Zymomonas mobilis has many desirable ethanogen attributes including producing near theoretical yields of ethanol and recombinants strains are able to ferment both C-5 and C-6 sugars. The availability of the Z mobilis ZM4 (ZM4) genome sequence and U.S. Department of Energy Joint Genome Institute plans to sequence the genomes of additional strains in the near future provide opportunities to gain fundamental insights into the organism's physiology leading to strain improvements. Towards this aim of strain improvements, we are using systems biology and genetic tools to elucidate ZM4 oxygen stress responses and process inhibitor tolerance mechanisms. Comparative genome resequencing of a classically derived acetate tolerant ZM4 strain (AcR) combined with transcriptomics, quantitative proteomics, metabolomics and genome reannotation form the basis of ongoing studies to elucidate key loci and targets for strain improvements. We have established a research platform that integrates the advantages of classical selection strategies and systems biology technologies to establish a new paradigm in industrial strain characterization and development.