Investigation of 2-oxoacid oxidoreductases in Methanococcus maripaludis and large scale growth of M. maripaludis

Abstract

With the emergence of Methanococcus maripaludis as a genetic model for methanogens, it becomes imperative to devise inexpensive yet effective ways to grow large numbers of cells for protein studies. Chapter 2 details methods that were used to cut costs of growing M. maripaludis in large scale. Also, large scale growth under different conditions was explored in order to find the conditions that yield the most cells. Chapter 3 details the phylogenetic analysis of 2-oxoacid oxidoreductase (OR) homologs from M. maripaludis. ORs are enzymes that catalyze the oxidative decarboxylation of 2-oxoacids to their acyl-CoA derivatives in many prokaryotes. Also in chapter 3, a specific OR homolog in M. maripaludis, an indolepyruvate oxidoreductase (IOR), was mutagenized, and the mutant was characterized. PCR and Southern hybridization analysis showed gene replacement. A no-growth phenotype on media containing aromatic amino acid derivatives was found.