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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11120-022-00945-4.