Fig. 4.

Bottom-up in vitro SCRaMbLE for β-carotene pathway optimization. a Phenotype–genotype correlation of the β-carotene pathway after bottom-up in vitro SCRaMbLE (strategy 1). A total of 17 SCRaMbLEd yeast strains were isolated for testing colony color and determination of SCRaMbLEd construct sequences. The pictures of yeast color for both SCRaMbLEd strains and strains with re-introduced SCRaMbLEd plasmid were taken after 3 days incubation at 30 °C on SC–Ura medium. The SCRaMbLEd pathway structure was initially analyzed by restriction enzyme digestion to check the number of insertions and PCR analysis to evaluate which genes were inserted. Primer walking sequencing was applied to verify the sequence of all recovered plasmids. yYW0339 with only a URA3 gene inserted was used as a control strain. b HPLC measurement of carotenoid production for SCRaMbLEd yeast strains. Quantification was performed in biological triplicate for each strain as shown. Error bars represent standard deviation from three replicates