Martin V J, Yoshikuni Y, Keasling J D
Department of Chemical Engineering, University of California, Berkeley, CA 94720-1462, USA.
Biotechnol Bioeng. 2001 Dec 5;75(5):497-503. doi: 10.1002/bit.10037.
Three plant genes encoding (+)-delta-cadinene, 5-epi-aristolochene, and vetispiradiene cyclases were expressed in Escherichia coli to evaluate the potential of this bacterium to synthesize sesquiterpenes in vivo. Various growth temperatures, carbon sources, and host strains were examined to optimize terpene production. The highest levels of sesquiterpene production occurred when the enzymes were expressed in strain DH5alpha from the trc promoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented with 0.2% (v/v) glycerol at 30 degrees C for 5-epi-aristolochene and vetispiradiene and 37 degrees C for (+)-delta-cadinene. The highest concentrations of sesquiterpenes observed were 10.3 microg of (+)-delta-cadinene, 0.24 microg of 5-epi-aristolochene (measured as (+)-delta-cadinene equivalents), and 6.4 microg of vetispiradiene (measured as (+)-delta-cadinene equivalents) per liter of culture. These sesquiterpene production levels are >500-fold lower than carotenoid production, both of which are synthesized from endogenous trans-farnesyl diphosphate (FDP) in E. coli. Based on these results, we conclude that the limiting factor for sesquiterpene synthesis in E. coli is the poor expression of the cyclase enzyme and not supply of the FDP precursor.
三个编码（+）-δ-杜松烯、5-表-马兜铃烯和菖蒲二烯环化酶的植物基因在大肠杆菌中表达，以评估该细菌在体内合成倍半萜的潜力。研究了各种生长温度、碳源和宿主菌株以优化萜类化合物的产量。当这些酶在高拷贝质粒pTrc99A的trc启动子（Ptrc）控制下，于补充了0.2%（v/v）甘油的M9培养基中，在30℃下表达5-表-马兜铃烯和菖蒲二烯环化酶，以及在37℃下表达（+）-δ-杜松烯环化酶时，菌株DH5α中倍半萜的产量最高。观察到的倍半萜最高浓度分别为每升培养物中10.3微克（+）-δ-杜松烯、0.24微克5-表-马兜铃烯（以（+）-δ-杜松烯当量计）和6.4微克菖蒲二烯（以（+）-δ-杜松烯当量计）。这些倍半萜的产量水平比类胡萝卜素的产量低500倍以上，二者均由大肠杆菌内源性反式法呢基二磷酸（FDP）合成。基于这些结果，我们得出结论，大肠杆菌中倍半萜合成的限制因素是环化酶的低表达，而非FDP前体的供应。
