Fig. 3

Engineering precursor supply to the shikimate pathway. a Overall view of the metabolic pathways giving rise to phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), and also the reactions that consume them. Reactions and genes in blue are heterologous, enzymes in green have their genes overexpressed, and other genes of interest are named. b Increasing PEP supply to the shikimate pathway heterologous using PEP synthases and pyruvate orthophosphate dikinases, and a native enolase. Enzymes of all three classes significantly increased levels of 2-PE, our proxy for phenylalanine production. c Increasing E4P supply to the shikimate pathway using fructose-6-phosphate phosphoketolases (Xfpk) from bacterial and fungal sources. d Attempts to simultaneously increase PEP and E4P supply to the shikimate pathway showed that the effects of increasing either metabolite individually are not additive, though in the end a modification was found that increased 2-PE production beyond what was achieved by individual precursors. Strains producing significantly higher amounts of 2-PE relative to that produced by ASR.004 as determined by an independent t-test are marked by an asterisk (*p < 0.05) or a hash (^#p < 0.005). All data are plotted as the mean ± s.d. of at least three biological replicates. Ec: Escherichia coli, Ct: Chaetomium thermophilum, Tt: Thielavia terrestris, Hm: Halofax mediterranei, An: Aspergillus nomius, At: Arabidopsis thaliana, Km: Kluyveromyces marxianus, Bb: Bifidobacter breve, Ll: Lactococcus lactis, Ca: Clostridium acetobutylicum, Rg: Rhodotorula graminis