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United States Department of Agriculture

Agricultural Research Service

Title: Genetic Stability of Metabolic Engineered Escherichia Coli Strains for Production of Ethanol

Authors
item Bothast, Rodney
item Dien, Bruce
item Wyckoff, Herbert - DEAN FOODS
item Hespell, Robert

Submitted to: Society of Industrial Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: August 7, 1997
Publication Date: N/A

Technical Abstract: Currently, almost all fuel ethanol is made by yeast fermentation of glucose from corn starch. In order to reduce the costs of fuel ethanol production, we are exploring the fermentation of other sugars derived from corn fiber and agricultural wastes plus developing new microorganisms to use these materials. Previously, Escherichia coli strains have been genetically engineered to contain the pet operon (Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase B genes) and to produce ethanol from multiple sugars. Strains carrying the pet operon in plasmid or in chromosomal sites require antibiotics in the media to maintain genetic stability and high ethanol productivity in continuous culture. To overcome this requirement, we used the conditionally lethal E. coli strain K12/FMJ39, which carries mutations for lactate dehydrogenase and pyruvate formate lyase and grows aerobically but is incapable of anaerobic growth unless these mutations are complemented. We have successfully transformed K12/FMJ39 with the pet operon plasmid, pLOI297. The resultant strain is capable of anaerobic growth, maintained the pet plasmid after 48 generations (23 with and 25 without antibiotic) in a chemostat and continued to produce ethanol at a constant rate. When grown in batch culture on either arabinose, glucose or xylose, ethanol yields were 0.40-0.44 g/g. The genetic stability conferred by selective pressure for pet-containing cells without requirement for antibiotics suggests potential commercial suitability for similar strains.

Last Modified: 9/2/2014
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