Difference between revisions of "Team:Stanford-Brown/SB16 BioMembrane Latex"
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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. | 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. | ||
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Revision as of 08:01, 19 October 2016
Abstract
With rising costs in synthetic rubber chemical synthesis, environmental blight, and deforestation diminishing the annual yield of natural rubber plantations, a new alternative for latex production is needed to address its global demand shortfall. To address this issue, we sought to transform the latex synthesis pathway into a single cell organism that could be grown in bioreactors, such as Escherichia coli. Due to its low doubling time and ability to be cultured in bulk, genetically modified E. coli capable of producing latex offer a promising solution for fast, high yield latex production. Through genetic manipulation of the endogenous methylerythritol phosphate (MEP/DOXP) pathway and transformation with rubber production genes from Hevea brasiliensis, we developed a transgenic single cell organism capable of converting glucose into cis-1,4-polyisoprene, the primary chemical constituent in latex. Not only is our modified organism capable of producing cis-polyisoprenes quickly, but also in high yield.
Figure 1: MEP/DOXP pathway. The rate limiting step is the first step, in which pyruvate and G3P are converted into DXP. Through constitutive expression of DXS we are able to accelerate the process and push the equilibrium of the reaction towards favoring the products, IPP and DMAPP.[20]
The problem with production
Produced by the rubber tree Hevea brasiliensis, natural rubber is an emulsion consisting of numerous proteins, starches, sugars, oils, resins, and alkaloids. From this emulsion latex is perhaps the most important product. Used in a wide variety of applications, latex accounts for the highest fraction of technically used elastomers, besides polyesters that consist of medium chain length hydroxyalkanoates (PHAMCL).[1, 2] Additionally, latex exhibits a large stretch ratio and high resilience to repeated stress, which makes it an ideal material for constructing flexible yet durable structures.[3] Because of its structural properties, latex is an ideal material for constructing flexible structures that need to adjust to variable mechanical stresses.
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.
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.
Rubber (Cis-1,4-Polyisoprene) Synthesis Pathway
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.
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.
DXS Synthase Optimization
Although IPP and DMAPP could be supplied to the cell by a media supplement, a more appealing and system incorporated approach is to produce both compounds within the host organism using only glucose. Since IPP and DMAPP are endogenous to many species, we identified the two IPP biosynthesis pathways: the mevalonate (MVA) pathway and methylerythritol phosphate (MEP/DOXP) pathway. While pathways generate IPP and DMAPP from ; however, only the MEP/DOXP pathway is endogenous in E. coli.[15] Through a six step process, the MEP/DOXP pathway converts molecules of pyruvate and glyceraldehyde 3-phosphate (G3P) into IPP and DMAPP (Figure 1). While incorporating the MVA pathway into E. coli would further enhance IPP and DMAPP output, we opted for optimizing the MEP/DOXP pathway to minimize the step count from converting a substrate into IPP.[16, 17] Additionally, since G3P and pyruvate are products of glycolysis, providing a glucose supplement to E. coli would allow for G3P and pyruvate use by the MEP/DOXP pathway without increasing cell stress.[18]
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.
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.
Incorporating rubber synthesis into E. coli
