Figure 4. Selection system for MtCM variants that can be activated by MtDS.

The selection system used is based on E. coli strain KA12 lacking the endogenous pheA and tyrA genes, which encode CM-prephenate dehydratase (PDT) and CM-prephenate dehydrogenase (PDH), respectively. Plasmid pKIMP-UAUC has the p15A-derived origin of replication (ori _p15A) and carries *tyrA for a monofunctional PDH and pheC for a monofunctional PDT, in addition to cat providing chloramphenicol resistance [21]. Plasmid pKIMP-ACG additionally contains the aroG gene encoding MtDS. (A) Performance of wild-type MtCM (encoded by aroQ_δ on plasmid pKTNTET) without MtDS (pKIMP-UAUC) or with MtDS (pKIMP-ACG) in comparison to a negative control (pKTCTET-0, lacking aroQ_δ). Growth was assessed on M9c-based minimal plates in the presence or absence of Phe (F) and Tyr (Y). Colony size was scored either as++(good growth),+(moderate growth), – (poor growth), or 0 (no trace of growth) as a function of the added Tet concentration, the inducer of the P_tet promoter upstream of aroQ_δ. (B) Schematic representation of the redesigned selection system. The aroQ_δ gene encoding an MtCM library variant is provided on plasmid pKT-CM that has otherwise the same structure as pKTNTET (bla, β-lactamase for ampicillin resistance; its ori _pUC is compatible with ori _p15A). Under stringent selection conditions (M9c), host cells transformed with both pKIMP-ACG and a pKT-CM library plasmid can only produce enough prephenate and consequently Phe and Tyr needed for growth if the encoded MtCM variant can engage in a productive complex with MtDS (wide green arrow). Transformants having insufficient CM activity (thin, light green arrow) require exogenously added F and Y for growing on M9c minimal plates.