| dc.description.abstract | Alginate is a naturally occurring polymer produced by brown algae and certain bacterial species – specifically those in the genera Azotobacter and Pseudomonas. This polymer has many applications including as a thickening agent in foods, as a wound dressing, as a 3-D scaffold for tissue engineering, and as an encapsulation device for drug delivery. Each use requires alginates with specific physiochemical properties, this being greatly influenced by the structural composition of the polymer. Currently, all industrial alginate is sourced from algae, however this method offers little opportunity to manipulate the polymer. Bacterial biosynthesis may offer an alternative production method, especially for highly specific, technological applications. In microbial synthesis, alginates are initially synthesized as poly-mannuron chains. Following polymerization, some mannuronic acids may be epimerized to guluronic acid via C-5 epimerases. These residues are organized in M-blocks, MG-blocks, and G-blocks. The concentration and length of blocks varies between species. G-rich alginates containing G-blocks are often desired due to their ability to form hydrogels. A. vinelandii secretes alginates constitutively and produces G-blocks. G-block formation in this species is achieved by secretion of C-5 epimerases, AlgE1-7, using a type I secretion system, EexDEF. Wild-type Pseudomonads do not produce alginates constitutively, although there are mutant strains which do, though they are not known to produce G-blocks. However, species such as P. fluorescens may be a desirable producer as it is nonpathogenic, easily manipulated, and mucoid strains can secrete large volumes of alginate. If P. fluorescens can express C-5 epimerases and the A. vinelandii secretion system, it may be able to produce G-rich alginates with G-blocks. P. savastanoi may also encode an epimerase secretion system (an EexDEF homolog) which may alternately be transferred. In this study, a P. fluorescens strain containing eexDEF was created and found to be stable. Plasmids carrying epimerase genes (algE4, algE6, and psmE) were also transferred. However, the production of G-rich alginates was neither conclusively verified nor refuted. The putative P. savastanoi secretion system was sequenced, inserted into a transposon vector (pCM11), and inserted into the P. fluorescens genome. The secretion system has yet to be verified. High G-content alginate production may also be attempted in P. savastanoi through the introduction of algU and psmE. Ultimately the efficacy of these species in producing industrially significant alginates requires further analysis. | |