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 population1,2. 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 bacteriochlorophyll6,7.
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
overcome20,21, 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 characterized22,23.
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
years6,7,31. 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.
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