Background

Waste Management

In 2012 the global population passed 7 billion. It is estimated that it will reach 8 billion in 2026 and 9 billion around 2040. The increasing population of the world brings with it a number of challenges, not the least of which is management of the increasing waste generation.

Europe, and Denmark in particular, have worked on this for many years. One of the major goals of the European Union is to work towards a circular economy. A circular economy, as the EU defines it, is an economy with zero waste. This, of course, doesn't mean that waste is eliminated, merely that the entirety of our waste is recovered and recycled.

Denmark has been recycling and recovering waste for a long time. For more than a century, Denmark has incinerated waste and for many of those years, it has been one of the major heat and electricity sources. In recent years, great strides have been made towards recycling still greater amounts of waste. In 2015, 35% of our waste was recycled¹. Exact numbers for recovery are unfortunately unavailable, as the Danish system of incinerating both for energy recovery and to get rid of waste muddies the picture significantly, but the number likely exceeds 50%¹.

The EU(28) recovers approximately half of their waste, excluding energy recovery⁴. The greater part of the remaining waste is incinerated⁴.

Diabetes

With the rising population, the number of diabetics worldwide is increasing as well. The International Diabetes Federation estimates that in 2015, there were 415 million adults with diabetes⁶. This is estimated to rise to 642 by 2040⁶.

Insulin demand

Assuming that an average diabetic needs 15 units of insulin daily to live symptomfree, the global insulin demand would yearly be approximately 79 thousand tons of pure crystaline insulin and rise to more than 120 thousand tons by 2040. $$\frac{\text{Number of diabetics }\cdot 15 \text{ units day}^{-1} \cdot 365 \text{days}}{288118443.804 \text{ units ton}^{-1}} = \text{ Insulin demand (tons)} $$

Insulin production

In their annual report, Novo Nordisk estimates that of the 415 million adults with diabetes, only 6% receives full care⁵. However, with increasingly broad access to drugs in areas of the world where expensive medication like insulin have previously been unavailable, this number can be expected to increase.

Insulin is produced using glucose as a substrate. Glucose is commonly refined from starch, which again is refined from eg. potatoes or corn. This means that arable land and food potentially fit for human consumption is being used to produce medication instead of feeding the ever increasing population. An approximation of the demand for arable land, starch and crops can be calculated with the expressions below^3,4:

$$ \frac{\text{Insulin Demand}}{0.05 \frac{\text{ton insulin}}{\text{ton glucose}}}\cdot 1 \frac{\text{ton starch}}{\text{ton glucose}} = \text{Starch demand} $$ $$ \frac{\text{Starch demand}}{0.17}\frac{\text{ton starch}}{\text{ton potato}} = \text{Potato demand (tons)}$$ $$ \frac{\text{Starch demand}}{0.34}\frac{\text{ton starch}}{\text{ton corn}} = \text{Corn demand (tons)} $$ $$ \frac{\text{Potato demand}}{17.4 \frac{\text{tons}}{\text{ha}}} = \text{Area demand (ha)} $$ $$ \frac{\text{Corn demand}}{5 \frac{\text{tons}}{\text{ha}}} = \text{Area demand (ha)} $$

Using these and the estimations of insulin production gives the following demands^3,4,5,6:

Year	Corn (kilotons)	Area (Corn) (km$$^2$$)	Potato (kilotons)	Area (Potato) (km$$^2$$)
2015	4637.808	9275.61	9275.616	5330.81
2040	7174.633	14349.26	14349.266	8246.70

Our Project

Yeastilization, this year's DTU Biobuilders project, focuses creating a combined solution of the worlds future excessive consumption of crops for non-feed purposes and increasing waste generation. By using a non-traditional yeast, Yarrowia liplytica, as chassis, we provide a new avenue for production of biotech products. Y. lipolytica has a broader substrate range than the traditional workhorse of the biotech industry, S. cerevisiae, which allows for the use of industrial biproducts instead of primarily produced glucose.

Using organic waste from industry instead of glucose from the agricultural industry to produce insulin (or other Biotech products) could potentially open up massive areas of arable land for food production. With a rising population, increase of food production is absolutely essential and every bit counts.

Aim

This project aims to develop the chassis for a versatile and efficient cell factory that can transform abundant waste streams into valuable products.

Methods

1. Substrate screening

To confirm the ability of Y. lipolytica for efficient utilization of an impure mixture of compounds, various waste streams will be investigated as a substrate. We chose mixtures of fats, present in biodiesel waste or vegetation waters from rapeseed oil production, as well as sugars, which are present in molasses or brewery waste. In order to demonstrate the versatility of Y. lipolytica, we are going even further and ferment homogenized household waste.

2. Product

As a proof of concept we aim to demonstrate the production of both an extracellular heterologous protein and an engineered metabolite, and show how a valuable product can be produced by our cell factory utilizing waste streams. We will implement a codon optimized version of the human proinsulin gene along with a native Y. lipolytica promoter and secretion signal into Y. lipolytica. Using an already constructed plasmid with S. cerevisiae optimized genes from the bacterium Erwinia uredovora encoding four enzymes, we will implement the biosynthesis pathway of beta-carotene in Y. lipolytica by using the K152005 biobrick.

3. Molecular toolbox

This project tries to solve the lack of the well-proven tools for Y. lipolytica. We will develop a standardized genetic toolbox, including CRISPR/Cas9-mediated genome editing. The molecular toolbox will bring new opportunities such as an introduction of new pathways, adjusting waste utilization and targeting genetic manipulations.

References

1. Miljøstyrelsen, Affaldsdatasystemet. Available at: ads.mst.dk [Accessed October 17, 2016].
2. Miljøstyrelsen, ISAG. Available at: http://www2.mst.dk/databaser/isag/Default.asp?advanced=No [Accessed October 17, 2016].
3. Food and Agriculture Organisation of the United Nation, FAOSTAT. Available at: http://faostat3.fao.org/home/E [Accessed October 18, 2016].
4. The European Union, Eurostat. Available at: http://ec.europa.eu/eurostat [Accessed October 17, 2016].
5. Novo Nordisk, 2015. Annual Report 2015.
6. International Diabetes Federation, 2015. Diabetes Atlas 7th ed.