Yi Jian, Draths K M, Li Kai, Frost J W
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Biotechnol Prog. 2003 Sep-Oct;19(5):1450-9. doi: 10.1021/bp0340584.
Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3-dehydroshikimic acid and byproducts 3-deoxy-d-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3-Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroF(FBR) and tktA inserts encoding, respectively, a feedback-insensitive isozyme of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3-dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf-encoded glucose facilitator and glk-encoded glucokinase resulted in the synthesis in 48 h of 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP-encoded galactose permease for glucose transport required 60 h to synthesize 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor-controlled conditions.
研究了不同的葡萄糖转运系统对磷酸烯醇丙酮酸可用性的影响，这可通过3-脱氢莽草酸的产量以及大肠杆菌从葡萄糖合成的副产物3-脱氧-D-阿拉伯庚酮糖酸、3-脱氢奎尼酸和没食子酸来反映。3-脱氢莽草酸是莽草酸途径中的一种高级中间体，可用于合成一系列商品、准商品和精细化学品。所有构建体均携带质粒aroF(FBR)和tktA插入片段，分别编码对反馈不敏感的3-脱氧-D-阿拉伯庚酮糖酸7-磷酸合酶同工酶和转酮醇酶。依赖天然大肠杆菌磷酸烯醇丙酮酸：碳水化合物磷酸转移酶系统进行葡萄糖转运，在48小时内导致合成3-脱氢莽草酸（49 g/L）和莽草酸途径副产物，总产率为33%（摩尔/摩尔）。使用异源表达的运动发酵单胞菌glf编码的葡萄糖转运蛋白和glk编码的葡萄糖激酶，在48小时内合成了3-脱氢莽草酸（60 g/L）和莽草酸途径副产物，总产率为41%（摩尔/摩尔）。利用天然大肠杆菌galP编码的半乳糖通透酶进行葡萄糖转运，需要60小时才能合成3-脱氢莽草酸（60 g/L）和莽草酸途径副产物，总产率为43%（摩尔/摩尔）。以前，由于不同的实验设计和培养条件，对改变葡萄糖转运对大肠杆菌合成莽草酸途径产物产量的影响进行直接比较受到了阻碍。在本研究中，使用来自同一祖代菌株的大肠杆菌构建体合成的相同产物和副产物混合物来比较提高磷酸烯醇丙酮酸可用性的策略。构建体在同一组发酵罐控制条件下培养。
