title: Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate aldolase to replace 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase.
abstract: Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase for microbial synthesis of shikimate pathway products provides an alternate strategy to circumvent the competition for phosphoenolpyruvate between 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase and the phosphoenolpyruvate:carbohydrate phosphotransferase system in Escherichia coli. E. coli KDPGal aldolase was evolved using a combination of error-prone polymerase chain reaction, DNA shuffling, and multiple-site-directed mutagenesis to afford KDPGal aldolase variant NR8.276-2, which exhibits a 60-fold improvement in the ratio kcat/KM relative to that of wild-type E. coli KDPGal aldolase in catalyzing the addition of pyruvate to d-erythrose 4-phosphate to form DAHP. On the basis of its nucleotide sequence, NR8.276-2 contains seven amino acid changes from the wild-type E. coli KDPGal aldolase. Amplified expression of NR8.276-2 in the DAHP synthase and shikimate dehydrogenase-deficient E. coli strain NR7 under fed-batch fermentor-controlled cultivation conditions resulted in synthesis of 13 g/L 3-dehydroshikimic acid in 6.5% molar yield from glucose. Increased coexpression of the irreversible downstream enzyme 3-dehydroquinate synthase increased production of 3-dehydroshikimic acid to 19 g/L in 9.7% molar yield from glucose. Coamplification with transketolase, which increases d-erythrose 4-phosphate availability, afforded 16 g/L 3-dehydroshikimic acid in 8.5% molar yield.
authors: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors: Ningqing
authors: N
authors: Ran
authors: John W
authors: JW
authors: Frost
mesh_terms: D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; C096387:3-dehydroshikimate; D012765:Shikimic Acid; D010728:Phosphoenolpyruvate; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases; C022366:6-phospho-2-keto-3-deoxy-galactonate aldolase; D005947:Glucose