(680e) Developing Metabolic Engineering Tools for a Cellulolytic Marine Bacterium Saccharophagus Degradans 2-40T

Saccharophagus degradans 2-40^T is a gram-negative marine bacterium with a rare capability of degrading many types of complex polysaccharides, including cellulose.  Analyses of its recently sequenced genome and subsequent experimental studies suggest that its cellulolytic system is unique in several aspects, notably the lack of exo-acting glucanases and significant surface features present only when metabolizing complex polysaccharides, signify a departure from known cellulolytic systems that rely on secreted soluble cellulases or complex cellulosome.  Thus the Saccharophagus system provides a unique model to study how gram-negative bacteria overcome transport limitations in accessing a polymeric substrate, a key aspect of cellulose metabolism. Additionally, the cellulolytic Saccharophagus degradans could be exploited for consolidated production of biofuel and other useful products. The fundamental and applied research requires reliable genetic tools, which are lacking for this marine bacterium.

We describe in this presentation our research toward establishing metabolic engineering tools in this microorganism. Starting from a broad range host plasmid, we succeeded in constructing a plasmid that was replicated and stably maintained by the bacterium. Electroporation protocols were developed and optimized, which led to transformation efficacy of about 1000 colonies per micrograms of DNA. Both kanamycin and chloramphenicol were found to be suitable as selection markers. We also tested several promoters for inducible expression of heterologous proteins, a key step in metabolic engineering.
Using red fluorescent protein as the first model protein, we are testing several promoters including those native to the organism, which will result in constitutive and inducible expression of heterologous proteins at varied levels.

To the best of our knowledge, this is the first expression plasmid system ever established for this microorganism. The genetic tools from the research could be used to uncover novel Saccharophagus cellulolytic mechanism diverged significantly from other known systems.  Our understanding on cellulose metabolism from this bacterium offers the first gram-negative template for synthetic biologists and metabolic engineers to develop more efficient biocatalysts for biofuel and other valuable products.