Fig. 4: A combination of the full-optimized designer three-enzyme pathway and the engineered E. coli cell factory leads to significant enhancement of Ψ titer.

a Ψ production by strains (CY1, CY2, CY3, CY4, and CY5) individually containing pRL01. The inactivation of Ψ kinase and nucleoside transporters was utilized to enhance Ψ titer. b Increasing Ψ production by introducing HDHD1 (coding for ΨMP specific phosphatase) (CY5/pRL02, CY6/pRL01), and blocking the precursor-consumption (CY7, CY8, CY9, CY10, and CY11). c Schematic diagram of plasmid pRL01 and pRL03. d The promoter engineering of EcpsuG and NmygdH on the expression plasmid, and HDHD1 on the chromosome, and the colors of promoters correspond to those in Supplementary Fig. 41. Left column, flask fermentation of related strains individually containing pRL01 was conducted with glycerol as carbon source. Right column, flask fermentation of related strains individually containing pRL03 was performed using glucose as carbon source. e Fed-batch fermentation monitoring (96 h) of the CY15/pRL01 strain with glycerol as carbon source. f Fed-batch fermentation monitoring (96 h) of the CY15/pRL03 strain utilizing glucose as carbon source. In (a) and (b), “+” denotes that the strategy has been conducted; and the denotation of “-” was conversely assigned. In (a), (b), and (d), “*” indicates p < 0.05, “**” indicates p < 0.01, “***” indicates p < 0.001, and “****” indicates p < 0.0001, and the values are the means ± s.d. measured from three biological replicates. The statistical test was performed using one-way ANOVA. In (e) and (f), Ψ titer, cell growth, and dry cell weight are shown as curve graph. The values (shown as related colored shades) were the means ± s.d. measured from three biological replicates. Source data are provided as a Source Data file.