Team:SDU-Denmark/Tour30

The production system

The micro-road to rubber

Natural rubber is a chain of several isoprene molecules. The rubber tree has a prenyltransferase PrenyltransferaseHRT2 enzyme, which links molecules of IPP IPPIsopentenyl pyrophosphate into the isoprene chain Isoprene chainnatural rubber. To start the extension of the chain, the prenyltransferase needs a DMAPP DMAPPDimethylallyl pyrophosphate molecule. Thus, in order to produce rubber, the bacteria needs the prenyltranferase, IPP, and DMAPP. Source: Light DR, Dennis MS. Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis. J Biol Chem. 1989 Nov 5;264(31):18589-97. (Link)

The basic idea is to convert sugar (glucose) into natural rubber through the glycolysis to yield pyruvate and GAP. These are further converted into IPP and DMAPP through the MEP pathway. A DMAPP and several IPP molecules can then be linked together into natural rubber through the action of the prenyltransferase (HRT2) from the rubber tree. IPP and DMAPP are molecules naturally present in E. coli, produced by the bacteria through the MEP pathway. MEP pathwayMethylerythritol phosphate pathway The MEP pathway converts molecules of GAP GAPGlyceraldehyde-3-phosphate and pyruvate - produced by the degradation of glucose (glycolysis) - into IPP and DMAPP. To increase the amount of IPP and DMAPP available for rubber production, we optimized the MEP pathway by elevating the levels of rate limiting enzymes in the pathway. Source: Zhao L, Chang WC, Xiao Y, Liu HW, Liu P. Methylerythritol phosphate pathway of isoprenoid biosynthesis. Annu Rev Biochem. 2013;82:497-530. doi: 10.1146/annurev-biochem-052010-100934. (Link)

Through gene manipulation, we codon-optimized and introduced the prenyltransferase gene from the rubber tree to E. coli. This enables the bacteria to express the prenyltransferase enzyme. In so far that the system is regulable, the bacteria should be able to convert sugar into natural rubber through; the glycolysis, the MEP pathway, and the prenyltransferase activity. Our experiments point towards this being possible. In fact, our data indicates that our bacteria do produce rubber, and we have proof of expression control. Source: Asawatreratanakul K, Zhang YW, Wititsuwannakul D, Wititsuwannakul R, Takahashi S, Rattanapittayaporn A, Koyama T. Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis. Eur J Biochem. 2003 Dec;270(23):4671-80. (Link)

Dig deeper to see the specification and models of our system, how we designed it in detail, and what thoughts we have about safety.
Or go to next chapter to learn more about the process.