Production of L -alanine by metabolically engineered Escherichia coli.
Escherichia coli W was genetically engineered to produce L: -alanine as the primary fermentation product from sugars by replacing the native D: -lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native D: -lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of L: -alanine to D: -alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l(-1) glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g(-1) glucose) with a chiral purity greater than 99.5% L: -alanine.
Zhang
Xueli
X
Department of Microbiology and Cell Science, University of Florida, Box 110700, Gainesville, FL 32611, USA.
Jantama
Kaemwich
K
Moore
J C
JC
Shanmugam
K T
KT
Ingram
L O
LO
D000409:Alanine / Q000096:biosynthesis*; D050788:Alanine Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D019143:Evolution, Molecular; D005285:Fermentation; D005818:Genetic Engineering / Q000379:methods*; D006019:Glycolysis
D003470:Culture Media; D050788:Alanine Dehydrogenase; D000409:Alanine