Engineered expansion of photosynthesis into the near-infrared

Photosynthesis is the ultimate source of all food and most energy resources on Earth. It is generally accepted that the earliest forms of photosynthesis relied on the visible light-absorbing chlorophyll molecules, and that expansion of this process into longer wavelength regions of the spectrum-the far-red and near-infrared-evolved later, permitted by the more structurally complex bacteriochlorophyll molecules. This expansion took place prior to the evolution of oxygenic photosynthesis, in which high-energy photons absorbed by chlorophylls are used to drive the thermodynamically-demanding extraction of electrons from water, releasing oxygen into the atmosphere. The expansion of anoxygenic photosynthesis arose via the duplication of the genes (bchBLN) encoding protochlorophyllide oxidoreductase (POR), the enzyme catalysing the penultimate step in chlorophyll biosynthesis. The duplicated genes (bchXYZ) encode chlorophyllide oxidoreductase (COR), catalysing the first committed step in bacteriochlorophyll biosynthesis, producing a pigment with a greatly red-shifted absorption maximum, permitting photosynthesis outside the visible range.
COR reduces a C=C bond on the tetrapyrrole ring of the chlorophyllide pigment, but it has recently been discovered that the isoform of this enzyme present in anoxygenic phototrophs using bacteriochlorophyll a has a surprising secondary activity reducing a vinyl side group; this pigment permits absorption of light between 780-900 nm. The isoform found in organisms employing bacteriochlorophyll b (CORb) lack this vinyl reductase activity, and produces a pigment that is used for photosynthesis > 1000 nm.
The two chlorophyll-containing photosystems of oxygenic phototrophs, such as higher plants, have overlapping absorption spectra, thus compete for photons of the same wavelength. It has been suggested that the reengineering of one of the photosystems to use bacteriochlorophylls-producing a complex absorbing in the near-infrared-could double the efficiency of photosynthesis, leading to improvement of crop plants with increased yields and season lengths. Expression of COR genes in oxygenic phototrophs has so-far been unsuccessful; COR is a ferredoxin-dependent reductase that uses oxygen-sensitive [4Fe-4S] cluster cofactors for catalysis, but produces deadly superoxide in oxygenic hosts. Solution of the structure of COR, and comparison to the published structures of POR, will inform the redesign of the bacteriochlorophyll-synthesising enzymes to tolerate oxygen, conditions under which the POR enzyme has evolved to operate, allowing for the production of bacteriochlorophylls in oxygenic hosts.
Our objectives are:
- To purify the substrate-bound catalytic components of CORa and CORb from the anoxygenic purple phototrophic bacteria Rhodobacter sphaeroides and Blastochloris viridis, respectively
- To perform structural analysis of the purified proteins via X-ray crystallography and cryo-EM
- To identify the residues responsible for the different catalytic activities of the two CORs
- Use a combination of published and project-generated structural data, and directed evolution experiments to design/engineer a COR enzyme able to operate in the presence of oxygen
- To introduce this modified enzyme into an oxygenic cyanobacterium, to produce bacteriochlorophyll in these hosts for the first time (cyanobacteria contain the same photosystems as higher plants so are limited in the same way as described above).
Extension of the chlorophyll biosynthesis pathway in a cyanobacterium towards the production of bacteriochlorophylls will permit the assembly of a near-infrared absorbing complex. The knowledge obtained can then be used to engineer plants for the same purpose.