Evaluation of genetic manipulation strategies on d-lactate production by Escherichia coli
Center for Bioresource and Bioenergy, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa; Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, P.O. Box 1334, Durban 4001, South Africa
In order to rationally manipulate the cellular metabolism of Escherichia coli for d-lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding d-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA-pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 (ackA-pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 (ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l d-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of d-lactate production are among the highest reported and the results have revealed which genetic manipulations improved d-lactate production by E. coli. © 2010 Springer Science+Business Media, LLC.
acetate kinase; acetic acid; alcohol; alcohol dehydrogenase; formic acid; fumarate reductase; lactate dehydrogenase; lactic acid; phosphate acetyltransferase; pyruvate oxidase; pyruvate water dikinase; succinic acid; synthetase; unclassified drug; ackA gene; adhE gene; aerobic fermentation; article; bacterial gene; bacterial growth; bacterial metabolism; bacterial strain; controlled study; dld gene; Escherichia coli; frda gene; gene deletion; gene mutation; genetic manipulation; nonhuman; pflB gene; poxb gene; pps gene; priority journal; pta gene; wild type; Aerobiosis; Anaerobiosis; Escherichia coli; Escherichia coli Proteins; Fermentation; Gene Deletion; Genetic Engineering; Lactic Acid; Metabolic Networks and Pathways; Mutation; Organisms, Genetically Modified; Escherichia coli