Fig. 2: Abolishment of substrate inhibition through protein engineering.

a Using a predicted protein model, several positions within the R domain (lycopene cyclase) of CarRP were identified as suitable locations for mutation in order to reduce substrate inhibition. Single substitutions are indicated by copper spheres whereas double substitutions are indicated by purple spheres. b β-carotene selectivity was tested on a total of 50 generated variants. Compared to wild type (WT), Y27R, V175W, and T31R-F92W (in red) showed the significantly increased β-carotene selectivity, suggesting a reduction in substrate inhibition. Data represent the mean value of two independent experiments. c, d Compared to the control strain YLMA03 harboring wild-type CarRP, the variants showed significantly increased production of β-carotene, along with a decrease in lycopene accumulation (c). In particular, the strain YLMA11 expressing CarRP (Y27R) achieved a titer of 2.38 g/L (c), in addition to a high selectivity of 98% (d). e The abolishment of substrate inhibition allowed higher fluxes to be channeled through the carotenoid synthesis pathway (through MVA and IUP overexpression), improving β-carotene titers while maintaining the high selectivity. Ultimately, 4.22 g/L β-carotene was produced in YLMA15 with ~98% selectivity. For cultures using strains containing IUP, 30 mM isoprenol (Supplementary Fig. 10) was added to the media post-glucose depletion. For c to e, the average and s.d. of three biologically independent experiments are shown. Source data are provided as a Source Data file.