Ran Ningqing, Frost John W
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
J Am Chem Soc. 2007 May 16;129(19):6130-9. doi: 10.1021/ja067330p. Epub 2007 Apr 24.
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.
通过2-酮-3-脱氧-6-磷酸半乳糖酸（KDPGal）醛缩酶的定向进化实现莽草酸途径产物的微生物合成，为规避大肠杆菌中3-脱氧-D-阿拉伯庚酮糖酸7-磷酸（DAHP）合酶与磷酸烯醇丙酮酸：碳水化合物磷酸转移酶系统之间对磷酸烯醇丙酮酸的竞争提供了一种替代策略。利用易错聚合酶链反应、DNA改组和多位点定向诱变相结合的方法对大肠杆菌KDPGal醛缩酶进行进化，得到KDPGal醛缩酶变体NR8.276-2，在催化丙酮酸与4-磷酸-D-赤藓糖加成形成DAHP的反应中，其kcat/KM比值相对于野生型大肠杆菌KDPGal醛缩酶提高了60倍。根据其核苷酸序列，NR8.276-2相对于野生型大肠杆菌KDPGal醛缩酶有七个氨基酸变化。在补料分批发酵罐控制的培养条件下，在缺乏DAHP合酶和莽草酸脱氢酶的大肠杆菌菌株NR7中扩增表达NR8.276-2，以葡萄糖为原料，6.5%的摩尔产率合成了13 g/L的3-脱氢莽草酸。不可逆的下游酶3-脱氢奎尼酸合酶的共表达增加，使3-脱氢莽草酸的产量从葡萄糖提高到19 g/L，摩尔产率为9.7%。与转酮醇酶共扩增，增加了4-磷酸-D-赤藓糖的可用性，以葡萄糖为原料，8.5%的摩尔产率得到了16 g/L的3-脱氢莽草酸。
