Fig. 1: Final metabolic network topology utilised in model development of wild-type E. coli engineered with the mevalonate (MVA) pathway to overproduce limonene.

There are several different metabolic pathways involved in limonene biosynthesis, including the glycolytic pathways, tricarboxylic acid cycle (TCA), the native deoxyxylulose 5-phosphate (DXP), and MVA pathways. Co-factor (in orange) consumption is represented by curved arrows. Intermediates: Glcex extracellular glucose, Glc glucose, G6P glucose-6-phosphate, 6PG 6-phosphogluconate, KDPG 2-keto-deoxy-6-phosphogluconate, X5P xylulose-5-phosphate, Ru5P ribulose-5-phosphate, R5P ribose-5-phosphate, F6P fructose-6-phosphate, F16BP fructose-1,6-biphosphate, GAP glyceraldehyde-3-phosphate, DHAP dihydroxyacetone phosphate, BPG 1,3-bisphosphoglycerate, 3PG 3-phosphoglycerate, PEP phosphoenolpyruvate, PYR pyruvate, DXP 1-deoxy-d-xylulose-5-phosphate, B flux to vitamin B6 pathway, MEP 2-C-methylerythritol-4-phosphate, CDPME 4-diphosphocytidyl-2-C-methylerythritol, CDPMEP 4-diphosphocytidyl-2-C-methylerythritol-2-phosphate, MEcPP 2-C-Methylerythritol-2,4-cyclodiphosphate, HMBPP hydroxymethylbutenyl 4-diphosphate, IPP isopentenyl diphosphate, DMAPP dimethylallyl diphosphate, GPP geranyl diphosphate, FPP farnesyl diphosphate, LIM limonene, LIMex extracellular limonene, AcCoA acetyl coenzyme A, AtAcCoA acetoacetyl-CoA, HMGCoA hydroxymethylglutaryl-CoA, MVA mevalonate, MVAP 5-phosphomevalonate, MVAPP 5-diphosphomevalonate, ACE acetic acid, ACEex extracellular acetic acid, ACTLD acetaldehyde, ETH ethanol, ETHex extracellular ethanol, LAC lactic acid, LACex extracellular lactic acid, AKG α-ketoglutarate, SucCoA, succinyl-CoA, SUC succinate, SUCex extracellular succinate, FUM fumarate, OAA oxaloacetate. Enzymes (in green): PTS phosphotransferase system, HK hexokinase, G6PDH lumped reactions of glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, PGDH 6-phosphogluconate dehydrogenase, KDPGA 2-keto-deoxy-6-phosphogluconate aldolase, Tkb transketolase, PGI phosphoglucose isomerase, PFK phosphofructokinase, FBA fructose-1,6-biphosphate aldolase, GDH glutamate dehydrogenase, PGK phosphoglycerate kinase, ENO enolase, PYK pyruvate kinase, DXS DXP synthase, DXR DXP reductase, ISPD CDPME synthase, ISPE CDPME kinase, ISPF MEcPP synthase, ISPG HMBPP synthase, ISPH HMBPP reductase, IDI isopentenyl diphosphate isomerase, ISPA farnesyl diphosphate synthase, LS limonene synthase, PDH pyruvate dehydrogenase, AtoB acetyl-CoA acetyltransferase, HMGS HMGCoA synthase, HMGR HMGCoA reductase, MK mevalonate kinase, PMK phosphomevalonate kinase, PMD diphosphate mevalonate decarboxylase, LDH lactate dehydrogenase, PoxB pyruvate oxidase, PCK phosphoenolpyruvate carboxykinase, PPC phosphoenolpyruvate carboxylase, ACS acetyl-CoA synthetase, PTACK lumped reactions of phosphate acetyltransferase and acetate kinase, ALDHB aldehyde dehydrogenase B, ALDH aldehyde dehydrogenase, ADH alcohol dehydrogenase, CSICD lumped enzymatic reactions of citrate synthase, aconitate hydratase A, aconitate hydratase B and isocitrate dehydrogenase, AKGDH α-ketoglutarate dehydrogenase, SCS succinyl-CoA synthetase, FRD fumarate reductase, MDH malate dehydrogenase. The illustration was created using Biorender.com.