Ran Ningqing, Draths K M, Frost J W
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
J Am Chem Soc. 2004 Jun 9;126(22):6856-7. doi: 10.1021/ja049730n.
The competition between the Escherichia coli carbohydrate phosphotransferase system and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase for phosphoenolpyruvate limits the concentration and yield of natural products microbially synthesized via the shikimate pathway. To circumvent this competition for phosphoenolpyruvate, a shikimate pathway variant has been created. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases encoded by Escherichia coli dgoA and Klebsiella pneumoniae dgoA are subjected to directed evolution. The evolved KDPGal aldolase isozymes exhibit 4-8-fold higher specific activities relative to that for native KDPGal aldolase with respect to catalyzing the condensation of pyruvate and d-erythrose 4-phosphate to produce DAHP. To probe the ability of the created shikimate pathway variant to support microbial growth and metabolism, growth rates and synthesis of 3-dehydroshikimate are examined for E. coli constructs that lack phosphoenolpruvate-based DAHP synthase activity and rely on evolved KDPGal aldolase for biosynthesis of shikimate pathway intermediates and products.
大肠杆菌碳水化合物磷酸转移酶系统与3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸（DAHP）合酶对磷酸烯醇丙酮酸的竞争限制了通过莽草酸途径微生物合成的天然产物的浓度和产量。为了规避对磷酸烯醇丙酮酸的这种竞争，已创建了一种莽草酸途径变体。对大肠杆菌dgoA和肺炎克雷伯菌dgoA编码的2-酮-3-脱氧-6-磷酸半乳糖酸（KDPGal）醛缩酶进行定向进化。相对于天然KDPGal醛缩酶，进化后的KDPGal醛缩酶同工酶在催化丙酮酸和D-赤藓糖-4-磷酸缩合以产生DAHP方面表现出高4至8倍的比活性。为了探究所创建的莽草酸途径变体支持微生物生长和代谢的能力，对缺乏基于磷酸烯醇丙酮酸的DAHP合酶活性并依赖进化后的KDPGal醛缩酶进行莽草酸途径中间体和产物生物合成的大肠杆菌构建体的生长速率和3-脱氢莽草酸的合成进行了研究。
