Difference between revisions of "Team:Stanford-Brown/SB16 BioMembrane Latex"

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<div class="col-sm-7 pagetext-L"><div class="text"><b>INSERT INTRO HERE</b></div>
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<div class="col-sm-7 pagetext-L"><div class="text"><b>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 <i>Escherichia coli</i>.  Due to its low doubling time and ability to be cultured in bulk, genetically modified <i>E. coli</i> 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 <i>Hevea brasiliensis</i>, 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.</b></div>
 
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<h1 class="sectionTitle-R">The problem with production</h1>
 
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<div class="col-sm-7 pagetext-R"><div class="text">In order to appropriate enhanced IPP and subsequently latex precursor production, we took advantage of <i>Escherichia coli</i>'s endogenous MEP pathway, and implemented an HMG-CoA reductase pathway for enhanced IPP production. Both pathways result in the production of IPP and its isomer, DMAPP. However, the HMG-CoA pathway is exogenous to <i>E. coli</i> and was needed for enhanced production of IPP. While the MEP pathway converts pyruvate and G3P into IPP/DMAPP through a seven step process, the HMG-CoA (MVA) pathway converts acetyl CoA into IPP/DMAPP through a six step process. Both pathways employ the use of metabolic products produced and used by glycolysis and cellular respiration (calvin cycle); hence enhanced expression of these pathways is expected to affect cellular growth and development.</div>
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<div class="col-sm-7 pagetext-R"><div class="text">Produced by the rubber tree <i>Hevea brasiliensis</i>, 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 (PHA<sub>MCL</sub>).[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.</div>
 
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<div class="col-sm-12 pagetext last">In the MEP pathway, the enzymes DXR and DXS are noted to be the rate limiting steps in the process, hence further enhanced production can be achieved via transfection of a high copy DXR/DXS gene. Since the rate limiting step in glycolysis is the conversion of oxaloacetate to phosphoenolpyruvate and CO<sub>2</sub> by phosphoenolpyruvate carboxylkinase (PEPCK), including a vector with a high copy number for PEPCK could also enhance production of IPP through provision of more substrate.<br><br>
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<div class="col-sm-12 pagetext last">Currently the only source of commercially usable natural rubber that can be processed into latex is available from the rubber tree <i>H. brasiliensis</i>.  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 <i>H. brasiliensis</i>.[4] In fact, <i>H. brasiliensis</i> 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. <i>H. brasiliensis</i>’ 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]<br><br>
  
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Since the MEP pathway is already present in <i>E. coli</i>, the purpose of transfecting enzymes critical for the MEP pathway into <i>E. coli</i> is to increase the enzyme concentration in the cytosol, which should result in more enzymatic reactions taking place. For this purpose, the plasmid pMevT will be used in conjunction with pMevB to allow for production of Mevalonate from Acetyl-CoA, then IPP from Mevalonate. The following two plasmids are pSB1C3 plasmids with a CAT selection marker and T7 promoter. pMevT encodes the rate limiting enzymes for conversion of acetyl-CoA into Mevalonate, pMevB encodes the rate limiting enzymes for conversion of mevalonate to IPP. These have been adapted from Martin et al. [20].  pSB1C3 DXS consists of the DXS gene (DXP synthase) which is the rate limiting step in the endogenous MEP pathway.<br><br>
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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.<br><br>
  
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For full realization of high throughput IPP production in <i>E. coli</i>, all three plasmids are to be transfected into the host to allow for high expression of all pathway elements. To verify all elements are property transfected, each plasmid must have a distinct selection markers such that with each subsequent transfection, un-transfected cells will be screened against. By transfecting one plasmid at a time into a population of cells with different selection assays, we can ensure that the final population of cells at the end of the process will contain all three genes.<br><br>
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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.<br><br>  
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Another item of concern is the metabolic strain each of these plasmids will place upon <i>E. coli</i>. IPP is a byproduct of glycolysis and cellular respiration; enhanced conversion of pyruvate and G3P or acetyl-CoA to IPP will decrease the amount of substrate available to the host for ATP production. Thus it is advisable to include a genetic switch, such as an IPTG inducible promoter into the plasmids. Other options include Pxtet tTA-On , lacR, lacO which can allow for regulated expression of downstream genes. For instance, lacR downstream of a Pxtet promoter on one construct and tTA-On downstream of a T7 promoter can allow for differential modulation of lacR downstream genes with Atc.<br><br>
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Using the same promoter sequence for each of the plasmids is also unadvisable, as identical promoters can result in homologous recombination (depending on host organism type), roadblocking, and RNAP leak through. The same issue applies to using the same terminators, in which RNAP leak through can occur and result in non-regulated expression of downstream genes. [33] Roadblocking is also an issue, in which a certain subset of repressors, when bound to a downstream promoter stopped by upstream transcription. This however can be easily addressed by moving regulatory elements upstream.<br><br>
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Since Martin et al. has already demonstrated use of  the pMevB and pMevT gene in <i>E. coli</i> for the production of OPP and artemisinin, the only step we need to accomplish is transfection of a metabolic pathway and its enzymes that can convert IPP to cis 1,4 poly(isoprene).
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Prior literature suggests in the rubber tree <i>Havea brasiliensis</i> utilizes a rubber transferase for synthesis of latex polymers. [24]  Additionally, <i>H. brasiliensis</i> rubber transferases require a supplement of glutathione, divalent magnesium ions, and SRPP proteins for production of rubber in detectable quantities. [29]<br><br>
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<i>References</i>
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Revision as of 07:26, 19 October 2016


Stanford-Brown 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.
Latex Production Pathway.

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.