Fig 2. Effect of PABA/PHB analogs on CoQ production.

(A) For the pre-culture, WT PR110 cells were cultivated in 10 mL medium for 1 day. The indicated amount (μg/mL) of benzoic acid (Bz), 4-nitrobenzoic acid (4-NB), 4-chlorobenzoic acid (4-ClBz), or 2,4-dihydroxybenzoic acid (2,4-DiHB, β-resorcylic acid) was added to the media containing ~1×10⁵ cells/mL and the cells were cultivated for two days with rotation at 30°C. Gray bars show the CoQ₁₀ content per 50 mL of medium, and white bars show CoQ₁₀ normalized against cell number. Diamonds show cell number. Five micrograms of CoQ₆ was used as an internal standard. Data are represented as the mean ± standard deviation (SD) of three measurements. Asterisks on bars denote statistically significant differences (**p<0.01) relative to the amount of CoQ in the medium or cells grown in YES (Student’s t-test). (B) CoQ₁₀ intermediate-like peak detected by HPLC analysis. (C) Quantitative analysis of the CoQ₁₀ intermediate-like peak (*) which is predicted to be 2-methoxy-4-hydroxy-5-decaprenyl-benzoic acid (2-methoxy-DHB). HPLC analysis was performed at 269 nm. (D) For LC-MS/MS analysis, the m/z 867 ion associated with [M+NH₄]⁺ selected as the precursor ion for a compound predicted to be 2-methoxy-DHB is shown (-R indicates the decaprenyl moiety).