Bioengineering a non-pathogenic bacteria to produce medically-relevant biopolymers

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 DESCRIPTION (provided by applicant): Alginate is a commercially-viable anionic biopolymer consisting of two monomeric subunits, ß-D-mannuronate and a-L-guluronate. Alginate is used in a variety of applications including textile manufacturing, waste water remediation food production, and a variety of medical applications such as wound care and drug delivery. Currently, commercial alginate is harvested from various genera of brown seaweeds. However, seaweed- derived alginate has several limitations including: seasonal growth, inconsistencies in yield, composition and quality between harvests, supply-side instability and the use of economically and environmentally costly processing methods. These limitations impact seaweed-derived alginate's use for biomedical and pharmaceutical application. Alginate is also produced by bacteria in the genera Azotobacter and Pseudomonas. Moreover, bacteria-derived alginate has several advantages; specifically, it is relatively easy, inexpensive, and environmentally-friendly to cultivate and harvest. Additionally, the bacteria can be easily manipulated to produce large amounts of alginate with specific customized properties for biomedical and pharmaceutical applications. However, there is little ongoing research to develop bacterial strains for commercial alginate production. Progenesis Technologies, LLC has patented a stable high alginate producing strain of Pseudomonas aeruginosa for use in recombinant alginate production. At a pre-petition consultation, the United States Federal Drug Administration (FDA) suggested abrogating the function of the secreted virulence factors exotoxin A and pyocyanin to decrease the relative pathogenicity of VE2. The FDA also suggested using the currently approved bacterial strains for alginate production. To date, we have abrogated the function of the virulence factors exotoxin A, pyocyanin, phospholipase C and endotoxin. In this proposal, we intended to design and evaluate a non- pathogenic bacterial strain for the production of the biopolymer alginate. We propose to accomplish this through the completion of two Specific Aims. In Aim 1, we will evaluate the pathogenicity of our recently modified P. aeruginosa strain PGN4 using a murine model. In Aim 2, we will determine the feasibility of producing alginate using currently approved E. coli strains. At the conclusion of ths project, we will seek the appropriate federal approval to use our newly-acquired, non-pathogenic bacterial strain as a platform for the production of biomedical and pharmaceutical grade alginate for use in various medical applications.