Fig. 5: Combinatorial CRISPRa programs can be applied to tune biosynthetic pathways.

a Tetrahydrobiopterin (BH4) production was tuned by delivering the combinatorial scRNA library to an E. coli strain harboring a BH4 pathway plasmid. BH4 is synthesized from GTP by expressing the gtpch gene from E. coli and the ptps and sr genes from M. alpina. BH4 then undergoes two oxidative decomposition steps yielding dihydrobiopterin (BH2) and biopterin. The BH4 pathway plasmid was constructed by placing the gtpch, ptps and sr genes under control of the J3, J5, and J6 promoters, respectively. b Tuning gene expression in new biosynthetic pathways only requires constructing a new pathway plasmid. The new plasmid is then cotransformed with the same scRNA library from Fig. 4. c Combinatorial tuning of BH4 pathway expression reveals that gtpch activity is limiting and that the sr gene is expressed in excess. Bars represent the average biopterins production of each strain in the combinatorial library harboring the BH4 pathway plasmid. Variations in biopterins production were measured by fluorescence (excitation: 340 nm, emission: 440 nm; see Supplementary Fig. 19). Baseline-subtracted normalized fluorescence values were converted into BH2 concentrations using the calibration curve in Supplementary Fig. 19a. The concentration values are given as BH2 concentration because >80% of the fluorescence signals generated from BH4 production strains have been previously shown to correspond to the BH2 oxidation state¹⁵. The x-axis heatmap is color coded to indicate the encoded promoter expression for each strain, as described on the bottom right. Values in panel c represent the average ± standard deviation calculated from n = 3 biologically independent samples. The sequence of the pathway plasmid containing J3-gtpch, J5-ptps and J6-sr is included in Supplementary Data 4. Source data are provided as a Source Data file.