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Title: ENGINEERING LACTIC ACID BACTERIA WITH PYRUVATE DECARBOXYLASE AND ALCOHOL DEHYDROGENASE GENES FOR ETHANOL PRODUCTION FROM ZYMOMONAS MOBILIS

Author
item Nichols, Nancy
item Dien, Bruce
item Bothast, Rodney

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/13/2003
Publication Date: 5/1/2003
Citation: NICHOLS, N.N., DIEN, B.S., BOTHAST, R.J. ENGINEERING LACTIC ACID BACTERIA WITH PYRUVATE DECARBOXYLASE AND ALCOHOL DEHYDROGENASE GENES FOR ETHANOL PRODUCTION FROM ZYMOMONAS MOBILIS. JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY. 2003. V. 30. P. 315-321.

Interpretive Summary: Ethanol is a renewable fuel that offers benefits including decreased dependence on imported petroleum, a cleaner environment, and economic development for rural communities. Currently, fuel ethanol is produced using yeast to ferment cornstarch. Agricultural biomass (i.e., cornstalks) is a potential low-cost alternative to cornstarch. However, biomass is different from starch because biomass contains a mixture of sugars, rather than pure glucose. At present, there is not a commercial process that can make ethanol from the types of sugars in biomass. We have developed new bacterial systems for ethanol production by genetically modifying several members of the lactic acid group of bacteria, which are commonly used in food production. In one case, production of the native fermentation product (lactic acid) decreased and was replaced by ethanol. This work allows further development of microorganisms that can efficiently make ethanol from inexpensive renewable resources.

Technical Abstract: Lactic acid bacteria are candidates for engineered production of ethanol from biomass because they are food-grade microorganisms which can, in many cases, metabolize a variety of sugars and grow under harsh conditions. Plasmids were constructed to express pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh), encoded by the pdc and adhB genes of Zymomonas smobilis, in lactic acid bacteria. Several strains were transformed with the plasmids, and transcription of pdc and adhB was confirmed by northern hybridization analysis of transformants. Pdc and Adh enzyme activities were low in these bacteria compared to Escherichia coli. The highest Pdc and Adh specific activities obtained in a specific strain were 0.13 U Pdc and 0.22 U Adh per mg protein in Pediococcus pentosaceous, compared to 1.2 U Pdc and 1.6 U Adh per mg protein in E. coli. Glucose fermentations were carried out using the transformed strains. Lactic acid was the primary fermentation product; however, in P. pentosaceous, the amount of lactate was reduced by more than 50% when the pet operon was present. The molar ratio of ethanol to lactate produced by transformants was 0.84 +/- 0.03, while no ethanol was produced in the parent strain. The transformed strains of P. pentosaceous produced 4.5 +/- 0.3 g ethanol 1**-1, with a yield of 0.20 +/- 0.01 g ethanol/g glucose consumed.