Fig. 3: Reprogramming lycopene biosynthesis by BETTER.

a Lycopene biosynthesis pathway and three artificial clusters equipped with constitutive promoters and tailored GGGGGGGG RBSs. Ten target genes are highlighted in red. CDP-ME 4-diphosphocytidyl-2-C-methyl-d-erythritol, CDP-MEP 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate, DMAPP dimethylallyl diphosphate, FPP farnesyl diphosphate, GGPP geranylgeranyl pyrophosphate, HMBPP (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate, IPP isopentenyl diphosphate, MEC 2C-methyl-d-erythritol-2,4-cyclodiphosphate, MEP 2-C-methyl-d-erythritol-4-phosphate. b Schematic illustration of two sets of double-gRNAs combinations and corresponding editing windows. The two Gs shaded in green are covered by two editing windows (blue and orange). c Components of RBS libraries of dxs gene generated by BETTER with the two sets of double-gRNAs combinations. Merged represents the sum of RBS variants generated by the aforementioned two editing events. Three colonies were used for base editing and the cells were mixed with an equal proportion before extraction of genomic DNAs, PCR amplification, and NGS. d Discreteness change of genetic combinations of RBS_ispE generated by BETTER with the two sets of double-gRNAs combinations. KL divergence was used to evaluate the discreteness of genetic combinations. e The matrixes show the coverage and discreteness of 256 G/A-containing RBS_ispE variants in the RBS libraries generated by BETTER with the two sets of double-gRNAs combinations. Scale bar represents log₂(relative proportion). f Screening lycopene overproduction cells from the libraries generated by BETTER. g Lycopene production and RBS strength assay of the ancestral strain (strain 0) and four screened strains with intense red pigmentation (strains 1-4). RBS strength was determined using a chromosomal GFP reporter. Scale bar represents GFP fluorescence normalized with OD_{600 nm}. Values and error bars reflect the mean ± s.d. of three biological replicates (n = 3). Statistical evaluation (P value) of the comparison between each screened strain and the ancestral strain was performed by a two-sided t test. ***P < 0.001, n = 3. Source data underlying Fig. 3d, e, g are provided as a Source Data file.