Engineering Photosystem I for Light-Driven Biocatalysis

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Reductive catalysis is a central element in a multitude of industrial and pharmaceutical processes, including 'functionalisation of bioactive compounds'1 and the reduction of alkenes2, but chemical catalysis is often unstable or functionally inert under standard conditions. This leads to a huge expenditure of energy and resources in maintaining the correct temperature and pressure environments for these reactions to be fruitful. In response to these limiters, there has been a great deal of successful research into alternative methods of catalysis, particularly in the fields of biocatalysis2 and photocatalysis3. Biocatalysis refers to the use of novel enzymes to catalyse reactions, while photocatalysis is a process in which photons are used to directly excite an inorganic semiconductor species, allowing it to catalyse the desired reaction4. Both approaches are currently used in industrial and pharmaceutical synthesis, however, greater reductive power could be achieved by merging these concepts. Photosystem I (PSI) is a biological molecular assembly for the ultimate photosynthetic reduction of NADP+ to NADPH, a reducing cofactor that powers metabolism in living systems. In recent years, there has been interest in engineering PSI/biocatalytic enzyme fusion proteins to harness the photocatalytic capabilities of PSI for use in novel reductive bio-photocatalysis5. Notably, PSI has been effectively fused with hydrogenase enzymes, resulting in highly reductive protein fusions capable of catalysing the reduction of H+ to elemental hydrogen using photons as the electron source6. Objectives To demonstrate that PSI can be used as a platform to develop novel light-driven enzymes that could be of biotechnological interest. To bioengineer a range of Photosystem I/reductive enzyme (PSI/RE) chimeric enzymes. To apply and develop directed evolution approaches to enhance the docking, substrate specificity, energetics, and/or kinetics of the PSI/RE fusions. Research A range of reductive enzymes will be fused with the PsaC subunit of a PSI from Synechocystis sp. PCC68036 to produce PSI/RE chimeras and undergo focused directed evolution. The reductive enzymes of interest will include industrial staples like members of the cytochrome p450 superfamily1, the ene-reductase group7, and a reductive dehalogenase8, as well as the carbon-fixing enzyme Crotonyl-CoA Carboxylase/Reductase (CCR)9 and a range of aldehyde/alcohol dehydrogenases of industrial interest10. In silico predictions of the pre- and post-fusion enzyme structures will be used to inform the best avenues for in vitro protein engineering, as well as suggest target regions for directed evolution. Diverse libraries of the PsaC/RE fusions will be produced using in vitro and in vivo mutagenesis techniques; error-prone PCR, site-saturation mutagenesis, and an EvolvR-derived11 approach developed in our lab. To this end, enzyme-specific approaches for screening and selection of light-driven chimera functionality will be developed, exploiting absorption, fluorescent probes, and colorimetry. Specifically, screening approaches will include Schiff's reagent12; substrate, product, or cofactor-specific spectrophotometry13,14; or photoactive PSI detection15. Ethical Considerations This research will necessitate genetic modification of Synechocystis. This could constitute an ethical problem should the modified cyanobacteria be released into the environment; however, the safety and operating procedures of our lab are compliant for work with genetically modified bacteria.