Investigating and Improving the Compatibility of the Photosynthetic Reaction Centre for Non-Native Redox Carrier Proteins

With current crops precipitously close to their theoretical productivity limits, considerable genetic engineering of photosynthesis will be required to keep pace with the increasing demands of a growing population. The light reactions of photosynthesis offer scope for improvement given chlorophyll-based phototrophs absorb poorly in the green and far-red regions of the electromagnetic spectrum. A synthetic biology approach can be envisaged that compensates for this spectral shortfall by combining the attributes of chlorophyll and bacteriochlorophyll-based photosystems. The long-term aim of this project is to re-engineer the far-red absorbing reaction centre–LH1 antenna (RC-LH1) complex from Rhodobacter sphaeroides to make it functionally compatible with the electron transport chain of the cyanobacterium Synechocystis sp. PCC 6803, an experimentally accessible chloroplast progenitor. This would represent a step towards a phototroph that can productively absorb all photosynthetically active radiation.
Bringing together what evolution has separated, a central design goal is to engineer the RC-LH1 complex to accept electrons from a different set of soluble redox carrier proteins, namely plastocyanin (Pc) and cytochrome c6 (cyt c6) rather than cytochrome c2 (cyt c2). Following a suppressor mutation in the rne gene that increases intracellular Pc levels, both Synechocystis redox carrier proteins can support photoheterotrophic growth in Rhodobacter sphaeroides, albeit with longer doubling times, particularly for Pc. The two non-native proteins were also capable of reducing the RC-LH1 complex in vitro, although kinetic assays showed greater compatibility under high salinities without the LH1 ring, which appears to have a functional role in improving the activity and selectivity of the RC. To invert its donor specificity, the periplasmic face of the RC-LH1 complex was redesigned to borrow key structural features from photosystem I, either the twin tryptophan or the aspartate-arginine motif, achieving up to 3-fold activity improvements that raise the prospects that a PSIII can be designed form RC-LH1.