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

Anagha Krishnan, Shuyi Zhang, Yang Liu, Kinan A. Tadmori, Donald A. Bryant, Charles G. Dismukes

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1448-1459
Number of pages12
JournalBiotechnology and bioengineering
Volume113
Issue number7
DOIs
StatePublished - Jul 1 2016

Fingerprint

Synechococcus
Cyanobacteria
Metabolism
NAD
Fermentation
Respiration
Anaerobiosis
Hydrogenase
Genes
Fluxes
Enzyme kinetics
Gene encoding
Carbon
Lactic Acid
Gene Components
Glucose
Reducing Agents
Gene Deletion
Regulator Genes
Operon

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Krishnan, Anagha ; Zhang, Shuyi ; Liu, Yang ; Tadmori, Kinan A. ; Bryant, Donald A. ; Dismukes, Charles G. / Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002. In: Biotechnology and bioengineering. 2016 ; Vol. 113, No. 7. pp. 1448-1459.
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abstract = "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.",
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Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002. / Krishnan, Anagha; Zhang, Shuyi; Liu, Yang; Tadmori, Kinan A.; Bryant, Donald A.; Dismukes, Charles G.

In: Biotechnology and bioengineering, Vol. 113, No. 7, 01.07.2016, p. 1448-1459.

Research output: Contribution to journalArticle

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