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J. Bacteriol. doi:10.1128/JB.00104-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Dihydrolipoamide Dehydrogenase Mutation Alters the NADH Sensitivity of Pyruvate Dehydrogenase Complex of Escherichia coli K-12

Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611

^* To whom correspondence should be addressed. Email: shan{at}ufl.edu.

	Abstract

Under anaerobic growth condition, an active pyruvate dehydrogenase (PDH) is expected to create redox imbalance in wild type Escherichia coli due to increased production of NADH (>2 NADH/glucose) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetylCoA into reduced fermentation products like alcohols during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA, pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried H322Y mutation (lpd102) while the other mutants carried E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homo-ethanol fermentation in an ldhA, pflB double mutant. Enzyme kinetic studies revealed that the LPD-E354K was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold higher Ki for NADH compared to the native PDH. Lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organism in a similar fashion.