Scaling-up and proteomic analysis reveals photosynthetic and metabolic insights toward prolonged H 2 photoproduction in Chlamydomonas hpm91 mutant lacking proton gradient regulation 5 (PGR5)

Clean and sustainable H 2 production is crucial to a carbon–neutral world. H 2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H 2 from H 2 O. However, it is currently not large-scalable because of lacking desirable strains with both optimal H 2 productivity and sufficient knowledge of underlying molecular mechanism. We hereby carried out extensive and in-depth investigations of H 2 photoproduction of hpm91 mutant lacking PGR5 (Proton Gradient Regulation 5) toward its up-scaling and fundamental mechanism issues. We show that hpm91 is at least 100-fold scalable (up to 10 L) with continuous H 2 collection of 7287 ml H 2 /10L-HPBR in averagely 26 days under sulfur deprivation. Also, we show that hpm91 is robust and active during sustained H 2 photoproduction, most likely due to decreased intracellular ROS relative to wild type. Moreover, we obtained quantitative proteomic profiles of wild type and hpm91 at four representing time points of H 2 evolution, leading to 2229 and 1350 differentially expressed proteins, respectively. Compared to wild type, major proteome alterations of hpm91 include not only core subunits of photosystems and those related to anti-oxidative responses but also essential proteins in photosynthetic antenna, C/N metabolic balance, and sulfur assimilation toward both cysteine biosynthesis and sulfation of metabolites during sulfur-deprived H 2 production. These results reveal not only new insights of cellular and molecular basis of enhanced H 2 production in hpm91 but also provide additional candidate gene targets and modules for further genetic modifications and/or in artificial photosynthesis mimics toward basic and applied research aiming at advancing solar-H 2 technology.