Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate: Ferredoxin oxidoreductase

Kelsey McNeely, Yu Xu, Gennady Ananyev, Nicholas Bennette, Donald A. Bryant, G Charles Dismukes

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. 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 by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H2 production in vivo was 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 H2 production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production 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 in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD+ ratio revealed that the reductant generated by PFOR contributing to the first phase of H2 production was not in equilibrium with bulk NADH/NAD+ and that the second phase corresponded to the equilibrium NADH-mediated process.

Original languageEnglish
Pages (from-to)2435-2444
Number of pages10
JournalApplied and Environmental Microbiology
Volume77
Issue number7
DOIs
Publication statusPublished - Apr 2011

Fingerprint

pyruvate synthase
Pyruvate Synthase
Synechococcus
NAD (coenzyme)
NAD
hydrogen production
fold
mutants
acetate
Ferredoxins
cells
ferredoxins
reducing agents
Reducing Agents
Pyruvic Acid
pyruvate dehydrogenase (lipoamide)
fluorometry
catabolism
Acetates
acetate-CoA ligase

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

Cite this

Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate : Ferredoxin oxidoreductase. / McNeely, Kelsey; Xu, Yu; Ananyev, Gennady; Bennette, Nicholas; Bryant, Donald A.; Dismukes, G Charles.

In: Applied and Environmental Microbiology, Vol. 77, No. 7, 04.2011, p. 2435-2444.

Research output: Contribution to journalArticle

McNeely, Kelsey ; Xu, Yu ; Ananyev, Gennady ; Bennette, Nicholas ; Bryant, Donald A. ; Dismukes, G Charles. / Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate : Ferredoxin oxidoreductase. In: Applied and Environmental Microbiology. 2011 ; Vol. 77, No. 7. pp. 2435-2444.
@article{f578c97803d54a22bf208a00a38b79d2,
title = "Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate: Ferredoxin oxidoreductase",
abstract = "The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. 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 by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H2 production in vivo was 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 H2 production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production 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 in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20{\%} in nifJ cells. Measurement of the intracellular NADH/NAD+ ratio revealed that the reductant generated by PFOR contributing to the first phase of H2 production was not in equilibrium with bulk NADH/NAD+ and that the second phase corresponded to the equilibrium NADH-mediated process.",
author = "Kelsey McNeely and Yu Xu and Gennady Ananyev and Nicholas Bennette and Bryant, {Donald A.} and Dismukes, {G Charles}",
year = "2011",
month = "4",
doi = "10.1128/AEM.02792-10",
language = "English",
volume = "77",
pages = "2435--2444",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "7",

}

TY - JOUR

T1 - Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate

T2 - Ferredoxin oxidoreductase

AU - McNeely, Kelsey

AU - Xu, Yu

AU - Ananyev, Gennady

AU - Bennette, Nicholas

AU - Bryant, Donald A.

AU - Dismukes, G Charles

PY - 2011/4

Y1 - 2011/4

N2 - The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. 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 by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H2 production in vivo was 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 H2 production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production 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 in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD+ ratio revealed that the reductant generated by PFOR contributing to the first phase of H2 production was not in equilibrium with bulk NADH/NAD+ and that the second phase corresponded to the equilibrium NADH-mediated process.

AB - The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. 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 by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H2 production in vivo was 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 H2 production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production 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 in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD+ ratio revealed that the reductant generated by PFOR contributing to the first phase of H2 production was not in equilibrium with bulk NADH/NAD+ and that the second phase corresponded to the equilibrium NADH-mediated process.

UR - http://www.scopus.com/inward/record.url?scp=79953282177&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79953282177&partnerID=8YFLogxK

U2 - 10.1128/AEM.02792-10

DO - 10.1128/AEM.02792-10

M3 - Article

C2 - 21317262

AN - SCOPUS:79953282177

VL - 77

SP - 2435

EP - 2444

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 7

ER -