The in vivo synthesis of plant sesquiterpenes by Escherichia coli

Three plant genes encoding (+)-delta -cadinene, delta -epi-aristolochene, a nd vetispiradiene cyclases were expressed in Escherichia coli to evaluate t he potential of this bacterium to synthesize sesquiterpenes in vivo, Variou s growth temperatures, carbon sources, and host strains were examined to op timize terpene production. The highest levels of sesquiterpene production o ccurred when the enzymes were expressed in strain DH5 alpha from the trc pr omoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented wit h 0.2% (v/v) glycerol at 30 degreesC for delta -epi-aristolochene and vetis piradiene and 37 degreesC for (+)-delta -cadinene. The highest concentratio ns of sesquiterpenes observed were 10.3 mug of (+)-delta -cadinene, 0.24 mu g of delta -epi-aristolochene (measured as (+)-delta -cadinene equivalents) , and 6.4 mug of vetispiradiene (measured as (+)-delta -cadinene equivalent s) per liter of culture. These sesquiterpene production levels are > 500-fo ld lower than carotenoid production, both of which are synthesized from end ogenous trans-farnesyl diphosphate (FDP) in E. coli. Based on these results , we conclude that the limiting factor for sesquiterpene synthesis in E. co li is the poor expression of the cyclase enzyme and not supply of the FDP p recursor. (C) 2001 John Wiley & Sons, Inc.