Abstract
Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction.
Highlights
Alginate is the collective term for a family of linear polysaccharides consisting of varying amounts of β-D-mannuronic acid (M) and α-L-guluronic acid (G) (Haug et al, 1966)
The production level is still sufficient to allow for identification of mutants with lowered alginate production, and the decrease could potentially be beneficial for identification of mutants with increased production
While the two-component response regulator AlgB is necessary for alginate production in P. aeruginosa, our study indicates that this is not the case in A. vinelandii
Summary
Alginate is the collective term for a family of linear polysaccharides consisting of varying amounts of β-D-mannuronic acid (M) and α-L-guluronic acid (G) (Haug et al, 1966). In nature alginates are produced by brown seaweeds and by several bacteria in the genera Pseudomonas and Azotobacter, among them the soil bacterium A. vinelandii (Gorin and Spencer, 1966). All commercial alginate production is currently based on extraction from brown algae, which yields complex mixtures of alginates with regard to both chain composition and molecular weight (Andersen et al, 2012). As most current applications rely on the gelling properties of G-blocks (stretches of consecutive G residues) (Andersen et al, 2012), A. vinelandii is an attractive candidate for strain engineering due to its innate ability to introduce G-blocks in the alginate chains (Ertesvåg et al, 1995; Svanem et al, 1999). No strains producing G-block alginates have been identified among the Pseudomonas species studied so far
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