Examination of 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

First Advisor

Paul Blum

Date of this Version

8-2017

Document Type

Dissertation

Citation

A dissertation presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Doctor of Philosophy

Major: Biological Sciences

Under the supervision of Professor Paul Blum

Lincoln, Nebraska, August 2017

Comments

Copyright 2017, Derrick White. 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 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