Rates of evolution of reaction center proteins and other proteins of photosynthesis

I have been doing molecular clock analysis of several proteins and enzymes relevant to photosynthesis. This has allowed me measure the rate of evolution. Several of this I have presented in my recent paper available in bioRxiv, which I have just submitted for peer-review. I present here a few more of other unpublished projects.

The rates are given as amino acid changes per site per billion years

Photosystem II core protein - D1: 0.088
Photosystem II core protein - D2: 0.12
Anoxygenic Type II RC core protein - L: 0.63
Anoxygenic Type II RC core protein - M: 0.60

Photosystem I core protein - PsaA: 0.13
Photosystem I core protein - PsaB: 0.15
Anoxygenic Type I RC protein of heliobacteria - PshA: 0.27
Anoxygenic Type I RC protein of the Chlorobi - PscA: 0.24

Mg-chelatase: ChlD/BchD: 0.60 (average from all phototrophs)
Putative Co-chelatase homolog to ChlD: 0.73 (average from archaea strains)

These were calculated using Phylobayes 3.3f under a CAT model using the birth-death model and soft bounds on the calibrations points, assuming an age for photosynthesis around 3.5 billion years ago.

If you want more details, please look at the paper above or send me an email.

Directed evolution of water oxidation catalysis for improved photosynthesis

Recently, a funding opportunity became available at Imperial open to all academic stuff at all levels. I participated with this little project about doing directed evolution on Photosystem II. The pre-proposal had to be really short so almost no considerations on the project can be really made. Only 1 proposal per department had to be put forward for the final round of selection. I ranked 3 out of 6...

The big issue I see with the project is that from an evolutionary perspective the rate of PSII water oxidation are limited not by the S cycle itself but by quinone exchange. The slow rates of quinone exchange at the same time are determined by the rates of quinone oxidation in the cytochrome b6f and other downstream processes.

It would be fun to prove that water oxidation can occur faster than it does. The directed evolution approach will probably have to also accelerate the rates of quinone exchange in PSII and also downstream in the thylakoid membrane and metabolic electron sink.

Summary of the project

Raising populations and greater incomes per capita will result in an unprecedented demand for food, fuel, and high-value products. This demand will not be met without an improvement of the efficiency of photosynthesis: the ultimate frontier in photosynthesis research. The engine that powers photosynthesis is called Photosystem II, a complex molecular machine that converts light into useful energy by decomposing water into protons, electrons, and oxygen. This chemical reaction is known as water oxidation and it is the source of all energy that sustains complex life and human societies. I hypothesise that Photosystem II has the potential to oxidise water several-fold faster than observed in known photosynthetic organisms. To test this hypothesis I will use directed evolution to select for variant Photosystem II with accelerated rates of water oxidation. The project aims to provide experimental support for the possibility of enhancing the catalytic efficiency of Photosystem II. The results of this innovative and high-risk project have the potential to be directly translated into strategies for the engineering of enhanced photosynthetic organisms.

Proposal 

It is likely that in the next decades the global demand for food, fibre, bioenergy, biopharmaceuticals, and other chemical precursors will not be met sustainably without significant improvements of the photosynthetic efficiency of crops and algae of biotechnological potential.^1,2

A radical and high-risk approach that could result in a significant enhancement of photosynthetic efficiency is the direct improvement of the rate of catalysis of Photosystem II, the light-driven water:plastoquinone oxidoreductase enzyme of oxygenic photosynthesis. I will employ directed evolution to screen and select for Photosystem II variants that display faster rates of water oxidation. The specific goal of the project is to demonstrate that faster rates of biological water oxidation are catalytically and thermodynamically possible.

Gene diversification will be accomplished using genome-wide random mutagenesis³ and iterative saturation mutagenesis⁴ of the core subunits of Photosystem II targeting the first and second coordination sphere of the Mn₄CaO₅ cluster, the exchangeable plastoquinone binding site, and the proton pathways. Cyanobacteria mutants will be screened for potential alterations in water oxidation photochemistry in a plate reader spectrometer using a range of oxygen sensitive dyes. Strains with potentially faster kinetics of water oxidation will be extensively characterised with the range of electrochemical, spectroscopic, and biochemical techniques available in my lab. Successful variants from both gene diversification strategies could be integrated using DNA shuffling.

                The accomplishment of improved catalytic efficiency of Photosystem II would be a tremendous breakthrough and should open a direct route for the technological realisation of enhanced photosynthesis in crops, eukaryotic algae, and cyanobacteria. Furthermore, it should expedite the development of artificial catalysts that mimic the water oxidation cycle, which still remains an outstanding technological challenge.^5,6

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

2. Tilman, D., Balzer, C., Hill, J. & Befort, B. L. Global food demand and the sustainable intensification of agriculture. PNAS, 108, 20260-20264, (2011).

3. Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat Rev Genet 16, 379-394, (2015).

4. Reetz, M. T. & Carballeira, J. D. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2, 891-903, doi:10.1038/nprot.2007.72 (2007).

5. Zhang, C. X. et al. A synthetic Mn4Ca-cluster mimicking the oxygen-evolving center of photosynthesis. Science 348, 690-693, (2015).

6. Schulze, M., Kunz, V., Frischmann, P. D. & Wurthner, F. A supramolecular ruthenium macrocycle with high catalytic activity for water oxidation that mechanistically mimics Photosystem II. Nat Chem 8, 577-584, (2016).

The uncanny connections between methanogenesis and photosynthesis

I have become quite interested in the evolutionary relationship between methanogenesis in archaea and photosynthesis in bacteria.

The first evolutionary link comes from the homology between the two enzymes of chlorophyll and bacteriochlorophyll synthesis, Protochlorophyllide reductase (BchLNB or ChLNB) and Chlorophyllide reductase (BchXYZ), and the enzyme required for the synthesis of the Ni-tetrapyrrole cofactor, coenzyme F430, of methyl-coenzyme M reductase, which is essential for methanogenesis. 

Another enzyme of chlorophyll synthesis, BciB (8-vinyl reductase) may also be related to another enzyme of methanogenesis (FrhB). See this paper.

Now a new paper by Kono et al (2017) seems to find that this evolutionary link goes much deeper, as it seems that methanogenic archaea not only have a proper rubisco, but also have phosphoribulokinase (PRK), previously considered to be unique to photosynthetic organisms that use the Calvin-Benson-Bassham cycle for carbon fixation.

I know that some enzymes of methanogenesis seem to trace back to the last universal common ancestor (LUCA), but the same isn't true for photosynthesis. From this perspective it would seem that photosynthesis emerged perhaps by borrowing a few components from methanogenesis.

Recently, Martin et al (2017) said in a recent perspective: "Let us presume, just for a moment, that the first bacteria and archaea were acetogens and methanogens respectively. On an uninhabited planet, they have no competitors, and life multiplies quickly given ample growth substrates. The founders of their respective domains would have bubbled off into the ocean bottom waters to be spread around by currents and eventually to be introduced back into hydrothermal systems in the crust [...]"

I would add to this, that life could have only spread around the globe and take a hold during the early Archaean after the evolution of photosynthesis. In fact, I would go as far as to say that it was the innovation of photosynthesis what allowed the early microbes to escape the hydrothermal vents and the crust... but other may disagree! :)

Stromatolitic-chert member, Strelley Pool Formation, middle to lower Paleoarchean, 3.35 to 3.46 Ga. Photo by James St. Johns.

Just thinking about things can be a source of scientific progress

I just read "One step beyond a ribosome: The ancient anaerobic core", by Filipa L. Sousa, Shijulal Nelson-Sathi, William F. Martin, published in Biochimica et Biophysica Acta 1857 (2016) 1027–1038

It is a fascinating paper. I particularly enjoyed the following concluding line:

"With regard to the most primitive forms of microbial physiology, microbiologists reached the same conclusion 45 years ago, namely that methanogens and acetogens probably represent the most ancient lineages. We required 2000 genomes and powerful computers for our conclusions, while Decker et al. just thought about it. Evidently, just thinking about things can be a source of scientific progress."

I like to think about things, I like to think especially about photosynthesis ;)

Fossil stromatolite

The letter that kick-started my science career

When I was finishing my undergraduate studies in Colombia, early 2004, I found myself quite confused because I didn't know what to do with my degree in biology... there used to be so little research done in Colombia that becoming a researcher in Colombia seemed like a incredibly remote possibility. I wasn't really into field-work and found slugs and other critters really disgusting to the touch, so I wasn't going to find myself in the jungle anytime soon. The only thing that seemed like a possibility was to teach biology in a high-school, but that wasn't really motivating me either. Nevertheless, I was incredibly interested in photosynthesis research!

Everything changed when I received some good advice from my final-year-project supervisor. He suggested that I should contact laboratories around the world and ask for postgraduate positions. That little piece of advice changed my life! I then proceeded to contact every laboratory in the world doing research in Photosystem II, my favorite subject. From many places I never heard back, but a couple of them were quite interested: Sweden in particular.

This is the unaltered letter that I sent to Prof. Stenbjörn Styring at Uppsala University. He then invited me to do a PhD in his lab. I traveled to Sweden two months after I got my Biology certificate.

My name is Tanai Cardona Londoño, I am a 21 years old Biologist from the University of los Andes, in Bogotá, Colombia. Next September I shall receive my undergraduate title. In consequence, I have begun to approach myself toward my professional and scientific goals: the study of the water oxidation by the oxygen-evolving complex. 

Throughout the four and a half years of universitary studies, I have been marveled with the amazing mechanism of photosystem II and its redox chemistry. Inevitably, I have had to meet with the extensive publications from your group, as well as those of other research centers around the world. Therefore, I have been reading about a great variety of related subjects, from the crystallographic structures and spectroscopic measurements to the evolutionary proposals of its appearance in nature. 

In that way, I decided to write for my graduation thesis a monograph about the recent advances in WOC-PSII structure and function, in order to familiarize me with the current investigations in the field. I took the decision to elaborate a bibliographic revision because there is neither anybody nor a laboratory interested and equipped in photosynthesis research around the whole boundaries of Colombia. However, I have been directed by a plant molecular biologist Ph.D.―who advise me to write you. 

Now that I am close to end my undergraduate studies, I am very interested to begin my postgraduate studies in the fields of structural biology or biochemistry; ideally centered in the investigation of the structure and function of metalloenzymes, especially PSII. That is the reason which motivate me to write this letter, namely to look for the possibility of join me with your research group. This would be invaluable for my scientific growth. At the same time, I would give all my effort and capabilities of working hard. 

I was wondering if there is a possibility for linking me with your research team and simultaneously begin my postgraduate studies with a fellowship or some other kind of economical support. If there is, please tell me what should I do to get this great opportunity. If there is not such opportunity, nevertheless, it has been a great pleasure to write you. 

Sincerely,

Tanai

Uppsala, Sweden

Structure and function of photochemical reaction centers

This is a research proposal that I submitted as part of an application for a position as an assistant professor at IST Austria. I was not shortlisted: evidently, they did not understand my awesomeness.

Structure and function of photochemical reaction centers

Research proposal for the position of Assistant Professor at IST Austria

Dr. Tanai Cardona

Department of Life Sciences, Imperial College London

London, UK

Why photosynthesis research at IST Austria

Life on earth is sustained by photosynthesis. Civilization depends on it because photosynthesis is the source of our food and gave rise to the fuels we burn to power our society. Scientists world-wide are trying to improve photo­synthesis to enhance crop productivity and thus feed a rapidly growing global population^1,2. At the same time, scientists are looking for ways to engineer photosynthetic organisms to produce clean energy alternatives to fossil fuels or high-value products, like plastics or pharmaceuticals³. Intensive research is also carried out to develop synthetic compounds that mimic natural photosynthesis to generate fuels directly from sunlight and water⁴, or to sequester carbon as a promising strategy to combat climate change⁵. As a result, the study of photosynthesis now is more relevant than ever before, as it is at the heart of many solutions to overcome current global challenges. However, our understanding of photosynthesis is enormously biased (Figure 1); our knowledge is based on just a handful of model plants and a few strains of bacteria. These are a very tiny fraction of the entire biodiversity of photosynthetic systems that abound in the planet and that still remains largely unexplored. It is this potential for breakthrough and discovery that excites me, it is the main reason I decided to pursue a career doing photosynthe­sis research when I was still an undergrad student, and it is the reason why I want to lead my own research group today.

Figure 1. This graph shows how little we know about photosynthetic systems. In the past 5 years there have been more than 20000 publications on Arabidopsis alone, a model plant system. Arabidopsis and Spinacia are plants; Synechocystis, Thermosynechococcus, and Gloeobacter, are model strains of Cyanobacteria. Rhodobacter and Blastochlororis are strains of Proteobacteria (purple bacteria). Together these strains make for 98.5% of all the publications accessible with PubMed by searches using the genus name of “the most popular” model organisms of each group. Chloroflexus and Roseiflexus are strains of Chloroflexi (green nonsulfur bacteria); Chlorobaculum and Chlorobium are strains of Chlorobi (green sulfur bacteria); Heliobacterium and Heliobacillus are strains of Firmicutes (heliobacteria); Chloracidobacterium and Gemmatimonas are newly discovered types of phototrophs belonging to the phylum Acidobacteria and Gemmatimonadetes, respectively. The data was retrieved September 2015.

Photosynthesis is possible thanks to photochemical reaction centers (Figure 2). These are molecular machines that transform the energy of light into chemical energy. In other words, nanoscaled photovoltaic solar panels found within the photosynthetic cell. They are fascinating because of their complexity, variety of forms, specialized chemistry, and incredible technological potential. As a result, I plan to build a multidisciplinary research group that combines biochemistry, biophysics, structural, evolutionary, and synthetic biology to study and exploit photochemical reaction centers and photosynthesis. In particular, focusing on those photosynthetic systems that are still very poorly understood. I propose here three research modules that I will develop at IST Austria:

1.       Functional and structural characterization of  photochemical reaction centers

2.       Design, remodeling, and enhancement of photosynthetic systems

3.       Reconstructing the origin and evolution of photosynthesis

Figure 2. Photochemical reaction centers from Cyanobacteria. Both are transmembrane multiprotein complexes carrying hundreds of cofactors, included chlorophylls, carotenoids, lipids, hemes, FeS clusters, among others. Type II reaction centers are distinguished because they lack FeS clusters. In this system after light absorption and charge separation, electrons are shuttled to quinones molecule (e.g. menaquinone, ubiquinone, plastoquinone). After it is reduced the quinone is released to the membrane to be oxidized by a Cytochrome bc₁ or b₆f complex. This contributes to the formation of a proton gradient and ATP synthesis. Among Type II reaction centers, Photosystem II is unique because of the water oxidizing complex, were oxygen evolution occurs. Type I reaction centers are characterized for having FeS clusters as terminal electron acceptors. After charge separation, the FeS clusters are oxidized by a ferredoxin, which then can go on to power metabolism (e.g. carbon or nitrogen fixation).

Photochemical reaction centers are thought to have originated only once in the domain Bacteria. This is because currently there are no described strains in the domain Archaea with photosynthesis based on protein complexes containing chlorophyll or bacteriochlorophyll^6,7. At the other end of the tree of life, eukaryotic algae and plants obtained photosynthesis via the endosymbiosis of a cyanobacterium. Within Bacteria, there are currently seven phyla known to have strains with reaction centers, these are: Cyanobacteria, Chloroflexi, Firmicutes, Chlorobi, Proteobacteria, and those recently found in Acidobacteria⁸ and Gemmatimonadetes⁹. Just a while ago, it was suggested that the phylum Actinobacteria might have been ancestrally capable of photosynthesis¹⁰, as some strains in this phylum seem to have a vestigial chlorophyll synthesis pathway. So today, 95% of research in photosynthesis is done in the Cyanobacteria/plant system, and less than 5% in the remaining types of phototrophic systems, as determined by the number of publications in the last five years (Figure 1). 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. Therefore, we know virtually nothing about most of the diversity of photosynthetic organisms currently inhabiting Earth.

Even though the less 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 Chlorobi¹¹, and potential applications of this quality may provide some insight to develop more efficient solar cells¹². 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 conditions¹³. Efforts to engineer this pathway in a model cyanobacterium have been attempted recently¹⁴. Heliobacteria are ubiquitous and powerful nitrogen fixers, commonly found in rice paddies around the world¹⁵. They are some of the fastest growing phototrophic 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.

Table 1. Phototrophic groups and some facts regarding crystal structures of reaction centers (RC).

PHYLUM	RC	STRUC-TURESa	SOURCES	BEST	INTERESTING TRAITS
Cyanobacteria and plastids	I	10	T. elongatus S. sp. 6803 Pea	2.5 Å16	Photosystem I comes in monomers, trimers, and tetramers
	II	15	T. elongatus T. vulcanus	1.9 Å17	The Mn4CaO5 cluster and water oxidation
Proteobacteria Purples	II	~100	R. sphaeroides B. viridis T. tepidum	1.8 Å18	Nobel Prize
Firmicutes Heliobacteria	I	0	―	―	It is made of a single subunit
Chlorobi Green sulfur	I	0	―	―	Chlorosomes and the Fenna-Matthews-Olson complex
Acidobacteria Chloracidobacterium	I	0	―	―	12 membrane-bound carotenoid protein subunits surround the complex
Chloroflexi Green non-sulfur	II	0	―	―	Three-pheophytin system
Gemmatimonadetes Gemmatinonas	II	0	―	―	Obtained RC via horizontal gene transfer recently
Actinobacteria Rubrobacter	―	―	―	―	Only has a vestigial chlorophyll synthesis pathway

^aCrystal structures with resolution better than 4.0 Å. Less than 1% of all protein structures in the PDB (Protein Data Bank) are from phototrophic organisms.

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 2). Today, there are crystal structures available for reaction centers in Cyanobcateria, plants, and in Proteo­bacteria, but none in the other phototrophic systems (Table 1). I will therefore focus on 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 could bring with me to Austria.

I have selected those three targets because their function and structure still remains to be elucidated. Moreover, they contain interesting 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.

This proposal is an expansion of an independent re­search program I started as a Research Fellow about two years ago here at Imperial. 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 I¹⁶ 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 re­leased¹⁹. This protocol could be further improved for the structure of Roseiflexus. Alternatively, modifications to available meth­ods to crystallize Photosystem II¹⁷ or the proteobacterial reaction center¹⁸ 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 low³. 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 overcome^20,21, 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 PAR (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^22,23.

            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 successfully²⁴. 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 be good at horizontally transferring genes (HGT) in nature: for exam­ple, Cyanobacteria of the marine Synechococcus/Prochlorococcus clade have obtained numerous photosynthetic genes from Gammaproteobacteria, including circadian clock components²⁵, carboxysome components and Ru­bisco²⁶, summed to chlorophyll synthesis genes²⁷, among many others²⁸. 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 centers⁹. Evidence for HGT of photosynthesis genes from Gammaproteobacteria to strains of Firmicutes of the genus Alkalibacterium has also been provided²⁹, but this result still awaits further experimental validation. HGT events between organisms of different phylum should be much more difficult to occur than within more closely related bacteria. 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 hydrogenase³⁰.

            If functional reaction center can be engineer 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. Combination of genes from different organisms could be mixed into novel gene clusters 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. Like this, it could be possible to test whether production of the desired compound can be enhanced by the acquisition of phototrophy. 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.

           

Reconstructing the origin and evolution of photosynthetic 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 my publications in the past four years^6,7,31. 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 catalysis³¹. 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. I am currently performing molecular clock analysis of reaction center proteins to time the origin of photosynthesis and to date important evolutionary events, such as the origin of the water oxidizing complex of Photosystem II. As structural and functional information from the targeted strains become available in my group, these will be used to create even more precise molecular evolutionary models.

          At IST Austria 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.

Although my research program at Imperial is set within an evolutionary context, I intend to develop and lead a more comprehensive and multidisciplinary research profile at the next stage of my career. I aim to branch from purely fundamental research into more applied biotechnological fields.

References

1        Price, G. D. & Howitt, S. M. Plant science: Towards turbocharged photosynthesis. Nature (2014) 513, 497-498.

2        Long, S.P., Marshall-Colon, A. & Zhu, X. G. Meeting the global food demand of the future by engineering crop photosynthesis and yield potential. Cell (2015) 161, 56-66.

3        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.

4        Faunce, T. et al. Artificial photosynthesis as a frontier technology for energy sustainability. Energ Environ Sci (2013) 6, 1074-1076.

5        Bensaid, S., Centi, G., Garrone, E., Perathoner, S. & Saracco, G. Towards artificial leaves for solar hydrogen and fuels from carbon dioxide. ChemSusChem (2012) 5, 500-521.

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

7        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,.

8        Bryant, D. A. et al. Candidatus Chloracidobacterium thermophilum: An aerobic phototrophic acidobacterium. Science (2007) 317, 523-526.

9        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.

10      Gupta, R. S. & Khadka, B. Evidence for the presence of key chlorophyll-biosynthesis-related proteins in the genus Rubrobacter (Phylum Actinobacteria) and its implications for the evolution and origin of photosynthesis. Photosynth res, doi:10.1007/s11120-015-0177-y (2015).

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

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

13      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.

14      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.

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

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

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