Looking for the Big Picture:Genome-Based Approaches to Improve Alginate Production in Azotobacter vinelandii

Alginates are commercially important polysaccharides with a wide range of industrial and technological applications. Polymer chain length and monomer distribution greatly affect the material properties, which makes different alginate types ideal for different areas of use. All commercial alginate manufacture is currently based on extraction from brown algae, but the polymers are also produced by bacteria in the genera Pseudomonas and Azotobacter. Bacterial bioproduction is technically possible, but is not yet economically competitive with algal alginates. A. vinelandii is an attractive candidate for development of bacterial bioproduction strains due to its potential for producing homogenous alginates with tailored monomer compositions, and thus high market value. Successful development of strains and cultivation conditions for bioproduction is however dependent on extensive knowledge of the factors affecting the biosynthetic process in question. Along with the availability of complete genome sequences, a multitude of new opportunities has arised with regard to investigations of gene functions, metabolic and regulatory relationships, environmental adaptations etc. Part of the work presented in this thesis is a contribution to the manual curation and annotation of the first published A. vinelandii genome, which provided the basis for our subsequent genomebased analyses of factors affecting alginate production in this organism. The genome annotation and analysis carried out as part of this thesis was mainly concerned with carbohydrate metabolism genes, and has provided a foundation for further investigations of sugar uptake and utilization in this organism. A notable outcome of the genome analysis was the discovery of numerous highly similar and apparently conserved intra-genome homologs among A. vinelandii core carbohydrate metabolism genes. Investigations of 943 bacterial and archaeal genomes confirmed that the number of such homologs is indeed unusually large in A. vinelandii. We propose that the retention of multiple gene copies confers adaptive benefits via gene dosage and/or increased regulatory flexibility. Genes, and thus cellular processes, affecting alginate production in A. vinelandii were investigated by construction and screening of a transposon insertion library comprising 4000 mutant strains. Abolished or diminished alginate production was confirmed for ~70 transposon insertion mutants and the disrupted genes were identified by sequencing. The disrupted genes included structural and regulatory genes involved in alginate biosynthesis, as well as genes involved in iron uptake, peptidoglycan recycling, motility and synthesis of several cofactors and central metabolites. Based on these results the effect of various medium supplements on alginate production in wild type A. vinelandii was investigated, and addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate was shown to result in significantly increased alginate levels. The screening results also revealed two possible new regulators of alginate biosynthesis; the fructose phosphotransferase system protein FruA and an IclR family transcriptional regulator. Two mutants were confirmed to have gained an increase in alginate production. For one of these the disrupted gene was identified as mucA, encoding the main negative regulator of alginate biosynthesis. Global effects of inactivating MucA were investigated by phenotypic characterization and transcriptome analyses of fermentor-grown A. vinelandii wild type and mucA strains. The mucA mutant has a lowered growth rate, elevated alginate production and diminished respiration rate compared to the wild type strain. Both medium composition and MucA inactivation had profound effects on carbon source utilization. The transcriptome analyses revealed new roles for the key regulators MucA/AlgU with regard to control of alginate composition, cell mass production, respiration and possibly nitrogen fixation. The redirection of carbon utilization in the mucA mutant was also reflected in transcriptional changes in genes involved in gluconeogenesis/glycolysis and energy production. The results presented in this thesis will have importance for further work towards the long-term goal of establishing bacterial systems for commercial bioproduction of alginates