STABILIZATION OF PET OPERON PLASMIDS AND ETHANOL PRODUCTION IN ESCHERICHIA COLI STRAINS LACKING LACTATE DEHYDROGENASE AND PYRUVATE-FORMATE LYASE ACTIVITIES

Authors: Hespell, Robert; Wyckoff, Herbert; Dien, Bruce; Bothast, Rodney

Submitted to: Journal of Applied & Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/9/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: 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 ethanol-producing microorganisms to use these materials. Escherichia coli bacteria have previously been genetically engineered to produce ethanol from multiple sugars. However, these strains require antibiotics in order to maintain stability. Use of antibiotics adds significant cost to fermentation and can cause environmental concerns. We have overcome the need for antibiotics by genetically engineering two new E. coli strains. Both strains produce ethanol in the absence of air. Antibiotics are not needed to maintain the new genes because cells which lose these genes die leaving only ethanol-producing cells as survivors. We are testing these strains for possible commercial use.

Technical Abstract: In the last decade, a major goal of biofuels research has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. Escherichia coli strains genetically engineered to contain the PET operon produce high levels of ethanol. Strains carrying the PET operon in plasmid or in chromosomal sites require antibiotics in the media to maintain genetic stability and high ethanol productivity. To overcome this requirement, we have used the conditionally lethal E. coli strain 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. E. coli strains FBR1 and FBR2 were created by transforming E. coli FMJ39 with the PET operon plasmids pLOI295 and pLOI297, respectively. Both strains were capable of anaerobic growth and displayed no apparent PET plasmid losses after 60 generations in serially transferred anaerobic batch cultures. In contrast, similar aerobic cultures rapidly lost plasmids. In high cell density batch fermentations, 3.8% to 4.4% wt/v ethanol (strain FBR2) was made from 10% glucose. Anaerobic, glucose-limited continuous cultures of strain FBR2 grown for 20 days showed no loss of antibiotic resistance. Anaerobic, serially transferred batch cultures and high density fermentations were inoculated with cells taken at 57 generations from the previous continuous culture. Both cultures continued high ethanol production in the absence of tetracycline. The genetic stability conferred by selective pressure for PET-containing cells without requirement for antibiotics suggests potential commercial suitability.