Currently the only source of commercially usable natural rubber that can be processed into latex is available from the rubber tree H. brasiliensis. While other plants are capable of producing rubber particles, these particles when processed are weaker, requiring less extension to break, compared to natural rubbers produced by H. brasiliensis.[4] In fact, H. brasiliensis is responsible for almost all of the world's natural rubber production through mostly rubber plantations or tree tapping.[2] Rubber farming however in recent years has been threatened by production shortfalls owing to diseases such as South American Leaf blight. H. brasiliensis’ narrow genetic base also signifies most large acreage farms plant genetically identical trees, making them prone to large crop failure.[5, 6] This problem is further exacerbated by deforestation and the growing land need for agriculture, which both decrease the amount of land available for rubber tree plantations, and consequently limit rubber production.[7, 8]

Due to the difficulties of harvest and acreage demand on latex plantations, chemical synthesis of synthetic latex is appealing alternative to natural latex. Although natural latex and synthetic latex have different chemical and physical properties, both materials are largely comprised of cis-1,4-polyisoprene polymers. While natural latex is difficult to handle and has diminished durability, resilience, and elasticity without vulcanization, synthetic latex does not require vulcanization and can be prepared using different proportions of isoprene monomers to yield a wide range of physical, mechanical, and chemical properties.[9, 10] By varying the mixture of isoprene and styrene butadiene polymers, synthetic rubbers have a unique advantage in that they can be tuned to a particular use. However, synthetic rubber lacks the mechanical and low temperature performance of its natural counterpart. Despite characteristic differences, both natural latex and synthetic latex rely heavily on cis-polyisoprene polymers as their primary constituent.

With global consumption of latex at over 11 million metric tons per annum, latex is an essential raw material worldwide.[11] Currently, natural rubber accounts for 40% of the global rubber demand, with the remaining 60% supplied from synthetic rubber.[12] However, the increasing price of petroleum has elevated prices in the synthetic rubber industry and consequently exacerbated the current market shortfall of natural rubbers. Additionally, butadiene, the primary monomer used in synthetic rubber synthesis, is facing a global shortage which is increasing the cost of synthetic rubber synthesis.[13] With an increasing demand of 5-6% per annum, the global latex economy cannot be sustained by the elevating cost synthetic rubber synthesis and dependence on shrinking latex farms.[8] For these reasons, an alternative method for latex production is desperately needed to sustain global demand and undercut material shortages.

Through our project, we sought to address the need for a faster and more economical alternative latex production system to mitigate the accumulating economic demand shortfall. To achieve our objective, we sought to design a transgenic organism capable of mimicking the natural rubber production process found in H. brasiliensis. Not only would a transgenic single cell organism allow for optimization of polymer synthesis, but also permit a high yield in polymer producing cells. A scaled up cell culture bioreactor would yield large volumes of both latex (cis-polyisoprene) producing cells and cis-polyisoprene polymers with minimal growth media input. For these reasons, we were motivated towards metabolic engineering Escherichia coli for high yield production of cis-polyisoprene compounds which could be used as a synthetic and natural latex replacement or precursor.

Our objective through this project was to enable E. coli to produce isoprene polymers (when supplied basic cell growth media and nutrients, such as glucose, magnesium, and nitrogen compounds. Our invention must also be scalable in culture to allow for high volume production of polyisoprene compounds. To develop the latex synthesis pathway in E. coli, we first examined the chemical constituents and the metabolic processes responsible for latex production.

Although there are a variety of polyisoprene polymer forms, natural rubber is largely composed of cis-1,4-polyisoprene, which is a polymer of isoprene units joined at the first and fourth carbon by a double bond. In order to produce the cis-1,4-polyisoprene polymer, rubber trees such as H. brasiliensis employ cis-prenyltransferase enzymes (also commonly referred to as rubber transferases) to link individual isoprene monomers into a polymer. The monomer used primarily in a cis-1,4-polyisoprene chain is isopentenyl pyrophosphate (IPP), which is also the isomer of its more reactive counterpart, dimethyl allyl pyrophosphate (DMAPP). In order to initialize chain elongation, prenyltransferase enzymes begin single DMAPP molecule and iteratively add IPP onto the chain, allowing for extension of the polymer. However, to be enzymatically active, cis-prenyltransferase enzymes require a magnesium(II) supplement and small rubber particle protein (SRPP) coenzyme that serve as activators.[14] Without a magnesium (II) supplement and SRPPs, prenyltransferases are incapable of polymer chain extension. Therefore, in order to polymerize rubber chains, the key xenogeneic components our host organism will need include (1) prenyltransferases, (2) IPP substrate, (3) DMAPP substrate, (4) a magnesium(II) supplement (such as MgSO₄), and (5) small rubber particle protein coenzymes.

To optimize the MEP/DOXP pathway, we overexpressed reaction rate limiting enzymes in the pathway, namely DXP synthase (DXS). Although we could have overexpressed all of the enzymes in the MEP/DOXP pathway, from prior research DXS was determined to be the rate limiting step in the metabolic pathway.[13] To control for DXS expression, we codon optimized and reintroduced the DXS enzyme into E. coli under an IPTG inducible constitutive promoter (DXS plasmid), allowing for regulated production of DXS (Figure 2). A supplement of IPTG would then activate high level expression of DXS, which in turn would override the pathway bottleneck at the beginning of the MEP/DOXP pathway. In accelerating the process limiting chemical conversion of G3P and pyruvate to 1-Deoxy-D-xylulose 5-phosphate (DXP or DOXP), we can subsequently receive a greater output of IPP and DMAPP downstream.

In order to design our optimized DXS synthase, we obtained the sequence for the DXS gene from EcoGene (EG13612). To allow for future protein purification and characterization, we also attached a FLAG, tetracysteine, and hexahistidine tag to the DXS gene. A double terminator, (BBa_B0010 and BBa_B0012) was also used to prevent RNA polymerase leak through.[19] Because the construct was too large to synthesize as one part, we fragmented the gene into two parts and used Gibson Assembly to insert them into linearized pSB1A3. This resulted in a plasmid with the full DXS Synthase gene and ampicillin resistance, which was then transformed into T7 express cells.