Team:SDU-Denmark/Tour31

Specifications

Rewiring of the E. coli machinery

MEP pathway The biosynthesis of isoprenoids in bacteria works through the methylerythritol phosphate pathway (MEP pathway). The pathway is initiated with a condensation reaction between D-glyceraldehyde 3-phosphate and pyruvate to produce DXP 1-deoxy-D-xylulose-5-phosphate and CO2 catalysed by Dxs. 1-deoxyxylulose-5-phosphate synthase

DXP is reductively isomerized to MEP 2-C-methyl-D-erythritol-4-phosphate by Dxr 1-deoxy-D-xylulose-5-phosphate reductoisomerase in a reversible NADPH-dependent reaction and subsequently converted to CDP-ME 4-diphosphocytidyl-2-C-methyl-D-erythritol in a CTP substrate inhibited reaction catalysed by IspD. 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase

DXP is not only a substrate in the isoprenoids biosynthesis, but also for the reversible synthesis of B1-vitamin and further synthesis of B6-vitamin in the thiamine biosynthesis pathway.

The next step in the MEP pathway is an ATP-dependent phosphorylation of the C2 hydroxyl group of CDP-ME, giving CDP-MEP 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate catalysed by IspE. 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase

CDP-MEP is cyclized to MEcPP 2-C-methyl-D-erythritol-2,4-cyclodiphosphat by IspF 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase and in the next reaction IspG 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase catalyzes the ring opening of the cyclic pyrophosphate and subsequent C3-reductive dehydration of MEcPP to HMBPP. 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate

The MEP pathway’s final step, the conversion of HMBPP to IPP isopentenyl diphosphate and DMAPP dimethylallyl diphosphate, is catalysed by IspH. 4-hydroxyl-3-methyl-butenyl-1-disphosphate reductase IPP and DMAPP are in equilibrium. The enzyme Idi isopentenyl diphosphat isomerase catalyses the reversible isomerization reaction between IPP and DMAPP in order to balance the ratio of IPP and DMAPP according to the cellular demands under various conditions 1, which in our system will cause dislocation of the equilibrium towards IPP production since IPP is the five carbon building block in the rubber biosynthesis. In accordance, the reversible formation of B1-vitamin from DXP, will be dislocated towards the production of DXP rather than B1-vitamin.