New insights into the biosynthesis of alginate in Pseudomonas fluorescens: from precursor synthesis to multiprotein factories
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Alginate is a biopolymer that has numerous industrial and medical applications due to its favorable material properties, which is greatly affected by the polymer chain length and monomer composition. Currently, all commercially available alginates are obtained from brown algae; however, the polymer is also produced by bacteria belonging to the two genera Pseudomonas and Azotobacter. Genetic engineering of bacteria opens up new possibilities for production of alginates with more defined properties and tailored composition than might be manufactured from seaweed. P. fluorescens is an attractive organism for establishment of the bacterial alginate production for industrial purposes due to its non-pathogenicity and the relative simplicity of cultivation and genetic manipulation. Successful development of recombinant strains with enhanced alginate production suited for industrial applications requires adequate knowledge and deep understanding of alginate biosynthesis procedure. The main part of this PhD project focuses on studying the connection between the central carbon metabolism and alginate production in P. fluorescens. The impact of key carbohydrate metabolism enzymes on growth and alginate synthesis of P. fluorescens grown on carbon sources glucose, fructose and glycerol was investigated. Mutational analyses of glucose-6- phosphate dehydrogenase isoenzymes (Zwf-1 and -2) revealed that Zwf-1 is the most important one for catabolism, while Zwf-2 indicated an anabolic role. Both isoenzymes preferred NADP+ as coenzyme, although NAD+ was also accepted. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production, while this mutation had no positive effect on growth in an alginate nonproducing strain. The strains deficient in both Zwf enzymes were hardly able to grow when fructose was used as the carbon source. It was postulated that accumulation of sugar-phosphates was detrimental to these mutants. A new hexose phosphate phosphatase enzyme was identified in P. fluorescens, which showed phosphatase activity when both glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) were used as substrates in vitro. The in vivo biological function of the phosphatase enzyme was further assessed, and the results revealed that this enzyme may play a role in the hypothesized sugar-phosphate stress response in P. fluorescens, particularly when alginate is produced. In order to obtain more insight regarding the factors affecting alginate production yield, the correlation between the number of alginate biosynthesis factories (multiprotein complexes) on the outer membrane of P. fluorescens and the alginate production level was investigated by developing an immunogold labeling procedure and using transmission electron microscopy (TEM) as a detection tool. This method enabled detection and counting of the alginate factories on the cell surface. The results obtained imply that the alginate production depends more on growth phase and precursor availability than on the number of alginate factories. Clustering study of the alginate factories revealed that they are not randomly distributed on the outer membrane of P. fluorescens. The results present in this thesis provide new fundamental knowledge and contribute to a better understanding of the bacterial alginate biosynthesis steps that could be important for development of engineered bacterial strains with enhanced alginate production suitable for industrial purposes.