Fig. 4
Induction and screening of pathway SCRaMbLE-in yeast variants. a A URA3-based counter selection strategy to facilitate the SCRaMbLE-in process. The SCRaMbLE-in device is based on a yeast centromeric plasmid with URA3 marker (pRS416). A BsaI site-flanked RFP cassette and a LEU2 expression cassette were placed between two loxPsym sites. LEU2 is used as positive selection marker for integration and URA3 is used as counter selection marker for non-integrated strains. After SCRaMbLE-in induction, successful integrated colonies were selected on SC-Leu + 5-FOA plates. b Violacein quantification in the violacein pathway SCRaMbLE-in variants. LWy137 is control strain with a single-copy violacein pathway inserted at the HO locus. LWy152, LWy238, and LWy239 are violacein SCRaMbLE-in strains. Error bar represents the standard deviation, n = 3. c β-carotene quantification in β-carotene pathway SCRaMbLE-in variants. LWy212 is control strain with a single-copy β-carotene pathway inserted at the HO locus. LWy215, LWy252, and LWy253 are β-carotene SCRaMbLE-in strains. Error bar represents the standard deviation, n = 3. d Color comparison of continuous SCRaMbLEd variants with violacein integrated pathway. Both darker colony color and lighter colony color were observed for the three SCRaMbLE-in strains with continuous SCRaMbLE. LWy152, LWy238, and LWy239 are SCRaMbLE-in strains; LWy256 and LWy257 are continuous SCRaMbLEd strains from LWy152; LWy258 and LWy259 are continuous SCRaMbLEd strains from LWy238; LWy260 and LWy261 are continuous SCRaMbLEd strains from LWy239. e Violacein quantification of continuous SCRaMbLEd variants with violacein integrated pathway. 152+: increased production compared with source strain LWy152; 152−: decreased production compared with source strain LWy152. Error bar represents the standard deviation, n = 3
