Contribution of Pentose Catabolism to Molecular Hydrogen Formation by Targeted Disruption of Arabinose Isomerase (araA) in the Hyperthermophilic Bacterium Thermotoga maritima

Authors

Derrick White, University of Nebraska - LincolnFollow
Raghuveer Singh, University of Nebraska - LincolnFollow
Deepak Rudrappa, University of Nebraska–LincolnFollow
Jackie Mateo, University of Nebraska-Lincoln
Levi Kramer, University of Nebraska-LincolnFollow
Laura Freese, University of Nebraska-Lincoln
Paul H. Blum, University of Nebraska-LincolnFollow

Date of this Version

2-2017

Citation

White D, Singh R, Rudrappa D, Mateo J, Kramer L, Freese L, Blum P. 2017. Contribution of pentose catabolism to molecular hydrogen formation by targeted disruption of arabinose isomerase (araA) in the hyperthermophilic bacterium Thermotoga maritima. Appl Environ Microbiol 83:e02631-16. https://doi.org/10.1128/AEM.02631-16.

Comments

Copyright © 2017 American Society for Microbiology. All Rights Reserved. Used by permission.

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 arabinose-supplemented 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.