Consequences of ccmR deletion on respiration, fermentation and H₂ metabolism in cyanobacterium Synechococcus sp. PCC 7002

CcmR, a LysR-type transcriptional regulator, represses the genes encoding components of the high-affinity carbon concentration mechanism in cyanobacteria. Unexpectedly, deletion of the ccmR gene was found to alter the expression of the terminal oxidase and fermentative genes, especially the hydrogenase operon in the cyanobacterium Synechococcus sp. PCC 7002. Consistent with the transcriptomic data, the deletion strain exhibits flux increases (30-50%) in both aerobic O₂ respiration and anaerobic H₂ evolution. To understand how CcmR influences anaerobic metabolism, the kinetics of autofermentation were investigated following photoautotrophic growth. The autofermentative H₂ yield increased by 50% in the CcmR deletion strain compared to the wild-type strain, and increased to 160% (within 20h) upon continuous removal of H₂ from the medium ("milking") to suppress H₂ uptake. Consistent with this greater reductant flux to H₂, the mutant excreted less lactate during autofermentation (NAD(P)H consuming pathway). To enhance the rate of NADH production during anaerobic metabolism, the ccmR mutant was engineered to introduce GAPDH overexpression (more NADH production) and LDH deletion (less NADH consumption). The triple mutant (ccmR deletion+GAPDH overexpression+LDH deletion) showed 6-8-fold greater H₂ yield than the WT strain, achieving conversion rates of 17 nmol 10⁸cells^-1h^-1 and yield of 0.87 H₂ per glucose equivalent (8.9% theoretical maximum). Simultaneous monitoring of the intracellular NAD(P)H concentration and H₂ production rate by these mutants reveals an inverse correspondence between these variables indicating hydrogenase-dependent H₂ production as a major sink for consuming NAD(P)H in preference to excretion of reduced carbon as lactate during fermentation.