Figure 5.

Evolution of esterase variants with activity on ABA esters. (a) Schematic of PACE for biocatalyst evolution. ABA derivative is preincubated with E. coli expressing ABA biosensor 3 h prior to transfer to a lagoon containing phage carrying BS2. Only BS2 phage that can hydrolyze the derivative to ABA will be able to replicate on the host cells and produce gIII to generate infectious progeny. (b) Biocatalyst PACE setup with a holding tank for host cell preincubation with ABA derivatives. (c) Phage competition in biocatalyst PACE. ABA ester 4a was preincubated with host cells, and phage carrying BS2 or HRV were mixed (1:500) as shown in part b. Phage samples were collected 0–48 h after addition to the lagoon and then analyzed by PCR. Bioconversions of BS2 and PACE BS2 variants on (d) ABA ester 4a or (e) ABA ester 4b.