Team:SDU-Denmark/Tour31

Specifications

Rewiring of the E. coli machinery

As mentioned, the nonpathogenic E. coli strain MG1655, only lacks the prenyltransferase enzyme from the rubber tree before it is able to produce natural rubber. We also wanted to enhance the IPP production of the cell for further rubber production. As a consequence of these goals, we studied the prenyltransferase and the MEP pathway to gain knowledge and subsequently enable us to reach our rubber-producing ambitions.

The MEP pathway

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 Source: Lishan Zhao, Wei-chen Chang, Youli Xiao, Hung-wen Liu and Pinghua Liu: Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis, Annu. Rev. Biochem. 2013. 82:497-530, page 500. , 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.

The rubber synthesis

Polyprenyltransferase

When used for rubber production in E. coli, the MEP pathway is followed by a prenyltransferase catalysed rubber polymerisation reaction, where one IPP molecule condenses with DMAPP followed by condensation products: GPP geranyl diphosphate, C₁₀, FPP farnesyl diphosphate, C₁₅ and GGPP. geranylgeranyl diphosphate, C₂₀ ² Source: Alaxander Steinbüchel: Production of rubber-like polymers by microorganism, Current Opinion in Microbiology 2003. 6:261-270. (book) In Hevea brasiliensis, the rubber polymerisation reaction is catalysed by a cis-1,4- polyprenyltransferase known as Hevea Rubber Transferase 2 (HRT2).

Notes:Ecocyc noted that overexpression of Dxs results in increased isoprenoid biosynthesis, but also yields a reduced growth rate for the whole cell. Ecocycs reference