Dr. Paul Blum
Date of this Version
White D, Singh R, Rudrappa D, Mateo J, Kramer L, Freese L, Blum P. 2016. Examination of Contribution of Pentose Catabolism to Molecular Hydrogen Formation by Targeted Disruption of Arabinose Isomerase (araA) in the Hyperthermophilic Bacterium, Thermotoga maritima. Appl Environ Microb.
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 between the pentose phosphate, Embden Myerhoff and Entner Doudoroff pathways. While values for H2 yield have been determined using pentose supplemented complex media (CM) and predicted by metabolic pathway reconstruction, quantitative in vivo measurements derived from pathway elimination have not been reported reflecting 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 was employed encoding synonymous nucleotide substitutions. Targeted inactivation of araA (arabinose isomerase) in pyrE mutant was accomplished using a divergent, codon optimized T. africanus pyrE allele fused to the T. maritima groES promoter. Mutants lacking araA were unable to catabolize arabinose in defined medium. The araA mutation was then repaired using targeted recombination. H2 synthesis using CM supplemented with arabinose was compared between wild type and araA mutant strains to provide a direct measurement of H2 production dependent on arabinose consumption. Development of a targeted recombination system for manipulation of T. maritima provides a new strategy to explore H2 formation and life at temperature extremes in the bacterial domain.
Advisor: Paul Blum