Directed Evolution of 2-Keto-3-deoxy-6-phosphogalactonate Aldolase To Replace 3-Deoxy-D-arabino-heptulosonic Acid 7-Phosphate Synthase
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- 作者：Ningqing Ran ; John W. Frost
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：May 16, 2007
- 年：2007
- 卷：129
- 期：19
- 页码：6130 - 6139
- 全文大小：147K
- 年卷期：v.129,no.19(May 16, 2007)
- ISSN：1520-5126
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.