Difference between revisions of "Team:Sydney Australia/Description"

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THE PROBLEM

In 1859 the French chemist Charles-Adolphe Wurtz first chemically synthesised ethylene oxide (also known as oxirane) from ethylene chlorohydrin and aqueous potassium hydroxide, unlocking a compound that has been extensively utilized in the pharmaceutical, medical, and manufacturing industry. From sterilising medical equipment to producing ethylene glycol (antifreeze), and as a versatile intermediate for pharmaceutical products, ethylene oxide is essential in many products we use. ¹ Indeed, in 1992, the world production of ethylene oxide was about 9.6 x 10⁶ metric tons, and that number has no doubt been increasing. ²

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Traditionally, ethylene oxide has been produced through a chlorohydrin process that was replaced by the complete oxidation, pioneered by Theodore Lefort in 1931.⁴ Since the production of ethylene oxide, chemists have successfully synthesised other epoxides including propylene oxide, and indene oxide. Epoxides are incredibly versatile due to the addition of the oxygen atom on the tetrahedral ring making the compound strained and unstable. ⁵ Consequently, the ring can be easily opened up releasing abundant amounts of energy that can be used in further reactions such as nucleophilic addition, hydrolysis and reduction.

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Unfortunately, the current chemical synthesis of epoxides are inefficient, expensive and uses hazardous and non-renewable reagents. The previous chlorohydrin process generated chlorine-containing by-products such as calcium chloride resulting in pollution. The present oxygen-based oxidation process, whilst more efficient than the chlorohydrin process has its downfalls. The silver catalyst is highly selective and thus ages quickly, the reaction requires incredibly pure oxygen (99% or greater), and toxic by-products such as formaldehyde are generated. ⁷ Furthermore, the main disadvantage is the lower yield or selectivity of ethylene oxide and the loss of 20-25% of the ethylene to carbon dioxide and water, and thus the conditions must be tightly controlled to ensure maximum selectivity, making this a high-maintenance process. ⁸

In a time where the demand for ethylene oxide is increasing we simply cannot afford to continue using this inefficient, environmentally damaging, and expensive method of chemical synthesis. Thus, the Sydney iGEM team is attempting to utilise the monooxygenase enzymes found in Mycobacterium Smegmatis to perform this reaction in a more efficient and safe manner, for a fraction of the cost.

THE SOLUTION

Monooxygenase enzymes are capable of performing this epoxide reaction, thus converting alkenes to epoxides in a safer and more efficient manner. These biological catalysts are renewable, non-toxic, biodegradable, and can be scaled up for large-scale production. Additionally, with larger alkenes, the product will be highly enantiomerically-enriched epoxides of predomoinantly only one optimal isomer, a trait crucial for the manufacture of bioactives such as pharmaceuticals.

We are focusing on the enzyme Ethene Monooxygenase, due to its ability to catalyse the ethylene to ethylene oxide reaction. To date it has only been found in Mycobacterium Smegmatis. Mycobacteirum is a difficult bacteria to work with as they are slow growing and difficult to work with. Consequently, the team is attempting to achieve high levels of expression in Escherichia Coli. If successful, with the oxidation of ethylene, we will attempt to oxidise indene creating extremely useful products.

PAST WORK

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AIMS

The aims of this project fall into two components –

1. To express high levels of ethene monooxygenase in Escherichia Coli

2. To show the oxidation of ethylene and other alkenes to their respective epoxides

DESIGN

In order to achieve successful expression of Ethene Monooxygenase in E. Coli, we decided to firstly use Pseudomonas Putida as a “stepping stone” to E. Coli. As previous work in the Coleman Laboratory showed, it is possible to get small amounts of expression in Pseudomonas, and thus our first step was to increase the level of expression already present. Once we achieve optimal expression levels then we will look at transferring this expression to E. Coli.

Furthermore, in the synthetic biology and iGEM world, Pseudomonas is a fairly different and novel cloning host, giving our project a unique aspect.

We hypothesised that the minimal expression in Pseudomonas was due to incorrect protein folding and thus our design sought to remedy this.

We took a three-pronged approach for optimising expression: First, we optimised protein folding by adding chaperones GroES and GroEL to encourage correct protein folding.; Second, we optimised translation through codon harmonisation. This was the major modelling component of our project and more information can be found on the modelling page.; Third, we optimised translation through adding weak ribosome binding sites. This was undertaken as we hypothesised that translation was occurring too fast and thus the protein did not have enough time to fold correctly.

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¹ American Chemistry Council, What is Ethylene Oxide?, 2015, ACC, USA, Accessed 20th of July 2015, http://www.americanchemistry.com/ProductsTechnology/Ethylene-Oxide/What-Is-Ethylene-Oxide.html.

² Sevas Educational Society, Manufacture of Ethylene Oxide, 2007, SES, Accessed 20th of July 2015, http://www.sbioinformatics.com/design_thesis/Ethylene_oxide/Ethylene-2520oxide_Methods-2520of-2520Production.pdf (p. 2).

³United States Environmental Protection Agency, 1986, Locating and estimating air emission from sources of ethylene oxide, viewed 20th of July 2015, http://www.epa.gov/ttnchie1/le/ethoxy.pdf (p. 11).

⁴ Wendt HD, Heuvels L, Daatselarr EV, and Schagen TV, 2014, “Industrial production of ethylene oxide” https://www.alembic.utwente.nl/wp-content/uploads/2014/04/LaTeX-example.pdf.

⁵ Wendt et al, “Industrial production of ethylene oxide”, p.2.

⁶ Rebsdat S, and Mayer, D, 2012, “Ethylene Oxide”, Ullmann’s Encyclopaedia of Industrial Chemistry, Vol 13, pp 548.

⁷ Wendt et al, “Industrial production of ethylene oxide”, p.2.

⁸ Rebsdat S, and Mayer, D, 2012, “Ethylene Oxide”, Ullmann’s Encyclopaedia of Industrial Chemistry, Vol 13, pp 559-554.

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@@ Line 74: / Line 74: @@
 We hypothesised that the minimal expression in Pseudomonas was due to incorrect protein folding and thus our design sought to remedy this.
-We took a three-pronged approach for optimising expression:
+; We took a three-pronged approach for optimising expression:
+: First, we optimised protein folding by adding chaperones GroES and GroEL to encourage correct protein folding.
-First, we optimised protein folding by adding chaperones GroES and GroEL to encourage correct protein folding.
+: Second, we optimised translation through codon harmonisation. This was the major modelling component of our project and more information can be found on the modelling page.
+: Third, we optimised translation through adding weak ribosome binding sites. This was undertaken as we hypothesised that translation was occurring too fast and thus the protein did not have enough time to fold correctly.
-Second, we optimised translation through codon harmonisation. This was the major modelling component of our project and more information can be found on the modelling page.
-Third, we optimised translation through adding weak ribosome binding sites. This was undertaken as we hypothesised that translation was occurring too fast and thus the protein did not have enough time to fold correctly.
 ''ADD: picture of designs --> Lizzie to add''