Fig. 5. Optimization of violacein production by tuning plasmid copy number.

A The action of 5 enzymes on two tryptophan molecules yields violacein (B) The complete VioABCDE pathway was inserted in the pUC-pTet plasmid. C Violacein production scales with aTc concentration when the pathway is inserted into the pUC-pTet plasmid. D A library of variable copy number plasmids containing the VioABCDE pathway was created by performing mutagenesis on the priming promoter of a ColE1-based plasmid. E Violacein production is can be scaled up or down by plasmid copy number. At very high copy number production is low due to increased metabolic burden. Inset: Lag time is drastically increased in constructs with high violacein production. Error bars = std. dev. (n = 4). F A two plasmid system was created in which the VioABC enzymes were placed on a single plasmid of fixed copy number and the VioDE were placed in our library of variable copy number plasmids. G The use of a two plasmid system outperforms a the single plasmid system in raw violacein yields. H Growth rate is dramatically improved with a two plasmid system. Our randomized approach leads to the creation of constructs with fast growth rates and high amounts of violacein production.