Modification of electron transfer proteins in the Chlamydomonas reinhardtii chloroplast for alternative fuel development

There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo.