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To increase the flux through the upstream MEP pathway, we targeted reported enzymatic bottlenecks (dxs, idi, ispD, and ispF) (gray) for overexpression by an operon (dxs-idi-ispDF) (Yuan et al., 2006). To channel the overflow flux from the universal isoprenoid precursors, IPP and DMAPP, toward Taxol biosynthesis, we constructed a synthetic operon of downstream genes GGPP synthase (G) and taxadiene synthase (T) (Walker and Croteau, 2001). Both pathways were placed under the control of inducible promoters in order to control their relative gene expression. In the E. coli metabolic network, the MEP isoprenoid pathway is initiated by the condensation of the precursors glyceraldehyde-3 phosphate (G3P) and pyruvate (PYR) from glycolysis. The Taxol pathway bifurcation starts from the universal isoprenoid precursors IPP and DMAPP to form geranylgeranyl diphosphate, and then the taxadiene. The cyclic olefin taxadiene undergoes multiple rounds of stereospecific oxidations, acylations, and benzoylation to form the late intermediate Baccatin III and side chain assembly to, ultimately, form Taxol.

O-Man glycan biosynthetic pathway. The biosynthetic pathways of representative O-Man glycans are shown. Genes that cause a disorder are indicated in red. Three main groups are identified: core M1–M3. All O-Man glycans are initiated on either a serine (S) or a threonine (T) in the acceptor protein using two enzymes protein-O-mannosyltransferase 1 and 2 (POMT1/2) and Dol-P-Man donor. Several genes in the biosynthesis of Dol-P-Man (GMPPB, DPM1–3) are deficient in some patients with dystroglycanopathy, whereas others (DOLK, DPM1, and PMM2) cause a more generalized congenital disorders of glycosylation (CDG). Mannose receives either GlcNAcβ1-2- to form core M1 or β1-4GlcNAc to form core M3. Mutations in both genes (POMGNT1 and POMGNT2) can cause a dystroglycanopathy. Core M2 can be formed if a branching β1-6GlcNAc is added. Core M1 and M2 are elongated by Gal and may terminate with Fuc, Sia, and GlcA with optional sulfation. None of the genes responsible for adding the terminal sugars have been associated with dystroglycanopathies. After the addition of the β1-4GlcNAc, core M3 is elongated with GalNAc, and then the Man is 6-O-phosphorylated via a specific kinase (POMK, also known as SGK196). With help of POMGNT1, FKTN and FKRP can act to add two ribitol-5-phosphate units to the GalNAc residue that is then extended with a single Xyl and GlcA by TMEM5 and B4GAT1, respectively. Ribitol-5-phosphate used by FKTN and FKRP is synthesized from CDP-ribitol by ISPD. Mutations in these genes (B3GALNT2, POMK, FKTN, FKRP, ISPD, TMEM5, and B4Gat1) can cause a dystroglycanopathy. The last defined step in core M3 biosynthesis is using this Xyl-GlcA primer for the stepwise addition of Xyl and GlcA to form a GAG-like repeating disaccharide termed matriglycan. This is catalyzed by the enzyme encoded by LARGE (or its homolog GYLTL1B) with dual glycosyltransferase activities. Matriglycan binds laminin to α-DG and its reduction or loss is believed to be the cause of most of the dystroglycanopathies (POMGNT1 mutations being the exception).

O-Man glycan biosynthetic pathway. The biosynthetic pathways of representative O-Man glycans are shown. Enzymes whose defects cause a disorder are indicated in red. Three main groups are identified: cores M1–M3. All O-Man glycans are initiated on either a serine (S) or a threonine (T) in the acceptor protein using two enzymes protein-O-mannosyltransferase 1 and 2 (POMT1/2) and Dol-P-Man donor. Several genes in the biosynthesis of Dol-P-Man (GMPPB, DPM1–3) are deficient in some patients with dystroglycanopathy, whereas others (DOLK, DPM1, and PMM2) cause a more generalized congenital disorder of glycosylation (CDG). Mannose receives either GlcNAcβ1-2- to form core M1 or β1-4GlcNAc to form core M3. Mutations in both genes (POMGNT1 and POMGNT2) can cause a dystroglycanopathy. Core M2 can be formed if a branching β1-6GlcNAc is added. Core M1 and M2 are elongated by Gal and may terminate with Fuc, Sia, and GlcA with optional sulfation. None of the genes responsible for adding the terminal sugars have been associated with dystroglycanopathies. After the addition of the β1-4GlcNAc, core M3 is elongated with GalNAc, and then the Man is 6-O-phosphorylated via a specific kinase (POMK, also known as SGK196). With help of POMGNT1, FKTN and FKRP can act to add two ribitol-5-phosphate units to the GalNAc residue that is then extended with a single Xyl and GlcA by TMEM5 and B4GAT1, respectively. Ribitol-5-phosphate used by FKTN and FKRP is synthesized from CDP-ribitol by CRPAA, also known as ISPD. Mutations in these genes (B3GALNT2, POMK, FKTN, FKRP, CRPPA, TMEM5, and B4Gat1) can cause a dystroglycanopathy. The last defined step in core M3 biosynthesis is using this Xyl-GlcA primer for the stepwise addition of Xyl and GlcA to form a GAG-like repeating disaccharide termed matriglycan. This is catalyzed by the enzyme encoded by LARGE1 (or its homolog GYLTL1B) with dual glycosyltransferase activities. Matriglycan binds laminin to α-DG and its reduction or loss is believed to be the cause of most of the dystroglycanopathies (POMGNT1 mutations being the exception).