Manipulation of Pseudomonas aeruginosa alginate pathway by varying the level of biosynthetic enzymes and growth temperature

Abstract

The manipulation of the alginate pathway in two Pseudomonas aeruginosa mucoid variants was attempted at growth temperatures within the range 20 degrees C-40 degrees C. This was carried out by increasing the level of either phosphomannose isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) or GDP-mannose dehydrogenase (GMD) encoded by algA or algD respectively, present in recombinant plasmids derived from the controlled expression vector pMMB24. The specific growth rate of cells expressing either algA or algD genes from recombinant plasmids was lower than that of cells harbouring the cloning vector only. Stimulation of alginate synthesis was observed when the expression of the alginate genes was low, in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction. The further increase of the level of alginate enzymes in induced cells, without the simultaneous increase of other limiting steps, had no positive effect on the strictly regulated alginate pathway. Temperature profiles for alginate synthesis were modified reflecting changes in rate limiting steps. Limitations on the polymerization ability and the competition between cell growth and alginate synthesis were possibly involved in the modification of the temperature profiles for alginate production, or in the decrease of the molecular weight of polymers produced by recombinants under conditions that led to highly active alginate synthesis. The acetyl content of alginates produced by the recombinants was higher than that of the biopolymer controls, possibly due to the higher acetyl-CoA availability in slower growing cells.