Fig. 3

Supplementation experiments indicate limiting S-adenosylmethionine (SAM) availability and demonstrate SAM biosynthesis bottleneck upstream of homocysteine. a Metabolic pathway diagram illustrating co-substrate requirement for reaction catalyzed by O-methyltransferase. SAM is generated from methionine, which in turn is generated from homocysteine. b Vanillate titers resulting from the presence or absence of 10 mM methionine supplementation to cultures 24 h after induction. Cultures receiving methionine produced nearly twofold higher vanillate titers. c Vanillate titers resulting from the presence or absence of 2.5 mM homocysteine supplementation to cultures 24 h after induction. Lower concentrations of homocysteine were used relative to methionine given the potential for homocysteine toxicity. Once again, cultures receiving supplement produced nearly twofold higher vanillate titers. d Vanillate specific yields resulting from homocysteine supplementation experiment. Higher specific yield upon homocysteine supplementation demonstrates that increased vanillate production is due to greater output per cell and not because of additional biomass. These pathway experiments used the PTS⁻ glu⁺ RAREʹ host and overexpression of aroG*,asbF _Bt , OMT _Hs , ppsA, and tktA