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

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 glyc...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2008-11, Vol.74 (21), p.6649-6655
Main Authors: Zhu, Yihui, Eiteman, Mark A, Altman, Ronni, Altman, Elliot
Format: Article
Language:English
Subjects:
Acetic acid
Acetic Acid - metabolism
Biological and medical sciences
E coli
Enzymes - genetics
Escherichia coli
Escherichia coli - genetics
Escherichia coli - growth & development
Escherichia coli - metabolism
Escherichia coli Proteins - genetics
Fundamental and applied biological sciences. Psychology
Gene expression
Glucose
Glucose - metabolism
Glycolysis - genetics
Metabolic Networks and Pathways - genetics
Metabolism
Microbiology
Models, Biological
Mutation
Nitrogen - metabolism
Phosphorus - metabolism
Physiology and Biotechnology
Pyruvic Acid - metabolism
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Summary: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.
ISSN:0099-2240
1098-5336
1098-6596
DOI:10.1128/AEM.01610-08