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Applied and Environmental Microbiology, November 2008, p. 6649-6655, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.01610-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain{triangledown}

Yihui Zhu, Mark A. Eiteman,* Ronni Altman, and Elliot Altman

Center for Molecular BioEngineering, Department of Biological and Agricultural Engineering, University of Georgia, Athens, Georgia 30602

Received 14 July 2008/ Accepted 9 September 2008

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h–1. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h–1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g.

* Corresponding author. Mailing address: Center for Molecular BioEngineering, Department of Biological and Agricultural Engineering, 408 Driftmier, University of Georgia, Athens, GA 30602. Phone: (706) 542-0833. Fax: (706) 542-8806. E-mail: eiteman{at}engr.uga.edu

{triangledown} Published ahead of print on 19 September 2008.

Applied and Environmental Microbiology, November 2008, p. 6649-6655, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.01610-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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