文摘
Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase for microbialsynthesis of shikimate pathway products provides an alternate strategy to circumvent the competition forphosphoenolpyruvate between 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase andthe phosphoenolpyruvate:carbohydrate phosphotransferase system in Escherichia coli. E. coli KDPGalaldolase was evolved using a combination of error-prone polymerase chain reaction, DNA shuffling, andmultiple-site-directed mutagenesis to afford KDPGal aldolase variant NR8.276-2, which exhibits a 60-foldimprovement in the ratio kcat/KM relative to that of wild-type E. coli KDPGal aldolase in catalyzing the additionof pyruvate to D-erythrose 4-phosphate to form DAHP. On the basis of its nucleotide sequence, NR8.276-2contains seven amino acid changes from the wild-type E. coli KDPGal aldolase. Amplified expression ofNR8.276-2 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 in6.5% 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 fromglucose. Coamplification with transketolase, which increases D-erythrose 4-phosphate availability, afforded16 g/L 3-dehydroshikimic acid in 8.5% molar yield.