Affiliation:
1. Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611
Abstract
ABSTRACT
The fermentative metabolism of
Escherichia coli
was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the dominant fermentation products from glycerol in wild-type
Escherichia coli
ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (
pflB
) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (
pck*
) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD
+
ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the
ptsI
gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of
pflB
in this background provided a further increase in the succinate yield. Together, these three core mutations (
pck
*,
ptsI
, and
pflB
) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
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