# Team:DTU-Denmark/description

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Project Description

# YeastilizationThe global population has been increasing constantly for the past several decades and this trend shows no sign of stopping. With this comes a number of problems, which we will describe in the following sections and seek to address in our project.

## 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, does not 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, the Danish insulin manufacturer, 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 below3,4:

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

Using these and the estimations of insulin production gives the following demands3,4,5,6:

Year Corn (kilotons) Area (Corn) (km2) Potato (kilotons) Area (Potato) (km2)
2015 4637.808 9275.61 9275.616 5330.81
2040 7174.633 14349.26 14349.266 8246.70

### Increasing Food Consumption

With the increasing population comes the obvious problem of how to deal with production of food for everyone. Since the 1960's the worldwide population has more than doubled. This has brought with it a natural increase in production of crops. This has traditionally been done through expansion of the arable land7.

Worldwide 1961 to 2005 2005 to 2050
Population $$\Uparrow 103\%$$ $$\Uparrow 38\%$$
Crop Production $$\Uparrow 148\%$$ $$\Uparrow 70\%$$
Arable Land $$\Uparrow 14\%$$ $$\Uparrow 5\%$$

Much of the population expansion has been centered around the developing countries, where the population has increased by 135% from 1961 to 2005 and the crop production has increased by 255% in the same period7.

Developing Countries 1961 to 2005 2005 to 2050
Population $$\Uparrow 135\%$$ $$\Uparrow 48\%$$
Crop Production $$\Uparrow 255\%$$ $$\Uparrow 97\%$$
Arable Land $$\Uparrow 34\%$$ $$\Uparrow 13\%$$

Presently around 12% of the global land surface is used for crop production. This represents approximately 36% of the land estimated to be to some degree suitable for crop production. According to the Food and Agriculture Organisation of the United Nation, this is estimated to increase by 0.1% yearly until 2050, which gives a total of 5% increase7.

Developed Countries 1961 to 2005 2005 to 2050
Population $$\Uparrow 34\%$$ $$\Uparrow 2\%$$
Crop Production $$\Uparrow 63\%$$ $$\Uparrow 23\%$$
Arable Land $$\Downarrow 1\%$$ $$\Downarrow 7\%$$

## Our Project

Yeastilization, this year's DTU Biobuilders project, focuses on creating a combined solution of the worlds future excessive consumption of crops for non-feed purposes and increasing waste generation. By using a non-conventional 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, Saccharomyces cerevisiae, which allows for the use of industrial biproducts instead of primarily produced glucose as substrate for fermentation.

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.

## Methods

1. Substrate screening
2. To confirm the the superior waste utilisation potential of Y. lipolytica over S. cerevisiae, we aim to analyse growth of both chassis on pure substrates and various waste streams from indsutry. We also aim to provide an insight into these waste sources and investigate the advantages and disadvantages of the substrates.

3. Molecular toolbox
4. In order to allow for heterologous protein production in Y. lipolytica, we aim to develop molecular tools for Y. lipolytica. We aim to develop a standardized genetic toolbox, including a BioBrick plasmid and CRISPR/Cas9-mediated genome editing. The molecular toolbox will bring new opportunities such as an introduction of single proteins and entirely new pathways into this non-conventional chassis. The toolbox aims to standardise synthetic biology in Y. lipolytica and facilitate a broad use of the chassis for waste utilisation and other applications.

5. Products
6. As a proof of concept, we aim to demonstrate the expression of a heterologous protein in Y. lipolytica by producing a codon-optimized version of Renilla reinformis green fluorescent protein (hrGFP) in Y. lipolytica under the regulation of a native Y. lipolytica promoter. Additionally, we aim to translate this into production of human proinsulin in Y. lipolytica.

7. Compute
8. To maximize the chance and extent of success at the products group in producing insulin and other heterologous compounds, we aim to analyze all the relevant sequences for restriction sites and suboptimal codons and return the sequences in codon-optimized form. Furthermore, we will use beta-carotene as an example for applying genome-scale modeling aiming to predict the optimal substrate conditions for maximization of production. Finally, we build a combined fermenter and OD600-measuring device controlled by an Arduino, in order to facilitate easy and fast inoculations with plenty of data to analyze, eventually helping the other groups obtain results.

## 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.
7. Conforti, P. ed., 2011. Looking ahead in world food and agriculture: Perspectives to 2050, Food and Agriculture Organisation of the United Nations.