Abstract
Nitrogenase-dependent H(2) production by photosynthetic bacteria, such as Rhodobacter capsulatus, has been extensively investigated. An important limitation to increase H(2) production using genetic manipulation is the scarcity of high-throughput screening methods to detect possible overproducing mutants. Previously, we engineered R. capsulatus strains that emitted fluorescence in response to H(2) and used them to identify mutations in the nitrogenase Fe protein leading to H(2) overproduction. Here, we used ultraviolet light to induce random mutations in the genome of the engineered H(2)-sensing strain, and fluorescent-activated cell sorting to detect and isolate the H(2)-overproducing cells from libraries containing 5 × 10(5) mutants. Three rounds of mutagenesis and strain selection gradually increased H(2) production up to 3-fold. The whole genomes of five H(2) overproducing strains were sequenced and compared to that of the parental sensor strain to determine the basis for H(2) overproduction. No mutations were present in well-characterized functions related to nitrogen fixation, except for the transcriptional activator nifA2. However, several mutations mapped to energy-generating systems and to carbon metabolism-related functions, which could feed reducing power or ATP to nitrogenase. Time-course experiments of nitrogenase depression in batch cultures exposed mismatches between nitrogenase protein levels and their H(2) and ethylene production activities that suggested energy limitation. Consistently, cultivating in a chemostat produced up to 19-fold more H(2) than the corresponding batch cultures, revealing the potential of selected H(2) overproducing strains.