Fig. 6

Chloroplastic metabolic engineering coupled with isoprenoid pool enhancement for committed Taxol intermediates in N. benthamiana cells. a Schematic representation of Taxol intermediate engineering in plant cells. Cytosolic engineering did not direct flux towards taxane biosynthesis, but enhanced a compound with similar retention time to taxadiene. The compound was speculated to be a native tobacco terpenoid. Chloroplastic engineering remarkably increased production of taxadiene and oxygenated taxanes, including taxadiene-5α-ol. MVA, mevalonate; AACT, acetoacetyl CoA thiolase; HMGS, hydroxymethylglutaryl coenzyme A synthase; HMGR, hydroxy 3 methylglutaryl coenzyme A reductase; MK, mevalonate kinase; PK, phosphomevalonate kinase; PMD, mevalonate pyrophosphate decarboxylase; MEP, 2-C-methyl-D-erythritol 4-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; IDI, IPP isomerase; T5αH, taxadiene 5α hydroxylase; CPR, Taxus cuspidata cytochrome P450 reductase; IspD, 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase; IspE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; IspH, isopentenyl/dimethylallyl diphosphate synthase. GGPPS, Geranylgeranyl diphosphate synthase. Lovastatin (10 μM) and Fosmidomycin (150 μM) were used to inhibit the MVA and the MEP pathways, respectively. b Gene constructs used to engineer oxygenated taxanes in the chloroplast. c The yield of taxanes produced in transient leaves at 5 dpi. d GC–MS analysis of tobacco leaf metabolites with GV3101 harboring empty vector; e GC–MS analysis of tobacco leaf metabolites with GV3101 harboring TS-tp(TS)/trT5αH-trCPR and DXS-GGPPS. Taxa-4(5),11(12)-diene (1) and three mono-oxygenated taxanes; taxadiene-5α-ol (2), OCT (3), and iso-OCT (4) are marked. Data in c represent the mean of n = 3 biologically independent samples (closed circles) and error bars show standard deviation. The source data of c are provided as a Source Data file