Patnaik R, Liao J C
Department of Chemical Engineering, Texas A&M University, College Station 77843-3122.
Appl Environ Microbiol. 1994 Nov;60(11):3903-8. doi: 10.1128/aem.60.11.3903-3908.1994.
Escherichia coli and many other microorganisms synthesize aromatic amino acids through the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP). It has been shown that overexpression of transketolase increases the production of DAHP in an aroB mutant strain (unable to further metabolize DAHP) with elevated DAHP synthase. However, the yield (percent conversion) of DAHP from glucose is still low. Stoichiometric analysis shows that many enzymes compete for intracellular PEP. In particular, the phosphotransferase system, responsible for glucose transport in E. coli, uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. This stoichiometric limitation greatly reduces the yield of aromatic metabolites. To relieve this limitation, we overexpressed PEP synthase in the presence of glucose and showed that it increased the final concentration and the yield of DAHP by almost twofold, to a near theoretical maximum. The PEP synthase effect is not observed without overproduced transketolase, suggesting that erythrose 4-phosphate is the first limiting metabolite. This result demonstrates the utility of pathway analysis and the limitation of central metabolites in the high-level overproduction of desired metabolites.
大肠杆菌和许多其他微生物通过磷酸烯醇丙酮酸（PEP）与4-磷酸赤藓糖之间的缩合反应合成芳香族氨基酸，形成7-磷酸-3-脱氧-D-阿拉伯庚酮糖（DAHP）。研究表明，转酮醇酶的过表达会增加DAHP合酶水平升高的aroB突变菌株（无法进一步代谢DAHP）中DAHP的产量。然而，葡萄糖转化为DAHP的产率（转化率百分比）仍然很低。化学计量分析表明，许多酶竞争细胞内的PEP。特别是负责大肠杆菌中葡萄糖转运的磷酸转移酶系统，利用PEP作为磷酸供体并将其转化为丙酮酸，而丙酮酸再循环回PEP的可能性较小。这种化学计量限制大大降低了芳香族代谢物的产率。为了缓解这种限制，我们在有葡萄糖存在的情况下过表达了PEP合酶，结果表明它将DAHP的最终浓度和产率提高了近两倍，接近理论最大值。在没有过量表达转酮醇酶的情况下未观察到PEP合酶的作用，这表明4-磷酸赤藓糖是第一种限制代谢物。这一结果证明了途径分析的实用性以及中心代谢物在所需代谢物高水平过量生产中的局限性。
