A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus

• Published in: Frontiers in microbiology 2022-08, Vol.13
• Main Authors: Barahona, Emma, Isidro, Elisa San, Sierra-Heras, Laura, Álvarez-Melcón, Inés, Jiménez-Vicente, Emilio, Buesa, José María, Imperial, Juan, Rubio, Luis M.
• Format: Article
• Language: English
• Subjects: biological hydrogen production; flow cytometry; hupA; hydrogenase; Microbiology; mutagenesis; nitrogenase
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2022.991123

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.