Biofuels Production projects

Engineering Industrially Important Clostridia for Adaptive Immunity to Phage

Large-scale bacterial fermentations are used for the production of a variety of resources such as food, pharmaceuticals, and the biofuel precursors acetone, butanol, and ethanol (ABE). Like all bacterial processes, these fermentations are vulnerable to attack by bacteriophage (phage), bacterial viruses that are easily introduced from outside sources or are already present in the genomes of the fermenting bacteria. Once introduced, phage can rapidly multiply by infecting and lysing increasing numbers of bacteria. This problem of "phage-out", in which a fermentation process is spoiled by phage-mediated cell lysis, has a dramatic impact on productivity. The overall objective of this proposal is to develop a new strategy to provide ABE fermenting bacteria with a defense system against phage. Solventogenic Clostridium strains of interest will be genetically engineered for phage resistance by introducing a resistance mechanism present in other bacteria. These strains will be tested for phage resistance in laboratory-scale fermentations, and the bacterial and phage population dynamics will be monitored in fermentations containing different combinations of strains. This strategy is expected to substantially increase productivity by reducing or eliminating the incidence of phage infection.

project Highlights

2014 Highlights

Large-scale bacterial fermentations including those used in ABE production are vulnerable to infection by bacteriophage, which has a dramatic effect on productivity. Our proposed approach to combat this problem is to engineer the bacterial strains to be resistant to phage by introducing a CRISPR-Cas adaptive immune system that will destroy phage DNA that has entered the cell. This approach will require the cloning and testing of multiple CRISPR-Cas systems, development of an improved genetic system for the ABE strains of interest, isolation and sequencing of numerous phages (to be performed by Dr. Miller’s group), and a more complete understanding of the relationship between phage sensitivity and growth phases relevant to ABE production. Work in all of these areas was underway in 2014.

Publications

 


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