Team:Manchester/Aimtest2

Introduction

Palm oil is a huge business. From food products such as margarine and chocolate, to cosmetics and even applications in biodiesel, palm oil is the most widely used vegetable oil in the world, and its demand is only increasing. But what most of the manufacturers of these products fail to publicly address is the massive devastation caused by ripping up rainforests in order to make room for oil palm plantations. Not only does this mass deforestation damage the planet by increasing carbon emissions and decreasing the amount of CO₂ taken up from the atmosphere, it also destroys the homes and habitats of thousands of endangered animals, including orangutans and tigers. Additionally, many rainforests are found growing atop peatlands, and so it follows that when they are ripped up or burned, colossal amounts of CO₂ are released. CO₂ is of course a greenhouse gas and so obviously this has a big impact on the extent and rate of global warming. We discuss the devastation associated with the palm oil industry extensively over on our Ethics pages.

Palm oil is made up of four major components: palmitic acid (44%), stearic acid (4.5%), oleic acid (39.2%), and linoleic acid (10.1%). These four fatty acids are fractionated from crude palm oil and used in various different applications according to their degree of saturation- stearic acid is used primarily in cosmetics, where oleic acid is an important oil used in cooking, for example. The biosynthesis of fatty acids has been studied extensively in E. coli, which makes this the ideal chassis for our project.

Our aim is to overexpress the pathway of naturally occurring saturated fatty acids (palmitic (C16:0) and stearic acid (C18:0)) to result in a net increase in their production, and then insert non-native genes to introduce double bonds in stearic acid, to yield oleic (C18:1, Δ9) and linoleic acid (C18:2, Δ9,12). Producing the most widely used and valuable components of palm oil in this way would render the widespread deforestation directly associated with the palm oil industry redundant, and so would halt this relentless destruction of the environment.

FAS module, Delta 9, Delta 12, fadD knockout

Homogenisation vs. exogenous feeding

One batch to rule them all?

Results

Future work: What’s next for Synthetic Palm Oil?

Whilst our iGEM Project is over, we looked at a variety of ways to build onto our project in the future.

Beta-Carotene Enhanced Palm Oil

It may be possible to optimise the palm oil produced to provide a healthier alternative to traditionally produced palm oil. One such example of this would be the introduction of beta-carotene into the palm oil component mix.
Beta-carotene is a precursor to vitamin A. Vitamin A is essential for a variety of health aspects – from eyesight to a strengthened immune system. Vitamin A can prove toxic at high levels, which is why introducing beta-carotene would be a safe alternative. The human body only converts sufficient beta-carotene to reach optimum vitamin A levels – preventing a potentially toxic excess of vitamin A.
This idea could potentially make use of the work Uppsala University (https://2013.igem.org/Team:Uppsala), who have been working on genetically engineering beta-carotene for their project this year.
Implications for the taste and appearance of this optimised palm oil alternative may however make this goal hard to achieve. Many food production companies would be keen to derive the benefits of beta-carotene enhanced palm oil but would be unlikely to implement such a product if changes to appearance and taste occurred as a result of using beta-carotene enhanced palm oil.

Waste paper as a source of Glucose

After attending the 2013 YSB 1.0 London conference and speaking to the UCL Academy team (https://2013hs.igem.org/Team:UCL_Academy) we were really interested in how we might find an alternative feed stock to glucose. Achieving this would dramatically decrease the running costs of any palm oil production plant, as glucose is one of the major cost factors in starting materials for production of our synthetic palm oil. The UCL Academy team worked to produce a cellulase BioBrick (due to be submitted to the registry in 2014) which would break down particular cellulose fibres in paper structure, producing glucose. Not only would this reduce the cost of producing our palm oil, but would also aid in the disposal of waste paper.

Delta 12

In 2012 the St Andrews iGEM Team (https://2012.igem.org/Team:St_Andrews) aimed to achieve a similar goal to our team, to produce a synthetic Omega-3 fatty acid. Their pathway included Delta 12 desaturase, an enzyme we required in the conversion of oleic acid to linolelic acid. St Andrews had produced the Delta 12 BioBrick and submitted this to the Registry – which provided an invaluable opportunity to use existing components from the Registry. Unfortunately when we requested this part from the iGEM Registry they were unable to send us this BioBrick as it would not transform, and after contacting the University they were also unable to provide us with this BioBrick.

As Delta 12 desaturase is an essential enzyme in the production of our Palm oil substituents we therefore attempted to re-produce the Delta 12 biobrick submitted by St. Andrews with improvements such as codon optimisation for E.coli BL21 (DE3).

Creating this system in Algae

During our project planning we evaluated the prospect of genetically engineering algae, such as cyanobacteria in order to produce our fatty acid products. The advantages of this would include being able to use light as a source of energy, significantly reducing the expense of media required - in particular glucose. The gene sequences for Delta 9 and Delta 12 desaturases are also not-native in E.coli, so these were obtained from a cyanobacteria. Whilst the cyanobacteria is prokaryotic, there was potential risk for incorrect protein folding when expressing these genes in E.Coli.
Unfortunately because of the slow growth rate of cyanobacteria we were unable to use this as a chassis for production of our fatty acids. Further, as an iGEM requirement all BioBricks must function in E.coli - so we would potentially have to repeat our project in two different organisms in order to satisfy this criteria.
In the future we would like to see our system created in algae to derive the potential benefits discussed.