Fig. 3: A GGPPS-mediated metabolic flow restrictor effectively relieves substrate inhibition.

a A GGPPS-mediated metabolic flow restrictor can vary the amount of flux through the carotenoid synthesis pathway, thus regulating lycopene formation rates. b Changes in GGPPS activity can be achieved by expressing enzymes from different organisms in Y. lipolytica, as indicated by the varying in vivo GGPP synthesis rate. In these experiments, a po1f background strain with no modifications other than GGPPS expression was used. c Compared to the strain expressing GGPPxd, other strains housing lower-activity GGPPSs mitigated the substrate-inhibition effect of lycopene cyclase. The slower formation rates of lycopene prevented its accumulation and hence nearly all lycopene was converted into β-carotene. ND, not detected. d Fermentation time courses indicated that a balanced pathway with the attenuated GGPPsa (YLMA25) led to minimal lycopene build-up throughout the experiment, consistent with YLMA11, which contained the Y27R variant of CarRP. On the contrary, rapid lycopene accumulation was observed in the strain with the exceedingly efficient GGPPxd (YLMA03). e Gene expression cassettes containing the balanced pathway (GGPPsa, CarB, and CarRP) was sequentially introduced into the po1f-T strain for β-carotene production. With higher copy numbers, β-carotene titers increased up to 2.13 g/L, and the selectivity of β-carotene was maximized. f Overexpressing MVA and IUP further improved β-carotene synthesis while maintaining its high selectivity. g Using the exceedingly efficient GGPPxd to deliberately trigger substrate inhibition, along with a mutated CarRP(E78K), a lycopene-producing strain was constructed, which reached a titer of 2.62 g/L. ND, not detected. For b to g, The average and s.d. of three biologically independent experiments are shown. Source data are provided as a Source Data file.