Synechococcussp. Strain PCC 7002nifJMutant Lacking Pyruvate:Ferredoxin Oxidoreductase

Abstract

ABSTRACTThenifJgene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). AnifJknockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacteriumSynechococcussp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (Fv/Fm). During autofermentation, the excretion of acetate and lactate products bynifJmutant cells decreased 2-fold and 1.2-fold, respectively. AlthoughnifJcells displayed higherin vitrohydrogenase activity than WT cells, H2productionin vivowas 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H2production bynifJcells compared to WT cells. Continuous electrochemical detection of dissolved H2revealed two temporally resolved phases of H2production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold innifJcells. The second phase was attributed to glycolytic NADH production and decreased 20% innifJcells. Measurement of the intracellular NADH/NAD+ratio revealed that the reductant generated by PFOR contributing to the first phase of H2production was not in equilibrium with bulk NADH/NAD+and that the second phase corresponded to the equilibrium NADH-mediated process.