each year as of 2013, equivalent to a rate of production of 9.51 tonnes per second.

If you spread the production of 0.138 tonnes of rubisco per second across countries all over the world, then it doesn't really seem like much.

The above example assumed that 1 rubisco could be active for an entire year, but in reality the half-life of rubisco is of several days, which is actually pretty stable. This means the production rate has to be higher than those values to compensate for the inactivation of the enzyme. On the other hand, there are also better rubiscos that can fix more than 3 CO₂ molecules per second, so it all balances itself out.

The big question is: can a hybrid system that combines genetic engineer, materials, surfaces, and other industrial technologies be implemented to use the reactions of the Calvin-Benson-Bassham cycle as a CO₂ sequestration mechanism? My colleagues at Imperial would probably say NO… but I’m hopeful that we’re only scratching the surface of the technologies that we’ll be able to develop in the near and far future. This system would have to be fully powered by renewables of course (e.g. solar) and it would have to be better than biomass accumulation.

What do you think?

Nature's carbon capture enzyme, also known as rubisco