Department of Chemistry, Stanford University, Stanford, CA 94305-5025, USA.
Metab Eng. 2010 Jul;12(4):378-86. doi: 10.1016/j.ymben.2010.02.003. Epub 2010 Feb 23.
Fatty acids are central hydrocarbon intermediates in the biosynthesis of diesel from renewable sources. We have engineered an Escherichia coli cell line that produces 4.5 g/L/day total fatty acid in a fed-batch fermentation. However, further enhancement of fatty acid biosynthesis in this cell line proved unpredictable. To develop a more reliable engineering strategy, a cell-free system was developed that enabled direct, quantitative investigation of fatty acid biosynthesis and its regulation in E. coli. Using this system, the strong dependence of fatty acid synthesis on malonyl-CoA availability and several important phenomena in fatty acid synthesis were verified. Results from this cell-free system were confirmed via the generation and analysis of metabolically engineered strains of E. coli. Our quantitative findings highlight the enormous catalytic potential of the E. coli fatty acid biosynthetic pathway, and target specific steps for protein and metabolic engineering to enhance the catalytic conversion of glucose into biodiesel.
脂肪酸是从可再生资源中生产柴油的生物合成中的重要碳氢化合物中间体。我们已经构建了一种大肠杆菌细胞系，可在分批补料发酵中每天生产 4.5 克/升的总脂肪酸。然而，进一步提高该细胞系中的脂肪酸生物合成证明是不可预测的。为了开发更可靠的工程策略，开发了一种无细胞系统，该系统可直接定量研究大肠杆菌中脂肪酸的生物合成及其调节。使用该系统，验证了脂肪酸合成对丙二酰辅酶 A 可用性的强烈依赖性以及脂肪酸合成中的几个重要现象。通过生成和分析大肠杆菌的代谢工程菌株，对无细胞系统的结果进行了验证。我们的定量发现突出了大肠杆菌脂肪酸生物合成途径的巨大催化潜力，并针对特定步骤进行蛋白质和代谢工程，以提高葡萄糖转化为生物柴油的催化转化率。
