Contribution of pentose catabolism to molecular hydrogen formation by targeted disruption of arabinose isomerase (araA) in the hyperthermophilic bacterium Thermotoga maritima

Derrick White, Raghuveer Singh, Deepak Rudrappa, Jackie Mateo, Levi Kramer, Laura Freese, Paul Blum

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H2). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H2 yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H2 using arabinosesupplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H2 production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H2 formation and life at an extremely high temperature in the bacterial domain.

Original languageEnglish (US)
Article numbere02631-16
JournalApplied and environmental microbiology
Volume83
Issue number4
DOIs
StatePublished - Jan 1 2017

Fingerprint

Thermotoga maritima
Pentoses
Isomerases
Arabinose
catabolism
pentoses
isomerases
arabinose
recombination
hydrogen
Hydrogen
Genetic Recombination
Bacteria
mutants
hydrogen production
bacterium
metabolism
bacteria
Thermosipho africanus
mutation

Keywords

  • Anaerobes
  • Biohydrogen
  • Extremophiles
  • Genetic systems
  • Homologous recombination

ASJC Scopus subject areas

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

Cite this

Contribution of pentose catabolism to molecular hydrogen formation by targeted disruption of arabinose isomerase (araA) in the hyperthermophilic bacterium Thermotoga maritima. / White, Derrick; Singh, Raghuveer; Rudrappa, Deepak; Mateo, Jackie; Kramer, Levi; Freese, Laura; Blum, Paul.

In: Applied and environmental microbiology, Vol. 83, No. 4, e02631-16, 01.01.2017.

Research output: Contribution to journalArticle

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