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.