Hal S. Alper, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-422, Cambridge, MA 02139, Gerald Fink, Whitehead Institute, Nine Cambridge Center, Cambridge, MA 02142, and Gregory N. Stephanopoulos, Department of Chemical Engineering, MIT, 66-264 MIT 77 Massachusetts Ave, Cambridge, MA 02139.
The engineering of a strain optimal for bioethanol production requires imparting multiple cellular phenotypes including tolerance to ethanol, high ethanol productivity, tolerance to inhibitory substrate compounds, and effective sugar utilization. Of these factors, one particularly difficult problem limiting prospects of effective ethanol production is improving the cellular tolerance to this toxic product. To approach this problem, we undertook the tool of global Transcription Machinery Engineering in which components of global cellular transcription machinery are engineered to elicit complex phenotypes controlled by multiple genes. This novel approach allows the high throughput probing of a vastly unexplored search space by evaluating multiple, simultaneous gene alterations which can span over multiple pathways within a cell. Specifically, engineering the sigma factors of Escherichia coli and TFIID components in Saccharomyces cerevisiae enabled the identification and optimization of strains with improved properties for bioethanol production. Here, we present recent results in the development of cellular systems with desired properties for successful, high-yield ethanol fermentations from lignocellulosic materials. These strains exhibit superior tolerance properties compared with both un-engineered strains as well as previously reported tolerant strains.