Bioprospecting for Genes That Confer Biofuel Tolerance Using a Genomic Library Approach

Bioprospecting for Genes That Confer Biofuel Tolerance Using a Genomic Library Approach
Microorganisms are capable of producing advanced biofuels that can be used as â??drop-inâ?? alternatives to conventional liquid fuels. However, the cells often become overwhelmed by the toxic effects of the biofuel product. In order to make microbial biofuels a competitive fuel source, mechanisms of improving resistance to the toxic effects of biofuel production is vital. Our investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand extreme environmental pressures.
Using a plasmid-based transgenic library approach, genes believed to impart resistance can be inserted and studied in Escherichia coli. Adaptions such as increased efflux pump efficiency and less permeable cell membranes could help to improve biofuel tolerance in the host organism.

Pseudomonas aeruginosa was studied initially because the bacterium has evolved mechanisms to survive attacks from many damaging compounds. A plasmid library from P. aeruginosa was created and transformed into E. coli. The resulting cells were then stressed with biofuel to determine if any of the genetically altered E. coli displayed improved tolerance. Our study was successful in identifying a specific gene from P. aeruginosa that help to significantly increase tolerance to limonene when expressed in E. coli. The identified gene was an organic hydroperoxide resistance gene (ohr) that helped to alleviate the oxidative stress that limonene imposed on the cells.

Additionally, the transgenic library approach is being applied to other microorganisms such as Alcanivorax borkumensis. A. borkumensis has adapted to survive in harsh, hydrocarbon rich environments, very similar to the conditions encountered by a biofuel producing strain of bacteria. This library is being expressed in E. coli and tested to see if any biofuel tolerance genes can be recovered.